Department of Molecular Genetics, The Weizmann Institute of Science, Rehovot, Israel
Author for correspondence (e-mail:
peter.lonai{at}weizmann.ac.il)
Accepted 29 July 2003
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SUMMARY |
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Key words: FGF signaling, Chimeras, Limb outgrowth, AER differentiation, Dorsoventral compartments
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Introduction |
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Fibroblast growth factors and their receptors have multiple roles in limb
development. They form epithelial-mesenchymal interaction loops, and are
involved in both the proximodistal and anteroposterior limb pattern (for
reviews, see Martin, 1998;
Niswander, 2003
). Fgf4,
Fgf8, Fgf9 and Fgf17 are expressed in the AER. Recent genetic
evidence for the role of Fgf4 and Fgf8 in mesenchymal
proliferation and limb outgrowth suggests that the proximodistal limb pattern
is established during the early stages of limb development
(Sun et al., 2002
).
AER-derived FGF signals also affect anteroposterior specification by
maintaining SHH signaling in the ZPA
(Niswander et al., 1994
).
Although the mesenchymal FGF receptor that processes AER-derived FGF signals
has yet to be identified, Fgfr1c, which is transcribed in the
progress zone (PZ) mesenchyme and is responsible for the shape and adhesion of
its cells (Saxton et al.,
2000
), is a likely candidate for this interaction loop.
A second interaction loop, which connects the PZ mesenchyme to the AER,
includes Fgf10 in the mesenchyme and Fgfr2b in the AER
(Ohuchi et al., 1997).
Targeted disruption of Fgf10 eliminated AER differentiation,
Fgf8 expression and limb outgrowth
(Min et al., 1998
;
Sekine et al., 1999
). Targeted
mutations of Fgfr2 (Arman et al.,
1999
; Xu et al.,
1998
), or Fgfr2b (De
Moerlooze et al., 2000
), exhibited phenotypes very similar to
those of Fgf10, suggesting that they function together as a
receptor-ligand pair. This ligand-receptor interaction, which leads from the
mesenchyme to the epithelium, complements the AER to mesenchyme loop, and the
two together contribute to AER formation, dorsoventral patterning, and
proximodistal limb outgrowth and patterning.
Despite a considerable body of evidence and a coherent scheme of gene interactions, the molecular and cellular mechanisms controlled by the FGF-FGFR system are poorly understood. Studying chimeric mouse embryos may help this analysis. Chimeras grown from mutant and wild-type cells can rescue the early lethality of certain mutations. They can reveal structures to which the activity of a receptor is directed, and if the chimera displays a mutant phenotype, interaction between its components may indicate whether the activity of a gene is intracellular, i.e. cell-autonomous, or extracellular, i.e. non-cell-autonomous.
We are interested in the mechanism of action of Fgfr2. Loss of
Fgfr2 causes amelia, the complete absence of all four limbs
(Xu et al., 1998;
Arman et al., 1999
;
De Moerlooze et al., 2000
);
therefore its null mutations are not suited for detailed study. We hoped that
chimera analysis would help to overcome this difficulty. Here, we report that
Fgfr2 contributes to ectodermal cell movement towards and into the
limb, to AER formation, and to certain aspects of dorsoventral ectodermal
polarity.
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Materials and methods |
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Generation and analysis of chimeric embryos
Small clumps (6-8 cells) of homozygous mutant
Fgfr22/
2
or control ES cells, expressing lacZ, were either aggregated with
E2.5 CD1 mouse morulae (Nagy et al.,
1993
), or were microinjected into blastocysts. Chimeric embryos
were transplanted into pseudopregnant CD1 female mice. Midgestation embryos
were isolated 6.5 to 8 days after embryo transfer, corresponding to
E9.0-E10.5; perinatal embryos were collected between day 14.5 and 16 after
transfer, which corresponds to E16.5 and E18.5. Colonization by mutant-derived
cells was estimated by whole-mount ß-galactosidase staining, which when
necessary was followed by Bouin's fixation, embedding in paraffin wax, and
Haematoxylin and Eosin staining. Calcified bone and cartilage was detected by
Alizarin and Alcian Blue staining, respectively
(Kaufman, 1992
).
BrdU and TUNEL assay
Pregnant female mice were injected with 10 mg/ml BrdU (100 µg/g body
weight). Embryos stained for ß-galactosidase were post-fixed in Bouin's
fixative and embedded in paraffin wax. Tissue sections were incubated with an
anti-BrdU antibody (Sigma, St. Louis), and visualized with HRP-conjugated goat
anti-mouse IgG (Jackson ImmunoResearch Laboratories, PA) and DAB substrate.
Four separate sections were analyzed from three different chimeric, and three
different control, limb buds using the ImagePlus software (Media Cybernetics,
MD). For TUNEL assay the apo TACS kit (R & D Systems, MN) was used.
Whole-mount in situ hybridization
Whole-mount in situ hybridization was performed according to established
methods (Conlon and Herrmann,
1992). The following probes were used: a 353 bp Fgf8
probe (nucleotides 591 to 943, GenBank Accession Number D38752); a 580 bp
Bmp4 probe (nucleotides 431 to 1010, GenBank Accession Number
X56848), and a 478 bp Cd44 probe (nucleotides 1105 to 1582, GenBank
Accession Number AJ251594). These probes were generated by PCR. The
Dlx2 probe was a gift from Dr J. Rubenstein (UCSF), the Msx1
probe was a gift from Dr B. Hill (University of Edinburgh), En1 was a
gift of Dr K. Schughart (GSF-Forschungszentrum), and Wnt7a and
Shh were donated by Dr A. McMahon (Harvard University). For
histological analysis after whole-mount in situ hybridization or
ß-galactosidase staining the embryos were post-fixed in 2%
glutaraldehyde, incubated in 15% sucrose overnight and then incubated in 30%
sucrose for 4 hours. They were then embedded in 10% gelatin. Frozen gelatin
blocks were sectioned into 25 µM sections. For double in situ hybridization
and ß-galactosidase staining, the embryos were fixed for 1 hour in
ice-cold 4% paraformaldehyde and stained overnight at room temperature with 1
mg/ml Salmon-gal (6-Chloro-3-indolyl-ß-D-galactopyranoside, Fluka Chemie
AG), a pink chromophore. Then they were post fixed in 4% paraformaldehyde
overnight and processed for in situ hybridization, omitting the methanol
dehydration and peroxide bleaching steps. Proteinase K treatment was reduced
to 5 µg/ml for 4 minutes at room temperature for both epithelial and
mesenchymal mRNAs.
Photography and image analysis
A Zeiss Axioplan or a Nikon DXM1200 microscope with a CCD camera was
used.
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Results |
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Defective limb morphogenesis in
Fgfr22/
2
Fgf2+/+
chimeras
First, to investigate the embryological effects of the mutation, we studied
late gestation (E16 and E17.5) stage chimeras. Of 47 pups, 20 had obvious limb
defects on at least one fore- or hindlimb, yielding 34 abnormal limbs.
Pre-axial polydactyly was the most frequent defect
(Fig. 1A-C), although some
limbs exhibited fewer than the normal number of digits
(Fig. 1D,F). Dorsally or
ventrally displaced digits were the most common
(Fig. 1A,C). In some limbs, the
dorsoventral polarity of the displaced extra digits was apparently reversed
(Fig. 1A,C,F). However, even in
these cases, the general polarity of the chimeric autopod was normal and no
dorsal footpads, or other signs of ventralization or dorsalization, were
observed. The chimeric autopod of most pups was fully developed and claws were
present (Fig. 1A-C).
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Skeletal preparations revealed defective morphogenesis in all three limb
components, the stylopod, zeugopod and autopod. The humerus of two chimeras
was partially duplicated, having a bony spike with a cartilage cap
(Fig. 2C,D), whereas the
zygopod of most mutant fore- and hindlimbs contained only one bone
(Fig. 2B-D,F). A similar defect
was observed in Fgf4-Fgf8 double mutants
(Sun et al., 2002). The
autopod of most chimeric limbs was poorly mineralized
(Fig. 2B-D,F), so the
abnormalities shown in Fig. 1 do not show up well in the skeletal preparations. The three components of the
limb develop consecutively; therefore, these phenotypes suggest that
Fgfr2 is active from early to late limb development.
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As AER signals induce proliferation of the limb mesenchyme, we reasoned that variation in the level of AER signaling might be responsible for the lobular structure of the chimeric limb. This could be explained either by local variation in the rate of cell proliferation, or by cell death. To distinguish between the two possibilities, cell proliferation and cell death was assessed by BrdU uptake and TUNEL assay, respectively, in sections cut in the anteroposterior plane (plane of the AER, if present; Fig. 5M,N). Immunochemical localization of BrdU revealed significantly fewer proliferating cells in the PZ under the trough areas than beneath the AER-containing peaks (Table 2). By contrast, there was no difference in the (low) levels of cell death exhibited by the limb buds of wild-type and chimeric mid-gestation embryos (Fig. 5M,N). We conclude that the lobular structure of the chimeric limb bud results from local reduction of mesenchymal proliferation under the AER-free areas, thereby diminishing proximodistal outgrowth.
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Discussion |
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The limb abnormalities observed in our chimeras included patterning defects
in all three proximodistal limb elements, i.e. in the stylopod, zeugopod and
autopod, indicating that Fgfr2b is required throughout the entire
period of limb outgrowth. Changes in the number and dorsoventral alignment of
digits indicated effects on dorsoventral patterning, whereas the position of
ectopic limb rudiments deviating from the site of normal limb outgrowth
indicated defects in the anteroposterior axis. These observations on late
gestation chimeras were consistent with the disrupted AER structure and with
disruption of the known functions of genes whose expression patterns were
altered at mid-gestation (Niswander,
2003). We therefore conclude that Fgfr2b is required for
normal patterning during the development of all three limb axes. This is in
accord with suggestions that the dorsoventral and proximodistal pre-pattern is
established during early limb development
(Dudley et al., 2002
;
Sun et al., 2002
). The
displaced mini-limbs additionally indicate that the mutation affects the
determination of limb position and polarity.
Fgfr2 activity is required for AER differentiation
Altabef et al. demonstrated that cells that form the AER originate from a
population scattered within a broad area of early ectoderm
(Altabef et al., 1997;
Altabef et al., 2000
). These
converge on the limb bud as it grows out from the flank and form the AER at
the dorsoventral compartment boundary of the trunk. It was therefore
interesting and significant that our high-grade mid-gestation chimeras showed
a lower level of mutant cells in the limb field than elsewhere in the trunk,
even than where limbs failed to develop. Moreover in the abnormal limb buds of
medium-grade chimeras, the proportion of mutant cells was lower in the limb
than in the trunk ectoderm, and there were almost no mutant cells in the AER.
This suggests that the movement of mutant ectodermal cells towards and into
the incipient limb bud and AER is inefficient compared with the wild type in
the absence of Fgfr2.
Although the surface ectoderm of the embryo transcribes Fgfr2
before limb outgrowth (Orr-Urtreger et
al., 1991), its loss in non-chimeric mutant embryos does not
affect the basic structure of the embryonic integument
(De Moerlooze et al., 2000
).
Absence of mutant cells from the AER fragments is consistent with the
interpretation that Fgfr2b is required for differentiation of the
simple squamous surface epithelium into the pseudostratified structure of the
AER. Double detection of mutant cells and AER-specific genes revealed that
patches of Fgf8 expression coincided with, and were restricted to,
patches of morphologically distinguishable AER. We therefore suggest that the
requirement for Fgfr2 in AER differentiation is cell-autonomous. AER
morphogenesis is correlated with the onset of AER-specific gene expression; Xu
et al. (Xu et al., 1998
)
observed that the loss of Fgfr2 results in failure of the mutant
ectoderm to express Fgf8 in response to Fgf10 signals from
the mesenchyme, and hence the failure of limb-bud formation.
Fgfr2 affects multiple aspects of the limb pattern
Many defects of our chimeras affected the dorsoventral limb pattern, which
depends on the dorsoventral compartment boundary. This boundary is established
in the prospective limb mesenchyme; it originates in the somatic or lateral
mesoderm, from where it is transferred to the prospective limb ectoderm before
limb outgrowth (Michaud et al.,
1997; Altabef et al.,
1997
) and its timing is in good agreement with the timing of
Fgfr2b expression (Orr-Urtreger
et al., 1993
). Thus, transcription of Fgfr2b in the
surface ectoderm is in synchrony with the formation of the dorsoventral
compartment boundary. Nevertheless, because the receptor is expressed in both
the dorsal and ventral ectoderm, Fgfr2 function may be necessary to
enable, but not to specify, boundary formation. Wnt7a is an early
effector of the distal dorsoventral limb pattern
(Altabef and Tickle, 2002
;
Parr and McMahon, 1995
). It
induces Lmx1b in the dorsal limb mesenchyme, whereas En1 in
the ventral ectoderm restricts Wnt7a expression to the dorsal side
(for a review, see Chen and Johnson,
1999
). Wnt7a expression in En1 mutants
(Cygan et al., 1997
), and
forced En1 expression in the entire limb bud
(Kimmel et al., 2000
), induced
AER displacements similar to those described here.
Defective AER differentiation in our chimeras was correlated with reduced
transcription of Wnt7a and En1 in the dorsal and ventral
ectoderm, respectively. Failure of expression of Wnt7a and
En1 in non-chimeric Fgfr2 mutant embryos, at and before
limb-bud outgrowth, suggests that FGFR2 signaling is upstream of the
expression of these two genes. This result is consistent with a previous
report on the role of FGF signaling in the control of Wnt7a
expression (Altabef and Tickle,
2002). Ectopic expression of En1 in the dorsal limb-bud
ectoderm, and its restriction to the wild-type component of the chimera,
indicates that Fgfr2b is required either to restrict En1
gene expression to the ventral surface of the limb bud, or to prevent cells
initiating on the ventral surface from migrating to the dorsal surface. We
assume that the observed dorsal and palmar outgrowth of digits, and the
reversal of dorsoventral polarity of some digits, were also due to these
effects.
In spite of abnormal dorsoventral gene expression, the epidermis (hair, nails, foot pads) of late gestation limbs did not exhibit dorsoventral patterning defects. This indicates that partial loss of Fgfr2 function did not affect later epidermal differentiation events. The abnormal position of the digits may have been due to the presence of mutant cells in the chimeric limb ectoderm. We have shown that mutant and wild-type cells form patches in the limb ectoderm. It stands to reason that patches of mutant ectoderm, which cannot form AER, when lodged at the ridge of the limb bud, hinder the accurate homing of wild-type cells to the dorsoventral boundary, and force them to differentiate at positions slightly dorsal or slightly ventral from it.
Defective En1 and Wnt7a activation, and the hindrance of AER positioning by the mutant component of the chimera, may explain defects in the distal limb bud and in the autopod of later limbs. However, they do not explain the frequent loss of the radius, the bifurcation of the humerus and the de novo generation of ectopic mini-limbs. The first two may be consistent with abnormal mesenchymal proliferation and patterning due to reduced signaling from the limb ectoderm and AER. The mechanism of generation of ectopic limb rudiments is more difficult to understand, but presumably involves displacement of normal AER-forming cells away from the normal limb-forming site.
The chimeric defects observed here affect a succession of
epithelial-mesenchymal interactions, which take place during limb development,
from its earliest phase until the formation of digits and phalanges. Their
failure in
Fgfr22/
2
Fgfr2+/+
chimeras is indicated by the gene expression abnormalities observed at
mid-gestation, and emphasizes the role of Fgfr2b in a whole series of
these interactions. The most significant contributions made by this study are
the findings: (1) that Fgfr2 is involved in ectodermal cell movement
towards and into the limb bud; (2) that it is required for the formation and
function of the AER; and (3) that Fgfr2 is upstream of En1
and Wnt7a, and is involved in ventral restriction of the En1
domain.
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ACKNOWLEDGMENTS |
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Footnotes |
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REFERENCES |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Altabef, M. and Tickle, C. (2002). Initiation of dorso-ventral axis during chick limb development. Mech. Dev. 116,19 -27.[CrossRef][Medline]
Altabef, M., Clarke, J. D. and Tickle, C.
(1997). Dorso-ventral ectodermal compartments and origin of
apical ectodermal ridge in developing chick limb.
Development 124,4547
-4556.
Altabef, M., Logan, C., Tickle, C. and Lumsden, A. (2000). Engrailed-1 misexpression in chick embryos prevents apical ridge formation but preserves segregation of dorsal and ventral ectodermal compartments. Dev. Biol. 222,307 -316.[CrossRef][Medline]
Arman, E., Haffner-Krausz, R., Gorivodsky, M. and Lonai, P.
(1999). Fgfr2 is required for limb outgrowth and lung-branching
morphogenesis. Proc. Natl. Acad. Sci. USA
96,11895
-11899.
Chen, H. and Johnson, R. L. (1999). Dorsoventral patterning of the vertebrate limb: a process governed by multiple events. Cell Tissue Res. 296, 67-73.[CrossRef][Medline]
Conlon, R. A. and Herrmann, B. G. (1992). Detection of messenger RNA by in situ hybridization to post implantation embryo whole mounts. Methods Enzymol. 225,361 -372.
Cygan, J. A., Johnson, R. L. and McMahon, A. P.
(1997). Novel regulatory interactions revealed by studies of
murine limb pattern in Wnt-7a and En-1 mutants.
Development 124,5021
-5032.
De Moerlooze, L., Spencer-Dene, B., Revest, J., Hajihosseini,
M., Rosewell, I. and Dickson, C. (2000). An important
role for the IIIb isoform of fibroblast growth factor receptor 2 (FGFR2) in
mesenchymal-epithelial signalling during mouse organogenesis.
Development 127,483
-492.
Drossopoulou, G., Lewis, K. E., Sanz-Ezquerro, J. J., Nikbakht,
N., McMahon, A. P., Hofmann, C. and Tickle, C. (2000).
A model for anteroposterior patterning of the vertebrate limb based on
sequential long- and short-range Shh signalling and Bmp
signalling. Development
127,1337
-1348.
Dudley, A. T., Ros, M. A. and Tabin, C. J. (2002). A re-examination of proximodistal patterning during vertebrate limb development. Nature 418,539 -544.[CrossRef][Medline]
Eswarakumar, V. P., Monsonego-Ornan, E., Pines, M., Antonopoulou, I., Morriss-Kay, G. M. and Lonai, P. (2002). The IIIc alternative of Fgfr2 is a positive regulator of bone formation. Development 129,3783 -3793.[Medline]
Haffner-Krausz, R., Gorivodsky, M., Chen, Y. and Lonai, P. (1999). Expression of FGFR2 during oogenesis, preimplantation and early postimplantation embryogenesis. Mech. Dev. 85,167 -172.[CrossRef][Medline]
Johnson, D. E. and Williams, L. T. (1993). Structural and functional diversity in the FGF receptor multigene family. Adv. Cancer Res. 60,1 -41.[Medline]
Kaufman, M. H. (1992). The Atlas of Mouse Development. London: Academic Press.
Kimmel, R. A., Turnbull, D. H., Blanquet, V., Wurst, W., Loomis,
C. A. and Joyner, A. L. (2000). Two lineage boundaries
coordinate vertebrate apical ectodermal ridge formation. Genes
Dev. 14,1377
-1389.
Loomis, C. A., Harris, E., Michaud, J., Wurst, W., Hanks, M. and Joyner, A. L. (1996). The mouse Engrailed-1 gene and ventral limb patterning. Nature 382,360 -363.[CrossRef][Medline]
Martin, G. R. (1998). The roles of FGFs in the
early development of vertebrate limbs. Genes Dev.
12,1571
-1586.
Michaud, J. L., Lapointe, F. and le Douarin, N. M.
(1997). The dorsoventral polarity of the presumptive limb is
determined by signals produced by the somites and by the lateral somatopleure.
Development 124,1453
-1463.
Min, H., Danilenko, D. M., Scully, S. A., Bolon, B., Ring, B.
D., Tarpley, J. E., DeRose, M. and Simonet, W. S.
(1998). Fgf-10 is required for both limb and lung
development and exhibits striking functional similarity to Drosophila
branchless. Genes Dev.
12,3156
-3161.
Mortensen, R. M., Conner, D. A., Chao, S., Geisterfer-Lowrance, A. A. and Seidman, J. G. (1992). Production of homozygous mutant ES cells with a single targeting construct. Mol. Cell Biol. 12,2391 -2395.[Abstract]
Nagy, A., Rossant, J., Abramow-Newerly, W. and Roder, J. C.
(1993). Derivation of completely cell culture-derived mice from
early passage embryonic stem cells. Proc. Natl. Acad. Sci.
USA 90,8424
-8428.
Niswander, L. (2003). Pattern formation: old models out on a limb. Nat. Rev. Genet. 4, 133-143.[CrossRef][Medline]
Niswander, L., Tickle, C., Vogel, A., Booth, I. and Martin, G. R. (1993). FGF-4 replaces the apical ectodermal ridge and directs outgrowth and patterning of the limb. Cell 75,579 -587.[Medline]
Niswander, L., Jeffrey, S., Martin, G. R. and Tickle, C. (1994). A positive feedback loop coordinates growth and patterning in the vertebrate limb. Nature 371,609 -612.[CrossRef][Medline]
Ohuchi, H., Nakagawa, T., Yamamoto, A., Araga, A., Ohata, T.,
Ishimaru, Y., Yoshioka, H., Kuwana, T., Nohno, T., Yamasaki, M. et
al. (1997). The mesenchymal factor, FGF10, initiates and
maintains the outgrowth of the chick limb bud through interaction with FGF8,
an apical ectodermal factor. Development
124,2235
-2244.
Orr-Urtreger, A., Givol, D., Yayon, A., Yarden, Y. and Lonai, P. (1991). Developmental expression of two murine fibroblast growth factor receptors, flg and bek. Development 113,1419 -1434.[Abstract]
Orr-Urtreger, A., Bedford, M. T., Burakova, T., Arman, E., Zimmer, Y., Yayon, A., Givol, D. and Lonai, P. (1993). Developmental localization of the splicing alternatives of fibroblast growth factor receptor-2 (FGFR2). Dev. Biol. 158,475 -486.[CrossRef][Medline]
Parr, B. A. and McMahon, A. P. (1995). Dorsalizing signal Wnt-7a required for normal polarity of D-V and A-P axes of mouse limb. Nature 374,350 -353.[CrossRef][Medline]
Riddle, R. D., Johnson, R. L., Laufer, E. and Tabin, C. (1993). Sonic hedgehog mediates the polarizing activity of the ZPA. Cell 75,1401 -1416.[Medline]
Saxton, T. M., Ciruna, B. G., Holmyard, D., Kulkarni, S., Harpal, K., Rossant, J. and Pawson, T. (2000). The SH2 tyrosine phosphatase shp2 is required for mammalian limb development. Nat. Genet. 4,420 -423.[CrossRef]
Sekine, T., Ohuchi, H., Fujiwara, M., Yamasaki, M., Yoshizawa, T., Sato, T., Yagishita, N., Matsui, D., Koga, Y., Itoh, N. et al. (1999). Fgf10 is essential for limb and lung formation. Nat. Genet. 21,138 -141.[CrossRef][Medline]
Sun, X., Mariani, F. V. and Martin, G. R. (2002). Functions of FGF signalling from the apical ectodermal ridge in limb development. Nature 418,501 -508.[CrossRef][Medline]
Xu, X., Weinstein, M., Li, C., Naski, M., Cohen, R. I., Ornitz,
D. M., Leder, P. and Deng, C. (1998). Fibroblast
growth factor receptor 2 (FGFR2)-mediated reciprocal regulation loop between
FGF8 and FGF10 is essential for limb induction.
Development 125,753
-765.