WO2002018545A1 - Genetic demonstration of requirement for nkx6.1, nkx2.2 and nkx6.2 in ventral neuron generation - Google Patents

Abstract

This invention provides a method of converting a stem cell into a ventral neuron which comprises introducing into the stem cell a nucleic acid which expresses homeodomain transcription factor Nkx6.1 or Nkx6.2 protein in the stem cell so as to thereby convert the stem cell into the ventral neuron. Provided are methods of diagnosing a motor neuron degenerative disease in a subject. Also provides is a method of treating neuronal degeneration in a subjet which comprises implanting in diseased neural tissue of the subject a neural stem cell which is capable of expressing homeodomain Nkx6.1 or Nkx6.2 protein under conditions such that the stem cell is converted into a motor neuron after implantation, thereby treating neuronal degeneration in the subject.

Description

GENETIC DEMONSTRATION OF REQUIREMENT FOR NKX6.1 , NKX2.2 AND NKX6.2 IN VENTRAL NEURON GENERATION

This application is a continuation-in-part of U.S. Serial No. 09/654,462, filed September 1, 2000, which is a continuation-in-part of U.S. Serial No. 09/569,259, filed May 11, 2000, the contents of which are hereby incorporated by reference into the present application.

Throughout this application, various references are referred to within parentheses. Disclosures of these publications in their entireties are hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains. Full bibliographic citation for these references may be found at the end of this application, preceding the claims.

BACKGROUND OF THE INVENTION

During the development of the embryonic central nervous system (CNS) the mechanisms that specify regional identity and neuronal fate are intimately linked (Anderson et al . 1997; Lumsden and Krumlauf 1996; Rubenstein et al . 1998). In the ventral half of the CNS, for example, the secreted factor Sonic hedgehog (Shh) has a fundamental role in controlling both regional pattern and neuronal fate (Tanabe and Jessell 1996; Ericson et al . 19976; Hammerschmidth et al . 1997) . Shh appears to function as a gradient signal. In the spinal cord, five distinct classes of neurons can be generated in vitro in response to two- to threefold changes in the concentration of Shh, and the position at which each neuronal class is generated in vivo is predicted by the concentration required for their induction in vivo (Ericson et al . 1997a; Briscoe et al . 2000). Thus, neurons generated in more ventral regions of the neural tube require progressively higher concentrations of Shh for their induction.

The genetic programs activated in neural progenitor cells in response to Shh signaling, however, remain incompletely defined. Emerging evidence suggests that homeobox genes function as critical intermediaries in the neural response to Shh signals (Lumsden and Krumlauf 1996; Tanabe and Jessell 1996; Ericson et al . 1997; Hammerschmidt et al . 1997; Rubenstein et al . 1998) . Several homeobox genes are expressed by ventral progenitor cells, and their expression is regulated by Shh. Gain-of-function studies on homeobox gene action in the chick neural tube have provided evidence that homeodomain proteins are critical for the interpretation of graded Shh signaling and that they function to delineate progenitor domains and control neuronal subtype identity (Briscoe et al . 2000). Consistent with these findings, the pattern of generation of neuronal subtypes in the basal telencephalon and in the ventral-most region of the spinal cord is perturbed in mice carrying mutations in certain Shh-regulated homeobox genes (Ericson et al . 1997; Sussel et al . 1999; Pierani et al . , unpublished) .

Members of the Nkx class of homeobox genes are expressed by progenitor cells along the entire rostro-caudal axis of the ventral neural tube, and their expression is dependent on Shh signaling (Rubenstein and Beachy 1998) . Mutation in the Nkx2. 1 or Nkx2. 2 genes leads to defects in ventral neural pattering (Briscoe et al . 1999; Sussel et al . 1999), raising the possibility that Nkx genes play a key role in the control of ventral pattering in the ventral region of the CNS. Genetic studies to assess the role of Nkx genes have, however, focused on only the most ventral region of the neural tube. A recently identified Nkx gene, Nkxβ . l , is expressed more widely by most progenitor cells within the ventral neural tube (Pabst et al . 1998; Qiu et al . 1998; Briscoe et al . 1999), suggesting that it may have a prominent role in ventral neural patterning. Here experiments show that in mouse embryos Nkxβ . l is expressed by ventral progenitors that give rise to motor (MN) , V2 , and V3 neurons. Mice carrying a null mutation of Nkxβ . l exhibit a ventral-to-dorsal switch in the identity of progenitor cells and a corresponding switch in the identity of the neuronal subtype that emerges from the ventral neural tube. The generation of MN and V2 neurons is markedly reduced, and there is a ventral expansion in the generation of a more dorsal VI neuronal subtype. Together, these findings indicate that Nkxβ . l has a critical role in the specification of MN and V2 neuron subtype identity and, more generally, that Nkx genes play a role in the interpretation of graded Shh signaling. SUMMARY OF THE INVENTION

This invention provides a method of converting a stem cell into a ventral neuron which comprises introducing into the stem cell a nucleic acid which expresses homeodomain transcription factor Nkx6.1 protein in the stem cell so as to thereby convert the stem cell into the ventral neuron.

This invention also provides a method of diagnosing a motor neuron degenerative disease in a subject which comprises: a) obtaining a nucleic acid sample from the subject; b) sequencing the nucleic acid sample; and c) comparing the nucleic acid sequence of step (b) with a Nkx6.1 nucleic acid sequence from a subject without motor neuron degenerative disease, wherein a difference in the nucleic acid sequence of step (b) from the Nkx6.1 nucleic acid sequence from the subject without motor neuron degenerative disease indicates that the subject has the motor neuron degenerative disease.

This invention provides a method of diagnosing a motor neuron degenerative disease in a subject which comprises: a) obtaining a nucleic acid sample from the subject; b) performing a restriction digest of the nucleic acid sample with a panel of restriction enzymes; c) separating the resulting nucleic acid fragments by size fractionation; d) hybridizing the resulting separated nucleic acid fragments with a nucleic acid probe (s) of at least 15 nucleotide capable of specifically hybridizing with a unique sequence included within the sequence of a nucleic acid molecule encoding a human Nkx6.1 protein, wherein the sequence of the nucleic acid probe is labeled with a detectable marker, and hybridization of the nucleic acid probe (s) with the separated nucleic acid fragments results in labeled probe- fragment bands; e) detecting labeled probe-fragment bands, wherein the labeled probe-fragment bands have a band pattern specific to the nucleic acid of the subject; and f) comparing the band pattern of the detected labeled probe- fragment bands of step (d) with a previously determined control sample, wherein the control sample has a unique band pattern specific to the nucleic acid of a subject having the motor neuron degenerative disease, wherein identity of the band pattern of the detected labeled probe- fragment bands of step (d) to the control sample indicates that the subject has the motor neuron degenerative disease.

This invention provides a method of treating neuronal degeneration in a subject which comprises implanting in diseased neural tissue of the subject a neural stem cell which comprises an isolated nucleic acid molecule which is capable of expressing homeodomain Nkx6.1 protein under conditions such that the stem cell is converted into a motor neuron after implantation, thereby treating neuronal degeneration in the subject.

This invention provides a method of converting a stem cell into a ventral neuron which comprises introducing into the stem cell a nucleic acid which expresses homeodomain transcription factor Nkx6.2 protein in the stem cell so as to thereby convert the stem cell into the ventral neuron.

This invention provides a method of converting a stem cell into a ventral neuron which comprises introducing into the stem cell a polypeptide which expresses homeodomain transcription factor Nkx6.1 in the stem cell so as to thereby convert the stem cell into the ventral neuron.

This invention provides a method of converting a stem cell into a ventral neuron which comprises introducing into the stem cell a polypeptide which expresses homeodomain transcription factor Nkx6.2 in the stem cell so as to thereby convert the stem cell into the ventral neuron.

This invention provides a method of diagnosing a neurodegenerative disease in a subject which comprises: a) obtaining a suitable sample from the subject; b) extracting nucleic acid from the suitable sample; c) contacting the resulting nucleic acid with a nucleic acid probe, which nucleic acid probe (i) is capable of hybridizing with the nucleic acid of Nkx6.1 or Nkx6.2 and

(ii) is labeled with a detectable marker; d) removing unbound labeled nucleic acid probe; and e) detecting the presence of labeled nucleic acid, wherein the presence of labeled nucleic acid indicates that the subject is afflicted with a chronic neurodegenerative disease, thereby diagnosing a chronic neurodegenerative disease in the subject. BRIEF DESCRIPTION OF THE FIGURES

Figures 1A-1U

Selective changes in homeobox gene expression in ventral progenitor cells in Nkxβ . l mutant embryos. (Figs. 1A-1C) Expression of Nkxβ. l in transverse sections of the ventral neural tube of mouse embryos E9.5. (Fig. 1A) Expression of Nkxβ . l is prominent in ventral progenitor cells and persists in some post-mitotic motor neurons at both caudal hindbrain, E10.5, (Fig. IB) and spinal cord, E12.5, (Fig. 1C) levels. (Fig. ID, and IE) Summary diagrams showing domains of homeobox gene expression in wild-type mouse embryos (Fig. ID) and the change in pattern of expression of these genes in Nkxβ . l mutants (Fig. IE), based on analyses at E10.0 - E12.5. (Figs. 1F-1I) Comparison of the domains of expression of Nkxβ . l (Figs. IF, 1J) Dbx2 (Figs.

1G, 1H, IK, IL) and Gshl (Figs. II, 1M) in the caudal neural tube of wild-type (Figs. 1F-1I) and Nkxβ . l mutant

(Figs. 1J-1H) embryos. (Fig. 1J) Horizontal lines, approximate position of dorsoventral boundary of the neural tube; vertical lines, expression of Dbx2 and Gshl .

Expression of Sonic hedgehog, Shh (Figs. IN, IR) , Pax7

(Figs. IN, IR) , Nkx2.2 (Figs. 10, IS), Paxβ (Figs. IP, IS),

Dbxl (Figs. IP, IT) and Nkx2. 9 (Figs. 1Q, 1U) in wild-type (Figs. 1N-1Q) or Nkxβ . l mutant (Figs. 1R-1U) embryos at spinal (Figs 1N-1P, 1R-1T) and caudal hindbrain levels (Figs 1Q, 1U) . Arrowheads, approximate position of the dorsal limit of Nkxβ . l expression. Scale bar shown in J= lOOμm (Figs. 1A-1C) ; 50μm (Figs. 1F-1M) or 60μm (Figs. 1N- 1U) . Ficfure 2A-2T

Disruption of motor neuron differentiation in Nkxβ . l mutant embryos. The relationship between the domain of Nkxβ . l expression (Figs. 2A-2C, green) by ventral progenitors and the position of generation of motor neurons and V2 interneurons (Figs. 2A-2D) in the ventral spinal cord of E10.5 wild-type embryos. (Fig. 2A) Isll/2 motor neurons; (Fig. 2B) HB9 motor neurons; (Fig. 2C) Lhx3 (Lim3) expression (red) by motor neurons, V2 interneurons and their progenitors is confined to the Nkxβ . l progenitor domain. (Fig. 2D) Chxl O (green) V2 interneurons coexpress Lhx3 (red). Expression of Isll/2 (Figs. 2E, 21), HB9 (Figs. 2F, 2J) , Lhx3 (Figs. 2G, 2K) and Phox2a/b (Figs. 2H, 2L) in the ventral spinal cord (Figs. 2E, 2F, 2G) and caudal hindbrain (Fig. 2H) of E10.5 wild-type (Figs. 2E-2H) and Nkxβ . l mutant (Figs. 2I-2L) embryos. Pattern of expression of Isll/2 and Lhx3 at cervical (Figs. 2M, 2N, 2Q, 2R) and thoracic (Figs. 20, 2P, 2S, 2T) levels of E12.5 wild-type (Figs. 2M-2P) and Nkx6.1 mutant (Figs. 2Q-2T) embryos. Arrows, position of Isll dorsal D2 interneurons. (Figs. 10Q-10T) Absence, position of Isll/2 dorsal D2 interneurons. Scale bar shown in I = 60μm (Figs. 2A-2D) ; 80μm (Figs. 2E-2L) ; 120μm (Figs. 2M-2T) .

Figures 3A-3J

Motor neuron subtype differentiation in Nkxβ . l mutant mice.

Depletion of both median motor column (MMC) and lateral motor column (LMC) neurons in Nkxβ . l mutant mice.

Expression of Isll/2 (red) and Lxh3 (green) in Ξ12.5 wilt- type (Figs. 3A, 3C) and Nkxβ . l mutant (Figs. 3B,3D) mice spinal cord at forelimb levels (Figs. 3E-3J) . Motor neuron generation at caudal hindbrain level (Figs. 3E, 3F) Nkxβ . l expression in progenitor cells and .visceral motor neurons in the caudal hindbrain (rhombomere [r] 7/8) of E10.5-E11 wild- type (Fig. 3E) Nkxβ. l mutant (Fig. 3F) mice. HB9 expression in hypoglossal motor neurons in E10.5-E11 wild- type mice (Fig. 3G) and Nkxβ. l mutant (Fig. 3H) mice. Coexpression of Isll (green) and Phox2a/b (red) in wild- type (Fig. 31) or Nkxβ . l mutant (Fig. 3J) mice. (h) hypoglossal motor neurons; (v) visceral vagal motor neurons. Scale bar shown in C = 50μm (Figs. 3A-3D) or 70μm (Figs. 3E-3J) .

Figures 4A-4L

A switch in ventral interneuron fates in Nkxβ . l mutant mice. Chxl ϋ expression in V2 neurons at rostral cervical levels of E10.5 wild-type (Fig. 4A) and Nkxβ . l mutant (Fig.

4B) embryos. Enl expression by VI neurons at rostral cervical levels of wild-type (Fig. 4C) and Nkxβ . l mutant

(Fig. 4D) embryos. Pax2 expression in a set of interneurons that includes VI neurons ( (Burrill et al . 1997) at caudal hindbrain levels of wild-type (Fig. 4E) and Nkxβ . l mutant (Fig. 4F) embryos. (Figs. 4G and 4H) Siml expression by V3 neurons in the cervical spinal cord of wild-type (Fig. 4G) and Nkxβ. l mutant (Fig. 4H) embryos. Evxl expression by V0 neurons at caudal hindbrain levels of wild-type (Fig. 41) and Nkxβ . l mutant (Fig. 4J) embryos. Enl (red) and Lhx3 (green) expression by separate cell populations in the ventral spinal cord of Ell wild-type (Fig. 4K) and Nkxβ . l mutant (Fig. 4L) embryos. Scale bar shown in B = 60μm (Figs. 4A-4D) ; 75μm (Figs. 4E, 4F) ; 70μm (Figs. 4G, 4J, 4H,

4J) , 35μm (Figs. 4K and 4L) . Fiqure 5A- 5B

Changes in progenitor domain identity and neuronal fate in the spinal cord of Nkxβ . l mutant embryos. (Fig. 5A) . In wild-type mouse embryos, cells in the Nkxβ . l progenitor domain give rise to three classes of ventral neurons : V2 neurons , motor neurons (MN) and V3 neurons . V3 neurons derive from cells in the ventral most region of Nkxβ . l expression that also express Nkx2. 2 and Nkx2. 9. VI neurons derive from progenitor cells that express Dbx2 but not Nkxβ . l . (Fig. 5B) . In Nkxβ . l mutant embryos the domain of Dbx2 expression by progenitor cells expands ventrally, and by embyonic day 12 [E12] occupies the entire dorsoventral extent of the ventral neural tube, excluding the floor plate . Checked area indicates the gradual onset of ventral Dbx2 expression. This ventral shift in Dbx2 expression is associated with a marked decrease in the generation of V2 neurons and motor neurons and a ventral expansion in the domain of generation of VI neurons. A virtually complete loss of MN and V2 neurons is observed at cervical levels of the spinal cord. The generation of V3 neurons (and cranial visceral motor neurons at hindbrain levels) is unaffected by the loss of Nkxβ . l or by the ectopic expression of Dbx2.

Figure 6 Human Homeobox Protein Nkx6.1. NCBI Accession No. P78426. (Inoue, H. et al . , "Isolation, characterization, and chromosomal mapping of the human Nkx6.1 gene (NKX6a) , a new pancreatic islet homeobox gene" Genomics 40 (2) : 367-370 , 1997) . Amino acid sequence of human homeobox protein Nkx6.1. Figure 7

Human NK Homeobox Protein (Nkx6.1) gene, exon 1. NCBI Accession No. U66797. Segment 1 of 3 (Inoue, H. et al . , "Isolation, character-ization, and chromosomal mapping of the human Nkx6.1 gene (NKX6a) , a new pancreatic islet homeobox gene" Genomics 40 (2) .-367-370, 1997). Nucleic acid sequence encoding human homeobox protein Nkx6.1, bases 1- 682.

Figure 8

Human NK Homeobox Protein (Nkx6.1) gene, exon 2. NCBI Accession No. U66798. Segment 2 of 3 (Inoue, H. et al . , "Isolation, character-ization, and chromosomal mapping of the human Nkx6.1 gene (NKX6a) , a new pancreatic islet homeobox gene" Genomics 40 (2) : 367-370 , 1997). Nucleic acid sequence encoding human homeobox protein Nkx6.1 , bases 1- 185.

Figure 9 Human NK Homeobox Protein (Nkx6.1) gene, exon 3 and complete eds. NCBI Accession No. U66799. Segment 3 of 3 (Inoue, H. et al . , "Isolation, character-ization, and chromosomal mapping of the human Nkx6.1 gene (NKX6a) , a new pancreatic islet homeobox gene" Genomics 40 (2) :367-370 , 1997) . Nucleic acid sequence encoding human homeobox protein Nkx6.1, bases 1-273. Protein encoded is shown in Fig. 7.

Figure 10 Expression of Nkx6.2 and Nkx6.1 in developing mouse and chick spinal cord. (A) At e8.5, Nkx6.2 and Nkx6.1 are expressed in a broad ventral domain of the mouse neural tube. (B) At e9.0, Nkx6.2 expression is largely confined to a narrow domain immediately dorsal to the domain of Nkx6.1 expression. A few scattered cells that co-express Nkx6.2 and Nkx6.1 are detected in more ventral positions at this stage. (C) At e9.5, Nkx6.2 is expressed in a narrow domain, dorsal to the Nkx6.1 boundary. (D-G) Comparative patterns of expression of Nkx6.2, Nkx6.1, Dbx2 , Dbxl and Pax7 in the intermediate region of el0.5 mouse spinal cord. (H-L) Expression pattern of Nkxβ . 2 , Nkxβ . l , Dbx2, Dbxl and Pax7 in HH stage 20 chick spinal cord. Panels on right indicate progenitor domains, defined according to Briscoe et al . , 2000.

Figure 11

Elevation in Nkxβ . 2 and Dbx2 expression in pi domain cells in Nkxβ . 2 mouse mutants. (A) Diagram of the targeting construct (i) used to replace the coding sequence of Nkxβ . 2 (ii) with a tau-lacZ PGK-neo cassette (iii) . Red bar indicates region used as probe in genotyping. (B-D) Sagital view of el0.5 spinal cord showing LacZ expression, detected by X-gal staining, in wild type (wt) (B) Nkx . 2^+/tlz (C) and Nkxβ . 2^{tlz tlz} (D) embryos. (E-G) Nkxδ .2 and LacZ expression in the pi domain of wt (E) , Nkx6. 2^+/tlz (F) , and Nkxβ . 2^{tlz tlz} (G) embryos at el0.5. (H-J) In situ hybridization with a 5'-UTR probe shows that expression of Nkxβ .2 is elevated in the pi domain of Nkxβ .2^tlz/tlz embryos (J) , compared with wt (H) or Nkxβ . 2*^/tlz (I) embryos. (K-M) Expression of Dbx2 is up regulated -2- fold in cells within the pi domain (yellow bracket) in Nkxβ .2^tlz/tlz embryos (M) , compared with wt (K) , or Nkxβ . 2^{+ tlz} (L) embryos. Abbreviations in (A) : H= Hindlll, B= BamHI , N= Ncol , S= Sphl , A=AccI .

Figure 12

A partial switch from VI to VO neuronal fate in Nkxβ . 2 mutant mice. (A-E) Expression of Nkx6.2 (A), Nkx6.1 (C, D) , Dbxl (B, C, E) , and Pax7 (B) appears normal at caudal hindbrain levels of elθ.5 Nkxβ . 2^+/tlz embryos. The expression of Nkx6.1 (D) and Dbxl (E) abuts the ventral and dorsal boundaries of LacZ expression. (F-J) In elθ.5 Nkxβ . 2^tlz/tlz embryos, expression of Nkx6.1 (H, I) and Pax7

(G) is unchanged but expression of Dbxl (F, G, H) is expanded ventrally into the pi domain. Many ventral ectopic

Dbxl⁺ cells in Nkx . 2^tlz/tlz embryos express LacZ (J) . (K-M)

Evxl/2⁺ VO neurons are generated dorsal to Enl⁺ VI neurons (K) and LacZ⁺ cells (M) in Nkxβ . 2^+/tlz embryos. Enl⁺ neurons express LacZ in Nkxβ . 2^+/tlz (L) and Nkxβ . 2^tlz/tlz (0) embryos.

(N-P) Evxl/2⁺ VO neurons are generated in increased numbers and at ectopic ventral positions in the caudal hindbrain of

Nkxβ . 2^tlz/tlz embryos. (N) The number of Enl⁺ VI neurons is reduced and the remaining Enl⁺ neurons are intermingled with ectopic Evxl/2⁺ cells. (P) Many Evxl/2⁺ neurons in Nkxβ . 2^tlz/tlz embryos co-express LacZ. (Q) Quantitation of Evxl/2⁺ VO, and Enl⁺ VI, neurons at the caudal hindbrain of Nkxβ . 2*^/tlz and Nkxβ .2^tlz/tlz embryos at elθ.5. Counts from 12 sections, mean + S.D. In panels (A-P) , the white arrowhead indicates the pO/pl boundary.

Figure 13

Deregulated expression of Nkx6.2 in Nkxβ. l mutant mice, and similar patterning activities of Nkx6 proteins in chick neural tube. (A) In elθ.5 wt embryos, Nkx6.2 expression is confined to the pi progenitor domain. (B) In Nkxβ . 1⁺ - embryos, scattered Nkx6.2⁺ cells are detected in the p2 , pMN and p3 domains. (C) In Nkx6. l^'embryos, Nkx6.2 is expressed in most progenitors in the p2 , pMN and p3 domains. (D-F) Misexpression of Nkx6.2 at high levels represses the expression of Dbxl (D) and Dbx2 (E) , but not Pax7 (F) . (G-P) Expression of Nkx6.2 in dorsal positions of the chick neural tube result in ectopic dorsal generation of motor neurons, as indicated by ectopic induction of Lim3 and HB9 expression (G-I, L-N) . Forced expression of Nkx6.2 at high levels in the pO and pi progenitor domains promotes the ectopic generation of Chxl0⁺ V2 neurons (J, K, O, P) and suppresses Evxl/2⁺ VO (K, P) and Enl⁺ VI (J, 0) neurons.

Figure 14

The deregulated expression of Nkx6.2 underlies motor neuron generation in Nkxβ . l mutants. (A) In elθ.5 wt embryos, Nkx6.2 expression is confined to the pi domain and Nkx6.1 is expressed in the p2 , pMN and p3 domains. (B) No change in the expression of Nkx6.1 is detected in Nkxβ . 2 ^tlz/tlz embryos. (C, D) In Nkxβ . l-^/- and Nkxβ . l-^/- ; Nkxβ . 2^{+ tlz} embryos, Nkx6.2 expression is derepressed in the p2 , pMN and p3 domains. (E) No expression of Nkx6.2 or Nkx6.1 protein is detected in Nkxβ . l-^/-; Nkxβ . 2^{tlz tlz} embryos. (F, G) HB9⁺, Isll/2⁺ motor neurons are generated in normal numbers in Nkxβ . 2^tlz/tlz embryos. The number of motor neurons is reduced by -60% in Nkxβ . I'^/' embryos (H) , by -80% in Nkxβ . 1^' /- ;Nkx6.2^+/tlz embryos (I) and by >90% in Nkxβ . r^/- ; Nkxβ . 2^{tlz tlz} at cervical levels of elθ.5 spinal cord (J) . (K-M) At el2, the number of motor neurons of medial (MMC) (Isll⁺, Lim3⁺) and lateral (LMC) (Isll⁺) subtype identity is reduced in similar proportions in Nkxβ. l-^/- and Nkxβ.2 ^_"; Nkxβ . 2 ^tlz/tlz embryos. Lim3⁺ V2 neurons are missing in Nkxβ . l-/- embryos and Nkxβ . l-^/- ; Nkxβ . 2^{tlz tlz} embryos at this stage. (N-P) Quantitation of HB9⁺ and Isll/2⁺ motor neurons at cervical and lumbar levels in wt, Nkxβ . 2 and Nkxβ . l single mutants and in Nkxβ . 2; Nkxβ . l compound mutants at elO and el2. Counts from 12 sections, mean + S.D.

Figure 15 Changes in class I protein expression and ventral interneuron generation in Nkxβ mutants. (A-E) Expression of Nkx6.1 and Nkx6.2 in the spinal cord in different Nkxβ mutant backgrounds at elθ.5. (F-J) Spatial patterns of Pax7 and Dbx2 expression in different Nkxβ mutant backgrounds. Note that the level of Dbx2 expression in the pMN domain of Nkxε.l-/-; Nkx6.2^{+ tlz} is very low, implying the existence of a pMN domain restricted gene that has the capacity to repress Dbx2 expression. Recent studies have provided evidence that the bHLH protein 0lig2 possesses these properties (Novitch et al . , 2001).

(K-0) Spatial patterns of expression of Pax7 and Dbxl in different Nkxβ mutant backgrounds. (P-T) Spatial patterns of generation of Evxl/2⁺ V0 neurons and Enl⁺ VI neurons in different Nkxβ mutant backgrounds. (Q) The generation of V0 neurons expands ventrally into the pi domain in Nkxβ . 2^tlz/tlz mutants at caudal spinal levels. (R, ') The number of Enl⁺ VI neurons increases ~3-fold in the ventral spinal cord of Nkxβ . 1-/"mutants, and ectopic Evxl/2⁺ cells are detected in position of the pMN domain in these mice (see also Sander et al . , 2000) . (S, T A") There is a progressive increase in Evxl/2⁺ V0 neurons and a loss of Enl⁺ VI neurons in the ventral spinal cord of Nkxβ .2"^_; Nkxβ .2 ^{+ t2z} and Nkxβ . l -/- ;Nkxβ . 2^{tlz tlz} embryos. (U,V,Z) The generation of Evxl/2* VO neurons correlates with the pattern of expression of Dbxl in progenitors in wt, Nkxβ . 2^{tlz tlz} and Nkxβ .2^{- _} _/Nkxβ . 2^tlz/tlz mutant backgrounds. Note that only the most lateral progenitor cells express Dbxl in Nkx6.1^{_ ~}; Nkx6.2^{tlz tlz} embryos, suggesting that expression of Dbxl in more medially-positioned progenitors is repressed by an as yet undefined gene. (X, Y) Ectopic ventral Evxl⁺ VO neurons derive from Dbxl^" progenitors in Nkxβ . l-/- and Nkxβ . l'/- ;Nkxβ . 2^+/tlz mutant embryos. Chxl0⁺ V2 neurons are generated at normal numbers in Nkxβ . 2^tlz/tlz mutants, but are missing at spinal cord levels in Nkxβ . l-^/- , Nkxβ . I'/^' ; Nkxβ . 2^+/tlz and Nkxβ . l-^/- ; Nkxβ . 2^tlz/tlz mutants ( '; Figure 5, see Sander et al. , 2000) .

Figure 16

Dissociation of Dbx expression and V0 neuronal fate in mice with reduced Nkx6 protein activity. (A) In elO.O wt embryos, pO progenitor cells express Dbxl and generate

Evxl/2⁺ V0 neurons. (B) In elO .0 Nkxβ .2^"A; Nkxβ . 2* ^tlz embryos there is no change in the domain of expression of Dbxl, but Evxl/2⁺ V0 neurons are generated in lateral positions, along much of the ventral neural tube. (C, D) In Nkxδ.l-/-; Nkx6.2^+/tlz embryos examined at elO.O many ectopic ventral Evxl/2⁺ neurons express LacZ. Framed area in (C) is shown at high magnification in (D) and indicates Evxl/2⁺ neurons that coexpress LacZ. (E) Evxl/2⁺ neurons located at the level of the pMN domain (bracket) derive from progenitors that express low or negligible levels of Dbx2 mRNA. (F) Summary of Dbxl expression and V0 neuron generation in wt, Nkxβ . 1^'/' ; Nkxβ . 2 ^{+ tI}&nd Nkxβ . l ;Nkxβ . 2^tlz/tlz embryos. The dissociation of Dbxl and Evxl/2 expression in Nkxβ .2^_-_/ Nkxβ .2^{+ l2} embryo suggests that reduced Nkx6 repressor activity is sufficient to repress Dbxl but insufficient to repress Evxl expression.

Figure 17

Genetic interactions between Nkx6 and Dbx proteins during the assignment of motor neuron and interneuron fate in the mouse neural tube. (A) Summary of domains of expression of Nkx6.1 (6.1), Nkx6.2 (6.2), Dbxl (Dl) and Dbx2 (D2) in the ventral neural tube of wild type (wt) and different Nkxβ mutant embryos. (B) Regulatory interactions between Nkx and Dbx proteins in the ventral neural tube. These interactions result in different levels of Nkx6 protein activity in distinct ventral progenitor domains, and thus promote the generation of distinct neuronal subtypes. For details see text.

Figure 18

Human NK Homeobox Protein (Nkx6.2) gene, complete eds. NCBI Accession No. AF184215.

Figure 19

Human Homeobox Protein Nkx6.2. NCBI Accession No. AAK13251. Amino acid sequence of human homeobox protein Nkx6.2.

Figure 20 Comparison of Amino Acid Sequences of Nkx6.2 Protein of Various Species with Other Nkx Protein Sequences . mNk6.3 == mouse amino acid sequence of Nkx6.3 protein; rNkx6.1 = rat amino acid sequence of Nkx6.1 protein; mNkx6.2 = mouse amino acid sequence of Nkx6.2 protein; and cNkx6.2 = chick amino acid sequence of Nkx6.2 protein.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the following standard abbreviations are used throughout the specification to indicate specific amino acids :

A=ala=alanine R=arg=arginine

N=asn=asparagine D=asp=aspartic acid

C=cys=cysteine Q=gln=glutamine

E=glu=glutamic acid G=gly=glycine

H=his=histidine I=ile=isoleucine

L=leu=leucine K=lys=lysine

M=met=methionine F=phe=phenylalanine

P=pro=proline S=ser=serine

T=thr=threonine =trp=tryptophan

Y=tyr=tyrosine V=val=valine

B=asx=asparagine or aspartic acid

Z=glx=glutamine or glut;amic acid

As used herein, the following standard abbreviations are used throughout the specification to indicate specific nucleotides: C=cytosine; A=adenosine; T=thymidine; G=guanosine; and U=uracil .

This invention provides a method of converting a stem cell into a ventral neuron which comprises introducing into the stem cell a nucleic acid which expresses homeodomain transcription factor Nkx6.1 protein in the stem cell so as to thereby convert the stem cell into the ventral neuron.

In an embodiment of the above-described method of converting a stem cell into a ventral neuron, the nucleic acid introduced into the stem cell incorporates into the chromosomal DNA of the stem cell . In a further embodiment of the method, the nucleic acid is introduced by transfection or transduction. In another further embodiment of the method, the ventral neuron is a motor neuron, a V2 neuron or a V3 neuron.

As used herein, the term "nucleic acid" refers to either DNA or RNA, including complementary DNA (cDNA) , genomic DNA and messenger RNA (mRNA) . As used herein, "genomic" means both coding and non-coding regions of the isolated nucleic acid molecule. "Nucleic acid sequence" refers to a single- or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5' to the 3' end. It includes both replicating vectors, infectious polymers of DNA or RNA and nonfunctional DNA or RNA.

The nucleic acids of the subject invention also include nucleic acids coding for polypeptide analogs, fragments or derivatives which differ from the naturally-occurring forms in terms of the identity of one or more amino acid residues (deletion analogs containing less than all of the specified residues; substitution analogs wherein one or more residues are replaced by one or more residues; and addition analogs, wherein one or more resides are added to a terminal or medial portion of the polypeptide) which share some or all of the properties of the naturally-occurring forms .

The nucleic acid sequences include both the DNA strand sequence that is transcribed into RNA, the complementary

DNA strand, and the RNA sequence that is translated into protein. The nucleic acid includes both the full length nucleic acid sequence as well as non-full length sequences. It being further understood that the sequence includes the degenerate codons of the native sequence or sequences which may be introduced to provide codon preference in a specific host cell .

As used herein, "protein", "peptide" and "polypeptide" are used to denote two or more amino acids linked by a peptidic bond between the α-carboxyl group of one amino acid and the -amino group of the next amino acid. Peptide includes not only the full-length protein, but also partial -length fragments . Peptides may be produced by solid-phase synthetic methods that are well-known to those skilled in the art . In addition to the above set of twenty-two amino acids that are used for protein synthesis in vivo, peptides may contain additional amino acids, including but not limited to hydroxyproline, sarcosine, and γ~ carboxyglutamate . The peptides may contain modifying groups including but not limited to sulfate and phosphate moieties. Peptides can be comprised of L- or D-amino acids, which are mirror-image forms with differing optical properties. Peptides containing D-amino acids have the advantage of being less susceptible to proteolysis in vivo.

Peptides may by synthesized in monomeric linear form, cyclized form or as oligomers such as branched multiple antigen peptide (MAP) dendrimers (Tarn et al . Biopolymers 51:311, 1999). Nonlinear peptides may have increased binding affinity by virtue of their restricted conformations and/or oligomeric nature. Peptides may also be produced using recombinant methods as either isolated peptides or as a portion of a larger fusion protein that contains additional amino acid sequences.

Peptides may be chemically conjugated to proteins by a variety of well-known methods. Such peptide-protein conjugates can be formulated with a suitable adjuvant and administered parenterally for the purposes of generating polyclonal and monoclonal antibodies to the peptides of interest. Alternatively, unconjugated peptides can be formulated with adjuvant and administered to laboratory animals for the purposes of generating antibodies. Methods for generating and isolating such antibodies are well-known to those skilled in the art.

The nucleic acids of the subject invention include but are not limited to DNA, RNA, mRNA, synthetic DNA, genomic DNA, and cDNA.

The nucleic acid sequence of the Nkx6.2 gene for various species may be found under the following NCBI Accession Nos.: human: AF184215; N55046; N50716N; H49739; H46204; H18874; mouse: BB449783; AV331479; BB358883; BB355466; L08074; and D .melanogaster : AF220236.

The amino acid sequence of the Nkx6.2 protein for various species may be found under the following NCBI Accession Nos.: AAK13251; MXKN2 ; MXKN1 ; S35304; T28492; AAF33780; P01524; P01523; 9GSSB; 17GSB; 1BH5D; 4GSSB; 1PGTB; 1GSUB; 1GN B; 2GLRB; 1AGSB . As used herein, the term "introducing into a cell" includes but is not limited to transduction and transfection. Transfection can be achieved by calcium phosphate co-precipitates, conventional mechanical procedures such as micro-injection, electroporation, insertion of a plasmid encased in liposomes, or virus vectors or any other method known to one skilled in the art. This invention provides an antibody produced by the above method.

This invention provides a method of diagnosing a motor neuron degenerative disease in a subject which comprises: a) obtaining a nucleic acid sample from the subject; b) sequencing the nucleic acid sample; and c) comparing the nucleic acid sequence of step (b) with a Nkx6.1 nucleic acid sequence from a subject without motor neuron degenerative disease, wherein a difference in the nucleic acid sequence of step (b) from the Nkx6.1 nucleic acid sequence from the subject without motor neuron degenerative disease indicates that the subject has the motor neuron degenerative disease.

In an embodiment of the above-described method of diagnosing a motor neuron degenerative disease in a subject the motor neuron degenerative disease is amyotrophic lateral sclerosis or spinal muscular atrophy.

As used herein, the term "sample" includes but is not limited to tonsil tissue, lymph nodes, spleen, skin lesions, blood, serum, plasma, cerebrospinal fluid, lymphocytes, urine, transudates, exudates, bone marrow cells, or supernatant from a cell culture. As used herein, "subject" means any animal or artificially modified animal. Artificially modified animals include, but are not limited to, SCID mice with human immune systems. The subjects include but are not limited to mice, rats, dogs, guinea pigs, ferrets, rabbits, chicken and primates. In the preferred embodiment, the subject is a human being.

This invention provides a method of diagnosing a motor neuron degenerative disease in a subject which comprises: a) obtaining a nucleic acid sample from the subject; b) performing a restriction digest of the nucleic acid sample with a panel of restriction enzymes; c) separating the resulting nucleic acid fragments by size fractionation; d) hybridizing the resulting separated nucleic acid fragments with a nucleic acid probe (s) of at least 15 nucleotide capable of specifically hybridizing with a unique sequence included within the sequence of a nucleic acid molecule encoding a human Nkx6.1 protein, wherein the sequence of the nucleic acid probe is labeled with a detectable marker, and hybridization of the nucleic acid probe (s) with the separated nucleic acid fragments results in labeled probe- fragment bands; e) detecting labeled probe-fragment bands, wherein the labeled probe-fragment bands have a band pattern specific to the nucleic acid of the subject; and f) comparing the band pattern of the detected labeled probe- fragment bands of step (d) with a previously determined control sample, wherein the control sample has a unique band pattern specific to the nucleic acid of a subject having the motor neuron degenerative disease, wherein identity of the band pattern of the detected labeled probe- fragment bands of step (d) to the control sample indicates that the subject has the motor neuron degenerative disease.

In an embodiment of the above-described method of diagnosing a motor neuron degenerative disease in a subject the nucleic acid is DNA. In a further embodiment of the above-described method the nucleic acid is RNA. In another embodiment the size fractionation in step (c) is effected by a polyacrylamide or agarose gel . In another embodiment the detectable marker is radioactive isotope, enzyme, dye, biotin, a fluorescent label or a chemiluminescent label. In yet another embodiment the motor neuron degenerative disease is amyotrophic lateral sclerosis or spinal muscular atrophy.

As used herein, "detectable marker" includes but is not limited to a radioactive label, or a calorimetric, a luminescent, or a fluorescent marker. As used herein, "labels" include radioactive isotopes, fluorescent groups and affinity moieties such as biotin that facilitate detection of the labeled peptide. Other labels and methods for attaching labels to compounds are well-known to those skilled in the art.

The phrase "specifically hybridizing" and the phrase "selectively hybridizing" describe a nucleic acid that hybridizes, duplexes or binds only to a particular target DNA or RNA sequence when the target sequences are present in a preparation of total cellular DNA or RNA. By selectively hybridizing it is meant that a nucleic acid binds to a given target in a manner that is detectable in a different manner from non-target sequence under high stringency conditions of hybridization. "Complementary", "antisense" or "target" nucleic acid sequences refer to those nucleic acid sequences which selectively and specifically hybridize to a nucleic acid. Proper annealing conditions depend, for example, upon a nucleic acid's length, base composition, and the number of mismatches and their position on the nucleic acid, and must often be determined empirically. For discussions of nucleic acid design and annealing conditions for hybridization, see, for example, Sambrook et al . (1989) Molecular Cloning: A Laboratory Manual (2nd ed. ) , Cold Spring Harbor Laboratory, Vols. 1-3 or Ausubel, F., et al . (1987) Current Protocols in Molecular Biology, New York. The above hybridizing nucleic acids may vary in length. The hybridizing nucleic acid length includes but is not limited to a nucleic acid of at least 15 nucleotides in length, of at least 25 nucleotides in length, or at least 50 nucleotides in length.

This invention provides a method of treating neuronal degeneration in a subject which comprises implanting in diseased neural tissue of the subject a neural stem cell which comprises an isolated nucleic acid molecule which is capable of expressing homeodomain Nkx6.1 protein under conditions such that the stem cell is converted into a motor neuron after implantation, thereby treating neuronal degeneration in the subject.

This invention provides a method of converting a stem cell into a ventral neuron which comprises introducing into the stem cell a nucleic acid which expresses homeodomain transcription factor Nkx6.2 protein in the stem cell so as to thereby convert the stem cell into the ventral neuron.

In one embodiment of the above method, the nucleic acid introduced into the stem cell incorporates into the chromosomal DNA of the stem cell. In another embodiment of the above method, the nucleic acid is introduced by transfection or transduction. In a further embodiment of the above method, the ventral neuron is a motor neuron.

This invention provides a method of converting a stem cell into a ventral neuron which comprises introducing into the stem cell a polypeptide which expresses homeodomain transcription factor Nkx6.1 in the stem cell so as to thereby convert the stem cell into the ventral neuron. In one embodiment of the above method, the ventral neuron is a motor neuron, a V2 interneuron or a V3 interneuron.

This invention provides a method of converting a stem cell into a ventral neuron which comprises introducing into the stem cell a polypeptide which expresses homeodomain transcription factor Nkx6.2 in the stem cell so as to thereby convert the stem cell into the ventral neuron. In one embodiment of the above method, the ventral neuron is a motor neuron.

This invention provides a method of diagnosing a neurodegenerative disease in a subject which comprises: a) obtaining a suitable sample from the subject; b) extracting nucleic acid from the suitable sample; c) contacting the resulting nucleic acid with a nucleic acid probe, which nucleic acid probe (i) is capable of hybridizing with the nucleic acid of Nkx6.1 or Nkx6.2 and (ii) is labeled with a detectable marker; d) removing unbound labeled nucleic acid probe; and e) detecting the presence of labeled nucleic acid, wherein the presence of labeled nucleic acid indicates that the subject is afflicted with a chronic neurodegenerative disease, thereby diagnosing a chronic neurodegenerative disease in the subject .

In one embodiment of the above method, the suitable sample is spinal fluid. In another embodiment of the above method, the nucleic acid is DNA. In a further embodiment of the above method, the nucleic acid is RNA.

This invention will be better understood from the Experimental Details which follow. However, one skilled in the art will readily appreciate that the specific methods and results discussed are merely illustrative of the invention as described more fully in the claims which follow thereafter.

FIRST SERIES OF EXPERIMENTS

EXPERIMENTAL DETAILS

A. Materials and Methods

Generation of Nkx6.1 null mutation

A null mutation in Nkxβ . l was generated by using gene targeting in 129-strain ES cells by excising an 800-bp Notl fragment containing part of exon 1 and replacing it by a

PGK-neo cassette (Sander and German, unpubl . ) Mutants were born at Mendelian frequency and died soon after birth; they exhibited movements only upon tactile stimulation.

Immunocytochemistry and in situ hybridization

Localization of mRNA was performed by in situ hybridization following the method of Schaeren- iemers and Gerfin-Moser

(1993) . The Dbx2 riboprobe comprised the 5' EcoRl fragment of the mouse cDNA (Pierani et al . 1999) . Probes for other cDNAs were cited in the text and used as described therein. Protein expression was localized by indirect fluorescence immunocytochemistry or peroxidase immunocytochemistry (Briscoe et al . 1999; Ericson et al . 1997). Nkxβ . l was detected with a rabbit antiserum (Briscoe et al . 1999). Antisera against Shh, Pax7, Isll/2, HB9, Lhx3 , ChxlO, Phox2a/b, Enl, and Pax2 have been described (Briscoe et al . 1999; Ericson et al . 1997). Fluorescence detection was carried out using an MRC 1024 Confocal Microscope (BioRad) . B . Results and Discussion

To define the role of Nkxβ . l in neural development, we compared patterns of neurogenesis in the embryonic spinal cord and hindbrain of wild-type mice and mice lacking Nkxβ . l (Sander et al . 1998). In wild-type embryos, neural expression of Nkxβ. l is first detected at spinal cord and caudal hindbrain levels at about embryonic day 8.5 (E8.5; Qiu et al . 1998; data not shown), and by E9.5 the gene is expressed throughout the ventral third of the neural tube (Figure 1A) . The expression of Nkxβ . l persists until at least E12.5 (Figures IB, 1C; data not shown). Nkxβ . l expression was also detected in mesodermal cells flanking the ventral spinal cord (Figures IB, 1C) . To define more precisely the domain of expression of Nkx6.1, we compared its expressions with that of ten homeobox genes - Pax3 , Pax7 , Gshl, Gsh2 , Irx3 , Paxβ, Dbxl , Dbxl, Dbx2 and Nkx2 . 9 - that have been shown to define discrete progenitor cell domains along the dorsoventral axis of the ventral neural tube (Goulding et al . 1991; Valerius et al . 1995; Ericson et al . 1997; Pierani et al . 1999; Briscoe et al . 2000).

This analysis revealed that the dorsal boundary of Nkxβ . l expression is positioned ventral to the boundaries of four genes expressed by dorsal progenitor cells: Pax3 , Pax7 , Gshl and Gsh2 (Figures II, IN; and data not shown) . Within the ventral neural tube, the dorsal boundary of Nkxβ . l expression is positioned ventral to the domain of Dbxl expression and close to the ventral boundary of Dbx2 expression (Figures IG, IH, and IP) . The domain of Paxβ expression extends ventrally into the domain of Nkxβ . l expression (Figure 10), whereas the expression of Nkx2 . 2 and Nkx2. 9 overlaps with the ventral-most domain of Nkxβ . l expression (Figures 10, 1Q) .

To address the function of Nkxβ. l in neural development, we analyzed progenitor cell identity and the pattern of neuronal differentiation in Nkxβ . l null mutant mice (Sander et al . 1998) . We detected a striking change in the profile of expression of three homeobox genes, Dbx2 , Gshl and Gsh2 , in Nkxβ . l mutants. The domains of expression of Dbx2, Gshl and Gsh2 each expanded into the ventral neural tube (Figures 1K-1M; data not shown). At E10.5, Dbx2 was expressed at high levels by progenitor cells adjacent to the floor plate, but at this stage ectopic Dbx2 expression was detected only at low levels in regions of the neural tube that generate motor neurons (Figure IK). By E12.5, however, the ectopic ventral expression of Dbx2 had become more uniform, and now clearly included the region of motor neuron and V2 neuron generation (Figure IL) . Similarly, in Nkxβ . l mutants, both Gshl and Gsh2 were ectopically expressed in a ventral domain of the neural tube, and also in adjacent paraxial mesodermal cells (Figure 1M; data not shown) .

The ventral limit of Pax6 expression was unaltered in Nkxβ . l mutants, although the most ventrally located cells within this progenitor domain expressed a higher level of Pax6 protein than those in wild-type embryos (Figures 10, IS) . We detected no change in the patterns of expression of Pax3 , Pax7, Dbxl , Irx3 , Nkx2 . 2 , or Nkx2. 9 in Nkxβ . l mutant embryos (Figures 1R-1U; data not shown) . Importantly, the level of Shh expression, by floor plate cells was unaltered in Nkxβ. l mutants (Figures IN and IR) . Thus, the loss of Nkxβ . l function deregulates the patterns of expression of a selected subset of homeobox genes in ventral progenitor cells, without an obvious effect on Shh levels (Figures ID, IE) . The role of Shh in excluding Dbx2 from the most ventral region of the neural tube (Pierani et al . 1999) appears therefore to be mediated through the induction of Nkx6.1 expression. Consistent with this view, ectopic expression of Nkxβ. l represses Dbx2 expression in chick neural tube (Briscoe et al . 2000) . The detection of sites of ectopic Gshl/2 expression in the paraxial mesoderm as well as the ventral neural tube, both sites of Nkxβ . l expression, suggests that Nkxβ . l has a general role in restricting Gshl/2 expression. The signals that promote ventral Gshl/2 expression in Nkxβ . l mutants remain unclear, but could involve factors other than Shh that are secreted by the notochord (Hebrok et al . 1998) .

The domain of expression of Nkxβ . l within the ventral neural tube of wild-type embryos encompasses the progenitors of three main neuronal classes: V2 interneurons, motor neurons and V3 interneurons (Goulding et al. 1991; Ericson et al . 1997; Qiu et al . 1998; Briscoe et a. 1999, 2000; Pierani et al . 1999; Figures 2A-2D) . We examined whether the generation of any of these neuronal classes is impaired in Nkxβ . l mutants, focusing first on the generation of motor neurons. In Nkxβ . l mutant embryos there was a marked reduction in the number of spinal motor neurons, as assessed by expression of the homeodomain proteins Lhx3 , Isll/2 and HB9 (Arber et al . 1999; Tsuchida et al. 1994; Figures 2E-2L) , and by expression of the gene encoding the transmitter synthetic enzyme choline acetyltransferase (data not shown) . In addition, few if any axons were observed to emerge from the ventral spinal cord (data not shown) . The incidence of motor neuron loss, however, varied along the rostrocaudal axis of the spinal cord. Few if any motor neurons were detected at caudal cervical and upper thoracic levels of Nkxβ . l mutants analyzed at E11-E12.5 (Figures 2M, 2N, 2Q, 2R) , whereas motor neuron number was reduced only by 50%-75% at more caudal levels (Figures 20, 2P, 2S, 2T; data not shown) . At all axial levels, the initial reduction in motor neuron number persisted at both E12.5 and pO (Figures 2M-2T; data not shown), indicating that the loss of Nkxβ . l activity does not simply delay motor neuron generation. Moreover, we detected no increase in the incidence of TUNEL⁺ cells in Nkxβ . l mutants (data not shown), providing evidence that the depletion of motor neurons does not result solely from apoptotic death.

The persistence of some spinal motor neurons in Nkxβ . l mutants raised the possibility that the generation of particular subclasses of motor neurons is selectively impaired. To address this issue, we monitored the expression of markers of distinct subtypes of motor neurons at both spinal and hindbrain levels of Nkxβ . l mutant embryos. At spinal levels, the extent of the reduction in the generation of motor neurons that populate the median (MMC) and lateral (LMC) motor columns was similar in Nkxβ. l mutants, as assessed by the number of motor neurons that coexpressed Isll/2 and Lhx3 (defining MMC neurons, Figures 3A, 3B) and by the expression of Raldh2 (defining LMC neurons, Sockanathan and Jessell 1998; Arber et al . 1999; Figures 3C, 3D) . In addition, the generation of autonomic visceral motor neurons was reduced to an extent similar to that .of somatic motor neurons at thoracic levels of the spinal cord of E12.5 embryos (data not shown) . Thus, the loss of Nkxβ . l activity depletes the major subclasses of spinal motor neurons to a similar extent .

At hindbrain levels, Nkxβ . l is expressed by the progenitors of both somatic and visceral motor neurons (Figures 3E, 3F; data not shown) . We therefore examined whether the loss of Nkxβ . l might selectively affect subsets of cranial motor neurons. We detected a virtually complete loss in the generation of hypoglossal and abducens somatic motor neurons in Nkxβ . l mutants, as assessed by the absence of dorsally generated HB9⁺ motor neurons (Figures 3G, 3H; data not shown, Arber et al . 1999; Briscoe et al . 1999). In contrast, there was no change in the initial generation of any of the cranial visceral motor neuron populations, assessed by coexpression of 2s22 and Phox2a (Briscoe et al . 1999; Pattyn et al . 1997) within ventrally generated motor neurons (Figures 31, 3J; data not shown) . Moroever, at rostral cervical levels, the generation of spinal accessory motor neurons (Ericson et al . 1997) was also preserved in Nkxβ . l mutants (data not shown). Thus, in the hindbrain the loss of Nkxβ . l activity selectively eliminates the generation of somatic motor neurons, while leaving visceral motor neurons intact. Cranial visceral motor neurons, unlike spinal visceral motor neurons, derive from progenitors that express the related Nkx genes Nkx2. 2 and Nkx2. 9 (Briscoe et al . 1999). The preservation of cranial visceral motor neurons in Nkxβ . l mutant embryos may therefore reflect the dominant activities of Nkx2. 2 and Nkx2. 9 within these progenitor cells.

We next examined whether the generation of ventral interneurons is affected by the loss of Nkxβ. l activity. V2 and V3 interneurons are defined, respectively, by expression of ChxlO and Siml (Arber et al . 1999; Briscoe et al . 1999; Figures 4A, 4G) . A severe loss of ChxlO V2 neurons was detected in Nkxβ . l mutants at spinal cord levels (Figure 4B) , although at hindbrain levels of Nkxβ . l mutants -50% of V2 neurons persisted (data not shown) . In contrast, there was no change in the generation of Siml V3 interneurons at any axial level of Nkxβ . l mutants (Figure 4H) . Thus, the elimination of Nkxβ . l activity affects the generation of only one of the two major classes of ventral interneurons that derive from the Nkx6.1 progenitor cell domain .

Evxl⁺, Pax2⁺ VI interneurons derive from progenitor cells located dorsal to the Nkxβ . l progenitor domain, (Figure 4B) within a domain that expresses Dbx2, but not Dbxl (Burrill et al. 1997; Matise and Joyner 1997; Pierani et al . 1999). Because Dbx2 expression undergoes a marked ventral expansion in Nkxβ . l mutants, we examined whether there might be a corresponding expansion in the domain of generation of VI neurons. In Nkxβ . l mutants, the region that normally gives rise to V2 neurons and motor neurons now also generated VI neurons, as assessed by the ventral shift in expression of the Enl and Pax2 homeodomain proteins (Figures 4B, 4C, 4E, 4F) . Consistent with this, there was a two- to threefold increase in the total number of VI neurons generated in Nkxβ . l mutants (Figures 4C, 4D) . In contrast, the domain of generation of Evxl/2 VO neurons, which derive from the Dbxl progenitor domain (Pierani et al . 1999), was unchanged in Nkxβ . l mutants (Figures 41, 4J) . Thus, the ventral expansion in Dbx2 expression is accompanied by a selective switch in interneuronal fates, from V2 neurons to VI neurons. In addition, we observed that some neurons within the ventral spinal cord of Nkxβ . l mutants coexpressed the VI marker Enl and the V2 marker Lhx3 (Figures 4K, 4L) . The coexpression of these markers is rarely if ever observed in single neurons in wild type embryos (Ericson et al . 1996). Thus, within individual neurons in Nkxβ . l mutants, the ectopic program of VI neurogenesis appears to be initiated in parallel with a residual, albeit transient, program of V2 neuron generation. This result complements observations in Hb9 mutant mice, in which the programs of V2 neuron and motor neuron generation coincide transiently within individual neurons (Arber et al . 1999; Thaler et al . 1999).

Taken together, the findings herein reveal an essential role for the Nkxβ . l homeobox gene in the specification of regional pattern and neuronal fate in the ventral half of the mammalian CNS. Within the broad ventral domain within which Nkxβ . l is expressed (Figure 5A) , its activity is required to promote motor neuron and V2 interneuron generation and to restrict the . generation of VI interneurons (Figure 5B) . It is likely that the loss of motor neurons and V2 neurons is a direct consequence of the loss of Nkxβ . l activity, as the depletion of these two neuronal subtypes is evident at stages when only low levels of Dbx2 are expressed ectopically in most regions of the ventral neural tube. Nonetheless, it can not be excluded that low levels of ectopic ventral Dbx2 expression could contribute to the block in motor neuron generation. Consistent with this view, the ectopic expression of Nkxβ. l is able to induce both motor neurons and V2 neurons in chick neural tube (Briscoe et al . 2000) . V3 interneurons and cranial visceral motor neurons derive from a set of

Nkxβ . l progenitors that also express Nkx2. 2 and Nkx2. 9

(Briscoe et al . 1999, Figure 5A) . The generation of these two neuronal subtypes is unaffected by the loss of Nkxβ . l activity, suggesting that the actions of Nkx2. 2 and Nkx2 . 9 dominate over that of Nkx6.1 within these progenitors. The persistence of some spinal motor neurons and V2 neurons in Nkxβ . l mutants could reflect the existence of a functional homologue within the caudal neural tube.

The role of Nkxβ. l revealed in these studies, taken together with previous findings, suggests a model in which the spatially restricted expression of Nkx genes within the ventral neural tube (Figure 5) has a pivotal role in defining the identity of ventral cell types induced in response to graded Shh signaling. Strikingly, in Drosophila, the Nkx gene NK2 has been shown to have an equivalent role in specifying neuronal fates in the ventral nerve cord (Chu et al . 1998; McDonald et al . 1998). Moreover, the ability of Nkxβ . l to function as a repressor of the dorsally expressed Gshl/2 homeobox genes parallels the ability of Drosophila NK2 to repress Ind, a Gshl/2-l k.e homeobox gene (Weiss et al . 1998) . Thus, the evolutionary origin of regional pattern along the dorsoventral axis of the central nervous system may predate the divergence of invertebrate and vertebrate organisms.

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Introduction

During the development of the vertebrate central nervous system, the assignment of regional identity to neural progenitor cells has a critical role in directing the subtype identity of post-mitotic neurons. Within the ventral half of the neural tube, the specification of progenitor cell identity is initiated by the long-range signalling activity of the secreted factor, Sonic hedgehog (Shh) (Briscoe et al . , 2001; Briscoe and Ericson, 2001). Shh signaling appears to establish ventral progenitor cell identities by regulating the spatial pattern of expression of homeodomain transcription factors of the Nkx, Pax, Dbx and Irx families (Ericson et al . , 1997; Pierani et al . , 1999; Briscoe et al . , 2000) . Members of all four gene families have been duplicated during evolution (Shoji et al., 1996; Wang et al . , 2000; Hoshiyama et al . , 1998, Peters et al . , 2001), and the resulting homeodomain protein pairs are typically expressed in overlapping or nested domains within the neural tube (Briscoe and Ericson, 2001) . Some of these homeodomain protein pairs have been proposed to have distinct, and others redundant, roles in spinal cord patterning (Mansouri and Gruss, 1998; Briscoe et al . , 1999; Pierani et al . , 2001), but the impact of such homeobox gene duplication on neuronal diversification has not been explored directly.

One unifying feature of this diverse array of progenitor homeodomain proteins is their subdivision into two general groups, termed class I and II proteins, on the basis of their mode of regulation by Shh signalling (Briscoe and Ericson, 2001) . The class I proteins are constitutively expressed by neural progenitor cells, and their expression is repressed by Shh signaling, whereas neural expression of the class II proteins requires exposure to Shh (Ericson et al . , 1997; Qiu et al . , 1998; Briscoe et al . , 1999; 2000; Pabst et al . , 2000) . Although the spatial pattern of expression of the class I proteins has revealed the existence of five ventral progenitor domains, class II proteins have been identified for only two of these domains

(Briscoe et al . , 2000), raising questions about the existence and identity of additional class II proteins.

There is, however, emerging evidence that the combination of class I and II proteins that is expressed by neural progenitor cells directs the fate of their neuronal progeny. In support of this, misexpression of individual progenitor homeodomain proteins in the chick neural tube promotes the ectopic generation of neuronal subtypes, with a specificity predicted by the normal profile of progenitor homeodomain protein expression (Briscoe et al . , 2000; Pierani et al . , 2001) . Conversely, the analysis of mouse mutants has provided genetic evidence that the activities of specific class I and II proteins are required to establish progenitor cell domains and to direct ventral neuronal fates (Ericson et al., 1997; Briscoe et al . , 1999; Sander et al . , 2000; Pierani et al . , 2001).

The participation of progenitor homeodomain proteins in the conversion of graded Shh signals into all-or-none distinctions in progenitor cell identity depends on cross- repressive interactions between selected pairs of class I and II protein (Ericson et al . , 1997; Briscoe et al . , 2000_; Sander et al . , 2000; Muhr et al . , 2001). In addition, most class I and II proteins have been shown to function directly as transcriptional repressors, through the recruitment of corepressors of the Gro/TLE class (Muhr et al . , 2001) . These findings have suggested a derepression model of neural patterning which invokes the idea that the patterning activities of individual class I or II proteins are achieved primarily through their ability to repress expression of complementary homeodomain proteins from specific progenitor domains. A central implication of this model is that homeodomain proteins direct progenitor cells to individual neuronal fates by suppressing alternative pathways of differentiation - a view that has strong parallels with proposed mechanisms of lineage restriction during lymphoid differentiation (Nutt et al . , 1999; Rolink et al . , 1999; Eberhard, et al . , 2000).

Much of the evidence that has led to this general outline of ventral neural patterning has emerged from an analysis of members of the Nkx gene family. Two closely-related Nkx repressor proteins, Nkx2.2 and Nkx2.9, function as class II proteins that specify the identity of V3 neurons (Ericson et al., 1997; Briscoe et al . , 1999, 2000). A more distantly related class II repressor protein, Nkxδ .1 , is expressed throughout the ventral third of the neural tube and when ectopically expressed, can direct motor neuron and V2 neuron fates (Briscoe et al . , 2000; Sander et al . , 2000). These gain-of-function studies are supported by an analysis of mice lacking Nkxβ . l function, which exhibit a virtually complete failure in V2 interneuron generation (Sander et al . , 2000) . Nkxβ. l null mice also show a reduction in motor neuron generation at rostral levels of the spinal cord, but at more caudal levels motor neurons are formed in near- normal numbers (Sander et al . , 2000). This observation reveals the existence of an Nkx6.1-independent program of spinal motor neuron generation, although the molecular basis of this alternative pathway is unclear.

A close relative of Nkxβ . l , termed Nkxβ . 2 (also known as NkxβB or Gtx) , has been identified (Komuro et al . , 1993; Lee et al . , 2001), and is expressed by neural progenitor cells (Cai et al . , 1999). In its alias of Gtx, Nkx6.2 has been suggested to regulate myelin gene expression (Komuro et al . , 1993) , but its possible functions in neural patterning have not been examined. The identification of an Nkxβ gene pair prompted us to address three poorly resolved aspects of ventral neural patterning. First, do closely related pairs of repressor homeodomain proteins serve distinct or redundant roles in ventral neural patterning? Second, are class I repressor proteins always complemented by a corresponding class II repressor, and if so, is Nkx6.2 one of the missing class II proteins? Third, to what extent is the generation of spinal motor neurons dependent on the activity of Nkx6 class proteins?

To address these issues we mapped the profile of expression of Nkx6.2 and Nkxδ .1 during neural tube development, and analysed mouse Nkxβ mutants to determine the respective contributions of these two genes to neural patterning. We show that Nkx6.2, like Nkx6.1 , functions as a class II repressor homeodomain protein. Our analysis of Nkxβ mutants further indicates that the duplication of an ancestral Nkxβ gene has resulted in the expression of two proteins that exert markedly different levels of repressor activity in the ventral neural tube. This differential repressor activity of these two proteins appears to provide both a fail-safe mechanism during motor neuron generation, and the potential for enhanced diversification of ventral interneuron subtypes. Moreover, we find that under conditions of reduced Nkxβ gene dosage, ventral neuronal subtypes can be generated from progenitor cells that lack the class I or class II proteins normally required for their generation. This finding supports one of the central tenets of the derepression model of ventral neural patterning - that progenitor homeodomain proteins direct particular neuronal fates by actively suppressing cells from adopting alternative fates.

The specification of neuronal fate in the vertebrate central nervous system appears to depend on the profile of transcription factor expression by neural progenitor cells, but the precise roles of such factors in neurogenesis remain poorly understood. A pair of closely-related homeodomain proteins that function as transcriptional repressors, Nkx6.2 and Nkxδ.l, are expressed by progenitor cells in overlapping domains of ventral spinal cord. We provide genetic evidence in the mouse that differences in the level of repressor activity of homeodomain proteins underlies the diversification of ventral interneuron subtypes, and provides a fail-safe mechanism during motor neuron generation. We also show that a reduction in Nkx6 protein activity permits VO neurons to be generated from progenitor cells that lack the homeodomain proteins normally required for their generation. This finding provides direct evidence for a model of neuronal fate specification in which progenitor homeodomain proteins direct specific neuronal fates by actively suppressing the expression of transcription factors that direct alternative fates.

EXPERIMENTAL DETAILS

A. Materials and Methods

Generation of Nkxβ . 2 mutant mice

Mouse Nkxβ . 2 genomic clones were isolated from a 129/θla mouse genomic library. A targeting construct was constructed by inserting a tau-lacZ/pGKneo cassette into a 5 kb 5 ' Hindlll-Ncol fragment and a 2.7 kb 3' Sphl-Accl fragment. The linearized targeting construct was electroporated into E14.1 (129/θla) ES cells. Cells were selected with G418 and screened by Southern blot analysis using a 200 bp 3' Accl fragment, which detected a 6 kb wild type band and a 2.9 kb mutant band. Recombinant clones were injected into C57BL/6J blastocysts to generate two chimeric founders, both of which transmitted the mutant allele. Mice homozygous for the mutant alleles were born at Mendelian frequency and survived through adulthood. All experiments involved mice maintained on a C57BL/6 background. The generation and genotyping of Nkxβ . l mutant mice have been described previously (Sander et al . 2000). Compound Nkxβ mutant mice were obtained by crossing Nkxβ .2^{+ t2z}; Nkxβ .2^+A double heterozygous mice. Genotyping was performed using Southern blot analysis.

Chick in ovo electroporation

Mouse Nkxβ. 2 was isolated by PCR (Komuro et al . , 1993) and chick Nkxβ . 2 from a chick spinal cord library (Basler et al . , 1993) using mouse Nkxβ . l and Nkxβ . 2 as probes. cDNAs encoding full-length mouse and chick Nkxβ . 2 were inserted into a RCASBP (B) retroviral vector and electroporated into the neural tube of stage HH (Hamburger and Hamilton, 1953) 10-12 chick embryos (Briscoe et al . , 2000). After 24-48h, embryos were fixed and processed for immunohistochemistry.

Immunohistochemistry and in situ hybridization histochemistry

Immunohistochemical localization of proteins was performed as described (Yamada et al . , 1993; Briscoe et al . , 2000). Guinea-pig antisera were generated against an 11 amino acid N-terminal sequence of mouse Nkx6.2. Other antibodies used were rabbit anti-Lim3 (Ericson et al . , 1997), mAb Hb9

(Tanabe et al . , 1998), rabbit anti-Isll/2 (Tsuchida et al . ,

1994), rabbit anti-ChxlO (Ericson et al . , 1997), rabbit anti-Enl (Davis et al . , 1991), mAb anti-Evxl/2, rabbit anti- Dbxl, rabbit anti-Dbx2 (Pierani et al . , 1999), rabbit anti-

Nkx6.1 (Jόrgensen et al . , 1999), mAb anti-Pax7 (Ericson et al . , 1996), rabbit anti-bgal (Cappel) and goat anti-bgal

(Biogeneseis) . Images were collected on a Zeiss LSM510 confocal microscope. In situ hybridisation was performed as described (Schaeren-Wiemers and Gerfin-Moser, 1993) , using chick probes for Dbxl , Dbx2 (Pierani et al . , 1999), Nkxβ . l (Briscoe et al . , 2000) and Nkxβ . 2. A mouse probe for the 5'UTR of Nkxβ . 2 comprised 346 bp upstream of the start ATG site. Whole-mount X-gal staining was performed as described (Mombaerts et al . , 1996).

B. Results

Distinct patterns of Nkx6.1 and Nkx6.2 expression in embryonic spinal cord

To examine the roles of Nkxβ class genes in ventral neuronal specification we compared the patterns of expression of Nkx6.2 and Nkx6.1 with that of other progenitor homeodomain proteins in the spinal cord of mouse and chick embryos . In the caudal neural tube of the mouse, the expression of Nkx6.2 was first detected at ~e8.5, in a broad ventral domain that largely coincided with that of Nkx6.1 (Figure 10A) . Between e8.5 and e9.5, the expression of Nkx6.2 was lost from most Nkx6.1⁺ cells in the ventral neural tube, although expression persisted in a narrow stripe of cells just dorsal to the limit of Nkx6.1 expression (Figure 10B, C) . At el0.0-el0.5, virtually all, Nkx6.2⁺ cells coexpressed Dbx2 (Figure 10E) , and the ventral limit of expression of both Nkx6.2 and Dbx2 coincided with the dorsal limit of Nkx6.1 expression at the pl/p2 domain boundary (Figure 10D, E) . Nkx6.2 was expressed predominantly within the pi domain, but scattered Nkx6.2⁺ cells were detected within the pO domain - the domain of expression of Pax7", Dbxl⁺ cells (Figure 10F) . Within the pO domain, however, individual Nkx6.2⁺ cells did not coexpress Dbxl, although they did express Dbx2 (Figure 10E-G) . Thus, the scattered

Nkx6.2⁺ cells found at the dorsoventral level of the pO domain exhibit a pi, rather than pO, progenitor cell identity. Studies in chick have similarly shown that pO and pi progenitors are interspersed in the most dorsal domain of the ventral neural tube (Pierani et al . , 1999).

In the chick neural tube, as in the mouse, Nkxβ. l and Nkxβ.2 are^' initially coexpressed in a broad ventral domain (Cai et al . , 1999; data not shown) . But in contrast to the mouse, Nkxβ.2 expression persists in ventral progenitor cells, with the consequence that the expression of Nkxβ . 2 and Nkxβ . l also overlaps at later developmental stages (Figure 10H, I) . Nevertheless, expression of chick Nkxβ . 2 is also detected in a thin stripe of cells dorsal to the limit of Nkxβ . l expression, within the pi domain (Figure 10H) . Thus, in both species, pi progenitors coexpress Nkx6.2 and Dbx2 and exclude Nkx6.1.

Nkx6.2 Regulates VO and VI Interneuron Fates by Repression of Dbxl Expression The establishment and maintenance of progenitor cell domains in the ventral neural tube has been proposed to depend on mutual repressive interactions between complementary pairs of class I and II homeodomain proteins (Briscoe et al . , 2000; Muhr et al . , 2001). But class II proteins have been identified for only two of the five known progenitor domain boundaries (the pl/p2 and pMN/p3 boundaries) (Ericson et al., 1997; Briscoe et al . , 1999, 2000; Sander et al . , 2000). The mutually exclusive pattern of expression of Nkx6.2 and Dbxl within pi and pO progenitors led us to consider whether Nkx6.2 might function as a class II protein that represses Dbxl expression, and thus help to establish the identity of pi progenitor cells and the fate of their Enl⁺ VI neuronal progeny.

To test this idea, we analysed the profile of expression of class I and II homeodomain proteins in Nkxβ. 2 mutant embryos. We inactivated the mouse Nkxβ.2 gene by homologous recombination in embryonic stem (ES) cells. A targeted Nkxβ . 2 allele {Nkxβ .2^tlz) was generated by replacing the coding sequence of Nkxβ . 2 with a tauLacZ cassette (Figure

11A) . In the spinal cord of Nkxβ . 2^+/tlz embryos analysed at el0.5, expression of LacZ and Nkx6.2 coincided within the pi progenitor domain (see Figure HE, F) . In Nkxβ . 2^tlz/tlz embryos, the location of LacZ⁺ cells was also similar to that in Nkxβ .2^+/tlz embryos (Figure HF, G) , but Nkx6.2 protein was not detected (Figure 11G) . These data provide evidence that the Nkxβ . 2^tlz allele generates a null mutation, and that disruption of the Nkxβ .2 locus does not perturb the normal spatial pattern of expression of this gene.

We did observe, however, that the level of LacZ expression was markedly elevated in Nkxβ . 2^{tlz tlz} , when compared with Nkxβ . 2^+/tlz, embryos (Figure 11B-D) . An elevation in level of expression of the residual 5' Nkxβ.2 transcript was also detected in Nkxβ .2^tlz/tlz embryos (Figure 11H-J) . These observations provide evidence that Nkx6.2 negatively regulates its own expression level within pi progenitor cells .

We next analysed the pattern of expression of class I and II homeodomain proteins in the spinal cord and caudal hindbrain of Nkxβ .2^tlz/tlz embryos . The domains of expression of the class II proteins Nkx2.2 and Nkx6.1, and of the class

I proteins Pax7, Dbx2 , Irx3 and Pax6 were similar in Nkxβ .2 ^ti^z/t^iz _f Nkxβ . 2^+/tlz , and wild type embryos (Figure 12B-D, G-I; data not shown) . In addition, normal patterns of expression of Dbx2 and Nkx6.1 were detected at the pl/p2 domain boundary (data not shown) , showing that establishment of the pi progenitor domain does not require Nkx6.2 function.

However, the level of Dbx2 expression in pi domain progenitors was increased -two-fold in Nkx6.2^{tlz tlz} mutants

(Figure 11K-M) , indicating that Nkx6.2 normally limits the level of Dbx2 expression in this domain.

We also detected a marked change in the pattern of expression of the pO progenitor cell marker Dbxl in Nkxβ . 2 ^tlz/tlz embryos. At caudal hindbrain levels, the number of ventral Dbxl⁺ progenitor cells increased 1.7- fold (Figure 12F) , and the domain of Dbxl⁺ cells expanded ventrally, extending through the pi domain to the dorsal limit of Nkx6.1 expression (Figure 12H) . Moreover, in Nkxβ . 2^{tlz tlz} embryos all of the ectopic Dbxl ⁺ cells found within the pi domain coexpressed LacZ (Figure 12J) . Thus, many progenitors within the pi domain initiate Dbxl expression in the absence of Nkx6.2 function. Nevertheless in Nkxβ . 2^{t2z tlz} embryos, numerous LacZ⁺ progenitors still lacked Dbxl expression (Figure 12J) , implying the existence of an Nkx6.2 -independent means of excluding Dbxl expression from pi progenitors . The ventral expansion of Dbxl was most prominent at caudal hindbrain and cervical spinal levels of the neural tube but a similar, albeit less marked, expansion of Dbxl expression was detected at caudal spinal levels

(data not shown; see Figure 15) . Taken together, these data imply that within pi domain progenitors Nkx6.2 functions as a weak repressor of Dbx2 expression and a more potent repressor of Dbxl expression.

We next analysed the generation of interneuron subtypes in the ventral neural tube. In wild type embryos, Dbxl⁺, Dbx2⁺, Nkx6.2" pO progenitors generate Evxl/2⁺ VO neurons

(Pierani et al . , 1999; 2001); Nkx6.2⁺, Dbxl", Dbx2⁺ pi progenitors give rise to Enl⁺ VI neurons (Burrill et al . ,

1997; Ericson et al . , 1997), and Nkx6.1⁺, Irx3⁺, p2 progenitors give rise to Chxl0⁺ V2 neurons (Ericson et al . , 1997; Briscoe et al . , 2000). Dbxl activity in pO progenitors is required to promote VO and suppress VI neuronal fates (Pierani et al . , 2001). The ventral expansion in Dbxl expression in Nkxβ . 2^tlz/tlz embryos therefore led us to examine whether the loss of Nkx6.2 function leads progenitor cells within the pi domain to adopt a V0 rather than VI neuronal fate .

In the caudal hindbrain of Nkxβ . 2 ^tIz/^tiz embryos examined at elθ.5, we detected a - two-fold increase in the number of

Evxl/2⁺ V0 neurons and the domain of V0 neuronal generation expanded ventrally the normal position of the pi domain

(Figure 12N) . Consistent with this, many Evxl/2⁺ neurons coexpressed LacZ (Figure 12P) , showing directly that some

V0 neurons derive from pi progenitors in the absence of

Nkx6.2 function. Conversely, the total number of Enl⁺ VI neurons generated in Nkxβ . 2 ^tlz/^tlz embryos was reduced by -50%

(Figure 12Q) . The dorsoventral position of generation of the remaining Enl⁺ VI neurons was similar in Nkxβ . 2^tlz/tlz embryos (Figure 12N) , and these neurons expressed LacZ

(Figure 120) showing directly that Nkx6.2⁺, Dbx2⁺ pi progenitor cells generate VI neurons. The total number of neurons generated from pi domain progenitors, defined by Cynl, TuJl and Liml/2 expression was similar in Nkxβ . 2^tlz/tlz and Nkxβ . 2^+/tlz embryos examined at elθ.5 (data not shown). In addition, the number of TUNEL⁺ cells was similar in Nkxβ . 2^tlz/tlz and Nkxβ . 2^+/tlz embryos (data not shown) . Chxl0⁺ V2 neurons and HB9⁺, Isll/2⁺ motor neurons were present in normal numbers and positions in Nkxβ . 2^tlz/tlz embryos (Figure 14; data not shown) . Together, these findings show that the activity of Nkx6.2 within pi progenitors promotes VI neuronal generation and helps to suppress the generation of VO neurons, a finding consistent with the proposed role of Nkx6.2 in repressing Dbxl expression from pi progenitors.

Repression of Nkx6.2 by Nkx6.1 underlies Nkxβ gene redundancy in spinal motor neuron generation We next addressed the respective contributions of Nkxβ. l and Nkxβ . 2 to motor neuron and V2 neuron generation. In the ventral neural tube, p2 and pMN progenitors express Nkx6.1 and give rise to V2 neurons and motor neurons respectively. Ectopic expression of Nkx6.1 is sufficient to induce motor neurons and V2 interneurons in dorsal regions of the neural tube, and in Nkxβ . l mutant mice V2 neurons are eliminated (Briscoe et al . , 2000; Sander et al . , 2000). Nevertheless, there is only a partial reduction in motor neuron generation in Nkxβ . l mutants (Sander et al . , 2000), revealing the existence of an Nkxβ . 1-independent pathway of motor neuron generation. Nkx6.2 does not normally contribute to motor neuron specification in the mouse, since its expression is extinguished from ventral progenitors well before the appearance of post-mitotic motor neurons (Figure 10A-C) , and there is no change in the number of motor neurons generated in Nkxβ . 2^tlz/tlz embryos (see Figure 14G) .

Three lines of evidence, however, led us to consider a cryptic role for Nkx6.2 in motor neuron generation. First, Nkx6.2 and Dbx2 share the same ventral limit of expression at the pl/p2 domain boundary, and the expression of Dbx2 is repressed by Nkx6.1 (Briscoe et al . , 2000; Sander et al . , 2000). Second, Nkx6.2 negatively regulates its own expression level within pi domain progenitors (Figure 11D, G, J) . Third, Nkx6.1 and Nkx6.2 possess similar Gro/TLE recruitment activities and DNA target site binding specificities (Muhr et al . , 2001). We reasoned therefore that under conditions in which Nkx6.1 activity is reduced or eliminated, Nkx6.2 expression might be derepressed in p2 and pMN progenitors .

In support of this idea, in Nkxβ . l^+/~ embryos examined at el0.5 we detected a marked increase in the number of Nkx6.2⁺ cells within the p2 and pMN domains (Figure 13B) . And in

Nkxβ . l^'/- embryos, expression of Nkx6.2 was detected in virtually all progenitor cells within the p2 and pMN domains

(Figure 13C) . Indeed, in Nkxβ . l-^/- embryos, the level of

Nkx6.2 expression in the nuclei of progenitor cells within the p2 and pMN domains was 1.9-fold greater than that in progenitor cells located within the pi domain (Figure 13C; data not shown) . Together, these data show that Nkx6.1 activity normally represses Nkx6.2 expression from p2 and pMN progenitors in the mouse embryo.

In turn, these findings raised the possibility that in Nkxβ . I'/' embryos, the derepression of Nkx6.2 expression substitutes for the loss of Nkx6.1 during motor neuron generation. If this is the case, Nkx6.2 would be predicted to mimic the ability of Nkx6.1 to induce motor neurons in vivo. Expression of chick or mouse Nkxβ.2 in the neural tube of HH stage 10-12 chick embryos repressed Dbx2 and Dbxl expression (Figure 13D-F) , and induced ectopic motor neuron differentiation (Figure 13G-I, L-N) with an efficacy similar to that of Nkxβ . l (Briscoe et al . , 2000) . These data show that Nkx6.2 can induce ectopic motor neurons when expressed at high levels in the dorsal neural tube, supporting the idea that both Nkx6 proteins can exert similar patterning activities in vivo (Figure 13D-0; Briscoe et al . , 2000). In addition, misexpression of Nkx6.2 in the pO and pi progenitor domains suppressed the generation of Evxl/2⁺ VO and Enl⁺ VI neurons and promoted the generation of Chxl0⁺ V2 neurons (Figure 13J, K, 0, P) . Thus, a high level of expression of Nkx6.2 is not compatible with the generation of either VO or VI neurons (Figure 130, P) .

Based on these findings, we examined whether Nkx6.2 has a role in motor neuron generation in Nkxβ. l mutant mice by testing the impact of removing Nkx6.2 as well as Nkx6.1 on the generation of spinal motor neurons. In Nkxβ . 2^tlz/tlz embryos there was no change in the number of motor neurons generated at any level of the spinal cord or hindbrain

(Figure 14G,N,0; data not shown) . In Nkxβ . l^~/^~ mutants, the number of spinal motor neurons was reduced by -60% at cervical levels, but by only 25% at lumbar levels (Figure 14H,N,0, Sander et al . , 2000). In Nkxβ . l-^/- ; Nkxβ . 2^+/tlz embryos, motor neuron generation was reduced to -25% of controls at both cervical and lumbar levels (Figure 141,N, O; data not shown) . In Nkxβ . l'^/' ; Nkxβ . 2^tlz/tlz embryos, the generation of motor neurons was reduced to <10% of wild type numbers, at all levels of the spinal cord (Figure 14J) . In these Nkxβ double mutant embryos, residual motor neurons were detected at elO.O, and no further increase in motor neuron number was evident at el2 (Figure 14M, P; data not shown) . Since there was no increase in apopototic cell death in the ventral neural tube over this period (data not shown) , we infer that the few spinal motor neurons present in Nkxβ double mutants are generated prior to elO . Together, these findings demonstrate that Nkx6.2 substitutes for the loss of Nkx6.1 in spinal motor neuron generation, and reveal a link between Nkxβ gene dosage and the incidence of motor neuron generation.

A Dissociation in Neuronal Fate and Progenitor Cell Identity in Nkxβ Mutant Mice

We next examined whether a reduction in Nkxβ gene dosage results in ectopic Dbx protein expression and VI and VO neuron generation in the p2 and pMN domains of the ventral spinal cord.

Enl⁺ VI neurons are normally generated from Dbx2⁺, Dbxl" pi progenitor cells, and we therefore analysed the relationship between Dbx2 expression and Enl⁺ VI neuronal generation in Nkxβ . l and Nkxβ . 2 compound mutants. As reported previously (Sander et al . , 2000), in Nkxβ . l-^/- embryos examined at elθ.5, ectopic ventral expression of Dbx2 was detected at high levels in the p2 and p3 domains, although cells in the pMN expressed only very low levels of Dbx2 (Figure 15H; see Sander et al . , 2000). Moreover, in Nkxβ . l-^/- embryos, ectopic Enl⁺ neurons were generated in the p2 and pMN domains of the ventral neural tube (Figure 15R) . In Nkxβ . l^'/- ; Nkxβ . 2^+/tlz embryos, Dbx2 expression was detected at intermediate levels in the pMN domain (Figure 151) , and in Nkxβ . l-/- ; Nkxβ . 2^tlz/tzl double mutant embryos, Dbx2 was detected at uniformly high levels in the p2 and pMN domains (Figure 15J) . Strikingly, in these Nkxβ . l and Nkx^β . 2 compound mutant backgrounds, and despite the enhanced ectopic expression of Dbx2 , the number of ectopic ventral Enl⁺ VI neurons was reduced rather than increased, when compared with the number generated in Nkxβ . l single mutants (Figure 15R, T) .

Since Evxl⁺ VO neurons are normally generated from Dbxl⁺, Dbx2⁺ pO progenitors, we examined whether the reduction in ectopic ventral Enl⁺ VI neuron generation at low Nkxβ gene dosage might reflect a change in the pattern of expression of Dbxl, and the ectopic generation of VO neurons. Consistent with this idea, in Nkxβ. l-^/-; Nkxβ . 2^tlz/tlz mutants, scattered Dbxl⁺ cells were detected in the p2 , pMN and p3 domains (Figure 150) , and ectopic ventral Evxl/2⁺ VO neurons were detected throughout the ventral neural tube (Figure 15T, Z) . Thus, in Nkxβ double mutants, the loss of VI neurons is associated with the ectopic ventral expression of Dbxl and the generation of ectopic VO neurons.

But in Nkxβ . l single and Nkxβ . l-/- ; Nkxβ . 2^+/tlz compound mutant backgrounds, the normal link between expression of Dbxl in progenitor cells and the generation of Evxl/2⁺ VO neurons was severed. In both these Nkxβ compound mutants backgrounds, the domain of expression of Dbxl was unchanged

(Figure 15M, N) : a result that can be accounted for by the maintained expression of Nkx6.2 within the pi domain, and the deregulated expression of Nkx6.2 within the p2 and pMN domains. Nevertheless, Evxl/2⁺ VO neurons were generated from progenitor cells in the position of p2 and pMN domains,

(Figure 15R, S, X, Y) .

We next considered whether these ectopic V0 neurons were generated from the position of the p2 and pMN domains, or whether they simply migrated ventrally from a more dorsal position of origin. Ectopic ventral Evxl/2⁺ VO neurons were detected as early as elO .0 (Figure 16B) , and many of them coexpressed LacZ (Figure 16C, D) , providing evidence that many of these neurons derive from progenitor cells within the position of the p2 and pMN domains. The finding that Evxl/2⁺ VO neurons are generated from the pMN domain in Nkxβ. l-^/-; Nkxβ .2^+/tlz embryos is especially significant, since these progenitors express negligible levels of Dbx2 (Figure 16E, 17) , arguing against the possibility that Dbx2 expression compensates for the absence of Dbxl during ectopic VO neuronal generation. These results therefore provide evidence that even though Dbxl activity is normally required for the generation of VO neurons (Pierani et al . , 2001) , under conditions in which Nkxβ gene dosage is markedly reduced, V0 neurons can be generated from progenitor cells that lack Dbxl expression.

Nevertheless, the pattern of ventral neurogenesis observed in Nkxβ . l-/- ; Nkxβ . 2^+/tlz mutants indicated that residual

Isll/2⁺, HB9⁺ neurons and ectopic Evxl⁺ neurons were each generated from progenitors located in the position of the pMN domain. This observation raised the question of whether these two neuronal populations are, in fact, distinct. Strikingly, we found that in this compound Nkxβ mutant background, many of the residual Isll/2⁺, HB9⁺ neurons transiently expressed Evxl (Figure 16H, I) . Thus, under conditions of reduced Nkxβ gene dosage, progenitor cells at the position of the pMN domain initially generate neurons with a hybrid motor neuron/VO neuron identity. c . Discussion

The patterning of cell types in the ventral neural tube depends on the actions of a set of homeodomain proteins expressed by neural progenitor cells. Duplication of many of these genes has resulted in the overlapping neural expression of pairs of closely-related homeodomain proteins, and raises the question of whether these proteins have distinct or redundant roles during ventral neurogenesis. We have used genetic approaches in mouse to examine the respective contributions of one such homeodomain protein pair, Nkx6.1 and Nkx6.2 , in ventral neural patterning. Our results imply that the duplication of an ancestral Nkxβ gene confers both redundant and distinct roles for Nkx6.1 and Nkx6.2 in ventral neuronal patterning. We discuss below how the specificity and efficacy of Nkx6-mediated transcriptional repression underlies the overlapping divergent patterning activities of the two proteins.

Redundant Activities of Nkx6 Proteins in Motor Neuron and VO Neuron Generation

Our genetic studies in mice indicate that Nkx6.1 and Nkx6.2 have qualitatively similar activities in promoting the generation of motor neurons and in suppressing the generation of VO neurons. How are these overlapping patterning activities achieved, given the distinct profiles of expression of these two genes?

Nkx6.1 has been shown to have a role in motor neuron generation (Sander et al . , 2000), but the finding that large numbers of motor neurons are generated at caudal levels of the spinal cord in Nkxβ . l mutant mice, points to the existence of an Nkx6.1-independent pathway of motor neuron generation. At face value, Nkx6.2 would appear a poor candidate as a mediator of the Nkx6.1-independent pathway of motor neuron specification, since it is not expressed by motor neuron progenitors, nor is motor neuron generation impaired in Nkxβ . 2 mutant mice. Nevertheless, the activity of Nkx6.2 is responsible for the efficient generation of spinal motor neurons in Nkxβ . l mutants. The basis of this redundant function resides in the derepression of Nkx6.2 expression in motor neuron progenitors in Nkxβ. l mutant mice. Strikingly, Nkx6.2 is even derepressed in Nkxβ .2^+/" embryos, whereas there is no change in the patterns of expression of Dbx2 and other homeodomain proteins implicated in the repression of motor neuron generation. The propensity for Nkx6.2 derepression thus appears to establish a "fail-safe" mechanism that ensures that the net level of Nkx6 protein activity is maintained in motor neuron progenitors under conditions in which Nkx6.1 levels decrease. A similar "fail-safe" regulatory mechanism may operate with other Nkx protein pairs . During pharyngeal pouch development, for example, the loss of Nkx2.6 expression appears to be compensated for by the up- regulation of Nkx2.5 (Tanaka et al . , 2000).

The finding that Nkx6.2 is derepressed in the absence of Nkx6.1 function also offers a potential explanation for the divergent patterns of expression of Nkx6.2 in the ventral neural tube of mouse and chick embryos . We infer that the chick Nkxβ . 2 gene is not subject to repression by Nkx6.1, permitting its persistent expression in p3 , pMN and p2 domain progenitor cells. Thus, in chick, the overlapping functions of Nkx6.1 and Nkx6.2 in motor neuron generation are associated with the coexpression of both genes by motor neuron progenitors, whereas in the mouse, Nkx6. activity is held in reserve, through its repression by Nkx6.1.

Nkx6.1 and Nkx6.2 also have an equivalent inhibitory influence on the generation of VO neurons, albeit through activities exerted in different progenitor domains. In pi progenitors, the repression of pO identity and VO neuron fate is accomplished by Nkx6.2. But ventral to the pl/p2 domain boundary it is Nkx6.1 that prevents Dbxl expression and VO neuronal generation. Thus, Nkx6.1 is a potent repressor of Dbxl expression, despite the fact that these two proteins lack a common progenitor domain boundary. The repression of genes that are normally positioned in spatially distinct domains has been observed with other class I and II proteins (Sander et al . , 2000) . This feature of neural patterning also parallels the activities of gap proteins in anteroposterior patterning of the Drosophila embryo, where the repressive activities of individual gap proteins are frequently exerted on target genes with which they lack a common boundary (Kraut and Levine, 1991; Stanojevic et al . , 1991).

Distinct Functions of Nkx6.1 and Nkx6.2 in Ventral Interneuron Generation

We now turn to the question of how Nkx6.1 and Nkxδ .2 can exert distinct roles in interneuron generation, given the similarities of the two proteins in DNA target site specificity (Jorgensen et al . , 1999; Muhr et al . , 2001), and their overlapping functions in the patterning of motor neurons and VO neurons .

One factor that contributes to the opponent influence of Nkx6.1 and Nkx6.2 on the specification of VI interneuron fate is a distinction in the dorsal limit of expression of the two proteins in the neural tube, presumably a reflection of differences in the regulation of expression the two proteins by graded Shh signalling. Nkx6.1 expression stops at the pl/p2 domain boundary. And within the p2 domain, Nkxδ .1 suppresses pi progenitor identity through repression of Dbx2 and Nkx6.2 expression, in this way ensuring the generation of Chxl0⁺ V2 neurons. Nkxδ .2 , in contrast, occupies the pi domain, where it is coexpressed with Dbx2. In pi domain cells, Nkx6.2 promotes the generation of Enl⁺ VI neurons by repressing the expression of Dbxl and Evxl, determinants of VO neuronal fate (Pierani et al . , 2001; Moran-Rivard et al . , 2001). Nevertheless, only a fraction of pi progenitors initiate Dbxl expression and acquire V0 neuron fate in the absence of Nkx6.2 function, raising the possibility that Dbx2 may also have a role in repressing Dbxl expression within pi progenitors (see Pierani et al . , 1999) .

The second major factor that underlies the opponent activities of Nkx6.1 and Nkx6.2 in VI interneuron specification appears to be a difference in the potency with which the two Nkx6 proteins repress a common set of target genes. This view is supported by several observations. Nkx6.1 completely represses Nkx6.2 , whereas Nkx6.2 exerts an incomplete negative regulation of its own expression in pl domain progenitors. Thus, Nkx6.1 is evidently a better repressor of Nkx6.2 than is Nkx6.2 itself. Similarly, Nkx6.2 is coexpressed with Dbx2 in pi domain progenitors, whereas Nkx6.1 excludes Dbx2 from p2 domain progenitors, indicating that Nkx6.1 also is a more effective repressor of Dbx2 expression than is Nkxδ .2. Consistent with this view, Nkx6.2 fails to repress Dbx2 expression completely from ventral progenitors in Nkxβ . l mutants. The fact that Nkx6.2 is only a weak repressor of Dbx2 is critical for the formation of the pi domain, since the maintained expression of Dbx2 in these cells ensures the exclusion of Nkx6.1 expression (Briscoe et al . , 2000).

Our results do not resolve why Nkx6.2 is a weaker repressor than Nkx6.1 in vivo. Differences in the primary structure of Nkx6.2 and Nkx6.1 (Cai et al . , 1999; Muhr et al . , 2001) could result in an intrinsically lower repressor activity of Nkx6.2 , when compared with that of Nkx6.1. But our findings are also consistent with the possibility that the two Nkx6 proteins have inherently similar repressor activities, and that the Nkx6.2 protein is merely expressed at a lower level. Indeed within pi progenitors, the level of Nkx6.2 expression is clearly subject to tight regulation, with significant consequences for neuronal specification. The selective expression of Nkx6.2 in pi progenitors, coupled with its weak negative autoregulatory activity, ensures a level of Nkx6 activity that is low enough to permit Dbx2 expression but is still sufficient to repress Dbxl expression, thus promoting the generation of VI neurons . Our findings therefore reveal that a gradient of extracellular Shh signalling is translated intracellularly into stepwise differences in the level of Nkx6 activity along the ventral-to-dorsal axis of the neural tube. Moreover, the different Nkx6 protein activity levels within ventral progenitor cells are a critical determinant of ventral neuronal fate. Cells that express low or negligible levels of Nkx6 activity (pO progenitors) are directed to a VO neuronal fate, cells that express an intermediate Nkx6 activity level (pi progenitors) are directed to a VI fate, and cells that express a high Nkx6 activity level (pMN and p2 progenitors) are directed to a motor neuron or V2 fate (Figure 17) .

Nkx6 Repressor Function and Neuronal Patterning by Derepression

The finding that many progenitor homeodomain proteins exert mutual-cross repressive interactions has led to a model of spinal neuronal patterning based on transcriptional derepression (Muhr et al . , 2001). Similar cross-repressive interactions may establish regional progenitor domains in more rostral regions of the developing CNS (Toresson et al . , 2000; Yun et al . , 2001). A premise of this model is that transcriptional repression is exerted at two sequential steps in neurogenesis. One repressive step operates at the level of the progenitor homeodomain protein themselves, but a second repressive step is exerted on neuronal subtype determinant factors that have a downstream role in directing neuronal subtype fates (Briscoe et al . , 2000; Muhr et al . , 2001) . Our analysis of Nkxβ compound mutant mice provides direct support for this two-step repression model, and in addition indicates that progenitor homeodomain proteins and neuronal subtype determinants differ in their sensitivity to repression by the same class II protein. Normally, the functions of Dbxl and Evxl are required sequentially during the generation of VO neurons (Pierani et al . , 2001; Moran- Rivard et al . , 2001). In Nkxβ . l-¹- ; Nkxβ . 2 ^+/tlz mutants, however, the generation of Evxl/2⁺ V0 neurons occurs in. the absence of expression of Dbxl by neural progenitor cells. Dbxl expression is therefore dispensable for V0 neuron generation under conditions of reduced Nkxβ gene dosage. From these results, we infer that the net level of Nkx6 protein activity in ventral progenitor cells is still above threshold for repression of Dbxl expression, but is below the level required for repression of Evxl expression. These data therefore support the idea that Nkx6 proteins normally inhibit V0 neuronal fate by repressing the class I progenitor homeodomain protein Dbxl, and independently by repressing expression of the V0 neuronal subtype determinant

Evxl.

A differential sensitivity of progenitor homeodomain proteins and neural subtype determinants to repression appears therefore to underlie the dissociation of progenitor cell identity and neuronal fate observed in Nkxβ mutants. Such two-tiered repression is, in principle, necessary to specify neuronal fate through transcriptional derepression. In the case of Nkx6.1, for example, repression of Dbxl and Dbx2 (and possible other unidentified repressors) should be sufficient to derepress motor neuron subtype determinants such as MNR2 and Lim3 in pMN progenitors. But, unless Nkx6.1 also represses the expression of VO determinants, Evxl expression would also be initiated in differentiating motor neurons, resulting in a hybrid neuronal phenotype. Indeed, under conditions in which Nkxβ gene dosage is reduced or eliminated, some of the neurons generated from the position of the pMN domain do transiently express a hybrid motor neuron/VO neuron phenotype .

The derepression model also invokes the idea that a major role of Nkx6 class proteins is to exclude the expression of Dbx2 and other proteins that inhibit motor neuron generation. This view offers a potential explanation of why a few residual motor neurons are generated in Nkxβ double mutants. We find that in the absence of Nkxβ gene function, residual motor neurons are generated only at early developmental stages, suggesting that progenitor cells within the position of the pMN domain have committed to a motor neuron fate prior to the onset of the deregulated ventral expression of Dbx2 and other motor neuron repressors . We note that a third Nkxβ-li^'k.e gene exists in the mouse, but this gene is not expressed in the spinal cord of wild type or Nkxβ mutant embryos (E. Anderson and J. Ericson, unpublished data) , and thus its activity appears not to account for the residual motor neurons generated in Nkxβ double mutants. Importantly, the detection of residual motor neurons in Nkxβ double mutants also provides evidence that Nkxδ proteins do not have essential functions as transcriptional activators during motor neuron specification, further supporting their critical role as repressors. Finally, the present studies and earlier work on neurogenesis in the ventral spinal cord (Ericson et al . , 1996; Thaler et al . , 1999; Arber et al . , 1999; Sander et al . , 2000) have provided evidence that newly-generated neurons can sometimes express mixed molecular identities. These observations raise the possibility that repressive interactions that select or consolidate individual neuronal identities are not restricted to progenitor cells. Consistent with this view, Evxl is required to establish VO and repress VI neuronal identity through an action in post- mitotic neurons (Moran-Rivard et al . , 2001), although it remains unclear whether Evxl itself functions in this context as an activator or repressor. Similarly, the homeodomain protein HB9 has been implicated in the consolidation of motor neuron identity, through repression of V2 neuronal subtype genes (Arber et al . , 1999; Thaler et al . , 1999). HB9 possesses an eh-1 Gro/TLE recruitment domain

(Muhr et al . , 2001), suggesting that HB9 controls the identity of post-mitotic motor neurons through a direct action as a transcriptional repressor. The consolidation of neuronal subtype identity in the spinal cord may therefore depend on transcriptional repressive interactions within both progenitor cells and post-mitotic neurons.

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Claims

What is claimed is:

1- A method of converting a stem cell into a ventral neuron which comprises introducing into the stem cell a nucleic acid which expresses homeodomain transcription factor Nkxδ .1 protein in the stem cell so as to thereby convert the stem cell into the ventral neuron.

2. The method of claim 1, wherein the nucleic acid introduced into the stem cell incorporates into the chromosomal DNA of the stem cell .

3. The method of claim 1, wherein the nucleic acid is introduced by transfection or transduction.

4. The method of claim 1, wherein the ventral neuron is a motor neuron, a V2 interneuron or a V3 interneuron.

5. A method of diagnosing a motor neuron degenerative disease in a subject which comprises: a) obtaining a nucleic acid sample from the subj ect ; b) sequencing the nucleic acid sample; and c) comparing the nucleic acid sequence of step (b) with a Nkx6.1 nucleic acid sequence from a subject without motor neuron degenerative disease, wherein a difference in the nucleic acid sequence of step (b) from the Nkx6.1 nucleic acid sequence from the subject without motor neuron degenerative disease indicates that the subject has the motor neuron degenerative disease.

6. The method of claim 5, wherein the motor neuron degenerative disease is amyotrophic lateral sclerosis or spinal muscular atrophy.

7. A method of diagnosing a motor neuron degenerative disease in a subject which comprises: a) obtaining a nucleic acid sample from the subj ect ; b) performing a restriction digest of the nucleic acid sample with a panel of restriction enzymes; c) separating the resulting nucleic acid fragments by size fractionation; d) hybridizing the resulting separated nucleic acid fragments with a nucleic acid probe (s) of at least 15 nucleotide capable of specifically hybridizing with a unique sequence included within the sequence of a nucleic acid molecule encoding a human Nkx6.1 protein, wherein the sequence of the nucleic acid probe is labeled with a detectable marker, and hybridization of the nucleic acid probe (s) with the separated nucleic acid fragments results in labeled probe- fragment bands ; d) detecting labeled probe-fragment bands, wherein the labeled probe-fragment bands have a band pattern specific to the nucleic acid of the subject; and f) comparing the band pattern of the detected labeled probe-fragment bands of step (d) with a previously determined control sample, wherein the control sample has a unique band pattern specific to the nucleic acid of a subject having the motor neuron degenerative disease, wherein identity of the band pattern of the detected labeled probe-fragment bands of step (d) to the control sample indicates that the subject has the motor neuron degenerative disease.

8. The method of claim 7, wherein the nucleic acid is DNA.

9. The method of claim 7, wherein the nucleic acid is RNA.

10. The method of claim 7, wherein the size fractionation in step (c) is effected by a polyacrylamide or agarose gel .

11. The method of claim 7, wherein the detectable marker is radioactive isotope, enzyme, dye, biotin, a fluorescent label or a chemiluminescent label .

12. The method of claim 7, wherein the motor neuron degenerative disease is amyotrophic lateral sclerosis or spinal muscular atrophy.

13. A method of converting a stem cell into a ventral neuron which comprises introducing into the stem cell a nucleic acid which expresses homeodomain transcription factor Nkx6.2 protein in the stem cell so as to thereby convert the stem cell into the ventral neuron.

14. The method of claim 13, wherein the nucleic acid introduced into the stem cell incorporates into the chromosomal DNA of the stem cell.

15. The method of claim 13, wherein the nucleic acid is introduced by transfection or transduction.

16. The method of claim 13, wherein the ventral neuron is a motor neuron.

17. A method of converting a stem cell into a ventral neuron which comprises introducing into the stem cell a polypeptide which expresses homeodomain transcription factor Nkx6.1 in the stem cell so as to thereby convert the stem cell into the ventral neuron.

18. The method of claim 17, wherein the ventral neuron is a motor neuron, a V2 interneuron or a V3 interneuron.

19. A method of converting a stem cell into a ventral neuron which comprises introducing into the stem cell a polypeptide which expresses homeodomain transcription factor Nkx6.2 in the stem cell so as to thereby convert the stem cell into the ventral neuron .

20. The method of claim 19, wherein the ventral neuron is a motor neuron.

21. A method of diagnosing a neurodegenerative disease in a subject which comprises: a) obtaining a suitable sample from the subject ,- b) extracting nucleic acid from the suitable sample; c) contacting the resulting nucleic acid with a nucleic acid probe, which nucleic acid probe (i) is capable of hybridizing with the nucleic acid of Nkx6.1 or Nkx6.2 and (ii) is labeled with a detectable marker; d) removing unbound labeled nucleic acid probe; and e) detecting the presence of labeled nucleic acid, wherein the presence of labeled nucleic acid indicates that the subject is afflicted with a chronic neurodegenerative disease, thereby diagnosing a chronic neurodegenerative disease in the subject.

22. The method of claim 21, wherein the suitable sample is spinal fluid.

23. The method of claim 21, wherein the nucleic acid is DNA.

24. The method of claim 21, wherein the nucleic acid is RNA.