1 Center for Molecular Neurobiology, Martinistrasse 85, 20251 Hamburg,
Germany
2 Department of Biomedical Sciences, Creighton University, Omaha, NE 68178,
USA
3 Institute of Neuroanatomy, University of Hamburg, Martinistrasse 52, 20246
Hamburg, Germany
Author for correspondence (e-mail:
msander{at}uci.edu)
Accepted 20 August 2003
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SUMMARY |
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Key words: Nkx6.1, Nkx6.2, Hindbrain, Facial nucleus, Motoneuron, Neuronal migration, Neuronal differentiation, Mouse
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Introduction |
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Although we are beginning to understand the genetic control of hindbrain
motoneuron specification, little is known about the molecular mechanisms that
control their subsequent development. A characteristic of bm neurons is their
complex pattern of migration in the vertebrate hindbrain
(Fritzsch, 1998). In mice, the
most extensive migration is observed in bm neurons of the facial (VIIth)
nerve, which are born in r4, and subsequently migrate within the mantle zone
tangentially along the ventral midline, through r5, and into r6
(Altman and Bayer, 1982
;
Ashwell and Watson, 1983
;
Auclair et al., 1996
;
Fritzsch and Nichols, 1993
).
In r6, facial branchio-motor (fbm) neurons first migrate dorsolaterally and
then radially to form the facial nucleus at the pial surface. However, not all
fbm neurons initiate their migration simultaneously. The first neurons cross
the r4/r5 boundary at embryonic day (E) 10.5, and reach their final
destination in r6 at E12, while the last neurons only exit r4 at E12.5 and
complete migration by E14. At E14, all fbm neurons are found in their final
location in r6. In contrast to fbm neurons, bm neurons of the trigeminal (Vth)
nerve remain within their rhombomere of origin, and translocate dorsolaterally
to the Vth nerve exit point in the dorsal half of r2/r3
(Studer et al., 1996
).
Given the extensive translocation of fbm neurons from r4 to r6, the
mechanisms of bm neuron migration have been predominantly explored in fbm
neurons. Several observations suggest that environmental factors, as opposed
to an underlying cell-intrinsic program, control the caudal migration of fbm
neurons. In support of this view, fbm neurons in kreisler (Mafb
Mouse Genome Informatics) and Krox20 (Egr2
Mouse Genome Informatics) mutant mice, which lack the entire r5, migrate out
of r4, and continue with a dorsolateral and not a caudal migration within the
anteriorly positioned r6 (Garel et al.,
2000; Manzanares et al.,
1999
; McKay et al.,
1997
; Schneider-Maunoury et
al., 1997
; Seitanidou et al.,
1997
; Swiatek and Gridley,
1993
). Further evidence for the role of environmental factors in
the initiation of caudal migration has been provided by homotopic
transplantation experiments between chick and mouse tissue
(Studer, 2001
). When chick r5
was replaced with mouse r5 or r6, chick fbm neurons, which normally lack a
caudal migration, redirected their cell bodies toward the ectopic mouse tissue
and followed a caudal migratory path, similar to mouse fbm neurons. Though
these data suggest that r5-derived cues are required to initiate fbm neuron
migration, it is largely unknown which cell-intrinsic factors enable fbm
neurons to appropriately respond to such cues.
In this study, we provide evidence that Nkx6.1, which is expressed in postmitotic motoneurons, is required for bm neuron migration. In the absence of Nkx6.1, fbm neurons are born in normal numbers, but fail to initiate caudal migration. We show that their migratory defect coincides with an ectopic expression of the netrin receptor Unc5h3, as well as the GDNF receptor Ret in fbm neurons in r4, suggesting a cell-autonomous role for Nkx6.1 in the control of fbm neuron development.
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Materials and methods |
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Immunohistochemistry and in situ hybridization
Embryos were fixed in 4% paraformaldehyde at 4°C overnight. Indirect
immunofluorescence analyses were performed on cryosections as described
previously (Briscoe et al.,
2000). The following primary antibodies were used: rabbit
anti-Nkx6.1 (Jensen et al.,
1996
); guinea pig anti-Nkx6.2 and mouse anti-Evx1
(Vallstedt et al., 2001
);
mouse anti-Islet1, mouse anti-2H3, mouse anti-Nkx2.2 and mouse anti-En1
(Developmental Studies Hybridoma Bank); and rabbit anti-Phox2b
(Pattyn et al., 2000
). Alexa
Fluor-conjugated secondary antibodies (Molecular Probes) were used.
In situ hybridizations on whole-mount preparations and frozen sections were
performed as described previously
(Gradwohl et al., 1996;
Wilkinson, 1992
). The mouse
neogenin (Neo1 Mouse Genome Informatics) riboprobe comprised
base pairs (bp) 3369-4538 (GenBank Accession Number Y09535), the probe for
mouse Nkx6.2 bp 253-1244 (GenBank Accession L08074). The following
other cDNA probes were used: peripherin
(Escurat et al., 1990
),
Hoxb1 (Murphy et al.,
1989
), Epha4
(Gilardi-Hebenstreit et al.,
1992
), Isl1 and Isl2
(Osumi et al., 1997
),
Dbx1 and Dbx2 (Sander et
al., 2000a
), Olig2
(Pabst et al., 2003
),
Irx3 (Stolt et al.,
2003
), Phox2b and Phox2a
(Pattyn et al., 1997
),
Ret (Pachnis et al.,
1993
), Tag1 (Cntn2 Mouse Genome
Informatics) (Garel et al.,
2000
), Unc5h3
(Ackerman et al., 1997
). For
photography, hindbrains were dissected from surrounding tissue, flattened on
microscope slides and mounted with 80% glycerol.
Retrograde labeling of cranial nerves
E11.5-E13.5 embryos were dissected and fixed in 4% paraformaldehyde. DiI
and DiA (Molecular Probes) injections were performed as previously described
(Fritzsch and Nichols, 1993).
For retrograde labeling, DiI- or DiA-soaked filter strips were applied to the
VIIth nerve lateral to the otocyst, to the mandibular branch of the Vth nerve
near the angle of the jaw, and to the glossopharyngeal/vagal (IX/Xth) nerves
near the jugular foramen, and allowed to diffuse for 2-5 days. Hindbrains were
dissected, mounted on glass slides in glycerol, and images captured with a
Biorad Radiance 2000 confocal system mounted onto a Nikon Eclipse 800
microscope. To visualize individual axons, the brains were gelatin embedded
and vibratome sectioned at 100 µm before photography.
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Results |
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The retrograde DiI labeling experiments not only revealed defects in the migratory behavior of bm neurons, but also ectopic axon projections in the hindbrain of Nkx6.1 mutants. When DiI was applied to the VIIth nerve in Nkx6.1 mutants, scattered cell bodies were retrogradely labeled in r2/r3, as well as in r6/r7 (inset in Fig. 3B; Fig. 3E,F). Such ectopically projecting neurons were not observed in wild-type embryos (Fig. 3A; data not shown). Double labeling through the VIIth nerve in conjunction with the Vth (data not shown) or IX/Xth nerve (Fig. 3G,H) showed colocalization of these ectopically projecting neurons with the trigeminal bm neurons or the bm neurons of the nucleus ambiguous (arrows in Fig. 3H), respectively. Together, these data suggest that a subset of motor axons in r2/r3 and r6/r7 fail to recognize their proper exit points, and leave the hindbrain with the VIIth nerve in r4 (Fig. 3K).
To test if motor axons in Nkx6.1 mutant embryos appropriately project their axons into the first, second and third branchial arches, respectively, we visualized the trajectories of these axons with an anti-neurofilament antibody on whole-mount embryos at E10.5. Analysis of the nerve branching patterns revealed accurate projections of the cranial nerves to the respective branchial arches in Nkx6.1 mutant embryos (Fig. 3I,J), indicating that Nkx6.1 is not required for the pathfinding of peripheral motor axons.
Hindbrain segment identity is unaffected by Nkx6.1
inactivation
Because the mechanisms which underlie bm neuron migration have been most
extensively studied in fbm neurons, we focused in our subsequent analyses
mainly on fbm neurons. Given the implication of environmental factors in the
control of fbm neuron migration (Garel et
al., 2000; Studer,
2001
), a possible mechanism by which Nkx6.1 might regulate their
migration is by altering the environment through which the neurons migrate. We
therefore tested if the segmentation and patterning of the r4/r5 region was
properly established in Nkx6.1 mutants. We used Hoxb1 as a
marker for the r4 territory and Epha4 for the r3 and r5 territories.
The expression of Hoxb1 (Fig.
4A,B) and EphA4 (Fig.
4C,D), as well as the relative size of the rhombomeres, appeared
normal in Nkx6.1 mutants, suggesting that the molecular patterning of
the r4/r5 region does not depend on Nkx6.1.
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We next investigated whether fbm neurons in r4 were correctly specified as
bm neurons. Between E9.5 and E11.5, when fbm neurons differentiate in r4,
wild-type and Nkx6.1 mutant embryos showed no difference in the
expression of the bm neuron markers Nkx2.2, Isl1, Phox2a and Phox2b
(data not shown, Fig. 5C,N,O).
As we have previously observed an expansion of V1 interneurons into the
motoneuron domain in the spinal cord of Nkx6.1 mutants
(Sander et al., 2000a), we
also tested if fbm neurons or their progenitors carry characteristics of V1 or
V0 interneurons. In contrast to spinal cord, we did not detected a ventral
expansion of the V1 or V0 progenitor markers Dbx2 or Dbx1,
respectively (Fig. 5J-M). Likewise, fbm neurons did not misexpress the V1 interneuron marker En1 or the
V0 interneuron marker Evx1 (Fig.
5P,Q).
Given that mice deficient for the transcription factor Ebf1 also
have a defect in fbm neuron migration
(Garel et al., 2000), we also
tested if fbm neurons in Nkx6.1 mutants have normal Ebf1
expression. The expression of Ebf1 in fbm neurons was not affected by
the Nkx6.1 mutation (Fig.
5R,S). Likewise, Nkx6.1 was normally expressed in fbm
neurons in Ebf1 mutant embryos (data not shown). Collectively, these
data suggest that r4 progenitors generate normal numbers of correctly
specified fbm neurons in the absence of Nkx6.1.
Migrating facial branchio-motoneurons show aberrant expression of
guidance receptors in Nkx6.1 mutant embryos
Based on these results, it seemed unlikely that defects in the early
specification of bm neurons or in the establishment of a correct rhombomeric
environment account for the aberrant migration of fbm neurons. We therefore
examined the possibility that Nkx6.1 regulates the expression of cell surface
molecules, and might thereby influence the ability of fbm neurons to interpret
guidance cues in their environment. First, to visualize fbm neurons at
different stages of their migration, we performed whole-mount in situ
hybridization with the vm/bm neuron marker Phox2b. Though most fbm
neurons were still located in r4 at E10.75, a few had progressed into the
rostral third of r5 in both wild-type and Nkx6.1 mutant embryos
(arrowhead in Fig. 6A,B),
suggesting that early during their migration a few fbm neurons cross the r4/r5
boundary in the absence of Nkx6.1. At E12.5, fbm neurons in wild-type embryos
were detected along their entire migratory path from r4 to r6, but in
Nkx6.1 mutants were clustered in ventral r4
(Fig. 6C,D). In Nkx6.1
mutants, the distribution of fbm neurons at E12.5 was almost indistinguishable
from their pattern at E10.75 (Fig.
6B,D), indicating that most fbm neurons have remained at their
point of origin in r4.
Previous work has shown that fbm neurons regulate the expression of
cell-surface receptors in a rhombomere-specific fashion
(Garel et al., 2000). In r4,
they strongly express the cell adhesion molecule Tag1, but become
Tag1-negative in r5 and r6 (Fig.
6E) (Garel et al.,
2000
). By contrast, the GDNF receptor subunit Ret is not
detected in fbm neurons in r4, but expressed in caudally and dorsolaterally
migrating fbm neurons in r5 and r6 (Fig.
6G) (Garel et al.,
2000
). To study if Nkx6.1 activity is required for the
rhombomere-specific expression of these cell surface molecules, we tested
Tag1 and Ret expression in Nkx6.1 mutants. Although
Tag1 was normally expressed in Nkx6.1 mutants
(Fig. 6F), fbm neurons showed
an ectopic expression of Ret in r4 at E11.5 and E12.5
(Fig. 6H, data not shown).
Notably, at E10.5, at the onset of fbm neuron migration, fbm neurons were
Ret negative in both wild-type and Nkx6.1 mutant embryos
(data not shown).
Recent studies have suggested that facial motoneurons in culture are
responsive to the diffusible guidance molecule netrin 1
(Varela-Echavarria et al.,
1997). Netrin 1 has been implicated in both neuronal migration and
axon guidance in vertebrates, and can either act as a chemoattractant or
chemorepulsive signal (Bloch-Gallego et
al., 1999
; Causeret et al.,
2002
; Finger et al.,
2002
; Przyborski et al.,
1998
). Through the interaction with the transmembrane receptor DCC
and its homologue neogenin netrin 1 mediates chemoattraction, while
interaction of netrin 1 with the Caenorhabditis elegans UNC5-related
receptors mediates a repulsive response
(Ackerman et al., 1997
;
Hong et al., 1999
;
Leonardo et al., 1997
;
Meyerhardt et al., 1997
).
To explore a possible role for netrin signaling in fbm neuron migration, we studied the expression of the three mammalian UNC5 homologues (UNC5H) UNC5H1, UNC5H2 and UNC5H3, as well as DCC and neogenin, in mouse embryos during fbm neuron migration. Although Unc5h1 and Unc5h2 were not detected in migrating fbm neurons, fbm neurons expressed Unc5h3. In wild-type embryos, Unc5h3 was not expressed before E12.0 (Fig. 6K). At E12.0, faint Unc5h3 expression was detected in r4, and a strong signal was seen in fbm neurons in r5 and r6 (Fig. 6I). At E13.5, when their migration is almost complete, Unc5h3 marked fbm neurons in r6 (Fig. 6J). Unc5h3 was also detected in dorolaterally migrating trigeminal motoneurons (Fig. 6I,J). Among the DCC homologues, we detected only neogenin in cranial motoneurons, which as Unc5h3 was localized in fbm neurons migrating away from the midline in r6 at E12.5 (Fig. 6O). Colocalization of Unc5h3 and neogenin was also observed in dorsolaterally migrating trigeminal motoneurons (Fig. 6I,J,O).
To test if Nkx6.1 is required for the coordinated expression of netrin receptors in fbm neurons, we studied Unc5h3 and neogenin expression in Nkx6.1 mutant hindbrains. Although no Unc5h3 expression was detected in wild-type embryos at E11.5 (Fig. 6K,M), strong expression of Unc5h3 was found in fbm neurons in Nkx6.1 mutants (Fig. 6L,N). This ectopic expression in r4 fbm neurons was maintained at E12.5 (data not shown). We observed that the onset of ectopic Unc5h3 expression coincided with the onset of the migratory defect. At E10.5, when early-born fbm neurons migrate into r5 in Nkx6.1 mutant embryos (Fig. 6B), no ectopic expression of Unc5h3 was observed (data not shown). However, at E11.5, when caudal migration has stopped in Nkx6.1 mutants, they expressed Unc5h3 ectopically (Fig. 6L,M). Fbm neurons in Nkx6.1 mutants did not express neogenin (Fig. 6P). These findings demonstrate that Nkx6.1 controls the cell surface characteristics of migratory fbm neurons, and reveal a temporal link between the ectopic expression of cell surface receptors in fbm neurons and their migratory defect.
Nkx6.1 and Nkx6.2 are co-expressed in r4
Given that early migratory fbm neurons progress into the r5 territory in
Nkx6.1 mutants, we considered that other factors with similar
function as Nkx6.1 might be present in r4. A close relative to Nkx6.1, Nkx6.2,
has previously been shown to have similar activity as Nkx6.1 in promoting the
generation of motoneurons in the spinal cord
(Vallstedt et al., 2001).
Based upon this finding, we examined if Nkx6.2 is expressed during fbm neuron
development. At E10.5, we detected Nkx6.2-positive cells in a broad ventral
domain in r4. Within this domain, the ventral and dorsal limit of expression
of Nkx6.2 coincided with the limits of Nkx2.2 expression, and virtually all
Nkx2.2-positive cells co-expressed Nkx6.2
(Fig. 7A). The most laterally
located Nkx6.2-positive cells produced Isl1
(Fig. 7B). As Nkx6.1 is
expressed in a similar domain as Nkx6.2
(Fig. 1C,D), we next tested if
these two factors are co-expressed in r4. At E10.5, essentially all
Nkx6.2-positive cells co-expressed Nkx6.1, but the domain of Nkx6.1-positive
cells extended beyond the dorsal limit of Nkx6.2 expression
(Fig. 7C). Nkx6.2
expression was also detected in trigeminal bm neurons in r2, and was
maintained in these neurons during their dorsolateral migration in r2/r3 (data
not shown; Fig. 7E,G). Although
Nkx6.1 was detected in the entire migratory stream of fbm neurons
from r4 to r6 between E11.5 and E12.5 (Fig.
1E,F), Nkx6.2 was confined to fbm neurons in r4, and
absent from fbm neurons in r5 and r6 (Fig.
7E,G). Notably, the level of Nkx6.2 expression in r4 fbm
neurons decreased markedly after E11, and Nkx6.2 was not detected in
postmigratory fbm neurons (Fig.
7G). This suggests that compensation by Nkx6.2 for Nkx6.1 might
only be effective during the early stages of fbm neuron development.
Consistent with such early compensatory function of Nkx6.2,
Nkx6.1/Nkx6.2 double mutant embryos show a complete lack of caudal
migration into r5 (Pattyn et al.,
2003
).
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Discussion |
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Nkx6.1 function in branchio-motoneuron development
Correct selection of a migratory path requires a constant crosstalk between
the migrating neuron and the environment through which the cell body
translocates (Hatten, 2002;
Nadarajah and Parnavelas,
2002
). The aberrant migration of trigeminal and facial bm neurons
in Nkx6.1 mutants could therefore either result from changes in the
surrounding environment, or from a cell-intrinsic defect in the neurons
themselves. As the mechanisms that underlie bm neuron migration have been most
extensively studied in fbm neurons, we performed a detailed analysis of the
defects in only these neurons. As illustrated by normal expression of
Hoxb1 and Epha4 in the r4 and r5 territories, we did not
find any alterations in the expression of markers that specify the regional
identity of the territory through which the neurons migrate. Although these
data argue against a role of extrinsic factors and favor a cell-autonomous
function for Nkx6.1 in the control of fbm neuron migration, we cannot exclude
that lack of Nkx6.1 in either the adjacent progenitors or in other cell types
leads to subtle changes in the environment that were not detected by our
markers.
As a cell-intrinsic mechanism, it is conceivable that defects in the early specification of bm neurons or in the timing of their differentiation could result in aberrant neuronal migration. Our analysis of fbm neuron development argues against such an early function of Nkx6.1, as we found no alterations in the timing of motoneuron generation, and normal expression of the bm neuron markers Nkx2.2, Isl1, Phox2b, Phox2a and Hoxb1. Moreover, fbm neurons did not co-express markers of more dorsally located interneurons, excluding that motoneurons have a mixed identity. Instead, it appears that Nkx6.1 has a cell-autonomous function in modulating the expression of cell-surface receptors in postmitotic fbm neurons. This hypothesis is supported by our finding, that fbm neurons in Nkx6.1 mutants ectopically express the guidance receptors Unc5h3 and Ret in r4 (Fig. 8B). As fbm neurons in Nkx6.1 mutants expressed Unc5h3 before any expression was detected in wild-type embryos, our data suggest that the aberrant Unc5h3 expression in r4 fbm neurons is not merely a result of their inability to migrate, but indeed indicates ectopic activation. However, it also needs to be considered that the misexpression of guidance receptors is a consequence and not the cause of the migratory defect.
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The role of guidance molecules in facial branchio-motoneuron
migration
The appropriate navigation of fbm neurons through the different substrates
along their migratory path from r4 to r6 most likely requires a
position-dependent modification of their cell surface characteristics. This
view is supported by the observation that fbm neurons regulate the expression
of Tag1, Ret and Cad8 in a rhombomere-specific fashion
(Garel et al., 2000). There is
substantial evidence that these cell surface molecules play a role in neuronal
migration in the CNS (Enomoto et al.,
2001
; Enomoto et al.,
2000
; Kyriakopoulou et al.,
2002
), but their direct involvement in fbm neuron migration
remains to be demonstrated.
Our present analysis shows that migrating fbm neurons also express the
netrin receptors Unc5h3 and neogenin in a rhombomere-specific
pattern. UNC5 netrin receptors have been shown to interact with DCC-type
receptors, transforming attraction as a response to netrin 1 mediated by DCC
into a repulsive function (Hong et al.,
1999). Further functional analyses in Drosophila revealed
that expression of Unc5 alone results in short-range repulsion, while
co-expression of Unc5 with the DCC homolog frazzled
mediates long-range repulsion (Keleman and
Dickson, 2001
). Our finding that dorsolaterally migrating fbm
neurons in r6, as well as dorsolaterally migrating trigeminal motoneurons,
co-express the attractant netrin receptor neogenin and the repulsive netrin
receptor Unc5h3, raises the possibility that floor plate derived
netrin 1 could be involved in driving these neurons away from the midline.
Consistent with this view, fbm neurons, which express Unc5h3, but
fail to express neogenin in Nkx6.1 mutants, remain close to the
ventral midline and do not complete a dorsolateral migration.
Control of facial branchio-motoneuron migration by different
transcription factors
Previous genetic studies in mice have shown that Nkx6.2 partially
compensates for Nkx6.1 in the development of spinal cord motoneurons
(Vallstedt et al., 2001). Our
finding that fbm neurons co-express Nkx6.1 and Nkx6.2 in r4, but only maintain
the expression of Nkx6.1 after crossing the r4/r5 boundary, raises
the possibility that Nkx6.2 might compensate for Nkx6.1 function in r4. As
Nkx6.2 is expressed at significant levels only until E11 and
downregulated thereafter, this hypothesis is consistent with our observation
that some fbm neurons progress into r5 at the onset of their migration. Direct
genetic evidence for a compensatory function of Nkx6.2 is provided by the
observation that fbm neurons in Nkx6.1/Nkx6.2 double mutant
embryos show a complete lack of caudal migration
(Pattyn et al., 2003
).
Strikingly, the downregulation of Nkx6.2 in fbm neurons after E11
temporally coincides with the onset of ectopic Ret and
Unc5h3 expression in r4 (Fig.
8), suggesting the possibility that in early development Nkx6.2
alone may be sufficient to prevent the expression of these receptors, and may
thereby maintain responsiveness of fbm neurons to r5-derived cues.
Three other transcription factors that have been implicated in the control
of fbm neuron migration are Hoxb1 (Goddard
et al., 1996; Studer et al.,
1996
) and the Gata factors, Gata2 and Gata3
(Nardelli et al., 1999
;
Pata et al., 1999
). In r4
progenitor cells, Hoxb1, Gata2 and Gata3 function in a
regulatory cascade, in which Hoxb1 is required to activate Gata2, and
Gata2 in turn to activate Gata3. We did not find any indication that
Nkx6.1 functions directly up- or downstream of this regulatory cascade, as we
observed no alterations in the expression of Hoxb1, Gata2 and
Gata3 in Nkx6.1 mutants (data not shown). Likewise,
Nkx6.1 shows a normal pattern and level of expression in
Gata3 mutant embryos (I. Pata and A. Karis, unpublished).
A late migratory defect of fbm neurons has been observed in mice, which are
mutant for the transcription factor Ebf1
(Garel et al., 2000). In
Ebf1 mutant mice, a subset of fbm neurons fails to migrate into r6
and undergo premature dorsolateral migration in r5. Similar to Nkx6.1, Ebf1 is
expressed in migrating fbm neurons, and the migratory defect in Ebf1
mutants is also associated with the premature expression of Ret in
fbm neurons in r4. These findings suggest that both transcription factors
might regulate similar targets. However, it appears that Ebf1 and Nkx6.1 do
not function in a regulatory cascade, as Ebf1 expression was not
affected in Nkx6.1 mutant embryos, and vice versa (data not
shown).
In summary, our study demonstrates a role for Nkx6.1 in the development of postmitotic motoneurons. Although Nkx6.1 is required for the correct specification of sm neuron progenitors, we show that the early specification and generation of vm/bm neurons is independent of Nkx6.1 function. Our data support a model in which Nkx6.1 functions cell-autonomously in postmitotic bm neurons to ensure their correct migration in the hindbrain. Given the ectopic expression of guidance receptors in pre-migratory fbm neurons of Nkx6.1 mutants, it will be interesting to further explore the role of cell-surface receptors in fbm neuron migration.
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ACKNOWLEDGMENTS |
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Footnotes |
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