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First published online August 14, 2006
doi: 10.1242/10.1242/dev.02529
1 Developmental Genetics, DKBW Centre for Biomedicine, University of Basel
Medical School, Mattenstrasse 28, CH-4058 Basel, Switzerland.
2 Department of Developmental Biology, Utrecht University, Padualaan 8, 3584 CH
Utrecht, The Netherlands.
Joint senior authors for correspondence (e-mail:
aimee.zuniga{at}unibas.ch;
rolf.zeller{at}unibas.ch)
Accepted 5 July 2006
 |
SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: BMP antagonist, Cyclopamine, Feedback signalling, FGF, Gremlin1, Hox gene expression, Limb development, Sonic hedgehog, SU5402
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Zeller and Deschamps,
2002). In particular, the expression of 5'Hoxd genes can be
divided in two phases (reviewed by
Deschamps, 2004): their
posteriorly nested early expression domains in the limb bud mesenchyme (phase
I) are established prior to activation of morphogenetic signalling by sonic
hedgehog (SHH) (Chiang et al.,
2001; Kraus et al.,
2001). In fact, Hoxd and Hoxa genes have been implicated in
Shh activation in the posterior limb bud mesenchyme
(Kmita et al., 2005;
Zakany et al., 2004). However,
subsequent upregulation and distoanterior expansion of 5'Hoxd gene
expression depends on SHH signalling and results in establishment of their
presumptive digit expression domains in the distal limb bud mesenchyme (late
domains/phase II). Spitz et al. (Spitz et
al., 2003) have identified the large cis-regulatory landscape that
regulates their expression in the digit-forming area, while the relevant
transacting signals and factors regulating their dynamic expression remained
largely unknown. Furthermore, extensive loss- and gain-of-function analysis in
the mouse has established that their expression in the presumptive digit
domains is indeed essential for patterning of the distal limb skeleton and
specification of digit identities
(Dollé et al., 1993;
Zakany et al., 1997).
Shh is expressed by the polarizing region (or ZPA)
(Riddle et al., 1993) and
mainly specifies identities along the anteroposterior limb bud axis. In limb
buds lacking Shh [e.g. mouse
(Chiang et al., 1996)] distal
development is disrupted and posterior identities are lost such that only one
zeugopodal element and one digit form. Furthermore, anterior grafts of
SHH-producing cells induce mirror-image digit duplications, in agreement with
the proposal that the distal limb buds is patterned by long-range
morphogenetic signalling (Riddle et al.,
1993; Yang et al.,
1997). However, genetic marking of Shh-expressing cells
and their descendants has revealed that the ulna and digits 3 to 5 derive
largely from descendants of cells that previously expressed Shh. In
addition, the study by Harfe et al. (Harfe
et al., 2004) revealed that an expansion-based temporal gradient
of exposure to SHH probably specifies digits 3 to 5. In particular, the
posterior mesenchymal cells that express Shh for the longest time
period were shown to give rise to digit 5. Interestingly, this and an earlier
study (Lewis et al., 2001) led
to the conclusion that only specification of digit 2 would require long-range
SHH signalling. Taken together, anteroposterior identities in the limb bud
mesenchyme seem to be largely specified by a kinetic memory that integrates
response to both autocrine and paracrine SHH signalling
(Harfe et al., 2004). In
addition, the cellular responsiveness to SHH signalling is modulated locally
as the cells exposed to the highest SHH levels reduce their sensitivity to SHH
over time (Ahn and Joyner,
2004). All these studies emphasize the importance of identifying
the molecular circuits that regulate the temporal and spatial kinetics of gene
expression during progression of vertebrate limb bud development (reviewed by
Zeller, 2004).
During limb bud morphogenesis, upregulation and maintenance of Shh
expression depends on the secreted BMP antagonist gremlin 1 (GREM1), which
promotes epithelial-mesenchymal (EM) feedback signalling by the apical
ectodermal ridge [AER, expressing several FGF genes
(Sun et al., 2002)]. GREM1
acts in the posteriordistal limb bud mesenchyme downstream of the initial,
GLI-mediated cellular response to SHH signalling
(Zuniga et al., 1999).
Experimental and genetic evidence indicates that the SHH/GREM1/FGF feedback
loop upregulates and maintains the expression of Shh, Grem1 and
5'Hoxd genes in the posterodistal mesenchyme and of FGF genes in the AER
(Haramis et al., 1995;
Laufer et al., 1994;
Niswander et al., 1994).
Analysis of mouse embryos lacking Grem1 showed that GREM1-mediated
BMP antagonism in the mesenchyme is essential to induce and/or upregulate the
expression of FGF and BMP genes in the overlying AER
(Khokha et al., 2003;
Michos et al., 2004). As a
consequence, the upregulation and maintenance of SHH signalling are disrupted,
which is indicative of the failure to establish EM feedback signalling
(Haramis et al., 1995;
Michos et al., 2004). These
molecular alterations result in the characteristic ld phenotype,
which includes loss of posterior digit identities and fusion (ulna with
radius) or loss (fibula) of the posterior zeugopodal element. By contrast,
single and compound mutant mouse embryos lacking FGF genes in the AER of their
limb buds did not display phenotypes, as would have been expected from
disrupting EM feedback signalling (see
Lewandoski et al., 2000;
Sun et al., 2000;
Sun et al., 2002). These
studies left some doubts with respect to the requirements of FGF signalling
from the AER for regulation of gene expression in the distal limb bud
mesenchyme and specification of digit identities.
In the present study, we analyse the interactions of SHH, GREM1 and FGFs in
the distal limb bud mesenchyme by combining analysis loss-of-function
mutations in the mouse with manipulation of mouse limb buds in culture. First,
we establish that SHH-dependent transcriptional upregulation of antagonists
and signals such as Grem1, Bmp2 and Jag1 are controlled by
localised and differential mesenchymal responsiveness to SHH signalling. The
BMP antagonist Grem1 is an early transcriptional target of SHH
signalling in the posterior limb bud mesenchyme, while the Notch ligand
Jag1 is identified as a relatively late SHH target in the
posterodistal mesenchyme. Grafts of SHH-producing cells into
Shh-/- mouse limb buds reveals that the spatially
restricted competence to express a particular target signal is an inherent,
SHH-independent property of the mesenchyme. Second, grafts of FGF producing
cells into Grem1 deficient limb buds restore the expression of
Shh and Hoxd13. In addition, blocking FGF signal
transduction with SU5402 in wild-type limb buds results alters gene expression
in a similar manner as is observed in Grem1-/- limb buds.
These results establish that GREM1-mediated BMP antagonism acts via FGF
signalling to propagate gene expression in the distal limb bud mesenchyme.
Third, SHH grafts are unable to restore Jag1 and Hoxd13
expression in the distal limb bud mesenchyme of Grem1-deficient
embryos. These results indicate that GREM1 is part of a timing mechanism that
regulates expression kinetics in response to SHH signalling. Finally, to
analyse the temporal requirement of SHH signalling, we block SHH signal
transduction from specific time points onwards by treating mouse limb buds
with cyclopamine. Rather unexpectedly, these studies reveal the differential
and limited dependence of the expression of particular genes on SHH
signalling. Upregulation of Grem1 expression requires SHH signalling,
while its anterior expansion in the distal mesenchyme appears to be SHH
independent. Jag1 expression depends on SHH only transiently during
transiently and its expression in the presumptive digit area is largely SHH
independent. Rather unexpectedly, only the establishment but not maintenance
of the Hoxd11 expression domain in the distal limb bud mesenchyme
requires SHH signalling. By contrast, establishment of the presumptive digit
expression domain of Hoxd13 requires SHH signal transduction for much
longer than Hoxd11. These studies show that the differential temporal
dependence of 5'Hoxd genes on SHH signalling correlates well with the
reverse co-linear establishment of their late expression domains (phase II)
(reviewed by Deschamps,
2004).
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
Mice homozygous for the Grem1-ldIn2 mutation
(Grem1In2) were intercrossed to generate embryos lacking
Grem1 expression specifically in limb buds
(Zuniga et al., 2004).
RNA in situ hybridization
Whole-mount in situ hybridization using digoxigenin-labelled antisense
riboprobes was performed as described by Haramis et al.
(Haramis et al., 1995). Three
or more independent embryos were analysed per stage and genotype, and yielded
comparable results.
In vitro grafting and culturing of mouse limb buds (trunk cultures)
Mouse forelimb buds were grafted and cultured as described
(Michos et al., 2004;
Zuniga et al., 1999) with the
following modifications. Trunks with forelimb buds attached were isolated from
wild-type, Grem1ln2/In2 and Shh-/-
mutant embryos from embryonic day E10.25 after counting their somites (32-34
somites). After isolation, spherical aggregates of cell beads expressing the
desired signalling molecule were grafted in forelimb buds. SHH signalling was
blocked by supplementing the culture medium with 10 µM cyclopamine (final,
dissolved in ethanol) and FGF signal transduction was blocked with 10 µM
SU5402 (final, dissolved in DMSO). Controls were treated with 0.16% ethanol
(final) or 0.03% DMSO (final), which is equal to the solvent content in the
respective experimental samples. Trunks were cultured between 3 and 32 hours
in serum-free, high-glucose DMEM medium (GIBCO-Invitrogen), supplemented with
penicillin/streptomycin, L-glutamine, non-essential amino acids, sodium
pyruvate, D-glucose, L-ascorbic acid, lactic acid, d-biotin, vitamin B12 and
PABA in 6.5% CO2 at 37°C. When culturing for 32 hours, the
medium was exchanged after 18-20 hours. Following culturing, samples were
rinsed in PBS and fixed overnight with 4% PFA at 4°C. Each result shown is
representative of minimally three independent embryos per genotype and type of
manipulation, the analysis of which yielded identical results (in many studies
significantly more embryos per result were analysed).
QT6 fibroblast cells expressing Shh, Grem1, Fgf4 or Fgf9
(full-length coding sequences cloned into the pRc/CMV vector, Invitrogen)
under control of the CMV promoter were generated by standard calcium phosphate
transfection. About 24 hours after transfection, cells were plated at high
density on bacterial plates, which results in formation of spherical cell
aggregates. The following day, cell aggregates were treated with mitomycin-C
to completely block their proliferation
(Zuniga et al., 1999) and
washed extensively before grafting into forelimb buds.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Like
Grem1, the expression of signals such as Bmp2 and
Jag1 is dependent on SHH signalling
(Laufer et al., 1994;
McGlinn et al., 2005). To gain insight into the temporal and spatial kinetics by which SHH regulates the expression of mesenchymal signals, SHH-producing cell aggregates were grafted into the forelimb bud mesenchyme of Shh-deficient and wild-type embryos (E10.25, 31-33 somites, Fig. 1). In response to SHH, an anterior ectopic ring of Gli1 expression is induced within 6 hours in wild-type forelimb buds (blue arrowheads, Fig. 1A). These results establish that the initial transcriptional response to SHH signalling is rapid and comparable with endogenous Gli1 expression levels (open arrowheads, Fig. 1A). Localized and strong ectopic Grem1 expression is detected within 9 hours in the mesenchyme distal to the graft (blue arrowhead, Fig. 1B). Ectopic Jag1 expression is also detected within 9 hours distally to the graft (blue arrowhead, Fig. 1C), but levels are much lower than the endogenous transcripts (big open arrowheads, Fig. 1C). After 15 hours the levels of ectopic Jag1 expression in the distal-anterior limb bud mesenchyme have increased significantly (blue arrowhead, Fig. 1D) and continue to rise (Fig. 4A and data not shown). These results reveal that mesenchymal cells respond to SHH signalling by differential and localized transcriptional upregulation of secondary signals. Indeed, Grem1 is expressed locally by the dorsal and ventral mesenchyme, while Jag1 (like 5'Hoxd genes) is expressed throughout the distal limb bud mesenchyme (see Fig. S1 in the supplementary material).
To analyse the responsiveness of nascent mesenchyme, SHH-expressing cells were grafted into the posterior limb bud mesenchyme of Shh-deficient limb buds. As a fraction of Shh-/- mouse embryos die prematurely, a series of pilot experiments established that culturing mutant limb buds for 15 hours allows reliable and reproducible assessment of gene expression levels (Fig. 1E-H; data not shown). As in wild-type limb buds (Fig. 1A), the general response to SHH signalling is revealed by upregulation of Gli1 expression around the graft (Fig. 1E). Despite this widespread initial response, the transcriptional upregulation of the BMP antagonist Grem1 and the Notch ligand Jag1 is always restricted to cells located distal to the SHH graft (blue arrowheads, Fig. 1F,G). By contrast, expression of Bmp2 is mostly upregulated in cells located proximally to the graft (blue arrowhead, Fig. 1H) and the AER (broken arrowhead, Fig. 1H). Restoration of SHH signalling in Shh-/- limb buds shows that the differential and spatial restricted competence to activate these SHH target signals is maintained in Shh-/- limb buds.
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; Zuniga et al.,
1999). Likewise, the expression of Jag1 in distal limb
bud core mesenchyme depends on GREM1-mediated EM feedback signalling (see Fig.
S1 in the supplementary material). However, the analysis of Htu
homozygous mouse embryos [carrying a point mutation in the Jag1 gene
(Kiernan et al., 2001)] shows
that JAG1 itself is does not regulate the expression of 5'Hoxd and other
morphoregulatory genes in the developing limb bud. Rather, this Notch ligand
seems to regulate aspects of cell proliferation in the limb bud mesenchyme
(L.P. and R.Z., unpublished). For the purpose of the present study,
Jag1 is used as an additional marker expressed within the
digit-forming territory of the distal limb bud mesenchyme (see Fig. S1 in the
supplementary material). To gain insight into the temporal and spatial kinetics by which GREM1-mediated EM feedback signalling regulates gene expression in the distal limb bud mesenchyme, GREM1 and FGF producing cell aggregates were grafted into the posterior limb bud mesenchyme of Gre1In2/In2 embryos (E10.25, 31-33 somites). These experiments establish that Shh expression in the posterior mesenchyme is restored (red arrowhead, Fig. 2A) and Fgf4 expression in the AER activated within 9 hours (red arrowhead, Fig. 2B). In limb buds lacking Grem1, grafts of FGF4 and FGF9 expressing cells restore Shh expression with identical kinetics (Fig. 2C; data not shown). This initial restoration of EM feedback signalling (Fig. 2A,B) is followed by upregulation of Jag1 expression in the distal mesenchyme within 15 hours (between graft and AER, Fig. 2D). The expression of 5'Hoxd genes in the distal mesenchyme is restored within 15 hours (Hoxd13, red arrowhead, Fig. 2E; Hoxd11 and Hoxd12: data not shown). Furthermore, grafts of FGF4 producing cells into Grem1 deficient limb buds restore Jag1 and Hoxd13 expression with identical kinetics (Fig. 2F,G).
|
). Culturing wild-type limb buds in the presence of 10 µM
SU5402 blocks FGF signal transduction (Fig.
3A-D). Such treatment interferes with transcriptional upregulation
of Jag1 and partly inhibits anterior expansion of its expression
(Fig. 3A,B). Similarly,
upregulation and anterior expansion of Hoxd13 expression, i.e.
establishment of its late, presumptive digit expression domain is disrupted by
treatment with SU5402 (Fig.
3C,D). By contrast, Grem1 expression is not significantly
altered by blocking FGF signal transduction
(Zuniga et al., 2004). In
summary, treatment of limb buds with SU5402 results in similar alterations of
the Jag1 and Hoxd13 expression domains, as observed in
Grem1-deficient limb buds at E11.0 (42 somites,
Fig. 3E,F; compare 3E with 3A,B
and 3F with 3C,D). These results further support the proposal that GREM1 acts
up-stream of and via FGF signalling during limb bud morphogenesis.
GREM1 is required to regulate the temporal kinetics of Jag1 and Hoxd13 expression in the distal limb bud mesenchyme
To further dissect the functional relevance of GREM1-mediated EM feedback
signalling with respect to SHH-dependent regulation of gene expression,
wild-type and Grem1In2/In2 limb buds (E10.25, 31-33
somites) received anterior grafts of SHH-producing cells
(Fig. 4) to clearly
discriminate induced (blue arrowheads) from endogenous gene expression (open
arrowheads). In wild-type limb buds, ectopic SHH signalling induces
significant anterior expansion and upregulation of Jag1 and
Hoxd13 gene expression in the distal mesenchyme within 15 to 22 hours
(Fig. 4A,C; see also
Fig. 1D and data not shown). By
contrast, no or only little transcriptional upregulation of ectopic
Jag1 and Hoxd13 transcripts is observed in the distal
mesenchyme of Grem1-deficient limb buds after 22 hours (blue
arrowheads, Fig. 4B,D; left
panels). In fact, the ectopic Jag1 expression levels in
Grem1-deficient limb buds after 22 hours are reproducibly lower than
the ones in wild-type limb buds after 9 hours (compare
Fig. 1C with
Fig. 4B). Only after 32 hours,
the levels of ectopic anterior Jag1 and Hoxd13 transcripts
(blue arrowheads, Fig. 4B,D;
right panels) become comparable with ones in wild-type limb buds (blue
arrowheads, Fig. 4A,C; right
panels). This indicates an at least 12 hours delay in efficient
transcriptional response to SHH signalling in Grem1-deficient limb
buds. This delay is probably due to the fact that grafts of SHH-producing
cells into Grem1-deficient limb buds do not restore FGF gene
expression by the mutant AER and thereby fail to restore EM feedback
signalling (Michos et al.,
2004; Zuniga et al.,
1999). These results establish that GREM1-mediated EM feedback
signalling regulates aspects of the temporal kinetics of the transcriptional
response to SHH signalling.
|
). This
approach was chosen as the genes of interest are either not expressed or
downregulated in limb buds of Shh-deficient embryos much prior to
establishing GREM1-mediated feedback signalling (Jag1, 5'Hoxd
genes) (Chiang et al., 2001;
Kraus et al., 2001). The
presence of 10 µM cyclopamine in the culture medium significantly reduces
Gli1 expression within 9 hours
(Fig. 5A), indicative of
blocking SHH signal transduction (E10.5, 34-36 somites). After 15 hours of
cyclopamine treatment, Gli1 is no longer detectable by whole-mount in
situ hybridization, while cellular apoptosis is not yet significantly
increased (Fig. 5A; data not
shown). Therefore, embryonic trunks were cultured for 15 hours in the presence
of cyclopamine prior to analysis (Fig.
5B,C and Figs
6,7).
Cyclopamine treatment induces loss of Fgf4 expression from the AER,
indicating that Fgf4 requires sustained SHH signalling
(Fig. 5B). By contrast, the
expression of Fgf8 is only slightly reduced in comparison with
wild-type controls (Fig.
5C).
|
).
Cyclopamine treatment from E10.25 (31-33 somites) onwards does not alter the
anterior limit of the Grem1 expression domain
(Figs 6A,B; broken lines
indicate the approximate anterior limits), while expression is lost from the
distal mesenchyme (brackets in Cyc panel in
Fig. 6A,B). This failure to
upregulate Grem1 expression in the distal-most mesenchyme is the
likely cause of the loss of Fgf4 expression from the overlying AER
(Fig. 5B). Furthermore,
concurrent inhibition of both SHH and FGF signal transduction neither alters
the anterior boundary of the Grem1 expression domain nor decreases
expression levels further (data not shown). In light of this rather unexpected
maintenance of the anterior expression boundary, Grem1 expression was
re-assessed in Shh deficient limb buds
(Fig. 6C). As previously
reported, Grem1 expression is rapidly downregulated in
Shh-deficient limb buds (Fig.
6C, compare panel Shh-/- with wild type)
(Zuniga et al., 1999).
Although overall expression levels are low, Grem1 transcripts are
reproducibly detected in the distoanterior mesenchyme of
Shh-deficient limb buds at E9.75
(Fig. 6C, anterior limit
indicated by broken line). By E10.5 (34-36 somites), Grem1 transcript
levels are further reduced, but expression remains throughout in the
distalmost mesenchyme in Shh-deficient limb buds
(Fig. 6C). These results show
that anterior expansion of the Grem1 expression domain occurs in the
absence of SHH, while SHH signalling is required to upregulate Grem1
expression in the distal limb bud mesenchyme.
|
|
),
requires SHH signal transduction for longer (i.e. beyond E10.75,
Fig. 7G,H). The results shown
in Fig. 7 reveal the graded
dependence of 5'Hoxd genes on SHH signal transduction. This differential
temporal dependence correlates well with the kinetics by which their
presumptive digit expression domains are established. In particular, the
expression of the 5' most Hoxd13 gene
(Fig. 7G,H) depends
significantly longer on SHH signal transduction than do the ones of the more
3' located Hoxd12 and Hoxd11 genes
(Fig. 7A-F), in agreement with
reverse co-linear establishment of their presumptive digit expression
domains.
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|
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). GLI3-mediated restriction of
Hand2 expression to the posterior limb bud mesenchyme seems to
regulate the activation of Shh, Grem1 and, possibly, 5'Hoxd
genes (te Welscher et al.,
2002a; Charite et al.,
2000; Yelon et al.,
2000; Zuniga et al.,
1999; Zuniga and Zeller,
1999). In addition, the transcriptional regulators Hoxd13
(Chen et al., 2004), Tbx3
(Rallis et al., 2005) and
Twist1 (Firulli et al., 2005)
interact with GLI and Hand2, respectively. In addition, genetic studies have
implicated retinoic acid, 5'Hoxa and 5'Hoxd genes in activation of
Shh expression in the posterior limb bud mesenchyme
(Kmita et al., 2005;
Mic et al., 2004;
Niederreither et al., 2002).
Subsequently, SHH signalling is required to upregulate and propagate
mesenchymal gene expression in the distal mesenchyme as the expression of many
genes is rapidly downregulated and/or lost in Shh-deficient limb buds
(Chiang et al., 2001;
Kraus et al., 2001;
Litingtung et al., 2002;
te Welscher et al., 2002b;
Zuniga et al., 1999). Our
study establishes that SHH is required for transcriptional upregulation but
not distoanterior expansion of Grem1 expression during limb bud
patterning. Therefore, the SHH-independent anterior expansion of
Grem1 expression in early limb buds could be regulated by the
pre-patterning mechanism acting upstream of SHH signalling (see before).
GREM1 acts via FGF-mediated EM signalling to regulate the temporal kinetics of gene expression in response to SHH signalling
In Grem1-deficient limb buds, the propagation of Shh
expression is disrupted and there is a significant temporal delay in
establishing the 5'Hoxd digit expression domains
(Haramis et al., 1995;
Michos et al., 2004). This
delay cannot be rescued by grafts of SHH-expressing cells into the posterior
limb bud mesenchyme (this study) and probably results in mesenchymal cells not
receiving their positional identities at the correct time. This achronism
provides a plausible explanation for the observed loss of posterior identities
in Grem1-deficient limbs (Michos
et al., 2004). In contrast to SHH, grafts of FGF-producing cells
upregulate mesenchymal gene expression in Grem1-deficient limb buds
with temporal kinetics comparable with GREM1-producing cells. Furthermore,
inhibition of FGF signal transduction in wild-type limb buds phenocopies
aspects of the molecular alterations observed in Grem1-deficient limb
buds. These results provide good evidence that GREM1-mediated BMP antagonism
regulates the mesenchymal response to SHH signalling indirectly via FGF
signalling from the AER. Hence, our studies support an essential role of
GREM1/FGF-mediated EM feedback signalling in regulation of the temporal
kinetics of gene expression and patterning. As GREM1 is required to induce
Fgf4, Fgf9 and Fgf17 expression in the posterior AER and to
upregulate Fgf8 expression
(Michos et al., 2004) (L.P.
and R.Z., unpublished), the overall strength of FGF signalling by the AER may
be most relevant to FGF-mediated EM feedback signalling. Indeed,
transgene-mediated overexpression of Fgf4 in the AER of mouse limb
buds lacking Fgf8 completely restores their development, which
indicates that FGF4 functionally replace FGF8 in the mutant AER
(Lu et al., 2006).
|
;
Harfe et al., 2004). In the
present study, we uncover the temporal requirement of SHH signal transduction
for establishment of the late, presumptive digit expression domains of
5'Hoxd genes. Their co-linear expression is reversed in the distal limb
bud mesenchyme, such that expression of the 5' most gene,
Hoxd13 extends most anterior and is essential for digit patterning
(Dollé et al., 1993).
Genetic manipulation of the Hoxd complex in mice has revealed the existence of
a large global control region (GCR) that regulates the establishment of their
late expression domains in the limb bud mesenchyme
(Spitz et al., 2003). A
distant digit enhancer is located far 5' to the Hoxd gene complex and
strongest enhances the expression of Hoxd13, while the expression of
the more 3' located Hoxd12 and Hoxd11 genes is
enhanced progressively less (Kmita et al.,
2002). Our study reveals that establishment but not maintenance of
the late 5'Hoxd expression domains requires SHH signalling. In good
correlation with their differential regulation by the digit enhancer,
Hoxd13 requires SHH signal transduction for significantly longer than
the more 3' located Hoxd12 and Hoxd11 genes.
Therefore, their differential dependence on SHH signalling could be mediated
by interaction of SHH targets such as the GLI transcriptional regulators with
the digit enhancer (Kmita et al.,
2002; Spitz et al.,
2003). Our study shows that 5'Hoxd genes are rendered
SHH-independent in a reverse co-linear fashion as their presumptive digit
expression domains are being established. This progressive stabilization and
SHH independent expression of 5'Hoxd genes may constitute a part of, or
at least mark the kinetic memory that mesenchymal cells may acquire as a
consequence of their overall exposure to SHH signalling
(Harfe et al., 2004).
SHH dependent and independent phases of vertebrate limb bud development
Our analysis, together with previous studies allows division of limb bud
patterning in three distinct phases. During the initial setup phase, the
AER-FGF and ZPA-SHH signalling centres and differential mesenchymal
responsiveness are established under the influence of the GLI3R-HAND2
pre-patterning mechanism (Fig.
8, phase I) (e.g. te Welscher
et al., 2002a). During this initial phase, the differential
responsiveness to SHH signalling is probably setup in the nascent mesenchyme
(this study) and Grem1 expression is activated in the posterior
mesenchyme (Zuniga et al.,
1999). In addition, the early posteriorly nested expression
domains of 5'Hoxd genes are established (e.g.
Zuniga and Zeller, 1999) and
participate in activation of Shh expression
(Kmita et al., 2005). The
second, dynamic phase is initiated by concurrent establishment of SHH and
GREM1/FGF-mediated EM feedback signalling, which coordinates temporal
progression with anterior expansion of gene expression in the distal
mesenchyme (Fig. 8, phase II;
yellow arrow). The present study leads us to conclude that SHH constitutes the
main `engine' for this dynamic phase, while the temporal kinetics are
regulated in concert with GREM1/FGF EM feedback signalling. During this
dynamic phase, the presumptive digit expression domains of Jag1 and
the 5'Hoxd genes are progressively established and rendered independent
of SHH signalling. Mesenchymal cells probably acquire their kinetic memory of
exposure to SHH signal transduction (Harfe
et al., 2004) and the expanding population of Shh
descendents displaces the posterior limit of the Grem1 expression
domain towards anterior (Scherz et al.,
2004). This widening gap between SHH-producing and
Grem1-expressing cells eventually terminates SHH/GREM1/FGF-mediated
feedback signalling and limb bud patterning
(Fig. 8, phase III). The
present study reveals the temporal and spatial kinetics by which mesenchymal
SHH signalling and GREM1-mediated BMP antagonism function together with FGF
signalling from the AER to pattern the distal limb bud mesenchyme. It will be
of particular interest to identify the BMP ligands that are antagonized by
GREM1 in the limb bud and participate in regulation of the gene expression
kinetics. Finally, it will be important to gain more insight into how these
highly dynamic morphoregulatory interactions are established
(Zuniga et al., 1999;
te Welscher, 2002a) and
terminated at the appropriate developmental time points
(Scherz et al., 2004).
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/133/17/3419/DC1
|
ACKNOWLEDGMENTS |
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|
Footnotes |
|---|
Present address: Ludwig Institute for Cancer Research, Department of Cell
and Molecular Biology, Karolinska Institute, Box 240, S-17177 Stockholm,
Sweden
Present address: NIOB/KNAW Hubrecht Laboratorium, Uppsalalaan 8, 3584 CT
Utrecht, The Netherlands
|
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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