1 Department of Embryology, Carnegie Institution of Washington, Baltimore,
Maryland 21210, USA
2 Craniofacial Developmental Biology and Regeneration Branch, NIDCR, National
Institutes of Health, Bethesda, Maryland 20892, USA
* Author for correspondence (e-mail: fan{at}ciwemb.edu)
Accepted 21 August 2002
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SUMMARY |
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Key words: Gas1, Fgf8, Fgf10, Limb, Growth, Apical ectodermal ridge, Mouse
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INTRODUCTION |
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There are many modulators of AER function. Shh, expressed in the
zone of polarizing activity (ZPA), regulates Fgf4 in the posterior
AER by acting through the intermediate component Gremlin, a BMP inhibitor
(Zuniga et al., 1999).
Inhibition of BMP signaling also can increase the thickness of the AER
(Pizette and Niswander, 1999
).
These studies indicate that BMP negatively modulates AER function and that SHH
counteracts this by modulating the BMP signal. However, at an earlier stage,
BMP signaling is necessary for AER formation
(Ahn et al., 2001
;
Pizette et al., 2001
).
Fgf10 appears to act through the Wnt/ß-catenin pathway
in the ectoderm to activate Fgf8 in the AER
(Kawakami et al., 2001
).
Consistently, the null mutant mouse embryos of Tcf1 and
Lef1, the Wnt downstream mediators, do not express
Fgf8 in the AER (Galceran et al.,
1999
). FGFs from the AER serve to maintain Fgf10
expression in the mesenchyme, but whether they are the sole and direct input
or require additional modulators and intermediate components is not yet
known.
Sufficient mesenchymal mass is required for the formation of the skeletal
elements of the appropriate number and size. The aforementioned growth
regulators are important in the generation of a defined amount of mesenchymal
mass. From this will be produced the mesenchymal condensations representing
the limb skeletal elements, including the proximal segment (stylopod;
femur/humerus), the medial segment (zeugopod) with two elements
(tibia-fibula/radius-ulna), and the distal segment (autopod) with, in the
mouse, the five elements (digits)
(Hinchliffe and Griffiths,
1983; Shubin and Alberch,
1986
). These skeletal elements arise by endochondral cartilage
formation, starting with a group of mesenchymal cells that condense and
differentiate into chondrocytes
(Erlebacher et al., 1995
). The
growth and size of each element are then coordinately regulated by successive
transitions in differentiation that are locally controlled, for example,
through the activities of BMP and of IHH, which activates a negative feedback
relay system by regulating PTHrP (a negative growth regulator)
(Ganan et al., 1996
;
Vortkamp et al., 1996
;
Zou et al., 1997
;
St-Jacques et al., 1999
).
We have previously proposed that GAS1, a GPI-anchored membrane glycoprotein
(Stebel et al., 2000), acts as
an inhibitor of SHH via direct physical interaction
(Lee et al., 2001a
). However,
Gas1 mutant mice do not display phenotypes related to those of SHH
overexpression (Lee et al.,
2001b
; Liu et al.,
2001
). Instead, we report here that Gas1 mutant limbs
have defects caused by reduced proliferation in the AER and mesenchyme, and
develop with small autopods, missing phalanges and anterior digit syndactyly.
We provide several lines of experimental evidence supporting the model that
Gas1 is a necessary mesenchymal factor that positively regulates
Fgf10 in a regional- and temporal-specific manner to maintain the
Fgf10/Fgf8 regulatory loop.
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MATERIALS AND METHODS |
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Skeletal preparation
Skin and internal viscera were removed and the bodies fixed overnight by
St. Marie's fluid, followed by standard Alcian Blue and Alizarin Red staining
procedures (Bancroft and Cook,
1994). Whole-mount fetal Alcian Blue staining was performed
according to the same protocol omitting the Alizarin Red.
In situ hybridization (ISH)
ISH on paraffin sections (8 µm) with 35S-UTP-labeled probes
was performed as described previously (Fan and Tesseir-Lavigne, 1994).
Photographs of the detected transcripts were taken as dark-field images with a
red filter. Phase images were taken with a blue filter and overlayed with the
dark-field images. Whole-mount ISH using DIG-labeled probes was performed
following standard procedures (Wilkinson,
1992). Probes used were: Gas1
(Lee and Fan, 2001
),
Shh (gift from Dr McMahon), Fgf8 (gift from Dr Martin),
Fgf4, Gli3 (gift from Dr Hui), Fgf9, Fgf17 (gift from Dr
Ornitz), Bmp2, Bmp4, Bmp7 (gift from Dr Hogan), Gremlin
(gift from Dr Zeller), Hoxb8, Hoxd13 (gift from Dr Duboule) and
Alx4 (gift from Dr Wisdom).
BrdU and TUNEL assays
Mice were injected with 10 mg/ml BrdU (Sigma) at 0.01 ml/g body weight, 1
hour before sacrifice. BrdU-positive cells were detected by using a BrdU
staining kit (Zymed). Cell death assays were performed using the In Situ Cell
Death Detection Kit, Fluorescein (Boehringer Mannheim) according to the
manufacture's protocol.
In vitro limb culture
Embryonic limbs were cultured as described previously
(Zuniga et al., 1999).
E9.75-E11.5 embryos from heterozygous mating were dissected in L-15 medium.
The heads and tails were removed for genotyping and the trunks including
forelimbs were used for injection. Recombinant FGF10 and FGF8 proteins
(Research Diagnostic Inc.) resuspended in PBS were delivered by glass needles.
1-25 µg/ml of FGF10 and 1-100 µg/ml of FGF8 were used for injection and
a pulse of 9.2 nl was injected into the right forelimb mesenchyme underneath
the AER. The left forelimb was not injected and served as an internal control.
Mock injection was performed using PBS. The injected limb buds were cultured
in BGJb medium with 0.2 mg/ml ascorbic acid (Gibco/BRL) at 37°C/5%
CO2. After overnight culturing, the trunk fragments were fixed and
subjected to whole-mount ISH.
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RESULTS |
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Gas1 mutant mice have limb abnormalities
To define the role of Gas1 in limb development, we analyzed the
limb phenotype of Gas1 null mutant (Gas1-/-) mice
(Lee et al., 2001b). Newborn
Gas1-/- mice have smaller fore- and hind-limb paws
(Fig. 2A-B'). Skeletal
preparation using the dyes Alcian Blue (for cartilage) and Alizarin Red (for
calcified bone) revealed that this is due to a size reduction of all
phalanges, metacarpals and metatarsals
(Fig. 2C-D'). The calcified
regions were of normal size but the chondrogenic regions were reduced in
proportion. Digits I-III were disproportionately reduced in size. In addition,
the second phalange of digit II was greatly reduced
(Fig. 2C') or absent
(Fig. 2D'), and there was a
high rate of soft tissue fusion between digits II and III
(Fig. 2A',B'). Syndactyly
between digits II and III was also observed by histology
(Fig. 2D') and by X-ray imaging
(not shown). The frequency of these defects is summarized in
Fig. 2E. The front and hind
paws of the adult Gas1-/- mice were almost of normal size
(not shown), indicating a compensatory growth postnatally. Carpals, tarsals
and long bones (radius, ulna, tibia, fibula) were either slightly shorter or
not affected. Macroscopic and histological analyses revealed no apparent
defects in the patterns of muscles and tendons (not shown). Thus,
Gas1 contributes to proper formation of the autopodial skeletal
elements, in particular the phalangeal elements and the anterior digits.
|
The Gas1 mutant has a delay in digit formation
We next determined the ontogeny of the mutant phenotype by histology
(Fig. 3). As early as E11.5,
the mutant limb bud width across the AP axis is noticeably reduced
(Fig. 4B'). The prechondrogenic
condensations of digits II-IV in the forelimb are normally visible by E12.5
(Fig. 3A). In the mutant,
condensations of digits III and IV are smaller and digit II is less evident
(Fig. 3A'); the autopod is also
narrower along the AP axis and shorter along the PD axis. At E13.5, the
metacarpals and first phalange are individualized by the onset of joint
formation (Fig. 3B). In the
mutant, separation of the phalanges is ill-defined and the phalanges of digit
II and metacarpal I are not apparent (Fig.
3B'). From E13.75 to E14.5, the third and second phalanges of all
digits become clearly defined (Fig.
3C,D); whereas in the mutant, the second phalanges of digits II
and III are not individualized (Fig.
3C',D').
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|
At E16.5, ossification of metacarpals III and IV is delayed in the mutant (Fig. 3E,E'). At E17.5, ossification of the first phalanges of digits III and IV (Fig. 3F,F') is clearly delayed. While ossification of digit III second phalange is delayed by about 24 hours, digit II second phalange does not appear to ever ossify (Fig. 3F' and not shown). Intriguingly, as soon as the ossification assumes, the bone segments appear normal in size at the expense of the chondrogenic domains (Fig. 2C', Fig. 2D', Fig. 3F'). The mutant hindlimb phenotype is similar (Fig. 2E). These manifestations suggest that Gas1-/- limbs have reduced mesenchymal mass as early as E11.5, which at least in part leads to the small and delayed condensations. Delayed or absent joint formation may affect the size of the chondrogenic region, but Gas1 is not essential for chondrogenic differentiation.
Reduced cell proliferation in the AER and distal mesenchyme in
Gas1-/- limbs
To detect alterations in cell proliferation, we assayed bromodeoxyuridine
(BrdU) incorporation in vivo. At E10.5, the rate of BrdU incorporation in the
limb mesenchyme adjacent to the AER (cells within 150 µm of the AER were
counted and compared) was not altered in the mutant
(Fig. 4A,A'). Note that the
mutant AER is present but thinner than normal. AER morphology was confirmed by
scanning electron microscopy (not shown). Importantly, the mutant AER has
fewer BrdU+ cells (Fig.
4A',D), indicating a non cell-autonomous effect of Gas1
on the AER. The most marked difference in the mesenchymal proliferation rate
is observed at E11.5 (Fig.
4B,B'D). The reduction is observed preferentially in the distal
mesenchyme at the anterior-to-central portion of the mutant limb: mild
anteriorly and severe in the central region
(Fig. 4D). This cellular mass
reduction prefigures the delay in digit formation. When the digit ray is
visible at E12.5, we found no marked difference in the rate of proliferation
between digit III/IV and digit IV/V of mutant and control limbs. However,
between digit II/III, there was a significant reduction (10%) in
proliferation rate in the mutant (Fig.
4C,C',D). This regional-specific defect is surprising given the
more general Gas1 expression in all interdigits at this stage. These
findings suggest that the main proliferation defect occurs around E11.5 and
the perduring smallness of the embryonic limb is due to this early deficiency
of precursor population.
Programmed cell death (PCD) is reduced in the
Gas1-/- limb
PCD is found in the following areas of the developing chick limb: anterior
and posterior necrotic zones, the opaque patch, the interdigital mesenchymes
and the joints (Hinchliffe,
1982; Hurle et al.,
1996
). Gas1 expression overlaps with the opaque patch at
E11.5, the interdigits at E12.5-13.5 (albeit weakly), and the joints
(E13.5-E15.5) (see also Lee and Fan,
2001
; Lee et al.,
2001c
). Furthermore, overexpression of Gas1 can trigger
PCD in cultured limb mesenchymal cells
(Lee et al., 2001c
). To assess
whether Gas1 normally plays a role in PCD, we performed
TUNEL-fluorescence labeling. At E11.5, fewer apoptotic cells were detected in
the central mesenchyme area (opaque patch) in the mutant compared to control
forelimb (Fig. 5A,A'), even
though the limb is already smaller. In the control E13.5 forelimbs, cell death
was observed in the interdigital zones and the prospective joints of the
digits (Fig. 5B). Interdigital
cell death in Gas1-/- forelimbs was relatively normal
posterior to digit III but greatly reduced anterior to digit III
(Fig. 5B'), correlating with
the anterior soft tissue syndactyly. Joint PCD was delayed in digits II and
III in the mutant, consistent with the delay in phalange separation seen
histologically. Since the mutant limbs appear delayed in development, we also
examined PCD at E14.0. There was still little PCD between digits II and III,
but PCD appeared relatively normal between other digits (compare
Fig. 5B,C,C'). By contrast, PCD
in the mutant joints at this time appeared at a higher rate than those of the
wild type at E13.5 and E14.0 (of digits II-IV in
Fig. 5C').
|
Reduced PCD between digits II and III suggests that Gas1 normally
facilitates PCD and supports the claim by Lee et al.
(Lee et al., 2001c), but in
other interdigits, PCD appeared to be relatively normal. However, more
apoptotic cells were detected in the mutant joints, suggesting that
Gas1 is anti-apoptotic. In other affected regions of the
Gas1 mutant, such as the eyes and the cerebellum
(Lee et al., 2001b
;
Liu et al., 2001
), the PCD
rate is not altered. Either Gas1 regulates PCD in a
cell-context-dependent manner or these region-specific alterations of PCD are
a secondary consequence of deregulated growth and heterochrony of the mutant
limb.
Expression of patterning genes is not obviously affected in the
Gas1-/- limbs
Owing to the fact that GAS1 can physically interact with SHH and IHH
(Lee et al., 2001a), we
examined whether there are patterning abnormalities related to deregulated SHH
signaling in the Gas1 mutant, using a battery of functional marker
genes in the SHH pathway. However, we did not observe expression pattern
changes. Shh expression in the ZPA
(Riddle et al., 1993
;
Echelard et al., 1993
) was
activated and maintained correctly at E10 and E10.5
(Fig. 6A' and not shown).
gremlin, a downstream target of Shh
(Zuniga et al., 1999
), was
expressed in the normal posterior domain albeit at apparently reduced levels
(Fig. 6B'). The expression of
Bmp2 and Bmp4 in the AER and the mesenchyme (reviewed by
Hogan, 1996
) was also
apparently normal in positions and levels
(Fig. 6C',D'). These results
suggest that the thinner AER is not due to misregulation of Bmps or
gremlin. Expression of Ptc1 (Ptch)
(Fig. 6E') (Marigo et al., 1996
) and
Gli1 (not shown) (Hui et al.,
1994
) also appeared normal in the posterior domain. Alx4
(Qu et al., 1997
) and
Gli3 (Hui et al.,
1994
) expression was confined to the anterior domain in the mutant
as in the control (not shown). These analyses were extended to E11.5 and E12.5
and no obvious alterations in these expression patterns were found. Lastly,
Hoxd13 (Dolle et al.,
1993
) (Fig. 6F')
and Hoxb8 (Charite et al.,
1994
) (not shown) expression was activated at a normal distal
position in the Gas1-/- limbs at E10.5 (not shown), E11.5
and E12.5 (not shown). The smaller domains of expression appear to be
proportional to the smaller size of the limb bud.
|
Since in the mutant autopod chondrogenesis is delayed, we examined the
expression patterns of Ihh, Bmp2, Bmp4, Bmp7, BmpRIA, BmpRIB and
PTHrP (reviewed by Hogan,
1996) in the developing cartilage. Their expression was delayed
corresponding to the delayed progression of the limb (by
12 hours). Once
initiated, these genes were expressed in normal patterns with proportionally
smaller domains (data not shown). As Gas1-/- displays
neither expanded nor reduced expression domains of Shh and
Ihh downstream reporters and the Gas1-/- limb
phenotype is unrelated to those of Shh-/-
(Chiang et al., 2001
) and
Ihh-/- (St-Jacques et
al., 1999
), Gas1 does not appear to modulate the activity
of the hedgehog pathway in the limb.
Gas1 mutant limbs are defective in maintaining Fgf8
expression
Because the Gas1 mutant AER is thinner
(Fig. 4A') and compromised AER
function is a potential cause of reduced mesenchymal mass (reviewed by
Martin, 1998), we reasoned
that expression of the AER-specific Fgfs may be affected in the
Gas1 mutant. Fgf4, Fgf9 and Fgf17 expression in the
AER at E10.5 (Fig. 7A-C) and
E11.5 (not shown) was normal in the mutants when compared to the controls (not
shown). In addition, Fgf8 expression at E9.5 was also normally
initiated (Fig. 7D,D').
However, at E10.0 and E10.5, AER-specific Fgf8 expression was lost in
the mutant (Fig. 7E',F'). At
E11.5, variable small patches of Fgf8 expression were regained in the
mutant AER (Fig. 7G'). This
Fgf8 reappearance was restricted: when observed, it was most frequent
in the posterior region, rarely in the anterior region, and never in the
central AER. Loss of the FGF8 input from the AER may be the main cause of the
Gas1 mutant limb defects.
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Fgf10 expression is reduced in the distal tip mesenchyme of
the mutant limb
Fgf10 is necessary and sufficient to initiate Fgf8
expression in the AER (Ohuchi et al.,
1997; Min et al.,
1998
; Sekine et al.,
1999
). Whether it is continuously required to maintain
Fgf8 in the AER has not been established. It is possible that
Gas1 maintains Fgf10, which in turn maintains Fgf8
in the AER. We therefore examined whether Fgf10 expression is altered
in the mutant. A small region of cells at the most distal tip mesenchyme
immediately underneath the AER showed a clear absence of Fgf10
expression at E9.5 both in the control and mutant forelimb
(Fig. 8A,A'). At E10.0 and
E10.5 (Fig. 8B,B',D,D'),
Fgf10 expression extended to the extreme tip of the control limb
mesenchyme, whereas mutant cells located at the distal tip mesenchyme lacked
Fgf10 expression. This reduction of Fgf10 expression could
also be observed by whole-mount ISH (Fig.
8C',E'), but only in very few mutant limbs (
8% of the mutant
limbs) in these cases, the Fgf10 loss appeared to be more
extensive than that seen in sections. We reasoned that most mutants had a
small affected domain (consistently detected by section ISH) which was not
easily discerned by whole-mount ISH. Note that the anterior Fgf10
expression domain was also slightly down regulated in the mutant. At E11.5,
there was a moderate recovery of Fgf10 expression in the mutant
distal mesenchyme (Fig. 8F'),
temporally corresponding to the reappearance of small patches of Fgf8
expression in the AER. These results support a model in which Gas1 is
required to activate Fgf10 expression in the distal tip mesenchyme
and Fgf10 in this distal tip mesenchyme is crucial for maintaining
Fgf8 expression in the AER.
|
FGF10 injection restores Fgf8 expression in Gas1
mutant limb
In the above model, GAS1 deficiency should be overcome by supplementing
FGF10 at the tip region. To test this, we applied FGF10 protein to the distal
tip region of the Gas1 mutant limb and examined restoration of
Fgf8 expression in the AER. The trunk segments containing the
forelimbs of embryos between E9.75 and E11.5 were cultured using an in vitro
system (Zuniga et al., 1999).
At E9.75-E10.0 (Fgf8 is already lost in the mutant AER), FGF10
protein ranging from 9.2 to 230 pg was delivered into the anterior-central
mesenchymal tip region (where Gas1 is normally expressed) underneath
the AER, by microinjection. Only the right limb was injected, hence the left
limb served as an internal control. The injected embryo trunks were cultured
for 16 hours before harvesting for assessment of Fgf8 expression
(diagram in Fig. 9A). Injection
of PBS into control (Fig. 9B)
and mutant (Fig. 9E) right
limbs did not alter their Fgf8 expression when compared to the
uninjected left side. Injection of 9.2 pg FGF10 protein (but not lower
amounts) into the mutant right forelimb rescued Fgf8 expression in
the AER (Fig. 9F), while the
uninjected side showed no Fgf8 expression. At this amount of FGF10,
only a weak and small domain of Fgf8 expression was observed in the
central AER of the mutant limbs. At 230 pg, FGF10 caused the entire length of
the mutant AER to express high levels of Fgf8 similar to the control
limb injected with the same amount of FGF10
(Fig. 9D,G). There was also a
FGF10 dosage-dependent increase in AER height in the injected mutant limbs.
Notably, high levels of FGF10 injected into control limbs caused an increase
in Fgf8 expression as well as AER height compared to the uninjected
side (Fig. 9D). At E11.5, after
the Fgf8 expression was lost for more than a day in the mutant, FGF10
injection still rescued its expression
(Fig. 9I), indicating that
Fgf8 expression in the AER requires continuous input of FGF10.
Injection of FGF10 to the proximal region (
200 µm from the AER) did
not rescue Fgf8 expression (not shown). Finally, injection of FGF8
protein (up to 1.8 ng) into the E9.75-E10.5 mutant limb did not rescue its own
expression in the AER (Fig. 9J and not shown), suggesting that FGF8 cannot restore Fgf10 expression
at the distal tip. Thus, supplementation with FGF10, but not FGF8, at the
distal tip mesenchyme can overcome the requirement of Gas1.
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DISCUSSION |
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Gas1 plays a role in regulating proliferation of the developing limb
through regulating Fgf8 and Fgf10
Gas1-/- limbs display reduced proliferation
preferentially in the anterior-to-central limb bud around E11.5. Gas1
is normally expressed in the anterior-to-central region between E9.5-E11.5.
Superficially, Gas1 appears to be a cell autonomous positive
regulator of proliferation. Paradoxically, Gas1 overexpression is
known to inhibit the cell cycle in cultured fibroblast
(Del Sal et al., 1992).
However, reduced proliferation in the Gas1-/- limb does
not completely correlate with the time and pattern of Gas1
expression. Reduced proliferation only became measurable after the
Fgf8 expression in the AER was lost, indicating that the
proliferation defect is more likely the consequence of compromised AER
function (reviewed by Johnson and Tabin,
1997
). The preferential reduction in the anterior-to-central
domain may reflect the fact that Fgf4, 9 and 17 are all
expressed in their normal posterior AER domain, whereas Fgf8 is lost
throughout the AER and thus the anterior-to-central domain does not continue
to receive the FGF signal. However, we cannot exclude the possibility that
Gas1 also directly contributes to anterior limb mesenchyme
proliferation.
The mutant AER also has a decreased rate of proliferation. Our data suggest that Gas1 acts indirectly to promote AER proliferation by establishing high levels of FGF10 at the distal tip mesenchyme. Consistently, injection of FGF10 at a high dosage can rescue the mutant AER such that it corresponds to wild-type AER in height and levels of Fgf8 expression (Fig. 9G). This increase of AER height leads us to propose that FGF10 regulates not only the level of Fgf8 expression in the AER but also the proliferation of the AER.
The continuous requirement of FGF10 for maintaining Fgf8
expression in the AER
Both gain-of-function (Ohuchi et al.,
1997) and gene inactivation studies
(Min et al., 1998
;
Sekine et al., 1999
) have
provided evidence that Fgf8 activation requires Fgf10.
However, it was not clear whether Fgf10 continues to be required to
maintain Fgf8 expression in the AER after initiation. The loss of
Fgf10/Fgf8 in the Gas1-/- limb and our FGF10
injection data in the Gas1-/- background strongly indicate
that Fgf10 at the distal region is continuously required for
Fgf8 expression and that the AER retains the potential to respond to
FGF10 long after the loss of Fgf8 expression.
The Gas1 mutant defines a domain of Fgf10 in the
distal tip mesenchyme required for Fgf8 maintenance
Gas1 is required for Fgf10 transcription at the distal
tip mesenchyme of the limb between E10 and E11.5. Normally, from E10,
Fgf10 is expressed in a broad contiguous domain directly underneath
and extending 150-200 µm away from the AER. In Gas1-/-
limbs, Fgf10 expression is lost in the distal-most 3-5 cell layers
(or more cell layers in rare cases) next to the AER
(Fig. 8B'-E'). In
the chick, FGF8 or AER signals including a combination of FGFs induce
Fgf10 expression over a broad domain
(Ohuchi et al., 1997). In
Gas1-/- limbs, the proximal Fgf10 domain is
relatively normal (though weaker anteriorly), suggesting that the remaining
AER FGFs can still act over a long distance. However, it also indicates that
these remaining FGFs are not sufficient to maintain Fgf10 at the
distal tip, even though FGFRI expression is normal (including the tip region,
not shown) in the mutant limb. Reciprocally, our data indicate that the distal
tip Fgf10 expression is necessary and sufficient, as shown by FGF10
rescue injection, to maintain Fgf8. Thus Gas1 is necessary
to maintain the distal Fgf10 domain and this is required to maintain
Fgf8 expression in the AER.
This finding provides several novel insights (Fig. 10). First, there are two distinct regulatory mechanisms for Fgf10 expression in the limb mesenchyme: the distal tip domain, which requires Gas1 function, and the proximal larger domain, which does not. Second, only this tip region of Fgf10 expression is responsible for maintenance of the Fgf8 expression in the entire AER. Although the proximal Fgf10 expression domain in the mutant extends to the anterior and posterior borders next to the ectoderm, it is not sufficient to maintain Fgf8 there. Third, Fgf4, 9 and 17 expression is present in the mutant, suggesting that either the FGF10 in the proximal region is sufficient to maintain their expression or their expression does not depend on FGF10. One possible mechanism whereby the three Fgfs are expressed in the absence of the distal FGF10 is that they are regulated by SHH/ZPA.
|
The relationship between Gas1 and the Fgf8 and
Fgf10 regulatory loop
Gas1 maintains Fgf10 expression at the tip mesenchyme,
either directly or indirectly. Gas1 is normally expressed in the
anterior two-thirds of the limb (Fig.
1H). It is possible that Gas1 directly controls distal
Fgf10 expression in this region
(Fig. 10, model a). In this
model, two signals are required to maintain Fgf10 at the tip,
Gas1 in the mesenchyme and the Fgf8 in the AER. In a model
of indirect control, Gas1 may help to mediate Fgf8's
feedback regulatory loop, which maintains the tip Fgf10 expression
(Fig. 10, model b). In this
model, GAS1 is an obligatory component of FGF8 signaling as injection of high
doses of FGF8 fails to overcome the Gas1-/- phenotype.
However, it should be noted that except for their similar limb defects,
Gas1 mutants and Fgf mutants do not share any common defects
in other tissues outside of the limb (Sun
et al., 1999), suggesting a specialized function of Gas1
in the limb in relation to Fgf8. In either model, it is intriguing
that the Fgf10 expression is more affected in the central tip than
the anterior region in the Gas1-/- limb and that this
causes the entire domain of Fgf8-AER expression to be lost.
Nonetheless, the discovery that Gas1 in the mesenchyme is an
additional component in the regulatory loop between Fgf10/Fgf8 adds a
new dimension to this molecular network.
The differences between Gas1 mutant and Fgf8
AER-knock-out mutants
The Gas1 mutant has phenotypes less severe than the two types of
Fgf8/AER-KO mutants reported. For simplicity, only the forelimb
phenotype is discussed. When Fgf8 is inactivated prior to its
expression in the AER (Moon and Capecchi,
2000), there is a severe growth defect and a loss of
Fgf10 expression in the anterior limb. When Fgf8 is
inactivated shortly after its initiation
(Lewandoski et al., 2000
), the
limb defect is milder and the Fgf10 expression is normal. In both
cases, the forelimbs are observably smaller at E10.5 and develop with
shortened or missing proximal bones in addition to the autopod defects. In
contrast, the Gas1 mutant's limb size reduction is not measurable
prior to E10.5 and the phenotype is restricted to the autopod. One possible
explanation is that the Gas1 mutant loses Fgf8 expression
later and has higher levels of residual FGF8 than both Fgf8/AER-KO
mutants. The three mutants may thus represent Fgf8 deficiency at
different stages and/or of different levels. Together, these data suggest a
progressively diminishing requirement of Fgf8 activity for the
proximal elements during the PD growth and patterning of the limb.
One difference between the Gas1 and the Fgf8/AER-KO
mutants is puzzling: high levels of Fgf4 are activated in the entire
AER in both types of Fgf8/AER-KO mutants
(Moon and Capecchi, 2000;
Lewandoski et al., 2000
). This
does not occur in the Gas1 mutant. While the precise reason for this
difference is unknown, we suggest that activation of compensatory
Fgf4 expression along the entire length of the AER still requires
Fgf10 at the specialized distal tip mesenchyme, which is missing only
in the Gas1 mutant.
Reduced proliferation and digit malformation
Missing phalanges and syndactyly, the phenotype of
Gas1-/-, can also be induced by chemical inhibitors of
proliferation, presumably because the autopod elements are most vulnerable as
they are formed late (Shubin and Alberch,
1986). The anterior digit condensations appear even later than the
posterior ones (Burke and Alberch,
1985
), making them more sensitive to growth disruption. The small
phalanges and metacarpals in the forelimb of Hoxd13-/-
mice (Dolle et al., 1993
) were
attributed to decreased proliferation
(Duboule, 1995
). We propose
that the Gas1-/- autopod defects are also a consequence of
insufficient precursor mesenchyme generated earlier, thereby causing delayed
chondrogenesis and small condensation sizes. The anterior digit defects may
thus be due to depletion of a smaller pool of mesenchymal cells by early
condensing posterior elements. Since the alterations of the proliferation and
PCD patterns in the mutant do not strictly correlate with Gas1
expression, we suggest that they reflect a secondary consequence of the
heterochrony of the mutant limb caused by the deregulation of Fgf8.
The loss of Fgf8 expression may also account for reduced PCD at E11.5
(Montero et al., 2001
).
Finally, the relatively normal patterns of the condensations and expression of
Bmp2, Bmp4, Bmp7, BmpRIB, Ihh, and PTHrP in the
Gas1-/- limb further argue for a defect in early
mesenchymal mass rather than a disruption in patterning or bone growth per se.
As the chondrogenic growth regulatory network appears relatively normal, this
may explain the post-natal compensatory growth of the mutant autopod
elements.
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ACKNOWLEDGMENTS |
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