Laboratory of Pathology, Center for Cancer Research, NCI, NIH, Bethesda,
MD 20892, USA
* Present address: Carnegie Inst Washington, Dept Embryol, Baltimore, MD 21210,
USA
Present address: 20/20 Gene Systems, Inc., Rockville, MD 20850, USA
Author for correspondence (e-mail:
smack{at}helix.nih.gov)
Accepted 18 December 2002
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SUMMARY |
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Key words: Brachyury, Limb development, AER formation, FGF, WNT
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INTRODUCTION |
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Limb outgrowth along the PD axis is regulated by FGF signals from a
specialized columnar ectoderm that normally forms along the DV boundary at the
limb apex, the apical ectodermal ridge (AER) (reviewed by
Martin, 1998;
Lewandoski et al., 2000
;
Moon and Capecchi, 2000
;
Sun et al., 2002
). Interacting
FGF and WNT cascades are critical in early limb induction, AER formation and
reciprocal epithelialmesenchymal interactions necessary for AER maintenance
(reviewed by Martin, 1998
;
Tickle and Munsterberg, 2001
).
Several WNTs and FGFs, acting directly or indirectly, are able to induce
ectopic limbs in the flank (e.g. Cohn et
al., 1995
; Ohuchi et al.,
1997
; Kawakami et al.,
2001
). Ultimately, FGF10 in the prospective limb lateral plate
induces ectodermal Wnt (Wnt3a in chick) and Fgf8
expression in the forming AER (Ohuchi et
al., 1997
; Kengaku et al.,
1998
; Galceran et al.,
1999
). FGF8 from the AER ectoderm also maintains high level
Fgf10 expression in limb mesoderm. Thus, reciprocal FGF8 and FGF10
signals form a positive regulatory loop by which AER and subridge mesoderm
functionally maintain each other (Ohuchi
et al., 1997
; Revest et al.,
2001
). Sonic hedgehog (Shh), expressed in
posterior limb bud mesoderm, regulates AP polarity and number of skeletal
elements. Activation of Shh also depends on ridge signals and
subsequently positive feedback between FGF4 from posterior ridge and SHH
maintains both signaling centers (reviewed by
Capdevila and Johnson,
2000
).
AER induction, as well as later maturation, is normally closely linked to
DV boundary formation although they can be uncoupled (reviewed by
Zeller and Duboule, 1997;
Tickle and Munsterberg, 2001
).
Early upstream BMP signaling regulates both processes in parallel
(Ahn et al., 2001
;
Pizette et al., 2001
);
contributing to AER induction by upregulating Msx genes and to
establishing DV polarity by activating En1 expression in ventral
ectoderm. EN1 represses Wnt7a expression in ventral ectoderm
(Loomis et al., 1996
;
Logan et al., 1997
), while
dorsal ectodermal WNT7a signals activate Lmx1b in the underlying
mesoderm to regulate dorsal fates (Parr
and McMahon, 1995
; Riddle et
al., 1995
; Vogel et al.,
1995
; Cygan et al.,
1997
; Chen et al.,
1998
).
The murine AER forms from a broad zone of Fgf8-expressing ventral
ectoderm in the prospective limb region, both by movement of pre-AER cells to
the DV border and loss of AER-gene expression in remaining ventral cells, to
form a sharply demarcated ridge during maturation
(Kimmel et al., 2000)
(reviewed by Tickle and Munsterberg,
2001
). This process is also regulated by En1 in the
ventral ectoderm, via repressing Wnt7a and setting a ventral border
for mature AER (Cygan et al.,
1997
; Loomis et al.,
1998
; Kimmel et al.,
2000
). Although the chick differs somewhat from mouse in that
Fgf8 expression is more restricted from its onset
(Crossley et al., 1996
), a
pseudostratified, columnar AER also forms from a broader zone of flat
ectodermal precursors that compact into a sharp ridge along the DV apex
(Altabef et al., 1997
;
Michaud et al., 1997
).
Regulation of maturation is likely to be conserved in chick; for example,
En1 also affects AER formation
(Laufer et al., 1997
;
Logan et al., 1997
;
Rodriguez-Estaban et al., 1997). The role of mesodermal signals in later
morphogenesis of a mature AER is unclear. Disruption of mesodermal FGF10
signaling interferes with early stages of AER induction, obscuring later
function (Min et al., 1998
;
Xu et al., 1998
;
Arman et al., 1999
;
Sekine et al., 1999
;
Revest et al., 2001
).
We previously noted expression of Brachyury (T) in early
limb buds on northern blots (Knezevic et
al., 1997a). T is the founding member of the T-box family
of transcription factors which share a conserved DNA binding domain and play
multiple roles during development (reviewed by
Papaioannou and Silver, 1998
),
several of which are expressed in developing limb
(Gibson-Brown et al., 1996
;
Gibson-Brown et al., 1998
;
Isaac et al., 1998
;
Logan et al., 1998
;
Ohuchi et al., 1998
).
T plays an essential role in primary mesoderm formation (reviewed by
Herrmann, 1995
) and has been
implicated in both WNT and FGF signaling pathways during gastrulation
(Smith et al., 1997
;
Yamaguchi et al., 1999a
;
Arnold et al., 2000
;
Tada and Smith, 2000
;
Galceran et al., 2001
), but
has not been reported to be expressed elsewhere. We found that T is
expressed at low levels in lateral plate at the onset of limb initiation and
persists in the subridge mesoderm of the limb bud, as well as several other
sites associated with WNT and FGF signaling. Retroviral misexpression of
T in chick causes anterior AER extension along the limb apex and
skeletal phenotypes consistent with extended AER function, whereas loss of
T in mutant mouse embryos disrupts normal AER morphogenesis. These
results suggest T functions in the mesoderm to direct AER maturation
along the DV limb border. Taken together, altered subridge Fgf10
expression levels in response to in vivo changes in T expression, and
the ability of ridge-specific WNT and FGF signals to induce T in
vitro suggest that T may also regulate maintenance of a mature AER as
a component of the reciprocal epithelial-mesenchymal signaling mediated by FGF
and WNT pathways.
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MATERIALS AND METHODS |
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In situ hybridization and immunostaining
Conditions for probe preparation and in situ hybridization were as
previously described (Knezevic et al.,
1997a; Knezevic et al.,
1997b
). For chick T probe, the color reaction contained
10% polyvinyl alcohol (Barth and Ivarie,
1994
) to enhance detection sensitivity, and was developed for
12-18 hours. Other chick probes used included Fgf4 (L. Niswander),
Fgf8 (J.-C. Izpisua-Belmonte), Fgf10 (S. Noji), Shh
(R. Riddle), and Wnt5a (T. Nohno). Mouse probes used included
Bmp4 (B. Hogan), Fgf8 (G. Martin), Fgf10 (D.
Ornitz), En1 (A. Joyner), Lmx1b (R. Johnson), Msx1,
Msx2 (R. Maas) and Shh, Wnt7a (A. McMahon). Whole-mount embryos
were paraffin embedded or frozen in OCT and sectioned at 5-10 µm, or
embedded in 3% agarose to cut 50 µm vibratome sections. Immunostaining of
cryosections or paraffin sections (after antigen retrieval by steaming) was
detected with peroxidase-linked secondary antibodies and Vectastain kit.
Affinity purified DLX-antibody was a gift from G. Panganiban (for details, see
(Panganiban et al., 1997
).
Proliferation and apoptosis assays
Mitosis-specific anti-phospho-histone H3 (Upstate Biotechnology) was used
to immunostain multiple sections. Mitotic cells in equal-sized areas were
counted and averaged for comparisons. Apoptosis was assessed on sections by
TUNEL assay (Apoptag kit, Intergen) using fluoro-dNTP incorporation and direct
fluorescence microscopy with propidium iodide counterstaining, or by
whole-mount staining of fresh embryos with 1/50,000 Nile Blue sulfate in
PBS.
Retrovirus preparation, infection and misexpression analysis
The chick T coding region
(Knezevic et al., 1997a) was
cloned as an NcoI-EarI fragment into the
NcoI-SmaI sites of Cla12Nco vector, and transferred into
RCASBP as a ClaI fragment. Virus production and infection were as
described by Morgan and Fekete (Morgan and
Fekete, 1996
), except that a Picospritzer II (Parker instruments)
was used for injection. Parallel control infections with alkaline phosphatase
(AP)-expressing virus (a gift from B. Morgan) were done to assess phenotype
specificity, and adequacy of hindlimb infection by AP staining.
T-virus infected embryos were also hybridized with a T probe
as a control to evaluate adequacy of infection. Overall, 55-75% of
T-infected embryos at early or late times post-infection had
abnormalities. The extents of viral T expression in hindlimb
suggested that the lack of visible effect of infection in the remaining
embryos was due to inherent variability in infection spread (data not shown).
Variability in amplification of T-virus also occurred in cell
culture. For skeletal analysis, infected day 9-10 embryos were fixed in 5%
TCA, stained with 0.1% Alcian Green 2GX in acid-alcohol, washed and dehydrated
in absolute alcohol, and cleared in methyl salicylate.
T-antibody production, purification and analysis
T/GST fusion protein from plasmid expressing N-terminal (amino acids 1-123)
T/GST fusion protein (a gift from B. Herrmann) was used to immunize rabbits
using standard techniques. Antibody was affinity purified as described
(Kispert and Herrmann, 1993).
Protein extracts were prepared from dissected embryonic tissues by lysis in
PBS containing protease inhibitor cocktail, 10 µg/ml AEBSF, 1% SDS, and 0.5
mM DTT. Proteins, electrophoresed on 4-12% NuPAGE gradient gels (Novex), were
blotted and probed with antibody using standard techniques. Monoclonal
anti-
-tubulin (Sigma) served as an internal protein loading control.
For immunohistochemistry, affinity-purified anti-T was pre-incubated with
blotted 100 kDa protein (24 hours) to remove cross-reacting epitopes.
Chick limb mesenchyme cell culture and northern analysis
Primary cell cultures from stage 19-20 chick limb buds, as described
(Knezevic et al., 1997b), were
incubated with recombinant FGF4 (a gift from Genetics Institute, Inc.) or
FGF8b (R&D systems) at 750 ng/ml and heparin sulphate (100 ng/ml).
Cultures infected with Wnt3a-expressing retrovirus (a gift from Cliff
Tabin) at a multiplicity of 3-5 IU/cell, were compared to RCASBP control.
Northern analysis with chick T 3'UTR and control probes was as
described previously (Knezevic et al.,
1997a
).
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RESULTS |
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T protein expression in limb was verified with immunoblots of protein from different chick and mouse tissues, probed with affinity-purified anti-T. T protein was detected in early stage chick and mouse limb buds, albeit at much lower levels than tailbud (Fig. 2A). A highly abundant, cross-reacting, ubiquitous 100 kDa protein (see Fig. 2A) hampered in situ immunostaining even after affinity purification, necessitating extensive pre-adsorption against blotted 100 kDa proteins to deplete cross-reacting epitopes. Depleted antibody revealed mouse T protein in subridge limb mesoderm and somites, as in chick (Fig. 2B). The intriguing proximity of T expression to the forming AER was investigated using retroviral misexpression to perturb T in developing limb.
|
T misexpression causes anterior AER extension and ensuing
late phenotypes
The prospective hindlimb lateral plate of stage 9-11 chick embryos was
infected with RCASBP retrovirus expressing full-length T protein. Changes in
limb bud morphology were discernible by stage 19-20 (48 hours
post-infection). The AER, visualized by Fgf8 expression as a
functional marker, extended farther anteriorly along the DV edge of infected
limb buds, accompanied by variable broadening of the anterior limb bud (50%,
7/15, Fig. 3A,B). In some
embryos, the `extended' AER was widened and irregular (e.g. see
Fig. 3B), but truly ectopic
AERs away from the DV limb margin were not seen, except for rare, small
isolated spots of Fgf8 expression (data not shown). Older infected
embryos (stage 24,
60 hours post-infection) also had anteriorly extended
AERs (40%, 12/29, Fig. 3C) and
some embryos without extension still showed stronger Fgf8 expression
in the AER on the infected side (20%, 6/29 embryos, not shown), which may
correlate with mild soft tissue phenotypes seen at day 10 (see below), perhaps
reflecting later infection onset or spread. Fgf4 expression, normally
restricted to posterior AER, was also extended anteriorly in T
infected limb buds (52%, 16/31, Fig.
3D), consistent with expanded AER extent and function.
|
At 10 days, T-infected embryos showed skeletal and soft tissue
phenotypes predicted by earlier AER changes (75%, 21/28; no abnormalities in
control-virus infections, 0/15). Skeletal abnormalities (54%, 15/28) included
anterior digit duplications (Fig.
4B-D), in some cases with posterior transformation of anterior
digits (Fig. 4B). Sometimes the
anterior-most metatarsal was also thickened
(Fig. 4C) or duplicated
(Fig. 4D). The proportion of
infected embryos with anterior digit changes correlated well with the
incidence of anterior AER extension at earlier stages. SHH plays a central
role regulating AP digit patterning and is often a factor in the production of
anterior polydactyly (discussed in
Knezevic et al., 1997b).
Moreover, changes in AER extent might also be associated with ectopic
Shh expression, driven by increased FGFs (reviewed by
Martin, 1998
). However, no
ectopic anterior Shh was detected in a set of infected limb buds
showing clear anterior broadening and AER extension (0/11,
Fig. 3C). This may not be
surprising since augmented AER function is not invariably linked to ectopic
Shh induction (e.g. Pizette and
Niswander, 1999
; Zhang et al.,
2000
) and digit I specification does not require SHH (see
Lewis et al., 2001
).
|
T misexpression caused milder interdigital soft tissue changes in
some embryos (21%, 6/28), including soft tissue broadening
(Fig. 4E,F), delayed loss of
webbing (Fig. 4E) and ectopic
cartilage condensations (Fig.
4D,E). Inhibition of AER regression is associated with
interdigital soft tissue overgrowth, apparently due to prolonged Fgf8
expression in the AER at late stages
(Pizette and Niswander, 1999).
Stronger late Fgf8 expression seen in some T-infected
embryos may have a similar effect, suggesting T may delay AER
regression.
Relationship of T to FGF and WNT signaling in limb
T is a transcription factor so we expected direct targets to be
expressed in the mesoderm, where T is normally expressed, and to
include secreted signals that regulate the AER. Since Wnts and
Fgfs are T targets during gastrulation
(Smith et al., 1997;
Tada and Smith, 2000
), we
checked Fgf10 and Wnt5a, both expressed in early distal limb
mesenchyme. Fgf10, which regulates limb initiation and AER formation
(Ohuchi et al., 1997
;
Min et al., 1998
;
Sekine et al., 1999
), was
increased in the distal subridge mesoderm in T-infected limb buds
(7/14 total, stage 19-24) and was already upregulated by stage 19-20 (4/7
embryos, Fig. 3E), suggesting
that Fgf10 may be an early target of T in the subridge zone.
Wnt5a, which plays a role in limb outgrowth
(Yamaguchi et al., 1999b
), was
unchanged in stage 19-21 infected embryos (0/8, data not shown) and later was
expanded slightly anteriorly (4/8, Fig.
3F), most likely as a very indirect effect.
Prominent T expression in apical subridge mesoderm suggested
regulation by AER signals. FGFs and WNTs also function upstream of T
during gastrulation (Smith et al.,
1997; Yamaguchi et al.,
1999a
; Arnold et al.,
2000
; Galceran et al.,
2001
), so AER-specific WNTs and FGFs were tested. Since limb
T expression was very weak, we evaluated quantitative effects on
T expression in primary cultures of stage 19/20 limb mesenchyme.
T was induced within 18 hours after infection with a
Wnt3a-expressing virus (Fig.
5). Recombinant FGF8b induced T within 8 hours
(Fig. 5), while recombinant
FGF4 protein had no effect early or later (data not shown). Hence, both FGF8b
and WNT3a ridge signals may participate in activating or maintaining subridge
T expression. Fgf4, expressed in posterior AER, functionally
overlaps with Fgf8 (Sun et al.,
2002
) but shows some differences in receptor binding
(Ornitz et al., 1996
) and
ability to regulate mesodermal genes (e.g.
Haramis et al., 1995
;
Mahmood et al., 1995
;
Kimura et al., 2000
), perhaps
explaining its failure to induce T.
|
Disrupted AER formation in mouse T-/-
embryos
To assess whether T plays a role in normal AER formation, we
analyzed mouse T-/- embryos. The T-/-
null mutant is an early embryonic lethal (E10.5), precluding skeletal
analysis, but allowing evaluation of forelimb AER formation. No hindlimb bud
forms owing to disrupted posterior mesoderm formation and ensuing posterior
truncations. T-/- forelimb buds were smaller than wild
type and often more rounded at mature AER stages, lacking a sharp DV edge (see
Figs 6,
7).
|
|
From its onset at E9, Fgf8 expression in pre-AER ectoderm was
mottled and weaker in T-/- embryos
(Fig. 6A). Later, Fgf8
continued to be irregular in the T-/- pre-AER, revealing
its failure to compact normally toward the DV apex
(Fig. 6B-E). At more mature
stages (E9.75-10) the T-/- AER was wavy, zigzagging into
both dorsal and ventral ectoderm, with variably weaker Fgf8 levels
(Fig. 6D,E). AER morphology was
assessed with anti-DLX, since Dlx gene expression marks AER
progenitors similarly to Fgf8
(Panganiban et al., 1997;
Loomis et al., 1998
). DLX
expression during AER morphogenesis (Fig.
7A-H') was irregular and sometimes less clearly restricted
to ventral ectoderm in T-/- embryos (e.g.
Fig. 7C,D), and revealed that
progression to a highly compact, pseudostratified AER was delayed and erratic.
Even in late stage T-/- limb buds with the mildest
phenotype, the extent of AER maturation varied dramatically in neighboring
sections (e.g. Fig. 7H vs
H').
Bmp4 is highly expressed in AER progenitors and BMP signalling has
been implicated in AER induction and DV patterning
(Ahn et al., 2001;
Pizette et al., 2001
).
Bmp4 expression in T-/- embryos was altered in
parallel with Fgf8, starting weak and patchy at E9
(Fig. 6F), and remaining broad
and irregular at later stages (Fig.
6G-J), consistent with abnormal AER maturation. Since
Bmp4 is expressed in ectoderm and in mesenchyme, the distribution was
examined on sections, revealing abnormalities in AER morphogenesis similar to
those seen with DLX antibody (Fig.
7I-N). Mesodermal Bmp4 expression, though comparatively
quite weak between E9-10, was detectable early and reduced in
T-/- limb buds (Fig.
7I,J). BMP downstream target genes Msx1 and Msx2
are expressed throughout ventral ectoderm as well as forming AER, and
underlying limb mesoderm (Ahn et al.,
2001
; Pizette et al.,
2001
). Despite reduced expression of Bmp4, Msx1 and
Msx2 expression was quantitatively preserved (data not shown), as was
BMP target En1 (below), indicating that the BMP pathway was largely
intact.
Pre-AER gene expression and morphology indicated disrupted maturation.
En1 in ventral ectoderm regulates compaction and positioning of
mature ventral AER borders by repressing Wnt7a
(Cygan et al., 1997;
Loomis et al., 1998
;
Kimmel et al., 2000
). In
T-/- embryos, Wnt7a and En1 were
expressed in dorsal and ventral limb bud ectoderm, respectively. However, the
distal ectodermal expression borders for each of these genes was not sharp as
it is in wild type (Fig. 6K-S),
and remained jagged and irregular even at late stages.
Fgf10 induces AER formation and was upregulated by T misexpression. Fgf10 expression declined progressively over time in T-/- embryos compared to wild type (Fig. 6T-X), but did not appear very different until E9.5, after changes in Fgf8 and Bmp4 expression were discernible.
Potential late mesenchymal effects of abnormal AER function in
T-/- embryos
Proliferation rates, assessed with the mitosis-specific antiphospho-histone
H3 (Gurley et al., 1978), were
similar in T-/- and wild-type limb buds at E9.5 and E9.75
(Fig. 8A, and not shown).
Normal mitotic rates are not inconsistent with reduced FGF8 in
T-/- limb buds; proliferation is unchanged even in
Fgf8/Fgf4 nulls despite reduced limb bud size
(Sun et al., 2002
). Levels of
apoptosis, analyzed in sections and whole mount, were similarly very low in
wild-type and T-/- limb buds from E9.5-E10
(Fig. 8B, and not shown), even
at E10 when extensive apoptosis was present in T-/- axial
tissues. Loss of ridge FGF survival signals causes mesenchymal apoptosis, but
does not become appreciable until about E10-10.5
(Moon and Capecchi, 2000
;
Revest et al., 2001
;
Sun et al., 2002
). Thus,
abnormalities in T-/- limb buds are not due to general
growth arrest and lost viability. They are also unlikely to be due to a
general developmental delay. Lmx1b, initially expressed uniformly and
later restricted to dorsal mesoderm after the AER forms
(Loomis et al., 1998
), showed
normal dorsal restriction in T-/- embryos at E10
(Fig. 8C). However, some
mesodermal gene expression was lost. Shh, which depends on ridge FGF
signals (reviewed by Capdevila and Johnson,
2000
), was absent in T-/- limb buds
(Fig. 8D). In
Fgfr2-IIIb null mutants, which fail to form a mature AER, loss of
Shh induction is evident before other mesodermal changes such as
decreased Msx1, or increased apoptosis
(Revest et al., 2001
). The
results suggest that T plays a role in AER morphogenesis and that
altered AER function in T-/- embryos causes some changes
in mesodermal gene induction. However, the possibility that AER changes in the
T-/- embryo are an indirect consequence of lost early
midline signals cannot be excluded at present (see
Dealy, 1997
).
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DISCUSSION |
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Restriction of T function to the limb apex DV boundary and
role in AER maturation
Normal T expression in limb bud is restricted to the immediate
subridge mesoderm. This may be due to a very stringent dependence of
T expression on signals from the ridge. In addition to maintenance by
ridge signals, lateral inhibitory signals may also serve to limit the zone of
T expression and/or function. In the ectoderm, Cux1 is
induced along the edges of the forming AER and seems to function to restrict
AER formation from spreading laterally (Taveres et al., 2000). Perhaps one of
the Sprouty genes, which are induced as feedback inhibitors during
FGF signaling, could serve a similar role in the limb mesoderm (see
Minowada et al., 1999). Even
when T is uniformly misexpressed throughout the early lateral plate
and limb mesoderm, its functional effects are nevertheless restricted to the
DV boundary along which AER formation normally occurs, resulting in AER
extension along the limb apex. In contrast, certain other factors can induce
ectopic AERs in any orientation in limb ectoderm (e.g.
Laufer et al., 1997
;
Rodriguez-Estaban et al., 1997; Kengaku et
al., 1998
; Pizette et al.,
2001
). This observed functional restriction could be due to
lateral inhibition of T function by other limb mesodermal factors.
Alternatively, T function may require positive mesodermal co-factors
that are also restricted to the DV boundary, and so are unavailable to
misexpressed T elsewhere in the limb. In either case, such
restrictions of T activity may provide a mesodermal mechanism to
ensure that normal AER formation occurs only along the DV boundary in the
ectoderm.
The consequences of loss of T are also consistent with such a
function, and suggest that mesodermal factors may play a role, along with
ectodermal factors such as En1
(Kimmel et al., 2000), in
regulating ridge maturation and positioning. However, unlike En1, T
is unlikely to function in AER formation by regulating DV boundary positions.
At present, it remains uncertain what mesodermal signal T may act
through to contribute to AER maturation. At very early stages, BMPs regulate
both DV polarity and AER formation in parallel, by inducing En1 in
ventral ectoderm and by activating Msx expression, respectively
(Ahn et al., 2001
;
Pizette et al., 2001
).
Although Bmp4 expression was reduced early in the
T-/- forelimb region, DV polarity was preserved in both
ectoderm and mesoderm. Likewise, expression of other BMP targets, Msx
genes, was unchanged. Consequently, we feel that the BMP signaling pathway is
not primarily affected by loss of T function; reduced Bmp4
and irregular En1 and Wnt7a distal expression borders are
more likely secondary to some other causative defect in AER morphogenesis.
FGF10 plays some role in formation of a mature AER, as well as in limb
initiation. In the complete absence of FGF10 no bud or pre-AER forms
(Min et al., 1998
;
Sekine et al., 1999
), but loss
of the ectodermal FGFR2-IIIb isoform, which FGF10 binds, disrupts AER
formation shortly after induction and the expression of Fgf8 and of
early ridge-dependent mesodermal genes is initiated but not maintained
(Revest et al., 2001
). In
T-/- limb buds, Fgf10 expression is not
substantially reduced until E9.5, but earlier quantitative changes in
distal/apical Fgf10 expression that are difficult to appreciate could
still contribute to a hypomorphic effect. However, whether decreased
Fgf10 is directly due to a loss of T, or is an indirect
effect of reduced AER function cannot be distinguished at present.
Restricted T expression along a narrow strip of apical mesoderm could strongly promote AER formation and morphogenesis along the DV boundary by ensuring high focal Fgf10 expression along the limb apex, or via another mesodermal signal. In the T-/- limb bud, Fgf8 expression and pre-AER compaction are not properly reinforced along this distal DV border and remain broad and irregular. Alternatively, T could play some other role in the mesoderm, such as changing properties of apical mesodermal cells to facilitate compaction of pre-AER ectoderm towards the DV edge.
Potential role of T in reciprocal mesenchymal-epithelial
signaling in the limb
During limb induction, WNT signals maintain high Fgf10 expression
in prospective limb and FGF10 activates ectodermal Wnt3a and
Fgf8 expression, initiating AER formation
(Kawakami et al., 2001). AER
signals subsequently also maintain mesodermal Fgf10 expression
(Ohuchi et al., 1997
).
T transcripts are first clearly detected at stage 15, at the onset of
Wnt3a and Fgf8 activation in the ectoderm
(Kengaku et al., 1998
). Both
the ability of WNT3a and FGF8 to induce T expression, and the ability
of T to increase subridge expression of Fgf10 early after
misexpression suggest that T may be a component of the mesodermal
response to developing AER signals that maintains high Fgf10 apically
and thereby also maintains the forming AER, establishing a regulatory loop
between ectoderm and mesoderm.
The T-box family oversees multiple aspects of limb development
Several T-box genes are expressed in developing limb
(Gibson-Brown et al., 1996;
Gibson-Brown et al., 1998
;
Isaac et al., 1998
;
Logan et al., 1998
;
Ohuchi et al., 1998
), raising
the question of specificity of T function. Tbx2 and
Tbx3 are expressed along the anterior and posterior borders of limb
mesoderm and in AER as well, but their expression and regulation suggest other
than a primary role in AER formation. Both Tbx2 and Tbx3 are
also expressed in the flank between limb buds and Tbx3 is
down-regulated upon ectopic limb bud induction by FGF
(Isaac et al., 1998
). Tight
regulation of Tbx3 by SHH (Tumpel
et al., 2002
) and loss of posterior forelimb elements in humans
with TBX3 mutations [Ulnar-Mammary Syndrome
(Bamshad et al., 1997
)] both
suggest a more direct role for Tbx3 in AP patterning. Tbx5
and Tbx4 are expressed selectively in forelimb or hindlimb mesoderm,
respectively, and regulate limb identity
(Rodriguez-Esteban et al.,
1999
; Takeuchi et al.,
1999
; Bruneau et al.,
2001
). Loss of Tbx5 in zebrafish interferes with pectoral
fin bud induction and Tbx5 misexpression in very early chick lateral
plate induces ectopic limbs (Ahn et al.,
2002
; Ng et al.,
2002
), showing that Tbx5 also regulates limb outgrowth.
However, misexpression of Tbx5 or Tbx4 in prospective limb
field cause transformations in limb identity and limb truncations
(Rodriguez-Esteban et al.,
1999
; Takeuchi et al.,
1999
) quite different from T misexpression phenotypes.
The identities of T-box family targets coordinated throughout limb formation
remain to be explored. Because of their different expression patterns and
distinct misexpression phenotypes, it is unlikely that T and other
T-box factors regulate targets redundantly. One interesting possibility is
that T and Tbx5 may cooperate in AER regulation, given the
dimerization potential of T-box proteins
(Muller and Herrmann, 1997
).
Such dimerization might regulate novel targets, since binding site analyses
indicate that different T-box proteins, including T and TBX5, recognize
distinct targets in vivo and in vitro
(Conlon et al., 2001
;
Ghosh et al., 2001
).
Expression of T in relation to several proposed FGF/WNT
signal relay sites
Intriguingly, T is expressed in association with several sites
proposed to `relay' signals for limb induction from midline to periphery
(reviewed by Martin, 1998)
including node/early notochord, somites and nephric duct epithelium adjacent
to nephrogenic mesoderm. Kawakami et al.
(Kawakami et al., 2001
)
propose that such relay sites may represent points of cross talk for FGF and
WNT signals, raising the possibility that T plays a role in signal
relay at these sites. Such questions are best addressed with genetic
approaches, such as a conditionally targeted T allele, currently
being developed.
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ACKNOWLEDGMENTS |
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Footnotes |
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