Note: Descriptions are shown in the official language in which they were submitted.
CA 02419851 2003-02-18
WO 02/18545 PCT/USO1/27256
-1-
GENETIC DEMONSTRATION OF REQUIREMENT FOR
NKX6.1, NKX2.2 AND NKX6.2 IN VENTRAh NEURON GENERATION
This application is a continuation-in-part of U.S. Serial
No. 09/654,462, filed September l, 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
CA 02419851 2003-02-18
WO 02/18545 PCT/USO1/27256
-2-
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
CA 02419851 2003-02-18
WO 02/18545 PCT/USO1/27256
-3-
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, Nkx6.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 Nkx6.1 is expressed
by ventral progenitors that give rise to motor (MN), V2,
and V3 neurons. Mice carrying a null mutation of Nkx6.1
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 Vl neuronal subtype. Together,
these findings indicate that Nkx6.1 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.
CA 02419851 2003-02-18
WO 02/18545 PCT/USO1/27256
-4-
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 probes) 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
CA 02419851 2003-02-18
WO 02/18545 PCT/USO1/27256
-5-
the nucleic acid probe is labeled with a detectable marker,
and hybridisation of the nucleic acid probes) 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
CA 02419851 2003-02-18
WO 02/18545 PCT/USO1/27256
-6-
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.
CA 02419851 2003-02-18
WO 02/18545 PCT/USO1/27256
BRIEF DESCRIPTION OF THE FIGURES
Figures 1A-lU
Selective changes in homeobox gene expression in ventral
progenitor cells in Nkx6.1 mutant embryos. (Figs. lA-1C)
Expression of Nkx6.1 in transverse sections of the ventral
neural tube of mouse embryos E9.5. (Fig. 1A) Expression of
Nkx6.1 is prominent in ventral progenitor cells and
persists in some post-mitotic motor neurons at both caudal
hindbrain, E10.5, (Fig. 1B) and spinal cord, E12.5, (Fig.
1C) levels. (Fig. 1D, and 1E) Summary diagrams showing
domains of homeobox gene expression in wild-type mouse
embryos (Fig. 1D) and the change in pattern of expression
of these genes in Nkx6.1 mutants (Fig. 1E), based on
analyses at E10.0 - E12.5. (Figs. lF-lI) Comparison of the
domains of expression of Nkx6.1 (Figs. 1F, 1J) D3ax2 (Figs.
1G, 1H, 1K, 1L) and Gshl (Figs. 1I, 1M) in the caudal
neural tube of wild-type (Figs. 1F-1I) and Nkx6.1 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. 1N, 1R), Pax7
(Figs. 1N, 1R), Nkx2.2 (Figs. 10, 1S), Pax6 (Figs. 1P, 1S),
Dbxl (Figs. 1P, 1T) and Nkx2.9 (Figs. 1Q, 1U) in wild-type
(Figs. 1N-1Q) or Nkx6.1 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 Nkx6.1 expression. Scale bar shown in J=
100~.m (Figs . lA-1C) ; 50~.m (Figs . 1F-1M) or 60~.m (Figs . 1N
1U) .
CA 02419851 2003-02-18
WO 02/18545 PCT/USO1/27256
_g_
Figure 2A-2T
Disruption of motor neuron differentiation in Nkx6.1 mutant
embryos. The relationship between the domain of Nkx6.1
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) Isl1/2 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 Nkx6.1 progenitor
domain. (Fig. 2D) ChxlO (green) V2 interneurons Coexpress
Lhx3 (red). Expression of Isll/2 (Figs. 2E, 2I), 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 Nkx6.1 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 Isl1/2 dorsal D2
interneurons. Scale bar shown in I - 60~,m (Figs. 2A-2D);
80~,m (Figs. 2E-2L) ; 120~.m (Figs. 2M-2T) .
Figures 3A-3J
Motor neuron subtype differentiation in Nkx6.1 mutant mice.
Depletion of both median motor column (MMC) and lateral
motor column (LMC) neurons in Nkx6.1 mutant mice.
Expression of Isll/2 (red) and Lxh3 (green) in E12.5 wilt-
type (Figs. 3A, 3C) and Nkx6.1 mutant (Figs. 3B,3D) mice
spinal cord at forelimb levels (Figs. 3E-3J). Motor neuron
CA 02419851 2003-02-18
WO 02/18545 PCT/USO1/27256
-9-
generation at caudal hindbrain level (Figs. 3E, 3F) Nkx6.1
expression in progenitor cells and.visceral motor neurons
in the caudal hindbrain (rhombomere [r] 7/8) of E10.5-E11
wild- type (Fig. 3E) Nkx6.1 mutant (Fig. 3F) mice. HB9
expression in hypoglossal motor neurons in E10.5-E11 wild-
type mice (Fig. 3G) and Nkx6.1 mutant (Fig. 3H) mice.
Coexpression of Isll (green) and Phox2a/b (red) in wild-
type (Fig. 3I) or Nkx6.1 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).
Fiaures 4A-4L
A switch in ventral interneuron fates in Nkx6.1 mutant
mice. ChxlO expression in V2 neurons at rostral cervical
levels of E10.5 wild-type (Fig. 4A) and Nkx6.1 mutant (Fig.
4B) embryos. En1 expression by V1 neurons at rostral
cervical levels of wild-type (Fig. 4C) and Nkx6.1 mutant
(Fig. 4D) embryos. Pax2 expression in a set of interneurons
that includes V1 neurons ((Burrill et al. 1997) at caudal
hindbrain levels of wild-type (Fig. 4E) and Nkx6.1 mutant
(Fig. 4F) embryos. (Figs. 4G and 4H) Siml expression by V3
neurons in the cervical spinal cord of wild-type (Fig. 4G)
and Nkx6.1 mutant (Fig. 4H) embryos. Evxl expression by VO
~5 neurons at caudal hindbrain levels of wild-type (Fig. 4I)
and Nkx6.1 mutant (Fig. 4J) embryos. Enl (red) and Lhx3
(green) expression by separate cell populations in the
ventral spinal cord of E11 wild-type (Fig. 4K) and Nkx6.1
mutant (Fig. 4L) embryos. Scale bar shown in B - 60~m
(Figs. 4A-4D); 75~.m (Figs. 4E, 4F); 70~.m (Figs. 4G, 4J, 4H,
4J) , 35~,m (Figs . 4K and 4L) .
CA 02419851 2003-02-18
WO 02/18545 PCT/USO1/27256
-10-
Ficrure 5A-5B
Changes in progenitor domain identity and neuronal fate in
the spinal cord of Nkx6. 1 mutant embryos . (Fig . 5A) . In
wild-type mouse embryos, cells in the Nkx6.1 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 Nkx6.1
expression that also express Nkx2.2 and Nkx2.9. V1 neurons
derive from progenitor cells that express Dbx2 but not
Nkx6.l. (Fig. 5B). In Nkx6.1 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 V1 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 Nkx6.1 or by the ectopic expression of Dbx2.
Figure 6
Human Homeobox Protein Nkx6.l. NCBI Accession No. P78426.
(moue, H. et al., "Isolation, characterization, and
chromosomal mapping of the human Nkx6.l gene (NKX6a), a new
pancreatic islet homeobox gene" Genomics 40(2):367-370,
1997). Amino acid sequence of human homeobox protein
Nkx6.l.
CA 02419851 2003-02-18
WO 02/18545 PCT/USO1/27256
-11-
Figure 7
Human NK Homeobox Protein (Nkx6.1) gene, exon 1. NCBI
Accession No. U66797. Segment 1 of 3 (moue, H. et al.,
"Isolation, character-ration, and chromosomal mapping of
the human Nkx6.l gene (NKX6a), a new pancreatic islet
homeobox gene" Genomics 40(2):367-370, 1997). Nucleic acid
sequence encoding human homeobox protein Nkx6.l, bases 1-
682.
Figure 8
Human NK Homeobox Protein (Nkx6.1) gene, exon 2. NCBI
Accession No. U66798. Segment 2 of 3 (moue, 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.l, bases 1-
185.
Figure 9
Human NK Homeobox Protein (Nkx6.1) gene, exon 3 and
complete cds. NCBI Accession No. U66799. Segment 3 of 3
(moue, 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.l, bases 1-273. Protein encoded is shown in
Fig. 7.
Figure 10
Expression of Nkx6.2 and Nkx6.l in developing mouse and
chick spinal cord. (A) At e8.5, Nkx6.2 and Nkx6.1 are
CA 02419851 2003-02-18
WO 02/18545 PCT/USO1/27256
-12-
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.l boundary. (D-G) Comparative patterns
of expression of Nkx6.2, Nkx6.l, Dbx2, Dbx1 and Pax7 in the
intermediate region of e10.5 mouse spinal cord. (H-L)
Expression pattern of Nkx6.2, Nkx6.l, Dbx2, Dbx1 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 Nkx6.2 and Dbx2 expression in p1 domain cells
in Nkx6.2 mouse mutants. (A) Diagram of the targeting
construct (i) used to replace the coding sequence of Nkx6.2
(ii) with a tau-lacZ PGK-neo cassette (iii). Red bar
indicates region used as probe in genotyping. (B-D) Sagital
view of e10.5 spinal cord showing LacZ expression, detected
by X-gal staining, in wild type (wt) (B) Nkx6.2+~tlZ (C) and
Nkx6. 2tlZ/tlZ (D) embryos . (E-G) Nkx6 . 2 and LacZ expression in
the p1 domain of wt (E) , Nkx6.2+~tlZ (F) , and Nkx6.2tlZ/tl~ (G)
embryos at e10.5. (H-J) In situ hybridization with a 5'-UTR
probe shows that Nkx6.2 is elevated
expression of in the p1
domain of Nkx6. 2tlz~tlz embryos(J) , compared with (H)
wt or
Nkx6.2~~tlZ (I) embryos. (K-M) Expression of Dbx2 is up
regulated ~2- fold in cells (yellow
within the
p1 domain
bracket) in Nkx6.2tlZ~tlZ s (M) , compared withwt (K)
embryo ,
or Nkx6.2+~tlZ (L) embryos. Abbreviations in ( A) :
H=
CA 02419851 2003-02-18
WO 02/18545 PCT/USO1/27256
-13-
HindIII, B= BamHI, N= NCOI, S= SphI, A=ACCI.
Fiaure 12
A partial switch from V1 to VO neuronal fate in Nkx6.2
mutant mice. (A-E) Expression of Nkx6.2 (A), Nkx6.1 (C, D),
Dbx1 (B, C, E), and Pax7 (B) appears normal at caudal
hindbrain levels of e10.5 Nkx6.2+~tlz embryos. The
expression of Nkx6.1 (D) and Dbx1 (E) abuts the ventral and
dorsal boundaries of LacZ expression.(F-J) In e10.5
Nkx6.2tlZ~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 p1 domain. Many ventral ectopiC
Dbx1+ cells in Nkx6.2t12~t1z embryos express LacZ (J) . (K-M)
Evx1/2+ VO neurons are generated dorsal to En1+ V1 neurons
(K) and LacZ+ cells (M) in Nkx6. 2+~tlZ embryos . En1+ neurons
express LaCZ in Nkx6. 2+~t'Z (L) and Nkx6. 2tlz/tlz (0) e~ryos .
(N-P) Evx1/2~ VO neurons are generated in increased numbers
and at ectopiC ventral positions in the caudal hindbrain of
Nkx6.2tlZ~tlZ embryos. (N) The number of Enl~ V1 neurons is
reduced and the remaining Enl+ neurons are intermingled
with ectopiC Evxl/2+ cells. (P) Many Evx1/2~ neurons in
Nkx6.2tlz/tlz e~ryos Co-express LaCZ. (Q) Quantitation of
Evx1/2+ V0, and En1+ V1, neurons at the caudal hindbrain of
Nkx6.2+~tlZ and Nkx6.2tlZ/tlZ embryos at e10.5. Counts from 12
sections, mean + S.D. In panels (A-P), the white arrowhead
indicates the p0/p1 boundary.
Figure 13
Deregulated expression of Nkx6.2 in Nkx6.1 mutant mice, and
similar patterning activities of Nkx6 proteins in chick
neural tube. (A) In e10.5 rNt embryos, Nkx6.2 expression is
CA 02419851 2003-02-18
WO 02/18545 PCT/USO1/27256
-14-
confined to the p1 progenitor domain. (B) In Nkx6.1+~-
embryos, scattered Nkx6.2+ cells are detected in the p2,
pMN and p3 domains. (C) In Nkx6.1-~-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 Dbx1 (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 p0 and p1 progenitor domains promotes
the ectopic generation of ChxlO~ V2 neurons (J, K, O, P)
and suppresses Evx1/2~ VO (K, P) and Enl+ V1 (J, O) neurons.
Figure 14
The deregulated expression of Nkx6.2 underlies motor neuron
generation in Nkx6.1 mutants. (A) In e10.5 wt embryos,
Nkx6.2 expression is confined to the p1 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 Nkx6.2
embryos . (C, D) In Nkx6. 1W and Nkx6. 1-W ; Nkx6. 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 Nkx6.1-~-; Nkx6.2tlZ~tlZ embryos. (F, G)
HB9+, Isl1/2~ motor neurons are generated in normal numbers
in Nkx6. 2tlZ~tlZ embryos . The number of triotor neurons is
reduced by w60% in Nkx6.1-~- embryos (H), by ~80o in Nkx6.1-
~-; Nkx6 . 2+~tlZ embryos ( I ) and by >90 o in Nkx6. 1-W ; Nkx6. 2~lZ~tlZ
at cervical levels of e10.5 spinal cord (J). (K-M) At e12,
the number of motor neurons of medial (MMC) (Isl1+, Lim3+)
and lateral (LMC) (Isll~) subtype identity is reduced in
CA 02419851 2003-02-18
WO 02/18545 PCT/USO1/27256
-15-
similar proportions in Nkx6. 1'~- and Nkx6. 1 '~-; Nkx6. 2 tlZltlZ
embryos . Lim3+ V2 neurons are missing in Nkx6. 1-~- embryos
and Nkx6. 1W'; Nkx6. 2tlZltlZ embryos at this stage . (N-P)
Quantitation of HB9+ and Isll/2+ motor neurons at cervical
and lumbar levels in wt, Nkx6.2 and Nkx6.1 single mutants
and in Nkx6.2; Nkx6.1 compound mutants at e10 and e12.
Counts from 12 sections, mean ~+- S . D .
Figure 15
Changes in class I protein expression and ventral
interneuron generation in Nkx6 mutants. (A-E) Expression of
Nkx6.l and Nkx6.2 in the spinal cord in different Nkx6
mutant backgrounds at e10.5. (F-J) Spatial patterns of Pax7
and Dbx2 expression in different Nkx6 mutant backgrounds.
Note that the level of Dbx2 expression in the pMN domain of
Nkx6.l'~-; Nkx6.2+~t~Z 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 Olig2 possesses these
properties (Novitch et al., 2001).
(K-O) Spatial patterns of expression of Pax7 and Dbxl in
different Nkx6 mutant backgrounds. (P-T) Spatial patterns
of generation of Evx1/2+ VO neurons and Enl+ Vl neurons in
different Nkx6 mutant backgrounds. (Q) The generation of VO
neurons expands ventrally into the p1 domain in Nkx6.2tlZltlZ
mutants at caudal spinal levels. (R, A') The number of En1+
V1 neurons increases ~3-fold in the ventral spinal cord of
lVkx6.lWmutants, and ectopic Evx1/2+ 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+ VO neurons and a loss of Enl+ V1 neurons in the
CA 02419851 2003-02-18
WO 02/18545 PCT/USO1/27256
-16-
ventral spinal cord of Nkx6.lW;Nkx6.2 */tlzand Nkx6.1 w
;Nkx6.2tlZltlZ embryos. (U, V, Z) The generation of Evxl/2+ VO
neurons correlates with the pattern of expression of Dbxl
in progenitors in wt, Nkx6. 2tlZ~tzZ and Nkx6. 1W-; Nkx6. 2tlz~tlz
mutant backgrounds. Note that only the most lateral
progenitor cells express Dbx1 in Nkx6.l'~'; Nkx6.2tlz~tlZ
embryos, suggesting that expression of Dbx1 in more
medially-positioned progenitors is repressed by an as yet
undefined gene. (X, Y) EctopiC ventral Evxl* VO neurons
derive from Dbx1- progenitors in Nkx6.lW and Nkx6.lW
Nkx6. 2+~tlz mutant embryos . ChxlO* V2 neurons are generated
at normal numbers in Nkx6.2tlZ~tl2 mutants, but are missing
at spinal cord levels in Nkx6.lW', Nkx6.lW';Nkx6.2*~tlZ and
Nkx6. 1-W ; Nkx6. 2tlZ~tlz mutants (A' ; Figure 5 , see Sander et
al., 2000).
Figure 16
Dissociation of Dbx expression and VO neuronal fate in mice
with reduced Nkx6 protein activity. (A) In e10.0 wt
embryos, p0 progenitor cells express Dbx1 and generate
Evxl/2* VO neurons. (B) In e10.0 Nkx6.1-~';Nkx6.2+~tlz
embryos there is no change in the domain of expression of
Dbxl, but Evx1/2* VO neurons are generated in lateral
positions, along much of the ventral neural tube. (C, D) In
Nkx6.1-~-; Nkx6.2*~tlz embryos examined at e10.0 many ectopiC
ventral Evx1/2* neurons express LacZ. Framed area in (C) is
shown at high magnification in (D) and indicates Evx1/2*
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 D.bx2
mRNA. (F) Summary of Dbxl expression and VO neuron
CA 02419851 2003-02-18
WO 02/18545 PCT/USO1/27256
-17-
generation in wt, Nkx6.1-l-;Nkx6.2 +/tl~nd Nkx6.1
Nkx6. 2t12/tlZ embryos . The dissociation of Dbx1 and Evxl/2
expression in Nkx6.1-l-;Nkx6.2+ltlZ embryo suggests that
reduced Nkx6 repressor activity is sufficient to repress
Dbx1 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 ) , Dbx1 (D1 ) and Dbx2 (D2 ) in the
ventral neural tube of wild type (wt) and different Nkx6
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 cds. 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 =
CA 02419851 2003-02-18
WO 02/18545 PCT/USO1/27256
-18-
mouse amino acid sequence of Nkx6.3 protein; rNkx6.l = 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.
CA 02419851 2003-02-18
WO 02/18545 PCT/USO1/27256
-19-
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 W=trp=tryptophan
Y=tyr=tyrosine V=val=valine
B=asx=asparagine or aspartic acid
Z=glx=glutamine or glutamic 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
CA 02419851 2003-02-18
WO 02/18545 PCT/USO1/27256
-20-
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
CA 02419851 2003-02-18
WO 02/18545 PCT/USO1/27256
-21-
protein. The nucleic acid includes both the full length
nucleic acid sequence as well as non-full length sequences.
Tt 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 a-carboxyl group of one amino acid and the
a-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 Y-
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 (Tam 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
CA 02419851 2003-02-18
WO 02/18545 PCT/USO1/27256
-22-
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; MXK~T2; MXKNl; 535304; T28492; AAF33780;
P01524; P01523; 9GSSB; 17GSB; 1BH5D; 4GSSB; 1PGTB; 1GSUB;
1GNWB; 2GLRB; lAGSB.
CA 02419851 2003-02-18
WO 02/18545 PCT/USO1/27256
-23-
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.
CA 02419851 2003-02-18
WO 02/18545 PCT/USO1/27256
-24-
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 probes) 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 probes) 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
CA 02419851 2003-02-18
WO 02/18545 PCT/USO1/27256
-25-
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
CA 02419851 2003-02-18
WO 02/18545 PCT/USO1/27256
-26-
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 a1.(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
CA 02419851 2003-02-18
WO 02/18545 PCT/USO1/27256
-27-
transcription factor Nkx6.2 protein in the stem cell so as
to thereby convert the stem cell into the ventral neuron.
Tn 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.l 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
CA 02419851 2003-02-18
WO 02/18545 PCT/USO1/27256
-28-
probe, which nucleic acid probe (i) is capable of
hybridizing with the nucleic acid of Nkx6.l 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
subj ect .
Tn 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.
CA 02419851 2003-02-18
WO 02/18545 PCT/USO1/27256
-29-
FIRST SERIES OF EXPERIMENTS
EXPERIMENTAL DETAILS
A. Materials and Methods
Generation of Nkx6.1 null mutation
A null mutation in Nkx6.1 was generated by using gene
targeting in 129-strain ES cells by excising an 800-by NotI
fragment containing part of axon 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-Wiemers and Gerfin-Moser
(1993). The 17bx2 riboprobe comprised the 5' EcoR1 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). Nkx6.1 was
detected with a rabbit antiserum (Briscoe et al. 1999).
Antisera against Shh, Pax7, Isl1/2, 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).
CA 02419851 2003-02-18
WO 02/18545 PCT/USO1/27256
-30-
B. Results and Discussion
To define the role of Nkx6.1 in neural development, we
compared patterns of neurogenesis in the embryonic spinal
cord and hindbrain of wild-type mice and mice lacking
Nkx6.1 (Sander et al. 1998). In wild-type embryos, neural
expression of Nkx6.1 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 Nkx6.1 persists until at
least E12.5 (Figures 1B, 1C; data not shown). Nkx6.1
expression was also detected in mesodermal cells flanking
the ventral spinal cord (Figures 1B, 1C) . To define more
precisely the domain of expression of Nkx6.l, we compared
its expressions with that of ten homeobox genes - Pax3,
Pax7, Gshl, Gsh2, Lrx3, Pax6, 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 Nkx6.1
expression is positioned ventral to the boundaries of four
genes expressed by dorsal progenitor cells: Pax3, Pax7,
Gshl and Gsh2 (Figures 1I, 1N; and data not shown). Within
the ventral neural tube, the dorsal boundary of Nkx6.1
expression is positioned ventral to the domain of Dbxl
expression and close to the ventral boundary of Dbx2
expression (Figures 1G, 1H, and 1P). The domain of Pax6
expression extends ventrally into the domain of Nkx6.1
CA 02419851 2003-02-18
WO 02/18545 PCT/USO1/27256
-31-
expression (Figure 10), whereas the expression of Nkx2.2
and Nkx2.9 overlaps with the ventral-most domain of Nkx6.1
expression (Figures 10, 1Q).
To address the function of Nkx6.1 in neural development, we
analyzed progenitor cell identity and the pattern of
neuronal differentiation in Nkx6.1 null mutant mice (Sander
et al. 1998). We detected a striking change in the profile
of expression of three homeobox genes, Dbx2, Gsh1 and Gsh2,
in Nkx6.1 mutants. The domains of expression of Dbx2, Gsh1
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 1K). 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 1L). Similarly, in
Nkx6.1 mutants, both Gsh1 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
Nkx6.1 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,
1S). We detected no change in the patterns of expression
of Pax3, Pax7, Dbxl, Irx3, Nkx2.2, or Nkx2.9 in Nkx6.1
mutant embryos (Figures 1R-lU; data not shown).
CA 02419851 2003-02-18
WO 02/18545 PCT/USO1/27256
-32-
Tmportantly, the level of Shh expression. by floor plate
cells was unaltered in Nkx6.1 mutants (Figures 1N and 1R).
Thus, the loss of Nkx6.1 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 1D, 1E). 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.l expression. Consistent with this view,
ectopic expression of Nkx6.1 represses Dbx2 expression in
chick neural tube (Briscoe et al. 2000). The detection of
sites of ectopic Gsh1/2 expression in the paraxial mesoderm
as well as the ventral neural tube, both sites of Nkx6.1
expression, suggests that Nkx6.1 has a general role in
restricting Gsh1/2 expression. The signals that promote
ventral Gsh1/2 expression in Nkx6.1 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 Nkx6.1 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 Nkx6.1 mutants, focusing first on
the generation of motor neurons. In Nkx6.1 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 (Artier et al. 1999; Tsuchida
CA 02419851 2003-02-18
WO 02/18545 PCT/USO1/27256
-33-
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 Nkx6.1 mutants
analyzed at E11-E12.5 (Figures 2M, 2N, 2Q, 2R), whereas
motor neuron number was reduced only by 50 0-75 o 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 p0 (Figures 2M-2T; data
not shown), indicating that the loss of Nkx6.1 activity
does not simply delay motor neuron generation. Moreover,
we detected no increase in the incidence of TUNEL+ cells in
Nkx6.1 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 Nkx6.1
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 Nkx6.1 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 Nkx6.1
mutants, as assessed by the number of motor neurons that
coexpressed Isll/2 and Lhx3 (defining MMC neurons, Figures
CA 02419851 2003-02-18
WO 02/18545 PCT/USO1/27256
-34-
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 Nkx6.1 activity depletes the maj or subclasses of
spinal motor neurons to a similar extent.
At hindbrain levels, Nkx6.1 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
Nkx6.1 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 Nkx6.1 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 Isl1 and Phox2a (Briscoe et al.
1999; Pattyn et al. 1997) within ventrally generated motor
neurons (Figures 3I, 3J; data not shown). Moroever, at
rostral cervical levels, the generation of spinal accessory
motor neurons (Ericson et al. 1997) was also preserved in
Nkx6.1 mutants (data not shown). Thus, in the hindbrain
the loss of Nkx6.1 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
CA 02419851 2003-02-18
WO 02/18545 PCT/USO1/27256
-35-
Nkx2.9 (Briscoe et al. 1999). The preservation of cranial
visceral motor neurons in Nkx6.1 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 Nkx6.1 activity. V2
and V3 interneurons are defined, respectively, by
expression of ChxlO and Siml (Artier et al. 1999; Briscoe et
al. 1999; Figures 4A, 4G). A severe loss of ChxlO V2
neurons was detected in Nkx6.1 mutants at spinal cord
levels (Figure 4B), although at hindbrain levels of Nkx6.1
mutants 500 of V2 neurons persisted (data not shown). In
contrast, there was no change in the generation of Sim1 V3
interneurons at any axial level of Nkx6.1 mutants (Figure
4H). Thus, the elimination of Nkx6.1 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~ V1 interneurons derive from progenitor cells
located dorsal to the Nkx6.1 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 Nkx6.1 mutants, we examined whether there
might be a corresponding expansion in the domain of
generation of V1 neurons. In Nkx6.1 mutants, the region
that normally gives rise to V2 neurons and motor neurons
now also generated V1 neurons, as assessed by the ventral
shift in expression of the Enl and Pax2 homeodomain
CA 02419851 2003-02-18
WO 02/18545 PCT/USO1/27256
-36-
proteins (Figures 4B, 4C, 4E, 4F). Consistent with this,
there was a two- to threefold increase in the total number
of V1 neurons generated in Nkx6.1 mutants (Figures 4C, 4D).
Tn contrast, the domain of generation of Evxl/2 VO neurons,
which derive from the Dbx1 progenitor domain (Pierani et
al. 1999), was unchanged in Nkx6.1 mutants (Figures 4I,
4J). Thus, the ventral expansion in Dbx2 expression is
accompanied by a selective switch in interneuronal fates,
from V2 neurons to V1 neurons. In addition, we observed
that some neurons within the ventral spinal cord of Nkx6.1
mutants coexpressed the V1 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
l5 neurons in Nkx6.1 mutants, the ectopic program of V1
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 Nkx6.1 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 Nkx6.1 is expressed (Figure 5A), its activity is
required to promote motor neuron and V2 interneuron
generation and to restrict the. generation of V1
interneurons (Figure 5B). It is likely that the loss of
motor neurons and V2 neurons is a direct consequence of the
CA 02419851 2003-02-18
WO 02/18545 PCT/USO1/27256
-37-
loss of Nkx6.1 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 Nkx6.1
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
Nkx6.1 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 Nkx6.1
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
Nkx6.1 mutants could reflect the existence of a functional
homologue within the caudal neural tube.
The role of Nkx6.1 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 Nkx6.1 to function as a repressor
of the dorsally expressed Gsh1/2 homeobox genes parallels
the ability of Drosophila NK2 to repress Ind, a Gshl/2-like
CA 02419851 2003-02-18
WO 02/18545 PCT/USO1/27256
-38-
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.
CA 02419851 2003-02-18
WO 02/18545 PCT/USO1/27256
-39-
REFERENCES
1. S.A. Anderson, D.D. Eisenstat, L. Shi, J.L.
Rubenstein, Science 278:474-476 (1997).
2. S. Arber, B. Han, M. Mendelsohn, M. Smith, T.M.
Jessell, S. Sockanathan, Neuron 23:659-674 (1999).
3. J. Brisooe, et al., Nature 398:622-627 (1999).
4. J. Briscoe et al., Cell 101:435-445 (2000).
5. J.D. Burrill, L. Moran, M.D. Goulding, H. Saueressig,
Development 124:4493-4503 (1997).
6. H. Chu; C. Parras; K. White; F. Jimenez, Genes & Dev.
12:3613-3624 (1998).
7. J. Ericson, et al., Cold Spring Harb. Symp. Quant.
Biol. 62:451-466 (1997a).
8. J. Ericson et al., Cell 87:661-673 (1996).
9. J. Ericson, et al., Cell. 90:169-180 (1997b).
10. M.D. Goulding et al., EMBO J. 10:1135-47 (1991).
11. M. Hammerschmidt, A. Brook, A.P. MCMahon Trends
Genet. 13:14-21 (1997)
12. M. Hebrok, S.K. Kim, D.A. Melton, Genes & Dev. 12:
CA 02419851 2003-02-18
WO 02/18545 PCT/USO1/27256
-40-
1705-1713 (1998).
13. A. Lumsden, R., and Krumlauf, R. Science 274: 1109-
1115 (1996) .
14. M.P. Matise, A.L. Joyner, J. Neurosci. 17:7805-7816
(1998) .
15. J. A. McDonald, S. Holbrook, T. Isshiki, J. Weirs,
C.Q. Doe, D. M. Mellerick. Genes & Dev. 12:3603-3612
(1998) .
16. O. Pabst, H. Herbrand, H. H. Arnold, Mech. Dev. 73:
85-93 (1998) .
17. A. Pattyn, X. Morin, H. Cremer, C. Goridis, J.F.
Brunet, Development 124:4065-4075 (1997).
18. A. Pierani, S. Brenner-Morton, C. Chiang, T. M.
Jessell, Cell 97:903-915 (1999).
19. M. Qiu, K. Shimamura, L. Sussel, S. Chen, J. L.
Rubenstein, Mech. Dev. 72:77-88 (1998).
20. J.L. Rubenstein and Beachy, P.A. Curr. Opin.
Neurobiol. 8:18-26 (1998).
21. J.L. Rubenstein et al., Annu Rev Neurosci. 21:445-477
(1998) .
22. Sander, M. et al. Keystone symposium on vertebrate
CA 02419851 2003-02-18
WO 02/18545 PCT/USO1/27256
-41-
development. Steamboat Springs, Colorado (1998).
23. Schaeren-Wiemers, N, and Gerlin-Moser, A.
Histochemistry 100:431-440 (1993).
24. Sockanathan, S. and Jessell, T.M. Cell 94:503-514
(1998) .
25. L. Sussel, O. Marin, S. Kimura, J.L. Rubenstein,
Development 126:3359-3370 (1999).
26. Y. Tanabe, and T.M. Jessell, Science 274:1115-23
(1996) .
27. J. Thaler et al., Neuron 23:675-687 (1999).
28. T. Tsuchida, et al., Cell 79:957-970 (1994).
29. M. T. Valerius, H. Li, J.L. Stock, M. Weinstein, S.
Kaur, G. Singh, S.S. Potter, Dev. Dyn. 203:337-51
(1995).
30. J. B. Weiss, T. Von Ohlen, D.M. Mellerick, G.
Dressier, C.Q. Doe, M.P.SCOtt, Genes & Dev. 12:3591
3602 (1998) .
CA 02419851 2003-02-18
WO 02/18545 PCT/USO1/27256
-42-
SECOND SERIES OF EXPERIMENTS
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
CA 02419851 2003-02-18
WO 02/18545 PCT/USO1/27256
-43-
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-
CA 02419851 2003-02-18
WO 02/18545 PCT/USO1/27256
-44-
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, Nkx6.l, 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 Nkx6.l function, which exhibit a virtually
CA 02419851 2003-02-18
WO 02/18545 PCT/USO1/27256
-45-
complete failure in V2 interneuron generation (Sander et
al., 2000). Nkx6.1 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-
s 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 Nkx6.l, termed Nkx6.2 (also known as
Nkx6B 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 Nkx6 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 Nkx6.l during neural tube development, and
analysed mouse Nkx6 mutants to determine the respective
contributions of these two genes to neural patterning. We
show that Nkx6.2, like Nkx6.l, functions as a class II
CA 02419851 2003-02-18
WO 02/18545 PCT/USO1/27256
-46-
repressor homeodomain protein. Our analysis of Nkx6
mutants further indicates that the duplication of an
ancestral Nkx6 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 Nkx6 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 Nkx6.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
CA 02419851 2003-02-18
WO 02/18545 PCT/USO1/27256
-47-
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.
CA 02419851 2003-02-18
WO 02/18545 PCT/USO1/27256
-48-
EXPERIMENTAL DETAILS
A. Materials and Methods
Generation of Nkx6.2 mutant mice
Mouse Nkx6.2 genomic clones were isolated from a 129/01a
mouse genomic library. A targeting construct was
constructed by inserting a tau-lacZ/pGKneo cassette into a
5 kb 5' HindIII-NcoI fragment and a 2.7 kb 3' SphI-AccI
fragment. The linearized targeting construct was
electroporated into E14.1 (129/01a) ES cells. Cells were
selected with 6418 and screened by Southern blot analysis
using a 200 by 3' AccI 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 Nkx6.1 mutant mice have been
described previously (Sander et al. 2000). Compound Nkx6
mutant mice were obtained by crossing Nkx6.2+~tlZ~ Nkx6.l~W
double heterozygous mice. Genotyping was performed using
Southern blot analysis.
Chick in ovo electroporation
Mouse Nkx6.2 was isolated by PCR (Komuro et al., 1993) and
chick Nkx6.2 from a chick spinal cord library (Basler et
al., 1993) using mouse Nkx6.1 and Nkx6.2 as probes. cDNAs
encoding full-length mouse and chick Nkx6.2 were inserted
into a RCASBP(B) retroviral vector and electroporated into
CA 02419851 2003-02-18
WO 02/18545 PCT/USO1/27256
-49-
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.
Tmmunohistochemistry and in situ hybridization
histochemistrv
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-Lima (Ericson et al.; 1997), mAb Hb9
(Tanabe et al., 1998), rabbit anti-Isl1/2 (Tsuchida et al.,
1994), rabbit anti-ChxlO (Ericson et al., 1997), rabbit
anti-En1 (Davis et al., 1991), mAb anti-Evx1/2, rabbit anti-
Dbxl, rabbit anti-Dbx2 (Pierani et al., 1999), rabbit anti-
Nkx6.1 (Jorgensen 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), Nkx6.1
(Briscoe et al., 2000) and Nkx6.2. A mouse probe for the
5'UTR of Nkx6.2 comprised 346 by 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 Nkx6 class genes in ventral neuronal
CA 02419851 2003-02-18
WO 02/18545 PCT/USO1/27256
-50-
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 e10.0-e10.5, 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.l expression at the p1/p2 domain boundary
l5 (Figure 10D, E). Nkx6.2 was expressed predominantly within
the p1 domain, but scattered Nkx6.2~ cells were detected
within the p0 domain - the domain of expression of Pax7-,
Dbx1+ cells (Figure 10F). Within the p0 domain, however,
individual Nkx6.2~ cells did not coexpress Dbxl, although
they did express Dbx2 (Figure l0E-G). Thus, the scattered
Nkx6.2+ cells found at the dorsoventral level of the p0
domain exhibit a p1, rather than p0, progenitor cell
identity. Studies in chick have similarly shown that p0 and
p1 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, Nkx6.1 and Nkx6.2
are~initially coexpressed in a broad ventral domain (Cai et
al., 1999; data not shown). But in contrast to the mouse,
Nkx6.2 expression persists in ventral progenitor cells, with
the consequence that the expression of Nkx6.2 and Nkx6.1
CA 02419851 2003-02-18
WO 02/18545 PCT/USO1/27256
-51-
also overlaps at later developmental stages (Figure 10H, I).
Nevertheless, expression of chick Nkx6.2 is also detected
in a thin stripe of cells dorsal to the limit of Nkx6.1
expression, within the p1 domain (Figure 10H). Thus, in
both species, p1 progenitors Coexpress Nkx6.2 and Dbx2 and
exclude Nkx6.l.
Nkx6.2 Regulates VO and V1 Interneuron Fates by Repression
of Dbx1 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 p1/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 p1 and p0 progenitors led us to consider whether
Nkx6.2 might function as a class II protein that represses
Dbx1 expression, and thus help to establish the identity of
p1 progenitor cells and the fate of their Enl~ V1 neuronal
progeny.
To test this idea, we analysed the profile of expression of
class I and II homeodomain proteins in Nkx6.2 mutant
embryos. We inactivated the mouse Nkx6.2 gene by homologous
recombination in embryonic stem (ES) cells. A targeted
Nkx6.2 allele (Nkx6.2tiZ) was generated by replacing the
coding sequence of Nkx6.2 with a tauLacZ cassette (Figure
11A) . In the spinal cord of Nkx6.2+~tlZ embryos analysed at
CA 02419851 2003-02-18
WO 02/18545 PCT/USO1/27256
-52-
e10.5, expression of LacZ and Nkx6.2 coincided within the
p1 progenitor domain (see Figure 11E, F) . In Nkx6.2tlZ/tlz
embryos, the location of LacZ+ cells was also similar to
that in Nkx6.2+~tlZ embryos (Figure 11F, G) , but Nkx6. 2
protein_was not detected (Figure 11G). These data provide
evidence that the Nkx6.2t12 allele generates a null mutation,
and that disruption of the Nkx6.2 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 Nkx6.2tjZ~tlz, when compared with
Nkx6.2+~tlz, embryos (Figure 11B-D) . An elevation in level
of expression of the residual 5' Nkx6.2 transcript was also
detected in Nkx6.2tlz/tlz e~ryos (Figure 11H-J) . These
observations provide evidence that Nkx6.2 negatively
regulates its own expression level within p1 progenitor
cells.
We next analysed the pattern of expression of class I and
II homeodomain proteins in the spinal cord and caudal
hindbrain of Nkx6. 2tlz/tlz embryos . The domains of expression
of the class II proteins Nkx2.2 and Nkx6.l, and of the class
I proteins Pax7, Dbx2, Irx3 and Pax6 were similar in Nkx6.2
Nkx6.2+~tl~, 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 p1/p2 domain
boundary (data not shown), showing that establishment of the
p1 progenitor domain does not require Nkx6.2 function.
However, the level of Dbx2 expression in p1 domain
progenitors was increased two-fold in Nkx6.2tlZ~tlZ mutants
(Figure 11K-M), indicating that Nkx6.2 normally limits the
CA 02419851 2003-02-18
WO 02/18545 PCT/USO1/27256
-53-
level of Dbx2 expression in this domain.
We also detected a marked change in the pattern of
expression of the p0 progenitor cell marker Dbx1 in
Nkx6.2tlZ/tlZ embryos. At caudal hindbrain levels, the number
of ventral Dbx1+ progenitor cells increased 1.7- fold
(Figure 12F), and the domain of Dbx1+ cells expanded
ventrally, extending through the p1 domain to the dorsal
limit of Nkx6.1 expression (Figure 12H). Moreover, in
Nkx6. 2tlz/tzz embryos all of the ectopiC Dbx1 * cells found
within the p1 domain coexpressed LacZ (Figure 12J). Thus,
many progenitors within the p1 domain initiate Dbxl
expression in the absence of Nkx6.2 function. Nevertheless
in Nkx6.2t1zlt~z embryos, numerous LacZ+ progenitors still
lacked Dbxl expression (Figure 12J), implying the existence
of an Nkx6.2-independent means of excluding Dbxl expression
from p1 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 Dbx1 expression was detected at caudal spinal levels
(data not shown; see Figure 15). Taken together, these data
imply that within p1 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 ira.terneuron subtypes in
the ventral neural tube. In wild type embryos, Dbx1+,
Dbx2+, Nkx6.2' p0 progenitors generate Evx1/2+ VO neurons
(Pierani et al., 1999; 2001); Nkx6.2+, Dbx1', Dbx2+ p1
progenitors give rise to En1+ Vl neurons (Burrill et al.,
1997; Ericson et al., 1997), and Nkx6.1+, Irx3+, p2
CA 02419851 2003-02-18
WO 02/18545 PCT/USO1/27256
-54-
progenitors give rise to ChxlO+ V2 neurons (Ericson et al.,
1997; Briscoe et al., 2000). Dbx1 activity in p0
progenitors is required to promote VO and suppress V1
neuronal fates (Pierani et al., 2001). The ventral
expansion in Dbx1 expression in Nkx6.2tlZ~tlz embryos
therefore led us to examine whether the loss of Nkx6.2
function leads progenitor Cells within the p1 domain to
adopt a VO rather than V1 neuronal fate.
In the caudal hindbrain of Nkx6.2 tlZ~tl~ embryos examined at
e10.5, we detected a ~ two-fold increase in the number of
Evxl/2+ VO neurons and the domain of VO neuronal generation
expanded ventrally the normal position of the p1 domain
(Figure 12N). Consistent with this, many Evx1/2+ neurons
coexpressed LacZ (Figure 12P), showing directly that some
VO neurons derive from p1 progenitors in the absence of
Nkx6.2 function. Conversely, the total number of En1+ V1
neurons generated in Nkx6.2tlZ~tlZ embryos was reduced by ~50%
(Figure 12Q). The dorsoventral position of generation of
the remaining En1+ V1 neurons was similar in Nkx6.2t3z/t3~
embryos (Figure 12N), and these neurons expressed LaCZ
(Figure 120) showing directly that Nkx6.2+, Dbx2* p1
progenitor cells generate V1 neurons. The total number of
neurons generated from p1 domain progenitors, defined by
Cynl, TuJ1 and Lim1/2 expression was similar in Nkx6.2t1zltlz
and Nkx6.2+~tlZ embryos examined at e10.5 (data not shown) .
In addition, the number of TLTNEL+ cells was similar in
Nkx6.2tlZltlZ and Nkx6.2+~tlZ embryos (data not shown) . ChxlO+
V2 neurons and HB9~, Isl1/2+ motor neurons were present in
normal numbers and positions in Nkx6.2tlZ~tlz embryos (Figure
14; data not shown). Together, these findings show that the
CA 02419851 2003-02-18
WO 02/18545 PCT/USO1/27256
-55-
activity of Nkx6.2 within p1 progenitors promotes Vl
neuronal generation and helps to suppress the generation of
VO neurons, a finding consistent with the proposed role of
Nkx6.2 in repressing Dbx1 expression from p1 progenitors.
Repression of Nkx6.2 by Nkx6.1 underlies Nkx6 gene
redundancy in spinal motor neuron generation
V~le next addressed the respective contributions of Nkx6.1 and
Nkx6.2 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 Nkx6.1 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 Nkx6.1 mutants (Sander et al., 2000), revealing the
existence of an Nkx6.1-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 l0A-C), and
there is no change in the number of motor neurons generated
in Nkx6.2t1z~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 p1/p2 domain boundary, and the expression of Dbx2 is
repressed by Nkx6.l (Briscoe et al., 2000; Sander et al.,
2000). Second, Nkx6.2 negatively regulates its own
CA 02419851 2003-02-18
WO 02/18545 PCT/USO1/27256
-56-
expression level within p1 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 Nkx6.1+~- embryos examined at
e10.5 we detected a marked increase in the number of Nkx6.2+
cells within the p2 and pMN domains (Figure 13B). And in
Nkx6.1-~- embryos, expression of Nkx6.2 was detected in
virtually all progenitor cells within the p2 and pMN domains
(Figure 13C). Indeed, in Nkx6.1-~- 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 p1 domain (Figure 13C;
data not shown). Together, these data show that Nkx6.l
activity normally represses Nkx6.2 expression from p2 and
pMN progenitors in the mouse embryo.
In turn, these findings raised the possibility that in
Nkx6.lw 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 Nkx6.2 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 Nkx6.1 (Briscoe et al., 2000). These data show
that Nkx6.2 can induce ectopiC motor neurons when expressed
CA 02419851 2003-02-18
WO 02/18545 PCT/USO1/27256
-57-
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 p0 and p1
progenitor domains suppressed the generation of Evx1/2+ VO
and En1+ V1 neurons and promoted the generation of ChxlO+ V2
neurons (Figure 13J, K, O, P). Thus, a high level of
expression of Nkx6.2 is not compatible with the generation
of either VO or V1 neurons (Figure 130, P).
Based on these findings, we examined whether Nkx6.2 has a
role in motor neuron generation in Nkx6.1 mutant mice by
testing the impact of removing Nkx6.2 as well as Nkx6.l on
the generation of spinal motor neurons . In Nkx6. 2tlZ~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 Nkx6.l~~- mutants, the
number of spinal motor neurons was reduced by ~60% at
Cervical levels, but by only 25o at lumbar levels (Figure
14H,N,0, Sander et al., 2000) . In Nkx6.1-~-; Nkx6.2+~tlZ
embryos, motor neuron generation was reduced to ~25% of
controls at both cervical and lumbar levels (Figure 14I,N,0;
data not shown) . In Nkx6.lW- ; Nkx6.2tlZ~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 Nkx6 double mutant embryos, residual motor neurons
were detected at e10 . 0 , and no further increase in motor
neuron number was evident at e12 (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
CA 02419851 2003-02-18
WO 02/18545 PCT/USO1/27256
-58-
in Nkx6 double mutants are generated prior to e10.
Together, these findings demonstrate that Nkx6.2 substitutes
for the loss of Nkx6.1 in spinal motor neuron generation,
and reveal a link between Nkx6 gene dosage and the incidence
of motor neuron generation.
A Dissociation in Neuronal Fate and Progenitor Cell Identity
in Nkx6 Mutant Mice
We next examined whether a reduction in Nkx6 gene dosage
results in ectopic Dbx protein expression and V1 and VO
neuron generation in the p2 and pMN domains of the ventral
spinal cord.
En1+ V1 neurons are normally generated from Dbx2+, Dbxl- p1
progenitor cells, and we therefore analysed the relationship
between Dbx2 expression and En1+ V1 neuronal generation in
Nkx6.1 and Nkx6.2 compound mutants. As reported previously
(Sander et al., 2000), in Nkx6.1-~- embryos examined at
e10.5, 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 Nkx6.IW embryos,
ectopic Enl~ neurons were generated in the p2 and pMN
domains of the ventral neural tube (Figure 15R). In Nkx6.1-~-
; Nkx6.2+~tlZ embryos, Dbx2 expression was detected at
intermediate levels in the pMN domain (Figure 15I), and in
Nkx6.1-W ; Nkx6.2tlZ~tz1 double mutant embryos, Dbx2 was
detected at uniformly high levels in the p2 and pMN domains
(Figure 15J). Strikingly, in these Nkx6.1 and Nkx6.2
compound mutant backgrounds, and despite the enhanced
ectopic expression of Dbx2, the number of ectopic ventral
CA 02419851 2003-02-18
WO 02/18545 PCT/USO1/27256
-59-
Enl+ V1 neurons was reduced rather than increased, when
compared with the number generated in Nkx6.1 single mutants
(Figure 15R, T).
Since Evxl+ VO neurons are normally generated from Dbx1+,
Dbx2+ p0 progenitors, we examined whether the reduction in
ectopic ventral En1+ V1 neuron generation at low Nkx6 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 Nkx6.1-~-; Nkx6.2tlZltlZ mutants,
scattered Dbxl~" cells were detected in the p2, pMN and p3
domains (Figure 150), and ectopic ventral Evx1/2+ VO neurons
were detected throughout the ventral neural tube (Figure
15T, ~). Thus, in Nkx6 double mutants, the loss of Vl
neurons is associated with the ectopic ventral expression
of Dbx1 and the generation of ectopic VO neurons.
But in Nkx6.1 single and Nkx6.1-~- ; Nkx6.2+~tlz compound
mutant backgrounds, the normal link between expression of
Dbx1 in progenitor cells and the generation of Evx1/2+ VO
neurons was severed. In both these Nkx6 compound mutants
backgrounds, the domain of expression of Dbx1 was unchanged
(Figure 15M, N): a result that can be accounted for by the
maintained expression of Nkx6.2 within the p1 domain, and
the deregulated expression of Nkx6.2 within the p2 and pMN
domains. Nevertheless, Evx1/2+ 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 VO neurons were
generated from the position of the p2 and pMN domains, or
CA 02419851 2003-02-18
WO 02/18545 PCT/USO1/27256
-60-
whether they simply migrated ventrally from a more dorsal
position of origin. Ectopic ventral Evx1/2+ VO neurons were
detected as early as e10.0 (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
Evx1/2+ VO neurons are generated from the pMN domain in
Nkx6.IW'; Nkx6.2+~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 Dbx1 during
ectopic VO neuronal generation. These results therefore
provide evidence that even though Dbx1 activity is normally
required for the generation of VO neurons (Pierani et al.,
2001), under conditions in which Nkx6 gene dosage is
markedly reduced, VO neurons can be generated from
progenitor cells that lack Dbx1 expression.
Nevertheless, the pattern of ventral neurogenesis observed
in Nkx6.1-~' ; Nkx6.2+~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 Nkx6 mutant
background, many of the residual Isl1/2+, HB9+ neurons
transiently expressed Evx1 (Figure 16H, I). Thus, under
conditions of reduced Nkx6 gene dosage, progenitor cells at
the position of the pMN domain initially generate neurons
with a hybrid motor neuron/VO neuron identity.
CA 02419851 2003-02-18
WO 02/18545 PCT/USO1/27256
-61-
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.l and Nkx6.2, in ventral neural patterning. Our
results imply that the duplication of an ancestral Nkx6 gene
confers both redundant and distinct roles for Nkx6.l and
Nkx6.2 in ventral neuronal patterning. Vale 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.l 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
CA 02419851 2003-02-18
WO 02/18545 PCT/USO1/27256
-62-
the spinal Cord in Nkx6.1 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 Nkx6.2 mutant mice. Nevertheless, the activity
of Nkx6.2 is responsible for the efficient generation of
spinal motor neurons in Nkx6.1 mutants. The basis of this
redundant function resides in the derepression of Nkx6.2
expression in motor neuron progenitors in Nkx6.1 mutant
mice. Strikingly, Nkx6.2 is even derepressed in Nkx6.1+~-
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 Nkx6.2 gene is not subject to repression by Nkx6.l,
permitting its persistent expression in p3, pMN and p2
CA 02419851 2003-02-18
WO 02/18545 PCT/USO1/27256
-63-
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, Nkx6.2 activity
is held in reserve, through its repression by Nkx6.l.
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 p1
progenitors, the repression of p0 identity and VO neuron
fate is accomplished by Nkx6.2. But ventral to the p1/p2
domain boundary it is Nkx6.1 that prevents Dbx1 expression
and ZTO neuronal generation. Thus, Nkx6.1 is a potent
repressor of Dbx1 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 Nkx6.2 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
CA 02419851 2003-02-18
WO 02/18545 PCT/USO1/27256
-64-
their overlapping functions in the patterning of motor
neurons and VO neurons.
One factor that contributes to the opponent influence of
Nkx6.l and Nkx6.2 on the specification of Vl 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,
Nkx6.1 suppresses p1 progenitor identity through repression
of Dbx2 and Nkx6.2 expression, in this way ensuring the
generation of ChxlO+ V2 neurons. Nkx6.2, in contrast,
occupies the p1 domain, where it is coexpressed with Dbx2.
In p1 domain cells, Nkx6.2 promotes the generation of Enl+
V1 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 p1 progenitors initiate Dbx1 expression and acquire VO
neuron fate in the absence of Nkx6.2 function, raising the
possibility that Dbx2 may also have a role in repressing
Dbxl expression within p1 progenitors (see Pierani et al.,
1999) .
The second major factor that underlies the opponent
activities of Nkx6.1 and Nkx6.2 in V1 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.l completely represses Nkx6.2, whereas Nkx6.2 exerts
an incomplete negative regulation of its own expression in
CA 02419851 2003-02-18
WO 02/18545 PCT/USO1/27256
-65-
p1 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 p1 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 Nkx6.2. Consistent with this
view, Nkx6.2 fails to repress Dbx2 expression completely
from ventral progenitors in Nkx6.1 mutants. The fact that
Nkx6.2 is only a weak repressor of Dbx2 is critical for the
formation of the p1 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.l. 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 p1 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 p1 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
Dbx1 expression, thus promoting the generation of V1
neurons.
CA 02419851 2003-02-18
WO 02/18545 PCT/USO1/27256
-66-
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 (p0 progenitors) are directed to a
VO neuronal fate, cells that express an intermediate Nkx6
activity level (p1 progenitors) are directed to a Vl 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
Dere~ression
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) .
CA 02419851 2003-02-18
WO 02/18545 PCT/USO1/27256
-67-
Our analysis of Nkx6 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 17bx1 and Evxl are required sequentially during
the generation of VO neurons (Pierani et al., 2001; Moran-
Rivard et al . , 2 0 O1 ) . In Nkx6. 1'~' ; Nkx6. 2 +/tl~ mutants ,
however, the generation of Evx1/2+ VO neurons occurs in. the
absence of expression of Dbx1 by neural progenitor cells.
Dbxl expression is therefore dispensable for VO neuron
generation under conditions of reduced Nkx6 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 Dbx1 expression, but is below
the level required for repression of Evx1 expression. These
data therefore support the idea that Nkx6 proteins normally
inhibit VO neuronal fate by repressing the class I
progenitor homeodomain protein Dbxl, and independently by
repressing expression of the VO 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 Nkx6 mutants.
Such two-tiered repression is, in principle, necessary to
specify neuronal fate through transcriptional derepression.
In the case of Nkx6.l, for example, repression of Dbx1 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
CA 02419851 2003-02-18
WO 02/18545 PCT/USO1/27256
-68-
also represses the expression of VO determinants, Evx1
expression would also be initiated in differentiating motor
neurons, resulting in a hybrid neuronal phenotype. Indeed,
under conditions in which Nkx6 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 Nkx6 double
mutants. We find that in the absence of Nkx6 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 Nkx6-like gene exists in
the mouse, but this gene is not expressed in the spinal cord
of wild type or Nkx6 mutant embryos (E. Anderson and J.
Ericson, unpublished data), and thus its activity appears
not to account for the residual motor neurons generated in
Nkx6 double mutants. Importantly, the detection of residual
motor neurons in Nkx6 double mutants also provides evidence
that Nkx6 proteins do not have essential functions as
transcriptional activators during motor neuron
specification, further supporting their critical role as
repressors.
CA 02419851 2003-02-18
WO 02/18545 PCT/USO1/27256
-69-
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 Vl neuronal identity through an action in post-
mitotic neurons (Moran-Rivard et al., 2001), although it
remains unclear whether Evx1 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.
CA 02419851 2003-02-18
WO 02/18545 PCT/USO1/27256
-70-
REFERENCES
Arber, S., Han, B., Mendelsohn, M., Smith, M., Jessell, T.
M., and Sockanathan, S. (1999). Requirement for the homeobox
gene Hb9 in the consolidation of motor neuron identity.
Neuron 23, 659-674.
Basler, K., Edlund, T.,. Jessell, T.M., and Yamada, T.
(1993). Control of cell pattern in the neural tube:
Regulation of cell differentiation by dorsalin-l, a novel
TGF beta family member. Cell 73, 687-702.
Briscoe, J., Sussel, L., Serup, P., Hartigan-O'Connor, D.,
Jessell, T. M., Rubenstein, J. L., and Ericson, J. (1999).
Homeobox gene Nkx2.2 and specification of neuronal identity
by graded Sonic hedgehog signalling. Nature 398, 622-622.
Briscoe, J., Pierani, A., Jessell, T.M., and Ericson, J.
(2000). A homeodomain code specifies progenitor cell
identity and neuronal fate in the ventral neural tube. Cell
101, 435-445.
Briscoe, J., and Ericson, J. (2001). Specification of
neuronal fates in the ventral neural tube. Curr Opin
Neurobiol.l, 43-49.
Briscoe, J., Chen, Y., Jesseil, T.M. and Struhl, G. (2001).
A hedgehog-insensitive form of patched provides evidence for
direct long-range patterning activity of Sonic hedgehog in
the neural tube. Molecular Cell, In Press.
CA 02419851 2003-02-18
WO 02/18545 PCT/USO1/27256
-71-
Burrill, J.D., Moran, L., Goulding, M.D. and Saueressig, H.
(1997). Pax2 is expressed in multiple spinal cord
interneurons, including a population of EN1+ interneurons
that require Pax6 for their development. Development 124,
4493-4503.
Cai, J., St Amand, T., Yin, H., Guo, H., Li, G., Zhang, Y.,
Chen, Y., and Qiu, M. (1999). Expression and regulation of
the chicken Nkx-6.2 homeobox gene suggest its possible
involvement in the ventral neural patterning and cell fate
specification. rev Dyn 216, 459-468.
Davis, C.A., Holmyard, D.P., Millen, K.J., and Joyner, A.L.
(1991) Examining pattern formation in mouse, chicken and
frog embryos with an En-specific antiserum. Development 111,
287-298.
Eberhard, D., Jimenez, G., Heavey, B. and Busslinger, M.
(2000). Transcriptional repression by PaxS (BSAP) through
interaction with corepressors of the Groucho family. EGO J.
19, 2292-2303.
Ericson, J., Morton, S., Kawakami, A., Roelink, H., and
Jessell, T. M. (1996). Two critical periods of Sonic
Hedgehog signaling required for the specification of motor
neuron identity. Cell 87, 661-673.
Ericson, J., Rashbass, P., Schedl, A., Brenner-Morton, S.,
Kawakami, A., van Heyningen, V., Jessell, T. M., and
Briscoe, J. (1997). Pax6 controls progenitor cell identity
and neuronal fate in response to graded Shh signaling. Cell
CA 02419851 2003-02-18
WO 02/18545 PCT/USO1/27256
-72-
90, 169-180.
Hamburger, H. and Hamilton, H.L. (1953). A series of normal
stages in the development of the chick embryo. J. Morphol.
88, 49-92.
Hoshiyama, D., Suga, H., Iwabe, N,, Koyanagi, M., Nikoh, N.,
Kuma, K., Matsuda, F., Honjo, T., and Miyata, T. (1998).
Sponge Pax CDNA related to Pax-2/5/8 and ancient gene
duplications in the Pax family. J. Mol. Evol. 47, 640-648.
Jorgensen, M.C., Vestergard Petersen, H., Ericson, J.,
Madsen, O,D., Serup, P. (1999). Cloning and DNA-binding
properties of the rat pancreatic beta-cell-specific factor
Nkx6.l. FEBS Lett. 461, 287-294.
Kraut, R. and Levine, M. (1991). Mutually repressive
interactions between the gap genes giant and Kruppel define
middle body regions of the Drosophila embryo. 111, 611-621.
Komuro, I., SChalling, M., Jahn, L., Bodmer, R., Jenkins,
N.A., Copeland, N.G., Izumo, S. (1993). 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. EMBO 12, 1387-1401.
Lee, S., Davison, J.A., Vidal, S.M., Belouchi, A. (2001).
Cloning, expression and chromosomal location of NKX6B to
10q26, a region frequently deleted in brain tumors.
Mammalian Genome 12, 157-162.
CA 02419851 2003-02-18
WO 02/18545 PCT/USO1/27256
-73-
Mansouri, A., and Gruss, P. (1998). Pax3 and Pax7 are
expressed in Commissural neurons and restrict ventral
neuronal identity in the spinal cord. Mech Dev 78, 171-178.
Moran-Rivard, L., Kagawa, T., Saueressig, H., Gross, M.,
Burrill, J., Goulding, M. (2001). Evxl is a postmitotiC
determinant of VO interneuron identity in the spinal cord.
Neuron 29, 385-399.
Mombaerts, P., T~lang, F., Dulac, C., Chao, S.K., Nemes, A.,
Mendelsohn, M., Edmondson.
J., Axel, R. (1996). Visualizing an olfactory sensory map.
Cell 87, 675-686.
Muhr, J. Andersson, E., Persson, M., Jessell, TM. Ericson,
J. (2001). Groucho-mediated transcriptional repression
establishes progenitor cell pattern and neuronal fate in the
ventral neural tube. Cell 104, 861-873.
Novitch, B., Chen, A.I. and Jessell, T.M. (2001).
Coordinate regulation of motor neuron subtype identity and
pan-neural properties by the bHLH repressor Olig2. Submitted
Nutt, S.L., Heavey, B., Rolink, A.G., and Busslinger, M.
(1999). Commitment to the B-lymphoid lineage depends on the
transcription factor Pax5. mature 401, 556-562.
Pabst, O., Herbrand, H., Takuma, N., and Arnold, H.H.
(2000). NKX2 gene expression in neuroectoderm but not in
mesendodermally derived structures depends on sonic hedgehog
in mouse embryos. Dev Genes Evol 210, 47-50.
CA 02419851 2003-02-18
WO 02/18545 PCT/USO1/27256
-74-
Peters, T., Dildrop, R., Ausmeier, K., and Ruther U. (2001).
Organization of mouse Iroquois homeobox genes in two
clusters suggests a conserved regulation and function in
vertebrate development. Genome Res. 10, 1453-62.
Pierani, A., Brenner-Morton, S., Chiang, C., and Jessell,
T. M. (1999). A sonic hedgehog-independent, retinoid-
activated pathway of neurogenesis in the ventral spinal
cord. Cell 97, 903-915.
Pierani, A., Moran-Rivard, L., Sunshine, M.J., Littman,
D.R., Goulding, M., and Jessell, T.M. (2001). Control of
interneuron fate in the developing spinal cord by the
progenitor homeodomain protein Dbxl. Neuron 29, 367-384.
Qiu, M., Shimamura, K., Sussel, L., Chen, S., and
Rubenstein, J. L. (1998). Control of anteroposterior and
dorsoventral domains of Nkx-6.1 gene expression relative to
other Nkx genes during vertebrate CNS development. Mech Dev
72, 77-88.
Rolink, A.G., Nutt, S.L., Melchers, F., and Busslinger, M.
(1999). Long-term in vivo reconstitution of T-cell
development by PaxS-deficient B-cell progenitors. Nature 401,
603-606.
Sander, M. , Payday, S. , Ericson, J. , Briscoe, J. , Berber,
E., German., M., Jessell T.M., and Rubenstein J.L. (2000).
Ventral neural patterning by Nkx homeobox genes: Nkx6.1
controls somatic motor neuron and ventral interneuron fates.
Genes Dev. 17, 2134-2139.
CA 02419851 2003-02-18
WO 02/18545 PCT/USO1/27256
-75-
SChaeren-Wiemers, N. and Gerfin-Moser, A. (1993). A single
protocol to detect transcripts of various types and
expression levels in neural tissue and Cultured cells: in
situ hybridization using digoxigenin-labeld CRNA probes.
Histochemistry 100, 431-440.
Shoji H, Ito T, Wakamatsu Y, Hayasaka N, Ohsaki K, Oyanagi
M, Kominami R, Kondoh H, Takahashi N. (1996) Regionalized
expression of the Dbx family homeobox genes in the embryonic
CNS of the mouse. Mech. Dev. 56, 25-39
StanojeviC, D., Small, S. and Levine, M. (1991). Regulation
of a segmentation stripe by overlapping activators and
repressors in the Drosophila embryo. Science 254, 1385-1387.
Tanabe, Y., William, C., and Jessell, T.M. (1998).
Specification of motor neuron identity by the MNR2
homeodomain protein. Cell 95, 67-80.
Tanaka, M., Yamasaki, N., Izumo, S. (2000). Phenotypic
Characterization of the murine Nkx2.6 homeobox gene by gene
targeting. Mol Cell Biol. 8, 2874-2879.
Thaler, J., Harrison, K., Sharma, K., Lettieri, K., Kehrl,
J., and Pfaff S.L. (1999). Active suppression of interneuron
programs rnrithin developing motor neurons revealed by
analysis of homeodomain factor HB9. Neuron 23, 675-687.
Toresson, H., Potter, S.S. and Campbell, K. (2000). Genetic
Control of dorsal-ventral identity in the telencephalon:
opposing roles for Pax6 and Gsh2. Development. 127, 4361-
CA 02419851 2003-02-18
WO 02/18545 PCT/USO1/27256
-76-
4371.
Tsuchida, T., Ensini, M., Morton, S.B., Baldassare, M.,
Edlund, T., Jessell, T.M., Pfaff, S.L. (1994). Topographic
organization of embryonic motor neurons defined by
expression of LIM homeobox genes. Cell 79, 957-970.
Wang, C.C., Brodnicki, T., Copeland, N.G., Jenkins, N.A.,
Harvey, R.P. (2000). Conserved linkage of NK-2-1/2-9 in
mammals. Mamm. Genome 11, 466-468.
Yamada, T., Pfaff, S.L., Edlund, T., and Jessell, T.M.
(1993). Control of cell pattern in the neural tube: motor
neuron induction by diffusible factors from notochord and
floor plate. Cell 73, 673-686.
Yun, K., Potter, S. and Rubenstein, J.L. (201. Gsh2 and Pax6
play complementary roles in dorsoventral patterning of the
mammalian telencephalon. Development 128, 193-205.
25
