Fibroblast Growth Factor Receptor 2-Iiib Acts Upstream of Shh And

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Developmental Biology 231, 47–62 (2001) doi:10.1006/dbio.2000.0144, available online at http://www.idealibrary.com on Fibroblast Growth Factor Receptor 2-IIIb Acts Upstream of Shh and Fgf4 and Is Required for Limb Bud Maintenance but Not for the Induction of Fgf8, Fgf10, Msx1, or Bmp4

Jean-Michel Revest, Bradley Spencer-Dene, Karen Kerr, Laurence De Moerlooze,1 Ian Rosewell, and Clive Dickson2 Imperial Cancer Research Fund, Lincoln’s Inn Fields, London WC2A 3PX, United Kingdom

Mice deficient for FgfR2-IIIb were generated by placing translational stop codons and an IRES-LacZ cassette into exon IIIb of FgfR2. Expression of the alternatively spliced receptor isoform, FgfR2-IIIc, was not affected in mice deficient for the IIIb ,isoform. FgfR2-IIIb؊/؊ lac Z mice survive to term but show dysgenesis of the kidneys, salivary glands, adrenal glands, thymus pancreas, skin, otic vesicles, glandular stomach, and hair follicles, and agenesis of the lungs, anterior pituitary, thyroid, teeth, and limbs. Detailed analysis of limb development revealed an essential role for FgfR2-IIIb in maintaining the AER. Its absence did not prevent expression of Fgf8, Fgf10, Bmp4, and Msx1, but did prevent induction of Shh and Fgf4, indicating that they are downstream targets of FgfR2-IIIb activation. In the absence of FgfR2-IIIb, extensive apoptosis of the limb bud ectoderm and mesenchyme occurs between E10 and E10.5, providing evidence that Fgfs act primarily as survival factors. We propose that FgfR2-IIIb is not required for limb bud initiation, but is essential for its maintenance and growth. © 2001 Academic Press Key Words: fibroblast growth factor receptor-2; IIIb isoform; Fgfs; limb development; mesenchymal-epithelial interactions.

INTRODUCTION have been conserved and adapted for multiple purposes during the evolution of complex animals. Intercellular signaling between epithelium and mesen- The Fgf family in mammals is composed of at least 20 chyme is a fundamental process for the induction and members that signal through transmembrane receptors patterning of animal organs. Several families of protein encoded by four genes (FgfR1 to FgfR4), although receptor molecules, originally identiﬁed as growth factors in vitro diversity is increased by alternative splicing which gener- (e.g., Fgfs, Egfs, Tgf-␤/Bmps), act as signaling molecules ates two distinct receptor isoforms for FgfR1, FgfR2, and during embryogenesis. In situ hybridization analyses with FgfR3 (reviewed in McKeehan et al., 1998; Ornitz, 2000; probes for the growth factors and their receptors have Yamaguchi and Rossant, 1995). The FgfRs are composed of shown temporal and spatial expression patterns consistent two or three Ig-like loops in the external domain, a trans- with paracrine signaling mechanisms (for reviews see membrane segment and a ligand-activated cytoplasmic ty- Cunha and Hom, 1996; Hogan and Yingling, 1998; Thesleff rosine kinase domain. The two membrane-proximal Ig and Sharpe, 1997). Interestingly, similar combinations of loops (Ig-II and Ig-III) comprise the ligand-binding domain, these molecules are expressed during the development of although alternative splicing of Ig-loop III can generate two diverse organs, suggesting that certain signaling networks isoforms (IIIb and IIIc) with distinct ligand binding properties and tissue distributions (Ornitz et al., 1996; Orr-

1 Urtreger et al., 1993; Peters et al., 1992). In addition, several Present address: SmithKline Beecham Biologicals, Rue de l’Institut 89, B-1330 Rixensart, Belgium. other splice variants of FgfR1 and FgfR2 have been de- 2 To whom correspondence should be addressed. Fax: 44-20- scribed including secreted forms and transmembrane forms 7269-3094. E-mail: [email protected]. lacking the ﬁrst Ig loop. It is not clear how these changes in

FIG. 1. Targeted disruption of the IIIb isoform of FgfR2. (A) Schematic depiction of the targeting construct used to disrupt FgfR2-IIIb. Translation termination codons in all three reading frames, and an IRES-LacZ gene were used to specifically disrupt exon IIIb. A neomycin resistance gene was inserted into the intron between exons IIIa and IIIb and flanked by LoxP sites to allow its excision by Cre recombinase after germ-line transmission. Primers P1, P2, and P3 were used to genotype mice by PCR. (B) Southern blot analysis indicates mice wild-type, or heterozygous, and homozygous for the mutant allele. The band migrating at 5.3 kb identifies the targeted recombinant allele. (C) Phenotype of mice homozygous and heterozygous for the disupted FgfR2-IIIb allele. The homozygous mutant mice shows an absence of limbs, eyelids, a tail dorsally curved, and abnormal skin. Skeletal preparation of heterozygous (D) and null embryos (E) at E16.5. White arrowheads indicate the region of fusion of caudal vertebrae (D and E). FgfR2-IIIbϪ/Ϫlac Z mice also show a delay in bone ossification (asterisk in D and E) and deformed scapulae (boxed area), respectively.

receptor structure affect their properties, but mice unable to Tabin, 1997; Martin, 1998; Ornitz, 2000; Yamaguchi and make the three Ig-loop form of FgfR1 die during develop- Rossant, 1995). Hence it is not surprising that mice null for ment (Xu et al., 1999). FgfR1 and FgfR2 die early in embryogenesis, since they each Targeted deletions of Fgfs have shown their importance act as receptors for several different Fgfs (Arman et al., during embryogenesis. Mice null for Fgf4, Fgf8, and Fgf10 1998; Deng et al., 1994; Xu et al., 1998; Yamaguchi et al., are not viable, while abrogation of other Fgfs (e.g., Fgf2, 1994). The importance of FgfR2 in the development of Fgf3, Fgf5, and Fgf7) yields viable mice with developmental several organs and tissues has been established with mice abnormalities of varying severity (reviewed in Johnson and expressing dominant negative FgfR2-IIIb receptors, a hypo-

FIG. 2. Expression of FgfR2-IIIc in mice deficient for FgfR2-IIIb. Radioactive in situ hybridization on E13.5 embryos was used to assess the expression pattern of FgfR2-IIIc in heterozygous and homozygous mutant mice. A probe against the FgfR2 tyrosine kinase domain (TK) and two isoform specific probes LacZ (for FgfR2-IIIb) and IIIc (for FgfR2-IIIc) were compared. (A) Choroid plexus of the fourth ventricle from heterozygous and homozygous mice shows a strong positive signal for TK and IIIc. This structure is negative for FgfR2-IIIb expression as shown using a probe directed against the LacZ gene. Additionally, the IIIc specific probe was used to assessed the expression of FgfR2-IIIc in different organs of heterozygous (B, C, F, G, J, K N, O, R, S) and homozygous (D, E, H, I, L, M, P, Q) -mutant mice. Disruption of the IIIb isoform did not detectably affect FgfR2-IIIc expression in the aortic valve (B–E), cartilage primordium of hyoid bone (F–I), primordium of incisor tooth (J–M), and vertebrae (N–Q). Lung epithelium which is a site of FgfR2-IIIb expression (LacZ probe) (T, U), remained negative for FgfR2-IIIc expression (IIIc probe) in heterozygous fetuses (R, S). Bars, 0.05 mm.

Copyright © 2001 by Academic Press. All rights of reproduction in any form reserved. 50 Revest et al. morphic allele of FgfR2, as well as FgfR2Ϫ/Ϫ mice partially in different reading frames were introduced upstream of the IRES. rescued from a placental defect by tetraploid ES cell fusion A neomycin-resistance gene, driven by the HSV-tk promoter and (Arman et al., 1999; Celli et al., 1998; Peters et al., 1994; Xu flanked by LoxP sites from plasmid pL2Neo, [a generous gift of H. et al., 1998). Gu (Gu et al., 1993)], was inserted into the intron between exons Vertebrate limb development is known to involve two IIIa and IIIb at the SpeI site. The complete construct of approxi- key signaling centers; the apical ectodermal ridge (AER) mately 10.4 kb in length, starting at an EcoRI site in the intron that regulates proximodistal growth, and a region of the upstream of exon IIIa and terminating at a SalI site in the intron posterior limb bud mesenchyme known as the zone of after exon IIIc was inserted into the exon trap vector pSPL3 (Church et al., 1994). A unique SacII site was introduced in the 5Ј part of the polarising activity (ZPA), which contributes to anterior– construct to linearize the plasmid before electroporation into posterior patterning (reviewed in Martin, 1998; Naski and 129P2/OlaHsd ES cells (Fig. 1A). Neomycin-resistant clones were Ornitz, 1998). Several members of the Fgf family including screened by Southern blotting, and candidate homologous recom- Fgf2, Fgf4, Fgf8, Fgf9, and Fgf17 are expressed in the AER, binant clones were rescreened by PCR with primers that would while Fgf10 is expressed in the mesenchyme. Mice deficient amplify the 5Ј and 3Ј junction fragments of correctly targeted DNA. for Fgf10 or its receptor FgfR2-IIIb are born without limbs, Three different lines of mice were used (2G1, 3G9, and 2G1 Cre). suggesting the importance of mesenchyme–ectodermal sig- The 2G1 Cre line was obtained by deletion of the neomycin- naling in the limb (DeMoerlooze et al., 2000; Min et al., resistance gene from the 2G1 line by injecting eggs derived from 1998; Sekine et al., 1999). These and many previous studies 2G1 heterozygous male crossed with wild-type Bl6 female with 10 have led to a model where expression of Fgf10 in the ng/ml of pMC-Cre [kindly provided by H. Gu (Gu et al., 1993)]. The mesenchyme is thought to induce Fgfs in the overlying resulting mice were then screened by PCR for the lack of the ectoderm, concomitant with the appearance of the apical neomycin resistance gene. No differences in phenotype between ectodermal ridge (reviewed in Martin, 1998; Naski and the various lines were observed. Ornitz, 1998). Continued Fgf10 signaling maintains the Genotyping. Genomic DNA was isolated from tail clips and AER, and in turn Fgfs in the AER stimulate the growth of genotype determined using different sets of primers P1/P2 and P3 the underlying mesenchyme, providing the basis of a posi- to discriminate between wild-type, heterozygous, and homozygous tive feedback loop for limb growth. In addition, a second null mice as shown in Fig. 1A. The PCR protocol using Expand o o loop is formed between Fgf4, and possibly other Fgfs, in the PCR System (Boehringer) was 94 C 2 min, then 10 cycles of 94 C 30 s, 61oC30s,68oC 5 min, followed by 17 cycles of 94oC30s, AER to induce and/or maintain Sonic hedgehog (Shh) ex- o o pression in the ZPA, which participates in patterning the 61 C30s,68C 5 min with an increasing time of 20 s for the elongation step at each cycle, then 68oC 7 min. anterior–posterior limb axis. A null mutation in FgfR2-IIIb In situ hybridization. Whole-mount in situ hybridization was provides a means of interrupting the mesenchymal-to- performed as described previously (Henrique et al., 1995). Briefly, ectoderm signal so that the dependence and timing of the following overnight hybridization at 70oC with the probes of different signaling events in early limb bud establishment interest, embryos were washed at high stringency in 50% form- can be further investigated. amide, 2ϫ SSC. Embryos were then washed in TBST (1.37 M NaCl, To investigate the specific role of the IIIb isoform of 0.027 M KCl, 0.25 M Tris–HCl pH 7.5, 1% Tween-20) and MABT FgfR2 in mouse development, we have generated mice (0.1 M maleic acid, 0.15 M NaCl, 1% Tween-20) and nonspecific deficient for this isoform using two strategies. The first binding of the antibody was blocked by incubation in 10% sheep approach employed a LoxP/Cre recombination to remove serum (Sigma) in MABT for 2 h at room temperature. Incubation in the specific IIIb exon (DeMoerlooze et al., 2000), while the anti-digoxygenin alkaline phosphatase conjugate antiserum (Boehr- second described here was created by the introduction of inger) was carried out overnight at 4°C in MABT. Post-antibody translational terminators and an IRES-LacZ gene into IIIb washing was carried out extensively in MABT. Embryos were then exon of FgfR2. Both types of FgfR2-IIIb-deficient mice give equilibrated in NTMT (0.1 M NaCl, 0.1 M Tris–HCl pH 9.5, 0.05 M a very similar phenotype, although the LacZ insertion is MgCl2, 0.1% Tween-20) and the color reaction was developed at slightly more severe for some of the visceral organ abnor- room temperature in NTMT containing 0.25 mg/ml NBT and 0.175 malities. We have used the FgfR2-IIIbϪ/ϪlacZ mice to inves- mg/ml BCIP (Roche, UK). When the desired intensity of staining tigate the function of FgfR2-IIIb and its relationship to other had been obtained, embryos were washed in PBT (phosphate buffer signaling molecules during limb bud initiation and mainte- saline 0.1% Tween-20). Whole-mount in situ sections were counterstained with 10% Nuclear fast red for 30 s and dehydrated nance. through alcohol washes before mounting. Radioactive in situ hybridization was performed on dewaxed tissue sections essentially as described previously (Kettunen et al., MATERIALS AND METHODS 1998). Probes to FgfR2-IIIc, Msx1, and Bmp4 were kindly provided by P. Kettunen (Kettunen et al., 1998). The LacZ probe was a Gene targeting. Recombinant lambda bacteriophage encom- PCR-generated fragment including nucleotides 2606–3349 cloned passing the alternatively spliced region of FgfR2 was isolated from in pGEM3Z and the FgfR2-TK probe was previously described a 129SvJ-mouse genomic library (a kind gift of A.-M. Frischauf). To (DeMoerlooze et al., 2000). The Fgf4 and Fgf8 probes were previ- construct the targeting vector, the IRES-LacZ gene was amplified ously described (Mahmood et al., 1995). The mouse Fgf10 and Sonic by PCR from the plasmid pGT1.8 IRES-␤GEO (a kind gift of P. S. hedgehog probes were kindly provided by S. Werner (Zurich) and A. Mountford) and introduced into exon IIIb. In addition, stop codons McMahon (Boston), respectively.

Histology and immunohistochemistry. Tissues were fixed TABLE 1 overnight in neutral buffered formalin, dehydrated through etha- Phenotypic Abnormalities in Mice Deficient for FgfR2-IIIb nol, embedded in paraffin wax, and 5-␮m sections were cut for staining in haematoxylin and eosin (H&E). Differential staining of Organ Dysgenesis Agenesis cartilage and bone was performed as described (McLeod, 1980). ߛ Apoptotic cells were detected by the incorporation of terminal Thyroid ߛ deoxynucleotidyltransferase-mediated dUTP end labeling Anterior pituitary ߛ (TUNEL) using the ApopTagRPlus In Situ Apoptosis Detection Kit Thymus ߛ (Oncor, USA) as recommended by the manufacturer. Pancreas Skin ߛ Hair follicles ߛ Lungs ߛ Tooth bud ߛ RESULTS Kidneys ߛ Salivary glands ߛ ߛ Targeted Disruption of the IIIb Isoform of FgfR2 Adrenal glands Eyelids ߛ ߛ An isoform-specific disruption of FgfR2 was created by Otic vesicle Glandular stomach ߛ introducing translation stop codons in all three reading Limbs ߛ frames and placing an IRES-LacZ gene cassette into exon IIIb which encodes the distal portion of Ig-loop III (Fig. 1A). Note. Dysgenesis is defined as malformation or reduction in In addition, a neomycin-resistance gene driven by HSV-tk number or size and agenesis as absence of organ. Embryos were and flanked by LoxP sites was introduced into the adjacent analyzed at E13.5, E16.5, and E18.5 and shown to exhibit the same intron for purposes of selecting recombinant ES cells. The abnormalities; n ϭ at least 3 for each stage studied. stop codons upstream of the IRES-LacZ cassette ensure that the receptor protein terminates early in exon IIIb and will therefore lack the kinase and transmembrane region, as well as part of the ligand-binding domain. The targeting bud development and was the subject of a more detailed construct was used to generate three different recombinant investigation. ES cell clones that were subsequently employed to generate chimeric mice. Offspring from the chimeras were used for interbreeding to obtain heterozygous and homozygous em- Effect of IIIb Inactivation on Receptor Splicing bryos (Figs. 1B and 1C). Homozygous embryos were ob- An important issue in the generation of isoform-specific tained at the expected ratio up to birth, but these mice null mutations is that the nontargeted isoform should could not survive outside the uterus due to agenesis of the function normally, and in tissues where the targeted iso- lungs (see below). At term, homozygous mutant fetuses form has been inactivated there is not a splicing switch to were smaller than their litter mates, lacked limbs, had a the nontargeted isoform, resulting in a potential gain-of- dorsally curved tail, no eyelids, and a thinner skin (Fig. 1C). function mutation. To evaluate receptor splicing in vivo, Although the targeting construct when expressed from an we used radioactive in situ hybridization on sections of exon trap vector in cell culture conferred ␤-galactosidase E13.5 heterozygous and mutant embryos with probes di- activity, such activity was not detected in sections from rected against the tyrosine kinase domain (TK), and the heterozygous or homozygous mutant mice. Consequently, IIIc exon of FgfR2, as well as LacZ to assess FgfR2-IIIb we have used in situ hybridization against the LacZ se- RNA expression. The TK probe detects both the IIIb and quences as a sensitive measure of FgfR2-IIIb expression (see IIIc isoforms of FgfR2, while the IIIc and LacZ are below). isoform-specific. We have examined a number of FgfR2- The several skeletal abnormalities found for FgfR2- IIIc expression sites to check they were not significantly Ϫ Ϫ IIIb / lacZ are similar to those previously described for the affected by the loss of the IIIb due to insertion of an Ϫ Ϫ FgfR2-IIIb / Cre mouse (DeMoerlooze et al., 2000). Briefly, IRES-LacZ cassette. Expression of FgfR2-IIIc in the cho- the heterozygotes are indistinguishable from wild-type lit- roid plexus was examined with the TK, IIIc, and LacZ ter mates, while the mutant homozygotes have no limbs, a probes and shows that FgfR2-IIIc expression is not de- rudimentary pelvic girdle, deformed scapulae, fusion of the tectably affected by inactivation of FgfR2-IIIb (Fig. 2A). caudal vertebrae causing the tail to curve dorsally (Figs. 1C Moreover, all other FgfR2-IIIc expression sites investi- and 1D), smaller otic capsules (Figs. 3C and 3D), cleft gated showed similar results (Figs. 2B–2Q). In a contrary palate, and a delay of membranous ossification (Figs. 1D situation, such as the lung epithelium, inactivation of and 1E, and data not shown). The term mice also demon- one FgfR2-IIIb allele does not result in activation of the strate a reduction in skull bone mass as seen by a compari- IIIc isoform (Figs. 2R and 2S). These results show that son of mandible size and shape (DeMoerlooze et al., 2000). homozygous inactivation of FgfR2-IIIb does not detect- The absence of limbs appears to result from a failure at limb ably affect the expression of FgfR2-IIIc, indicating that

FIG. 3. Disruption of the IIIb isoform of FgfR2 affects the normal development of several organs. Sections through E16.5 wild-type (A, C, E, G, I, K, M, O, Q, S) and null embryos (B, D, F, H, J, L, N, P, R, T) were sectioned and stained with H&E. We observed abnormalities of the anterior pituitary gland (A, B), otic vesicle (C, D), salivary glands (E, F), teeth (G, H), thyroid (I, J), thymus (K, L), pancreas (M, N), kidney (O, P), adrenal glands (Q, R), skin (S, T). White arrowhead indicates a hair follicle in (S). ad: adrenal gland; ot: otic vesicle/capsule; p: parathyroid; thy: thyroid; i: infundibular recess; r: Rathke’s pouch; tm: thymus; s: salivary glands; k: kidney; pa: pancreas; mol: molar teeth. Bar, 0.1 mm except forS&Tbar, 0.5 mm. splicing of FgfR2-IIIc occurs normally in FgfR2-IIIbϪ/ϪlacZ agenesis of the lungs, anterior pituitary gland (Figs. 3A mice. and 3B and data not shown), limbs, and eyelids (Figs. 1C–1E), as well as dysgenesis of the otic vesicle and capsule (Figs. 3C and 3D, and data not shown), salivary Visceral Organ Agenesis and Dysgenesis glands (Figs. 3E and 3F), and glandular stomach (see Table Consistent with previous observations on FgfR2- 1 and DeMoerlooze et al., 2000). Interestingly, several of IIIbϪ/ϪCre mice, FgfR2-IIIbϪ/ϪlacZ mice show essentially the phenotype abnormalities reported for FgfR2-IIIbϪ/ϪCre

FIG. 4. Detection of the AER in FgfR2-IIIbϪ/Ϫlac Z mice. Sections through the limb bud region of heterozygous (A, C, E) and homozygous (B, D, F) mutant mice were prepared from embryos at E9.5, E10, and E10.5 and stained with H&E. At E9.5 there was no detectable AER in either heterozygous and homozygous mutant embryos, and the presumptive limb bud region was a single layer of ectodermal cells (A, B). By E10 and E10.5 a discernible AER was present in the heterozygous embryos (C, E), and a thickening of the ectoderm layer morphologically similar to an AER was present in the FgfR2-IIIbϪ/Ϫlac Z mice (D, F). The poorly formed AER of homozygous mutant embryos contained many cells with features of apoptotic death, suggesting its rapid loss through degeneration. Bar, 0.1 mm.

mice are more severe in the FgfR2-IIIbϪ/ϪlacZ mice. For also show a more severe, or previously unseen, dysgen- example, dysgenesis of the teeth appears as complete esis of the thymus (Figs. 3K and 3L), pancreas (Figs. 3M agenesis (Figs. 3G and 3H), and there is also agenesis of and 3N), kidneys (Figs. 3O and 3P), adrenal glands (Figs. the thyroid (Figs. 3I and 3J). The FgfR2-IIIbϪ/ϪlacZ mice 3Q and 3R), and skin (Figs. 3S and 3T). However, the

FIG. 5. Whole-mount in situ hybridization on heterozygous (A, C, E, G, H) and FgfR2-IIIbϪ/Ϫlac Z (B, D, F) mice at E9.5, E10.5 and E11.5 using a TK (A, B) and LacZ probe (C–H). We were not able to detect any staining in the presumptive limb bud in heterozygous and FgfR2-IIIbϪ/Ϫlac Z mice at E9.5 (A and B). Asterisks indicate the position the presumptive forelimbs. White and black arrows indicate the AER in heterozygous (C, E) and the presumptive AER in FgfR2-IIIbϪ/Ϫlac Z mice (D, F). Sections of heterozygous limb buds at E10.5 probed with LacZ, then sectioned and counterstained with Nuclear fast red (G, H). The staining reveals the IIIb isoform of FgfR2 in the apical ectodermal ridge and as a band of ectodermal cells delineating the limbs from the body trunk (G, H). G, bar, 0.5 mm and C, bar, 0.1 mm.

genetic background of these two homozygous mutant strains are slightly different, with FgfR2-IIIbϪ/ϪlacZ mice maintained by backcrossing heterozygotes with C57BL mice, as opposed to intercrossing the 129 ϫ C57 (ICRF) heterozygote/wild-type littermates. The relative reduction in organ size was determined by serially sectioning entire organs and comparing the length of the major and minor axes. Care was taken to ensure that the tissues were cut in the same plane, which could in some cases be conﬁrmed by the arrangement of the internal tissue architecture. The size reduction of the kidney from null mice was accompanied by a reduction in the number of presumptive nephrons.

Limb Bud Development .Presence of a discernible AER in FgfR2-IIIb؊/؊lacZ mice To determine whether the homozygous mutant mice initi- ated a recognizable AER, stained transverse sections of embryos were examined from E9.5 to E10.5. At E9.5 there was no morphologically detectable AER, and no difference was seen between the presumptive limb buds on the trunk of heterozygous or homozygous mutant embryos (Figs. 4A and 4B). By E10 to E10.5 the morphological appearance of an AER was present in the heterozygous embryos, but was only discernible as a thickening of the ectoderm on FgfR2- IIIbϪ/ϪlacZ embryos (Figs. 4C–4F). Moreover, the presence of cells with contracted nuclei was suggestive of considerable death by apoptosis. FgfR2-IIIb expression in the AER. Recent evidence suggests that Fgf10 expressed in the lateral plate mesoderm of the limb ﬁeld signals to FgfR2-IIIb in the overlying ectoderm to induce the formation of the apical ectodermal ridge and to induce AER speciﬁc genes (see Martin, 1998; Naski and Ornitz, 1998; Ohuchi et al., 1997; Xu et al., 1998). To directly test this model, wild-type, heterozygous, and homozygous embryos were taken from E9.5 to E11.5 and hybridized with several marker probes that are expressed in the AER or the underlying mesenchyme of the progress zone of the limb bud. At each stage of development, several embryos were examined, and for Fgf4 expression, embryo somites were counted to ensure better monitoring through early limb bud development. In the initial set of experi- ments, we used a probe against LacZ as a sensitive method to detect FgfR2-IIIb RNA expression, although this RNA cannot be translated into a normal receptor.

FIG. 6. Whole-mount in situ hybridization using heterozygous ished in FgfR2-IIIbϪ/Ϫlac Z mice by E10.5 (D) and undetected at E11.5 (A, C, E, G, I, K) and FgfR2-IIIbϪ/Ϫlac Z (B, D, F, H, J, L) embryos at (F). An asterisk indicates the position of the presumptive limb bud, E9.5, E10.5, and E11.5, with probes against Fgf10 or Fgf8. At E9.5, and black arrows indicate the location of stained cells. At E9.5, Fgf8 Fgf10 is expressed by the mesenchymal cells of the presumptive is expressed in the presumptive AER as a thin line of ectodermal limb bud of heterozygous (A), and at a lower level in the limb bud cells in heterozygotes (G) and FgfR2-IIIbϪ/Ϫlac Z mice (H). At E10.5 of FgfR2-IIIbϪ/Ϫlac Z mice (B). At E10.5 and E11.5 heterozygous mice and E11.5, Fgf8 is strongly expressed in the AER of heterozygous show strong expression of Fgf10 in the mesenchyme constituting mice (I and K), but is only weakly detected in the presumptive the limb bud progress zone (C and E), but expression in the forelimb bud of FgfR2-IIIbϪ/Ϫlac Z mice at E10.5 (J) and only detected mesenchyme of the presumptive limb bud was considerably dimin- in the hindlimb bud by E11.5 (L).

At E9.5, FgfR2 RNA expression was not detected in the ectoderm of the presumptive limb bud or in the underlying mesoderm of heterozygous or homozygous mutant embryos (Figs. 5A and 5B). At E10.5 and E11.5, heterozygous mice showed clear expression of FgfR2-IIIb in the AER, while homozygous mutants showed weak expression as an ectodermal patch at the site of prospective limb bud development (Figs. 5C, 5D and 5E, 5F). The use of a pan-FgfR2 probe derived from the receptor tyrosine kinase domain gave essentially the same result as the LacZ probe confirming the ectodermal expression as FgfR2-IIIb (data not shown). By sectioning E10.5 heterozygotes previously hybridized with an antisense LacZ probe, FgfR2-IIIb expression was found to be exclusively in the ectodermal cells of the limb bud (Figs. 5G and 5H). Interestingly, we were unable to detect expression of either FgfR2 isoforms in the mesenchyme using a pan-specific TK probe, although it was strongly positive using an FgfR1 probe (Peters et al., 1992; Orr-Urtreger et al., 1993 and data not shown). Heterozygous mice also showed a ring of ectodermal FgfR2-IIIb expression around the base of the limb bud at E10.5-E11.5 that was stronger in the anterior and posterior positions, although this may reflect the ectodermal fold around the limb bud base (Fig. 5G). Fgf8 and Fgf10 precede expression of FgfR2-IIIb. The major ligand for FgfR2-IIIb is Fgf10 and it was detected in the presumptive limb bud mesoderm at E9.5 in both heterozygous and FgfR2-IIIbϪ/ϪlacZ embryos (Figs. 6A and 6B). In the heterozygote embryos, Fgf10 expression was maintained in the progress zone at all stages examined (Figs. 6C and 6E), but in FgfR2-IIIbϪ/ϪlacZ embryos expression was reduced at E10.5 and undetectable by E11.5 (Figs. 6D and 6F). Strong expression of Fgf8 in the ectoderm coincides with the morphological appearance of the AER. However, it was detected at a low level in the ectoderm of the presumptive forelimb buds at E9.5 in both heterozygous and homozygous mutants, which is prior to the appearance of the AER and expression of FgfR2-IIIb (Figs. 6G and 6H). From E10.5 to E11.5, Fgf8 expression was dramatically increased in the AER of heterozygous embryos (Figs. 6I and 6K), while its expression in the presumptive forelimb bud of FgfR2- IIIbϪ/ϪlacZ embryos remained low at E10.5 (Fig. 6J), and it was not detectable at E11.5. Interestingly, at E11.5, the hindlimb bud showed a discrete line of Fgf8 expression in the prospective hindlimb bud region of FgfR2-IIIbϪ/ϪlacZ embryos (Fig. 6L). Hence, expression of Fgf8 is detected in the ectoderm of the presumptive forelimb bud, and at 1 day later in hindlimb bud, consistent with the later time of hindlimb development. As Fgf8 is detected in the presumptive limb buds of FgfR2-IIIbϪ/ϪlacZ embryos, its induction is FIG. 7. Expression of Msx1 and Bmp4 in limb buds of heterozy- not dependent on FgfR2-IIIb activation. gous and FgfR2-IIIbϪ/Ϫlac Z mice. From E9.5 to E11.5, Msx1 is highly expressed by the mesenchymal cells underlying the ectoderm/AER in heterozygous mice (A, C, E). In FgfR2-IIIbϪ/Ϫlac Z mice, Msx1 expression was similar at E9.5 (B), but was reduced at E10.5 and lost by E11.5 (D and F). A similar temporal pattern of expression was position of the presumptive location of the limb buds. White and seen for Bmp4, although spatially it was found in the ectoderm as black arrows indicate the limb region in heterozygous mice and the well as the adjacent mesenchyme (G–L). Asterisk indicates the presumptive limb region in FgfR2-IIIbϪ/Ϫlac Z mice.

Expression of Bmp4 and Msx1 in the AER Two other genes associated with limb bud initiation are Msx1, which is expressed in the mesenchyme, and Bmp4, which is detected in both the ectoderm and mesenchyme of the limb bud. Msx1 shows an area of expression that is restricted to the progress zone and helps to deﬁne a bound- ary between undifferentiated proliferating cells and the more proximal differentiated cells (Davidson et al., 1991). Interestingly, removal of the AER from mouse limb buds in explant culture does not cause down regulation of Msx1 (Wang and Sassoon, 1995), suggesting that its expression is independent of the AER. Using a probe to Msx1, it was detected at a similar level in the limb bud mesenchyme of heterozygous and FgfR2-IIIbϪ/ϪlacZ mice at E9.5, prior to formation of a discernible AER (Figs. 7A and 7B). However, while it continued to be expressed at high levels in the mesenchyme of heterozygous embryos (Figs. 7C and 7E), its expression was diminished at E10.5 and was hardly detectable by E11.5 in the FgfR2-IIIb null mutants (Figs. 7D and 7F). Bone morphogenic proteins (Bmps) are thought to be negative regulators of limb bud growth, since their ectopic application antagonizes AER function, while their inhibi- tion enhances Fgf8 and Fgf4 expression (Ganan et al., 1998; Pizette and Niswander, 1999). Expression of Bmp4 can be seen as a patch at the site of the presumptive forelimb bud in heterozygous and homozygous mutant embryos at E9.5 (Figs. 7G and 7H). Bmp4 expression then normally increases to much higher levels in the AER and the progress zone mesenchyme, with the highest level of expression in the anterior and posterior limb bud mesenchyme (Figs. 7I and 7K). However, the null mutants showed patches of Bmp4 at a considerably reduced level in the presumptive forelimb bud site at E10.5 and expression was only just detectable at E11.5 (Figs. 7J and 7L). As for Msx1, Bmp4 is induced but its level of expression rapidly decreases in limb buds lacking FgfR2-IIIb. Sonic hedgehog and Fgf4 act downstream to FgfR2-IIIb signaling. Sonic hedgehog is expressed in the mesenchyme of the limb bud posterior margin in the ZPA region (Riddle et al., 1993). It is thought that Fgf4 expression in the AER induces Shh, and Shh maintains Fgf4, although Fgf2, Fgf8, Fgf9, and Fgf17 may be able to compensate for Fgf4 (Laufer et al., 1994; Moon et al., 2000; Niswander et al., 1994; Sun et al., 2000; Vogel and Tickle, 1993). Heterozy- gous mice showed a low level of Shh expression at E9.5 at the site of the presumptive forelimb bud, and subsequently higher levels in the ZPA at E10.5 and E11.5 (Figs. 8A, 8C, FIG. 8. Shh and Fgf4 expression in the limb bud of heterozygous and and 8E). Expression of Shh was not detected in FgfR2- Ϫ/ϪlacZ FgfR2-IIIbϪ/Ϫlac Z mice. In heterozygotes, Shh is expressed in a few cells IIIb mice at any of the stages examined (Figs. 8B, 8D, located in the posterior part of the presumptive limb bud at E9.5 (A). and 8F). In an analogous manner, Fgf4 expression was not By E10.5 strong expression is detected in the posterior region of the forelimb bud mesenchyme (ZPA) and in the hindlimb at E11.5 (C and E). Shh was not detected in the limb of FgfR2-IIIbϪ/Ϫlac Z mice between E9.5 to E11.5 (B, D, and F). Fgf4 was not detected at E9.5 in the heterozygotes (G) or at any time in mutant limb buds (H, J, and L), but of the limbs in FgfR2-IIIbϪ/Ϫlac Z mice and arrowheads indicate the was strongly expressed in the AER of heterozygotes at E10.5 and E11.5 posterior part of the limb region in heterozygous mice. Arrows (I and K). Asterisks indicate the position of the presumptive location show position of Fgf4 in the branchial arches.

detected in heterozygotes at E9.5 (Fig. 8G), although it was strongly expressed at E10.5 and E11.5 (Figs. 8I, 8K, and Bueno and Heath, 1996; Niswander and Martin, 1992). Fgf4 expression was not detected in the limb buds of FgfR2- IIIbϪ/ϪlacZ mice at several stages examined between E9.5 and E11.5 (Figs. 8H, 8J, 8L, and data not shown), although expression of Shh and Fgf4 was detected at other known expression sites (e.g., Shh in the neural floor plate, notochord and hindgut; Fgf4 in the branchial arches; Bitgood and McMahon, 1995; Echelard et al., 1993; Roelink et al., 1994). These results indicate that FgfR2-IIIb function is required for the induction of Shh and Fgf4 during early limb bud growth. Limb bud apoptosis correlates with loss of FgfR2-IIIb expression. Histological sections through the presumptive limb buds of FgfR2-IIIb null mice showed a heteroge- neity of nuclear size indicative of apoptosis (Fig. 4). To assess the degree of apoptosis directly, sections were stained using a TUNEL assay. At E9.5 there was little evidence for apoptosis in either heterozygous or FgfR2- IIIbϪ/ϪlacZ mice (Figs. 9A and 9B), but by E10-E10.5 the mutant mice showed extensive apoptosis in the presumptive AER and limb bud mesenchyme (Fig. 9D). The more intense TUNEL staining in the mesenchyme was not in the progress zone but at more proximal and posterior region adjacent to the presumptive ZPA (Fig. 9D). At E11.5 the level of apoptosis was less but still substantial (Fig. 9F). These findings could explain the low expression level of limb bud marker genes assessed, although a reduction in gene transcription cannot be excluded as an accompanying mechanism. Hence a major function of FgfR2-IIIb activation is the survival of the AER and indirectly the survival of the underlying mesenchyme. Cell proliferation in the early limb bud of FgfR2-IIIb؊/؊lacZ mice. The loss of FgfR2-IIIb signaling could result in a significant decrease in limb bud cell proliferation, as well as a reduction in cell survival as demonstrated above. At E9.5, there were no apparent differences in proliferating cell numbers. However, in sections from homozygous mutant embryos at E10.5 and E11.5, there was a noticeable increase in nonproliferating cells that largely correlated with those cells showing characteristics of apoptosis (data not shown).

DISCUSSION FIG. 9. Detection of apoptosis in the early limb bud of heterozy- Ϫ Ϫlac Z Visceral Organ Agenesis and Dysgenesis gous and FgfR2-IIIb / mice. Apoptotic cells were detected using a TUNEL assay in heterozygous (A, C, E) and FgfR2-IIIbϪ/Ϫlac Z (B, D, The phenotype of FgfR2-IIIbϪ/ϪCre mice and FgfR2- F) mice at E9.5 (A, B), E10.5 (C, D), and E11.5 (E, F). No difference IIIbϪ/ϪlacZ are very similar and consistent with the dominant in the level of apoptosis was seen between limb buds (lb) of negative FgfR2-IIIb receptor studies (Celli et al., 1998; heterozygous and homozygous mutant mice at E9.5. Open arrow- DeMoerlooze et al., 2000; Peters et al., 1994; Werner et al., heads indicate the position of apoptotic cells. At E10.5 FgfR2- Ϫ Ϫ 1994). Perhaps most surprising is that abrogation of one IIIb / lac Z mice show extensive brown staining of apoptotic cells in receptor isoform has such drastic consequences for so many the ectoderm and the proximal mesenchyme which is also present to a lower degree at E11.5. Small arrowheads indicate the position tissues (Table 1). However, an analysis of receptor expres- of apoptotic cells and large arrowheads apoptotic zones. All sec- sion patterns has demonstrated the presence of FgfR2-IIIb tions were cut parallel to the dorsal/ventral axis of the limb buds. in the epithelial component of all the affected organs and a Bar, 0.2 mm. complementary expression pattern for its two major ligands

Fgf10 and/or Fgf7, which are usually detected in the adja- Sekine et al., 1999; Xu et al., 1998). However, the results cent mesenchyme (Finch et al., 1995; Mason et al., 1994; reported here demonstrate that Fgf8 can be detected at Orr-Urtreger et al., 1993; Peters et al., 1992; Yamasaki et similar levels in E9.5 embryos from both heterozygous and al., 1996 and unpublished data). This suggests that the FgfR2-IIIb null embryos prior to AER detection, which is developmental abnormalities probably result from the loss consistent with the findings of Vogel et al. (Figs. 6G and 6H; of a mesenchymal to epithelial signal during the early Vogel et al., 1996). Interestingly, Fgf8 RNA was detected in stages of organogenesis. This interpretation is supported by control embryos, before a morphological AER, and before the phenotypes of mice lacking Fgf3, Fgf7,orFgf10 (Guo et expression of FgfR2-IIIb RNA (Figs. 5 and 6). In addition, al., 1996; Mansour et al., 1993; Min et al., 1998; Sekine et Fgf8 expression was detected in the presumptive forelimb al., 1999). Fgf3-deficient mice show a curved tail due to the and hindlimb bud sites despite a failure to maintain an AER fusion of the caudal vertebrae and a partially penetrant ear (Figs. 4D, 4F and 6J, 6L). Expression of Fgf10 in the mesen- defect that correlates with a failure of the otic vesicle to chyme also precedes FgfR2-IIIb expression in the ectoderm form the endolymphatic duct (Mansour et al., 1993). This but was reduced at E9.5 in FgfR2-IIIbϪ/ϪlacZ embryos. Thus, indicates that although Fgf3 can activate both FgfR1-IIIb maintenance of Fgf10 expression appears in part to be AER and FgfR2-IIIb, the latter receptor is the one that is crucial dependent since its expression diminishes when the ridge for the response to Fgf3 during inner ear development fails to maintain (Figs. 6D and 6F). A similar finding holds (Mathieu et al., 1995). Mice lacking Fgf7 show a hair for Bmp4, a gene expressed in both the mesenchyme and follicles defect and smaller kidneys with fewer nephrons AER (Figs. 7I–7L). Although faint, Bmp4 is detected in the (Guo et al., 1996; Qiao et al., 1999). Again, these abnormali- presumptive limb bud ectoderm as a narrow band of expres- ties are at least in part reflected in the FgfR2-IIIb null sion, suggesting that the epithelial cells destined to form a strains (Figs. 3O, 3P, and data not shown). Finally, Fgf10- morphological AER are already specified prior to FgfR2-IIIb deficient mice show major defects strikingly similar to expression. Previous finding have shown that Msx1 is a those in the FgfR2-IIIbϪ/Ϫ mice, including agenesis of the good marker for the progress zone of the early limb bud lungs and limbs (Min et al., 1998; Sekine et al., 1999). (Wang and Sassoon, 1995). Interestingly, its expression is dependent on AER in the chick but not in the mouse (Lizarraga et al., 1999; Wang and Sassoon, 1995). Expression Limb Bud Development of Msx1 was found in the mesenchyme of control and The role Fgfs play in limb development has been exten- FgfR2-IIIbϪ/ϪlacZ embryos, but despite not requiring an AER sively studied, and this has led to a widely accepted model for synthesis, its expression was diminished in the homozy- for early limb bud initiation and maintenance with poten- gous mutant mice. Reduced expression of the genes de- tial roles postulated for Fgf2, Fgf4, Fgf8, Fgf9, Fgf10, as well tected in the limb bud ectoderm and mesenchyme can in as FgfR1 and FgfR2 (Arman et al., 1999; Crossley et al., part be explained by the extensive loss of cell numbers 1996; DeMoerlooze et al., 2000; Deng et al., 1997; Fallon et through apoptosis (Fig. 9). However, we cannot exclude a al., 1994; Niswander et al., 1993; Ohuchi et al., 1997; Vogel change in the transcription level of the genes as an accom- et al., 1996; Xu et al., 1998). According to the accepted panying mechanism for the apparent decrease in expres- model, Fgf10 in the lateral plate mesoderm signals to sion. It is interesting that apoptosis is most apparent in the FgfR2-IIIb in the overlying ectoderm to induce Fgf8 con- proximal mesenchyme and not the region underlying the comitant with formation of the apical ectodermal ridge AER, suggesting that survival factor signaling from the AER (Arman et al., 1999; Crossley et al., 1996; Min et al., 1998; must be mediated over many cell diameters. Hence, the Ohuchi et al., 1997; Sekine et al., 1999; Vogel et al., 1996; survival signals imparted by FgfR2-IIIb activation presum- Xu et al., 1998). The AER is maintained by Fgf8, and the ably function as an autocrine/intracrine response in the underlying mesenchyme receives growth signals through a ectoderm and as a paracrine signal for the underlying positive feedback loop probably involving multiple Fgfs. In mesenchyme. It is possible that Fgf8 is a mediator of the addition, a second feedback loop was suggested to occur paracrine signal, since the growth of limb bud explants can between Fgf4 in the AER and Shh expression in the ZPA, be maintained in the absence of the AER by Fgf8 (Crossley which is involved in determining the anterior–posterior et al., 1996; Mahmood et al., 1995; Vogel et al., 1996). limb axis (Laufer et al., 1994; Niswander et al., 1994). The Shh is induced and maintained in the ZPA through the importance of Fgf signaling is most dramatically illustrated action of Fgf8/Fgf4 signaling from the AER (Laufer et al., by the induction of ectopic limbs when Fgf-coated beads or 1994; Moon et al., 2000; Niswander et al., 1994; Sun et al., expressing cells are placed in the chick or mouse flank 2000; Vogel and Tickle, 1993). In the FgfR2-IIIbϪ/ϪlacZ mice, (Cohn et al., 1995; Crossley et al., 1996; Ohuchi et al., 1997, Shh was not detected in the limb bud at several different 1995). stages (Fig. 8), while control embryos showed strong Shh The lack of limb development in mice deficient for Fgf10 expression as expected. These data are consistent with the demonstrates a key role for this molecule. Moreover, these results of Ohuchi et al. that showed removal of the AER studies indicated that mice either lacking Fgf10 or deficient suppresses Shh expression as well as Fgf10 (Ohuchi et al., for FgfR2 fail to form limb buds and do not express Fgf8 in 1997). Thus, it appears that Shh induction is dependent the ectoderm of the presumptive AER (Min et al., 1998; either directly or indirectly on FgfR2-IIIb activity, consis-

FIG. 10. A model showing the proposed function of FgfR2-IIIb in limb bud development. Signals from the lateral plate mesoderm induce expression of ectodermal and mesenchymal genes associated with limb bud initiation (Fgf8, Fgf10, Msx1, and Bmp4) independently and prior to FgfR2-IIIb expression. Expression of FgfR2-IIIb allows Fgf10, already expressed in the underlying mesenchyme, to signal through FgfR2-IIIb and maintains cell survival in the ectoderm and proximal mesenchyme. FgfR2-IIIb signaling induces Fgf4 expression in the AER, which is maintained by a positive feedback loop with Shh in the ZPA. Presumably other Fgfs can undertake this function since targeted inactivation of Fgf4 in the AER is phenotypically silent (Moon et al., 2000; Sun et al., 2000). Shh can also maintain Fgf10 expression in the Progress Zone (PZ) (Ohuchi et al., 1997) and could therefore potentiate the activation of FgfR2-IIIb by Fgf10. This facilitates the high level of expression of the induced genes, such as Fgf4, Fgf8, Fgf10, Msx1, and Bmp4. Fgf4/Fgf8 acting through FgfR1-IIIc in the mesenchyme may function as indirect survival factors for the mesenchyme. In the absence of FgfR2-IIIb, Fgf4, Shh, and other genes downstream FgfR2-IIIb in the AER and the PZ are not activated and cannot maintain the expression of Fgf10 via Shh. The presumptive limb bud ectoderm and proximal mesenchyme undergo apoptosis causing a dramatic reduction in Fgf10, Fgf8, Msx1, Bmp4 levels that leads to loss of the limb bud.

tent with the previous ﬁnding that Fgf10 can induce Shh and Fig. 10). In brief the following points are taken into (Ohuchi et al., 1997). Fgf4 expression was also dependent account: (1) Fgf10 signaling via FgfR2-IIIb is not required for upon FgfR2-IIIb signaling and most likely through an auto- expression of the early AER or mesenchymal limb bud crine induction since they are expressed in the same cells. marker genes such as Fgf8, Fgf10, Bmp4, and Msx1. This In agreement with others, Fgf4 was detected subsequent to suggests that the pattern of gene expression associated with Shh, suggesting that Fgf4 is unlikely to be the inducer of limb bud initiation is already speciﬁed in the region of the Shh (Bueno and Heath, 1996; Niswander and Martin, 1992). ectoderm and mesenchyme destined to become the limb Moreover, conditional Fgf4 gene deletions in the AER bud; (2) signaling to FgfR2-IIIb in the ectoderm is essential develop normal limbs (Moon et al., 2000; Sun et al., 2000). to maintain the AER; (3) FgfR1-IIIc rather than FgfR2-IIIc in The results of the present study make several new points the underlying mesenchyme must be the main receptor to which modify previously described models (Martin, 1998; receive the Fgf signals from the AER (Peters et al., 1992; and Naski and Ornitz, 1998; Ohuchi et al., 1997; Xu et al., 1998 data not shown). This notion is supported by exclusion of

FgfR1Ϫ/Ϫ cells from the progress zone in null/wild-type Deng, C. X., Bedford, M., Li, C. L., Xu, X. L., Yang, X., Dunmore, J., chimeras (Deng et al., 1997); (4) the initiation of Shh and and Leder, P. (1997). Fibroblast growth-factor receptor-1 (FGFR-1) Fgf4 expression is dependent on FgfR2-IIIb expression, is essential for normal neural-tube and limb development. Dev. since neither was detected in the mutant limb buds. Biol. 185, 42–54. Finally, the absence of FgfR2-IIIb leads to a lack of limb Deng, C. X., Wynshaw-Boris, A., Shen, M. M., Daugherty, C., bud growth which is mainly due to massive levels of Ornitz, D. M., and Leder, P. (1994). Murine FGFR-1 is required for Genes apoptosis, with an apparent small reduction in prolifera- early postimplantation growth and axial organization. Dev. 8, 3045–3057. tion. These findings show that Fgf10/FgfR2-IIIb signaling is Echelard, Y., Epstein, D., St-Jacques, B., Shen, L., Mohler, J., necessary to maintain the integrity of limb bud. McMahon, J., and McMahon, A. (1993). Sonic hedgehog, a member of a family of putative signaling molecules, is impli- ACKNOWLEDGMENTS cated in the regulation of CNS polarity. Cell 75, 1417–1430. Fallon, J., Lopez, A., Ros, M., Savage, M., Olwin, B., and Simandl, B. (1994). Apical ectodermal ridge growth signal for chick limb We thank the ICRF transgenic animal unit and the Histolopa- thology Unit for their expert technical help. We also thank Dr. development. Science 264, 104–107. Julian Lewis for critically reading the manuscript. This work was Finch, P., Cunha, G., Rubin, J., Wong, J., and Ron, D. (1995). Pattern supported in part by a TMR Marie Curie Research training grant to of keratinocyte growth factor and keratinocyte growth factor J.-M.R. receptor expression during mouse fetal development suggests a role mediating morphogenetic mesenchymal-epithelial interactions. Dev. Dyn. 203, 223–240. REFERENCES Ganan, Y., Macias, D., Basco, R. D., Merino, R., and Hurle, J. M. (1998). Morphological diversity of the avian foot is related with Arman, E., Haffner-Krausz, R., Chen, Y., Heath, J. K., and Lonai, P. the pattern of msx gene expression in the developing autopod. (1998). Targeted disruption of fibroblast growth-factor (FGF) Dev. Biol. 196, 33–41. receptor-2 suggests a role for FGF signaling in pregastrulation Gu, H., Zou, Y.-R., and Rajewsky, K. (1993). Independent control of mammalian development. Proc. Natl. Acad. Sci. USA 95, 5082– immunoglobulin switch recombination at individual switch 5087. regions evidenced through Cre-loxP-mediated gene targeting. Arman, E., Haffner-Krausz, R., Gorivodsky, M., and Lonai, P. Cell 73, 1155–1164. (1999). Fgfr2 is required for limb outgrowth and lung-branching Guo, L., Degenstein, L., and Fuchs, E. (1996). Keratinocyte growth morphogenesis. Proc. Natl. Acad. Sci. USA 96, 11895–11899. factor is required for hair development but not for wound Bitgood, M., and McMahon, A. (1995). Hedgehog and Bmp genes are healing. Genes Dev. 10, 165–175. coexpressed at many diverse sites of cell-cell interaction in the Henrique, D., Adam, J., Myat, A., Chitnis, A., Lewis, J., and mouse embryo. Dev. Biol. 172, 126–138. Ish-Horowicz, D. (1995). Expression of a Delta homologue in Bueno, D., and Heath, J. (1996). Co-expression pattern analysis of prospective neurons in the chick. Nature 375, 787–790. Fgf4, Fgf8 and Shh gene expression at diverse signaling centers Hogan, B., and Yingling, J. (1998). Epithelial/mesenchymal interac- during mouse development. Int. J. Dev. Biol. Suppl 1, 79S-0–80S. tions and branching morphogenesis of the lung. Curr. Opin. Celli, G., Larochelle, W. J., MacKem, S., Sharp, R., and Merlino, G. Genet. Dev. 8, 481–486. (1998). Soluble dominant-negative receptor uncovers essential Johnson, R., and Tabin, C. (1997). Molecular models for vertebrate roles for fibroblast growth-factors in multiorgan induction and limb development. Cell 90, 979–990. patterning. EMBO J. 17, 1642–1655. Kettunen, P., Karavanova, I., and Thesleff, I. (1998). Responsiveness Church, D., Stotler, C., Rutter, J., Murrell, J., Trofatter, J., and of developing dental tissues to fibroblast growth factors: Expres- Buckler, A. (1994). Isolation of genes from complex sources of sion of splicing alternatives of FGFR1, -2, -3, and of FGFR4; and mammalian genomic DNA using exon amplification. Nat. stimulation of cell proliferation by FGF2, -4, -8, and -9. Dev. Genet. 6, 98–105. Genet. 22, 374–385. Cohn, M. J., Izpisua-Belmonte, J. C., Abud, H., Heath, J. K., and Laufer, E., Nelson, C. E., Johnson, R. L., Morgan, B. A., and Tabin, Tickle, C. (1995). Fibroblast growth-factors induce additional C. (1994). Sonic hedgehog and FGF4 act through a signaling limb development from the flank of chick-embryos. Cell 80, 739–746. cascade and feedback loop to integrate growth and patterning of Crossley, P. H., Minowada, G., MacArthur, C. A., and Martin, G. R. the developing limb bud. Cell 79, 993–1003. (1996). Roles for FGF8 in the induction, initiation, and mainte- Lizarraga, G., Ferrari, D., Kalinowski, M., Ohuchi, H., Noji, S., nance of chick limb development. Cell 84, 127–136. Kosher, R., and Dealy, C. (1999). FGFR2 signaling in normal and Cunha, G., and Hom, Y. (1996). Role of mesenchymal-epithelial limbless chick limb buds. Dev. Genet. 25, 331–338. interactions in mammary gland development. J. Mamm. Gland Mahmood, R., Bresnick, J., Hornbruch, A., Mahony, C., Morton, Biol. Neopl. 1, 21–35. N., Colquhoun, K., Martin, P., Lumsden, A., Dickson, C., and Davidson, D., Crawley, A., Hill, R., and Tickle, C. (1991). Position- Mason, I. (1995). A role for FGF8 in the initiation and mainte- dependent expression of two related homeobox genes in devel- nance of vertebrate limb bud outgrowth. Curr. Biol. 5, 797–806. oping vertebrate limbs. Nature 352, 429–431. Mansour, S. L., Goddard, J. M., and Capecchi, M. R. (1993). Mice DeMoerlooze, L., Spencer-Dene, B., Revest, J. M., Hajihosseini, M., homozygous for a targeted disruption of the protooncogene int-2 Rosewell, I., and Dickson, C. (2000). An important role for the have developmental defects in the tail and inner-ear. Develop- IIIb isoform of fibroblast growth factor receptor 2 (FGFR2) in ment 117, 13–28. mesenchymal-epithelial signaling during mouse organogenesis. Martin, G. (1998). The roles of FGFs in the early development of Development 127, 483–492. vertebrate limbs. Genes & Dev. 12, 1571–1586.

Mason, I., Fuller-Pace, F., Smith, R., and Dickson, C. (1994). FGF7 Pizette, S., and Niswander, L. (1999). BMPs negatively regulate (keratinocyte growth factor) expression during mouse develop- structure and function of the limb apical ectodermal ridge. ment suggests roles in myogenesis, forebrain regionalisation and Development 126, 883–894. epithelial-mesenchymal interactions. Mech. Dev. 45, 15–30. Qiao, J., Uzzo, R., Obara-Ishihara, T., Degenstein, L., Fuchs, E., and Mathieu, M., Chatelain, E., Ornitz, D., Bresnick, J., Mason, I., Herzlinger, D. (1999). FGF7 modulates ureteric bud growth and Kiefer, P., and Dickson, C. (1995). Receptor-binding and mito- nephron number in the developing kidney. Development 126, genic properties of mouse fibroblast-growth-factor-3— 547–554. modulation of response by heparin. J. Biol. Chem. 270, 24197– Riddle, R., Johnson, R., Laufer, E., and Tabin, C. (1993). Sonic 24203. hedgehog mediates the polarizing activity of the ZPA. Cell 75, McKeehan, W. L., Wang, F., and Kan, M. (1998). The heparan- 1401–1416. sulfate fibroblast growth-factor family—diversity of structure Roelink, H., Augsburger, A., Heemskerk, J., Korzh, V., Norlin, S., and function. Prog. Nucl. Acid Res. Mol. Biol. 59, 135–176. Ruiz i Altaba, A., Tanabe, Y., Placzek, M., Edlund, T., and Jessell, McLeod, M. (1980). Differential staining of cartilage and bone in T. (1994). Floor plate and motor neuron induction by vhh-1, a whole mouse fetuses by alcian blue and alizarin red S. Teratology vertebrate homolog of hedgehog expressed by the notochord. Cell 22, 299–301. 76, 761–775. Min, H., Danilenko, D., Scully, S., Bolon, B., Ring, B., Tarpley, J., Sekine, K., Ohuchi, H., Fujiwara, M., Yamasaki, M., Yoshizawa, T., DeRose, M., and Simonet, W. (1998). Fgf10 is required for both Sato, T., Yagishita, N., Matsui, D., Koga, Y., Itoh, N., and Kato, limb and lung development and exhibits striking functional S. (1999). Fgf10 is essential for limb and lung formation. Nat. similarity to Drosophila branchless. Genes Dev. 12, 3156–3161. Genet. 21, 138–141. Moon, A., Boulet, A., and Capecchi, M. (2000). Normal limb Sun, X., Lewandoski, M., Meyers, E. N., Liu, Y. H., Maxson, R. E., development in conditional mutants of Fgf4. Development 127, and Martin, G. R. (2000). Conditional inactivation of Fgf4 reveals 989–996. complexity of signaling during limb bud development. Nat. Naski, M., and Ornitz, D. (1998). FGF signaling in skeletal devel- Genet. 25, 83–86. opment. Front. Biosci. 7, 81–94. Thesleff, I., and Sharpe, P. (1997). Signaling networks regulating Niswander, L., Jeffery, S., Martin, G., and Tickle, C. (1994). A dental development. Mech. Dev. 67, 111–123. positive feedback loop coordinates growth and patterning in the Vogel, A., Rodriguez, C., and Izpisua-Belmonte, J. C. (1996). In- vertebrate limb. Nature 371, 609–614. volvement of FGF8 in initiation, outgrowth and patterning of the Niswander, L., and Martin, G. (1992). Fgf4 expression during vertebrate limb. Development 122, 1737–1750. gastrulation, myogenesis, limb and tooth development in the Vogel, A., and Tickle, C. (1993). FGF4 maintains polarizing activity mouse. Development 114, 755–768. of posterior limb bud cells in-vivo and in-vitro. Development Niswander, L., Tickle, C., Vogel, A., Booth, I., and Martin, G. 119, 199–206. (1993). FGF4 replaces the apical ectodermal ridge and directs Wang, Y., and Sassoon, D. (1995). Ectoderm-mesenchyme and outgrowth and patterning of the limb. Cell 75, 579–587. mesenchyme-mesenchyme interactions regulate Msx1 expres- Ohuchi, H., Nakagawa, T., Yamamoto, A., Araga, A., Ohata, T., sion and cellular differentiation in the murine limb bud. Dev. Ishimaru, Y., Yoshioka, H., Kuwana, T., Nohno, T., Yamasaki, Biol. 168, 374–382. M., Itoh, N., and Noji, S. (1997). The mesenchymal factor, Werner, S., Smola, H., Liao, X., Longaker, M., Krieg, T., Hofschnei- FGF10, initiates and maintains the outgrowth of the chick limb der, P., and Williams, L. (1994). The function of KGF in morpho- bud through interaction with FGF8, an apical ectodermal factor. genesis of epithelium and reepithelialization of wounds. Science Development 124, 2235–2244. 266, 819–822. Ohuchi, H., Nakagawa, T., Yamauchi, M., Ohata, T., Yoshioka, H., Xu, X. L., Li, C. L., Takahashi, K., Slavkin, H. C., Shum, L., and Kuwana, T., Mima, T., Mikawa, T., Nohno, T., and Noji, S. Deng, C. X. (1999). Murine fibroblast growth factor receptor 1 (1995). An additional limb can be induced from the flank of the alpha isoforms mediate node regression and are essential for chick-embryo by FGF4. Biochem. Biophys. Res. Commun. 209, posterior mesoderm development. Dev. Biol. 208, 293–306. 809–816. Xu, X. L., Weinstein, M., Li, C. L., Naski, M., Cohen, R. I., Ornitz, Ornitz, D. M. (2000). FGFs, heparan sulfate and FGFRs: Complex D. M., Leder, P., and Deng, C. X. (1998). Fibroblast-growth-factor- interactions essential for development. Bioessays 22, 108–112. receptor-2 (FGFR2)-mediated reciprocal regulation loop between Ornitz, D. M., Xu, J. S., Colvin, J. S., McEwen, D. G., MacArthur, FGF8 and FGF10 is essential for limb induction. Development C. A., Coulier, F., Gao, G. X., and Goldfarb, M. (1996). Receptor 125, 753–765. specificity of the fibroblast growth-factor family. J. Biol. Chem. Yamaguchi, T. P., Harpal, K., Henkemeyer, M., and Rossant, J. 271, 15292–15297. (1994). FGFR-1 is required for embryonic growth and mesoder- Orr-Urtreger, A., Bedford, M., Burakova, T., Arman, E., Zimmer, Y., mal patterning during mouse gastrulation. Genes Dev. 8, 3032– Yayon, A., Givol, D., and Lonai, P. (1993). Developmental 3044. localization of the splicing alternatives of fibroblast growth- Yamaguchi, T. P., and Rossant, J. (1995). Fibroblast growth-factors in factor receptor-2 (FGFR2). Dev. Biol. 158, 475–486. mammalian development. Curr. Opin. Genet. Dev. 5, 485–491. Peters, K., Werner, S., Chen, G., and Williams, L. (1992). Two FGF Yamasaki, M., Miyake, A., Tagashira, S., and Itoh, N. (1996). receptor genes are differentially expressed in epithelial and Structure and expression of the rat messenger-RNA encoding a mesenchymal tissues during limb formation and organogenesis novel member of the fibroblast growth-factor family. J. Biol. in the mouse. Development 114, 233–243. Chem. 271, 15918–15921. Peters, K., Werner, S., Liao, X., Wert, S., Whitsett, J., and Williams, L. (1994). Targeted expression of a dominant negative FGF Received for publication September 18, 2000 receptor blocks branching morphogenesis and epithelial differen- Accepted December 8, 2000 tiation of the mouse lung. EMBO J. 13, 3296–3301. Published online January 26, 2001