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First published online December 7, 2007
doi: 10.1242/10.1242/dev.009225
1 Department of Human Genetics, University of Utah, Salt Lake City, UT 84112,
USA.
2 Department of Neurobiology and Anatomy, University of Utah School of Medicine,
20 North 1900 East, Salt Lake City, UT 84132, USA.
3 Howard Hughes Medical Institute, University of Utah, Salt Lake City, UT 84112,
USA.
Author for correspondence (e-mail:
mario.capecchi{at}genetics.utah.edu)
Accepted 10 October 2007
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Hoxc10, Hoxd10, Motoneuron specification, Spinal cord, Mouse
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Price et al., 2002), yet all
are derived from a common progenitor pool.
Considerable progress has been made in defining the mechanisms that control
specification and differentiation of spinal cord motoneurons. Early in
development the spinal cord becomes patterned along both the dorsoventral and
rostrocaudal axis, with motoneuron generation occurring in a restricted
ventral domain (Jessell,
2000). Once generated, motoneurons become highly organized into
lateral and medial motor columns (LMC and MMC, respectively), subdivisions
(lateral and medial) of the columns, and motor pools
(Fig. 1A), each with
characteristic peripheral targets and a unique position in spinal cord
(reviewed by Landmesser,
2001). Research carried out over the last decade, primarily at
brachial levels, has implicated a regulatory network of Hox genes in
establishing the columnar, divisional, and pool specification of motoneurons
(Dasen et al., 2003;
Dasen et al., 2005;
Vermot et al., 2005). Much
less is known about motoneuron specification and diversification at lumbar
levels.
Hox10 genes are expressed in lumbar spinal cord in both chick
(Lance-Jones et al., 2001) and
mouse (Choe et al., 2006). In
chick, ectopic expression of Hoxd10 induces thoracic motoneurons to
express markers characteristic of lateral LMC (lLMC) neurons and innervate
limb muscles (Shah et al.,
2004). Conversely, targeted disruption of Hoxa10 and/or
Hoxd10 has been reported to perturb locomotor behavior, vertebral
column segmentation, and peripheral nerve projections in a manner that
suggests one or more lumbar segments have been transformed into thoracic
segments (Carpenter et al.,
1997; Lin and Carpenter,
2003; Rijli et al.,
1995; Tarchini et al.,
2005; Wahba et al.,
2001). These results indicated a role for Hoxa10 and/or
Hoxd10 in the generation of lumbar LMC motoneurons, but provide
little insight into the cellular, developmental, or molecular mechanisms
regulated by Hox10 genes. With these earlier studies as background, we
disrupted both Hoxc10 (whose function has not been previously
analyzed in mice) and Hoxd10, and examined the expression and
function of these Hox genes during development and differentiation of
motoneurons, nerve innervation and muscle morphogenesis. We show that
Hoxc10 and Hoxd10 are expressed in the right time and place
to function in motoneuron patterning. In the absence of Hoxc10 and
Hoxd10 function, motoneurons in rostral lumbar segments fail to
establish an LMC and instead differentiate as thoracic neurons. Surprisingly,
in more caudal segments the LMC consists almost entirely of medial LMC (mLMC)
neurons with few, if any, motoneurons differentiating into lLMC neurons. Since
nearly all thigh muscles become innervated by mLMC neurons from a reduced
number of spinal segments, motor pools are also clearly disrupted in mutant
animals. Together, our results show that Hoxc10 and Hoxd10
play major roles in specifying the columnar, divisional and motor pool
identities of lumbar motoneurons. In addition, mutations in these genes have
minor, but consistent, effects on hindlimb muscle development.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). A
Hoxc10RFP knockout allele was generated by replacing the
first exon with the red fluorescent protein (RFP) gene and a
Hoxd10hrGFP knockout allele was generated by replacing the
first exon with the humanized Renilla green fluorescent protein (hrGFP) gene
(Fig. 2A). To generate the
Hoxc10RFP knockout allele, an 8.9 kb
XhoI-SalI genomic fragment spanning the two Hoxc10
exons was used to construct the targeting vector. A reporter neo
cassette containing the RFP, DsRed2 (Clontech) cDNA and a self-excision
neomycin resistance gene (neo), was used to replace the first exon of
Hoxc10, the first five amino acids of Hoxc10 being left intact and
in-frame with RFP. To construct the Hoxd10 targeting vector, a 9.3 kb
EcoRI-SpeI genomic fragment spanning the two Hoxd10
exons was used. A reporter-neo cassette containing an hrGFP
(Stratagene) cDNA and a self-excision neo was used to replace the
first exon of Hoxd10, the first three amino acids of Hoxd10 being
left intact and in-frame with hrGFP. In addition, the thymidine kinase gene
(Tk1) was included in the final targeting vectors of Hoxc10
and Hoxd10. To allow removal of the neo selection cassette
subsequent to its use for isolation of targeted ES cell lines, the selectable
gene neo, is embedded in a Cre/loxP-based self-excision
cassette, in which Cre expression is mediated by a promoter, ACE, that is not
expressed in ES cells, but is expressed in the male germline of mouse chimeras
derived from these ES cells during spermatogenesis
(Bunting et al., 1999).
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Immunohistochemistry and in situ hybridization
Immunostaining and in situ hybridization were performed as previously
described (Boulet and Capecchi,
1996; Huber et al.,
2005; Wang and Scott,
2007). The following primary antibodies were used: mouse anti-Isl1
[1:50, 39.4D5, Developmental Studies Hybridoma Bank (DSHB)]; rabbit anti-Isl1
(1:2000, provided by Dr S. Pfaff, Salk Institute, San Diego, CA); rabbit
anti-Hb9 (also known as Mnx1 - Mouse Genome Informatics; 1:8000, provided by
Dr S. Pfaff); rabbit anti-Lim3 (also known as Lhx3 - Mouse Genome Informatics;
1:2000, provided by Dr S. Pfaff); rabbit anti-Lim1 (also known as Lhx1 - Mouse
Genome Informatics; 1:20,000, provided by Dr T. M. Jessell, Columbia
University, New York); rabbit anti-nNOS (1:5000, ImmunoStar, Hudson, WI);
rabbit anti-Olig2 (1:20,000, provided by Dr J. Alberta, Harvard University,
Boston, MA); sheep anti-Chx10 (also known as Vsx2 - Mouse Genome Informatics;
1:1000, Exalpha, Maynard, MA); mouse anti-myosin (1:4000, my32, Sigma); rabbit
anti-MyoD (1:50, Santa Cruz); mouse anti-neurofilament 165 (1:50, 2H3, DSHB);
mouse anti-βIII-tubulin (1:1000, Sigma); mouse anti-BrdU antibody (1:5,
G3G4, DSHB). Species-specific Alexa Fluor 488- and Alexa Fluor 546-conjugated
secondary antibodies (Invitrogen) were used at 1:1000. A Raldh2 (also
known as Aldh1a2 - Mouse Genome Informatics) probe (provided by Dr
Song Wang from our laboratory) was transcribed from a 603 bp cDNA fragment
(1571-2173 bp; NM_009022). Hoxa10 probe was transcribed from a 1043
bp cDNA fragment (1199-2241 bp; NM_008263). ER81 (also known as
Etv1 - Mouse Genome Informatics) and Pea3 (also known as
Etv4 - Mouse Genome Informatics) plasmids were provided by Dr S.
Arber, University of Basel, Switzerland; the Sema3E plasmid was
provided by Dr T. M. Jessell. Other template plasmids (Hoxc10, Hoxd10,
Hoxc9, Hoxd9, Hoxc11 and Hoxc11) were created in our laboratory
(Hostikka and Capecchi,
1998).
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Cell death in motoneurons was assessed by TUNEL (Cell Death Detection Kit; Roche) on Isl1- or Hb9-labeled sections. Mitotic cells were labeled with bromodeoxyuridine (BrdU; Sigma) injected intraperitoneally (50 µg/g body weight) into E9.5-E10.75 timed pregnant mice, and subsequently identified with motoneuron markers and anti-BrdU.
Anterograde and retrograde labeling
Axon projections from motoneurons in specific spinal cord levels were
labeled with DiI
(1,1'-dioctadecyl-3,3,3'3-tetramethylindocarbocyanine perchlorate;
2.5 mg/ml dimethylformamide; Molecular Probes, Eugene, OR) in
paraformaldehyde-fixed embryos (Carpenter
et al., 1993). Retrograde labeling of motor pools was achieved by
injecting tetramethylrhodamine dextran [(3000 MW; Invitrogen) in Tris-buffered
saline (TBS) with 10% lysophosphatidyl choline (Sigma)] into individual
muscles (Vrieseling and Arber,
2006).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Lin and Carpenter, 2003;
Tarchini et al., 2005). All
three Hox10 transcripts were first detected in lumbar spinal cord at E10.5,
and the rostrocaudal expression domain of each did not change in any of the
ages examined (data not shown). By E13.5 Hoxc10 and Hoxd10
were expressed most intensely in segments L2-L4, with lower levels of
expression in L1 and segments caudal to L4
(Fig. 1B). Whereas
Hoxc10 and Hoxd10 were expressed only in lumbar regions,
Hoxa10 expression extended from T10 to lumbar levels
(Fig. 1B)
(Choe et al., 2006).
Transverse serial sections confirmed the timing and rostrocaudal extent of
expression of all three Hox10 transcripts
(Fig. 1C-E). Furthermore, by
staining these sections with antibodies to distinguish different motor columns
(Fig. 1A), we were able to
determine the cell types that expressed different Hox10 transcripts, and show
that each transcript had a unique, highly dynamic pattern of expression.
Postmitotic motoneurons, identified by Islet1 (Isl1) expression, first
appeared in lumbar spinal cord around E10.0-E10.5 (data not shown), slightly
later than reported for brachial spinal cord
(Arber et al., 1999), and by
E11 most, if not all, postmitotic motoneurons in segments L2-L4 expressed
Hoxc10 and Hoxd10 (Fig.
1C). By contrast, Hoxa10 was expressed in a very focal
ventral domain, overlapping the characteristic position of the V3 interneuron
domain (Fig. 1C)
(Briscoe et al., 1999), and
extending rostrally into thoracic spinal cord (data not shown).
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The posterior expression limits of Hox10 genes are more easily defined in
section in situ hybridizations than in whole-mount preparations.
Hoxc10 and Hoxd10 expression in motoneurons extends only
through L5 (data not shown). These genes are also expressed at extremely low
levels in intermediolateral regions in more caudal spinal cord, which could
account for the caudal expression of Hox10 genes observed in whole-mount
preparations (Carpenter, 2002)
(Fig. 1B). Taken together, our
data show that Hoxc10 and Hoxd10 are expressed in
motoneurons exclusively in lumbar spinal cord. The fact that both
Hoxc10 and Hoxd10 are initially expressed in almost all
newly generated motoneurons but later become restricted to subpopulations of
motoneurons, primarily to lLMC and mMMC neurons, suggests they may play
sequential roles in specifying or maintaining motoneuron identity at different
stages. Conversely, the absence of Hoxa10 from motoneurons at early
stages suggests that this paralog may have relatively little influence on
motoneuron specification. Moreover, because Hoxc10 and
Hoxd10 have similar, although not identical, expression patterns in
the developing spinal cord, they may share redundant functions in motoneuron
development.
Generation of Hoxc10 and Hoxd10 double mutants
To examine the roles of Hox10 genes in the development of hindlimb
motoneurons, we analyzed different combinations of Hox10 double and
triple-mutant mice. The Hoxa10 allele has been reported previously
(Wahba et al., 2001). Here we
describe mice carrying new alleles of Hoxc10 and Hoxd10 that
lacked the neo cassette (Fig.
2), which we created because the presence of neo can
affect the mouse phenotype by altering the expression of nearby genes
(Greer and Capecchi, 2002;
Manley et al., 2001). This is
particularly a problem with Hox genes, since the density of genes is high
within this complex. Homozygous mutant mice were found to lack Hoxc10
or Hoxd10 transcripts when examined by in situ hybridization (data
not shown), and thus appear to be null mutants. Both single and double-mutant
animals were viable and had an apparently normal lifespan, although the double
mutants were sterile. Double heterozygotes and single homozygous mutants did
not have any obvious alteration in gait, and therefore heterozygotes were
sometimes used with wild-type (WT) animals as controls in this study. The lack
of an aberrant phenotype in double heterozygous and single homozygous mutants
differs from the phenotypes of previously generated Hoxd10 null
mutants, which had obvious defects in locomotor behavior
(Carpenter et al., 1997;
Tarchini et al., 2005). The
more normal behavior observed in our single mutants most likely results from
the lack of the neo gene in our alleles. The lack of apparent
locomotor phenotypes associated with mutations in either Hoxc10 or
Hoxd10 alone also emphasizes the redundant functions of these Hox
genes.
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We next compared Hoxc10-/-/Hoxd10-/- double-mutant animals with Hoxa10-/-/Hoxc10-/- and Hoxa10-/-/Hoxd10-/- double mutants. Locomotor defects in Hoxc10-/-/Hoxd10-/- mutants were significantly more severe than in the latter two groups. Surprisingly, locomotor defects in the Hox10 triple-mutant animals (n=3) seemed to be less severe than in Hoxc10-/-/Hoxd10-/- double mutants, although the triple-mutant mice died around weaning, as a result of kidney defects. Thus, it appears that the loss of Hoxa10 does not significantly contribute to defects in locomotor behavior, most likely because Hoxa10 is not expressed within the motoneuron domain at early stages (Fig. 1C). Given the less prominent expression pattern of Hoxa10 in the lumbar motor column, and the negligible additional contribution of the Hoxa10 mutation to the Hoxc10-/-/Hoxd10-/- mutant phenotype, we focused further analysis primarily on Hoxc10-/-/Hoxd10-/- double-mutant animals.
Hindlimb muscle morphology and innervation patterns in Hox10 mutants
Severe locomotion defects could result from either motoneuron projection
errors and/or altered muscle patterning. Analysis of the overall pattern of
muscle (Greene, 1935) and
nerve innervation in cross-sections through the hindlimbs of E14.5-E15.0
mutant and WT embryos revealed that the anterior head of the biceps was
missing from the thigh in four out of seven
Hoxc10-/-/Hoxd10-/- mutant embryos. In the
shank, the extensor hallucis longus was missing from the anterior group in
four out of four double-mutant embryos, and two more muscles were missing from
the lateral group in three out of four double-mutant embryos
(Fig. 3A and see Fig. S1 in the
supplementary material). The loss of these muscles was confirmed by dissecting
P0 hindlimb muscles stained with AP-conjugated anti-myosin (data not shown).
To our knowledge, this is the first report of muscle defects associated with
Hox10 mutant animals.
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The lack of innervation to anterior and lateral shank muscles in the double mutants was confirmed in whole-mount embryos stained with neurofilament antibody at E12. Importantly, neurofilament staining also revealed striking abnormalities in the contribution of spinal nerves to hindlimb innervation. In WT mice, segments L1-L3 contribute axons to the rostral lumbar plexus and segments L3-L5 contribute to the caudal sacral plexus (Fig. 3B). In the Hoxc10-/-/Hoxd10-/- mutant mice, however, L1 and L2 did not project to the hindlimb, and instead appeared to innervate the body wall, and L3 and L4 contributed axons to the lumbar plexus and L4 and L5 contributed to the sacral plexus (n=6; 100% penetrance; Fig. 3B). At the level of the lumbar plexus, both the dorsal and ventral branches were present but significantly reduced in size, most likely because of the reduced number of segments projecting to this plexus. By contrast, the dorsal branch of the sacral plexus (the peroneal nerve), which normally supplies the anterior and lateral groups of muscles in the shank, was totally absent, whereas the ventral branch (the tibial nerve) was only slightly smaller than in WT embryos (Fig. 3B,C). The near total lack of innervation of anterior and lateral shank muscles, which normally extend and abduct the limb, could be a major reason for the crossed-limb phenotype observed in Hoxc10-/-/Hoxd10-/- double-mutant mice.
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L1 and L2 motoneurons become thoracic motoneurons in Hoxc10-/-/Hoxd10-/- double mutants
The above observations indicated that the L1 and L2 spinal nerves projected
to the body wall rather than to the limb in
Hoxc10-/-/Hoxd10-/- mutants. This was confirmed
by DiI injections into spinal cord segments L1 and L2 in double mutants
(Fig. 4A). In addition, we
observed many features consistent with the hypothesis that motoneurons in
segments L1 and L2 actually differentiated into thoracic motoneurons in
Hoxc10-/-/Hoxd10-/- mutants, and never
acquired characteristics of LMC neurons. For example, the columnar
organization of motoneurons in the ventral horn of segments L1 and L2
resembled that of thoracic rather than lumbar spinal cord in double mutants
(Fig. 4B). Normally at thoracic
levels in WT animals, there are two motor columns in the spinal cord: the MMC
in ventral cord, which has a medial and lateral division (mMMC and lMMC), and
the visceral sympathetic preganglionic motor column (PGC) in the
intermediolateral cord (Fig.
1A). By contrast, at lumbar levels motoneurons occupy the large
LMC and smaller mMMC. In
Hoxc10-/-/Hoxd10-/- mutants, however,
staining of spinal cord sections for Isl1 and Lim3 showed that the arrangement
of motor columns characteristic of thoracic cord extended caudally to the
rostral part of L3 (Fig. 4B),
with an apparent LMC first appearing only at L3. Furthermore, neuronal form of
nitric oxide synthase (nNOS)+/Isl1+ staining in
intermediolateral cord, which is characteristic of visceral motoneurons
(Thaler et al., 2004),
extended caudally into rostral L3 in the double mutants
(Fig. 4C). These ectopic
nNOS+/Isl1+ motoneurons behave like those in the
thoracic region, projecting their axons to sympathetic ganglia
(Fig. 4D). Thus, in
Hoxc10-/-/Hoxd10-/- mutants,
motoneurons characteristic of thoracic cord were present in segments L1 and
L2.
By contrast, markers characteristic of LMC neurons were absent from L1 and
L2 in these mutant embryos. Retinaldehyde dehydrogenase 2 (Raldh2), a generic
marker for LMC motoneurons, is expressed in LMC motoneurons throughout all
lumbar segments in WT animals (Sockanathan
and Jessell, 1998). In
Hoxc10-/-/Hoxd10-/- mutant animals,
however, Raldh2 expression was absent from segments L1 and L2
(Fig. 5A,A'), and was
reduced in more caudal lumbar segments. Further evidence that LMC neurons were
missing is that hindlimb muscles normally innervated by LMC neurons in these
segments, such as the adductor (not shown) and quadriceps
(Fig. 6E), were innervated by
motoneurons in segment L3. Finally, several markers characteristic of specific
LMC motor pools were not expressed in segments L1 and L2 in double mutants.
For example, Pea3 (Arber et al.,
2000) and Sema3E
(Livet et al., 2002;
Messersmith et al., 1995) are
normally expressed in several LMC motor pools at lumbar levels from L1 to L5,
but not in thoracic regions. In double-mutant embryos, neither gene was
expressed in segments L1 and L2, but both were still expressed in more caudal
segments (Fig. 5B,B' and
data not shown). In mice, ER81 is normally expressed in both thoracic
(Fig. 5C,C') and lumbar
spinal cord (Arber et al.,
2000), with a clear gap between the thoracic domain and the two
lumbar pools (Fig. 5C,C',
Control). By contrast, in the double mutants, the thoracic domain of
ER81 expression extended into segments L1 and L2, and expression of
ER81 was lost in segments L3 and L4
(Fig. 5C,C').
Because Hox9 genes, especially Hoxc9, are suggested to be
thoracic motoneuron determinants in chick
(Dasen et al., 2003;
Dasen et al., 2005), we asked
whether altered expression of Hoxc9 and Hoxd9 in
Hoxc10-/-/Hoxd10-/- mutants could account for
the switch of L1 and L2 motoneurons to a thoracic identity. In WT embryos,
Hoxc9 and Hoxd9 were expressed in thoracic and lumbar spinal
cord, with expression terminating around lumbar segment L5. There was no
obvious change in expression of either Hoxc9 or Hoxd9 in the
double knockouts (see Fig. S2 in the supplementary material; data not shown)
(Carpenter et al., 1997).
Hoxc11 and Hoxd11 transcripts were observed from L3 through
the sacral segments in control embryos, and expression of these Hox genes was
also not obviously altered in the double mutants (see Fig. S3 in the
supplementary material; data not shown)
(Tarchini et al., 2005).
Taken together these data show that motoneurons in segments L1 and L2 in Hoxc10-/-/Hoxd10-/- double mutants differentiate as thoracic, rather than lumbar, motoneurons. Moreover, these findings identify Hoxc10 and Hoxd10 as having important roles both in establishing the border between thoracic and lumbar segments of spinal cord, and in defining the identity of motoneurons in these segments.
Lim1+ lateral LMC motoneurons are absent in segments L3-L5 of double mutants
Whereas motoneurons from L1 and L2 were converted to thoracic phenotypes,
motoneurons from L3-L5 maintained their LMC identity, defined by
Raldh2 expression, and innervated the hindlimb in double mutants.
However, neurons in segments L3-L5 are clearly impacted by deletion of
Hoxc10 and Hoxd10 function. Since most hindlimb muscles
received some innervation (Fig.
3A and see Fig. S1 in the supplementary material), motoneurons in
segments L3-L5 must have taken over some functions normally mediated by
neurons in L1 and L2. Despite this, there appeared to be fewer LMC neurons in
L3-L5 in double mutants; Raldh2
(Fig. 5A') and Hb9
(Fig. 6C) expression were
greatly reduced, and the peroneal nerve was missing entirely
(Fig. 3B,C). To resolve these
discrepancies, with an eye toward elucidating additional functions of Hox10
genes in motoneuron differentiation, we examined the subtype identity of LMC
neurons in double mutants. Surprisingly, lLMC motoneurons, as defined by Lim1
staining, were severely reduced or absent in eight out of ten double-mutant
embryos at E13.5, and noticeably reduced in the other two embryos
(Fig. 6A). Lim1+
lLMC neurons were also not observed in lumbar segments at earlier stages (see
Fig. S5 in the supplementary material), indicating that Lim1+ LMC
neurons failed to differentiate in double mutants, rather than having
differentiated and died. Instead, neurons in ventrolateral spinal cord, the
usual location of the lLMC, expressed Isl1, characteristic of mLMC, and
Pea3 was restricted to Isl1+ neurons, instead of being
expressed in both Lim1+ and Isl1+ motoneurons
(Fig. 5B' and data not
shown), as in WT mice (Arber et al.,
2000; Wang and Scott,
2007).
The number of Hb9+ neurons was reduced (Fig. 6C and see Fig. S6 in the supplementary material), demonstrating a paucity of LMC neurons. However, the number of mLMC neurons in L3-L5 was the same in control and Hoxc10-/-/Hoxd10-/- mutants, based on counts of Isl1+ cells in serial sections of seven embryos of each genotype (Fig. 6B). Similarly, the number of Lim3+ neurons was not obviously affected in double mutants (data not shown). Thus, the missing Lim1+ motoneurons do not appear to have become Isl1+ or Lim3+ neurons. Instead, the lLMC appears to be missing entirely, with Isl1+ mLMC neurons being displaced to the most lateral part of the spinal cord in its absence. By contrast, the lLMC appeared to form normally in embryos homozygous for the individual new Hoxc10 or Hoxd10 mutant alleles (see Fig. S4 in the supplementary material).
Because Lim1+ lLMC motoneurons normally project their axons to
dorsally derived hindlimb muscles in WT animals, the loss of the
Lim1+ LMC neurons is most likely responsible for the absence of the
peroneal nerve (Fig. 3B,C).
This differs from the loss of the peroneal nerve in EphA4 mutants, which
results from misrouting of lLMC neurons into ventral branches
(Helmbacher et al., 2000). We
verified that neurons in segments L3-L5 did not project in any dorsal nerve by
injecting DiI into motoneurons in these segments
(Fig. 6D). Thus, the loss of
the Lim1+ lLMC neurons explains the lack of innervation in anterior
or lateral shank muscles, which are normally innervated by axons in the
peroneal nerve (Fig. 3A and see
Fig. S1 in the supplementary material).
Intriguingly, however, dorsal thigh muscles, which are also normally
innervated by Lim1+ lLMC neurons projecting in a dorsal nerve,
clearly received some innervation (Fig.
3A) even though their usual motor pools appeared to be missing. To
determine which neurons supplied dorsal thigh muscles, we retrogradely labeled
quadriceps motoneurons with tetramethylrhodamine dextran. As expected
(McHanwell and Biscoe, 1981),
the quadriceps in WT animals was innervated by
Lim1+/Isl1- lLMC motoneurons in segment L2, with a
smaller contribution from L1. By contrast, quadriceps muscles in double-mutant
embryos were innervated by Lim1-/Isl1+ mLMC motoneurons
in segment L3 (Fig. 6E). Thus,
in the absence of Hoxc10 and Hoxd10 function some mLMC
motoneurons became misrouted and innervated dorsal-derived thigh muscles. The
aberrant innervation of extensor muscles in the thigh, such as the quadriceps,
by motoneurons that normally innervate flexor muscles in WT animals most
likely contributed significantly to the locomotor defects in double mutants.
If motoneurons receive their usual complement of central connections, as
occurs when they innervate inappropriate muscles following surgical
manipulations (Landmesser and O'Donovan,
1984; Vogel,
1987), then both extensors and flexors would be activated at the
same time, leading to the rigid extended posture of limbs in mutant
animals.
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;
Ericson et al., 1992;
Thaler et al., 2004). As with
motoneuron progenitors, the numbers of newly generated Isl1+
postmitotic motoneurons were similar in control and double knockout embryos at
early stages (E10.0-E11.0; Fig.
7B). These observations suggest that Hoxc10 and
Hoxd10 function are dispensable for the generation and initial
specification of motoneurons during early embryogenesis.
Late-born motoneurons survive, but are misplaced in double mutants
The striking reduction in lLMC neurons in double mutants was not brought
about by increased apoptosis. TUNEL staining of serial sections of lumbar
spinal cord was nearly identical in controls and double mutants at all ages
examined (E10.5-E14.0; see Fig. S6 in the supplementary material). This
differs from a previous study in which increased apoptosis of neurons was
suggested as a reason for forelimb locomotor defects in Hoxc8 mutants
(Tiret et al., 1998).
Motoneurons were initially generated in normal numbers and did not die in excess of normal, yet the entire Lim1+ lLMC was absent in double mutants. Where are these missing neurons? To address this question, we compared the fate of Hoxd10-expressing cells, the cells we expected to be most directly affected by loss of the Hoxd10 gene product, in control and double-mutant animals. Because the Hoxd10 mutant allele was generated by replacing the first exon of the Hoxd10 gene with the hrGFP gene, we could follow the fate of cells in the double mutants by analyzing hrGFP expression in sections of heterozygous and double-mutant embryos. At E13.5 in Hoxc10+/-/Hoxd10+/- heterozygous control embryos, hrGFP+ motoneurons were located in the most lateral part of ventral horn (Fig. 8A), closely resembling the pattern of endogenous Hoxd10 expression in the lLMC in WT embryos (Fig. 1E). By contrast, in the double-mutant mice, hrGFP+ cells were no longer tightly clustered laterally, but instead were scattered throughout the entire ventral horn area (Fig. 8A). This finding suggests that the inactivation of Hoxc10 and Hoxd10 alters migration of Hoxd10-expressing cells in lumbar spinal cord.
The altered distribution of Hoxd10-expressing cells in the ventral
horn raised the possibility that the missing lLMC motoneurons had changed
their identity, but the numbers of motoneurons in the mLMC and mMMC had not
increased in double mutants (Fig.
6B) and the numbers of Hb9+ cells had decreased
(Fig. 6C). Further, there was
no obvious change in the expression of Chx10, a V2 interneuron marker
(Arber et al., 1999;
Ericson et al., 1997) in
double mutants (data not shown). Instead, it appeared that cells fated to be
lLMC neurons were born in normal numbers, but failed to acquire or retain
markers characteristic of other populations of mature motoneurons or
interneurons.
Motoneuron generation starts at E10.0 at the hindlimb level and is mostly
completed by E11.0. Prospective lLMC motoneurons exit the cell cycle later
than prospective mLMC motoneurons. These late-born motoneurons emerge from the
ventricular zone, migrate laterally past the earlier-born mLMC motoneurons,
acquiring their lLMC identity during the migration process, and eventually
settle in the lateral part of the ventral horn
(Sockanathan and Jessell,
1998). The difference in timing of generation of mLMC and lLMC
neurons allowed us to investigate the fate of the late-born motoneurons in
more detail. We labeled late-born cells by injecting BrdU into pregnant
females at E10.5, a time by which the early-born motoneurons in WT embryos
have already exited cell cycle and no longer incorporate BrdU, and analyzed
motoneuron identity and distribution at E12.0. In the control embryos, most
BrdU+ cells in the ventral horn settled laterally and were
Lim1+/Isl1-, suggesting that late-born cells were indeed
lLMC motoneurons (Fig. 8B). By
contrast, in double-mutant embryos, BrdU+ cells were scattered
throughout the ventral horn and intermingled with Isl1+ cells.
Importantly, most BrdU+ cells expressed neither Isl1 nor Lim1.
Thus, the loss of Hoxc10 and Hoxd10 appears to cause late-born motoneurons to
differentiate incompletely. These neurons downregulated expression of Isl1 on
schedule, but failed to migrate to their appropriate location or acquire other
markers characteristic of mature motoneurons, consistent with the observed
decrease in Hb9+ cells. It is possible that some of the late-born
motoneurons in double mutants differentiated into interneurons, but
investigation of this possibility must await discovery of additional markers
of mature interneurons.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Landmesser,
2001). Here we show that Hoxc10, which had not been
previously studied, and Hoxd10 together play essential roles in
patterning lumbar motoneurons at all three levels of organization, and
elucidate some of the cellular and molecular processes governed by these
genes. We also show that these genes have minor, but consistent, effects on
patterning hindlimb muscles.
Hoxc10 and Hoxd10 determine the rostral boundary of lumbar motor columns
Our analysis of Hoxc10-/-/Hoxd10-/-
double-mutant embryos showed conclusively that Hox10 gene products govern the
patterning of lumbar versus thoracic motor columns. Motoneurons in segments L1
and L2 differentiated as thoracic motoneurons in double mutants, expressing
nNOS, but failing to express markers of LMC neurons, such as Raldh2.
The remaining LMC in segments L3-L5 innervated the entire hindlimb, indicating
that the LMC in Hoxc10-/-/Hoxd10-/-
mutants was compressed from five to three segments, rather than simply being
shifted posteriorly, as suggested previously
(Lin and Carpenter, 2003).
Several lines of evidence suggest that Hoxc10 and Hoxd10
play primary roles in the patterning of thoracic versus lumbar motor columns
within the spinal cord, with Hoxa10 playing a lesser role. For
example, Hoxa10, unlike Hoxc10 and Hoxd10, is not
expressed in prospective motoneurons during their early genesis, and therefore
is unlikely to be involved in the early steps of their specification.
Furthermore, locomotor defects were more severe in
Hoxc10-/-/Hoxd10-/- mutants than in
Hoxa10-/-/Hoxc10-/- or
Hoxa10-/-/Hoxd10-/- mutants.
Importantly, ectopic expression of Hoxd10 in thoracic motoneurons in
chick is sufficient to convert them into lumbar-like motoneurons
(Shah et al., 2004). The
conversion of prospective LMC neurons in L1 and L2 to thoracic motoneurons in
the Hoxc10-/-/Hoxd10-/- mutants may
result from the persistence of Hox9 gene function in these neurons in
the absence of normal Hoxc10 and Hoxd10 expression [i.e. a
case of elimination of posterior prevalence
(Duboule and Morata,
1994)].
Thus, it appears that Hoxc10 and Hoxd10, together with
more rostrally expressed Hox genes, determine the rostral border between
thoracic and lumbar motor columns in the spinal cord. The failure to convert
more caudal segments to thoracic cord as well as the persistence of an LMC in
more caudal segments in
Hoxc10-/-/Hoxd10-/- mutants is most
likely due to the presence of Hox11 genes, which are expressed in the
caudal spinal cord from segment L3 in both WT
(Carpenter, 2002) and
Hoxc10-/-/Hoxd10-/- mutant embryos
(see Fig. S3 in the supplementary material). Interestingly, the LMC in caudal
lumbar segments normally consists predominantly of Isl1+ mLMC
neurons in both chick and mouse (data not shown), similar to the LMC in
Hoxc10-/-/Hoxd10-/- mutants.
Hox11 genes appear to be important in generating these motoneurons,
since misexpression of Hoxd11 in rostral lumbar motoneurons induces
an overabundance of Isl1+ mLMC neurons relative to Lim1+
lLMC neurons (Misra et al.,
2005). In summary, Hoxc10 and Hoxd10 are
required for proper columnar specification of the lumbar motoneurons.
Hoxc10 and Hoxd10 determine divisional specification in the LMC
In addition to establishing the boundary between thoracic and lumbar motor
columns, our results reveal a novel role for Hox10 genes in the divisional
specification of LMC. Hoxc10 and Hoxd10 are essential for
development of the lateral division of the LMC in lumbar cord. The lLMC was
nearly eliminated in
Hoxc10-/-/Hoxd10-/- mutants as
evidenced by the reduction or loss of lateral Lim1+ neurons and the
dorsal nerve branches of the lumbar and sacral plexii. Our results differ from
the reported milder phenotype for
Hoxa10-/-/Hoxd10-/- mice, in which
both divisions of LMC neurons were present in neonates but reduced in size;
although the spatial relationships between the two groups of cells were
retained, the groups were clustered together
(Lin and Carpenter, 2003).
Perturbation of any number of developmental processes could produce a lack
of lLMC neurons in Hoxc10-/-/Hoxd10-/-
double mutants. We show here that the loss of the lLMC did not result from a
decreased production of motoneuron precursors or from the increased apoptosis
of Lim1+ motoneurons. Instead, presumptive lLMC neurons failed to
migrate to their normal position and never acquired markers characteristic of
known populations of motoneurons or interneurons. The observed migratory
defect resembles the effects of perturbing cadherin expression on motoneuron
sorting (Price et al., 2002),
suggesting a mechanism by which Hox genes could govern migration of
prospective lLMC neurons. Thus, in the absence of Hoxc10 and Hoxd10,
motoneuron precursors appear to be generated normally, but late-born neurons
fail to differentiate into lLMC neurons or into any other clearly recognizable
neuron population.
Hoxc10 and Hoxd10 affect motor pool specification and limb muscle development
Motor pools represent specific groups of motoneurons in the LMC that
establish functional connections with individual muscles in the limb. Our
findings show that Hoxc10 and Hoxd10 influence lumbar motor
pool formation, although these effects may be indirect as a consequence of
Hox10 function in columnar and divisional specification. Motor pools were
clearly aberrant in
Hoxc10-/-/Hoxd10-/- mutants. There was
no LMC in segments L1 and L2 and no lLMC in more caudal segments in
double-mutant embryos, yet most hindlimb muscles were innervated. The
remaining Isl1+ mLMC neurons in segments L3-L5 must, therefore,
have distributed themselves among many more muscles than normal, clearly
altering motor pools. Retrograde labeling showed that at least one muscle, the
quadriceps, was innervated inappropriately by Isl1+ neurons in the
absence of Lim1+ neurons. We expect that other dorsal muscles in
the thigh were similarly innervated by Isl1+ neurons, which
normally innervate ventral muscles.
Further evidence that Hox10 genes influence motor pool formation is that
the normal expression patterns of the ETS transcription factor genes,
ER81 and Pea3, which are restricted to specific motor pools
in WT animals (Arber et al.,
2000), were altered in
Hoxc10-/-/Hoxd10-/- mutant embryos
(Fig. 5B,B' and
5C,C'). Some change in
ETS expression was expected, since some motoneurons that usually express ETS
factors were missing in double mutants. In addition, innervation of
inappropriate muscles by the remaining motoneurons may also contribute to
altered expression of ER81 and Pea3, since ETS expression is
normally initiated and shaped by signals from the periphery
(Lin et al., 1998;
Wang and Scott, 2004). In
addition, the peripheral signals themselves may be perturbed in double
mutants.
Hox10 genes are expressed in the developing hindlimb
(Morgan and Tabin, 1994;
Nelson et al., 1996;
Wellik and Capecchi, 2003) as
well as in the lumbar spinal cord. Thus, the disruption of Hox10 genes in the
periphery may have contributed to the observed perturbations in muscle
innervation. For example, we have previously shown that restricted
inactivation of Hoxb1 in the periphery resulted in the failure of
these motoneuron axons to innervate the facial muscles
(Arenkiel et al., 2004).
Therefore, the locomotor and innervation mutant phenotypes in the
Hoxc10-/-/Hoxd10-/- double mutants
reported here are likely to have resulted from contributions of Hox function
in both motoneuron specification and in motoneuron targeting in the periphery.
These potential contributions should be separable through the use of
conditional mutagenesis. Importantly however, the motoneuron specification
defects discussed in this paper are not likely to have been affected by the
functions of Hoxc10 and Hoxd10 in the periphery, since this
specification occurs before the axons grow into the limb and indeed before the
motoneurons are born (Matise and
Lance-Jones, 1996).
In conclusion, we have elucidated novel functions of Hoxc10 and Hoxd10 in the patterning of lumbar motoneurons. We showed that disruption of Hoxc10 and Hoxd10 causes rostral lumbar motoneurons to adopt a thoracic phenotype, and prevents the differentiation of Lim1+ lateral LMC neurons. Most hindlimb muscles in double mutants become innervated by the remaining medial LMC neurons. Together, these results show that Hoxc10 and Hoxd10 are important in establishing the columnar, divisional and motor pool identity of lumbar motoneurons. The downstream cascades of genes activated and repressed by Hoxc10 and Hoxd10, which ultimately govern the differentiation of lumbar motoneurons, remain to be determined.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/1/171/DC1
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ACKNOWLEDGMENTS |
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Footnotes |
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