1 Institut de Génétique et de Biologie Moléculaire et
Cellulaire, CNRS/INSERM/Université Louis Pasteur, BP 10142, 67404
Illkirch Cedex, C.U. de Strasbourg, France
2 Unité d'Embryologie Moléculaire, Institut Pasteur, Bat. J.
Monod, 25 rue du Dr Roux, 75724 Paris Cedex 15, France
3 E. Kennedy Shriver Center, University of Massachusetts Medical School,
University of Massachusetts, Waltham, MA 02452, USA
4 Departments of Medicine and Molecular and Cellular Biology, Center for
Cardiovascular Development, Baylor College of Medicine, One Baylor Plaza,
Houston, TX 77030, USA
* Authors for correspondence (e-mail: isalr{at}igbmc.u-strasbg.fr and dolle{at}igbmc.u-strasbg.fr)
Accepted 5 January 2005
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SUMMARY |
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Key words: Motoneurons, Retinoic acid, Spinal cord, Raldh2, Hox genes, Lim1, Islet1, Mouse
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Introduction |
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In addition to its function during early spinal neurogenesis
(Appel and Eisen, 2003), RA
signaling plays a major role in the specification of chicken motoneuron
subtypes, soon after these cells withdraw from the cell cycle
(Sockanathan and Jessell,
1998
; Sockanathan et al.,
2003
). These sequential roles highlight the importance of the
spatiotemporal control of local RA levels, which mainly results from regulated
expression of synthesizing enzymes, the retinaldehyde dehydrogenases (Raldh),
and metabolizing enzymes, the cytochrome P450s Cyp26
(Niederreither et al., 1999
;
Abu-Abed et al., 2001
;
Sakai et al., 2001
). Raldh2
acts as the main RA-synthesizing enzyme during early embryogenesis
(Niederreither et al., 1999
).
This enzyme is first expressed in the mesoderm adjacent to the node during
early gastrulation; its expression expands to paraxial mesoderm during the
phase of early neuronal specification, and at later stages Raldh2 is expressed
in LMC neurons (Zhao et al.,
1996
; Niederreither et al.,
1997
; Sockanathan and Jessell,
1998
; Diez del Corral et al.,
2003
). In agreement with this expression pattern, transgenic mice
that serve as in-vivo reporters of RA signaling have revealed RA activity in
early paraxial mesoderm and later in postmitotic cells of the ventral spinal
cord at brachial and lumbar levels
(Rossant et al., 1991
;
Colbert et al., 1995
;
Solomin et al., 1998
). As
motoneurons exit the cell cycle, they begin to express the homeoprotein Islet1
(Sharma et al., 1998
). This
expression is rapidly downregulated in lateral LMC neurons when these cells
begin to express Lim1, thus generating a molecular distinction between lateral
and medial LMC cells (Tsuchida et al.,
1994
). Lim1 participates to the regulation of the adhesion
molecule Epha4, both proteins being required for the proper establishment of
topographic projections (Helmbacher et
al., 2000
; Kania et al.,
2000
; Kania and Jessell,
2003
). The switch from Islet1+ to Lim1+ cells appears to depend on
RA provided by early born LMC neurons, as in-vitro and in-vivo exogenous RA
exposure of chicken LMC neurons represses Islet1 and promotes Lim1 expression
(Sockanathan and Jessell,
1998
).
An additional earlier role has been assigned to RA produced by paraxial
mesoderm at limb levels in the specification of brachial versus thoracic motor
columns, as a blockade of RA signaling prevented the acquisition of an LMC
identity by newly generated chick brachial motoneurons, which instead acquired
a thoracic character (Sockanathan et al.,
2003). Parallel studies have shown that Hoxc proteins, which are
expressed at specific AP levels of the spinal cord in both neural progenitors
and postmitotic cells, regulate the expression of molecular markers of either
thoracic or brachial columnar fate. In particular, Hoxc6 is required for the
expression of Raldh2 in the brachial LMC
(Dasen et al., 2003
).
Conversely, regulation of Hox genes via RA has been described, although at an
earlier stage in neural progenitors (Liu
et al., 2001
). Thus, there seem to be complex interactions between
the regulation of RA signaling and Hox gene expression during spinal
neurogenesis. Targeted disruptions of Hoxa10, Hoxc8, Hoxd9 and
Hoxd10 led to aberrant patterns of motor axon connectivity in the
limbs, suggesting roles for Hox proteins in the determination of motoneuron
subtypes (Rijli et al., 1995
;
Carpenter et al., 1997
;
Tiret et al., 1998
;
de la Cruz et al., 1999
).
However, the cellular basis for these defects remains unknown. Retrograde
labelings performed on Hoxc8/ mice revealed
a reverse mediolateral position of a subset of motoneurons with respect to
their target muscles, suggesting that Hoxc8 is required for establishing the
distinction between medial and lateral LMC
(Tiret et al., 1998
).
To investigate the role of local RA synthesis during the specification of LMC neurons, we have generated mice bearing a conditional mutation that ablates Raldh2 function essentially in developing brachial motoneurons and later in mesenchymal cells at the base of the forelimb. Analysis of these mice indicates that RA synthesis within the LMC plays a crucial role in the brachial LMC in the specification of a subset of lateral cells, in the position of motor cell bodies as well as in the regulation of the expression of transcription factors and adhesion molecules required for correct axonal projections to target muscles. We also report striking similarities in the molecular defects observed in Raldh2 conditional mutants and Hoxc8 knockout mice, suggesting that RA signaling and Hox gene functions are essential for the specification of LMC cells.
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Materials and methods |
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Immunohistochemistry and in-situ hybridization
In-situ hybridization on cryosections was performed as described
(Chotteau-Lelièvre et al.,
2004). The Raldh2 probe used to detect both the wild-type
and Raldh2L alleles hybridized with 75 bp of the
fourth exon and the subsequent 3' coding sequence. Other template
plasmids were produced in our laboratory (RARß) or kindly provided by M.
Petkovich (Cyp26B1), R. Behringer (Lim1), C-C. Hui
(Islet1), S. Pfaff (Lhx3), C. Henderson (Sema3e and
cadherin 7), D. Wilkinson (Epha4), R. Klein (Ephb2), M.
Capecchi (Hoxc6 and Hoxc8), X. Desbiens (ER81 and
Pea3), M. Kmita (lacZ) and A. Esquela Kerscher
(Gdnf).
Vibratome (100 µm) sections were processed for immunochemistry
(Scardigli et al., 2003) using
rabbit polyclonal anti-Epha4 (kindly provided by P. Charnay)
(Becker et al., 1995
) and mouse
monoclonal anti-neurofilament (2H3; Developmental Studies Hybridoma Bank,
DSHB) antibodies. X-Gal staining was performed as described
(Scardigli et al., 2003
).
Double in-situ hybridization and immunolabeling experiments were performed
on predissected spinal cords fixed overnight in 4% paraformaldehyde and
processed for in-situ hybridization with digoxigenin-labeled probes
(Chotteau-Lelièvre et al.,
2004), using an InsituPro (Intavis) robot and Fast Red (Roche) to
reveal alkaline phosphatase activity. Subsequent immunofluorescence was
performed as described (Scardigli et al.,
2003
) with mouse monoclonal anti-ß-galactosidase (Promega),
anti-Hoxc8 (C952-7E, Babco), anti-Islet1/2, anti-Lim1/2 and anti-Lim3 (40.2D6,
4F2, and 67.4E12, DSHB), and rabbit polyclonal anti-Raldh2
(Berggren et al., 1999
),
anti-ß-galactosidase (5 prime-3 prime Inc.) and anti-Islet1 (K5, kindly
provided by T. Jessell) (Tsuchida et al.,
1994
) antibodies. Although the latter antibody has been reported
to cross-react with Islet2 (Tsuchida et
al., 1994
), it recognized only Islet1 in our conditions (1.5% of
the K5+ cells were Lim1+ per hemisection, whereas 25% of Islet2+ cells were
Lim1+, average of 15 sections from five controls). Alexa 488-Alexa 594- and
Cy5-coupled secondary antibodies were used (Molecular Probes and Jackson
ImmunoResearch Laboratories, Inc.). Both the sectioned and whole-mount spinal
cords were analyzed using a Leica Sp2MP confocal microscope, except for
Fig. 8A-C and
Fig. 2C,D which were obtained
using a Leica macrofluo and macro confocal (D.H. and J. L. Vonesch,
unpublished), respectively. Images of cryosectioned spinal cords represent a
single focal plan, whereas those of whole-mounts and vibratome sections were
obtained by the projection of an average of 20 stacks of pictures
(representing 40 µm in thickness). Analysis of dissected spinal cords was
performed on a minimum of five control and
Raldh2L/ embryos. Analysis of Hoxc8
mutants was done on two embryos for each genotype; no variation for any marker
was observed among each group. Whole-mount anti-neurofilament staining was
performed as described (Maina et al.,
1997
), and embryos were documented on a Leica M420 macroscope.
Comparisons between control and mutant spinal cords were performed in all
cases on littermate embryos.
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Results |
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We then examined the efficiency of the RarbCre transgenic line to
excise the Raldh2L2 allele by monitoring Raldh2
immunoreactivity in mutants. At E9.5, Raldh2 expression was not significantly
altered in mutants compared with controls
(Fig. 2C,D). However, Raldh2
protein was never observed in the brachial spinal cord of mutants at all
subsequent stages (E10.5-15.5); residual protein was still present in
scattered meningeal and mesenchymal cells surrounding the spinal cord
(Fig. 2E-J; and data not
shown). Raldh2 expression at E11.5 was also markedly diminished at the level
of the brachial plexus (Fig.
2G,H), a region in which motoneurons make pathfinding decisions
(Landmesser, 2001). At the
lumbar level, a reduced number of Raldh2+ motoneurons was observed in the
E12.5 mutant spinal cord (Fig.
2I,J, insets). This residual RA activity can explain the lack of
abnormal hind limb phenotype in Raldh2L/
mutants.
Thus, excision of the Raldh2L2 allele using the
RarbCre transgenic line led to a complete absence of Raldh2 protein
in brachial motoneurons. The pattern of excision of
Raldh2L2, especially in the mesoderm, was clearly distinct
from the pattern of excision described using the same RarbCre
transgenic mice in the RNA polymerase II large subunit locus
(Moon et al., 2000) or the
ROSA26 locus (J.V. and I.L.R., unpublished). Our result supports the finding
that LoxP recombination is locus-position-dependent (Voojs et al.,
2001). In summary, the Raldh2L/ mice
constitute a model to study the early requirement for local RA synthesis
during the early phase of motoneuron specification, and a default in these
developmental decisions could be the cause of the adult phenotype.
Altered patterns of RA signaling in brachial Raldh2L/ spinal cord cells
We first examined the distribution of RA-responsive cells within
Raldh2L/ spinal cord using an RA-responsive
reporter transgenic line (RARE_hsp68_lacZ)
(Rossant et al., 1991). The
patterns of ß-galactosidase (ß-Gal) expression were analyzed by
immunofluorescence in control and Raldh2L/
transgenic embryos. In E10.5 control embryos, ß-Gal+ cells were scattered
along the DV axis of the brachial spinal cord
(Fig. 3A). In
Raldh2L/ mutants, fewer ßGal+
cells were found in the ventral horns and in the dorsalmost region of the
brachial spinal cord (Fig. 3B).
We further showed that in the E11.5 control ventral spinal cord, some
Islet1/2+ cells expressed ß-Gal (Fig.
3C, arrows), whereas no Islet1/2+ cells were ß-Gal+ in
Raldh2L/ spinal cords
(Fig. 3D), indicating the
depletion of RA-responsive motoneurons. At E12.5, ß-Gal+ cells were
distributed along the dorsal and intermediate regions of control flat-mounted
spinal cords (Fig. 3E). By
contrast, in Raldh2L/ mutants, ß-Gal+
cells were essentially found in a broad longitudinal band within the
intermediate region of the spinal cord
(Fig. 3F, brackets). This
pattern is reminiscent of RA activity in Raldh2 null mutants rescued
by RA supplementation (Mic et al.,
2002
; Niederreither et al.,
2002
).
|
Loss of Lim1+ cells and redistribution of Islet1+ cells in Raldh2L/ spinal cord
We then analyzed the identity of the brachial LMC neurons in
Raldh2L/ mutants. We first quantified the
number of Islet1/2+ motoneurons in the ventral spinal cord at E11.5. Islet1/2+
cells were counted on serial transverse sections spanning the entire
Raldh2 domain, which was mapped by in-situ hybridization (see
Materials and methods). A significant 10% decrease of the number of Islet1/2+
cells was found in mutants (Fig.
4A). Double IHC was performed on adjacent control and mutant
sections using: (1) an anti-Lim1/2 antibody; and (2) an anti-Islet2 antibody
(Fig. 4B) or an anti-Islet1
antibody (K5 antibody; Fig. 4C;
see Materials and methods) to quantify Lim1+ or Islet1+ motoneurons. We found
that, within the Raldh2 domain, the number of Islet1+ cells (medial
LMC and MMC cells) was unchanged in Raldh2L/
mutants compared with controls, whereas the number of Lim1+/Islet2+
motoneurons (lateral LMC cells) was significantly decreased by about 20% in
mutants (Fig. 4B).
|
Alterations of the Lim1+ and Pea3+ motor pools in Raldh2L/ brachial LMC
We next analyzed the expression of selective markers of LMC motor pools.
The overall organization of the LMC was not disrupted in
Raldh2L/ spinal cord. Indeed, the expression
domain of Ephb2, which closely matches that of Raldh2 in controls
(Fig. 5A), was not spatially
altered in mutants (Fig. 5A-D).
In addition, the organization of the MMC (Lim3+ cells) and V2 columns
(Chx10+ cells) were not modified in mutants, indicating that no
change of the identity of LMC neurons toward MMC neurons or V2 interneurons
had occurred in the mutants (data not shown). By contrast, we found a
consistent decrease of the expression of cadherin 7 and Epha4 in the
Lim1+ lateral LMC posterior domain, confirming the loss of a subset of Lim1+
cells (data not shown, and Fig.
5E-H).
|
Impaired dorsal motor axonal projections in Raldh2L/ embryos
In an attempt to establish a link between the LMC molecular defects
described above with the adult
Raldh2L/phenotype, we investigated the
development of forelimb motor axonal projections. We first analyzed by double
IHC at E11.5 the expression of neurofilament and Epha4, the latter being
preferentially expressed in axons projecting dorsally in the wild-type limb
bud (Helmbacher et al., 2000;
Kania and Jessell, 2003
).
Neurofilament distribution revealed no obvious defect in motor axonal
projections in mutants (Fig.
6A,B). By contrast, the expression of Epha4 was significantly
diminished in the dorsal axonal projections in
Raldh2L/ forelimb
(Fig. 6C,D, thin arrows). Epha4
expression in the proximal-dorsal limb mesenchyme was unaffected in mutants
(Fig. 6C,D, arrowheads),
suggesting that the lack of this protein in dorsal axons is linked to the
corresponding motor pool defect (Fig.
5E-H).
|
Downregulation of Hoxc6 and Hoxc8 expression in Raldh2L/ LMC
The forelimb defect observed in Raldh2L/
mice is reminiscent of the Hoxc8 knockout mouse phenotype
(Le Mouellic et al., 1992;
Tiret et al., 1998
). We
hypothesized that decreased local RA signaling could affect Hox gene
expression in the brachial LMC. Hoxc6 and Hoxc8 expression
were therefore analyzed at E12.5. While in controls Hoxc6 was broadly
expressed in the Raldh2 domain (Fig.
7A), in Raldh2L/ spinal cord its
expression was abnormally low, especially in the central LMC domain (brackets,
Fig. 7A,B). In controls,
Hoxc8 was expressed at the anterior level of the LMC in the
Lim1+ interneuron column, while at the posterior level of the LMC it
was additionally expressed in the Islet1+ and Lim1+ motor
columns (Fig. 7C,E). In
mutants, the Hoxc8 interneuron domain was comparable to that of controls,
whereas the anterior boundary of Hoxc8 expression in the motoneurons
was shifted posteriorly at the transcript and protein levels
(Fig. 7C-L). Hoxc8
downregulation was most prominent in the Lim1+ motor column, as shown
in dissected spinal cords co-labeled with Lim1 and on serial
transverse sections (red arrows, Fig.
7E-L). Taken together, these data show that decreased RA signaling
affects Hox gene expression within LMC neurons, which may in turn play a role
in the molecular alterations described in the
Raldh2L/ LMC.
|
In Hoxc8+/ and Hoxc8/ mutants, expression of Lim1 was downregulated in the posterior LMC (Fig. 8G-I) and Islet1+ cells were abnormally distributed (Fig. 8J-L). In Hoxc8+/ mice, Islet1 was continuously distributed in two thick longitudinal columns throughout the LMC (Fig. 8K, arrowheads). In Hoxc8/ mutants, Islet1+ cells spanned a uniform domain throughout most of the LMC (Fig. 8L). The analysis of Islet1 expression on serial transverse sections revealed ectopic dorsal Islet1+ cells at the level of the posterior LMC in Hoxc8+/ mice, and this abnormality was more pronounced in Hoxc8/ embryos (Fig. 8K,L). Analysis of Pea3 expression showed a mislocation of these cells in Hoxc8+/ mutants: the distance between the ventral midline and the Pea3+ cells was larger in mutants (Fig. 8N, horizontal bars). Pea3 expression was greatly diminished in Hoxc8/ mutants, and the ventral migration of these cells did not occur properly (Fig. 8O). In both Hoxc8+/ and Hoxc8/ mutants, the distribution and levels of Gdnf transcripts in the brachial plexus and the CM muscle was comparable to those of wild-type embryos (Fig. 8P-R).
Taken together, the analysis of the Hoxc8 mutant spinal cords revealed striking similarities with the abnormalities found in Raldh2L/ mutant mice, including a decrease in RA activity monitored by the expression of Rarb, a downregulation of Lim1 expression within the motor columns, a redistribution of Islet1+ cells and a mispositioning of Pea3+ cells. It was found, however, that Hoxc8+/ heterozygous mutants most closely phenocopied the Raldh2L/ spinal cord phenotype, whereas Hoxc8/ homozygous mice clearly exhibited more severe cellular defects.
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Discussion |
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RARß, a key component of the RA signaling pathway during motoneuron specification
Our findings show that the regulation of RA signaling in the LMC cells
depends at least on the activity of two molecules: Raldh2 and RARß. It is
noteworthy that the skeletal analysis of the forepaw of compound
RARß/RAR mutant mice revealed a retraction of the digits that
resembles the Raldh2L/ phenotype
(Ghyselinck et al., 1997
).
Unlike Raldh2, which is uniformly expressed in the LMC cells, Rarb
expression is dynamic, suggesting that this receptor plays a key role in the
spatiotemporal regulation of RA activity during the specification of
motoneurons. At E11.5, Rarb expression in the spinal cord was similar
to that of the RARE_hsp68_lacZ reporter transgene, whereas one day
later it became restricted to specific motor pools
(Fig. 3, and data not shown).
These expression patterns most probably reflect the sequential roles of
RARß-mediated RA signaling during motoneuron differentiation.
Local RA synthesis within the spinal cord is required for the specification of a subset of Lim1+ LMC cells
The analysis of Raldh2L/ mutant mice did
not reveal any re-specification of the LMC cells toward another cell fate; for
example, no re-specification of the LMC cells toward thoracic motoneuron fates
such as the MMC cells or preganglionic motoneurons (PrGG) was observed
(I.L.R., unpublished). As RA signaling provided by the paraxial mesoderm has
been shown to specify a brachial versus thoracic motor column fate
(Sockanathan et al., 2003),
our data are in agreement with the fact that RarbCre-mediated
excision of Raldh2L2 allele mostly occurs within the
spinal cord and does not significantly deplete Raldh2 function in paraxial
mesoderm during the phase of brachial versus thoracic fate decision.
We observed in Raldh2L/ mutant mice a
decrease of 10% in the number of motoneurons at E11.5
(Fig. 4). This decrease is cell
type specific, as it only affects the Lim1+ population. A decrease in
the proliferation of motoneuron progenitors or a premature cell death of
postmitotic Lim1+ cells could explain this phenomenon. We favor the
second hypothesis, because no reduction in RA-responsive cells was observed in
ventral neural progenitors at E9.5 and 10.5 in the
Raldh2L/ mutants
(Fig. 3B). Our results thus
confirm previous findings that demonstrated the role of RA signaling in the
specification of chick lateral LMC neuronal identity
(Sockanathan and Jessell,
1998).
The whole range of later developmental (from E12.5) and postnatal defects observed in Raldh2L/ mice cannot, however, be explained by the early death of a subset of Lim1+ motoneurons, or by an increased rate of the normally occurring programmed cell death, as TUNEL experiments performed at E13.5 did not show significant differences between controls and mutants (I.L.R., unpublished). The abnormal axonal arborization of a branch of the radialis nerve and the decreased Epha4 protein expression in the growing dorsal limb axons indicate sustained functional defects in specific motor pools (Fig. 6). One possibility is that RA deficiency leads to the acquisition of an incomplete motoneuron identity and to the presence of motoneurons that neither expressed Islet1 nor Lim1 and, therefore, cannot properly express differentiation markers.
RA synthesis is required for the correct positioning of Islet1+ motoneurons, including Pea3+ cells
We found that Pea3+ motoneurons were mispositioned in the
Raldh2L/ posterior LMC. Although the
expression of Pea3 in motoneurons and the migration of these cells
are both regulated by Gdnf, this trophic factor is unlikely to be responsible
for the migratory defect observed in the
Raldh2L/ mutants
(Fig. 5I-L). Indeed, we did not
detect any change in the distribution and level of Gdnf transcripts
in mutants. This result contrasts with the marked decrease in RA activity at
the level of the brachial plexus and the hypaxial LD and CM muscles, i.e. at
the same sites as Gdnf expression, in
Raldh2L/ mutants
(Fig. 5). Possible explanations
are that RA activity does not regulate Gdnf in the tissues, or that
the remaining RA activity present in mutants is sufficient to allow normal
expression levels of this factor. In any event, these data strengthen the idea
that most of the molecular defects described in
Raldh2L/ mutants are linked to an RA
deficiency within the LMC.
The mispositioning of Pea3+ cells, which are constituted by over 95% of
Islet1+ cells at segmental levels C7/8
(Livet et al., 2002), is very
probably a consequence of the redistribution of Islet1+ cells within the
Raldh2L/ LMC, as observed at E12.5
(Fig. 4). The early loss or the
incomplete specification of some Lim1+ cells and the change in the expression
patterns of Epha4, cadherins 8 and 7
(Fig. 5), which encode for
adhesion molecules regulating motor pool segregation
(Price et al., 2002
;
Coonan et al., 2003
), may be
responsible for the mispositioning of Islet1+ cells. It is noteworthy
that the positioning of motor cell bodies can influence peripheral axonal
projections, as it has been demonstrated by single cell transplantation
experiments in zebrafish embryos or by retrograde labeling in Hoxc8
mutant mice (Eisen, 1991
;
Tiret et al., 1998
). The
alteration of Islet1+ cell distribution in
Raldh2L/ mutants could, therefore, lead to
subtle aberrant axonal projections.
Lateral LMC specification requires RA signaling and Hox gene function
We found that the loss of Raldh2 function in the lateral LMC was
dispensable for the specification of a large subset of lateral LMC cells, as
shown by the relatively subtle alterations of Lim1 expression in the
developing spinal cord and the subsequent correct innervation of most dorsal
limb muscles in Raldh2L/ mutants. Although
the present conditional knockout does not completely rule out a contribution
of RA produced by paraxial mesoderm or meningeal cells in the process of
lateral LMC specification, our data show that RA was not the only signal to
specify this cell fate. Strikingly, the molecular features of the
Raldh2L/ mutants, such as the loss of
Lim1+ motoneurons, the redistribution of Islet1+ cells or
the downregulation of Rarb in the posterior medial LMC, were shared
with Hoxc8 mutants (Fig.
8). We thus demonstrated that Hoxc8 is a key molecule, together
with RA signaling, in directing a lateral LMC fate. This finding, along with
work on the function of Hoxc6 and Hoxc9 in the specification of a brachial or
thoracic motoneuron fate (Dasen et al.,
2003), unveils novel roles for Hox proteins within postmitotic
differentiating neurons.
The closest phenocopy of the Raldh2L/
phenotype was found in Hoxc8+/ heterozygous
mutants, indicating that a decrease in the level of Hoxc8 protein is
sufficient to modify the specification of a lateral LMC cell type. This
finding underlines the importance of the regulation of Hoxc8 gene
expression in postmitotic cells of the ventral spinal cord. At the protein
level, Hoxc8 activity is regulated by the presence of co-factors such as the
Meis proteins (Popperl et al.,
1995). Interestingly, we found that Meis2 was expressed in the
Lim1+ posterior LMC, and was excluded from the Islet1+
posterior domain (J.V. and I.L.R., unpublished), suggesting that Hoxc8 and
Meis2 co-expression spatially restricts Hoxc8 function to the lateral LMC.
Hoxc8/ mutants clearly display more drastic
brachial spinal cord abnormalities than
Raldh2L/ mutants. This may reflect an
absolute requirement for Hoxc8 in the regulation of some target genes and/or
an early requirement for Hoxc8 in neural progenitors or paraxial mesoderm,
which would set up the anterior boundary of the LMC.
In conclusion, our results provide new insights into the specification of
LMC cell fates. Raldh2 expressed in the LMC provides local RA activity that is
transduced by RARß and activates the expression of the lateral LMC marker
Lim1 and allows a correct positioning of cell bodies within the LMC. A recent
study demonstrated a bi-directional regulation between Hox genes
(Hoxb4 and Hoxd4) and RARß during the formation of
rhombomere boundaries, and showed that the regulatory sequences of each gene
contain both active RAREs and Hox consensus binding sites
(Serpente et al., 2005). Our
results suggest that a similar bi-directional regulation between Hoxc8 and
RARß occurs during the specification and early differentiation of the LMC
cells.
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ACKNOWLEDGMENTS |
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