Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
  • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
  • C07K14/4702—Regulators; Modulating activity
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

• Nkx2 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, Nkx ⁇ .
• Nkx ⁇ . 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.
• Nkx ⁇ . l is expressed by ventral progenitors that give rise to motor (MN) , V2 , and V3 neurons.
• Mice carrying a null mutation of Nkx ⁇ . 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.
• Nkx ⁇ . 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.
• 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
• 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
• FIG. 1A-1C Expression of Nkx ⁇ . l in transverse sections of the ventral neural tube of mouse embryos E9.5.
• FIG. 1A Expression of Nkx ⁇ . 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 Nkx ⁇ .
• FIG. 1J-1H embryos.
• Fig. 1J Horizontal lines, approximate position of dorsoventral boundary of the neural tube; vertical lines, expression of Dbx2 and Gshl .
• MMC median motor column
• LMC lateral motor column
• HB9 expression in hypoglossal motor neurons in E10.5-E11 wild- type mice (Fig. 3G) and Nkx ⁇ . l mutant (Fig. 3H) mice. Coexpression of Isll (green) and Phox2a/b (red) in wild- type (Fig. 31) or Nkx ⁇ . l mutant (Fig. 3J) mice. (h) hypoglossal motor neurons; (v) visceral vagal motor neurons. Scale bar shown in C 50 ⁇ m (Figs. 3A-3D) or 70 ⁇ m (Figs. 3E-3J) .
• 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 Nkx ⁇ . 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 Nkx ⁇ . l mutant (Fig. 4H) embryos. Evxl expression by V0 neurons at caudal hindbrain levels of wild-type (Fig. 41) and Nkx ⁇ . l mutant (Fig. 4J) embryos.
• 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
• 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.
• FIG. 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.
• Elevation in Nkx ⁇ . 2 and Dbx2 expression in pi domain cells in Nkx ⁇ . 2 mouse mutants (A) Diagram of the targeting construct (i) used to replace the coding sequence of Nkx ⁇ . 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 Nkx ⁇ . 2 tlz tlz (D) embryos.
• Evxl/2 + VO neurons are generated dorsal to Enl + VI neurons (K) and LacZ + cells (M) in Nkx ⁇ . 2 +/tlz embryos.
• Enl + neurons express LacZ in Nkx ⁇ . 2 +/tlz (L) and Nkx ⁇ . 2 tlz/tlz (0) embryos.
• Nkx ⁇ . 2 tlz/tlz embryos 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 Nkx ⁇ . 2 tlz/tlz embryos co-express LacZ.
• Q Quantitation of Evxl/2 + VO, and Enl + VI, neurons at the caudal hindbrain of Nkx ⁇ . 2* /tlz and Nkx ⁇ .2 tlz/tlz embryos at el ⁇ .5. Counts from 12 sections, mean + S.D. In panels (A-P) , the white arrowhead indicates the pO/pl boundary.
• Nkx6.2 Deregulated expression of Nkx6.2 in Nkx ⁇ . l mutant mice, and similar patterning activities of Nkx6 proteins in chick neural tube.
• A In el ⁇ .5 wt embryos, Nkx6.2 expression is confined to the pi progenitor domain.
• B In Nkx ⁇ . 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.
• Nkx6.2 underlies motor neuron generation in Nkx ⁇ . l mutants.
• Nkx6.2 expression In el ⁇ .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 Nkx ⁇ . 2 tlz/tlz embryos.
• C, D In Nkx ⁇ . l- / - and Nkx ⁇ . l- / - ; Nkx ⁇ . 2 + tlz embryos, Nkx6.2 expression is derepressed in the p2 , pMN and p3 domains.
• N-P Quantitation of HB9 + and Isll/2 + motor neurons at cervical and lumbar levels in wt, Nkx ⁇ . 2 and Nkx ⁇ . l single mutants and in Nkx ⁇ . 2; Nkx ⁇ . 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 Nkx ⁇ mutants.
• A-E Expression of Nkx6.1 and Nkx6.2 in the spinal cord in different Nkx ⁇ mutant backgrounds at el ⁇ .5.
• F-J Spatial patterns of Pax7 and Dbx2 expression in different Nkx ⁇ mutant backgrounds. Note that the level of Dbx2 expression in the pMN domain of Nkx ⁇ .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).
• P-T Spatial patterns of generation of Evxl/2 + V0 neurons and Enl + VI neurons in different Nkx ⁇ mutant backgrounds.
• Q The generation of V0 neurons expands ventrally into the pi domain in Nkx ⁇ . 2 tlz/tlz mutants at caudal spinal levels.
• R, ' The number of Enl + VI neurons increases ⁇ 3-fold in the ventral spinal cord of Nkx ⁇ . 1-/"mutants, and ectopic Evxl/2 + cells are detected in position of the pMN domain in these mice (see also Sander et al . , 2000) .
• 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, Nkx ⁇ . 1 '/' ; Nkx ⁇ . 2 + tI &nd Nkx ⁇ . l ;Nkx ⁇ . 2 tlz/tlz embryos.
• Nkx6 and Dbx proteins 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 Nkx ⁇ 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.
• Nkx6.2 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
• cNkx6.2 chick amino acid sequence of Nkx6.2 protein.
• C cytosine
• A adenosine
• T thymidine
• G guanosine
• 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.
• the nucleic acid introduced into the stem cell incorporates into the chromosomal DNA of the stem cell .
• the nucleic acid is introduced by transfection or transduction.
• the ventral neuron is a motor neuron, a V2 neuron or a V3 neuron.
• nucleic acid refers to either DNA or RNA, including complementary DNA (cDNA) , genomic DNA and messenger RNA (mRNA) .
• 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.
• 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
• 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 .
• protein As used herein, "protein”, “peptide” and “polypeptide” are used to denote two or more amino acids linked by a peptidic bond between the ⁇ -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 ⁇ 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.
• MAP branched multiple antigen peptide
• 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.
• 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 .
• 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.
• the motor neuron degenerative disease is amyotrophic lateral sclerosis or spinal muscular atrophy.
• 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.
• 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 nucleic acid is DNA.
• the nucleic acid is RNA.
• the size fractionation in step (c) is effected by a polyacrylamide or agarose gel .
• the detectable marker is radioactive isotope, enzyme, dye, biotin, a fluorescent label or a chemiluminescent label.
• the motor neuron degenerative disease is amyotrophic lateral sclerosis or spinal muscular atrophy.
• detectable marker includes but is not limited to a radioactive label, or a calorimetric, a luminescent, or a fluorescent marker.
• 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.
• 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.
• 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.
• 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.
• the nucleic acid introduced into the stem cell incorporates into the chromosomal DNA of the stem cell.
• the nucleic acid is introduced by transfection or transduction.
• 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.
• 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.
• 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 .
• the suitable sample is spinal fluid.
• the nucleic acid is DNA.
• the nucleic acid is RNA.
• a null mutation in Nkx ⁇ . 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.
• 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). Nkx ⁇ . l was detected with a rabbit antiserum (Briscoe et al . 1999).
• Nkx ⁇ . l To define the role of Nkx ⁇ . l in neural development, we compared patterns of neurogenesis in the embryonic spinal cord and hindbrain of wild-type mice and mice lacking Nkx ⁇ . l (Sander et al . 1998). In wild-type embryos, neural expression of Nkx ⁇ . 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 Nkx ⁇ . l persists until at least E12.5 ( Figures IB, 1C; data not shown). Nkx ⁇ .
• Nkx ⁇ . l has a general role in restricting Gshl/2 expression.
• the signals that promote ventral Gshl/2 expression in Nkx ⁇ . l mutants remain unclear, but could involve factors other than Shh that are secreted by the notochord (Hebrok et al . 1998) .
• Nkx ⁇ . 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) .
• V2 interneurons et al. 1991
• Ericson et al . 1997 Qiu et al . 1998
• Pierani et al . 1999; Figures 2A-2D We examined whether the generation of any of these neuronal classes is impaired in Nkx ⁇ . l mutants, focusing first on the generation of motor neurons. In Nkx ⁇ .
• Nkx ⁇ . l mutants The persistence of some spinal motor neurons in Nkx ⁇ . 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 Nkx ⁇ . 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 Nkx ⁇ .
• MMC median
• LMC lateral
• Nkx ⁇ . l is expressed by the progenitors of both somatic and visceral motor neurons ( Figures 3E, 3F; data not shown) .
• Figures 3E, 3F We therefore examined whether the loss of Nkx ⁇ . l might selectively affect subsets of cranial motor neurons.
• 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 Nkx ⁇ . l mutant embryos may therefore reflect the dominant activities of Nkx2. 2 and Nkx2. 9 within these progenitor cells.
• 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 Nkx ⁇ . l mutants at spinal cord levels ( Figure 4B) , although at hindbrain levels of Nkx ⁇ . l mutants -50% of V2 neurons persisted (data not shown) .
• the elimination of Nkx ⁇ . 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 Nkx ⁇ . 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 Nkx ⁇ . l mutants, we examined whether there might be a corresponding expansion in the domain of generation of VI neurons. In Nkx ⁇ .
• the ventral expansion in Dbx2 expression is accompanied by a selective switch in interneuronal fates, from V2 neurons to VI neurons.
• some neurons within the ventral spinal cord of Nkx ⁇ . 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).
• 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).
• Nkx ⁇ The role of Nkx ⁇ . 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 Nkx ⁇ .
• 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) .
• the evolutionary origin of regional pattern along the dorsoventral axis of the central nervous system may predate the divergence of invertebrate and vertebrate organisms.
• class I and II 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) .
• Shh Shh signalling
• mice mutants have 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).
• 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).
• 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) .
• Nkx2.2 and Nkx2.9 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, Nkx ⁇ .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).
• mice lacking Nkx ⁇ . l function which exhibit a virtually complete failure in V2 interneuron generation (Sander et al . , 2000) .
• Nkx ⁇ . 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.
• Nkx ⁇ . l also known as Nkx ⁇ B or Gtx
• Nkx ⁇ B also known as Nkx ⁇ B
• 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 Nkx ⁇ gene pair prompted us to address three poorly resolved aspects of ventral neural patterning.
• Nkx6.2 functions as a class II repressor homeodomain protein.
• Our analysis of Nkx ⁇ mutants further indicates that the duplication of an ancestral Nkx ⁇ 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.
• 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.
• Nkx6.2 and Nkx ⁇ .l A pair of closely-related homeodomain proteins that function as transcriptional repressors, Nkx6.2 and Nkx ⁇ .l, are expressed by progenitor cells in overlapping domains of ventral spinal cord.
• Nkx6.2 and Nkx ⁇ .l A pair of closely-related homeodomain proteins that function as transcriptional repressors, Nkx6.2 and Nkx ⁇ .l, are expressed by progenitor cells in overlapping domains of ventral spinal cord.
• 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.
• 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
• Mouse Nkx ⁇ . 2 genomic clones were isolated from a 129/ ⁇ la 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/ ⁇ la) 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.
• mice 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 Nkx ⁇ . l mutant mice have been described previously (Sander et al . 2000). Compound Nkx ⁇ mutant mice were obtained by crossing Nkx ⁇ .2 + t2z ; Nkx ⁇ .2 +A double heterozygous mice. Genotyping was performed using Southern blot analysis.
• Mouse Nkx ⁇ . 2 was isolated by PCR (Komuro et al . , 1993) and chick Nkx ⁇ . 2 from a chick spinal cord library (Basler et al . , 1993) using mouse Nkx ⁇ . l and Nkx ⁇ . 2 as probes.
• cDNAs encoding full-length mouse and chick Nkx ⁇ . 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.
• 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-
• Nkx ⁇ 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 .
• 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) .
• 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) .
• Nkx6.2 + cells coexpressed Dbx2 ( Figure 10E)
• 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) .
• individual Nkx6.2 + cells did not coexpress Dbxl, although they did express Dbx2 ( Figure 10E-G) .
• 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).
• Nkx ⁇ . l and Nkx ⁇ .2 are ' initially coexpressed in a broad ventral domain (Cai et al . , 1999; data not shown) .
• Nkx ⁇ .2 expression persists in ventral progenitor cells, with the consequence that the expression of Nkx ⁇ . 2 and Nkx ⁇ . l also overlaps at later developmental stages ( Figure 10H, I) .
• expression of chick Nkx ⁇ . 2 is also detected in a thin stripe of cells dorsal to the limit of Nkx ⁇ . l expression, within the pi domain ( Figure 10H) .
• 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).
• 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).
• 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.
• I proteins Pax7, Dbx2 , Irx3 and Pax6 were similar in Nkx ⁇ .2 t i z /t iz f Nkx ⁇ . 2 +/tlz , and wild type embryos ( Figure 12B-D, G-I; data not shown) .
• 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.
• V0 neurons derive from pi progenitors in the absence of
• Nkx6.2 function. Conversely, the total number of Enl + VI neurons generated in Nkx ⁇ . 2 tlz / tlz embryos was reduced by -50%
• Nkx6.1 Repression of Nkx6.2 by Nkx6.1 underlies Nkx ⁇ gene redundancy in spinal motor neuron generation
• Nkx ⁇ . l and Nkx ⁇ . 2 To motor neuron and V2 neuron generation.
• 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 Nkx ⁇ . l mutant mice V2 neurons are eliminated (Briscoe et al . , 2000; Sander et al . , 2000).
• 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 Nkx ⁇ . 2 tlz/tlz embryos (see Figure 14G) .
• 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).
• Nkx6.2 negatively regulates its own expression level within pi domain progenitors ( Figure 11D, G, J) .
• 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 .
• Nkx ⁇ . l '/ - embryos expression of Nkx6.2 was detected in virtually all progenitor cells within the p2 and pMN domains
• 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.
• 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).
• 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) .
• a high level of expression of Nkx6.2 is not compatible with the generation of either VO or VI neurons ( Figure 130, P) .
• Nkx6.2 has a role in motor neuron generation in Nkx ⁇ . l mutant mice by testing the impact of removing Nkx6.2 as well as Nkx6.1 on the generation of spinal motor neurons. In Nkx ⁇ . 2 tlz/tlz embryos there was no change in the number of motor neurons generated at any level of the spinal cord or hindbrain
• 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 Nkx ⁇ . l and Nkx ⁇ . 2 compound mutants.
• Nkx ⁇ . l- / - embryos examined at el ⁇ .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).
• Nkx ⁇ .
• Nkx ⁇ . l single and Nkx ⁇ . l-/- ; Nkx ⁇ . 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 Nkx ⁇ compound mutants backgrounds, the domain of expression of Dbxl was unchanged
• 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 Nkx ⁇ mutant background, many of the residual Isll/2 + , HB9 + neurons transiently expressed Evxl ( Figure 16H, I) . Thus, under conditions of reduced Nkx ⁇ 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.
• Our results imply that the duplication of an ancestral Nkx ⁇ gene confers both redundant and distinct roles for Nkx6.1 and Nkx6.2 in ventral neuronal patterning.
• 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 Nkx ⁇ . l mutant mice, points to the existence of an Nkx6.1-independent pathway of motor neuron generation.
• 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 Nkx ⁇ . 2 mutant mice. Nevertheless, the activity of Nkx6.2 is responsible for the efficient generation of spinal motor neurons in Nkx ⁇ . l mutants.
• 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 .
• the chick Nkx ⁇ . 2 gene is not subject to repression by Nkx6.1, permitting its persistent expression in p3 , pMN and p2 domain progenitor cells.
• 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.
• the repression of pO identity and VO neuron fate is accomplished by Nkx6.2.
• ventral to the pl/p2 domain boundary it is Nkx6.1 that prevents Dbxl expression and VO neuronal generation.
• 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).
• Nkx6.1 and Nkx ⁇ .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 .
• Nkx6.1 and Nkx6.2 are 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.
• Nkx ⁇ .1 suppresses pi progenitor identity through repression of Dbx2 and Nkx6.2 expression, in this way ensuring the generation of Chxl0 + V2 neurons.
• Nkx ⁇ .2 in contrast, occupies the pi domain, where it is coexpressed with Dbx2.
• 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) .
• Nkx6.1 completely represses Nkx6.2
• Nkx6.2 exerts an incomplete negative regulation of its own expression in pl domain progenitors.
• Nkx6.1 is evidently a better repressor of Nkx6.2 than is Nkx6.2 itself.
• 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 Nkx ⁇ .2. Consistent with this view, Nkx6.2 fails to repress Dbx2 expression completely from ventral progenitors in Nkx ⁇ . 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).
• 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.
• 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.
• the different Nkx6 protein activity levels within ventral progenitor cells are a critical determinant of ventral neuronal fate.
• Nkx6 activity pO progenitors
• pi progenitors cells that express an intermediate Nkx6 activity level
• pMN and p2 progenitors cells that express a high Nkx6 activity level
• Figure 17 motor neuron or V2 fate
• 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 Nkx ⁇ 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 .
• Nkx ⁇ . l- 1 - ; Nkx ⁇ . 2 +/tlz mutants 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 Nkx ⁇ 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
• 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 Nkx ⁇ mutants.
• Such two-tiered repression is, in principle, necessary to specify neuronal fate through transcriptional derepression.
• repression of Dbxl and Dbx2 should be sufficient to derepress motor neuron subtype determinants such as MNR2 and Lim3 in pMN progenitors.
• 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 Nkx ⁇ double mutants.
• 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 .
• Nkx ⁇ -li ' k.e gene exists in the mouse, but this gene is not expressed in the spinal cord of wild type or Nkx ⁇ mutant embryos (E. Anderson and J. Ericson, unpublished data) , and thus its activity appears not to account for the residual motor neurons generated in Nkx ⁇ double mutants.
• the detection of residual motor neurons in Nkx ⁇ double mutants also provides evidence that Nkx ⁇ proteins do not have essential functions as transcriptional activators during motor neuron specification, further supporting their critical role as repressors.
• the present studies and earlier work on neurogenesis in the ventral spinal cord (Ericson et al . , 1996; Thaler et al . , 1999; Arber et al .
• 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.
• 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
• 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.
• a homeodomain code specifies progenitor cell identity and neuronal fate in the ventral neural tube.
• Pax6 controls progenitor cell identity and neuronal fate in response to graded Shh signaling.
• Gtx a novel murine homeobox-containing gene, expressed specifically in glial cells of the brain and germ cells of testis, has a transcriptional repressor activity in vitro for a serum- inducible promoter.
• Evxl is a postmitotic determinant of VO interneuron identity in the spinal cord. Neuron 29, 385-399.

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