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First published online 21 June 2006
doi: 10.1242/dev.02478
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Department of Neuroscience, University of Virginia, 409 Lane Road, MR4, Room 5032, Charlottesville, VA 22908, USA.
e-mail: jl7nf{at}virginia.edu
Accepted 1 June 2006
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Gdf11, Smad, Spinal cord, Hox, Rostrocaudal patterning
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Helms and Johnson, 2003;
Jessell, 2000), while retinoic
acid (RA) originating from the somites, and fibroblast growth factors (Fgfs)
and Wnt proteins originating from the regressing primitive streak/Hensen's
node region are the major rostralizing and caudalizing signals acting along
the RC axis (for a review, see Diez del
Corral and Storey, 2004). Incorporation of the DV and the RC
signals will then induce the expression of different transcription factors in
neural progenitor cells located at different positions of the spinal cord, and
these transcription factors in turn, determine neuronal identity.
Motoneurons are generated in the ventral spinal cord in response to Shh
signaling (for reviews, see Briscoe and
Ericson, 2001; Jessell,
2000), and their cell bodies segregate into different columns and
pools at different RC positions according to their targets of innervation
(Hollyday, 1980a;
Hollyday, 1980b;
Landmesser, 1978a;
Landmesser, 1978b). Thus,
lateral motor column (LMC) motoneurons that innervate limb musculature are
present only at cervical/brachial and lumbar levels, while various motoneuron
pools that innervate individual muscles are located at different RC positions.
Hox family transcription factors are expressed in different RC domains in the
spinal cord and they control the generation of specific motoneuron subtypes at
each RC level (for reviews, see Carpenter,
2002; Krumlauf et al.,
1993). Thus, Hoxc6, Hoxc9 and Hoxa10/Hoxd10 define motoneuron
columnar identities at the brachial, thoracic and lumbar levels, respectively
(Carpenter et al., 1997;
Dasen et al., 2003;
Lin and Carpenter, 2003;
Shah et al., 2004), while
Hoxc8 determines motor pool identities
(Dasen et al., 2005;
Vermot et al., 2005).
Moreover, manipulating Hox gene expression also results in changes in spinal
nerve projections (Burke and Tabin,
1996; Carpenter et al.,
1997; Dasen et al.,
2003; de la Cruz et al.,
1999; Shah et al.,
2004; Tiret et al.,
1998).
Spinal cord stem cells are located around Hensen's node in chick, and the
node in mouse embryos (Mathis and Nicolas,
2000; Schoenwolf,
1992). Progenitor cells designated for rostral spinal cord leave
the stem zone prior to the progenitor cells designated for caudal spinal cord.
Although the RC identities of these progenitor cells are specified when they
leave the stem zone, their identity is still modifiable to a certain degree
prior to somite formation (Ensini et al.,
1998; Lance-Jones et al.,
2001; Liu et al.,
2001). In addition to the signaling factors, including Fgfs, Wnts
and RA, that have been shown to control Hox gene expression in neural tissues
(Bel-Vialar et al., 2002;
Dupe et al., 1997;
Marshall et al., 1994;
Shimizu et al., 2005;
Zhang et al., 1997), we have
identified an additional activator of Hox gene expression,
growth/differentiation factor 11 (Gdf11), using an in vitro assay system
(Liu et al., 2001). Gdf11 is a
transforming growth factor-ß (TGF-ß) family member and its
expression begins at HH stage 10
(Hamburger and Hamilton, 1951)
in chick embryos around Henson's node, caudal paraxial mesoderm and caudal
neural plate (Liu et al.,
2001). Similarly, Gdf11 expression begins in the tail bud
and caudal neural plate region in mouse embryos around E8.5
(Nakashima et al., 1999).
Mouse mutants lacking Gdf11 die shortly after birth and exhibit six extra
thoracic vertebrae, caudally displaced lumbar vertebrae and truncated tails
(McPherron et al., 1999). A
caudal displacement of Hox gene expression domains associated with the
vertebral phenotype was observed in Gdf11-/- embryos,
suggesting that Gdf11 has a role in patterning caudal structures
(McPherron et al., 1999). In
the olfactory epithelium, Gdf11 controls the number of olfactory receptor
neurons by inhibiting the proliferation of their progenitors
(Wu et al., 2003). However, in
the developing retina, Gdf11 controls the number of retinal ganglionic cells
by affecting the competence of progenitor cells but not their proliferation
(Kim et al., 2005), indicating
Gdf11 has a function in cell fate specification in addition to cell
proliferation.
In this study, we investigate the function of Gdf11 in patterning spinal
tissue in vivo. We manipulated Gdf11 levels in the developing spinal cord by
expressing either Gdf11 or its antagonist follistatin (Fst)
(Gamer et al., 2001), using in
ovo electroporation in early chick embryos. Our data demonstrate that Gdf11
has the ability to caudalize neural tissues by inducing caudal Hox gene
expression and suppressing rostral Hox gene expression. This results in
rostral displacements of Hox expression domains followed by changes in
motoneuron identity and peripheral projection. These observations are further
corroborated by analyses of the spinal cord phenotype in Gdf11
loss-of-function mouse embryos - without endogenous Gdf11, these embryos
exhibit caudal displacement and expansion of Hox expression domains, as well
as a caudal shift in motoneuron columnar and pool positions. The severity of
this phenotype increases towards the caudal end of the spinal cord, suggesting
that Gdf11 function is more important for caudal neural identity. We also
provide evidence that Gdf11 induces phosphorylation of Smad2 and activated
Smad2 is able to control Hox gene expression. These data demonstrate that
Gdf11 signaling through Smad2 plays a crucial role in determining Hox gene
expression domains and neuronal identity in the caudal spinal cord.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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)
containing a splicing substrate, multiple cloning sites and an IRES-eGFP
cassette into the pNES1689tk vector
(Zimmerman et al., 1994)
containing the human nestin intron 2 enhancer followed by the HSV thymidine
kinase promoter. Coding regions of the following genes were cloned into the
pNES-IRES-GFP vector: human Gdf11 (provided by S.-J. Lee), human
follistatin (provided by M. Matzuk) and a constitutively activated
Smad2-Smad2E (provided by M. Funaba).
In ovo electroporation
Electroporation was performed as described
(William et al., 2003).
Observation under a fluorescence microscope (Nikon SMZ1500) was made the day
after electroporation and only embryos with visible GFP expression were used
for analysis. Stages of embryos used for electroporation and the time of
harvesting are indicated in the `results' section. The Gdf11
construct was used at 2-5 mg/ml; Fst and Smad2-2E constructs
were used at 1-5 mg/ml.
Immunohistochemistry and in situ hybridization
For immunohistochemistry, mouse embryos were harvested at E12.5, and chick
embryos were harvested 3 days after electroporation (
HH stage 25).
Fixation, tissue preparation and antibody staining were performed as described
(Liu and Jessell, 1998;
Liu et al., 2001), and images
were collected using a Nikon C-1 confocal microscope.
For in situ hybridization, chick embryos were harvested at various stages
after electroporation, and mouse embryos were harvested at E12.5-E13.5.
Whole-mount in situ hybridization was performed as described for chick
(Thery et al., 1995), and for
mouse embryos (Garces et al.,
2000). A BCIP/NBT kit (Vector Labs) was used for the color
reaction. Chick Hoxc6-Hoxc10 probes were provided by C.
Tabin. Mouse and chick Raldh2, Pea3 and Er81 probes, as well
as mouse Hoxc6 and Hoxc10 probes were provided by T. Jessell
and J. Dasen. All protocols for animal use were approved by the Institutional
Animal Care and Use Committee of the University of Virginia and were in
accordance with NIH guidelines.
For neurofilament staining, chick embryos were harvested 4-5 days after electroporation (HH stage 27-29), fixed overnight in 4% paraformaldehyde in 0.1 M phosphate buffer pH 7.4 at 4°C, then washed three times with PBS (1 hour/wash) before incubating with 0.15% H2O2 in PBS at 4°C overnight. Afterwards, the embryos were washed in PBS, and blocked in a solution of 10% heat-inactivated goat serum, 0.1% Triton X-100 in PBS (blocking buffer) for 6 hours. Embryos were then incubated with 3A10 antibody (DSHB) (1:150 dilution in blocking buffer) at 4°C overnight. Six 1-hour washes followed by a 3-hour blocking was performed the next day prior to the incubation with HRP-conjugated goat anti-mouse secondary antibody (Jackson Immuno) at 4°C overnight. The following day, six 1-hour washes with PBS were performed prior to detecting the signals using a VIP kit (Vector Labs).
Western analyses
Ventral neural plate explants were harvested from somite 5-8 chick embryos,
prior to the onset of endogenous Gdf11 expression, as described
(Liu et al., 2001).
Recombinant Gdf11 (R&D Systems) was added at various concentrations to F12
culture media supplemented with 2 mM glucose, N2 supplement, L-glutamine and
penicillin/streptomycin (Invitrogen). After culturing for 1 hour at 37°C,
total protein was extracted using extraction buffer [50 mM Tris pH 7.5, 400 mM
NaCl, 1%NP40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM EDTA, 5 mM NaF, and
protease inhibitors (Roche)]. Protein from 11 explants was used for each
sample analysis.
Gdf11 electroporated embryos were harvested the day after electroporation (HH stage 14-15) and a 10-12 somite-long segment with high levels of GFP expression was isolated from each embryo. Each segment was separated in the midline and protein was extracted from both the electroporated and control sides for western analysis. Total protein from individual E9.5 mouse embryo was extracted and one-half of the protein from each embryo was used for western analysis. At least two sets of samples were collected for each experiment.
Phospho-Smad2/3, phospho-Smad1/5/8, Smad1 (for chick), Samd2 and Smad5 (for
mouse) antibodies (Cell Signaling Technology) were used at 1:1,000 dilutions.
SuperSignal West Femto Maximun sensitivity substrate (Pierce) was used as
directed. Each blot was probed, stripped with Restore western blot stripping
buffer (Pierce) and reprobed with a different antibody in the following order:
pSmad1/5/8, Smad1 (chick) or Smad5 (mouse), pSmad2 and Smad2. Quantification
of signals was performed using NIH ImageJ software
(Abramoff, 2004).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The rostral expression boundary of this caudal
expression domain recedes as the embryos ages and by HH stages 21, the
expression is confined to the tip of the tail bud (see Fig. S1A-D in the
supplementary material). Around HH stage 15, Gdf11 begins to express
in the rostral spinal cord, coincident with the commencement of neurogenesis.
The boundary of this expression domain extends caudally as the embryo ages and
reaches the hindlimb level HH stage 21 (see Fig. S1A-D in the
supplementary material). By this stage, Gdf11 is expressed highly in the
differentiated neurons located at lateral positions in the spinal cord (see
Fig. S1D in the supplementary material).
To examine the effect of Gdf11 on RC patterning of the neural tube, we
expressed a cDNA encoding the human Gdf11 protein in somite 10-14 (HH stage
10-11) chick embryos using in ovo electroporation. To confine gene expression
to neural progenitor cells, when their RC identity is being defined, we
constructed an expression vector (pNES-IRES-eGFP) controlled by a human nestin
intron 2 enhancer (Zimmerman et al.,
1994). An internal ribosomal reentry sequence (IRES) followed by
an eGFP-coding region was placed into the same transcription unit to identify
the cells that express the electroporated DNA. Using this construct, GFP
expression can be detected 4-6 hours after electroporation.
We first examined the effect of Gdf11 on Hoxc protein expression in HH
stage 25 embryos 3 days after electroporation
(Fig. 1A-H). In longitudinally
sectioned spinal cords, a one to two segment rostral displacement in the
expression domains of Hoxc6-Hoxc10 was observed in the electroporated sides in
all embryos with strong GFP expression, an indicator of high electroporation
efficiency (Fig. 1A-D). The
shifts in Hoxc protein expression domains are also apparent in cross-sectioned
spinal cords (Fig. 1E-H). We
noticed a reduction in size of the electroporated side in the cross-sectioned
spinal cord (Fig. 1E-H), which
could reflect an anti-proliferative function of Gdf11 that was observed
previously in the olfactory epithelium (Wu
et al., 2003). To examine whether only motoneurons are affected,
we assessed LH2, Lim1/2 and Isl 1/2 expression in HH stage 25 embryos 3 days
after electroporation (see Fig. S2A-C in the supplementary material). The
numbers of LH2+(dI1), dorsal Isl1+(dI3) and
Lim1+(dI2, dI4, dI6) neurons, as well as motoneurons were reduced
to a similar degree (61%-67% of the controls) in the electroporated side
when compared with the control side, indicating that Gdf11 electroporation has
a similar effect on cell proliferation at different DV positions.
To avoid possible complications generated by the proliferation effect of Gdf11 on analyzing the RC patterning phenotype, we examined Hox gene expression by in situ hybridization in HH stage 14-17 embryos 1 day after Gdf11 electroporation, when motoneuron differentiation has just begun. We observed no difference in either the length of the neural tube or the number and size of the somites between the electroporated side and the control side (Fig. 1J-M'). Cross-sections through neural tubes of these embryos showed little difference in size between the electroporated and the control sides (data not shown). Nevertheless, a rostral expansion of Hoxc6-Hoxc10 expression domain is clearly visible in the electroporated side (Fig. 1J-M), indicating that in the spinal cord, the function of Gdf11 in patterning/cell fate determination is independent from its function in proliferation.
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).
Therefore, we next examined if the changes in Hox expression domains induced
by Gdf11 are followed by changes in motoneuron identity. Spinal cords from HH
stage 29 embryos 5 days after Gdf11 electroporation were isolated,
and the expression pattern of a lateral motor column (LMC) marker,
Raldh2 (Sockanathan and Jessell,
1998), was examined by in situ hybridization
(Fig. 2A). A rostral shift of
Raldh2 expression domains was observed at both brachial and lumbar
levels in the electroporated side (Fig.
2A), indicating that the rostrally displaced Hox expression
domains are able to induce a corresponding shift in motoneuron columnar
identity. Loss of Hoxc8 function in both chick and mouse embryos results in
the loss of Pea3 expression in motoneuron pools located in the
brachial level spinal cord (Dasen et al.,
2005; Vermot et al.,
2005). Therefore, we also examined the expression of Pea3
and another motor pool marker Er81
(Lin et al., 1998) in spinal
cords isolated from Gdf11 electroporated embryos. Rostral
displacements of Pea3 and Er81 expression domains were also
observed in the electroporated side at both brachial and lumbar levels
(Fig. 2B; data not shown).
To determine if the axons originating from the rostrally displaced LMC
motoneurons are able to project to appropriate targets, we used an antibody
against neurofilament (3A10) to visualize spinal nerve projections in HH stage
29 embryos 5 days after Gdf11 electroporation. In the control side of
the embryos, spinal nerves 13-16 and a small region of spinal nerve 17
innervate the forelimb (Hollyday and
Jacobson, 1990). However, a one-segment rostral shift in the
spinal nerves that project to the forelimb was observed in the electroporated
side (Fig. 2C). At the hindlimb
level, spinal nerves that innervate the crural plexus originate normally from
lumbosacral (LS) level 1-3, and to a lesser extent from thoracic (T) level 7
(Lance-Jones and Dias, 1991),
as seen in the control side (Fig.
2D). An approximately one-segment rostral shift in spinal nerves
that innervate the crural plexus and the spinal nerves that innervate the
thoracic region was observed in the electroporated side
(Fig. 2C,D). The extent of the
rostral shift of the spinal nerve projections depends upon the efficiency of
electroporation - in most cases an approximately one-segment rostral shift was
observed in embryos harvested at HH stage 29. Surprisingly, a rostral shift in
limb positions at the electroporated side was also observed in 85%
(24/28) of the Gdf11 electroporated embryos. The shift is more
apparent at the hindlimb level (Fig.
2D) but can also occur in the forelimbs (data not shown).
Depending upon the extent of Gdf11 expression, independent forelimb
or hindlimb shift was also detected.
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Expression of follistatin causes caudal displacement of Hox expression domains and motoneuron identity in the spinal cord
If increased levels of Gdf11 are able to caudalize the spinal cord, we
reasoned that reducing the level of Gdf11 should have the opposite effect.
Therefore, we expressed an antagonist of Gdf11 - follistatin (Fst)
(Gamer et al., 2001) in neural
progenitor cells to suppress the function of endogenous Gdf11. A cDNA encoding
human Fst was cloned into the pNes-IRES-eGFP expression vector and
electroporated into somite 10-17 chick embryos. Immunohistochemical analyses
for Hoxc protein expression were performed on HH stage 25 embryos 3 days after
electroporation. In contrast to the rostral shift of Hoxc expression domains
observed in Gdf11 electroporated embryos, a one to two segment caudal
displacement in the expression domains of Hoxc6-Hoxc10 was observed in the
Fst electroporated side (Fig.
3A-H), indicating that appropriate levels of Gdf11 signaling are
required in the neural tube to define proper Hox expression domains.
As Fst is also an antagonist to BMP family proteins that pattern dorsal
spinal cord, we therefore examined LH2, Lim1/2, and Isl 1/2 expression in
cross-sectioned spinal cords of HH stage 25 embryos 3 days after
electroporation (see Fig. S2D-F in the supplementary material). There is no
significant change in the number of motoneurons, but a 35% reduction of
LH2+ neurons, a 45% reduction of dorsal Isl+
neurons, and a 27% reduction of Lim1+ neurons was detected in
the electroporated side (see Fig. S2D-F in the supplementary material; data
not shown).
Associated with the displaced Hox expression domains, Raldh2, Pea3
and Er81 expression in spinal cords isolated from embryos 5 days
after Fst electroporation also exhibit a corresponding caudal shift
in the electroporated side (Fig.
4A,B; data not shown). Examination of spinal nerve projections in
embryos electroporated with Fst revealed an approximately one-segment
caudal shift of the spinal nerves projecting into the forelimbs, the hindlimbs
and the intervening thoracic regions in the electroporated side
(Fig. 4C,D). In addition, a
caudal shift of limb positions (either the forelimb, the hindlimb or both) was
observed in 70% of the electroporated embryos (20/28)
(Fig. 4D).
These results suggest that the amount of Gdf11 a neural progenitor receives helps to determine the RC identity of its neuronal descendents. A higher concentration of Gdf11 at a given RC position will drive the progenitor cell towards a more caudal identity, while a lower concentration of Gdf11 ensures a more rostral identity. The RC identity change in the progenitor cells is reflected by changes in Hox gene expression domains, and results in the generation of motoneuron columns and pools at new positions along the RC axis.
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). Hoxc6-Hoxc10 gene expression in spinal cords isolated from E12.5 Gdf11-/- embryos and their control littermates was examined by whole-mount in situ hybridization (Fig. 5A-D and data not shown). The overall length of the spinal cord is very similar between mutants and their control littermates at this stage (Fig. 5A-D), albeit the truncated tails are clearly visible in the mutants (data not shown). The position of the rostral Hoxc8 expression boundary is very similar between the mutants and controls, but the caudal boundary extends more caudally in the mutants (Fig. 5A,B). By contrast, the entire expression domain of Hoxc10 is shifted caudally in the Gdf11 mutants (Fig. 5C,D).
To examine the changes in Hox expression in more detail, we performed immunohistochemical analyses of Hoxc5-Hoxc10 protein expression in spinal cords isolated from E12.5 Gdf11-/- and control littermates at different RC levels (Fig. 6A-J and data not shown). We did not detect a significant difference in the Hoxc5 expression domain, nor in the rostral expression boundaries of Hoxc6 and Hoxc8 between Gdf11-/- and control embryos (data not shown). However, the caudal boundary of Hoxc6 expression shifted approximately one segment caudally (Fig. 6A,B), while the caudal boundary of Hoxc8 expression domain shifted approximately five segments caudally (Fig. 6C,D) in the Gdf11-/- spinal cords when compared with the controls. The rostral and caudal expression boundaries of Hoxc9 exhibit an approximately one-segment and a six-segment caudal shift, respectively (Fig. 6E,F and data not shown), while the entire expression domain of Hoxc10 shifted approximately six segments caudally (Fig. 6G-J).
These results demonstrate that Hox expression patterns at the cervical level are essentially normal in Gdf11-/- embryos. However, a caudal expansion of Hoxc6-Hoxc10 expression was observed starting from the thoracic level, and the degree of expansion increases progressively towards the caudal end of the neural tube. This results in an approximately one-segment expansion of the cervical level spinal cord, an approximately one-segment caudal displacement and approximately five-segment expansion of the thoracic level spinal cord, and a approximately six segment caudal displacement and elongation of the lumbar level spinal cord at the expense of the sacral spinal cord.
Along with the caudal expansion of Hoxc expression domains in the Gdf11-/- spinal cord, changes in the expression domains of the LMC marker Raldh2 and motoneuron pool marker Pea3 are also observed (Fig. 5E-H). Whole-mount in situ hybridization performed on isolated E13.5 spinal cords revealed that the rostral boundaries of cervical LMC and Pea3+ motoneuron pools are maintained at similar RC positions in the control and the mutant embryos. However, the caudal boundaries of cervical LMC and Pea3 expression domain extend more caudally in the Gdf11-/- spinal cord when compared with that of the controls (Fig. 5E-H). By contrast, not only the entire Raldh2 and Pea3 expression domains at the lumbar level shift caudally but the length of these domains are also increased in the Gdf11-/- spinal cords (Fig. 5E-H).
These results demonstrate that Gdf11 plays a role in assigning RC identity to the spinal cord in both chick and mouse embryos. However, endogenous Gdf11 function is most probably required for patterning the caudal spinal cord from thoracic to sacral levels, as the RC identity of the cervical spinal cord is essentially normal in Gdf11-/- embryos, while the severity of the rostralization phenotype increases towards the caudal end of the spinal cord.
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; Shi and Massague,
2003). Activin type II receptor 2A and 2B were shown to mediate
the function of Gdf11 in axial patterning in mouse, and Gdf11 was shown to
induce the phosphorylation of Smad2 and reduce the level of phospho-Smad1 in
Xenopus ectodermal explants (Oh
et al., 2002). We therefore, examined if Smad proteins are
involved in mediating the function of Gdf11 in inducing caudal Hox gene
expression.
We first harvested ventral neural plate from five- to eight-somite chick
embryos (Liu et al., 2001),
prior to the expression of endogenous Gdf11, and cultured them in serum-free
medium for 1 hour with or without Gdf11. Total protein extracted from these
explants was then used in western analyses to detect phospho-Smad 1/5/8
(p-Smad1/5/8) and phospho-Smad 2/3 (p-Smad2/3) levels
(Fig. 7A). A
concentration-dependent induction of p-Smad2/3 was observed with the addition
of Gdf11: a 60-fold induction by 10 ng/ml Gdf11; a 100-fold
induction by 25 ng/ml Gdf11; and a 230- fold induction by 50 ng/ml Gdf11
(Fig. 7A; data not shown). By
contrast, p-Smad1/5/8 was reduced to 20% of the control when treated with
10ng/ml Gdf11 (Fig. 7A), and to
50% of the control with 25 ng/ml Gdf11 (data not shown), while 50 ng/ml
Gdf11 increases p-Smad1/5/8 levels approximately threefold over the control
(Fig. 7A). These results
demonstrate that in vitro, Gdf11 activates the phosphorylation of Smad2/3 very
efficiently, but, by contrast, it inhibits the phosphorylation of Smad1/5/8 at
lower concentrations while inducing their phosphorylation at higher
concentrations.
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50% reduction in p-Smad2/3 but no apparent
change in p-Smad1/5/8 level was detected in Gdf11-/-
embryos when compared with their wild-type littermates at E9.5
(Fig. 7C), a stage when
Gdf11 is only expressed in the caudal region (see Fig. S3B in the
supplementary material).
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Glu changes at amino acid 465 and 467 of the Smad2 protein
(Funaba and Mathews, 2000). A
construct expressing Smad2-2E was electroporated into somite 9-14
chick embryos, and Hoxc6-Hoxc10 protein expression was analyzed 3 days later
at HH stage 24-25. As Smad2 is a transcription factor, we did not anticipate
significant Hox expression domain shifts as observed in the Gdf11 and
Fst electroporated embryos. Nevertheless, a caudalizing effect on Hox
protein expression was observed in cells with strong GFP expression
(indicating high levels of Smad2-2E expression)
(Fig. 7D-M). Hoxc9 and Hoxc10
induction is clearly visible in cells expressing high levels of Smad2-2E
(Fig. 7F,G,K,L), while Hoxc6
and Hoxc8 expression was simultaneously suppressed
(Fig. 7D,E,I,J). Smad2-2E
induced Hoxc9-expressing cells never co-express Hoxc6, although they are
located in the Hoxc6 expression domain
(Fig. 7H,M), as observed in
Fgf-induced Hoxc9 expression (Dasen et al.,
2003). This result demonstrates that Gdf11 can affect Hox gene
expression through a Smad2-mediated pathway. |
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Dasen et al.,
2003; Liu et al.,
2001); and second, although a rostralization phenotype was
observed in the Gdf11-/- embryos, a rostral to caudal
difference still exists suggesting that even without Gdf11, Fgf signals are
able to pattern the spinal cord to a certain degree.
Spinal cord stem cells are located around Hensen's node in chick, and the
node in mouse embryos (Mathis and Nicolas,
2000; Schoenwolf,
1992), where high concentrations of Fgf are present. The function
of Fgf is important in initiating and maintaining the stem zone identity and
in inducing caudal neural properties
(Delfino-Machin et al., 2005;
Diez del Corral et al., 2003;
Mathis et al., 2001). The
expression of Gdf11 begins at HH stage 10 in and around Hensen's node
where high levels of Fgf signals are present. At this stage, most of the
progenitor cells designated for cervical/brachial levels have already left the
stem zone. Therefore, the combined activities of Fgf and Gdf11 will most
probably determine the expression patterns of Hox genes and the RC identity of
caudal spinal neurons (Fig.
8A).
The RC identities of the spinal progenitor cells are specified when they
leave the stem zone; however, their identity is still modifiable to a certain
degree prior to somite formation (Ensini
et al., 1998; Lance-Jones et
al., 2001; Liu et al.,
2001). Therefore, the function of Gdf11 in controlling RC identity
is most probably exerted upon the progenitor cells prior to somite formation
and neurogenesis. When Gdf11 is expressed in the neural tube by in
ovo electroporation, progenitor cells are exposed to ectopic Gdf11 in addition
to pre-existing Fgf/Gdf11 signals, resulting in a rostral displacement of the
Hox expression domains, as well as motoneuron columns and pools
(Fig. 8B). By contrast, when
Fst is expressed in the neural tube, a fraction of endogenous Gdf11
signaling is inhibited, leading to caudal displacement of Hox expression
domains and motoneuron columns and pools.
In a mouse mutant lacking Gdf11, Fgf signals are able to pattern the neural tube to a certain degree with a slight caudal expansion of cervical identity, in contrast to the significant caudal displacement and expansion of thoracic and lumbar identities. We do not have spinal cord markers that are expressed in a more caudal position than Hoxc10; however, judging from the caudal extent of Raldh2 and Pea3 expression, the sacral region in these embryos is most probably transformed into lumbar identity (Fig. 8C). These results suggest that Gdf11 has a function in determining Hox gene expression and neuronal identity in the caudal spinal cord.
Both Fgfs and Gdf11 have functions in controlling Hox gene expression and
our previous results have demonstrated that low concentrations of Fgf are
required for Gdf11 to induce caudal Hox gene expression in vitro
(Liu et al., 2001), suggesting
Gdf11 could increase response of the progenitor cells to Fgf signals. Further
studies will be required to elucidate the level of interaction between the Fgf
and Gdf11 signaling pathways and the molecules involved.
|
Misexpression of a constitutively activated form of Smad2 (Smad2-2E) in
neural tissue is able to mimic the function of Gdf11 in inducing Hoxc9/Hoxc10
and inhibiting Hoxc6/Hoxc8 expression, demonstrating more directly that Smad2
mediates the function of Gdf11 in controlling Hox gene expression in vivo
(Fig. 8D). Smad transcription
factors could act directly on Hox genes to induce/repress their expression,
and several potential Smad-binding elements (SBE, 5'-CAGAC-3' or
5'-GTCT-3') are present in the Hoxc9-Hoxc8 intergenic
region, as well as in the intron of Hoxc8. Alternatively, Smad2 could
control Hox gene expression indirectly through the Cdx genes, which are key
mediators involved in integrating Fgf, RA and Wnt signals in inducing Hox gene
expression (for a review, see Lohnes,
2003).
|
), and BMP1 can activate a latent form of Gdf11
(Ge et al., 2005), thus
further increasing the complexity of the potential signaling pathways
involved. Unfortunately, Smad1 loss-of-function mouse mutants die around E9.5
(Lechleider et al., 2001),
while Smad2 mouse mutants die around gastrulation
(Waldrip et al., 1998) before
their function in RC patterning of the spinal cord can be assessed.
Conditional mutants of these genes will help to confirm the participation of
Smad proteins in mediating the function of Gdf11 function.
Limb position changes associated with changes in neural Gdf11 levels
We observed an unexpected rostral shift in limb positions in embryos
electroporated with Gdf11 and a caudal shift in limb positions in
embryos electroporated with Fst using an enhancer that directs gene
expression in neural progenitor cells. The observed limb position change may
be induced by Gdf11 secreted from the neural tube, or by Gdf11 secreted from
neural crest cells that have migrated into the somites in the electroporated
side. Alternatively, an as yet unknown factor that is controlled by the neural
Hox code and secreted by the neural tube could act as a feedback mechanism to
ascertain that the limb positions are in register with the positions of the
LMC motoneurons. Previous studies have demonstrated that axial and paraxial
mesoderm influence rostrocaudal patterning of the neural tube
(Ensini et al., 1998;
Lance-Jones et al., 2001).
However, there is no evidence so far for the existence of a feedback signal
from the neural tissue to the adjacent mesoderm. Although
Gdf11-/- embryos exhibit a caudal displacement in their
hindlimb positions along with the caudal displacement of neuronal identity,
Gdf11 may be functioning independently in both mesoderm and neural tissues in
this case. Further studies are required to reveal the mechanism that underlie
the shift of limb positions observed in these electroporated embryos.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/133/15/2865/DC1
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ACKNOWLEDGMENTS |
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