WO2010053522A2 - Methods for identifying and producing neural stem and progenitor cells and their progeny - Google Patents

Abstract

Methods of identifying and producing subpallial progenitor cells are provided. Methods are also provided for differentiating subpallial progenitor cells. Therapeutic uses of the generated cells are further provided.

Description

METHODS FOR IDENTIFYING AND PRODUCING NEURAL STEM AND PROGENITOR CELLS AND THEIR PROGENY

CROSS-REFERENCE

[0001] This application claims the benefit of U.S. Provisional Application No. 61/109,373 filed October 29, 2008 that is incorporated herein by reference in its entirety.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with government support under Contract numbers F32 MH070211 , K05 MH065670, and ROl MH49428 awarded by the National Institutes of Health (NIH). The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

[0003] Gene expression and function studies in mouse and chick provide evidence for a common organization of the developing telencephalon in vertebrates (Smith-Fernandez et al., 1998; Puelles et al., 2000). GABAergic interneurons have a common origin in the subpallium and common mechanisms govern their migration. In human it has been estimated that 65% of GABAergic neurons are born locally in the cortical germinal zone (Letinic and Rakic, 2001). Based on the conserved expression of homeobox-containing and other transcription factors it has been proposed that the subpallium contains four subdivisions: the lateral ganglionic eminence, subdivided into dorsal and ventral part, dLGE, vLGE, respectively, the medial ganglionic eminence (MGE), the caudal ganglionic eminence (CGE), and the septum contributing to the generation of striatum, pallidum and the telencephalic stalk, respectively (reviewed by Marin and Rubenstein, 2001).

[0004] The combination of transcription factors (TFs) that are expressed in a cell are a fundamental signature of its identity. Knowledge of the identities and order of expression of TFs and other genes during organogenesis is essential for understanding the transcriptional networks that operate to control the phenotypic state and developmental potency of the constituent cells, whether during development or in maturity. The TF codes expressed in stem/progenitor and their derivatives in the developing basal ganglia of the subpallium in the mammalian brain, as well as in their progeny and derivatives, are incompletely understood. An understanding the identity and order of influence of this transcriptional hierarchy would provide information useful both for the engineering of stem and progenitor cells to become cells of specific phenotypes and as a means of identifying cells as belonging to a specific developmental stage and/or having a specific developmental potency.

SUMMARY OF THE INVENTION

[0005] Methods of identifying and producing subpallial progenitor cells are provided herein. [0006] Methods are provided for identifying subpallial progenitor cells, subtypes of subpallial progenitor cells, and their progeny. In some aspects, the methods include determining the level of expression in a cell of one or more genes, where the level of expression of the one or more genes in indicative of the presence of a type of subpallial progenitor cell or its progeny. In some aspects, the genes encode transcription factors. [0007] Also provided are methods of determining the developmental status of a cell. In some aspects, the methods include determining the level of expression in a cell of one or more genes. In some aspects, the methods include determining the expression in the cell of one or more genes in a list or table which identifies a pattern of gene expression in a cell during the maturation of a subpallial progenitor.

[0008] Further provided are methods of promoting the differentiation of a stem or progenitor cell of the subpallium. In some aspects, the methods include determining the expression in the cell of one or more genes provided in a list or table which identifies a pattern of gene expression in a cell during the maturation of a subpallial progenitor and exposing the cell to factors in sequence according to the table to promote the differentiation or further differentiation of the stem or progenitor cell.

[0009] Also provided herein are methods of producing subpallial progenitor cells or their progeny. In some aspects, the methods include modulating the level of expression in a cell of one or more genes. In some aspects, the methods include modulating the expression in the cell of one or more genes provided in a list or table which identifies a pattern of gene expression in a cell during the maturation of a subpallial progenitor.

[0010] Methods of identifying an agent that modulates differentiation of a subpallial neural progenitor cell are also provided. In some aspects, the methods include contacting a stem or progenitor cell, in which gene expression has been modulated, with a candidate agent and detecting any alteration in the developmental status of said cell relative to that of a stem or progenitor cell treated according to the same methods, but which is not contacted with the candidate agent, where any alteration is indicative that the agent modulates the differentiation of a subpallial neural progenitor cell.

[0011] Cells produced by any of the herein-described methods are further provided.

[0012] In one aspect in the invention, a method of differentiating a subpallial progenitor cell is provided, the method comprising forcing expression of Gsxl, Gsx2, Ascll, Dlxl, Dlx2, combinations or homologs thereof in said cell. In one embodiment, the method further comprises forcing the expression of one or more additional genes or homologs thereof, selected from Tables 2-4.

[0013] In another aspect in the invention, a method of differentiating a subpallial progenitor cell is provided, the method comprising exposing said progenitor cell to one or more transcription factors produced by the genes Gsxl,

Gsx2, Ascll, Dlxl and Dlx2 or homologs thereof. In an embodiment, the method further comprises exposing said pregenitor cell to one or more additional transcription factors produced by one or more genes selected from Tables

2-4 or homologs thereof. In another embodiment, the progenitor cell is human. In yet another embodiment, said differentiated cell is a LGE-derived nueron, striatal neuron, CGE-derived interneuron, VIP+ , calretinin+/somatistatin- , NPY+ .

[0014] In another aspect, also provided herein is a differentiated cell produced by the methods described above. In one embodiment, the differentiated cell is a LGE-derived nueron, striatal neuron, CGE-derived interneuron, VIP+, calretinin+/somatistatin-, NPY+.

[0015] In a further aspect, provided herein is a composition comprising a differentiated cell derived by the forced expression of Gsxl, Gsx2, Dlxl, Dlx2, Ascll combinations or homologs thereof in a subpallial progenitor cell. In one embodiment, said differentiated cells is further derived by the forced expression of one or more additional genes selected from Tables 2-4 or homologs thereof.

[0016] In an aspect, provided herein is a composition comprising a differentiated cell derived by exposing a subpallial progenitor to one or more transcription factors produced by Gsxl, Gsx2, Dlxl, Dlx2 and Ascll or homologs thereof. In an embodiment, differentiated cells is further derived by exposure to one or more additional transcription factors produced by one or more genes selected from Tables 2-4 or homologs thereof.

[0017] Also provided herein is a method for treating a disorder arising from the loss of number or function of a striatal intemeuron comprising administering to a patient in need thereof, differentiated cells made by the method herein, wherein said cells increase the number or function of striatal interneurons. In one embodiment, said disorder is Huntington's disease, epilepsy, schizoprenia, autism, stroke, Parkinson's Disease, Tourette's Syndrome, dystonia, or Alzheimer's disease.

[0018] In another aspect of the invention, provided is a method for treatment or amelioration of symptoms caused by the imbalance of the excitory/inhibitory neuronal circuitry comprising administering cells produced by the methods described above.

INCORPORATION BY REFERENCE

[0019] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

DESCRIPTION OF THE DRAWINGS

[0020] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

[0021] Figure 1 : £⁾£c/<£2-Dependent Expression of Transcription Factors in LGE Progenitors. In situ hybridization of coronal hemisections through the El 5.5 telencephalon of control and Dlxl&2-/- mutant animals demonstrates •D/x7<£2-dependent expression in the SVZ and MZ of the LGE. For some genes the effect is either exclusive, or predominant, to the dLGE, leaving expression in the vLGE relatively normal (indicated by arrows). In other cases, however, vLGE expression is reduced, (a-d') Note the loss of Dlx5&6 expression in the LGE, but not in the septum. Arrowheads mark the defects in the dSe, despite the maintenance of several transcription factors. dLGE, dorsal lateral ganglionic eminence; dSe, dorsal septum; vLGE, ventral lateral ganglionic eminence; vSe, ventral septum. Scale bars a-r', 500 μm.

[0022] Figure 2: Ectopic Expression of Ventrolateral Cortical Markers in the dLGE. In situ hybridization of coronal hemisections through the E15.5 telencephalon of control and DIxI &2-/- mutants, (a-c') Arrows mark the ectopic expression of three ventrolateral cortical markers (Ebβ, Id2, NHLH2) in the dLGE of the DIxI &2-/- mutant, (d-e'); Id2 is also ectopically expressed in the septum (arrowhead). Two cortical markers (Tbrl, Vglut2) do not show ectopic expression. VPs, ventral pallidum superficial; VPd, ventral pallidum deep. Scale bars a-e', 500 μm. [0023] Figure 3: Dlxl &2 Repress Expression of Transcription Factors in LGE Progenitors. In situ hybridization of coronal hemisections through the El 5.5 telencephalon of control and Dlxl&2-/- mutants show transcription factors whose expression is up-regulated in the LGE, particularly in the SVZ. (a-e'): Genes whose expression is normally detected only in the VZ; (f-n'): Genes whose expression is normally detected in the VZ and SVZ of the LGE. Scale bars a-n', 500 μm.

[0024] Figure 4: Dlxl &2-/- Mutants Have Ectopic LGE and Septal Expression of Transcription Factors That Normally Mark the MGE and/or Diencephalon. In situ hybridization of coronal hemisections through the El 5.5 telencephalon of control and Dlxl&2-/- mutants, (a-e') Several transcription factors that are normally not expressed in the LGE or Septum are ectopically expressed in the Dlxl&2-/- mutant animals. This includes markers of the MGE {Gbxl, Gbx2, and Gshl) and diencephalon (Otp). Scalebars a-e', 500 μm.

[0025] Figure 5: D/x-Dependent and Independent Transcription Factor Expression in Maturing Striatal Neurons. In situ hybridization of coronal hemisections through the E15.5 telencephalon of control and Dlxl&2-/- mutants, (a-f ) Transcription factors whose expression is severely reduced in the DIx I &2-/- mutants, (g-v') Transcription factors whose expression is partially reduced/maintained. (h',i') Arrowheads show reduced expression in the dorsal septum. dStr, dorsal striatum; OT, olfactory tubercle, Se, septum; vStr, ventral striatum. Scale bars a-v', 500 μm. [0026] Figure 6: Dϋx-Dependent and Independent Expression of Non-Transcription Factor Markers of Striatal Differentiation and Migration. In situ hybridization of coronal hemisections through the E 15.5 telencephalon of control and DIx 1 &2-A mutants, (a-o') Non transcription factors whose expression is decreased in the striatum, (p-w') Non transcription factors whose expression is maintained or slightly increased in the striatum. dStr, dorsal striatum; OT, olfactory tubercle, Se, septum; vStr, ventral striatum. Scale bars a-w', 500 μm. [0027] Figure 7: DIxI &2-/--JrfashI-/- Compound Mutants Define Epistatic Relationships in dLGE, vLGE and Septal Differentiation. In situ hybridization of coronal hemisections through the E15.5 telencephalon of control, DIxI &2-/-, Mashl-I- mutants and DM&.2 -I -Mashl-I- mutants, (a'-p') The Dlxl&2-I- mutant shows severe dLGE differentiation defects and mild vLGE and septum defects (e.g. Sp8 n, n')- (a"-p") The Mashl-I- mutant animal shows severe septal vLGE defects and mild dLGE defects (e.g. Sp9 b,b"). (a'"-p'") The Dlxl&2-I--Jrfashl-I- mutant animal demonstrates the cooperative roles of by DIxI &2 and Mash 1 in dLGE, vLGE and septal development due to the aggravation of the individual mutant phenotypes. Arrows mark remnants of normal LGE gene expression in the Dlxl&2-I-M- ^hl-I- mutants. dStr, dorsal striatum; OT, olfactory tubercle, Se, septum; vStr, ventral striatum. Scale bars a-ppp"', 500 μm.

[0028] Figure 8: Z)ZxV dL2-Dependent Expression in the LGE and Septum at E12.5. In situ hybridization of coronal sections through the E12.5 rostral telencephalon of control and Dlxl&2-/- mutant animals. dLGE, dorsal lateral ganglionic eminence; dSe, dorsal septum; vLGE, ventral lateral ganglionic eminence; vSe, ventral septum. Scale bars a-bb', 500 μm.

[0029] Figure 9: Dlxl&2 and Mashl Dependent Expression in the LGE and Septum at E15.5. In situ hybridization of coronal hemisections through the E15.5 rostral telencephalon of control, Dlxl&2-/- and Mashl-/- animals. dLGE, dorsal lateral ganglionic eminence; dSe, dorsal septum; vLGE, ventral lateral ganglionic eminence; vSe, ventral septum. Scale bars a-q'", 500 μm.

[0030] Figure 10: Transcription factors whose expression is reduced in either the LGE/MGE (left pair) or CGE (right pair), in the DIxI &2-I- mutants as shown by in situ hybridization on coronal hemisections from E15.5 forebrains. Control: left section; DIxI &2-I-: right section. Abbreviations: CGE, caudal ganglionic eminence; LGE, lateral ganglionic eminence; MGE, medial ganglionic eminence; POA, pre-optic area; Magnification Bar: 500μm. [0031] Figure 11 : Transcription factors whose expression is increased in either the LGE/MGE (left pair) or CGE (right pair), in the Dlxl&2-I- mutants as shown by in situ hybridization on coronal hemisections from E15.5 forebrains. Control: left section; Dlxl&2-/-: right section. Abbreviations CGE, caudal ganglionic eminence; LGE, lateral ganglionic eminence; MGE, medial ganglionic eminence; POA, pre-optic area; Magnification Bar: 500μm. [0032] Figure 12: Non-transcription factors whose expression is altered in either the LGE/MGE (left pair) or CGE

(right pair), in the Dlxl&2-I- mutants as shown by in situ hybridization on coronal hemisections from E15.5 forebrains. Control: left section; Dlxl&2-/-: right section. Magnification Bar: 500μm.

[0033] Figure 13: Genes whose expression marks ectopia in the CGE or MGE, in the Dlxl&2-I- mutants, as shown by in situ hybridization on coronal hemisections from E15.5 forebrains. Control: left section; DM&2-I-: right section. Magnification Bar: 250μm.

[0034] Figure 14: Genes whose expression is altered in either the LGE/MGE (left set of sections) or CGE (right set of sections) DIxISH-I-, Mashl-I-, or Dlxl &2\Mash 1-1- mutants as shown by in situ hybridization on coronal hemisections from El 5.5 forebrains. Magnification Bar: 500μm.

[0035] Figure 15: Ascll expression in E 15.5 Gsx2^~'^~, DIx 1/2 ^~'^~ single and Gsxl'^'; Dlxl/2^'A compound mutants.

Coronal hemisections through the telencephalon. Top tier: rostral telencephalon, SE/LGE level; Middle tier: middle telencephalon, LGE/MGE level; Bottom Tier: caudal telencephalon, CGE level. Abbreviations: CGE, LGE and

MGE: caudal, lateral and medial ganglionic eminence, respectively. D: diencephalon; NCx: neocortex; SE: septum.

[0036] Figure 16: Ascll expression in E15.5 Gsxl^'1', Dlxl/2 ^A single and Gsxl^''^'; Dlxl/2^'1' compound mutants. See legend to Figure 2 for figure organization and labeling. VZ: ventricular zone, SVZl: subventricular zone 1; SVZ2: subventricular zone 2; MZ; mantle zone.

[0037] Figure 17: Dlxl expression in E15.5 GsxZ'^', Dlxl/2 ^~'^~ single and Gsx2^~'^'\ Dlxl/2^'1' compound mutants. See legend to Figure 2 for figure organization and labeling.

[0038] Figure 18: Dlxl expression in E15.5 Gsxl '^', Dlxl/2 ^A single and Gsxl '^'; Dlxl/2^''^' compound mutants. See legend to Figure 2 for figure organization and labeling.

[0039] Figure 19: GADl expression in E 15.5 GsxX^1', Dlxl/2 ^''^' single and Gsxl^1'; Dlxl/2^''^' compound mutants.

See legend to Figure 2 for figure organization and labeling.

[0040] Figure 20: GADl expression in E15.5 Gsxl '^', Dlxl/2 ^''^' single and Gsxl"'^'; DM/2^'1' compound mutants.

See legend to Figure 2 for figure organization and labeling.

[0041] Figure 21 : Schema proposing a transcriptional circuit/hierarchy in the LGE. Arrows: positive regulation; red box: inhibition. Asterisk (^♦): Strongly upregulated in dorsal LGE.

DETAILED DESCRIPTION OF THE INVENTION

[0042] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are described in the literature. See, for example, Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1989); DNA Cloning, Volumes I and II (D. N. Glover ed., 1985); Oligonucleotide Synthesis (M. J. Gait ed., 1984); Mullis et al. U.S. Pat. No. 4,683,195; Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds. 1984); Transcription And Translation (B. D. Hames & S. J. Higgins eds. 1984); Culture Of Animal Cells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); the treatise, Methods In Enzymology (Academic Press, Inc., N. Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Methods In Enzymology, VoIs. 154 and 155 (Wu et al. eds.), Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes HV (D. M. Weir and C. C. Blackwell, eds., 1986); Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y., 1986).

[0043] It must be noted that as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to "a cell" includes a plurality of such cells and so forth. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any materials and methods similar or equivalent to those described herein can be used to practice or test the present invention, the preferred materials and methods are now described.

[0044] Overview

[0045] Described herein are methods of identifying and producing precursor cells and their progeny. Precursor cells as defined herein, include stem cells and progenitor cells. Progeny cells include cells that naturally differentiate from precursor cells once the precursor cells are introduced into the appropriate cellular environment. Progeny cells further include cells that are further generated in vitro from precursor cells. Also provided are methods of determining the developmental status of a cell and methods of differentiating a precursor cell to form a differentiated cell. Methods of identifying an agent that modulates differentiation of a precursor cell into a differentiated cell are also included. As described herein, the cells and agents are also useful in method of treatment for various disorders, diseases, or conditions using the precursor cells, differentiated cells, and agents that modulate differentiaion.

[0046] Methods of identifying precursor cells

[0047] In one aspect herein, methods of identifying and producing precursor cells are provided. Precursor cells include but are not limited to subpallial progenitor cells, subtypes of subpallial progenitors such as those found in the septum, large ganglionic eminence (LGE), medial ganglionic eminence (MGE), caudal ganglionic eminence (CGE), and preoptic areas (POA), each of which has multiple subdivisions, and their progeny. Precursor cells also include stem cells and progenitor cells. In one embodiment, the forebrain basal ganglia is a type of stem cell. [0048] In some embodiments, the methods include determining the level of expression in a precursor cell of one or more genes, where the level of expression of the one or more genes in indicative of the presence of a type or subtype of precursor cell (e.g. subpallial progenitor cell or its progeny). In some aspects, the genes encode gene products for transcription factors. In some embodiments, the genes comprise genes listed in Tables 2-4. [0049] All subpallial progenitors share a common default transcription factor code which establishes the potency to produce the subtypes of subpallial progenitors including those of the septum, large ganglionic eminence (LGE), medial ganglionic eminence (MGE), and preoptic area, each of which has multiple subdivisions (Yun et al., 2001; Campbell, 2003; Flames et al., 2007; Long et al., 2007). Each subpallial progenitor subtype can further be specified by the expression of additional transcription factors. As disclosed herein, in the default subpallial progenitor state, the expression of transcription factors Mashl (equivalent to Ascll), Foxgl, Otx2, Olig2, Arx, Dlxl/2, Sp9 and Vaxl are evident. Dorsal LGE progenitors further express elevated levels of transcription factors Brn4, Dlxl/2 Er81, Esrg, Gsh2 (equivalent to Gsx2), Meisl, Oct6, Pbxl, Pbx3 Six3, Sp8, Sp9 and Tshzl, whereas ventral LGE progenitors further express elevated levels of GIi 1, Gshl (equivalent to Gsxl), Mashl, Otx2 and Vaxl. MGE progenitors further express Nkx2.1, Nkx6.2, Lhx6 and Lhx7(8). Preoptic area progenitors additionally express Nkx2.1, Nkx5.1 and Nkx5.2. Septal progenitors express Zicl, Mashl and Vaxl in addition to the default subpallial transcription factor profile.

[0050] In some embodiments of the present method, the expression of one or more transcription factors listed in Tables 2-4 is indicative of the presence of a precursor cell (such as a subpallial progenitor or subtype of subpallial progenitor). In some embodiments, the level of expression of at least two, three, four, five, six, seven, eight, nine, ten, fifteen or twenty transcription factors herein identified is characteristic of a precursor cell. In some embodiments, determining the level of expression includes detecting RNA level of the genes. Any suitable qualitative or quantitative methods known in the art for detecting specific mRNAs can be used. mRNA can be detected by, for example, hybridization to a microarray, in situ hybridization in tissue sections, by reverse transcriptase-PCR, or in Northern blots containing poly A+ mRNA. One of skill in the art can readily use these methods to determine differences in the size or amount of mRNA transcripts between two samples. For example, the level of particular mRNAs in cells contacted with agent is compared with the expression of the mRNAs in a control sample or a sample isolated from a different developmental stage or anatomical region. [0051] In some embodiments, determining the level of expression includes detecting the level of gene product, e.g. the polypeptide expressed from the genes. Accordingly, in another method of detecting level of expression, the cell is assayed at the protein level. For example, detection may utilize staining of cells or histological sections with labeled antibodies, performed in accordance with conventional methods. The presence or absence of antibody binding may be determined by various methods, including flow cytometry of dissociated cells, microscopy, radiography, scintillation counting, etc. Any suitable alternative methods of qualitative or quantitative detection of levels or amounts of differentially expressed polypeptide can be used, for example ELISA, western blot, immunoprecipitation, radioimmunoassay, etc. [0052] Methods of determining the developmental status of a cell

[0053] Also provided are methods of determining the developmental status of a cell. In some embodiments, the methods include determining the level of expression in a cell of one or more genes selected from the genes listed in Tables 2-4. In some embodiments, the methods include determining a pattern of gene expression in a cell. Gene expression may vary temporally during the maturation of a precursor cell, such as a subpallial progenitor, from a stem cell.

[0054] In some embodiments, tables are provided which categorize the genes expressed early and late during the developmental trajectory of subpallial progenitors. The cell kinetics of the developing central nervous system is determined by both proliferation and apoptosis. In the human neocortex, for example, at week 6 of gestation, proliferation is confined to the ventricular zone, where mitotic figures and nuclear immunoreactivity for proliferating cell nuclear antigen (PCNA) are detectable. Cell division is symmetric, with both daughter cells reentering mitosis. At week 7, the subventricular zone, a secondary proliferative zone, appears. It mainly gives rise to local circuit neurons and glial cells. Around week 12, the ventricular and subventricular zones are thickest, and the nuclear PCNA label is strongest, indicating that proliferation peaks at this stage. Thereafter, asymmetric division becomes the predominant mode of proliferation, with one daughter cell reentering mitosis and the other one migrating out. During the early stages of development, the lateral wall of the ventricle forms a continuous semicircular sheet with no obvious regional specialization. T he first evidence of differentiation is the ventrolateral appearance of a dome-shaped elevation protruding into the ventricular cavity. This elevation becomes divided by a sulcus into a lateral and a medial part, the LGE and MGE, respectively (Smart and Sturrock, 1979; Lammers et al., 1980). The sulcus separating the ganglionic eminences from the dorsal part of the ventricle (corticostriatal sulcus) represents the boundary separating two major compartments of the germinal zone. Dorsal to the sulcus, proliferating cells in the ventricular zone (VZ) will give rise to the laminated neocortex. In the embryonic brain, neural stem cells in the ventricular walls feed proliferative zones in the subventriculum. Ventral to the sulcus, the ventricular and subventricular zones will produce cells destined to give rise to the striatum and other paleocortical formations. The DIx homeobox genes, has attracted interest due to their patterns of expression in the forebrain during development. Early in gestation, DIx gene expression in the telencephalon is restricted to the primordia of the basal ganglia, and is excluded from the cerebral cortex (Porteus et al. (1991) Neuron 7:221-229; Bufone et al. (1993) J. Neurosci. 13(7):3155-3172), where its expression is co-extensive with cells producing GABA (Anderson et al. (1997) Neuron 19:27-37). Then, beginning around E12.5, Dlx+/GABA+ cells are found migrating along two tangential pathways that introduce these cells into cortical regions of the telencephalon: a lateral and a medial pathway. The lateral migratory pathway originates in both the lateral and medial ganglionic eminences of the basal ganglia and introduces specific types of Dlx+/GABAergic interneurons in the striatum, olfactory cortex, neocortex and hippocampus (Porteus et al. (1994) J. Neurosci. 14(l l):6370-6383; Anderson et al. (1997) Science 278:474-476). Mice lacking DIx 1 and Dlx2 have a four-fold reduction in the numbers of GABAergic neocortical neurons (Anderson et al. (1997) Science 278:474-476). Others have also identified this lateral pathway, but had not demonstrated GABAergic interneurons in this pathway prior to our discovery (deCarlos et al. (1996) J. Neurosci. 16:6146-6156; Tamamaki et al. (1997) J. Neurosci. 17:8313-8323). Since then other groups have also reported migration along the lateral pathway (Lavadas et al. (1999) J. Neurosci. 19:7881-7888; Wichterle et al. (1999) Nat. Neurosci. 2:461-466.

[0055] The medial migratory pathway (also known as the rostral migratory stream), appears to originate in the region of the lateral ganglionic eminence and septum, and is the source for GABAergic interneurons of the olfactory bulb and perhaps subsets of cortical interneurons. See, Gadisseux et al. (1992) J. Comparative Neurol. 324:94-114; Luskin (1993) Neuron 11:173-189); DeDiego et al. (1994) Eur. J. Neurosci. 6:983-997; Lois and Alvarez-Buylla (1994) Science 271:264:1145-1148; and Meyer et al. (1998) J. Comp. Neurol. 397:493-518. This pathway contains DIx+ cells (Porteus et al. (1994) J. Neurosci. 14(11):6370-6383); Dlxl and Dlx2 mutants have a >95% reduction in the number of GABAergic neurons of the olfactory bulb (Bulfone et al. (1998) Neuron 21:1273-1282). [0056] The lateral pathway, and perhaps the medial as well, seed the proliferative zone (subventricular zone) of the postnatal rodent brain with DIx+ cells. This proliferative zone is known to be the source of postnatal neurogenesis. In particular, this proliferative zone is a source of GABAergic interneurons of the olfactory bulb (Luskin (1993) Neuron 11: 173-189; and Lois and Alvarez-Buylla, (1994) Science 264: 1145-1148).

[0057] As such, the developmental progression of LGE, MGE, CGE, POA, and septal progenitors can be mapped to their serial anatomical presence in ventricular, subventricular and mantle zones. Disclosed herein are tables containing genes encoding transcription factors and other polypeptides expressed in the progenitor zones ordered such that the left two columns contain genes expressed in the proliferative zones (VZ and SVZ, respectively); the right columns contain genes expressed at later developmental stages (SVZ and MZ). See Tables 3, 4, 8 and 9, by way of example. Expression of the genes has been assessed in DIx 1&2, Mashl, or double mutants to establish their epistasis to genes known to be crucial in subpallial specification. Accordingly, the relative levels of perturbation of a given gene in these mutants over the course of a cell's anatomical localization/developmental progression is an indicator of the role of the gene product, e.g., a transcription factor or other polypeptide, in establishing the state of differentiation. The collection of these genes and their results for different subpallial progenitors at different developmental stages thereby constitutes a combinatorial transcription factor code, or signature, which characterizes each progenitor at each stage.

[0058] Accordingly, it is envisioned that, in some embodiments of the presently disclosed methods, the developmental status of a cell can be determined by assessing the level of expression in a cell of one or more genes. In some embodiments the one or more genes are provided in a list, figure or table which identifies a pattern of gene expression in a cell during the maturation of a subpallial progenitor. The level of expression of one or more, up to and including all of the genes in the table or list can be assessed by methods as known in the art. In this way, the anatomical origin and developmental stage of a cell can be determined. For example, identification of a subpallial progenitor is accomplished by consulting, e.g. one or more of Figure 1, Table 3 and Table 4 as provided herein, identifying genes whose expression occurs at the VZ, SVZ, and MZ anatomical/developmental phases of the cell's maturity, and determining the level of expression of those genes in a cell, in addition to those genes which are expressed in all subpallial progenitors, so as to identify e.g. an LGE or septal progenitor. [0059] Methods of producing progenitor cells

[0060] Also provided herein are methods of producing precursor cells such as subpallial progenitor cells or their progeny. In some embodiments, the methods include modulating the level of expression in a cell of one or more genes. In some embodiments, the expression of one or more genes and homologs thereof is forced. Forced expression may include introducing expression vectors encoding polypeptides of interest into cells, introducing exogenous purified polypeptides of interest into cells, or contacting cells with a non-naturally occuring reagent that induces expression of an endogenous gene encoding a polypeptide of interest. In some embodiments, the modulation is positive modulation, i.e., an elevation in the level of expression. In some aspects, the methods include modulating the expression in the cell of one or more genes provided in Tables 2-4 which identifies a pattern of gene expression in a cell during the maturation of a subpallial progenitor, exposing the cell to factors in sequence according to the table to promote the differentiation of the stem or progenitor cell, for example, to a mature GABAergic neuron or other neural cell. The cell can be exposed to gene products in sequence according to the table which will differentiate the cell. For example, consultation of Table 3 reveals distinct groups of transcription factors which are expressed early, during maturation, and late; early LGE genes include those expressed in the stem cell compartment (Erml, GIi 1, Hesl, Hesrl), progressing through TFs expressed in multiple anatomical/developmental compartments, through to genes expressed solely in the medial zone in mature cells (Egr3, Emx2, Evi3, Ikaros, Mef2c, Rarβ, Rxrγ. Therefore, a stem/progenitor cell can be differentiated according to serial exposure to the transcription factors listed in Tables 2-4. In this way, differentiation of a cell can be performed in vivo or in vitro using a cell which occurs in an organism; using a cell, such as a stem cell, which is isolated from an organism; using a cell which is derived in culture from an embryonic stem cell or embryoid body, from an induced pluripotent stem cell, and the like.

[0061] In some embodiments, methods of generating neural cells with an elevated level of efficiency are provided. Protocols which permit the isolation of some specific neuronal cells are known in the art. Such protocols typically involve differentiating stem cells or neural precursors under conditions such that multiple neural subtypes are generated, for example, neurons, multiple neuronal subtypes, astrocytes and oligodendrocytes, microglia, and the like. In contrast, according to the present methods, a stem/progenitor cell can be efficiently directed to differentiate to a specific cell type according to the present methods using serial exposure to transcription factors as provided herein. As such, in the present method the progeny derived from a neural stem/progenitor is substantially the desired cell type; e.g. the present methods yield the desired cell type at a frequency greater than a differentiation protocol wherein the expression of herein-described transcription factors is not modulated. In this way, cells may be derived at a high level of efficiency, where the progeny resulting from the directed differentiation can be at least 10%, sometimes 20%, 30%, 40%, 50% or more, as much or more than 60%, 70%, sometimes 80%, 90%, up to 100% of a desired neural cell subtype.

[0062] In the context of cell ontogeny, the adjective "differentiated" is a relative term. A "differentiated cell" is a cell that has progressed further down the developmental pathway than the cell it is being compared with. Thus, pluripotent embryonic stem cells can differentiate to lineage-restricted precursor cells, such as neural progenitor cells, which are multipotent for neural cell types; and various types of neural progenitors. These in turn can be differentiated further to other types of precursor cells further down the pathway, or to an end-stage differentiated cell, which plays a characteristic role in a certain tissue type, and may or may not retain the capacity to proliferate further. Neurons, astrocytes, and oligodendrocytes are all examples of terminally differentiated cells. Unless explicitly indicated otherwise, the techniques of this invention can be brought to bear without restriction on any type of progenitor cell capable of differentiating into neuronal or glial cells. [0063] Precursor cells

[0064] As used herein, precursor cells comprise progenitor cells and stem cells. Progenitor cells and stem cells can be produced using methods described herein or be obtained from various methods known in the art. In one embodiment, the progenitor and stem cells are derived from humans. Stem cells can be embryonic stem (ES) cells or induced pluripotent/multipotent stem (iPS) cells. ES or iPS cells are obtained using methods known in the art. Pluripotent stem cells have the ability to differentiate into cells of all three germ layers (ectoderm, mesoderm and endoderm). In contrast, multipotent stem cells can give rise to one or more cell-types of a particular germ layer(s), but not necessarily all three.

[0065] Embryonic stem (ES) cells are both self-renewing and pluripotent. The induced cells may also be self- renewing and pluripotent. However, in contrast to ES cells, the induced cells can be derived from a wide range of cells and tissue, including non-embryonic tissue.

[0066] A precursor cell which finds use in the present methods can be, by way of example and without limitation, any stem or progenitor cell with a developmental potency which includes a capacity to generate, i.e. differentiate into the target brain cell of interest. Such cells may include, without limitation, neural progenitors, neural stem cells, neuroepithelial progenitors, embryonic stem cells; induced multipotent/pluripotent stem cells; any multipotent neural stem cell or pluripotent stem cell obtained from primary tissue or from an individual, or produced by the induction of pluripotency in a previously more-differentiated cell, as is known in the art. Except where otherwise required, the invention can be practiced using stem cells of any vertebrate species. Included are stem cells from humans; as well as non-human primates, domestic animals, livestock, and other non-human mammals. This invention can be practiced using stem cells of various types, which may include the following non-limiting examples.

[0067] U.S. Pat. No. 5,851,832 reports multipotent neural stem cells obtained from brain tissue. U.S. Pat. No. 5,766,948 reports producing neuroblasts from newborn cerebral hemispheres. U.S. Pat. No. 5,654,183 and 5,849,553 report the use of mammalian neural crest stem cells. U.S. Pat. No. 6,040,180 reports in vitro generation of differentiated neurons from cultures of mammalian multipotent CNS stem cells. WO 98/50526 and WO 99/01159 report generation and isolation of neuroepithelial stem cells, oligodendrocyte-astrocyte precursors, and lineage- restricted neuronal precursors. U.S. Pat. No. 5,968,829 reports neural stem cells obtained from embryonic forebrain and cultured with a medium comprising glucose, transferrin, insulin, selenium, progesterone, and several other growth factors.

[0068] Among the stem cells suitable for use in this invention are mammalian pluripotent and multipotent stem cells derived from tissue formed after gestation, such as a blastocyst, or fetal or embryonic tissue taken any time during gestation. Non-limiting examples are primary cultures or established lines of embryonic stem cells or embryonic germ cells.

[0069] Embryonic stem cells can be isolated from blastocysts of members of the primate species (Thomson et al., Proc. Natl. Acad. Sci. USA 92:7844, 1995). Human embryonic stem (hES) cells can be prepared from human blastocyst cells using the techniques described by Thomson et al. (U.S. Pat. No. 5,843,780; Science 282:1145, 1998; Curr. Top. Dev. Biol. 38:133 ff., 1998) and Reubinoff et al, Nature Biotech. 18:399,2000.

Induced pluripotent stem (iPS) cells can also be used. Methods for the induction of pluripotency in somatic tissue are also known in the art. The process of inducing cells to become multipotent or pluripotent is based on forcing the expression ("forced expression") of polypeptides, particularly proteins that play a role in maintaining or regulating self-renewal and/or pluripotency of ES cells. Examples of such proteins are the Oct3/4, Sox2, Klf4, and c-Myc transcription factors, all of which are highly expressed in ES cells. Forced expression may include introducing expression vectors encoding polypeptides of interest into cells, transduction of cells with recombinant viruses, introducing exogenous purified polypeptides of interest into cells, contacting cells with a non-naturally occuring reagent that induces expression of an endogenous gene encoding a polypeptide of interest (e.g., Oct3/4, Sox2, Klf4, or c-Myc), or any other biological, chemical, or physical means to induce expression of a gene encoding a polypeptide of interest (e.g., an endogenous gene Oct3/4, Sox2, Klf4, or c-Myc). iPS cells can also be used to form ES cells and methods for accomplishing this are known in the art.

[0070] The multipotent or pluripotent cells may be induced from a wide variety of mammalian cells. Examples of suitable populations of mammalian cells include those that include, but are not limited to: fibroblasts, bone marrow- derived mononuclear cells, skeletal muscle cells, adipose cells, peripheral blood mononuclear cells, macrophages, hepatocytes, keratinocytes, oral keratinocytes, hair follicle dermal cells, gastric epithelial cells, lung epithelial cells, synovial cells, kidney cells, skin epithelial cells or osteoblasts.

[0071] As understood in the art, differentiation of a cell is the process by which cells become structurally and functionally specialized, for example, during embryonic development or in vitro. Correspondingly, dedifferentiation, as known in the art, is the process whereby differentiated, (i.e., adult, somatic, or specialized) cells are restored to an unspecialized state. Dedifferentiation allows for respecialization into other cell types distinct from that of the cell which has undergone the dedifferentiation. Once obtained, cells may be dedifferentiated by exposure to transcription factors such as, for example OCT4, SOX2, NANOG, and LIN28. See, e.g., Takahashi. K., et al, Induction of pluripotent stem cells from adult human fibroblasts by defined factors, Cell 131, 861-72 (2007) The iPS cells may be identified by staining for markers as is known in the art, for example, by staining for the absence of stage-specific embryonic antigen (SSEA)-I and the presence of hES cell-specific surface antigens(Adewumi et al., 2007), including SSEA-3, SSEA-4, tumor-related antigen (TRA)-l-60, TRA-1-81 and TRA-2-49/6E (alkaline phosphatase), NANOG protein, and the like. [0072] Differentiation of precursor cells

[0073] Further provided are methods of promoting the differentiation of a subpallium precursor cell (stem or progenitor cell). In an embodiment, iPS cells can be differentiated into ES cells or into forebrain basal ganglia cells. In one embodiment, stem cells can be differentiated into forebrain basal ganglia cells, which can be further differentiated to form progenitor cells. In an embodiment, the progenitor cells produce LGE neurons such as striatal neurons. In another embodiment, the progenitor cells produce CGE-derived interneurons. In some embodiments, the methods include inducing the expression in the cell of one or more genes provided in Tables 2-4. The cell can further be exposed to gene products sequentially, either singularly or in combination, to further differentiate the cell. In some embodiments, the expression of one or more genes is forced. In some embodiments, the expression of Gsxl, Gsx2, Ascll, Dlxl, Dlx2, homologs or combinations thereof, is forced, resulting in differentiation of a subpallial progenitor cell. In another embodiment, the expression of additional genes or homologs thereof are forced. In a further embodiment, other genes known in the art for generating differentiated cells are also forced to express. One of skill in the art will readily appreciate that this method can be performed at any stage of the differentiation of a subpallial progenitor cell; i.e. in vitro at the equivalent time of embryonic day 12.5, day 15 or day 18 in mice. In some embodiments, modulating the level of gene expression includes introducing one or more polynucleotides into the stem or progenitor cell. The introducing allows for the expression of the introduced polynucleotides in the cell, e.g. by expression of messenger RNA and its translation into polypeptides. [0074] Many nonviral techniques for the delivery of a polynucleotide into a cell can be used, including direct naked DNA uptake (e.g., Wolffs al, Science 247: 1465-1468, 1990), receptor-mediated DNA uptake, e.g., using DNA coupled to asialoorosomucoid which is taken up by the asialoglycoprotein receptor in the liver (Wu and Wu, J. Biol. Chem. 262: 4429-4432, 1987; Wu etal, J.Biol.Chem. 266: 14338-14342, 1991), liposome-mediated delivery (e.g., Kaneda et al., Expt. Cell Res. 173: 56-69, 1987; Kaneda et al., Science 243: 375-378, 1989; Zhu et al. Science 261: 209-211, 1993), by use of iontophoresis, electroporation and other pharmacologically sound methods of delivery. Many of these physical methods can be combined with one another and with viral techniques; enhancement of receptor-mediated DNA uptake can be effected, for example, by combining its use with adenovirus (Curiel et al, Proc. Natl. Acad. ScL USA 88: 8850- 8854, 1991; Cristiano et al, Proc. Natl. Acad. Sci. USA 90: 2122- 2126, 1993).

[0075] Many vectors useful for transferring exogenous genes into target mammalian cells are available. The vectors may be episomal, e.g. plasmids, virus derived vectors such cytomegalovirus, adenovirus, etc., or may be integrated into the target cell genome, through homologous recombination or random integration, e.g. retrovirus derived vectors such MMLV, HIV-I, ALV, etc. Combinations of retroviruses and an appropriate packaging line may be used, where the capsid proteins will be functional for infecting the target cells. Usually, the cells and virus will be incubated for at least about 24 hours in the culture medium. The cells are then allowed to grow in the culture medium for short intervals in some applications, e.g. 24-73 hours, or for at least two weeks, and may be allowed to grow for five weeks or more, before analysis. Commonly used retroviral vectors are "defective", i.e. unable to produce viral proteins required for productive infection. Replication of the vector is accomplished by growth in the packaging cell line. [0076] For some uses such as, for example, temporally specific and developmentally appropriate expression of one or more transcription factors, it is desirable to have a regulatable promoter driving expression. Where such a promoter is included, the promoter function of the LTR can be inactivated. This can be accomplished by a deletion of the U3 region in the 3'LTR, including the enhancer repeats and promoter, which is sufficient to inactivate the promoter function. After integration into a target cell genome, there is a rearrangement of the 5' and 3' LTR, resulting in a transcriptionally defective provirus, termed a "self-inactivating vector". [0077] The vectors may include genes that must later be removed, e.g. using a recombinase system such as Cre/Lox, or the cells that express them destroyed, e.g. by including genes that allow selective toxicity such as herpesvirus TK, bcl-xs, etc.

[0078] Suitable inducible promoters are activated in a desired target cell type, either the transfected cell, or progeny thereof. By transcriptional activation, it is intended that transcription will be increased above basal levels in the target cell by at least 10 fold, often about 100 fold, more usually by at least about 1000 fold, etc. [0079] To prove the generation of progenitor and differentiated cells or to identify differentiated cells surronding brain tissue, a reporter gene can be used that is under the control of a cell type specific promoter. In a specific embodiment, the hygromycin B phosphotransferase-EGFP fusion protein is expressed in a cell type specific manner. The method of purifying comprises sorting the cells to select green fluorescent cells and reiterating the sorting as necessary, in order to obtain a population of cells enriched for cells expressing the construct (e.g., hygromycin B phosphotransferase-EGFP) in a cell-type-dependent manner. Selection of desired sub-populations of cells may also be accomplished by negative selection of proliferating cells with the herpes simplex virus thymidine kinase/ganciclovir (HSVtk/GCV) suicide gene system or by positive selection of cells expressing a bicistronic reporter, e.g., Anderson et al. (2007) MoI Then (1 l):2027-2036.Cells can be further induced by co-culturing the cells with another cell type such as an oligodendrocyte or astrocyte.

[0080] The subpallial progenitor cells can form five major subtypes: LGE, CGE, MGE, POA, septum. Differences in fibroblast growth factor (FGF) signaling, which is mediated in part by CoupTFl&2, can be used to influence the differentiation of the progenitors into the various subtypes.FGF signaling causes repression of CoupTFl&2 and is likely critical in making septum LGE and CGE. For example, high FGF signaling leads to the formation of septum, low or no FGF signaling leads to the formation of CGE, and medium FGF signaling leads to the formation of LGE. In one embodiment, FGF signaling is modulated to for septum LGE. In another embodiment, FGF signaling is modulated to form CGE. In another embodiment, CGE can be formed from LGE by decreasing the FGF signaling. [0081] The septum expresses FGF8 and FGF17. By using FGF8 ligands or antagonist, the cells progenitors can be moved into different subtypes and states. Commericially available antagonists and ligands are known in the art. [0082] To generate MGE and POA, expression of Mkx2.1 transcription factor is needed, which can be done through sonic hedgehog signaling, or through tranduction of the transcription factors in the cell. MGE can be formed from subpallial progenitor cells by introducing Nkx2.1 transcripton factor. POA can be formed from subpallial progenitor cells by introducing Nkx2.1 and/or Nkx5.1 transcription factors.

[0083] In one embodiment, the precursor cells can be differentiated into LGE-derived neurons such as, but not limited to, striatal neurons. LGE produces projection neurons that migrate to the striatum (striatal projection neurons). LGE can also produces olfactory bulb interneurons as well as central nucleus of the amygdala. In another embodiment, the precursor cells can be differentiated into CGE-derived interneurons such as, but not limited to, VIP+, calretinin+/somatistatin-, and NPY+. CGE produces interneurons that migrate to the cortex (cortical interneurons). MGE cells can also be derived by LGE and CGE.

[0084] In one embodiment, stem cells can be used to make LGE and CGE.

[0085] Provided herein is also a composition comprising a differentiated cell that is derived by forced expression of Gsxl, Gsx2, Dlxl, Dlx2, Ascll, or combinations thereof. The differentiated cell can also be derived by additionally forcing expression of one or more genes from Tables 2-4. In one embodiment the differentiated cell is a striatal neuron. In another embodiment, the differentiated cell is a CGE-derived interneuron.

[0086] Provided herein is also a composition comprising a differentiated cell that is derived by exposing a precursor cell such as a subpallial progenitor and its subtypes to one more transcription factors produced by Gsxl,

Gsx2, Dlxl, Dlx2, Ascll, or combinations thereof. The differentiated cell can also be derived by additionally exposure to one or more genes from Tables 2-4. In one embodiment the differentiated cell is a striatal neuron. In another embodiment, the differentiated cell is a CGE-derived interneuron.

[0087]

[0088] Soluble factors

[0089] In some embodiments, differentiation of a subpallial progenitor by modulating the level of expression of one or more genes according to the presently claimed methods may be accomplished by exposure of the cell to a soluble factor, e.g., a molecule which is introduced into a cell culture system or into an in vivo system which includes the cell. In some embodiments, the soluble factor contacts the cell or is taken up into, e.g. the cytoplasm of the cell. In some embodiments, such soluble factors include, but are not limited to, organic compounds; polypeptides; proteins; polynucleotides; cells; small molecules; chemokines, cytokines; antisense molecules; antibodies and fragments thereof; genetic agents including, for example, mRNA, shRNA, siRNA, a virus or genetic material in a liposome; an inorganic molecule including salts; and the like.

[0090] Methods of identifying an agent that modulates differentiation of a subpallial neural progenitor cell are also provided. In some aspects, the methods include contacting a stem or progenitor cell, in which gene expression has been modulated, with a candidate agent and detecting any alteration in the developmental status of said cell relative to that of a stem or progenitor cell treated according to the same methods, but which is not contacted with the candidate agent, where any alteration is indicative that the agent modulates the differentiation of a subpallial neural progenitor cell. In some embodiments, a stem, progenitor or subpallial progenitor cell is exposed to conditions which modulate the expression of one or more genes, as disclosed in the present methods. Additionally, the cell is exposed to an agent under experimental condition, or, under control conditions, the cell is not exposed to the agent but is otherwise treated as under the experimental conditions. Thereafter, the cells can be assayed for changes in subsequent expression levels of genes which are disclosed herein as characteristic of developmental progression or lack thereof. Any change or lack of change between the experimental and control cell is therefore indicative of an ability of the agent to modulate the differentiation of a subpallial neural progenitor cell. In this way, agents are identified which affect the differentiation of a subpallial neural progenitor cells.

[0091] Agents may include any molecule, such as soluble factors as described above, or others. Generally a plurality of assay mixtures are run in parallel with different agent concentrations to obtain a differential response to the various concentrations. Typically, one of these concentrations serves as a negative control, i.e., at zero concentration or below the level of detection. Screening may be directed to known bioactive compounds and chemical analogs thereof. In some embodiments, the agent may modulate or be predicted to modulate the expression of molecules in the experimental cell such as, i.e., transcription factors, kinases, homeobox genes, cytokines, neurotransmitters, or any other intracellular or secreted molecule which affects the developmental and/or migrational fate of the cell. Useful concentrations of soluble factors range from 0.1 nM to 100 mM, 1 nM to 10 mM, or 10 nM to 1 mM. Exposure times can be at least 10 minutes, 30 minutes, 60 minutes, 90 minutes, two hours, three hours, four hours, six hours, eight hours, ten hours, twelve hours, sixteen hours, twenty hours, 1 day, 2 days, 3 days, 4 days, 6 days, 8 days, 10 days, 12 days or combinations thereof.

[0092] In one embodiment, a soluble factor is a transcription factor. Soluble factors, such as transcription factors (TFs), can be used during any stage of differentiation of precursor cells. For example, TFs can be used to in the differentiation process of iPS cells to form ES cells, or in the differentiation process of ES or iPS cells to form forebrain basal ganglia cells, or in the differentiation process of forebrain basal ganglia cells to form progenitor cells. TFs can also be used to produce stem cells. Stem cells, both ES and iPS, can be directed to differentiate with the use of transcription factors in combination with other known factors and methods. In one embodiment, according to known methods, ES and iPS cells are first differentiated into forebrain basal ganglia, which are then exposed to Gsxl&l, Dlxl&2, and Ascll to produce progenitors.

[0093] Drugs and other pharmaceutical agents that mimic the effects of the transcription factors described herein are also useful in the disclosed methods. For example, sonic hedgehog signaling, FGF Signaling, and TGFβ signaling have been shown to be required for production of subpallial progenitor cells. It has been shown that if TGFβ signaling is inhibited, the development of subpallial progenitor cells is also inhibited. There are known drugs and other agnonists that affect these signaling pathways and therefore, can be useful in the methods described herein. For example, activitin and bone morphogenetic proteins (BMPs) are non-limiting examples of TGFβ signaling ligands and can be useful for production of progenitors. [0094] Methods of assessment

[0095] In some embodiments, any difference between the experimental and control cells is assessed by staining for a marker and observing a change. Nonlimiting examples of a change or lack of change include a change or lack of change in cell morphology, gene expression, gene product secretion, and cell surface molecule presence. Cell stains are known to those of skill in the art. Typically a candidate compound will be added to the cells, and the response of the cells monitored through evaluation of cell surface phenotype, functional activity, patterns of gene expression, and the like. In some embodiments, assays are used to identify agents that have a low toxicity in human cells. Detection of change or lack of change in the cells may utilize staining of cells, performed in accordance with conventional methods. For example, antibodies of interest are added to the cell sample, and incubated for a period of time sufficient to allow binding to the epitope, for example, at least about 10 minutes. The antibody may be labeled with a label, for example, chosen from radioisotopes, enzymes, fluorescers, chemiluminescers, or other labels for direct detection. Alternatively, a second stage antibody or reagent is used to amplify the signal. Such reagents are well known in the art. For example, the primary antibody may be conjugated to biotin, with horseradish peroxidase-conjugated avidin added as a second stage reagent. Final detection uses a substrate that undergoes a color change in the presence of the peroxidase. The absence or presence of antibody binding may be determined by various methods, including flow cytometry of dissociated cells, microscopy, radiography, scintillation counting, etc. [0096] Cellular gene expression may be assessed following exposure to a candidate agent. The expressed set of genes may be compared with control cells of interest, e.g., cells also derived according to the present methods but which have not been contacted with the agent. Any suitable qualitative or quantitative methods known in the art for detecting specific mRNAs can be used. mRNA can be detected by, for example, hybridization to a microarray, in situ hybridization in tissue sections, by reverse transcriptase-PCR, or in Northern blots containing poly A+ mRNA. One of skill in the art can readily use these methods to determine differences in the size or amount of mRNA transcripts between two samples. For example, the level of particular mRNAs in cells contacted with agent is compared with the expression of the mRNAs in a control sample.

[0097] In addition to or instead of in situ analysis, mRNA expression levels can be determined using hybridization analysis, which is based on the specificity of nucleotide interactions. Oligonucleotides or cDNA can be used to selectively identify or capture the DNA or RNA of specific sequence composition, and the amount of RNA or cDNA hybridized to a known capture sequence determined qualitatively or quantitatively, to provide information about the relative representation of a particular RNA message within the pool of cellular RNA messages in a sample. Hybridization analysis may be designed to allow for concurrent screening of the relative expression of hundreds to thousands of genes by using, for example, array-based technologies having high density formats, including filters, microscope slides, or microchips, or solution-based technologies that use spectroscopic analysis (e.g., mass spectrometry).

[0098] In another method, the test sample is assayed at the protein level. Methods of analysis may include 2- dimensional gels; mass spectroscopy; analysis of specific cell fraction, e.g., lysosomes; and other proteomics approaches. For example, detection may utilize staining of cells or histological sections (e.g., from a biopsy sample) with labeled antibodies, performed in accordance with conventional methods. Cells can be permeabilized to stain cytoplasmic molecules. In general, antibodies that specifically bind a differentially expressed polypeptide are added to a sample, and incubated for a period of time sufficient to allow binding to the epitope, usually at least about 10 minutes. The antibody can be detectably labeled for direct detection (e.g., using radioisotopes, enzymes, fluorescers, chemiluminescers, and the like), or can be used in conjunction with a second stage antibody or reagent to detect binding (e.g., biotin with horseradish peroxidase-conjugated avidin, a secondary antibody conjugated to a fluorescent compound, e.g., fluorescein, rhodamine, Texas red, etc.). The presence or absence of antibody binding may be determined by various methods, including flow cytometry of dissociated cells, microscopy, radiography, scintillation counting, etc. Any suitable alternative methods can of qualitative or quantitative detection of levels or amounts of differentially expressed polypeptide can be used, for example ELISA, western blot, immunoprecipitation, radioimmunoassay, etc. [0099] Homology

[00100] Described herein are neuronal transcription factors useful for the generation of progenitor cells or differentiated cells used in the methods described herein, and exogenous genes encoding such transcription factors. In some embodiments, a neuronal transcription factor has a naturally occurring amino acid sequence, e.g., that of: human or mouse Gsx2, human or mouse Gsxl , human or mouse Dlxl/2, or human or mouse Ascll . In other embodiments, the amino acid sequence of a neuronal transcription factor is a non-naturally occurring amino acid sequence variant of a neuronal transcription factor that is, nevertheless, functionally or structurally homologous to a neuronal transcription factor amino acid sequence, as described herein. In some embodiments, the transcription factors or genes encoding the transcription factors have at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology to a respective mouse or human transcription factor or gene listed in Tables 2-4. [00101] Evaluating the structural and functional homology of two polypeptides generally includes determining the percent identity of their amino acid sequences to each other. Sequence identity between two or more amino acid sequences is determined by conventional methods. See, for example, Altschul et al., (1997), Nucleic Acids Research, 25(17):3389-3402; and Henikoff and Henikoff ( 1982), Proc. Natl. Acad. ScL USA, 89:10915 (1992). Briefly, two amino acid sequences are aligned to optimize the alignment scores using a gap opening penalty of 10, a gap extension penalty of 1, and the "BLOSUM62" scoring matrix of Henikoff and Henikoff (ibid.). The percent identity is then calculated as: ([Total number of identical matches]/[length of the longer sequence plus the number of gaps introduced into the longer sequence in order to align the two sequences])(100).

[00102] Those skilled in the art will appreciate that there are many established algorithms available to align two amino acid sequences. The "FASTA" similarity search algorithm of Pearson and Lipman is a suitable protein alignment method for examining the level of identity shared by an amino acid sequence of a neuronal transcription factor disclosed herein and the amino acid sequence of another peptide. (Pearson and Lipman (1988), Proc. Nat'l Acad. Sci. USA, 85:2444, and by Pearson (1990), Meth. Enzymol., 183:63).

[00103] Also described herein are nucleic acids (e.g., exogenous genes) encoding Gsxl, Gsx2, Dlxl, Dlx2, or Ascll, as described herein, that hybridize specifically under low, medium, or high stringency conditions to a probe of at least 100 nucleotides from a nucleic acid encoding the amino acid sequence any of the transcription factors listed in Table 2-4. Low stringency hybridization conditions include, e.g., hybridization with a 100 nucleotide probe of about 40% to about 70% GC content; at 42 ⁰C in 2XSSC and 0.1% SDS. Medium stringency hybridization conditions include, e.g., at 50 ⁰C in 0.5X SSC and 0.1% SDS. High stringency hybridization conditions include, e.g., hybridization with the above-mentioned probe at 65 ⁰C in 0.2X SSC and 0.1% SDS. Under these conditions, as the hybridization temperature is elevated, a nucleic acid with a higher homology can be obtained. Such nucleic acids encoding Gsxl, Gsx2, Dlxl, Dlx2, or Ascll are useful in the forced expression of these progenitors as described herein. In some embodiments, nucleic acid sequences encoding variants of transcriptions factors have at least medium stringency hybridization to genes listed in Table 2-4

[00104] Non-naturally occurring sequence variants of the neuronal transcription factors can be generated by a number of known methods. Such methods include, but are not limited to, "Gene Shuffling," as described in U.S. Patent No. 6,521,453; "RNA mutagenesis," as described in Kopsidas et al., (2007), BMC Biotechnology, 7: 18-29; and "error-prone PCR methods." Error prone PCR methods can be divided into (a) methods that reduce the fidelity of the polymerase by unbalancing nucleotides concentrations and/or adding of chemical compounds such as manganese chloride (see, e.g., Lin-Goerke et al., (1997), Biotechniques, 23:409-412), (b) methods that employ nucleotide analogs (see, e.g., U.S. Patent No. 6,153,745), (c) methods that utilize 'mutagenic' polymerases (see, e.g., Cline, J. and Hogrefe.H.H. (2000), Strategies (Stratagene Newsletter), 13:157-161 and (d) combined methods (see, e.g., Xu et al., (1999), Biotechniques, 27: 1102-1108. Other PCR-based mutagenesis methods include those, e.g., described by Osuna et al., (2004), Nucleic Acids Res., 32(17):el36 and Wong et al., (2004), Nucleic Acids Res.,10;32(3):e26), and others known in the art.

[00105] Confirmation of the retention, loss, or gain of function of the amino acid sequence variants of neuronal transcription factors can be determined in various types of assays according to the protein function being assessed. For example, where the neuronal transcription factor is a transcriptional activator, function is readily assessed using cell-based, promoter-reporter assays, where the reporter construct comprises one or more cognate target elements for the transactivator polypeptide to be assayed. Methods for generating promoter-reporter constructs, introducing them into cells, and assaying various reporter polypeptide activities, can be found in detail in, e.g., Current Protocols in Molecular Biology, John Wiley & Sons, N. Y. (2005), 3.16-3.17 and 9.1-9.14, respectively). Promoter activity can be quantified by measuring a property of the reporter polypeptide (e.g., enzymatic activity or fluorescence), reporter polypeptide expression (e.g., by an ELISA assay), or reporter mRNA expression (e.g., by a fluorescent hybridization technique). Suitable reporter polypeptides include, e.g., firefly luciferase, Renilla luciferase, fluorescent proteins (e.g., enhanced green fluorescent protein), β-galactosidase, β lactamase, ALP, and horseradish peroxidase. [00106] In view of the guidance provided herein, a broad range of neuronal transcription factor sequence variants (e.g., Gsxl, Gsx2, Dlxl, Dlx2, or Ascll sequence variants), operable in the methods described herein, can readily be identified by those of ordinary skill in the art without undue effort. [00107] CeU Therapeutics

[00108] The neuronal progenitor cells, or cells differentiated from the neuronal progenitor cells, may be used as a therapy to treat diseases, conditions, or disorders. The therapy may be directed at treating the cause of the diseases; or alternatively, the therapy may be to treat the effects of the diseases, conditions or disorders. The progenitor cells may be transferred to, or close to, an injured site in a subject; or the cells can be introduced to the subject in a manner allowing the cells to migrate, or home, to the injured site. The transferred cells may advantageously replace the damaged or injured cells and allow improvement in the overall condition of the subject. In some instances, the transferred cells may stimulate tissue regeneration or repair.

[00109] The transferred cells may be cells differentiated from progenitor cells. In some cases, the transferred cells may be progenitor cells that have not been differentiated.

[00110] The number of administrations of treatment to a subject can vary. Introducing the induced and/or differentiated cells into the subject can be a one-time event. In certain situations, such treament can elicit improvement for a limited period of time and require an on-going series of repeated treatments. In other situations, 'multiple administrations of the cells can be required before an effect is observed. The exact protocols for administration depend upon the disease or condition, the stage of the disease and parameters of the individual subject being treated.

[00111] The cells may be introduced to the subject at the site of damaged or diseased tissue, or at a remote location. In some embodiments, the progenitor or differentiated cells can migrate at least 0.01 mm, 0.05 mm, 0.10 mm, 0.50 mm, 1.0 mm, 5.0 mm or 10.0 mm from the introduction site. In some embodiments, the progenitor cells may also be engineered to respond to cues that can target their migration into lesions for brain or spinal cord repair, e.g., Chen et al., (2007), Stem Cell Rev. , 3(4):280-288. The progenitor or differentiated cells can exhibit functional integration within host circuits. In further embodiments, the progenitor or differentiated cells can functionally integrate into excitory/inhibitory circuitry.

[00112] The progenitor or differentiated cells can be transferred to subjects suffering from a range of neurologic diseases, conditions or disorders, e.g., Alzheimer's disease, Parkinson's disease, Huntington's Disease, Tourette's Syndrome, dystonia, multiple sclerosis, or other central nervous system disorder. For the treatment of multiple sclerosis, neural progenitor cells may be differentiated into oligodendrocytes or progenitors of oligodendrocytes, which are then transferred to a subject suffering from MS.

[00113] The cells can be introduced to the subject via injection or implantation into target sites or the cells described herein can be inserted into a delivery device which facilitates introduction by, injection or implantation, of the cells into the animals. Such delivery devices include tubes, e.g., catheters, for injecting cells and fluids into the body of a subject. In one embodiment, the tubes additionally have a needle, e.g., a syringe, through which the cells described herein can be introduced into the animal at a desired location. [00114] The cells described herein can be inserted into such a delivery device, e.g., a syringe, in different forms. For example, the cells can be suspended in a solution or embedded in a support matrix when contained in such a delivery device. As used herein, the term "solution" includes a pharmaceutically acceptable carrier or diluent in which the cells described herein remain viable. Pharmaceutically acceptable carriers and diluents include saline, aqueous buffer solutions, solvents and/or dispersion media. The use of such carriers and diluents is well known in the art. The solution is preferably sterile and fluid to the extent that easy syringability exists. Preferably, the solution is stable under the conditions of manufacture and storage and preserved against the contaminating action of microorganisms such as bacteria and fungi through the use of, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. Solutions of the invention can be prepared as described herein in as a pharmaceutically acceptable carrier or diluent and, as required, other ingredients enumerated above, followed by filter sterilisation.

[00115] The induced cells can be differentiated into cells and then subsequently transferred into subjects suffering from a wide range of diseases or disorders. Subjects suffering from neurological diseases or disorders could especially benefit from stem cell therapies. In some approaches, the induced cells can be differentiated into neural stem cells or neural cells and then transplanted to an injured site to treat a neurological condition, e.g., Huntington's disease, Tourette's syndrome, dystonia, Alzheimer's disease, Parkinson's disease, multiple sclerosis, cerebral infarction, spinal cord injury, or other central nervous system disorder, see, e.g., Morizane et al., (2008), Cell Tissue Res., 331(l):323-326; Courts and Keirstead (2008), Exp. Neurol., 209(2):368-377; Goswami and Rao (2007), Drugs, 10(10):713-719.

[00116] The differentiated cells produced using the methods described herein are useful for treatment of disorders or diseases that have a deficiency in the number or function of LGE-derived neurons, striatal neurons, or CGE- derived interneurons. Non-limiting examples include Huntington's disease, Tourette's syndrome, dystonia, epilepsy, schizoprenia, autism, stroke, Alzheimer's disease, and the like. The differentiated cells can also be useful for treatment or amelioration of symptoms caused by the imbalance of the excitory/inhibitory neuronal circuitry. Huntington's disease (HD) is a neurodegenerative disorder that mainly affects the projection neurons of the striatum and cerebral cortex. In one embodiment, the differentiated cells produced using the methods described herein are useful for treatment of Huntington's disease.

[00117] For the treatment of Parkinson's disease, the induced cells may be differentiated into dopamine-acting neurons and then transplanted into the striate body of a subject with Parkinson's disease. For the treatment of multiple sclerosis, neural stem cells may be differentiated into oligodendrocytes or progenitors of oligodendrocytes, which are then transferred to a subject suffering from MS.

[00118] In some embodiments, the present invention is useful in the treatment of degenerative diseases. A degenerative disease is a disease in which the decline (e.g., function, structure, biochemistry) of particular cell type, e.g., neuronal, results in an adverse clinical condition. Examples of degenerative diseases of the central nervous system include those that affect the basal ganglia and the striatum. Degenerative diseases that can be treated with the substantially homogenous cell populations of the invention include, for example, Parkinson's disease, multiple sclerosis, epilepsy, Huntington's, dystonia, (dystonia musculmusculorum deformans) and choreoathetosis. [00119] In some embodiments, the present invention is useful in the treatment of conditions caused by an acute injury. An acute injury condition is a condition in which an event or multiple events results in an adverse clinical condition. The event which results in the acute injury condition can be an external event such as blunt force or compression or an internal event such as sudden ischemia (e.g., stroke or heart attack). Acute injury conditions that can be treated with the substantially homogenous cell populations of the invention include, for example, spinal cord injury, traumatic brain injury, brain damage resulting from myocardial infarction and stroke. [00120] In some embodiments, the invention includes a method of treating a human suffering from a neurological condition, comprising the step of administering to the human a substantially homogenous cell population of the present invention. "A neurological condition," as used herein, refers to any state of the nervous system (central or peripheral nervous system) which deviates in any manner from a normal nervous system or nervous system of a mammal, e.g., human, not affected by a neurological condition. The neurological condition can be a condition of the central (brain or spinal cord) or peripheral nervous system. The neurological condition can be, for example, the result or consequence of a disease, e.g., Parkinson's disease or multiple sclerosis, acute injury condition, e.g., stroke, brain injury, spinal cord injury, or a combination of disease and acute injury condition. Other neurological conditions which can be treated with the substantially homogenous population of cells of the invention include, for example, chronic or intractable pain.

[00121] In some cases, the induced cells are transferred into an immunocompromised animal, e.g., SCED mouse, and allowed to differentiate. The transplanted cells may form a mixture of differentiated cell types and tumor cells. The specific differentiated cell types of interest can be selected and purified away from the tumor cells by use of lineage specific markers, e.g., by fluorescent activated cell sorting (FACS) or other sorting method, e.g., magnetic activated cell sorting (MACS). The differentiated cells may then be transplanted into a subject (e.g., an autologous subject, HLA-matched subject) to treat a disease or condition. The disease or condition may be a hematopoietic disorder, an endocrine deficiency, degenerative neurologic disorder, hair loss, or other disease or condition described herein.

The cells and their level of gene expression described herein can also be used for diagnostic methods and research tools. For example, changes in gene expression can be used to detect or diagnose a disorder, disease, or condition. Changes in gene expression can also be used to predict the likelihood for developing a disorder, disease, or condition. In addition, changes in gene expression can be used for monitoring the course of treatment for a disorder, disease, or condition. [00122] Storage of CeUs

100123] The precursor and differentiated cells described herein may be stored. Thus, cells or materials from any point during the processes may be stored for future completion of the process or modification for use. [00124] The methods of storage may be any method including the methods described herein, e.g., using cryopreservation medium. Some exemplary cryopreservation media include the "Cryopreservation Medium For Primate ES Cells" (ReproCELL, Tokyo, Japan) or mFreSR™ (StemCell Technologies, Vancouver, CA). The cells preferably are rapidly frozen in liquid nitrogen, and stored in a liquid nitrogen storage vessel. Other suitable cryopreservation media and methods for cryopreservation/thawing of cells generated by the methods described herein are provided in, e.g., U.S. Patent Application Serial Nos: 10/902,571 and 11/142,651. See also, Ha et al., (2005), Hum. Reprod., 20(7): 1779- 1785.

[00125] The invention will now be further described by way of reference only to the following non- limiting examples. It should be understood, however, that the examples following are illustrative only, and should not be taken in any way as a restriction on the generality of the invention described herein. In particular, while the invention is described in detail in relation to the use of mouse cells, it will be clearly understood that the findings herein are not limited to these-types of cells, but would be useful in identifying and producing subpallial progenitors and progeny from any animal. The examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the subject methods, and are not intended to limit the scope of what is regarded as the invention.

EXAMPLES

Example 1: DIx 1&2 and Mashl transcription factors control striatal patterning and differentiation through parallel and overlapping pathways

[00126] Here is defined the expression of approximately 100 transcription factors in progenitors and neurons of the developing basal ganglia. The transcriptional hierarchy of these genes with respect to the DIx homeodomain genes, which are essential for differentiation of most GABAergic projection neurons of the basal ganglia, are elucidated. This analysis identifies DZx-dependent and £>Zx-independent pathways. The D/x-independent pathway depends in part on the function of the Mashl b-HLH transcription factor. These analyses define core transcriptional components that differentially specify the identity and differentiation of the striatum, nucleus accumbens, and septum. [00127] The basal ganglia have fundamental roles within cortical-basal ganglia-thalamic networks that control progressively higher-order types of learning: limbic, associative, and sensorimotor (Yin and Knowlton, 2006). The principal telencephalic constituents of the basal ganglia include the striatum (caudate, putamen, nucleus accumbens), and the globus pallidus, whose embryonic anlage are the lateral and medial ganglionic eminences (LGE, MGE, respectively) (Campbell, 2003; Puelles et al., 2000). The progenitor zone of the LGE is the source for striatal projection neurons and olfactory bulb interneurons, whereas the progenitor zone of the MGE is the source for pallidal projection neurons and cortical and striatal interneurons (Campbell, 2003; Marin and Rubenstein, 2003). The septal anlage (Se) lies adjacent to the LGE and MGE, and is the source of septal projection neurons and is thought to generate some olfactory bulb interneurons (Long et al., 2003, 2007; Kohwi et al., 2007). The progenitor domain of the nucleus accumbens is poorly defined.

[00128] Current efforts are aimed at elucidating the genetic circuits that regulate the specification, differentiation, and function of LGE-, MGE-, and Se-derived cells, with particular emphasis on defining the transcription factors and relevant signaling pathways. Sonic hedgehog (Shh) and fibroblast growth factor (FGF) signaling coordinately are essential for specification and patterning the basal ganglia (Gutin et al., 2006; Storm et al., 2006) — both pathways converge on expression of Nkx2.1, a homeobox transcription factor that is essential for MGE specification (Sussel et al., 1999). For instance, severe Fgβ hypo-morphs fail to establish Nkx2.1 and Shh expression in the anlage of the MGE. However, these mutants express DIx homeobox transcription factors (Storm et al., 2006), whose function is essential in perhaps all differentiating basal ganglia neurons including striatal projection neurons (Anderson et al., 1997a,b; Lobo et al., 2006). The vast majority of basal ganglia neurons are GABAergic and the DIx genes are sufficient to promote GABAergic differentiation (Anderson et al., 1999; Stuhmer et al., 2002). Herein is provided evidence that DIx 1&2 work in concert with other transcription factors to specify GABAergic fate. [00129] Specification of the striatum depends on the function of the Gshl&2 homeobox genes, which are expressed in the LGE ventricular zone (VZ) (Corbin et al., 2000; Toresson et al., 2000; Toresson and Campbell, 2001; Yun et al., 2001, 2003); there is evidence that these genes drive LGE expression of Mashl and Dlxl&2. Mashl encodes a b-HLH transcription factor that autonomously promotes neurogenesis and nonautonomously represses differentiation of adjacent progenitors through Notch-signaling (Casarosa et al., 1999; Horton et al., 1999; Yun et al., 2002). Furthermore, it forms a complex with Brnl, POU-homeobox protein, which promotes neural differentiation (Castro et al., 2006). Mashl also promotes GABAergic fate (Fode et al., 2000). [00130] Dlxl&2 repress Mashl expression and Notch signaling, thereby driving later steps in LGE development (Yun et al., 2002). Dlxl&2^~'^~ mutants have reduced LGE expression of the Arx homeobox gene (Cobos et al., 2005a). ArK is required for migration of late-born striatal projection neurons (Colombo et al., 2007) and interneurons destined for the olfactory bulb (Yoshihara et al., 2005). These phenotypes are also found in the Dlxl&2^'A mutants (Anderson et al., 1997b; Long et al., 2007). However, striatal development is not fully blocked in the Dϊxl&I^1' mutants, demonstrating that parallel and/or redundant pathways continue to promote the generation and migration of some striatal neurons. Other transcription factors that control LGE specification and differentiation are herein identified.

[00131] Herein is described a comprehensive analysis of transcription factors that are expressed at various stages of differentiation in the embryonic LGE and the effect of loss otDlxl&2 function on their expression. Thereby we define transcription factors that are genetically downstream oϊDlxl&2, as well as transcription factors that are candidates to function upstream, redundantly and in parallel.

[00132] Mashl is a key candidate to function with Dlxl&2 to promote striatal differentiation. Through analysis of Dlxl όύ ' ^Mashl^''^' triple mutants we demonstrate that most striatal differentiation depends on their combined function. Furthermore, we have defined the unique and combined function of DIx 1&2 and Mashl in regulating development of distinct dorsoventral domains with the LGE and adjacent parts of the septum — this provides novel insights in development of the accumbens nucleus. As a whole, this study enables deciphering of the transcription factor circuitry that controls development of the basal ganglia.

MATERIALS AND METHODS

RNA preparation and gene expression array analysis

[00133] RNA was isolated from both the cortex and the lateral and medial ganglionic eminences and their mantle of embryonic day (E)15.5 mouse basal ganglia by dissection with fine forceps. We paid particular attention to avoiding contamination from the adjacent ventrolateral cortex in the basal ganglia samples. We identified Dhd&2^~'^~ mutants based on their cleft palate and subsequently by polymerase chain reaction (PCR) genotyping. RNA was pooled independently from the cortex and the subpallium of two Dlxl&2~^/~ and two Dlxl&2+/- mutants (= 20 μg). The sex of the specimens was not determined.

[00134] RNA was purified from forebrain tissue by first homogenizing in 1 mL TRIzol Reagent (Invitrogen, Carlsbad, CA) using Teflon homogenizer and then incubated at room temperature for 5 minutes. Then 200 μL of chloroform (Fisher Scientific, Pittsburgh, PA) was added, samples shaken vigorously, and spun in microcentrifuge at 12,00Og for 15 minutes at 4°C. The colorless upper phase was removed and RNA precipitated at room temperature for 10 minutes after the addition of 0.5 mL of isopropyl alcohol (Fisher Scientific). The samples were spun in microcentrifuge at 12,00Og for 10 minutes, washed with 70% ethyl alcohol (Fisher Scientific), and resuspended in 10 μL of nuclease-free water. The purified total RNA was shipped to the NINDS/NIMH Microarray Consortium (http://arrayconsortium.tgen.org/) where biotin-labeled cRNA hybridization probes were generated using the Affymetrix's GeneChip FVT Labeling Kit (Santa Clara, CA), which simultaneously performs in vitro transcription (linear = 20-60-fold amplification) and biotin-labeling. Briefly, the provided RNA was added to 4 μL of 10 X rVT Labeling Buffer, 12 μL of IVT Labeling NTP Mix, 4 μL of IVT Labeling Enzyme Mix, and nuclease-free water and incubated for 16 hours at 37°C. The samples were then stored at -8O⁰C until use in hybridization. Amplifications and hybridizations (in triplicate) using the Affymetrix Mouse Genome 430 2.0 array (which has coverage for 39,000 transcripts) were performed. cRNA was fragmented into 35-200 bp fragments using magnesium acetate buffer (Affymetrix). Ten μg of labeled cRNA was hybridized to Mouse Genome 430 2.0 array for 16 hours at 45⁰C. The GeneChips were washed and stained according to the manufacturer's recommendations using the Gene-Chips Fluidics Station (Model 450; Affymetrix). Each expressed gene sequence is represented by 11 probe pairs on the array and each oligonucleotide probe is 25mer. TGEN uses GeneChip Operating Software (GCOS) to scan the arrays and to perform statistical algorithm that determines the signal intensity of each gene. The data were presented using two different primary analyses: iterative comparisons and analyses performed in Gene-spring v. 6.2. For more in-depth analysis, we considered two populations of genes: the first being those genes obtained from the array that showed at least 2-fold change in expression between the BG of control and

-A

Dlxl&2 mutants with /"-value <0.05, and the second those genes that we determined were important based on our knowledge and literature searches.

Animals and tissue preparation

[00135] Mice were maintained in standard conditions with food and water ad libitum. All experimental procedures were approved by the Committee on Animal Health and Care at the University of California, San Francisco (UCSF). Mouse colonies were maintained at UCSF in accordance with National Institutes of Health and UCSF guidelines. Mouse strains with null allele of Dlxl&2 and Mashl were used in this study (Guillemot et al., 1993; Anderson et al., 1997b; Qiu et al., 1997). These strains were maintained by backcrossing to C57BL/6J mice. For staging of embryos, midday of the vaginal plug was calculated as embryonic day 0.5 (EO.5). PCR genotyping was performed as described (Anderson et al., 1997b; Parras et al., 2004). Since no obvious differences in the phenotypes of DU1&2+/+ and Dlxl&2+/- and Mashl+I+ and Mashl+I- brains have been detected, they were both used as controls. Embryos were anesthetized by cooling, dissected, and immersion fixed in 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS, pH 8.0) for 4-12 hours. Samples were either cryoprotected in a gradient of sucrose to 30%, frozen in embedding medium (OCT, Tissue-Tek, Torrance, CA), and cut using cryostat or dehydrated in ethanol, embedded in paraffin, and cut using microtome.

In situ hybridization

[00136] In situ hybridization experiments were performed using digoxigenin riboprobes on 20 μm frozen sections cut on cryostat. Tables Ia and Ib list the nucleotide sequences of in situ hybridization probes used in the in situ analyses. The sections were subsequently postfixed in 4% paraformaldehyde (PFA; Fisher Scientific) for 15 minutes. After three washes in 1 X PBS, sections were treated with 10 μg/mL proteinase K (Roche, Indianapolis, IN) in 1 X PBS for 15 minutes, transferred to 4% PFA for 5 minutes, and then washed three times for minutes each in 1 X PBS. Subsequently, sections were acetylated for 10 minutes (1.3% triethanolamine, 0.25% acetic anhydride, 17.5 mM HCl). Slides were then transferred to hybridizing chamber (Thermo-Shandon, Pittsburgh, PA) where they were incubated for 1 hour at room temperature with 500 μL of hybridization solution (50% formamide [Ambion, Austin, TX], 10% dextran sulfate, 0.2% tRNA [Invitrogen], 1 X Denhardt's solution [from 50 x stock; Sigma, St. Louis, MO], 1 X salt solution [from 10 X stock containing 2M NaCl, 0.1 M Tris, 50 mM NaH₂PO₄, 5OmM Na₂HPO₄, 50 mM EDTA, pH 7.5]). Digoxigenin (DIG)-labeled RNA probes were heated to 80⁰C for 10 minutes, cooled in ice, and added to prewarmed (62⁰C) hybridization solution to final concentration of 200 -400 ng/mL (typically 0.2 μL of probe in 100 μL of hybridization solution). Then 200 μL of hybridization solution containing the appropriate probe was added to each slide, which was subsequently covered with coverslip and incubated overnight at 62⁰C. The next day the coverslips were gently removed and the slides were washed three times for 20 minutes each with 50% formamide (Ambion), 0.5 X SSC, and 0.1% Tween 20 at 62⁰C. Slides were then washed three times in MABT (0.1 maleic acid, 0.2 NaOH, 0.2M NaCl, 0.01% Tween 20, incubated for hour in blocking solution [10% blocking solution (Roche) and 10% sheep serum (Sigma) in MABT]). Slides were then incubated overnight with anti-DIG antibody (1:5,000; Roche) diluted in solution containing 1% sheep serum and 1% blocking solution in MABT. Slides were next washed three times for 60 minutes each in MABT. The slides were then washed three times for minutes each in reaction buffer (0.1 Tris, pH 9.5, 0.1M NaCl, and 50 mM MgCl₂) and incubated in the dark in nitroblue tetrazolium (NBT)/5-bromo-4-chloro-3-indolyl phosphate (BCIP) solution (3.4 μL/mL from NBT stock and 3.5 μL/mL from BCIP stock in reaction buffer [100 mg/mL NBT stock in 70% dimethylformamide; 50 mg/mL BCIP stock in 100% dimethylformamide; Roche]). Slides were checked periodically and the reaction was stopped in 10 mM Tris pH 8.0, 0.05 M EDTA buffer. Finally, slides were dried overnight, dehydrated, and coverslipped with Permount (Fisher Scientific). Statements about the levels of expression are based on interpretation by at least two independent observers; whenever possible domains of expression outside of the DIx expression zone are used as internal controls. Images of stained sections were captured using Zeiss AxioCam MR (Thornwood, NY) and saved as TIFF files. These files were then opened with Adobe Photoshop (San Jose, CA) and adjusted using the auto contrast menu item and compiled into Adobe Illustrator to create the figures. [00137] RESULTS

Identification of transcription factors differentially expressed in the embryonic basal ganglia and cortex [00138] To understand the mechanisms that regulate patterning and differentiation of the mouse embryonic basal ganglia and cortex, we used gene expression array analysis and informative methods to identify the transcription factors (TFs) expressed at E15.5 and E18.5. Table 2A lists alphabetically TFs identified by the microarray analysis on RNA prepared from E 15.5 basal ganglia and cortex. Table 2 A further lists the transcription factor gene name, basal ganglia/cortex (BG/Ctx) ratio of expression in wild type E15.5 embryos, the expression levels in the wild type cortex (Ctx) and basal ganglia (BG), and the expression level in the BG of the Dlxl&2-/- mutants (BG -/-) (expression levels are in arbitrary units generated by the analysis of gene expression array data). Also shown are the investigators from who we received the plasmid used for in situ hybridization (Origin of Plasmid). Genes shown in green are expressed primarily in the basal ganglia. Genes shown in aqua are expressed both in the basal ganglia and cortex, but have a 2-fold bias towards the basal ganglia. Genes shown in yellow are expressed in both the basal ganglia and cortex at roughly the same level. Genes shown in lavender are expressed primarily in the cortex. The gene in orange is expressed in the diencephalon and only a small part of the amygdala. The columns on the left indicate whether we performed in situ hybridization (ISH) at E12.5 and E15.5. NR1H4 (FXR), FoxOl, NR4A1, and Nolzl are genes expressed in the striatum at E18.5 (Chang et al., 2004; Gray et al., 2004); in situ analysis did not detect El 5.5 expression. Table 2B lists transcription factors that remain strongly expressed at El 8.5 in mouse striatum. The list is ordered by normalized expression level and shows descriptions of the gene products. [00139] We generally restricted our analysis to TFs whose expression was above the level of 30 units (we cannot usually detect expression by in situ hybridization of most genes below 30 units). We compared TF expression in basal ganglia and cortex (Table 2A). Using these results, we defined three classes of TFs. [00140] Class 1. The expression of these TFs is largely restricted to the El 5.5 basal ganglia; they are indicated in green: ATBFl, Brn4, Dlxl/2/5/6, Ebfl, ESRG, Gbxl/2, Gshl/2, Ikaros, Islet 1, Lhx6/7, Liml, Med6, Meisl, Nkx2.1, Nkx2.2, Nkx5.1, Nkx6.2, Nolzl, Npasl, Otx2, Pbx3, Peg3, Proxl, RARβ, RXRy₁ Six3, Vaxl, and Zicl. These TFs are responsible for regulating regional identity or phenotypes specific to basal ganglia neurons, such as gene programs responsible for making GABAergic medium spiny neurons of the striatum or GABAergic local circuit neurons of the cortex and olfactory bulb.

[00141] Class 2. These TFs are expressed in both El 5.5 cortical and basal ganglia cells, but show at least 2-fold bias toward basal ganglia expression; they are indicated in aqua: Arx, Asb4, BrnS, COUP-TFII, Egr3, ER81, Evi3, FoxPl, FoxP2, Lmo4, Mqβ, Mashl, Oct6, OHgI 12, Soxl, Sox 10, Sp8, Sp9, and TCF 4. These TFs may share similar functions within the cortical and subcortical telencephalon, but can also influence processes specific to the basal ganglia.

[00142] Class 3. These TFs are expressed at roughly equal levels in the El 5.5 cortex and basal ganglia, or are expressed at higher levels in the cortex and are indicated in yellow: BFl, Brn2, COUP-TFl, Ctipl, Ctip2, Cux2, antisense Dlx6, Emxl, Emx2, Erm, FoxGl, FoxP4, GUI, Hesl, HesRl, Hes5, Id4, Lhx2, Lmol, Lmo3, Meβc, Meis2, Nexl, NHLH2, Nur77, Otxl, Pax6, Pbxl, RORβ, Sall3, Sox4, Soxll, TIx, TLE4. These TFs have general roles in regulating developmental processes common to both parts of the telencephalon.

[00143] In addition to regional specificity, we sorted some of these TFs according to their expression within proliferative zones. Table 3 lists TFs that are expressed in the T LGE in the Dlxl&2-I- Mutants, and defines their expression in primary and secondary progenitors (VZ and SVZ) and in postmitotic neurons of the striatum (MZ). The ventricular zone (VZ), subventricular zone (SVZ), and mantle zone (MZ) of the LGE of an E15.5 embryo are depicted as discrete boxes. The effect of the Dlxl&2-I- mutation on gene expression in each box is indicated using a color code: Gray represents unchanged gene expression. White represents no detectable expression. Magenta represents severe reduction in expression. Orange represents moderate/mild reduction in expression. Blue represents an increase of gene expression. Green represents ectopic expression. The genes are ordered as follows: left column are genes expressed in the proliferative zones (VZ and SVZ); right column are genes expressed at later developmental stages (SVZ, SVZ and MZ, MZ). The genes are arranged alphabetically within each grouping. A "d" represents the effect is primarily in the dorsal part of the LGE and a "v" represents the ventral part. Numerous expression patterns were noted, for instance, Dlxl&2 and Mashl are expressed in progenitors, whereas Ikaros and RXRy are expressed in postmitotic neurons. Below we describe how loss of either DIx 1&2 ox Mashl function affects the expression of many of these TFs..

Identification of transcription factors whose expression is altered in the subcortical telencephalon of Dlxl&T'^' mutants

[00144] The DIx family of transcription factors is preferentially expressed in the basal ganglia at E15.5 (Table 2A) (Bulfone et al., 1993a,b; Porteus et al., 1994; Liu et al., 1997; Eisenstat et al., 1999). Analysis of mice with targeted null mutations in both DIx 1 and Dlxl (Dlxl&2^~/~) show that the DIx genes are necessary for differentiation and migration of basal ganglia GABAergic neurons (Anderson et al., 1997a; Yun et al., 2002; Cobos et al., 2007). To identify Dώc-regulated TF genes we used gene expression microarray analysis to compare TF expression in the basal ganglia of El 5.5 control and DIx 1&2^''^' mutants. Of the genes listed in Table 2 A, 15 genes showed greater than 2-fold reduced expression, 8 genes showed greater than 2-fold increased expression, and the expression of 72 genes did not change significantly in the Dlxl&I^1' basal ganglia (Table 2A).

[00145] The microarray data do not indicate how the TF expression changed within the different cellular subtypes of the basal ganglia. For example, changes in expression could reflect alterations in progenitors and/or postmitotic cells. Therefore, to obtain spatial resolution of TF gene expression we performed in situ hybridization on E15.5 control and Dlxl&T^1' mutant coronal sections (Figs. 1-6). Because of the complexity of the basal ganglia, we concentrated this study's analysis on rostral telencephalic regions that contain the LGE and septum. Table 3 summarizes the expression patterns of 60 TFs in the LGE, and defines how their expression changes in primary and secondary progenitors (VZ and SVZ, respectively) and in postmitotic neurons of the striatum (MZ) of Dh.l&2'^' mutants. Details of the in situ hybridization analysis are described below.

Dlxl&2 specify the molecular identity of the SVZ in the dLGE by positively regulating expression of a set of transcription factors

[00146] Dlxl&2 are expressed in a dorsoventral gradient in progenitor cells of the LGE at E12.5 and E15.5 (Fig. la,b; Fig. 5). Their expression is particularly high in the dorsal LGE (dLGE), where they are detected in most cells in both the ventricular and subventricular zones beginning around E10.5 (Yun et al., 2002). They also show similar dorsoventral gradient in the septum (Fig. la,b). Dlxl&2^'/' mutants have clear defect in LGE development, whereas the septal deficits are subtle (Fig. la-r¹) (Anderson et al., 1997a,b). DIxS and Dlxβ expression is lost in the LGE and maintained or increased in septal neurons (Fig. lc-d'; Table 3) (Anderson et al., 1997a,b). Thus, in contrast to the septum, the LGE ofDlxl&2^'A mutants lack expression of all DLX proteins expressed in the brain (DLX1,2,5,6) (Fig. 1) (Eisenstat et al., 1999).

[00147] Truncated Dlxl and Dlx2 transcripts, which do not encode functional proteins, are produced in Dhd&2^~'^~ mutants (Fig. la,b') (Zerucha et al., 2000; Long et al., 2007). Using in situ probes to the truncated DIxX and Dlx2 transcripts, we investigated the population of DIx- lineage cells that persist in Dlxl&2^'/' mutants. Dlxl RNA expression continues at low levels throughout the SVZ of the subpallium in the Dlxl&2^~'^~ mutants. Thus, we conclude that Dlxl expression is, at least in part, independent of DIx function and cells in the DIx lineage are present in primary and secondary progenitor populations (Fig. Ia'; Fig. 5). However, Dlxl expression in the mantle zone is not detectable in the mutant, indicating that mantle neurons generated from the LGE progenitors fail to activate and/or maintain Dlxl RNA expression.

[00148] Unlike Dlxl, Dlxl RNA expression is not detectable in the dorsal LGE (dLGE) and dorsal septum (dSe). However, its expression is maintained, albeit at low levels, in the SVZ of the ventral LGE (vLGE) and ventral septum (vSe) (Fig. Ib', Fig. 5). Lack of Dlxl RNA in the dLGE and dSe indicates that these progenitor zones are the most severely affected by loss of Dlxl &2 function. This is supported by the greatly reduced expression of several TFs in the dLGE: ATBFl, Brn4, ER81, ESRG, Meisl, Meis2, Oct6, Pbxl, Six3, Sp& and Vaxl at E15.5 (Fig. le-i', 11-r'; Table 2A) (E12.5 analysis of a subset of these TFs support this conclusion; Fig. 5). [00149] While the dLGE shows the greatest reduction in TF gene expression, the vLGE also is defective in the Dlxl &2^'1' mutants, as exemplified by reduced Brn4, GUI, and Oct6, expression (Fig. lf,f Ij j'ln,n'). Similar to the LGE, dorsal parts of the septum are preferentially affected by loss of Dlxl &2 function, as exemplified by reduced ATBFl, Brn4, Ctipl, ER81 and Pbxl expression in the dSe (Figs. le,e' lf,f 5i,i' lh,h' lo,o') Ectopic cortical marker expression in the dLGE of Dlxl&2?^~ mutants

[00150] Disruption oiDlxl&2 function, perhaps through the loss of DLX1,2,5,6 expression, has profound effect on specification of dLGE SVZ cells. Another TF expressed in the developing basal ganglia, Gsh2, has been shown to be important for specifying dorsoventral fate in the LGE (Corbin et al., 2000; Toresson et al., 2000; Yun et al., 2001). To test whether Dlxl&2 have similar function as Gshl in specifying dLGE identity, we studied whether there is ectopic expression of ventrolateral cortical markers in the dLGE. In the Dlxl&I^1' mutants, we previously observed ectopic expression of Neuropilinl in the dLGE, which is normally strongly expressed in the ventral cortex (Fig. 6q,q') (Marin et al., 2001; Le et al., 2007). Consistent with this, the TFs Ebβ, IdI and NHLH2, which ordinarily mark cells of ventrolateral cortex, all show ectopic expression in the SVZ of the dLGE (Fig. 2a-c'; Tables 1, 3). This further indicates that some dLGE SVZ cells have shifted from subcortical identity toward cortical one. Gshl expression is maintained in the Dlxl&Z^1' mutants (Table 2A), indicating that the shift in subcortical identity occurs through D/x7cδ2-dependent and Gsλ2-independent pathway.

[00151] To more fully explore the extent of the subcortical to cortical shift in gene expression, we examined expression of other cortical markers, including both TFs (Dbxl, Emxl, Emx2, Otxl, Pax6, Tbrl and Tbr2) and non-TFs (vesicular glutamate transporters: Vglutl and Vglut2). None of these genes were ectopically expressed in the dLGE (Fig. 2d-e and data not shown). Furthermore, analysis at E12.5 failed to show ectopic Ebβ, IdI and NHLH2, providing evidence that this phenotype appeared during further maturation of the dLGE/striatum (Fig. 5). Thus, although the E15.5 Dlxl&2^~/~ dLGE SVZ has ectopic expression of some ventrolateral cortical TFs, it has not fully shifted its fate to produce cortical neurons with glutamatergic features (Vglutl &2), and it maintains expression OΪGAD67, although at lower level (Fig. 6gg,gg')These results indicate that the Dlxl&2^" dLGE maintains its subpallial identity likely through the expression of subcortical TFs necessary for dLGE specification.

dLGE molecular identity is partially maintained in the Dlxl&I^^' mutant: DLx 1&2 negatively regulate a subset of transcription factors expressed in the VZ and SVZ of the LGE

[00152] To identify TFs that might compensate for loss of Dlxl&2 function and maintain dLGE identity, we further characterized TFs that are upregulated va Dlxl&2^~/~ basal ganglia. Loss of DIx 1&2 function leads to increased SVZ expression of some TFs that mark LGE progenitors. This includes TFs that normally are expressed in the VZ of the LGE (COUP-TFI, Erm, Lhx2, Otx2, Pax6, RORb and 7Zx) and TFs that are expressed in both the VZ and SVZ, or SVZ, of the LGE (ESRG, Foxgl, Gsh2, Hes5, Liml, Lmol, Mashl, Sall3, Soxll and Sp9) (Figs. 1, 3; see Fig. 5 for E12.5 data) (Yun et al., 2002). Thus, while loss of DIx expression results in downregulation of some TFs in the dLGE SVZ (Fig. le-r') a separate class of TFs may be responsible for maintaining dLGE molecular properties (Fig. 3), explaining why the dLGE does not fully take on cortical properties (Fig. 2).

[00153] It merits mention that the expression of some TFs, whose expression marks VZ cells throughout the telencephalon (Hesl, Hesrl), do not appear to be altered in the Dlxl&2^~'^~ mutant (data not shown) (Yun et al., 2002). Thus, while DIx 1&2 function is required to repress expression of several VZ TFs and maintain LGE identity, expression of a distinct set of progenitor cell regulators is not under DIx control, thereby contributing to the DIx independent specification of the LGE.

Ectopic expression of selected MGE and diencephalic TFs in the SVZ of the LGE

[00154] Our data indicate that DIx 1&2 promote LGE differentiation through repression of LGE progenitor TFs

(COUPTFI, Erm, Foxgl, Gsh2, Hes5, Lhx2, Lmol, Mashl, Otx2, Pax6, ROR-b, Sall3, Sp9, and TJx), and ventral cortical TFs (Ebβ, Id2, and NHLH2). To determine if Dlxl&2 might regulate LGE identity through repression of TFs expressed in other forebrain domains, we examined the expression of TFs that are restricted to the MGE and diencephalon. Indeed, DIx 1&2 repress TFs that are normally restricted to the E 12.5 and E 15.5 MGE (Gshl, Gbxl, and Gbx2), and the progenitor cells of small domain of the amygdala and diencephalon (Otp) (Fig. 4, and Fig. 5). DIx repression is specific to these ventral genes, as other ventral telencephalic TFs are not ectopically expressed (Nkx2.1, NkxS.l, Nkx6.2, Lhx6, and Lhx7/8; Fig. 5 and data not shown). Thus, Dlxl&2 have a fundamental role in specifying the properties of LGE SVZ progenitors by repressing certain MGE TFs, diencephalic TF, ventrolateral cortical TFs and selected TFs expressed in the VZ of the LGE.

Expression of some TFs is partially maintained in LGE SVZ and in differentiating striatal cells in the Ωbcl&f^' mutant

[00155] To determine which TFs act in parallel with Dlxl&2 for LGE formation, we next examined which aspects of LGE differentiation are maintained in the Dlxl&T^1' mutants. Although progenitor cells of the Dlxl&2^';' mutant LGE have ectopic expression of cortical and MGE TFs, they still express Gshl and Mashl, TFs that are essential for LGE development (Casarosa et al., 1999; Horton et al., 1999; Corbin et al., 2000; Toresson et al., 2000; Toresson and Campbell, 2001; Yun et al., 2001, 2002, 2003). Thus, to address what aspects of LGE development are preserved in Dϊxl&2^~'^~ mutants, we studied the expression of TFs that mark the El 5.5 LGE SVZ and F5 mantle zone (MZ; striatum and olfactory tubercle) (Fig. 5). This analysis identified two types of TFs: 1) those whose expression is strongly reduced in the SVZ and/or MZ (particularly in the dLGE), and 2) those whose expression is mildly reduced and/or maintained.

[00156] There were several TFs whose expression was either eliminated (Dlx5&6) or greatly reduced in the SVZ/MZ of the dLGE (Egr3, Evi3, Dcaros, MeGc, RARβ and RXRγ) (Figs, lc-d' 5a-f) Note that ATBFl, Egr3, Ikaros, RARβ and RXRγ expression are nearly specific for striatal and septal cells. The striatal expression of these TFs is reduced particularly in parts related to the dLGE (ATBFl, Meis2, Pbx3) (Fig. 5h,h',r,s'). Furthermore, expression of several TFs in striatal-related structures, such as the olfactory tubercle (OT), appears to be lost (ATBFl, FoxPl, FoxP2, FoxP4, Islet 1, Lmo4, RXRγ, Six3, and Soxl) (Fig. 5e,e',h,h¹,k-m',p,p',s-t') [00157] A larger set of LGE TFs continue to be expressed to varying degrees in the LGE SVZ and/or MZ: Arx, ATBFl, Ctipl, Ebfl, ESRG, Evi3, FoxGl, FoxPl, FoxP2, FoxP4, Isletl, Lmo3, Lmo4, Meisl, Meis2, Pbx3, Six3, Soxl, Sox4, Soxl 1, and Tle4 (Figs. li,i',l,l'; 3f,f ,m,m'5b,b',g-v') (Cobos et al., 2005a). Expression of some TFs remains strong in both the dLGE and vLGE, such as Ctipl, Ebfl, FoxPl, FoxP2, FoxP4, Isletl, Lmo3, Lmo4, Soxl, Sox4, and Tle4 (Fig. 5i-p',u-v').

Expression of some effector genes is partially maintained in the LGE SVZ and in differentiating striatal cells in the Dlxl&T mutant

[00158] Thus, based on TF expression in DIx 1 &2^~'^~ mutants, some programs of striatal differentiation are attenuated, while others are partially maintained. To characterize which aspects of striatal differentiation are affected in Dlxl&l^1' mutants, we examined expression of genes that mark the striatal differentiated state: dopamine, receptor 1 (DlR), dopamine receptor 2 (D2R), glutamic acid decarboxylase 61 (GAD61 or GADl), preprotachykinin (Substance P), and vesicular GABA transporter, as well as other markers of the developing striatum (Cad8, Golf, Gucyla3, Neurexin3, PK2, PKRl, Robol, Robo2, Sema3a, Tiam2, and TrkB) (Fig. 6 and data not shown) (Long et al., 2007). In some cases, expression of striatal genes was not detectable in the Dlxlδt2^~'^~ mutant (i.e., CadS and Tiam2; Fig. 6a,a',m,m'). However, in most cases residual expression was seen in superficial parts of the striatal mantle (DlR, D2R, Enk, GAD67, Golf, Gucyla3, SubP, Robo2, Sema3a, and TrkB), demonstrating that the TF programs for striatal histogenesis are partially preserved (Fig. 6). Note, however, that the expression of these genes is particularly attenuated in parts of the striatum related to the dLGE, highlighting the evidence that DIx 1&2 function is critical for this region.

[00159] Loss oϊDlxl&2 function does not strongly affect some genes (Robol) and in some cases leads to overexpression of other genes (CyclinD2, Pak3, PK2, PKRl, Slitl) (Fig. 6) (Cobos et al., 2007). These findings further support the idea that aspects of LGE identity are maintained by TFs, such as Mashl, which continue to be expressed in the Dlxl&l'^' mutants (Figs. 1, 3, 5) (Cobos et al., 2007; Long et al., 2007). Next, we tested this hypothesis by studying the LGE and striatal phenotype of Dlxl&2^''^'; Mashl'^' triple mutants.

Dlxl&T' ^'',MaShI^''^' compound mutants define genes epistatic only to Dlx\&2 or epistatic to both Dlxl&2 and Mashl

[00160] While many aspects of LGE/striatal differentiation are lost in the Dlxl&2^~'^~ mutants, many aspects are also maintained (Figs. 1, 3, 5, 6). The maintained characteristics may be regulated by the expression of TFs whose expression persists in mutant LGE progenitors (Figs. 1, 3, 5). A good candidate of this type of TF is Mashl, due to its overexpression in the Dlxl&2^~'^~ mutants (Fig. 3j j'). As MASHl and DLX2 proteins are coexpressed in progenitors of the dLGE (Porteus et al., 1994; Yun et al., 2002), they have the potential to regulate the developmental programs of these cells in parallel and/or in series. Here we explored the hypothesis that Mash 1 has a critical role in maintaining certain aspects of LGE/striatal differentiation in the Dlxl&2^~'^~ mutants. [00161] We studied the expression of TFs and selected other genes in the LGE and striatum in Dlxl&2^~'^~, Mashl^'1' and Dlxl&2^~'^' -Jdashl'^' mutants at El 5.5, concentrating on genes whose expression persists in Dlxl&2^~'^' mutants (Fig. 7; Table 4). In Table 4, the ventricular zone (VZ), subventricular zone (SVZ), and mantle zone (MZ) of the LGE and septum of an E15.5 embryo are depicted as discrete boxes. The effect of the Dlxl&2-/- (D), Mashl-/- (M), and Dlxl&2-/-; Mashl-/- (DM) mutations on gene expression in each box is indicated using a color code: Gray represents unchanged gene expression. White represents no detectable expression. Magenta represents severe reduction in expression. Orange represents moderate/mild reduction in expression. Blue represents an increase of gene expression. Green represents ectopic expression. The genes are ordered according to when their expression begins; genes at the beginning are expressed in the VZ, whereas genes at the end are expressed only in the MZ. The genes are arranged alphabetically within each generalized grouping. The left columns correspond to the LGE and the right columns correspond to the septum. A "d" represents the effect is primarily in the dorsal part of the structure and a "v" represents the ventral part.

[00162] Expression of these genes fell into two general classes (Table 6): I) epistatic only to Dlxl&2^~'^' or II) epistatic to both Dlxlά.2^'1' and Mashl^'1'. Expression of Class I genes (ER81, GHl, Gshl, Sp8) is reduced or lost in the Dlxl&2^~'^~ mutants, is not overtly affected in the Mashl^'1' mutants, and the triple mutant phenocopies the Dlxl&2^~'^~ mutant. Expression of Class II genes is altered in both the Dlxl&2^'A and Mashl'^' mutants, and in most cases these phenotypes are exacerbated in the triple mutants. There are six subtypes of Class II genes based on their expression changes (described in Table 6). We hypothesize three classes of responses to DIx 1&2 that appear to be independent of Mashl function (Class Ia,b,c), and six classes of responses to Dlxl&2 and Mashl (Class πa,b,c,d,e,f). Class I genes are therefore epistatic only to DIx 1&2, whereas Class II genes are epistatic to both Dlxl&2 and Mash 1. The types of responses are described in the table.

[00163] Finally, several transcription factors continue to be expressed, albeit generally at lower levels, in the LGE oϊDlxl&Z'-; Mashl^1' mutants (Arx, Gshl, Gsh2, Isletl, Lmo4, O!ig2, Pax6, and Pbxl) (Figure 7; Table 4), demonstrating that some fundamental aspects of LGE specification are independent of DIx and Mashl. Some of these genes may be responsible for maintenance of the remaining striatal differentiation in these mutants. [00164] The expression study further revealed that the expression of numerous other genes, (non-transcription factors) were affected in the Dlxl&2-/- Mutants. Table 5 alphabetically lists the gene name, basal ganglia/cortex (BG/Ctx) ratio of expression in wild type El 5.5 embryos, the expression levels in the wild type cortex (Ctx) and basal ganglia (BG), and expression level in the BG of the Dlxl&2-/- mutants (BG -/-) (expression levels are in arbitrary units generated by the analysis of gene expression array data). Also shown is the NCBI Accession Number for the gene used in the in situ hybridization (Origin of Plasmid). The column on the left indicates whether we performed in situ hybridization (ISH) at E15.5.

Mashl and Dlxl&2 regulation of septum and vLGE differentiation

[00165] While dLGE development is more dependent on DIx 1&2 than Mashl, the septum and the vLGE are more dependent on Mashl (Fig. 7; Table 4). The septum in DlxlSύ.^'1' mutants is relatively normal, most likely preserved through the continued expression of Mashl and Dlx5&6 (Figs. lc',d'; 3j j')However, in Mashl^'1' mutants the septum (particularly the ventral septum) is hypoplastic and lacks almost all expression of Er81, Hes5, Isletl, Olig2, Pbxl, Sp8, and Sp9 (Fig. 7; Fig. 9). Despite these dramatic reductions, Mashl^'1' mutants maintain expression of Arx, Dlxl, Dlx5, and GAD61 (Fig. 7). Septal size in Dlxl&2^''^~Mashϊ'^~ mutants is further reduced, and Arx and GAD61 expression are substantially decreased (Fig. 7h",l")showing that this aspect of septal identity is determined by DIx function. Also note in the Mαshl^''^' mutants that vLGE expression of Arx, Isletl, Lmo4, Pbxl, Preprotαchykinin, Six3, and Sp9 is attenuated (Fig. 7, Fig. 9).

DISCUSSION

[00166] In this study we have provided foundation for defining the transcription factor (TF) circuitry that controls development of the LGE and its product, the striatum. Table lists all of the TFs that we could reliably identify that are expressed at E15.5 in the developing mouse basal ganglia (factors that are part of the core transcriptional machinery are not listed). Based on gene expression array and in situ hybridization we have identified 53 TFs that have higher expression levels in the basal ganglia than in the cortex (TFs colored in green and aqua in Table 2A); these are likely to have roles in defining features that are specific to basal ganglia neurons, such as GABAergic fate. [00167] Among these TFs, Dlxl&2 and Mαshl are known to have central roles in basal ganglia differentiation (Anderson et al., 1997b; Casarosa et al., 1999; Horton et al., 1999; Fode et al., 2000; Yun et al., 2002). We systematically defined the role oiDlxl&2 in regulating the expression of TFs listed in Table 2, identifying TFs whose expression is dependent and independent of Dlxl &2 function (Table 3). We provide evidence that some of

-A the Dlxl&2 independent TFs depend on Mαshl function, and vice versa (Table 4). Based on analysis of Dlxl &2

-I- -I- -I-

Mαshl and Dlxl&2 J - vlαshl mutants we propose epistatic relationships between these TFs (Table 4; Table 6). Subdivisions of the basal ganglia

[00168] The progenitor domains of the embryonic basal ganglia consists of the septum, LGE, MGE, and preoptic area, each of which has multiple subdivisions (Yun et al., 2001; Campbell, 2003; Flames et al., 2007; Long et al., 2007). In these experiments we have focused on rostral parts of the LGE and the septum, each of which has dorsal and ventral progenitor domains (Fig. 1). In subsequent experiments we focus on analysis of the caudal LGE (which includes most of the CGE), MGE, and preoptic area.

[00169] The dLGE contains progenitors for both the striatum and olfactory bulb interneurons (Toresson et al., 2000; Corbin et al., 2000; Yun et al., 2001 ; Stenman et al., 2003), whereas vLGE progenitors are currently thought to produce primarily striatal neurons (Toresson and Campbell, 2001; Yun et al., 2003). Given its proximity to the septum, our data indicates that rostral parts of the vLGE produce accumbens neurons.

[00170] DIx 1&2 are expressed in dorsoventral gradient in both the LGE and septal progenitor domains (Fig. la,b; Fig. 8) (Eisenstat et al., 1999; Yun et al., 2002). In the dLGE, Dlxl&2 are expressed in most cells of the VZ. Previously, we demonstrated that DLX2 and MASHl are coexpressed in most dLGE progenitors (VZ and SVZ), whereas in the vLGE there is much less DLX2 expression, particularly in the VZ (Yun et al., 2002). Here we present evidence that Dlxl&2 function is more important in the dLGE than the vLGE, whereas Mashl function is more important in the vLGE and the septum.

Dlxl&2 specify the fate and differentiation of dLGE neurons

-/- [00171] Previous analyses of the Dlxl&2 mutants demonstrated that these TFs regulate differentiation and migration of LGE-derived progenitors in part through repressing expression of Mashl and the Λfo/cΛ-signaling pathway (Anderson et al., 1997b; Yun et al., 2002). Here we demonstrate more profound defects in the dLGE — the

SVZ of the Dlxl&2 mutants ectopically express ventral pallial (Ebβ, Id2), MGE (Gbxl&2; Gshl), and diencephalic (Otp) TFs (Figs. 2, 4). The neurons generated in the Dlxl&2 mutant dLGE express low levels of GAD61 and vesicular GABA transporter (Fig. 6) (Long et al., 2007). Thus, Dlxl&2 are essential for repressing both dorsal (pallial) and ventral (MGE) TFs from the dLGE, in addition to promoting GABAergic fate. This is consistent with previous evidence that the DIx genes are sufficient to induce expression of GABAergic markers as a result of ectopic expression oϊDlxl and Dlx5 in cortical progenitors (Anderson et al., 1999; Stuhmer et al., 2002). [00172] Loss of Dlxl&2 greatly reduces dLGE expression of Arx, ATBFl, Brn4, DIxS, Dlx6, ER81, Meisl, Meis2, Oct6, Pbxl, Six3, Sp8, and Vax\ (Figs. 1, 5; Table 3). Currently, the function of only Arx, Sp8, and Vax\ has been defined in the dLGE. Arx, Sp8, and Vax\ promote development of interneurons that migrate rostrally from this zone to the olfactory bulb (Soria et al., 2004; Yoshihara et al., 2005; Waclaw et al., 2006). Dlxl&2 mutants fail to produce olfactory bulb interneurons due to combination of molecular specification and migration defects, which include reduced expression of Arx, Sp8, and Vaxl (Figs. 1, 5) (Bulfone et al., 1998; Long et al., 2007).

-I-

[00173] Dlxl&2 mutants also show severe defects in striatal and olfactory tubercle development. Previously, we provided evidence that early LGE differentiation and migration to the striatum were preserved (El 1.5-E12.5) relative to those processes at E15.5 (Anderson et al., 1997b; Yun et al., 2002). However, early LGE development is not normal; most of the molecular defects observed at E15.5 can be appreciated at El 1.5 and E12.5 (Fig. 5) (Cobos et al., 2005b; Long et al., 2007). Furthermore, there is reduction in the numbers of neurons that express markers of both the striatonigral (dopamine receptor 1; DlR and preprotachykinin) and striatopallidal (dopamine receptor 2;

D2R and enkephalin) medium spiny neurons (Fig. 6; data not shown). -/- [00174] Reduced expression of several TFs is likely to contribute to the DIx 1&2 striatal hypoplasia and molecular defects. Reduced Arx expression (Fig. 5g,g') (Cobos et al., 2005b) may result in migration defects of

LGE-derived cells, as Arx mice have related phenotype (Colombo et al., 2007). Likewise, reduced expression of retinoid nuclear receptors (RARβ and RXRγ) could contribute to the striatal phenotype (Fig. 5e,e',f,f ). Retinoid signaling through these receptors is implicated in regulating striatal differentiation (Toresson et al., 1999; Waclaw et al., 2004) and the expression of DlR and D2R (Krezel et al., 1998; Wang and Liu, 2005).

-/- [00175] Despite the severe molecular defects in the LGE of the Dlxl&2 mutants, some features of the LGE and even the striatum are preserved (Figs. 3, 5, 6, 7; Tables 2, 3). This is most likely due to set of TFs that continue to be expressed in LGE progenitors (Figs. 3, 5; Tables 2, 3). For instance, expression of the neurogenic TFs Sox4 and Soxll (Bergsland et al., 2006) is preserved (Figs. 3m,m'; 5u,u'), consistent with the preservation of core features of neurogenesis in the LGE, such as MAP2 and β-III-tubulin expression (Anderson et al., 1997b; Cobos et al., 2007).

-/- Furthermore, partial LGE identity may be maintained in DIx 1 &2 mutants by virtue of Gshl , Gsh2, Mash 1 , and TIx expression in progenitor cells. These TFs contribute to striatal development (Horton et al., 1999; Casarosa et al., 1999; Corbin et al., 2000; Toresson et al., 2000; Toresson and Campbell, 2001; Yun et al., 2001, 2002, 2003; Stenman et al., 2003). [00176] Indeed, striatal expression of certain TFs is maintained at relatively high levels (Fig. 5). This includes

Ebfl, aTF that regulates prenatal striatal development (Garel et al., 1999). In the postnatal brain, Ebfl is

-/- preferentially expressed in striatonigral neurons. Consistent with this, the Ebfl mutant mouse shows defects in gene expression (preprotachykinin) and projections of striatal neurons to the substantia nigra (Lobo et al., 2006).

-/- The continued expression of Ebfl in the DIx 1&2 mutants indicates that differentiation of striatonigral neurons may be preserved. However, other molecular features of these neurons (DlR, preprotachykinin, and Evβ [also known as Zfp521]) are more reduced than Ebfl expression (Figs. 5b,b'6c,c'jj')This indicates that Dlxl &2 differentially regulate expression of distinct sets of genes within immature striatonigral neurons. [00177] Expression of separate set of genes is repressed by Dlxl&2and is promoted by Mαshl [Hes5, Olig2, and

Sp9) (Fig. 7; Table 4; Table 6). Previously, we provided evidence that elevations inMαshl expression in the

-/- Dlxl&2 mutants leads to increased Notch-signaling that increases HesS expression (Yun et al., 2002). Current work provides evidence that Dlxl &2 repression oiOligl is central to promoting neurogenesis and blocking oligodendrogenesis in the telencephalon (Petryniak et al., 2007).

Mαshl has a prominent role in differentiation of vLGE and septal neurons

[00178] While dLGE development is severely derailed by loss of Dlxl&2, vLGE and septal development are relatively preserved (Figs. 1, 5-7; Fig. 9; Table 4), perhaps because Dlxl&2 are not as strongly expressed in the VZ of these progenitor domains (Fig. la,b) (Eisenstat et al., 1999; Yun et al., 2002) and because Dlx5&6 expression are maintained (Fig. Ic' ,d'). On the other hand, vLGE and septal differentiation and growth are strongly affected in the

-/- Mαshl mutant (Fig. 7; Fig. 9; Table 4). In particular, expression of Islet 1, Lmo4, Meis2, Pbxl, Six3, Sp9, and Vαxl are greatly reduced in these progenitor domains (Fig. 7; Fig. 9; Table 4). Despite these defects in the septum, expression of Arx, Dlxl, and Dlx5 are preserved, which may explain why GAD61 continues to be expressed in the

-/- septum ofMαshl mutants. Thus, while the dLGE and vLGE/septum express many of the same TFs, their development has distinct dependence on them. Furthermore, this indicates divergent programs for development of the striatum (regulated by the dLGE) and the nucleus accumbens (regulated by the vLGE).

DIx and Mashl have parallel and overlapping functions in LGE/striatal differentiation

[00179] DLX2 and MASHl are coexpressed in VZ and SVZ cells of the dLGE — this has allowed us to test whether they cooperate in regulating differentiation of these cells by comparing the phenotype of single and

-/- -/- -/- -/- compound mutants. Based on in situ hybridization analysis of DIx 1 &2 Mashl and DIx 1&2 ;Mashl mutants, we

-/- -/- -/- have identified genes that are epistatic only to Dlxl&2 (Class I) or epistatic to both Dlxl&2 and Mashl (Class

II) (Fig. 7; Table 4; Table 6). We have defined six subtypes of Class II genes based on their expression changes (described in Table 6). It is striking that LGE progenitors in the triple mutant continue to express Arx, Gsh, Islet 1, Lmo4, Olig2, and Pbx\ (Fig. 7). Thus, LGE specification (and GAD61 expression) has not been fully eliminated in the triple mutants, which implicates one or several of these TF genes in maintaining aspects of LGE identity. [00180] In addition to regulating overlapping transcriptional pathways in the LGE, Dlxl&2 and Mashl regulate parallel transcriptional pathways. For example, whereas Mashl promotes expression of genes involved in neurogenic differentiation such as expression of general neural markers such as Soxl Αnά Mapl (Fig. 9, data not shown) (Yun et al., 2002), Dlxl&2 are not required for induction of these genes (Fig. 9) (Yun et al., 2002; Cobos et al., 2007).

[00181] Our study provides a foundation for: 1) performing computational analyses of gene expression networks; 2) designing enhancer analyses; 3) further in vivo genetic analyses of single and compound mutants; and 4) deciphering transcriptional codes that can be used to drive immature progenitor cells to differentiate into striatal medium spiny neurons.

[00182] Finally, we elucidated how the DIx genes regulate the fate and function of LGE-derived neurons by identifying changes in the expression of effector genes (Fig. 6). For instance, Dlxl&2 have a profound role in defining the GABAergic fate through promoting expression of GAD61 and vGAT (Fig. 6g,g'; Figure 8) (Anderson et al., 1999; Stuhmer et al., 2002; Long et al., 2007). Dlxl&2 also regulate neuronal migration and neurite morphogenesis; recently we presented evidence that this is in part mediated through Dlxl&2 repression of Pak3 (Cobos et al., 2007). However, as shown in Figure 6, there are major changes in the expression of other genes that regulate the cytoskeleton through modulating intracellular signaling (Cad8, CXCR4, Golf, GucylaS, PK2, PKRl, RDC, Robol, Robo2, Sema3a, SHtI, Tiam2, TrkB). Finally, the DIx genes regulate receptors and neuropeptides that are central modulators of striatal function (DlR, D2R, enkephalin, andpreprotachykinin). Therefore, by virtue of their expression in progenitors (VZ and SVZ), immature neurons and mature neurons, the DIx genes have central roles in transcriptional hierarchies that specify the differentiation and function of striatal neurons and in initiating and maintaining the GABAergic state.

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Example 2: Dlxl&2 and Mashl transcription factors control MGE and CGE patterning and differentiation through parallel and overlapping pathways

[00238] Here we define the expression of approximately 100 transcription factors in progenitors and neurons of the developing mouse medial and caudal ganglionic eminences (MGE and CGE), anlage of the basal ganglia and pallial interneurons. We elucidated the transcriptional hierarchy of these genes with respect to the DIx homeodomain genes, which are essential for differentiation of most GABAergic projection neurons of the basal ganglia. This analysis identified Die-dependent and Die-independent pathways. The Dfcc-independent pathway depends in part on the function of the Mashl b-HLH transcription factor. These analyses define core transcriptional components that differentially specify the identity and differentiation of the globus pallidus, basal telencephalon and pallial interneurons.

[00239] The combination of transcription factors (TFs) that are expressed in a cell are a fundamental signature of its identity. This information is essential for understanding the transcriptional networks that are operating to control the state of the cell, whether during development or in maturity. Furthermore, understanding the transcriptional hierarchy provides useful information for engineering stem and progenitor cells to become cells of specific phenotypes. In elucidating the TF codes expressed in stem/progenitor and their derivatives in the developing basal ganglia and their derivatives, including cortical interneurons, we have systematically identified and characterized the expression of TFs in the prenatal mouse subpallium, defining those TFs that are expressed in stem/progenitors, and those expressed in postmitotic cells. We identified two major transcriptional pathways in the developing subpallium, regulated by the Dlxl&2 and Mashl genes (Anderson et al., 1997a,b; Casarosa et al., 1999; Yun et al., 2002; Castro et al., 2006; Long et al., 2007). Here we evaluate the effects of null mutations of either Dlxl&2, or Mashl, or Dlxl&2 and Mashl on the expression of many of these subpallial TFs. In previous studies we focused on the TF codes, and effect of the Dlxl&2 and Mashl mutations on the developing septum, lateral ganglionic eminence (LGE), and olfactory bulb.

[00240] Here we investigated these parameters in the medial ganglionic eminence (MGE) and caudal ganglionic eminence (CGE). The MGE is the anlage for the pallidum (globus pallidus are related pallidal cell groups), interneurons that tangentially migrate to the pallium (cortex and hippocampus), and striatum (Sussel et al., 1999; Marin and Rubenstein, 2001; Wonders and Anderson 2006; Xu et al., 2008) and oligodendrocytes (Kessaris et al., 2006; Petryniak et al., 2007). The CGE is the anlage for distinct subtypes of pallial interneurons (Xu et al., 2004; Butt et al., 2005; Wonders and Anderson, 2006; Myoshi et al., 2007); it is currently unknown whether the CGE also produces neurons that remain in the subpallium. [00241] Our analysis, based on gene expression array data, followed by in situ hybridization, provides a nearly comprehensive description of the TFs expressed in stem/progenitor cells and their derivatives of the embryonic day (E) 15.5 MGE and CGE, in mice with different dosages of Dlxl&2 and Mashl.

Materials and Methods

[00242] RNA preparation and gene expression array analysis RNA was isolated from El 5.5 mouse embryos using either the dissected cortex, the combined lateral and medial ganglionic eminences and their mantle, or the medial ganglionic eminence from control (mixture of wild-type and Dlxl/2 ; ratio not known) or Dlxl/2 brains (Cobos et al., 2007; Long et al., 2008). RNA was purified and shipped to the NINDS/NIMH Microarray Consortium (TGEN; http://arrayconsortium.tgen.org/) where biotin-labeled cRNA hybridization probes were generated using the Affymetrix's GeneChip IVT Labeling Kit (Santa Clara, CA), which simultaneously performs in vitro transcription (a linear ~20-60-fold amplification) and biotin-labeling. The samples were hybridized to the Affymetrix Mouse Genome 430 2.0 array. TGEN uses GeneChip Operating Software (GCOS) to scan the arrays and to perform a statistical algorithm that determines the signal intensity of each gene (see Cobos et al., 2007; Long et al., 2008 for details).

Animals

[00243] Mice were maintained in standard conditions with food and water ad libitum. All experimental procedures were approved by the Committee on Animal Health and Care at the University of California, San Francisco (UCSF). Mouse colonies were maintained at UCSF, in accordance with National Institutes of Health and UCSF guidelines. Mouse strains with a null allele of Dlxl&2 and Mashl were used in this study (Anderson et al., 1997b; Casarosa et al., 1999). These strains were maintained by backcrossing to C57BL/6J mice. For staging of embryos, midday of the vaginal plug was calculated as embryonic day 0.5 (E0.5). PCR genotyping was performed as described (Anderson et al., 1997b; Casarosa et al., 1999). Since no obvious differences in the phenotypes of

+/+ +/- +/+ +/-

DIx 1&2 and DIx 1&2 and Mashl and Mashl brains have been detected, they were both used as controls.

Tissue Preparation and In Situ Hybridization

[00244] Preparation of sectioned embryos and in situ hybridization were performed using digoxigenin riboprobes on 20μm frozen sections cut on a cryostat using methods described in Long et al., 2007.

Results

Identification of Transcription Factors Expressed in Cells of the Embryonic Mouse MGE and CGE [00245] To understand mechanisms that regulate patterning and differentiation of the mouse embryonic basal ganglia and its derivatives, such as the striatum, globus pallidus and telencephalic interneurons, we have identified most of the transcription factors (TFs) that have key roles in regulating its development at E 15.5. We used gene expression array analysis of RNA prepared from E15.5 mouse basal ganglia (LGE, MGE and CGE combined), MGE and neocortex, followed by informatic methods to identify known TFs. We then compared the expression of the basal ganglia vs. the cortex, control vs. Dlxl&2 basal ganglia, and control vs. Dlxl&2 MGE; this is an extension of the analysis of the LGE and septum that we recently reported (Long et al., 2008). [00246] Table 7 lists TFs that are expressed in the E 15.5 basal ganglia, MGE and cortex identified by gene expression array analysis (not including general transcription factorsAn asterisk in columns 1 and 2, indicates if the results were verified by in situ hybridization. Columns to the right indicate the raw hybridization scores for the individual genes for hybridization using RNA isolated from control El 5.5 cortex, combined LGE, MGE and CGE (basal ganglia; BG), MGE, or from DU1&2-I- basal ganglia (BG) (see Long et al., 2008 for details) or Dlxl&2-I- MGE (see Cobos et al., 2007 for details).

[00247] We have eliminated TFs whose expression was below the level of 30 units (we usually can not detect expression by in situ hybridization of most genes below this level), and general transcriptional components. In this paper we have focused on TFs expressed in the MGE and CGE. We do not have array results for the CGE; however, as it is composed of both LGE and MGE parts (Flames et al., 2007), the basal ganglia sample approximates the CGE. It is likely biased towards the dorsal CGE (dCGE) (also see Supplemental Table 1 for LGE/dCGE differences). Flames et al., (2007) proposed subdivisions of the LGE, MGE, CGE and POA - these were based on analysis at El 1.5 and E 13.5; those subdivisions are more difficult to discern at El 5.5, so herein we have not used this nomenclature.

[00248] To evaluate the array results, we used in situ hybridization on coronal sections of E 15.5 forebrain as indicated by an asterisk in Table 7 (Figs. 10,11, 12, 13, 14). These results confirm the TF expression in the subpallium, and allowed us to identify TFs that are expressed in progenitor (VZ and SVZ) and mantle (MZ) zones of the LGE, MGE and CGE. For example, this analysis identifies several TFs that are expressed in the globus pallidus (GP) [Arx, Dlxl, ER81 (Etvl), Gbxl, Lhx6, Lhx7(8), Oct6 (POUiFl), ROR-beta, TCF4, Tshz2, and ZJp521 (Evi30)] (Figs. 10, 11). It also enabled us to evaluate differential expression between the LGE, MGE and CGE. [00249] TFs that are specifically expressed in progenitor cells of MGE are Lhx6, Lhx7(8), Nkx2.1 ; those preferentially expressed in the MGE (compared to the LGE) include ER81 (Etvl), Sox4 and Sox 11 (also see Flames et al., 2007). TFs that are expressed in progenitor cells of the LGE and not detected in the MGE by this assay include ESRG, FoxPl, FoxP2, FoxP4, Sp8; TFs preferentially expressed in the LGE (compared to the MGE) include ATBFl (Zβιx3), COUP-TFl (NR2F1), CTIP2 βclllb), Ebfl, Islet 1, Meisl, Meis2, Oct6 (POUSFV), Pbxl, Pbx3, Six3 and TCF4. Of note, the MGE expression of many of these genes is within a narrow corridor between SVZ and mantle, and may correspond to the ventral migration of LGE cells (Lopez-Bendito et al., 2006). [00250] The CGE contains at least two subdivisions; the ventral part is a caudal extension of the MGE, and the dorsal part is a caudal extension of the LGE (Flames et al., 2007). However, whereas the LGE is largely dedicated to generating projection neurons of the striatum, accumbens, olfactory tubercle, and interneurons of the olfactory bulb (Long et al., 2007, 2008), the dorsal CGE (dCGE) is known to generate interneurons of the neocortex and hippocampus (Xu et. al., 2004; Butt et al., 2005; Miyoshi et al., 2007). Thus, there must be molecular differences in the progenitors of the LGE and CGE. Therefore, we qualitatively compared TF expression in the VZ/SVZ of these regions using in situ hybridization (Figs. 10 and 11). Table 8 summarizes our conclusions. We found qualitative evidence for a rostrocaudal expression gradient on in situ hybridization experiments. Some TFs are preferentially expressed in the LGE (green color; i.e. ATBFl (Zfhx3) and Isletl), others equally expressed in the LGE and dCGE (yellow color; i.e. DIx genes), and still others preferentially expressed in the dCGE (red color; i.e. Arx, COUP-TFI (NR2F1), Mashl, Proxl, SaIU, Soxl, Sox4, and Sp9).

[00251] Below we describe how loss of either Dlxl&2 or Mashl function affects the expression of many of these TFs and selected non-TFs. The analysis was performed using in situ hybridization on E 15.5 coronal sections; in general the gene expression array results (Table 7) were in accord with the histological analysis. Analysis at E 12.5

-/- was also performed in the DIx 1&2 mutants for selected genes (column 1 in Table 7), and showed similar results

(data not shown)(for the LGE and septum, see Long et al., 2008).

Dlxl&2 functions in the MGE

[00252] Dlxl&2 are required, to varying degrees, to promote expression of several TFs in MGE progenitors (VZ and SVZ), including Arx, bMaf, Brn4, Cux2, Dlxl, Dlx2, DhS, Dlx6, ER81 (Etvl), GUI, Lhx6, Lhx7, Pbxl, Peg3,

Sox4, Soxll and Vaxl (Fig. 1) and non-TFs, including CXCR4, CXCR7 (RDCl), CyclinD2, GAD67, Gucyla3, Shb,

Tiam2 and Thbs (Fig. 12).

[00253] Dlxl&2 repress the expression of a set of TFs, including antisense-D£c6, COUPTFl, Ctip2, Gbxl, Gshl,

Gsh2, Id2, Id4, Dcaros, Islet 1, Lhx2, Mashl, Nkx2.l, Olig2, Otp, Proxl, Sall3, Six3 and Soxl (Fig. 11) and non-TFs including Dactl and PKRl (Fig. 12). Some TFs do not show a discernable expression change, such as Sp9 (Fig. lOHHH-III'). The MGE produces several types of cells including projection neurons of the globus pallidus and

-/- interneurons that migrate to the cortex and hippocampus. Dlxl&2 mutants produce a small globus pallidus, but with reduced numbers of neurons expressing ER81 (Etvl), Gbxl, Gbx2, Lhx6, Lhx7/8, Lmo3, Meisl, Oct6 (POU3F1), Pbx3, RORb, Tcf4, Sema3a, Tshz2 and Zpf521 (Fig. 10), and non-TFs Cad8, Gad67 (Gadl), Robo2 and Sema3a (Fig. 12). On the other hand, some TFs show increased expression in the MGE mantle zone including ATBFl, Ebfl, ESRG, Fez, FoxP2, Isletl and Pbx3 (Fig. 11); this may be due to ectopic accumulation of cells from striatal and/or POA migrations (Lopez-Bendito et al., 2006), or ectopic expression of these TFs in the pallidal MZ.

[00254] Most interneuron precursors fail to migrate into to the cortex in Dlxl&2 mutants (Anderson et al., 1997a; Pleasure et al., 2000; Cobos et al., 2005), and appear to remain as ectopia in the basal ganglia, some of which express neuropilin2 (NP2) (Marin et al., 2001). Here we show that these ectopia form in a caudal position within the CGE, and continue to express many TF and non-TF markers characteristic of immature interneurons, including bMaf, ErbB4, Lhx6, NP2 and Nphx (Fig. 11). Surprisingly, there also are large ectopia that express globus pallidus markers: ER81 (Etvl) and Nkx2.1 (Ttfl) (Fig. 13). Finally, there are ectopia expressing other genes {Pbxl, Proxl and Soxll) that currently are not known to mark specific cell types (Fig. 13).

Dlxl&2 function in the CGE

[00255] Dlxl&2 are required to promote expression of several TFs in the CGE including Arx, Brn4, Dlxl,2,5,6, ESRG, FoxPl, FoxP4, Meisl, Meis2, Oct6 (POU3F1), Pbxl, Pbx3, Proxl, Six3, Sox4, Soxl 1, Sp8, Tle4, Tshzl and Vaxl, and non-TFs including CXCR4, CXCR7 (RDCl), ErbB4, Gad67 (Gadl), Gucyla3, Robo2, Shb, Tiam2 and Thbs. The reduction of some genes likely corresponds to the block of MGE-derived interneuron tangential migration (i.e. bMaf, Cux2, Lhx6; Fig. IC-D', G-H', CC-DD').

[00256] Dlxl&2 represses the expression of several TFs including antisense-D£c6, COUPTFI (NR2F1), Ctip2 (Bell Ib), Gbxl, Gshl, Gsh2, Id2, Ikaros, Isletl, Lmol, Mashl, Olig2, Otp and SaIB, and several non-TFs including Dactl and PKRl (Fig. 11). Several genes show little change in expression including FoxP2, Hes5, Id4, Lhx2, Otx2, Pax6, Soxl and Sp9 (Figs. 10, 11); this is unlike the LGE, which has increased Hes5, Lhx2 and Sp9 expression (Figs. 10, 11; Table 9; Anderson et al., 1997b, Yun et al., 2002; Long et al., 2007, 2008). Table 9 depicts, as discrete boxes, the ventricular zone (VZ), subventricular zone (SVZ) and mantle zone (MZ) of the LGE, MGE and CGE of E 12.5 and El 5.5 embryos. The effect of the Dlxl&2-/- mutation on transcription factor expression in each box is indicated using a color code: Yellow represents that expression was not analyzed by in situ hybridization at E 12.5 and represents expression that was uninterpretable at E15.5. Gray represents that expression was not clearly changed in the mutant. White represents no detectable expression. Magenta represents severe reduction in expression. Orange represents moderate/mild reduction in expression. Green represents ectopic expression. Blue represents increased expression. The genes are ordered as follows: left column are genes expressed in the proliferative zones (VZ and SVZ); right column are genes expressed at later developmental stages (SVZ, SVZ&MZ, MZ). The genes are arranged alphabetically within each grouping. A 'd' represents the effect is primarily in the dorsal part of the LGE/MGE/CGE and a V represents the ventral part.

[00257] Finally, as noted above, there are ectopic accumulations of cells in the CGE expressing several markers characteristic of the globus pallidus or cortical interneurons (i.e. bMaf, ER81 (Etvl), ErbB4, Lhx6, Nkx2.1 (Ttfl), NP2, Nphx, Pbxl, Proxl and Sox 11; Fig. 13).

DIxI &2 ;Mashl compound mutants define genes epistatic to DIxI &2, Mashl or both Dtxl&2 and Mashl

-A

[00258] While many aspects of MGE and CGE differentiation are lost in the DIx 1&.2 mutants, many aspects are maintained (Figs. 10, 11, 12; Table 9). The maintained characteristics may be regulated by TFs whose expression persists in mutant LGE progenitors (Fig. 11). A good candidate of this type of TF is Mashl, due to its

-/- over-expression in the DIx 1&2 mutants (Fig. 2)(Yun et al., 2002). As MASHl and DLX2 proteins are co-expressed in progenitors of the dLGE (Porteus et al., 1994; Yun et al., 2002), they have the potential to regulate the developmental programs of these cells in parallel and/or in series. Here, we explored the hypothesis that Mashl has

-/- a critical role in maintaining certain aspects of MGE and CGE differentiation in the DIx 1&2 mutants, as it does in the LGE.

-A -/-

[00259] We studied the expression of TFs and selected other genes in the MGE and CGE in DIx 1 &2 , Mashl and

Dlxl&2 \Mashl mutants at E15.5, concentrating on genes whose expression persists in Dlxl &2 mutants (Fig. 14).

Expression of these genes fell into four general classes as shown in Table 10. :

-/- [00260] Class I genes appear to be epistatic only to Dlxl&2 . Expression of Class Ia genes (ER81, GUI, Gshl,

SpS) is reduced or lost in the DIx 1&2 mutants, is not overtly affected in the Mashl mutants and the triple mutant

-/- phenocopies the DIx 1&2 mutant.

-/- [00261] Class Ib genes are ectopically expressed in the Dlxl&2 mutants, and are not overtly modified by loss of

-/- Mashl .

-A CGE

[00262] Class II genes appear to be epistatic only to Mashl (i.e. Hes5 : i.e. in only the CGE).

-/- -/-

[00263] Class III genes appear to be altered in both the Dlxl &2 and Mashl mutants, and in most cases these phenotypes are exacerbated in the triple mutants. There are five subtypes of Class III genes based on their

-A -A MCE CCE differentiation responses in the Dlxl &2 and Mashl mutants: IHa (A rx , Dlxl, Dlx5, Gad67 ): decreased in

-A -A CCE -I-

Dlxl&2 ; increased in Mash 1 ; HIb (Arx , Meis2, Six3, Soxl, Sp9, Vaxl): decreased in both Dlxl &2 and

-I- MCE -A -A

Mαshl ; IIIc {Dαctl, Hes5 , Olig2, Soxl): increased in Dlxl&2 ; decreased in Mαshl ; HId {Islet I): increased in the VZ of the CGE (and LGE; but reduced in the SVZ and MZ of the LGE); not greatly modified by Mash I

-I- -I- dosage; HIe (Tshz2): increased in Dlxl&2 ; ectopic in Mashl .

[00264] Class IV (ER81 in the MGE) genes show a modest decrease in the number of labeled globus pallidus

-I- -I- -/- -I- neurons in Dlxl&2 , Mashl and Dlxl&2 ;Mashl mutants.

[00265] Of note, several TFs continue to be expressed in the DIx 1&2 M^shl mutants, albeit generally at lower levels, in the CGE (Gshl, Isletl, Olig2, Sp9) and MGE (ER81, Isletl, Olig2, Sp9) (Fig. 14), demonstrating that some fundamental aspects of CGE and MGE specification, such as GAD67 expression (Fig. 12) are not fully dependent on DIx and Mashl.

[00266] Cell counting analysis data from a double immunofluorescent labeling of Gsx2/Dlx2; Dlx2/Ascll; Gsx2/Ascll in the LGE of the E10.5 and E12.5 mouse brain is shown in Table 11. This table shows cells expressing Ascll, Dlx2, Gsx2 in the E10.5 and E12.5 dorsal LGE. Asterisk (^♦) indicates that Gsx2⁺ cell density was too high to count with accuracy, but clearly was greater than Ascll density; VZ: ventricular zone; SVZl : deep subventricular zone; SVZ2: superficial subventricular zone; MZ: mantle zone.

Discussion

[00267] We report a comprehensive analysis of the TFs, excluding general transcription factors, that are expressed in the developing (E15.5) mouse MGE and CGE. This study complements our TF analysis in developing septum and LGE, and the work of Flames et al. (2007), which used a subset of these TFs to define E13.5 subpallial progenitors zones. We defined the response of many of these genes in mice lacking expression of Dlxl&2, Mashl, or DIx 1&2 and Mashl . This analysis enables decipherment of the transcriptional circuitry that controls cell fate and differentiation of MGE and CGE derivatives. This information will be extremely useful in understanding normal pathways that control the development and evolution of the basal ganglia and pallial interneurons, and will help predict the effect of mutations, whether they are in experimental animals of in humans. Furthermore, understanding transcriptional hierarchies enables engineering of stem cells.

TF Profile in the VZ and SVZ of the dCGE

[00268] Flames et al. (2007) demonstrated that the CGE is a composite of the caudal LGE (the dorsal part of the CGE) and MGE (the ventral part). Here we show that while the dorsal CGE (dCGE) does share many properties with the LGE, there are several important differences (Table 8). We find TFs that are preferentially expressed in the LGE [green color; i.e. ATBFl (Zflιx3) and Isletl], equally expressed in the LGE and dCGE (yellow color; i.e. DIx genes), and preferentially expressed in the dCGE [red color; i.e. Arx, COUP-TFI (NR2F1), Mashl, Proxl, SaIB, Soxl, Sox4, and Sp9]. As such, 1) the TFs preferentially expressed in the LGE are important in development of striatal projection neurons and olfactory bulb interneurons; 2) the TFs preferentially expressed in the dCGE are important in development of cortical interneurons (subsets of NPY, CR and VIP-expressing pallial interneurons; see Zhao et al., 2008); 3) TFs that are equally expressed in the LGE and dCGE have general roles in regulating the development of telencephalic GABAergic neurons.

[00269] Within the dCGE, there is a VZ and SVZ, but a MZ is not clearly distinct; this feature is exemplified by the expression of the DIx genes whose combinatorial expression define these three differentiation zones in the LGE and MGE (Fig. 10). The CGE may produce subpallial nuclei, although a caudal nucleus, such as the central nucleus of the amygdala, is also a possibility (Carney et al., 2006; Garcia-Lόpez et al., 2008). [00270] It is likely that the CGE primarily consists of a large SVZ where pallial interneurons are produced and

+ partially mature. In addition, many MGE-derived interneurons (Lhx6 ) migrate through the CGE; it is likely that local CGE factors regulate their development; this idea is discussed below in the section of subpallial neuronal

-/- ectopia in the DIx 1&2 mutants.

Role of Dlxl&2 in dCGE development

[00271] DU1&2 have a profound role in promoting differentiation of the dCGE, as exemplified by the reduced expression of Arx, Brn4, Dlx5, Dlx6, ESRG, FoxP4, Meisl, Meis2, Pbxl, Pbx3, Proxl, Six3, Sox4, Soxll, Sp8, Tle4,

-/- Tshzl, and Vaxl in the Dlxl&2 mutants. These findings are similar to the phenotype of dorsal parts of the LGE and septum, but not ventral parts of these primordia. Thus, the dLGE and dCGE share similar dependence on DIx 1&2. [00272] In the dLGE and dCGE, DIx 1&2 apparently have their strongest functions in the SVZ, as exemplified by reduced Sp9 expression in the SVZ and not in the VZ. Furthermore, Dlxl&2 are required to repress the expression of COUP-TFI, Ctip2, Mashl and SaIB, supporting the model that Dlxl&2 promote the maturation of SVZ progenitors (see Yun et al., 2002; Long et al., 2007; 2008). Below we discuss the role of Mashl in CGE

-/- development, and the effect of removing Mashl function in DIx 1&2 mutants. Elevated levels of anti-sense Dlx6 transcripts are intriguing (Fig. 11), indicating the DIx 1&2 are required to repress this potential inhibitor of Dlx6 function (Faedo et al., 2004; Feng et al., 2006).

[00273] Several genes are ectopically expressed in the mutant dCGE, including markers of the MGE (Gshl and Gbxl), the ventral pallium {Id2 and Lmol), and dLGE (Ikaros and Isletl), and diencephalon (Otp) (Fig. 11). These results show that DIx 1&2 are required to specify the identity of dCGE progenitors, a finding that is also apparent in the dLGE.

MGE TFs and the function of Dlxl&2

[00274] Several TFs appear to preferentially, or exclusively, mark MGE progenitors, and their derivatives: Cux2, ER81, Gbxl, Gbx2, Gshl, Lhx6, Lhx7, Nhc2.1 (TTF-I), Nkx6.2, Proxl, ROR-beta and TCF4. The MGE also shares molecules features with the LGE/dCGE, such as expression of Arx, Brn4 (POU3/4), Dlxl&2/5/6, Mashl, Sp9 and Vaxl (Figs. 10, 11; Flames et al., 2007).

[00275] The preoptic progenitor and mantle zones (pPOA) are rostroventral to the MGE (Flames et al., 2007), and express many of the same genes as the MGE, but also have their distinct molecular features, including expression of COUP-TFI, Dbxl, Lhx2, NkxS.l and Nkx5.2 (Hmx2 and Hmx3) and Nkx6.2 (Wang et al., 2004;

Flames et al., 2007).

-/- [00276] While regional identity of DIx 1&2 MGE is not greatly disturbed, its differentiation of the globus pallidus and pallial interneurons is impeded, likely secondary to increased notch signaling, as reflected by increased Mashl and Hes5 expression (Fig. 11; see Yun et al., 2002). Increased expression of the Pak3 kinase has also been implicated in cytoskeletal dysregulation leading to premature neurite extension and inhibition of migration (Cobos et al., 2007).

[00277] Regional identity of the mutant MGE is likely preserved by the continued expression of Nkx2.1 (which appears to be increased; Fig. T), a TF required from MGE specification (Sussel et al., 1999). However, despite preserved Nkx2.1 expression, expression of Cux2 andLhx7(8) are clearly reduced (Fig. 10). Cux2 is expressed in tangentially migrating interneurons (Cobos et al., 2006), and its function is linked to the development of reelin-expressing intemeurons (Cubelos et al., 2008). Lhx7(8) is expressed in the SVZ of the ventral MGE (Flames et al., 2007), and its derivatives in the pallidum and striatal intemeurons, where it is required for the cholinergic phenotype (Zhao et al., 2003; Fragkouli et al., 2005; Mori et al., 2004). [00278] Lhx6 is expressed in MGE progenitors, and in pallidal neurons, striatal interneurons, and pallial intemeurons, and it promotes tangential migration, integration into the cortical plate and differentiation of

+ + somatotstatin and parvalbumin intemeurons (Sussel et al., 1999; Marin et al., 2000; Alifragis et al., 2004; Liodis et

-/- al., 2007; Zhao et al., 2008). Its expression is reduced in the MGE of the Dlxl&2 mutant (Fig. 1; Petryniak et al.,

2007), and is reduced in the cortex, secondary to reduced intemeuron migration (Cobos et al., 2006).

[00279] Here we report a large Lhx6 ectopia in a superficial part of the dCGE (Fig. 10, 13), indicating that these

+ cells correspond to intemeurons that have failed to migrate to the cortex. However, this ectopia is also Nkx2.1 (Fig.

11, 13). As Nkx2.1 is not expressed in pallial interneurons (Sussel et al., 1999), this indicates several interesting possibilities, including: 1 ) Dlxl &2 are required to repress Nkx2.1 in intemeurons - perhaps persistent Nkx2.1 expression contributes to the defect in tangential migration; 2) this ectopia could be a misplaced globus pallidus. We think that this hypothesis is less likely because the ectopia also expresses neurexophilin-1 (Nphx), a marker of tangentially migrating intemeurons and not of the globus pallidus, and because the ectopia does not express the following globus pallidus markers: Arx, Dlxl, Gbxl, Lhx6, Lhx7(8), Oct6 (POU3F1), ROR-beta, TCF4, Tshz2, and Zfp521 βvi30) (Fig. 10). There are other ectopia in the CGE and MGE, that are located outside of the Lhx6/Nkx2.1/Nphx ectopia, that express ER81, ErbB4, NP2, Proxl and Sox 11 (Fig. 13); these may correspond to distinct subtypes of neurons that failed to disperse (see Marin et al., 2001 for NP2).

Transcriptional and neurogenic pathways downstream of Dlxl&2 and Mashl

-/- [00280] We studied the expression of TFs and selected other genes in the MGE and CGE in Dlxl &2 ,

Mashl and DIx 1&2 Jrfashl mutants at El 5.5, concentrating on genes whose expression persists in

-/- Dlxl&2 mutants (Fig. 14). Expression of these genes fell into four general classes (Table 10). Several TFs continue

-/- -/- to be expressed in the Dlxl&2 M^hI mutants, albeit generally at lower levels, in the CGE {Gshl, Islet I, Olig2,

Sp9) and MGE (ER81, Isletl, Olig2, Sp9) (Fig. 14); in addition, GAD67 expression is weakly maintained. Thus, ER81, Gshl, Isletl, Olig2, Sp9, or other TFs (i.e. Gsh2 or Nkx2.1) are likely maintaining the fundamental features of the embryonic basal ganglia in the triple mutant. Furthermore, while some telencephalic cell types are reduced in

Dlxl&2 , Mashl and Dlxl &2 ; Mashl mutants (striatal, pallidal and pallial GAB Aergic, dopaminergic, cholinergic neurons) (Marin et al., 2000; Long et al., 2007, 2008), oligodendrocyte generation is promoted (Petryniak et al., 2007). Finally, early stages of neurogenesis are dependent on Mashl (i.e. Soxl), and not Dlxl&2 (Fig. 14). Thus, this analysis dissects the complementary roles ofDlxl&2 and Mashl in promoting the differentiation of the subpallium. v- Non-TF gene dysregulation in Dlxl&2 mutants

[00281] The subpallial progenitor zones produce GABAergic, cholinergic and dopaminergic neurons, oligodendrocytes and astrocytes. The DIx genes are essential for the differentiation of many of these neurons (Marin et al., 2000; Yun et al., 2002; Long et al., 2007), and repress glial differentiation (Yun et al., 2002; Petryniak et al, 2007). We have identified key effector genes whose expression is down-stream (directly/indirectly) of Dlxl &2. Supplemental Fig. 11 shows examples; others are described in Cobos et al. (2007) and Long et al. (2008). [00282] Dlxl&2 promote GABAergic differentiation through promoting expression of the enzymes that synthesize GABA: GAD67 (Gadl) and GAD65 (Gad2), and the pump that concentrates GABA in synaptic vesicles (vGAT) (Fig. 12 and Anderson et al., 1999; Stuhmer et al., 2002; Long et al., 2007; 2008; Eisenstat, Cobos and Rubenstein, unpublished).

[00283] Alterations in migration may be contributed by reduced expression of cytokine receptors (CXCR4, CXCR7) and the neuregulin receptor, ErbB4. Migration defects may also be contributed by alterations in Gucyla3, NP2, Robo2, Shb, Thbs and Tiam2 expression (Fig. 12). Defective differentiation of striatal and pallidal neurons is indicated by reduced expression of Cad8, Robo2 and Semala and several other genes.

[00284] Dlxl &2 represses several non-TFs in progenitor cells including Dactl and PK2 (Fig. 12; for additional

-/- genes, see Cobos et al., 2007 and supra); some of these are complementary to changes in the Mashl mutant (Fig.

14). These changes in progenitor properties are associated with persistent CyclinD2 expression in the LGE/dCGE SVZ (Fig. 12), and elevated proliferation in the SVZ of the LGE/dCGE (Anderson et al., 1997b). On the other hand, there is reduced CyclinD2 expression in the MGE SVZ, which is consistent with the reduce proliferation of this region (Anderson et al., 1997b; Petryniak et al., 2007).

[00285] This work was supported by the research grants to JLRR from: Nina Ireland, Larry L. Hillblom Foundation, NIMH ROl MH49428-01, NIMH R37 MH049428-16A1 and K05 MH065670; Inma Cobos: MIRG-CT-2007-210080 and National Alliance for Research on Schizophrenia and Depression Young Investigator Award; Greg Potter: NIMH F32 MH070211.

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Comp Neurol. 506:16-29.

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The Notch Signaling By Mashland Dlxl&2 Regulates Sequential Specification And Differentiation Of Progenitor

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LIM-homeobox gene Lhx8 is required for the development of many cholinergic neurons in the mouse forebrain.

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Example 3: Gsxl and Gsx2 Have Opposite Interactions With Dlxl&2 in Controlling Basal Ganglia

Differentiation

[00326] Transcription programs that define regional and cell type identity in the forebrain are beginning to be elucidated. Described herein is the development of mouse subcortical telencephalic domains, known as the lateral, medial and caudal ganglionic eminences (LGE, MGE and CGE), and the septum, that largely generate GABAergic neurons (Flames et al., 2007). These regions employ over 100 transcription factors to mediate their development

(Long et al., 2009a,b), including the Dlxl&2 and Gsxl&2 (formerly labeled Gshl&2) homeodomain encoding genes. These genes are expressed in primary and secondary progenitors in the ventricular and subventricular zones

(VZ and SVZ); subsets of these cells continue to express DIx 1 and/or Dlx2 (Cobos et al., 2005, 2007b and unpublished). Gsxl&2 together promote LGE regional fate (Corbin et al., 2000; Toresson et al, 2000; Toresson and

Campbell, 2001; Yun et al., 2001, 2003; Waclaw et al. 2004), whereas Dlxl&2, which are linked genes, promote later steps in subcortical differentiation, in part through inducing the expression of the Dlx5&6 gene pair (except in the septum) (Anderson et al., 1997a; Long et al., 2007, 2009a,b).

[00327] Different DIx gene dosages controls different processes. DIx 1&2 mutants lack expression of

DIx 1/2/5/6; loss of expression of these 8 alleles defines the most fundamental Dlx-mediated programs, which include: 1) repressing Notch signaling and glial differentiation, through decreasing Ascll (Mashl) expression (Yun et al., 2002; Petryniak et al., 2007); 2) promoting GABAergic neural differentiation including the expression of glutamic acid decarboxylase (GAD)(Anderson et al., 1997a; Long et al., 2009a,b); 3) promoting neuronal migration through repressing neurite outgrowth and Pak3 kinase expression (Anderson et al., 1997b; Long et al., 2007; Cobos et al., 2007). Dlxl-/-;Dlx2+/- mutants show defects in synapse development (Cobos and Rubenstein, unpublished). DIx 1-/- mutants show defects in survival of a subset of subcortically-derived neurons (dendrite innervating interneurons) (Cobos et al., 2005).

[00328] The Gsx2 and DIx 1&2 genes mediate their subcortical transcriptional programs in combination with the Ascll (Mashl) bHLH gene. Subcortical development in the Gsx2;Ascll and Dlxl/2;Ascll compound mutants is much more abnormal than in the individual mutants (Long et al., 2009a,b; Wang et al., 2009). [00329] A feature of the Dlxl/2 mutants is their over-expression of Gsxl and Gsx2 (Yun et al., 2002; Long et al., 2009a,b). Herein we explored the effect of reducing Gsx expression in the Dlxl/2 mutants, concentrating on the expression of a panel of transcription factors that are abnormally expressed in the Dlxl/2 mutants (Long et al., 2009a,b). We found that the compound mutants generally showed an exacerbation of the Gsx and DIx mutant phenotypes in the CGE, LGE, MGE and Septum. Furthermore, we found that removing Gsxl from the Dlxl/2 mutants further increased Ascll expression, whereas removing Gsx2, decreased Ascll expression. Thus, despite their redundant properties, Gsxl and Gsx2 have distinct interactions with DIx 1&2. From these studies, and earlier ones, we present a model of the transcriptional network that regulates subcortical development.

Materials and Methods

[00330] Mice were maintained in standard conditions with food and water ad libitum. All experimental procedures were approved by the Committee on Animal Health and Care at the University of California, San Francisco (UCSF).

Mouse colonies were maintained at UCSF, in accordance with National Institutes of Health and UCSF guidelines.

Mouse strains with a null allele of Dlxl&2, Gsxl and Gsx2 were used in this study (Anderson et al., 1997b;

Casarosa et al., 1999). These strains were maintained by backcrossing to CD-l?check with kenny For staging of embryos, midday of the vaginal plug was calculated as embryonic day 0.5 (E0.5). PCR genotyping was performed as described (Anderson et al., 1997b; Casarosa et al., 1999). Gsxl&2 genotyping was performed as in Yun et al.,

2003 and Wang et al., 2009.

[00331] Tissue Preparation, In Situ Hybridization and Immunofluoresence

[00332] Preparation of sectioned embryos, immunofluoresence and in situ hybridization were performed using digoxigenin riboprobes on 20μm frozen sections cut on a cryostat using methods described in Long et al., 2007 and

2009a,b. We used a rabbit polyclonal anti-GSX2 antibody (diluted: 1 :5000; Toresson et al., 2000), a guinea pig polyclonal anti-DLX2 (diluted 1 :3000; Kuwajima et al., 2006), and a mouse monoclonal anti-MASHl (diluted:

1:500; BD Phaπningen). Riboprobes are described in Long et al., 2009a,b.

[00333] Microscopy

[00334] Images of in situ hybridization results were captured using a Zeiss AxioCam MR (Thornwood, NY) and saved as TIFF files. Images of immunofluoresence were captured using a Zeiss LSM 510 confocal microscope. The images were then processed in Adobe Photoshop CS3 (San Jose, CA).

[00335] Statistical Analysis of the Number of DLX2-, GSX2-, and ASCLl-Expressing Cells

[00336] DLX2-, GSX2-, and MASHl -expressing cells were visualized on 20 μm, coronal forebrain sections from

E10.5 and E12.5 wild-type mice by immunofluorescence confocal microscopy. The images were imported into

Adobe Photoshop CS3, and a rectangle encompassing the VZ and SVZ domains was placed orthogonal to the ventricular surface (see Figure 1). The labeled cells in the VZ, SVZl, and SVZ2 within each rectangle were manually counted using Photoshop counting tool. [00337] Subpallial Expression of DLX2, GSX2 and ASCLl Protein

[00338] To compare DLX2, GSX2 and ASCLl protein expression at the cellular level in the developing subpallium, we used double immunofluorescence at E10.5, E12.5 and E15.5 (Fig. 1; Sup. Figs. 1,2,3). These analyses complement previous studies that examine expression of GSX2 (Corbin et al., 2000; Yun et al., 2003;

Wang et al., 2009), DLX2 (Porteus et al., 1994; Eisenstat et al., 1999; Yun et al., 2002), DLX2&ASCL1 (Porteus et al., 1994; Yun et al., 2002), DLX2& OLIG2 and ASCL1&OLIG2 (Petryniak et al., 2007).

[00339] At E10.5, most ventricular zone (VZ) progenitors in the dorsal LGE (dLGE) strongly express

GSX2, with weaker expression spreading ventrally through the ventral LGE (vLGE) and the MGE (Fig. 1 ; Sup. Fig.

1). ASCL1+ cells are scattered throughout the VZ and mantle zone (MZ) of the LGE and MGE. DLX2+ cells in the

VZ are most concentrated in the dLGE, whereas the thin mantle zone (MZ) has many DLX2+ cells. In the VZ (of the LGE), most ASCL1+ and DLX2+ cells co-express GSX2, whereas in the MZ, most ASCL1+ and DLX2+ cells are GSX2- (Table 1).

[00340] Expression at E12.5 and E15.5 showed similar results (Fig. 1; Sup. Figs. 2,3). The main difference is the formation of the subventricular zone (SVZ). Previously, we presented evidence that the SVZ consists of two layers: SVZl (adjacent to the VZ) and SVZ2 (Yun et al., 2002; Petryniak et al., 2007). DLX2 is robustly expressed in nearly all cells in SVZl and SVZ2. The intensity of GSX2 expression decreases as cells move from the VZ to

SVZl, although most cells continue to express detectable GSX2 and co-express DLX2 and ASCLl (Table 1).

However, in SVZ2, GSX2 and ASCLl expression is at background levels except in occasional cells.

[00341] In sum, GSX2 expression in the VZ is temporally upstream of DLX2 expression; as progenitors mature to the SVZl state, there generally is co-expression of GSX2, ASCLl and DLX2. Therefore, analysis of

Gsx2-/-;Dlxl/2-/- (Gsx2;Dlxl/2) compound mutants can assess the cell-autonomous effects of losing expression these transcription factors in the same cells.

[00342] Organization and Presentation of the In Situ Hybridization Data

[00343] We deemed it essential to maintain the anatomical integrity of the data by showing a rostro-to-caudal series of sections through the telencephalon in all six genotypes analyzed in this paper: wild type, Gsxl, Gsx2, Dlxl/2,

Gsxl;Dlxl/2 and Gsx2;Dlxl/2 mutants for the 20 in situ probes analyzed. Thus, each figure shows coronal sections representing three rostrocaudal positions: rostral telencephalon, SE/LGE level; middle telencephalon, LGE/MGE level; caudal telencephalon, CGE level. This resulted in the generation of a large number of figures. We decided to show 6 figures in the body of the paper, and place the other 48 as supplemental figures. The regular figures where chosen to highlight some of the major differences between the Gsxl;Dlxl/2 and Gsx2;Dlxl/2 mutants at El 5.5; they show opposite effects on the expression of Ascll, DIx 1, and GADl, providing evidence that although Gsxl&2 have redundant functions in LGE patterning, Gsxl and Gsx2 individually have opposite functions in regulating Ascll expression. Figure 8 is a schema which integrates the data from the current study with results from previous work on the Gsxl;Gsx2, Ascll;Dlxl/2, Ascll; Olig2, Dlxl/2;Olig2 single and compound mutants (Petryniak et al., 2007;

Long et al., 2009a,b; Wang et al., 2009). The schema, which proposes a regulatory circuit in the LGE, serves as a useful framework consider when examining the gene expression results described below.

[00344] Transcription Factor Expression in LGE Progenitor Cells at E12.5 and E15.5 in Gsx2, Dlxl/2 and

Gsx2;Dlxl/2 mutants: Combined Functions in Regional Patterning and Opposite Roles in Ascll Regulation. [00345] Previous studies showed that Gsx2 promotes the expression of the DIx genes (Corbin et al., 2000; Toresson et al., 2000, 2001; Yun et al., 2001; 2003), whereas Dlxl/2 repress Gsx2 expression (Yun et al., 2002; Long et al.,

2009a). Both Gsx2 and Dlxl/2 promote LGE (dLGE) identity. Loss of Gsx2 results in transformation of the dLGE towards ventral pallial fate (Corbin et al., 2000; Toresson et al, 2000; Toresson and Campbell, 2001; Yun et al.,

2001, 2003; Waclaw et al. 2004); loss of Dlxl/2 transforms neurons in the rostrodorsal striatal region towards mixed palllial/subpallial properties (Long et al., 2009a). Here we further investigated Gsx2 function towards understanding the ramifications of its over-expression in the Dlxl/2 mutants (Long et al., 2009a,b); thus we studied the phenotype of the Gsx2;Dlxl/2 compound mutants to determine whether some of the Dlxl/2 phenotype is reversed by removing expression of Gsx2.

[00346] In addition to the alteration in dLGE specification, Gsx2 mutants also have vLGE defects. At El 2.5 there is reduced expression of LGE progenitor (VZ and SVZ) regulators/markers, including Arx, Ascll, DIx 1, Hes5, Olig2,

Pbxl, Six3, Sp9 and Vaxl (Sup. Figs. 4,5,6,13,18,20,21,22,23). Gsx2 mutants show increased Gsxl expression in the VZ (Sup. Fig. 11; Toresson et al, 2000; Toresson and Campbell, 2001; Yun et al., 2001, 2003).

[00347] Loss of Dlxl/2 likewise results in reduced Arx, Olig2, Pbxl, Six3, Sp9 and Vaxl expression (Sup. Figs.

4,18,20,21,22,23; Cobos et al., 2005; Long et al., 2009a); whereas it leads to increased Ascll and Gsx2 WZJSVZ expression (Sup. Figs. 5,12), increased Gsxl, Hes5 and Isletl SVZ expression (Sup. Figs. 11,13,14), and ectopic Otp

SVZ expression (Sup. Fig. 19; Yun et al., 2002; Long et al., 2007, 2009a). Unlike Gsx2 mutants, Sp9 expression is only modestly reduced (Sup. Fig. 22).

[00348] Combined loss of Dlxl/2 and Gsx2 function (Gsx2;Dlxl/2 compound mutants) further reduce LGE properties based on the further reduction of Ascll, DIx 1 and Vaxl expression, as well as the more ventral expansion of Ngn2 (cortical) expression (Sup. Figs. 6,5,23,15). Weakened LGE properties remain in the vLGE based on the expression of DIx 1 and Gsxl.

[00349] Therefore, at E 12.5 the Gsx2;Dlxl/2 compound mutants have a severe defect in regional specification of the LGE that is more severe than in either the Dlxl/2 or Gsx2 mutants. On the other hand, the level of Hes5 SVZ expression (Notch signaling) appeared reduced compared to the Dlxl/2 mutants (Sup. Figs.13).

[00350] Analysis at E15.5 confirmed previous studies of the Gsx2 and Dlxl/2 mutants. Gsx2 mutants at E15.5 show recovery of LGE properties that is dependent on Gsxl function (Toresson and Campbell, 2001; Yun et al.,

2003).

[00351] The E15.5 Gsx2;Dlxl/2 compound mutants do not show the full recovery of LGE properties seen in the

Gsx2 mutants, showing that Dlxl/2 contribute to the recovery. Furthermore, the size of the LGE is appears reduced compared to either single mutant, and compared to the size of the MGE (as defined by Nkx2.1 expression; Sup. Fig.

33). This hypoplasia is clearer at E12.5 than E15.5, and clearer in mid-telencephalic regions where normally the

LGE and MGE are of similar size (Sup. Figs. 16).

[00352] Gsx2;Dlxl/2 compound mutants exhibit greatly reduced expression of Arx, Gadl, Pbxl, Sp8 and Sp9 (Sup.

Figs. 24, 36, 39, 40), and reduced Dlxl and Vaxl VZ expression (Fig. 4, Sup. Fig. 41). Gsxl and Otp continued to be ectopically expressed (Sup. Figs. 28,35), showing that this property is independent of Gsx2 (fully epistatic to loss of Dlxl/2). On the other hand, Ascll, Hes5, Olig2, Sall3 and Six3 expression, are only modestly altered in the

Gsx2;Dlxl/2 compound mutants, and have an phenotype that appeared intermediate between that of the Gsx2 or

Dlxl/2 mutants (Fig. 2, Sup. Figs. 30, 34, 37, 38). Furthermore, Ngn2 expression remains restricted to the pallium in the Gsx2;Dlxl/2 compound mutants (Sup. Figs. 32) [00353] Thus in Gsx2;Dlxl/2 compound mutants, while important features of LGE progenitors are disrupted (Arx, Gadl, Gsxl, Pbxl, Otp, Sp8, Sp9, Vaxl), some fundamental features (Ascll and Olig2) are preserved. While removing Gsx2 function in the Dlxl/2 mutants rescues the elevated level of Hes5 expression (Notch-signaling), it does not rescue other major phenotypes, suggesting the Gsx2 over-expression is not the key mechanism underlying most of the Dlxl/2 mutant phenotype.

[00354] Molecular Properties of LGE Neurons (Striatum) in Gsx2, Dlxl/2 and Gsx2;Dlxl/2 mutants: Loss of GADl and Pbxl; Preservation of FoxP4 and Islet 1.

[00355] The changes in the LGE progenitor properties describe above are reflected by altered expression of markers of LGE neurons (striatum) at E12.5: Arx, Ebfl, FoxP4, Isletl, Six3 (transcription factors) and GADl (enzyme). Each of these markers is reduced in the Gsx2 and Dlxl/2 mutants, and each is almost eliminated in the Gsx2;Dlxl/2 compound mutants, except for FoxP4 and Isletl, whose expression persists in the mantle and SVZ, respectively (Sup. Figs. 4,7,8,14,21,9).

[00356] At E 15.5 the Gsx2;Dlxl/2 compound show remarkable preservation of some striatal properties, particularly of FoxP4 and Isletl. However, expression of Ebfl, FoxP4 and Six3 are clearly reduced, Sp9 expression is severely reduced, and Gadl and Pbxl expression in the dorsal striatum is not detectable (Sup. Figs. 26,31,25,38,40,36). Preserved expression of FoxP4 and Isletl show that some aspects of striatal differentiation are preserved in the Gsx2;Dlxl/2 compound mutants.

[00357] Transcription Factor Expression in dorsal CGE Progenitor Cells at E12.5 and E15.5 in Gsx2, Dlxl/2 and Gsx2;Dlxl/2 mutants: Similarity to the LGE.

[00358] Progenitors in the dorsal CGE largely share properties with the LGE, its rostral extension (Flames et al., 2007; Long et al., 2009a,b). Likewise analyses of the Gsx2, Dlxl/2 and Gsx2;Dlxl/2 mutants shows that the LGE and dCGE share nearly identical molecular responses. Two additional points merit mention: 1) the dCGE may not exist in the E12.5 Gsx2;Dlxl/2 compound mutant, suggesting that it is most similar to the dLGE; 2) the dCGE does not express markers found in striatal differentiation (Ebfl and Isletl) (Long et al., 2009b). [00359] Transcription Factor Expression in MGE Progenitor and Mantle Cells in Gsx2, Dlxl/2 and Gsx2;Dlxl/2 mutants.

[00360] While the focus of this study was on gene expression of the LGE/dCGE, some analysis of the MGE was performed, and deserves attention, particularly as previous studies did not detect a clear MGE defect in Gsx2 mutants (Yun et al., 2003).

[00361] At E12.5, Gsx2 mutants show reduced MGE progenitor cell expression of Ascll, Arx, DIx 1, GADl, Hes5, Nkx2.1, Olig2, Pbxl, Sp9 and Vaxl. While cellular expression of Ascll isn't clearly reduced, the Ascll + VZJSVZ is thinner (Sup. Figs. 4,6,9,13,5,16,18,20,22,23). Gsxl VZ expression is increased (Sup. Fig. 11). Despite these changes, Nkx6.2 expression in the dorsal-most MGE, and Gbxl expression in the mantle zone appear normal (data not shown).

[00362] At E12.5 Dlxl/2 mutants have clear MGE defects as previously described (Long et al., 2009b). Briefly, here we confirm the severe reduction in Arx, GADl, Pbxl and Sp9 expression. Furthermore, Gsx2 VZJSVZ expression is increased (Sup. Figs. 4,6,20,22,12), and Gsxl, Isletl, Ascll, Olig2 and Otp SVZ expression is increased (Sup. Figs. 11,14,5,18,19). Vaxl expression appears unchanged (Sup. Fig. 23). [00363] Removing Gsx2 function in the Dlxl/2 mutants rescues the elevated level of Hes5 expression (Notch- signaling) in E12.5 Gsx2;Dlxl/2 mutants (Sup. Fig. 13). Despite this "rescue", the MGE of Gsx2;Dlxl/2 mutants is more abnormal than the single mutants for many properties. There is reduced expression of Ascll, DIx 1, GADl,

Gbxl and Vaxl (Sup. Figs. 5,6,9,10,23), and there is increased expression of Nkx2.1 and Olig2 in the VZ, Arx,

Gsxl and Sp9 in the SVZ, and Nkx2.1 in the MZ (Sup. Figs. 16,18,4,11,22). Isletl and Otp SVZ ectopic expression remains similar to the Dlxl/2 mutants (Sup. Figs. 14,19). Nkx6.2 expression in the dMGE (and POA) is preserved

(not shown).

[00364] Transcription Factor Expression in Septal Progenitor and Mantle Cells at E12.5 and E15.5 in Gsx2,

Dlxl/2 and Gsx2;Olxl/2 mutants.

[00365] Gsx2 function in septal development has previously not been reported. At E 12.5 Gsx2 mutants show reduced expression of Arx, Hes5, Isletl, Olig2, Vaxl (Sup. Figs. 4,13,14,18,23), whereas Ascll, Dlxl, Foxp4,

GADl, Ngn2 and Six3 expression does not appear modified (Sup. Figs. 5,6,8,9,15,21). Pbxland Sp9 septal expression is just beginning, and therefore it is difficult to discern a phenotype (Sup. Figs. 20,22).

[00366] By E15.5, Arx expression appears restored (as in the LGE), and Gsxl is increased (Sup. Figs.

24,28). However, expression is reduced for Ascll (thinner VZ), FoxP4, Isletl, Olig2 (ventral VZ), Sp8 (SVZ &

MZ), Sp9 (MZ) and Vaxl (Sup. Figs. 2,26,31,34,39,40,41). Expression appears normal for Dlxl, GADl, Gbxl,

Ngn2 and Pbxl (Figs. 4,5, Sup. Figs. 27,32,36). Expression of Six3 may be increased (Sup. Fig. 38) .

[00367] We previously reported septal defects in the Dlxl/2 mutants (Long et al., 2009a), and here confirm them at E12.5 and E15.5. At E12.5 there is: reduced expression of Arx, Olig2, Six3 (Sup. Figs. 4,18,21); increased expression of Ascll, Gsxl, Gsx2, Isletl and Sp9 expression (Sup. Figs. 5,11,12,14,22); no obvious change in expression of Dlxl, FoxP4, GADl, Pbxl and Vaxl (Sup. Figs. 6,8,9,20,23). At E15.5 there is: reduced expression of Dlxl, Gbxl (MZ) Olig2 (slight), Pbxl and Six3 (Fig. 4; Sup. Figs. 27,34,36,38); increased expression of Ascll,

Gsxl, Gsx2, Isletl and Sp9 (Fig. 2; Sup. Figs. 28,29,31,40); no obvious change in expression of GADl, Arx, FoxP4, and Vaxl (Fig. 6; Sup. Figs. 24,26,41); ectopic expression of Otp (Sup. Fig. 35).

[00368] Gsx2;Dlxl/2 compound mutant septum showed complex and time dependent phenotypes. At

E12.5 the phenotype is more severe for Ascll (reduced), Dlxl (reduced), GADl (reduced), Gsxl (increased), Ngn2

(increased) and Vaxl (reduced) (Sup. Figs. 5,6,9,11,15,23). For other genes, the compound mutant phenocopies the

Dlxl/2 mutant: Arx (decreased), Isletl (increased), Six3 (decreased), Sp9 (increased); or the phenocopies the Gsx2 mutant: Arx (decreased), Ngn2 (ectopic, but more severe), Vaxl (decreased) (Sup. Figs. 4,14,21,22). There may be partial rescue of the Olig2 expression in the compound mutant (Sup. Fig. 18).

[00369] At E15.5 the Gsx2;Dlxl/2 compound mutant's septum shows a more severe reduction of GADl,

Arx, FoxP4, Pbxl, Sp8, Sp9 and Vaxl (Fig. 6; Sup. Figs. 24,26,36,39,40,41). For other genes, the compound mutant phenocopies the Dlxl/2 mutant: Dlxl (reduced), Gbxl (reduced), Gsxl (increased), Isletl (increased), Otp

(increased), Sp9 (increased) (Fig. 4; Sup. Figs. 27,28,31,35,40).; or phenocopies the Gsx2 mutant: Six3 (increased),

Vaxl (reduced) (Sup. Figs. 38,41). There is an intermediate phenotype for Isletl : increased in the SVZ (like

Dlxl/2) and decreased in the MZ (like Gsx2). Ngn2 was not ectopically expressed (Sup. Fig. 32). Ascll and Hes5 expression appears intermediate between the Gsx2 mutant and Dlxl/2 mutant phenotypes (Fig.2; Sup. Fig. 30).

Nkx2.1 (medial septum MZ) and Olig2 (VZ) expression did not appear clearly changed in the compound mutant

(Sup. Figs. 23,34).

[00370] Transcription Factor Expression in LGE Progenitor Cells at E 15.5 in Gsxl, Dlxl/2 and Gsxl;Dlxl/2 mutants: Opposite effect on Ascll expression compared with the Gsx2;Dlxl/2 mutants. [00371] Previous studies have failed to identify strong molecular or cellular defects in Gsxl mutant basal ganglia (Toresson et al., 2001; Yun et al., 2003). Here we investigated Gsxl function because its expression is increased in the Dlxl/2 mutants (Long et al., 2009a,b); thus we studied the phenotype of the Gsxl;Dlxl/2 compound mutants to determine whether some of the Dlxl/2 phenotype is caused by over-expression of Gsxl. Analysis was performed only at E15.5.

[00372] Removing Gsxl expression in the Dlxl/2 mutants did not clearly rescue any phenotype associated with Dlxl GADl, Gbxl, Gsx2, Otp, Sall3 and Six3 expression (Long et al., 2009a,b). On the contrary, the Gsxl ;Dlx 1/2 compound mutants showed an exacerbation of the Dlxl/2 mutant phenotype in the following ways: 1) further increase in Ascll expression in the SVZ of the LGE and dCGE (Fig. 3); 2) increase in Ascll (LGE and MGE) and Gsx2 (MGE) progenitor expression (Fig. 3; Sup. Fig.44); 3) increased GADl expression in the MGE (arrow in Fig. 7), and ectopic GADl and Sp9 expression in the ventral CGE MZ (not the most ventral part) (arrow in Fig. 7; Sup. Fig. 48); 4) loss of Gsx2, Sall3 and Sp9 expression in the rostral vLGE (Sup. Figs. 44,46,48); 5) no change in Dlxl, Dlx2 and GADl LGE/Septum expression (Fig.5; Sup. Fig.42; Fig.7). [00373] Discussion

[00374] Elucidating transcriptional networks will be required to understand the mechanisms the define development of the brain. Here we have focused on the roles of the Gsxl&2 and DIx 1&2 genes in regulating the expression of 21 transcription factors during patterning and differentiation of the mouse subcortical telencephalon. Based on the results we have obtained in this study, and in previous publications on basal ganglia phenotypes in DIx 1&2 (Anderson et al., 1997; Petryniak et al., 2007; Long et al., 2009a,b), Gsxl&2 (Corbin et al., 2000; Toresson et al, 2000; Toresson and Campbell, 2001; Yun et al., 2001, 2003; Waclaw et al. 2004), Ascll (Mashl) (Casarosa et al., 1999; Horton et al., 1999; Yun et al., 2002; Castro et al., 2006; Long et al., 2009a,b), and Olig2 (Petryinak et al., 2007) mouse mutants, we have generated a provisional model of the transcriptional circuit in the LGE/dCGE (Fig. 8); a definitive model will require additional data, including demonstration of direct transcription regulation at each step. In the discussion, we will address the basis for this model.

[00375] GSX2, ASCLl (MASHl) and DLX2 expression define their temporal hierarchy in the LGE. [00376] While ASCLl and DLX2 proteins are strongly expressed throughout the subpallium, GSX2 expression is most easily detected in the LGE and septum, although it is expressed in the MGE. Here were focused on LGE expression at E10.5-E15.5 (Fig. 1; Sup. Figs. 1,2,3).

[00377] Double-immunofluorescence analysis of GSX2, ASCLl and DLX2 protein expression in the LGE provides evidence for a temporal hierarchy of their expression. At E 10.5 the most immature cells (VZ cells) express only GSX2. As the VZ cells mature, scattered cells express ASCLl and DLX2, most of which co-express GSX2. By E12.5, the vast majority of LGE progenitors (VZ+SVZ) co-express the GSX2, ASCLl and DLX2 (Table 1). Co- expression is strongest in SVZl, the part of the SVZ adjacent to the VZ.

[00378] Previous studies of the Dlxl/2;Ascll mutants showed that subpallial progenitors and neurons in the triple mutant have much more severe defects than either the Dlxl/2 or Ascll mutants (Long et al., 2009a,b). Likewise Gsx2&Ascll double mutants show more severe defects than the single mutants (Wang et al., 2009). Here we demonstrated a functional interaction between Gsxl and Gsx2 with Dlxl/2, and provide evidence for the functional hierarchy of Gsx2, Gsxl, Ascll and Dlxl/2. We suggest that these phenotypes are caused in large part to cell autonomous defects, particularly in the SVZl, where GSX2, ASCLl and DLX2 are co-expressed. [00379] Gsx2 Homeodomain: Top of the hierarchy of dLGE/dCGE identity. [00380] We propose that Gsx2 promotes the identity of primary progenitors in the VZ of the dLGE and dCGE. Gsx2 null mutants fail to specify dorsal parts of the LGE and CGE, showing reduced expression of other transcription factors that mark the VZ of these regions (Ascll, Dlx2, Olig2). Therefore, we hypothesize that Gsx2, with Gsxl (see below), promotes the expression of Ascll, Dlx2, and Olig2, from which emanate three major pathways (Fig. 8): 1) Neural differentiation driven by DIx 1&2; 2) Lateral inhibition to promote the maintenance of multipotent progenitors driven by Ascll promoting Delta expression which in turn increases Notch signaling and Hes5 expression; 3) Progenitor cell maintenance through Hes5 and competence to produce oligodendrocytes through Olig2.

[00381] Gsxl Homedomain: Redundant with Gsx2 for vLGE specification, and Repressed by Gsx2 and Dlxl&2. [00382] Previous studies showed that Gsxl mutants have a very mild telencephalic phenotype. They have ectopic expression of Dbxl, a marker of the ventral cortex and preoptic area; the ramifications of this are not known. Gsx2 and Dlxl&2 are negative regulators of Gsxl (Sup. Figs. 11,28; Toresson et al., 2000; Yun et al., 2001; Long et al., 2009a,b). Thus, Gsx2 mutants are partially rescued by Gsxl; Gsxl/2 mutants have misspecification of the dorsal and ventral LGE (Toresson et al., 2001; Yun et al., 2003). Dlxl&2 repression of Gsxl was explored herein by making Gsxl;Dlxl/2 mutants. Loss of Gsxl did not rescue Dlxl/2 mutant phenotypes. However, because Gsx2 is also repressed by Dlxl/2, and because Gsxl and Gsx2 have redundant functions, it may be necessary to generate Gsxl/2;Dlxl/2 mutants to observe a rescue.

[00383] Ascll (Mashl) bHLH: Promotes the subcortical progenitor state through notch signaling, and with Gsx2 and Dlxl/2 promotes subcortical differentiation

[00384] Previous studies demonstrated that Ascll promotes the subcortical progenitor state through increasing Notch signaling (via Deltal expression) and cell non-autonomously (through lateral inhibition) repressing DIx expression (Casarosa et al., 1999; Horton et al., 1999; Yun et al., 2002; Castro et al., 2006); this has the effect of repressing neurogenesis and promoting gliogenesis, including oligodendrogenesis (Parras et al., 2007; Petryniak et al. 2007). Ascll mutants continue to express Gsx2 at roughly normal levels at E12.5 (Wang et al., 2009) and E15.5 (Long and Rubenstein, unpublished).

[00385] Ascll ;Gsx2 compound mutants have a severe reduction in LGE differentiation (Wang et al.,

2009). This is despite continued expression of Gsxl, providing evidence that Gsx2 and Ascll together contribute to specifying the LGE developmental program.

[00386] Analysis of Ascll ;Dk 1/2 individual and compound mutants provided evidence for distinct Ascll and Dlxl/2 dependent pathways of LGE/dCGE development; we proposed that the Ascll pathway operates through the expression of Hes5, Olig2 and Sp9; the DIx pathway components are described below (Long et al., 2007; Long et al., 2009a,b). Gsx2 also is a positive regulator of Sp9 (which could be through Ascll; Sup. Fig. 40), Hes5 and Ascll (Sup. Figs. 30; Fig. 2; Wang et al., 2009). Thus, Gsx2 and Ascll share common regulatory functions for Notch signaling (based on Hes5 expression), and Sp9 expression which distinguish them from Dlxl/2. [00387] Ascll ;Dlx 1/2 compound mutants have greatly reduced subcortical differentiation, but continue to express limited aspects of subcortical identity, based on expression of GADl, and truncated Ascll and DIx 1 RNAs; we postulated that subcortical identity is maintained in these mutants through the function of a few key transcription factors, including those encoded by Gsxl&2 and Isletl (Long et al., 2009a,b). [00388] Gsx2;Dlxl/2 mutants, maintain Ascll expression (albeit weakened); their LGE also continues to express Dlxl, GADl, Gsxl, Foxp4, Isletl, Olig2, and Six3. This provides evidence that Ascll, alone, or with other transcription factors, is able to maintain basic aspects of subcortical GAB Aergic fate. [00389] DIx 1&2 have opposite interactions with Gsxl and Gsx2 in regulating Ascll expression [00390] The DIx genes promote LGE/dCGE development through controlling the expression of multiple transcription factors (Fig. 8; Long et al., 2009a,b). Generally, they repress the expression of transcription factors that promote the progenitor and/or glia cell state, including Ascll, Gsxl&2, Hes5, and Olig2. The block in subcortical neural differentiation in DIx 1&2 mutants may be due, in part, to persistent expression of transcription factors that promote progenitor cell properties. For instance, the over-expression of Olig2 in Dlxl&2 mutants is linked to their over-production of oligodendrocytes (Petryniak et al., 2007). This phenotype is reversed in Ascll;Dlxl/2 compound mutants (Petryniak et al., 2007). On the other hand, compound Gsxl;Dlxl/2 or Gsx2;Dlxl/2 mutants do not show a rescue of most of DIx 1/2 phenotypes. The exception is for Ascll and Hes5 (Notch signaling) in the Gsx2;Dlxl/2 mutants (Fig. 2; Sup. Fig. 30).

[00391] DIx 1&2 promote the expression of transcription factors that direct specific pathways of neural differentiation including Arx, Dlx5&6, EBF, Pbxl, Six3, Sp8 and Vaxl. Thus, compound Gsx2;Dlxl/2 and Ascll ;Dlxl/2 mutants have greatly weakened subcortical development (herein and Long et al., 2009a,b). [00392] At E 12.5 removing Gsx2 function from the DIx 1/2 mutants further weakened regional specification of the dLGE and dCGE (Sup. Figs. 4,5,6,9,11,14,18,20.21,22,23). Gsx2;Dlxl/2 mutants have reduced Ascll expression, compared with the individual mutants (Sup. Fig. 5). We postulate that reducing Ascll levels in the context the Gsx2;Dlxl/2 null state is an important mechanism that contributes to the more severe phenotype of these compound mutants.

[00393] At E15.5, removing Gsx2 function from the Dlxl/2 mutants also reduces the level of Ascll expression (Fig. 2; although not as much as at E 12.5). This result contrasts with the Gsxl ;Dlx 1/2 mutants, which show increased Ascll expression in the SVZ of the E15.5 LGE, MGE and CGE (Fig. 3), suggesting that Gsxl, like Dlxl/2, promotes subcortical differentiation through repression of Ascll expression and Notch signaling. [00394] Gsxl;Dlxl/2 compound mutants also show reduced Gsx2, Sall3 and Sp9 expression in the rostral vLGE (Sup. Figs. 44,46,48). This is interesting because this region, which we postulated generates nucleus accumbens, is preferentially spared in the Dlxl/2 mutants (Long et al., 2009a). Ascll mutants preferentially affect the vLGE compared with the dLGE (Long et al., 2009a). [00395] MGE-specific phenotypes in Gsx;Dlxl/2 compound mutants

[00396] While the Gsx/Mash/Dlx subcortical program promotes MGE differentiation, regional and cellular fate specification in this region also operates through a parallel/overlapping program mediated by the Nkx2.1 and the Lhx6/7(8) genes (Sussel et al., 1999; Liodis et al., 2008; Zhao et al., 2009).

[00397] We noted some MGE-specific phenotypes in the Gsx;Dlxl/2 compound mutants. Gsxl;Dlxl/2 mutants show an increase in GADl and Gsx2 expression in MGE progenitor domains, and an ectopic collection of cells expressing GADl and Sp9 in the mantle of the ventral CGE (not the most ventral part); these cells appear to emanate from the MGE (based on a trail of expression connecting the MGE to the ectopia (Fig. 7; Sup. Figs. 44,48 ). [00398] Removing Gsx2 function in the Dlxl/2 mutants rescues the elevated level of Hes5 expression (Notch- signaling) in the MGE (as in the LGE). Despite this "rescue", the MGE of Gsx2;Dlxl/2 mutants is more abnormal than the single mutants for many properties (see results). The preserved expression of Ascl 1 and Nkx2.1 are probably responsible for maintaining much of the MGE developmental program.

[00399] Septal phenotypes in Gsx2 and DIx 1&2 mutants

[00400] Our previous analysis of the Ascll and DIx 1/2 mutants discovered specific roles for these genes in septal development (Long et al., 2009a). For instance Dlxl&2 are positive regulators of ER81 and Pbxl, while Ascll promotes expression of Arx, Hes5, Isletl, Olig2, Sp9 and Vaxl. Here we extended our analysis of the Dlxl/2 mutants and provided the first analysis of septal development in the Gsx2 mutants. We provided evidence for a

Gsx2 dependent septal pathway mediated through expression of Ascll, FoxP4, Isletl, Olig2, Sp9 and Vaxl (Fig. 2;

Sup. Figs. 5,8,14,18,22,23,26,31,34,40,41).

[00401] We confirmed that Dlxl&2 promote expression of Dlxl, Gbxl (MZ), Olig2 (slight), Pbxl and

Six3, and repress expression of Ascll, Gsxl, Gsx2, Isletl, Sρ8 and Sp9. Unlike in the LGE, expression of GADl and Vaxl do not seem to change, perhaps because Dlx5&6 continue to be expression in the septum of Dlxl/2 mutants (Anderson et al., 1997; Long et al., 2007, 2009a). In Gsx2;Dlxl/2 compound mutants, Islet expression showed a phenotype intermediate between the Gsx2 and Dlxl/2 mutants.

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Example 4: Production of a progenitor cell from a stem cell (prophetic example)

A subpallial progenitor cell is produced from ES or iPS cells by forced expression (induction) of the genes under the control of the transcription factors Gsx2, Ascll (Mashl) and Dlx2. Introduction of the transcription factors will be performed directly in the ES and iPS cells, or after the ES/iPS cell have been induced to an embryonic forebrain or ventral forebrain state. Transcription factor introduction can be accomplished either using transfection/transduction, such as via viral vector gene delivery (retroviral/lentiviral/AAV/sendai) or through incubation with extracellular transcription factor protein. For the latter, vectors that express Gsx2, Ascll and Dlx2 fusion proteins that have N-termini signal sequence to enhance cell membrane permeability are prepared as described in Becker-Hapak et al, (2003), Curr. Protocols Cell Biol., Unit 20.2, John Wiley & Sons. Following the expression and purification of the fusion proteins, forced expression is initiated by replacing the culture medium with 2 ml of MC-ES medium containing purified fusion proteins (100 nM each). The iPS cells are incubated for about three hours at 37 °C. Afterwards, the medium is replaced with MC-ES medium supplemented with 10 μM Y- 27632 (Calbiochem), which helps to prevent cell apoptosis triggered by the passaging procedures.

The following 3 days, the induction procedure is repeated, followed by the replacement of media. Two days later, cells are harvested and in-situ hybridization assays are performed to identify subpallial progenitor cell candidates. Example 5: Differentiation of a LGE progenitor cell to form striatal neurons (prophetic example)

LGE progenitor cells are differentiated into striatal neurons by forced expression of the genes (e.g. GADl, vgat, RXRg, Ikaros) activated by the transcription factors, Dlxl and Dlx2.Transcription factor introduction can be accomplished either using transfection/transduction, such as via viral vector gene delivery

(retroviral/lentiviral/AAV/sendai) or through incubation with extracellular transcription factor protein. For the latter, this is accomplished by incubating a LGE progenitor cell in the presence of Dlxl and Dlx2 fusion proteins (100 nM each). The fusion proteins have a signal sequence that make the fusion proteins more permeable to the cell membrane of the progenitor cells. After incubation for about three hours at 37 ⁰C, the medium is replaced with MC- ES medium supplemented with 10 μM Y-27632 (Calbiochem). During the induction period the medium is replaced daily with MC-ES medium containing 100 nM of each of the fusion proteins for one hour, and the medium is then replaced with MC-ES medium free of fusion proteins until the following day. The following 2 days, the induction procedure is repeated, followed by the replacement of media. Four days later, cells are harvested for RT-PCR, western blotting, flow cytometry, and immunocytochemistry analysis that confirms that the LGE progenitor cells differentiated into striatal neurons.

Example 6: Transplantation of Human iPS Cell-Derived striatal neurons in a human patients with Huntington's Diseasel (prophetic example)

Striatal neurons are prepared according to methods described herein and are used in a Phase I clinical trial, which is designed to assess the safety and preliminary efficacy of striatal neurons as a treatment for Hungtinton's disease in humans. The trial enrolls at least one patient with Hungtinton's disease. All patients are transplanted with striatal neurons via injection, and are immunosuppressed for nine months. Following transplantation, the patients are evaluated regularly over a 12-month period in order to monitor and evaluate the safety and tolerability of the striatal neuron implants and the immunosuppression. In addition, magnetic resonance imaging (MRI) of the brain post- transplant may enable the measurement of new striatal formation. Results show that patients who have the striatal implants demonstrate moderate improvement in symptoms such as rigidity, writhing motions or abnormal posturing in comparison to patients who have control implants.

H

B> a^*

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TABLE 1. Nucleotide Sequences of In Situ Hybridization Probe

Insert Size in cDNA Nucleotides of Probe

Plasmid Name Source airs or Nucleotides from 3' End

Arx Kunio Kitamura 1000 1000 from 3' end

ATBFI Dino Leone 464 11887-11423

Bm4 Bryan Crenshaw 573 513-1086

Cad8 Chris Redies 460 1251 to 1711

CoupTFi Ming Tsai 1500 full coding sequence

Ctipi MarkLeid 766 full coding sequence

CXCR4 Dan LJttman 580 full coding sequence

CyclinD2 A, Malamacci 475 1329-1804

D1R Josh Corbin 534 3604-4138

Is) D2R Josh Coitin 804 517-1321

DIxI John Rubenstein 2800 2800 from 3' end

Dlx2 John Rubenstein 1700 1700 from 3' end

DIxS John Rubenstein 1600 1600 from 3' end

Dlx6 John Rubenstein 210 210 from 3' end

Ebfi R. Grosschedl 737 193-930

Ebf3 Sonla Garel 1750 1750 from 3' end

Egr3 J.D. Powell 1416 full coding sequence

Enk Josh Cortsn 1441 full coding sequence

ER81 Thomas Jessell 2000 full coding sequence

EώB4 Cary Lai 920 Amino Adds 326-633

Erm; Etv5 Anne Chotteau-Lelievre 1998 full coding sequence

ESRG; NR3B3 Paul Gray 720 302-1022

Evi3; Zfp521 RZPD; ImaGenes 3936 full coding sequence

H

TABLE 1. Nucleotide Sequences of In Situ Hybridization Probe

Insert Size in cDNA Nucleotides of Probe

Plasmid Name Source Basepairs or Nucleotides from 3' End

FoxGi E. Lai 1563 2094-3657

FoxP1 Russell Ferland 1900 full coding sequence

FoxP2 Russell Ferland 2000 full coding sequence

FoxP4 Russell Ferland 2058 2058 from 3' end

GAD67 Bryan Condie 2000 full coding sequence

Gbxi Mike Frohman 550 334884

Gbx2 Mike Frohman 650 650 from 3' end

GIiI AlexJoyner 1683 945-2628

Golf; Gna1 Richard Axel 1540 M540

Gsh1 Steve Potter 2200 full coding sequence

Gsh2 Steve Potter 460 1038-1498

Gucyia3 ATCC 9841394 2000 full coding sequence

HesS Francois Guillemot 548 73-621

Id2 Mark Israel 260 546-806 lkaros 1093 243-1336 lsleti TomJesseil 1510 31-1541

Lhx2 Juan Botas 585 1172-175?

Lhx6 Vassilis Pachπis 1342 1208-2550

Lmo1, human Gordon Gill 469 61-530

Lmo3 TH Babbitts 2000 full coding sequence

Lmo4 Gordon Gill 500 full coding sequence

σ^*

TABLE 1. Nucleotide Sequences of In Situ Hybridization Probe

Insert Size in cDNA Nucleotides of Probe

Plasmid Name Source Base pairs or Nucleotides from 3' End

Mashi Francois Guillemot 2100 full coding sequence

MeSc Eric Olson 416 1-416

Meisi Kenny Campbell 2300 full coding sequence

Meis2 Kenny Campbell 2400 full coding sequence

NHLH2 Debora Good 1900 full coding sequence

Nkx2.1 John Rubenstein 2200 full coding sequence

Nkx6.2 John Rubenstein 1300 1300 from 3' end

Neuropilin2, NP2 M Tessier-Lavigne 1200 Amino Adds 536-911

Oct6; Pou3f1 M.G. Rosenfeld 2300 full coding sequence

Otp Antonio Simeone SOO 500from 3' end

01x2 John Rubenstein 2100 2100 from 3' end

Pax6 Peter Gross 302 656-958

Pbxi Heike Pόpperi 1294 358-1652

PK2 QYZhou 509 1-509

PKRI QYZhou 975 975 from 3' end

Preprotachykinin Invitrogen 1034 Image Clone 1166182

RAR-beta Kenny Campbell 2025 1036-3061

RDCI ATCC MGC-18378 1897 63-1960

Roboi M Tessier-Lavigne 1000 1000 from 3' end

Robo2 M Tessier-Lavigne 1700 1700 from 3' end

ROR-beta Dennis O'Leary 2007 638-2645

RXR- gam ma Kenny Campbell 1226 907-2133

H

8-

TABLE 1. Nucleotide Sequences of In Situ Hybridization Probe

Insert Size in cDNA Nucleotides of Probe

Plasmid Name Source Basepairs or Nucleotides from 3' End

Sall3 P Monaghan 870 2883-3753

Sema3a M Tessier-Lavigne 1182 1-1182

Six3 Peter Gfuss 650 650 from 3' end

Slid M Tessier-Lavigne 746 2353-3099

Sox1 R Lovell Badge 940 1851-2791

Soxil John Rubenstein 2592 2592 from 3' end

Sox4 John Rubenstein 1900 1900 from 3' end

Os Sp8 Kenny Campbell 880 1622-2502

Sp9 Kenny Campbell 2400 full coding sequence

Tbii John Rubenstein 264 264 from 3' end

Tιam2 RZPD; ImaGeπes 5148 full coding sequence

Tle4 Johan Ericson 2300 full coding sequence

TIx Paula Monaghan 1700 1700 from 3' end

TrkB FR Klein 487 1270-1757

Tsh1 X. Cauttt 827 2844-3671

Tsh2 X. CatM 1152 M152

Vaxi Peter Gruss 950 950 from 3' end

VGAT Brian Conde 730 1295-2025

V$lut2 Robert Edwards 305 305 from 3' end

Zid JunAruga 333 333 from 3' end

The name of the plasmid donor and nucleotide sequence range for each in situ hybridization probe used in our analysis.

Table 2A

E12 ISH E15 ISH TF Name Color Code BG/Cortex Ctx BG BG -/- BG-/BG+ BG+/BG-

Aqua 4.64 487 2262 618 0.27 3.66

Aqua 4.81 16 77 125 1.62 0.62

Green 30.44 18 548 503 0.92 1.09

Yellow 0.75 5121 3856 3116 0.81 1.24

Yellow 0.56 149 83 93 1.12 0.89

Green 7.18 28 201 114 0.57 1.76

Aqua 0.16 19 3 10 3.33 0.30

CoupTFl (NR2F1) Yellow 1.31 2414 3154 3561 1.13 0.89

⁽SsπffiOMOl) Aqua 3.38 102 345 339 0.98 1.02

Ctipl (Bcllla, Evi9) Yellow 1.06 1522 1616 1052 0.65 1.54

Ctip2 (Bcll lb, Rit-lb) Yellow 1.43 1106 1579 792 0.50 1.99

Cux2 Yellow *

Lavender 1.83 18 33 27 0.82 1.22

Green 9.84 120 1181 7 0.01 168.71

Green 7.06 50 353 15 0.04 23.53

Green 7.76 96 745 80 0.11 9.31

Green 9.80 15 . 147 21 0.14 7.00

Yellow 4.00 11 44 4 0.09 11.00

Green 21.00 576 132 0.23 4.36

Lavender 0.66 74 49 198 4.04 0.25

Aqua 2.05 21 43 18 0.42 2.39

Yellow 0.31 100 75 0.75 1.33

Yellow 0.43 356 152 177 1.16 0.86

66 213 137 0.64 1.55

21 33 26 0.79 1.27

149 388 122 0.31 3.18

34 86 90 1.05 0.96

56 43 0.77 1.30

9 48 63 1.31 0.76

7 53 156 2.94 0.34

13 192 205 1.07 0.94

167 227 290 1.28 0.78

Yellow 1.10 106 117 144 1.23 0.81

Lavender 0.12 1870 230 686 2.98 0.34

Yellow 0.98 197 194 196 1.01 0.99

Green 2.27 U 25 25 1.00 1.00

Green 254.00 6 1524 1318 0.86 1.16

Green 0.36 14 5 22 4.40 0.23

Yellow 0.31 2120 650 774 1.19 0.84

Green 2.67 46 123 98 0.80 1.26

Green 252.50 2 505 224 0.44 2.25

Yellow 0.62 569 351 362 1.03 0.97 Lmo3 (Rbtn3) Yellow * tas3 Aqua 2.19 360 789 581 0.74 1.36

Aqua 1.07 122 131 167 1.27 0.78 SMifl ({fe89j) Aqua 4.51 63 284 330 1.16 0.86 Table 2A (cont'd)

* I I Green 6.00 386 229 0.59 1.69

* MeOc Yellow 0.44 398 174 151 0.87 1.15

I Green 4.67 49 229 154 0.67 1.49

* Meis2 (MRGlB) Yellow 1.23 1489 1827 1275 0.70 1.43

* Nexl (NeuroDδ) Yellow 0.12 5163 620 881 1.42 0.70

* Yellow * Green 0.46 65 30 47 1.57 0.64

* Green 7.00 3 21 30 1.43 0.70

* Green 1.67 3 5 3 0.60 1.67

* Green 1.24 100 124 112 0.90 1.11

* Green *

* Green 0.50 12 6 2 0.33 3.00

* Green 1.18 65 77 51 0.66 1.51 Aqua 1.30 74 96 25 0.26 3.84

* Aqua 5.73 79 453 638 1.41 0.71

* Aqua 5.66 29 164 281 1.71 0.58

*

Orange 0.88 8 7 16 2.29 0.44

* Otxl Yellow 0.31 40 53 1.33 0.75

^H '-. I Green 0.46 35 71 2.03 0.49

* Pax6 Yellow 0.42 200 83 103 1.24 0.81

Yellow 1.03 180 186 126 0.68 1.48 Green 7.28 76 553 671 1.21 0.82 Green 1.22 381 463 818 1.77 0.57

Green 3.07 14 43 56 1.30 0.77

Green 14.00 271 40 0.15 6.78

* RORb Yellow 0.70 42 83 1.98 0.51

I Green 21.29 14 298 48 0.16 6.21

* SaIB (msall, spalt) Yellow 0.84 68 57 145 2.54 0.39

I Green 9.70 33 320 153 0.48 2.09

I Aqua 3.60 15 54 35 0.65 1.54

* Sox 4 Yellow 0.73 827 602 865 1.44 0.70

Sox 5 Yellow 0.17 1023 172 223 1.30 0.77

55 62 77 1.24 0.81

123 85 0.69 1.45

6142 4716 3765 0.80 1.25

152 81 0.53 1.88

646 215 270 1.26 0.80

1030 169 111 0.66 1.52

2771 818 1245 1.52 0.66

16 23 18 0.78 1.28

* 124 102 80 0.78 1.28

» Green 2.69 13 35 22 0.63 1.59

593 859 1592 1.85 0.54

89 39 32 0.82 1.22 Table 2B

Table 3

Table 4

LGE SEPTUM

VZ SVZ MZ VZ svz MZ

Gray

White Magenta Orange Blue Green Table 5 5 ISH TF Name BG/Cortex Ctx BG BG -/- BG-/BG + Origin c

Adamts5 1.39 31 43 28 0.65 NMJ

BcIl Ib 1.43 1106 1579 792 0.50 NMJ

Camk2a 10.26 23 236 122 0.52 AW4

Capl 1.13 460 518 309 0.60 NMJ

* Cmkorl 3.96 408 1614 708 0.44 BCO

Crabpl 6.88 68 468 116 0.25 NMJ

* Crym 0.35 329 114 41 0.36 NMJ

* Cxcr4 0.88 307 271 81 0.30 D8

DIcI 1.79 78 140 73 0.52 BQl

* Drdla 1.32 31 41 17 0.41 BE9

Gcnt2 1.90 50 95 42 0.44 BM2

Gpr88 110.50 2 221 50 0.23 BBO

Granulin 1.35 287 388 194 0.50 BBO

* Gucyla3 2.62 188 492 179 0.36 BGO

Histlhlc 1.99 139 277 128 0.46 BB5

Htr3a 2.07 92 190 62 0.33 NMJ

Ivd 0.87 229 199 96 0.48 AV2

Lck 2.80 10 28 15 0.54 AA8

Lgalsl 1.96 266 522 274 0.52 AI 6-

Mbp 2.10 70 147 105 0.71 LO

Myh6 13.08 13 170 89 0.52 NMJ

Ncdn 1.89 323 610 309 0.51 BCO

Penkl 13.31 13 173 79 0.46 Ml

* Pla2g4b 0.93 126 117 52 0.44 BCO

Purg 1.61 57 92 48 0.52 AF4

Rbpl 9.70 213 2066 889 0.43 NMJ

Rnasepl 1.61 62 100 43 0.43 R7

Rpl22 1.70 2909 4952 2182 0.44 NMJ

Rrbpl 1.08 37 40 7 0.18 NM_

Rrp4 2.14 14 30 15 0.50 AW4

SlOO calcium binding 2.04 103 210 103 0.49 AV2

* Shb 1.31 274 358 178 0.50 BI4'

Slcolal 2.36 11 26 5 0.19 ABO

Snx6 1.46 174 254 129 0.51 BCO

Sppl 4.84 31 150 57 0.38 NMJ

Syndecan 1 1.75 193 338 160 0.47 BB5

Syt6 1.73 48 83 41 0.49 ABO

* Tacl 86.55 11 952 457 0.48 NMJ

* Thbsl 0.87 204 178 69 0.39 AVO

* Tiam2 0.27 895 239 55 0.23 BM2

Top2b 13.95 19 265 44 0.17 BB2

* Viaat 10.76 42 452 38 0.08 NMJ

* Wnt5a 0.83 82 68 38 0.56 BBO

Wnt7a 2.01 160 321 158 0.49 AKO

Zfpl45; PLZF 2.10 31 65 29 0.45 Z4

Table 6

Transcription Factors E12 ISH E15 ISH TF Name BG/ Cortex Ctx BG Expr BG Expr -/- MGE Expr MGE Expr -/- t ~

Arx 4.64 487 2262 618 3648 948

Asb2 3.18 11 35 32 11 14

Asb4 4.81 16 77 125 419 992

ATBFl 30.44 18 548 503 124 246

BFl (FoxGl) 0.75 5121 3856 3116 4142 4781

BhthbS 0.32 3822 1219 1053 28 189

Bm2 (POU3F2) 0.56 149 83 93 89 86

Brn4 (POU3F4) 7.18 28 201 114 501 209

Bm5 (P0U6F1) 0.16 19 3 10 1 6

CoupTFl (NR2F1) 1.31 2414 3154 3561 1479 3857

CoupTFII (NR2F2) 3.38 102 345 339 73 110

Ctipl (Bdlla, Evi9) 1.06 1522 1616 1052 891 692

Ctip2 (BdIIb₁ RiMb) 1.43 1106 1579 792 1441 1748

Cux2

Dach2 2.79 33 92 171 153 277

Dbxl 1.83 18 33 27 17 28

Olxl 9.84 120 1181 7 3278 2

01x2 7.06 50 353 15 1069 15

D 1x5 7.76 96 745 80 751 49

Dlx6 9.80 15 147 21 172 5

Dlx6 Antisense (Evfl, Evf2) 4.00 11 44 4 22 2

DIxIn

Ebfl 21.33 27 576 132 144 96

Ebf2 1.59 17 27 42 17 29

Ebf3 0.66 74 49 198 12 50

Egr3 2.05 21 43 18 45 25

Emxl 0.31 323 100 75 33 4

Emx2 0.43 356 152 177 105 94

ER81 (Etvl) 3.23 66 213 137 820 283

ESRG (ESRRG, NR3B3) 1.57 21 33 26 20 25

Evi3 (Zfp521, EHZF) $

Fah 3.55 22 78 42 59 36

Fez (FezFl) *

Fez -I (FezF2) *

Fox03a 0.75 4 3 8 17 19

FoxO4 0.46 13 6 36 17 23

FoxPl 2.60 149 388 122 96 81

FoxP2 2.53 34 86 90 31 14

FoxP4 (mFKHLA) 1.30 43 56 43 30 38

FXRlalphal (NR1H4) 2.10 10 21 14 30 21

Transcription Factors E12 ISH ElS ISH TF Name BG/ Cortex Ctx BG Expr BG Expr -/- MGE Expr MGE Expr -/- H ft

Gbxl

Gbx2 5.33 48 63 15 124

GIiI (ZfpS)

Gshl 7.57 7 53 156 112 484

Gsh2 14.77 13 192 205 185 308

Hesl 0.64 171 110 88 131 93

Hes5 1.36 167 227 290 702 689

HesRl (Heyl) 1.10 106 117 144 137 107

HIxI 3.00 11 33 27 2 11

Id2 0.12 1870 230 686 122 729

Id4 0.98 197 194 196 214 294

Ikaros (ZNFNlAl) 2.27 11 25 25 36 31

Isletl 254.00 6 1524 1318 1339 2264

Klf4 1.33 36 48 45 54 88

KIfS 7.14 21 150 92 62 298

LhXl (UmI) 0.36 14 5 22 8 15

Lhx2 0.31 2120 650 774 428 853

Lhx6 2.67 46 123 98 130 167

Lhx7 252.50 2 505 224 1513 166

Uix9 0.68 200 135 200 4 123

Lmol 0.62 569 351 362 1764 1048

Lmo3 (Rbtn3)

Lm o4 2.19 360 789 581 697 761

Maf A ♦

Maf B 1.07 122 131 167 53 72

Maf C 1.04 28 29 20 18 3

Mashl (Asdl) 4.51 63 284 330 536 941

Med6 6.03 64 386 229 344 275

Mef2c 0.44 398 174 151 48 26

Melsl 4.67 49 229 154 115 142

Meis2 (MRGlB) 1.23 1489 1827 1275 1067 1078

Msc (MyoR) 3.21 14 45 49 2 4

Mytll 0.74 1018 757 933 276 566

Neurog2 0.23 1508 341 211 9 66

Nexl (NeuroDδ) 0.12 5163 620 881 1 78

NHLH2 (Hen2, NSCL2)

Nkx2.1 (Ttfl) 0.46 65 30 47 31 24

Nkx2.2 7.00 3 21 30 1 20

Nkx5.1 (Hmxl) 1.67 3 5 3 4 2

Nkx5.2

Transcription Factors H E12 ISH E15 ISH TF Name /Corteii Ox BG Expr BG Expr-/- MGE Expr MGE Expr -/-

ST

Nkx6.1 15.00 1 15 8 14 16 O

Nkx6.2 1.24 100 124 112 76 74

NoIzI (Zfp5O3)

Npasl 0.50 12 6 2 16 5

Nr4a2 0.72 132 95 114 37 50

Nur77 (NR4A1) 1.18 65 77 51 36 26

Oct6 (P0U3F1) 1.30 74 96 25 76 62

OIigl 5.73 79 453 638 318 896

Olig2 5.66 29 164 281 654 859

Otp 0.88 8 7 16 9 15

Otxl 0.31 129 40 53 2 22

Otx2 0.46 76 35 71 151 359

Pak3 1.33 179 238 526 230 395

Pax6 0.42 200 83 103 27 16

Pbxl 1.03 180 186 126 191 137

Pbx3 7.28 76 553 671 209 700

Peg3 (EntM, Gcap4, PwI, ZfplO2) 1.22 381 463 818 512 568

Phox2a (Arix, Pmx2) 0.73 22 16 22 4 10

Proxl 3.07 14 43 56 34 94

RALDH 3 (ALDH6)

RARB 14.26 19 271 40 56 28

RORβ 0.70 60 42 83 31 39

RXRγ (NR2B3) 21.29 14 298 48 57 26

Sall3 (msall. spalt) 0.84 68 57 145 90 171 oo O Siml 1.19 37 44 44 35 38

S 1x3 9.70 33 320 153 188 253

SoIt 1.09 238 260 200 759 340

Soxl 3.60 15 54 35 105 140

Soxll 0.77 6142 4716 3765 5005 3203

Sox4 0.73 827 602 865 507 1046

Sox5 0.17 1023 172 223 243 347

Sox6 1.14 258 294 406 711 836

Sox8 4.24 29 123 85 63 96

Sp8 (Btd) 2.41 63 152 81 44 6

Sp9

Tbrl 0.33 646 215 270 29 5

Tbr2 0.16 1030 169 111 2 1

TCF4 0.30 2771 818 1245 2066 2272

TIe4

TIx 1.44 16 23 18 9 4

Tox 0.86 182 156 257 215 532

Trp53 0.82 124 102 80 109 120

Tφ53bpl 0.66 212 140 153 127 91

Tshzl *

Tshz2 *

Vaxl 2.69 13 35 22 31 17

Zbtb20 0.76 507 385 746 139 359

Zfhxlb 0.31 1037 321 388 310 140

Zfp618

Zicl 1.45 593 859 1592 613 604

Non Transcription Factors

E12 ISH E15 BH TF Name BG/ Cortex Qx BG Expr BG Expr -/- MGE Expr MGE Expr-/- K

* Adamtsδ 1.39 31 43 28 133 31 o-

Adrenergic Receptor, alpha 2a 7.38 13 96 102 41 50

Ankyrin Repeat and SOCS box-containing protein 4 7.13 15 107 152 784 1425

B3gait5 12.00 5 60 29 94 72

BdI Ib 1.43 1106 1579 792 1441 1748

Bdkrbl 1.69 16 27 10 17 21

Cad7, type 2 0.44 55 24 159 29 38

* Cad8 *

CaIbI 9.41 34 320 177 236 174

Calσ 3.20 5 16 91 42 88

Camk2a 10.26 23 236 122 50 136

Capl 1.13 460 518 309 222 369

Carnitine Deficiency-Associated Gene expressed in ventride 3 0.14 63 9 60 3 15

* Cσ4 0.23 22 5 23 3 3

Cd69 1.63 8 13 4 1 5

Cfh 2.00 10 20 11 13 5

Clcal 1.60 5 8 39 24 35

Clca2 1.50 10 15 31 4 18

Coatomer Protein Complex, subunit gamma 2, antisense 2 4.23 242 1024 1680 74 363

Cobl 9.40 10 94 87 24 25

* Crabpl 6.88 68 468 116 113 12

* Crym 0.35 329 114 41 18 33

* CXCR4 0.88 307 271 81 415 211

* CXCR7 3.96 408 1614 708 4602 1651

* CydinD2 1.18 1802 2126 1631 3051 1700

* Dactl *

Did 1.79 78 140 73 45 40

* Drdla 1.32 31 41 17 5 4

Egfl6 0.29 21 6 21 13 15

Erbb2ip 0.42 95 40 150 73 47

* ErbB4 4.67 3 14 7 12 2

Ta^b Non Transcription Factors ia" E12 ISH E15 ISH TF Name ./Cortex : Qx BGExpr BG Expr-/- MGE Expr MGE Expr-/-

Gabral 9.57 7 67 94 18 40

GADl (GAD67) 5.46 321 1753 1358 583 520

GABA-A Receptor, subunit gamma 1 6.11 9 55 78 18 12

Gcnt2 1.90 50 95 42 70 58

Gng4 5.00 15 75 77 14 39

Gpr88 110.50 2 221 50 23 18

Granulin 1.35 287 388 194 360 402

Gucyla3 2.62 188 492 179 655 174

H2-K 2.77 13 36 12 2 13

H2-Q1 1.96 26 51 17 32 18

Histlhlc 1.99 139 277 128 473 288

Htr3a 2.07 92 190 62 223 113

Ivd 0.87 229 199 96 144 183

Kcnj9 10.67 3 32 24 9 1

KnjppeHike factor 5 7.14 21 150 92 62 298 oo Lck 2.80 10 28 15 10 19

N) Lgalsl 1.96 266 522 274 1212 589

Lor 13.50 2 27 35 10 16

Mbp 2.10 70 147 105 115 119

M oxdl 6.60 5 33 25 7 16

Myh6 13.08 13 170 89 59 95

Ncdn 1.89 323 610 309 269 390

NP2 1.18 150 177 248 17 57

Nphx φ

Npy2r 7.75 4 31 30 36 26

0lfm3 32.00 4 128 159 78 63

Omg 12.25 4 49 65 37 91

Ostb 9.33 3 28 18 23 5

Penkl 13.31 13 173 79 39 34

Phkal 0.44 36 16 38 32 17

PK2 *

PKRl *

Pla2g4b 0.93 126 117 52 121 124

Plaa 0.48 33 16 78 21 25

Non Transcription Factors S- E12 ISH E15 ISH TF Name /Cortex Ox BG Expr BGExpr-/- MGE Expr MGE Expr -/- a"

Pre B-cell Leukemia Transcription Factor 3 15.65 122 1909 2013 696 2110

Presenilin 1 0.50 10 5 11 9 7

Prok2 11.80 5 59 63 27 108

Protease, cysteine, 2 (NEDD8 specific) 0.33 69 23 86 118 82

Purg 1.61 57 92 48 52 58

Pyruvate Carboxylase 1.34 41 55 17 10 17

Rbpl 9.70 213 2066 889 2917 1452

Resplβ 129.50 2 259 245 33 61

Rnasepl 1.61 62 100 43 107 106

Robo2 _$

Rp)22 1.70 2909 4952 2182 7650 5885

R rbpl 1.08 37 40 7 18 21

Rφ4 2.14 14 30 15 13 11

SlOO caldum binding protein AlO (calpactin) 2.04 103 210 103 520 189

Scmhl 0.35 98 34 212 43 33

Sema3a 0.93 195 181 185 59 76

Semaβd 0.61 33 20 84 38 26 oo Shb 1.31 274 358 178 410 213

Sicolal 2.36 11 26 5 6 7

Snx6 1.46 174 254 129 258 110

Sppl 4.84 31 150 57 32 17

Syndecan 1 1.75 193 338 160 470 301

Syt6 1.73 48 83 41 54 21

Tad 86.55 11 952 457 310 174

Thbsl 0.87 204 178 69 166 86

Tiam2 0.27 895 239 55 438 42

Top2b 13.95 19 265 44 56 19

Trtir 0.40 45 18 28 4 5

Uty 0.77 135 104 7 45 80

VlraS 0.50 2 1 8 2 9

Viaat 10.76 42 452 38 617 24

Vsnll 7.82 28 219 255 54 133

Wnt5a 0.83 82 68 38 110 87

Wπt7a 2.01 160 321 158 513 441

Zfpl45; PLZF 2.10 31 65 29 2 2

Table 8

Differential TF Expression Between the LGE and CGE

Table 10

CGE

Claims

What is Claimed:

1. A method of differentiating a subpallial progenitor cell comprising forcing expression of Gsx 1 , Gsx2, Ascl 1 , DIx 1, Dlx2, combinations or homologs thereof in said cell.

2. The method of claim 1, further comprising forcing the expression of one or more additional genes or homologs thereof, selected from Tables 2-4.

3. A method of differentiating a subpallial progenitor cell comprising exposing said progenitor cell to one or more transcription factors produced by the genes Gsxl, Gsx2, Ascll, Dlxl and Dlx2 or homologs thereof.

4. The method of claim 3, further comprising exposing said pregenitor cell to one or more additional transcription factors produced by one or more genes selected from Tables 2-4 or homologs thereof.

5. The method of claim 1 or 3, wherein said progenitor cell is human.

6. The method of claim 1 or 3, wherein said differentiated cell is a LGE-derived nueron, striatal neuron, CGE- derived interneuron, VIP+ , calretinin+/somatistatin- , NPY+ .

7. A differentiated cell produced by the method of claim 1 or 3.

8. A differentiated cell of claim 7 wherein the differentiated cell is a LGE-derived nueron, striatal neuron, CGE- derived interneuron, VIP+, calretinin+/somatistatin-, NPY+.

9. A composition comprising a differentiated cell derived by the forced expression of Gsxl, Gsx2, Dlxl, Dlx2, Ascll combinations or homologs thereof in a subpallial progenitor cell. .

10. The composition of claim 6, wherein said differentiated cells is further derived by the forced expression of one or more additional genes selected from Tables 2-4 or homologs thereof.

11. A composition comprising a differentiated cell derived by exposing a subpallial progenitor to one or more transcription factors produced by Gsxl, Gsx2, Dlxl, Dlx2 and Ascll or homologs thereof.

12. The composition of claim 9, wherein said differentiated cells is further derived by exposure to one or more additional transcription factors produced by one or more genes selected from Tables 2-4 or homologs thereof.

13. A method for treating a disorder arising from the loss of number or function of a striatal interneuron comprising administering to a patient in need thereof, differentiated cells made by the method of claim 1 or 3, wherein said cells increase the number or function of striatal interneurons.

14. The method of claim 11 wherein said disorder is Huntington's disease, epilepsy, schizoprenia, autism, stroke, Parkinson's Disease, Tourette's Syndrome or Alzheimer's disease.

15. A method for treatment or amelioration of symptoms caused by the imbalance of the excitory/inhibitory neuronal circuitry comprising administering cells produced by the method of claim 1 or 3.