Fibroblast growth factors (FGFs) can influence the growth and differentiation of cultured cells derived from neuroectoderm, ectoderm or mesenchyme. The FGFs interact with a family of at least four closely related receptor tyrosine kinases that are products of individual genes. To investigate the role of FGFs in the growth and differentiation of embryonic tissues and to determine whether the individual FGF receptor genes might have specific functions, we compared the localization of mRNA for two FGF receptor genes, FGFR1 (the fig gene product) and FGFR2 (the bek gene product), during limb formation and organogenesis in mouse embryos (E9.5-E16.5). Although the two genes were coexpressed in some tissues, the differential expression of FGFR1 and FGFR2 in most embryonic tissues was striking. FGFR1 was expressed diffusely in mesenchyme of limb buds, somites and organ rudiments. In contrast, FGFR2 was expressed predominantly in the epithelial cells of embryonic skin and of developing organs. The differential expression of FGFR1 and FGFR2 in mesenchyme and epithelium respectively, suggests the receptor genes are independently regulated and that they mediate different functions of FGFs during development.

The fibroblast growth factors (FGFs) constitute a family of at least seven polypeptide growth factors that influence cell growth and differentiation in vivo and in vitro (for review see Burgess and Maciag, 1989; Rifkin and Moscatelli, 1989; Gospodarowicz, 1990). FGFs appear to play a role in mesoderm induction and early embryonic pattern formation (Kimelman and Kirschner, 1987; Slack et al., 1987). Moreover, recent studies showed that members of the FGF family are also expressed in later stages of embryonic development (Hebert et al., 1990; Wilkinson et al., 1989; Caday et al., 1990; Joseph-Silverstein et al., 1989; Haub and Goldfarb, 1991; Schnurch and Risau, 1991). Basic FGF and acidic FGF, for example, are broadly distributed in the embryo (Gonzalez et al., 1990 and Fu et al., 1991). Furthermore, high affinity binding sites for the FGFs can be detected in chick embryos at stages consistent with a role in organogenesis (Olwin and Hauschka, 1990). These properties suggest FGFs are active in embryos during tissue differentiation and organ development.

We have recently cloned an FGF receptor, FGF receptor 1 (FGFR1), from chicken embryos. The chicken FGFR1 cDNA clone encoded a receptor tyrosine kinase that bound both basic and acidic FGF (Lee et al., 1989). Subsequently, three other FGF receptor genes (FGFR2, the bek. gene product; FGFR3; FGFR4) were cloned and found to be closely related to FGFR1 (Reid et al., 1990; Safran et al., 1990; Ruta et al., 1988; Dionne et al., 1990; Kombluth et al., 1988; Pasquale, 1990; Hattori et al., 1990; Houssaint et al., 1990; Keegan et al., 1991; Partanen et al., 1991). Whether the individual FGF receptor genes have specific functions in development has not been addressed. FGFR1 is known to be expressed in the developing CNS and in embryonic mesenchyme, but the distribution of the other FGF receptors in development is not known (Heuer et al., 1990; Wanaka et al., 1991).

In this study, we compared the distribution of mRNA for FGFR1 and FGFR2 in mouse embryos during limb formation and organogenesis. In accordance with previous studies, we found that FGFR1 was expressed in the CNS and in many mesenchymal populations. Surprisingly, however, FGFR2 was expressed primarily in embryonic epithelia.

Murine FGFR1 and FGFR2 cDNA clones

A 2.9 kb Sal-Sal fragment of human FGFR1 was used to screen a mouse embryonyl carcinoma cell library (kindly provided by Gail Martin, UCSF). A 3.5 kb mouse cDNA was isolated and subcloned and a restriction map was made prior to sequencing. For the generation of riboprobe, a 300 bp HincII-Sacl restriction fragment encoding a small portion of the extracellular domain, the transmembrane domain and most of the juxtamembrane domain was subcloned into the HincII/Sacl site of pBluescript KS vector. The sequence of the restriction fragment was identical to the mouse FGFR1 sequence reported by Reid and coworkers (Reid et al., 1990).

The FGFR2 clone was generated by PCR using oligonuceotide primers based on the published mouse bek sequence and designed to encompass the first kinase domain and the kinase insert (nucleotides 61 to 93 and 332 to 359). The expected 298 bp fragment was amplified from single-stranded cDNA synthesized by reverse transcription from adult mouse brain RNA using MM-LV reverse transcriptase. The fragment was subcloned into the EcoRI/Smal site of pBluescript KS + for sequencing and in vitro transcription. The nucleotide sequence was identical to the reported mouse bek sequence (Kombluth et al., 1988) except for the deletion of a guanine residue 4 bases from the 5’ end of the clone.

Probe generation

Radiolabeled antisense and sense transcripts were made for both FGFR1 and FGFR2 by in vitro transcription. Plasmids were linearized with the appropriate restriction enzyme and transcribed using either T3 (Stratagene) or T7 (Promega) RNA polymerase. For use in protection assay, transcriptions were done in the presence of 32P-labeled UTP (800 Ci/mmol, Amersham) and for in situ hybridization transcription reactions were labeled using 35S-labeled LHP (1200 Ci/mmol, Amersham).

RNase protection assay

Swiss-Webster mice were killed by cervical dislocation at various stages of gestation (9.5,10.5,12.5, 14.5 and 16.5 days post coitus) and the embryos were dissected from the uterus. Newborn mice were killed by cervical dislocation. The tissues were frozen immediately in liquid nitrogen for storage. Total cellular RNA was isolated from embryonic and newborn tissues as described by Chirgwin et al., 1979.

50 μg samples of total RNA were hybridized with 1×105 c.p.m. of 32P-labeled antisense riboprobe at 42°C overnight. Hybrids were digested with RNase A and T1 for 40 minutes at 30°C (Melton et al., 1984). The digests were resolved on 5% polyacrylamide/8 M urea gels and visualized by autoradiography.

In situ hybridization

Embryos were collected as described above except that after dissection, they were fixed overnight in 4% paraformaldehyde in phosphate-buffered saline. Following fixation, the embryos were dehydrated in ethanols, cleared with toluene and embedded in paraffin. The paraffin embedded embryos were sectioned at 10 microns and mounted on aminoalkylsilane treated slides. In situ hybridization experiments were performed according to Wilkinson (Wilkinson et al., 1987) with the modifications described by Frohman (Frohman et al., 1990).

Controls for the specificity of the hybridizations

It is unlikely that the probes used for these studies crosshybridize to homologous mRNA species, because when used to analyze RNA from adult tissues by northern analysis, they detect differential expression of FGFR1 and FGFR2 in heart, brain and liver (S. W. unpublished data). Further evidence for the specificity of these probes was the striking differential hybridization of the probes in serial embryo sections. No specific hybridization was seen using sense riboprobes.

To determine the relative levels of expression of FGFR1 and FGFR2 at various stages of development, RNase protection assays were performed using total RNA isolated from mouse embryos at 9.5, 10.5, 12.5, 14.5, 16.5, and 18.5 days p.c. (post coitus) and from newborn mice. Both FGFR1 and FGFR2 were expressed at relatively high levels from 9.5 to 14.5 days p.c. and then fell sharply between 14.5 and 16.5 days p.c. (see Fig. 1). These results correlate well with previous ligand binding studies (Olwin and Hauschka, 1990) and with the in situ hybridization studies described below.

Fig. 1.

RNase protection assays showing relative expression of FGFR1 (Panel A) and FGFR2 (Panel B) during mouse development. :P-labeled antisense FGFR1 and FGFR2 transcripts were used to hybridize 50 micrograms of total RNA isolated from mouse embryos at 9.5, 10=5, 12.5, 14.5 and 16.5 days p.c. and from newborn mice. After RNase digestion, protected fragments were resolved by polyacrylamide gel electrophoresis and gels were exposed overnight to Hyperfilm-MP (Amersham).

Fig. 1.

RNase protection assays showing relative expression of FGFR1 (Panel A) and FGFR2 (Panel B) during mouse development. :P-labeled antisense FGFR1 and FGFR2 transcripts were used to hybridize 50 micrograms of total RNA isolated from mouse embryos at 9.5, 10=5, 12.5, 14.5 and 16.5 days p.c. and from newborn mice. After RNase digestion, protected fragments were resolved by polyacrylamide gel electrophoresis and gels were exposed overnight to Hyperfilm-MP (Amersham).

The cellular distribution of FGFR1 and FGFR2 mRNA in embryonic tissues was studied by in situ hybridization of 35S-labeled transcripts to serial sections of mouse embryos at 9.5, 10.5, 12.5 and 16.5 days p.c. The results of these experiments are described in the following discussion. In the figures that accompany the discussion (Figs 210), the left panel is a brightfield photomicrograph showing the region being examined, and the middle and right panels are darkfield photomicrographs of serial sections hybridized with antisense transcripts for FGFR1 and FGFR2, respectively.

Fig. 2.

FGF receptor expression in developing limb. Serial embryo sections were hybridized with antisense transcripts for FGFR1 (B,E) or FGFR2 (C,F). (A-C) sagittal sections at 10.5 days p.c.; (D-F) transverse sections at 12.5 days p.c. li, limb; h, heart; 1, lung; s, somite; n, neural tube. Magnification 100×.

Fig. 2.

FGF receptor expression in developing limb. Serial embryo sections were hybridized with antisense transcripts for FGFR1 (B,E) or FGFR2 (C,F). (A-C) sagittal sections at 10.5 days p.c.; (D-F) transverse sections at 12.5 days p.c. li, limb; h, heart; 1, lung; s, somite; n, neural tube. Magnification 100×.

FGF receptor expression in limb bud development

The early limb bud is formed by the accumulation of lateral plate mesenchyme under the flank ectoderm. The limb bud mesenchyme then induces the ectoderm covering the apical surface of the limb bud to thicken forming the apical ectodermal ridge (AER). Removal of the AER at any time results in the cessation of limb growth. During early limb differentiation (9.5-10.5 days p.c.), FGFR1 was expressed diffusely in the mesenchyme of the limb bud, while FGFR2 was expressed in the ectoderm, including the AER as it becomes morphologically distinct (Fig. 2A-C). A cross section of a forelimb at 12.5 days p.c. shows continued expression of FGFR2 in the ectoderm and coexpression of FGFR1 and FGFR2 in the cartilage blastema (Fig. 2D-F). This pattern of coexpression in prebone/precartilage elements is a theme which recurs in the prevertebral column and in cranial-facial bone formation (Fig. 6 and data not shown).

FGF receptor expression in developing skin and hair

Initially, the embryo is covered by a single layer of ectodermal cells. As discussed above, from 9.5 to 12.5 days p.c. FGFR2 was expressed prominently in the ectoderm and FGFR1 was expressed in the underlying mesenchyme (Fig. 2). By 16.5 days, when a multilayered epidermis has developed, FGFR2 was expressed in the proliferating basal layer but was not detected in the more mature kératinocytes of the upper layers of the epidermis (Fig. 3C, E). FGFR1 expression, however, was confined to a tight band of dermal mesenchymal cells just below the epidermis (Fig. 3B, D). In hair follicles at 16.5 days, FGFR1 was expressed conspicuously in the dermal papilla, but was not detected in the hair bulb (Fig. 3B). FGFR2 was detected in both the hair bulb and the dermal papilla (Fig. 3C).

Fig. 3.

FGF receptor expression in developing skin and hair. Embryo sections (16.5 days p.c.) including skin from the snout (A-C) and lower extremity (D-E) hybridized with antisense probes for FGFR1 (B, D) or FGFR2 (C, E). Panels D and E were photographed at high magnification (400×) using epi-illumination to visualize the silver grains and transmitted light to visualize the tissue, e, epidermis; d, dermis; b, hair bulb; p, dermal papilla; arrowheads indicate the basal cell layer of the epidermis. Magnification (A-C), 200×.

Fig. 3.

FGF receptor expression in developing skin and hair. Embryo sections (16.5 days p.c.) including skin from the snout (A-C) and lower extremity (D-E) hybridized with antisense probes for FGFR1 (B, D) or FGFR2 (C, E). Panels D and E were photographed at high magnification (400×) using epi-illumination to visualize the silver grains and transmitted light to visualize the tissue, e, epidermis; d, dermis; b, hair bulb; p, dermal papilla; arrowheads indicate the basal cell layer of the epidermis. Magnification (A-C), 200×.

KGF (keratinocyte growth factor), a member of the FGF family, is synthesized by dermal fibroblasts and stimulates the growth of cultured keratinocytes (Finch et al., 1989). Recently, the receptor for KGF was identified as an isoform of FGFR2 that should be detected by the probe used in this study (Miki et al., 1991 and Johnson et al., 1991). Thus, dermal fibroblasts could influence the proliferation and differentiation of the epidermis by the action of KGF on epidermal cells expressing this isoform of FGFR2.

FGF receptor expression in the developing gut and lung

Lung development is initiated by the sprouting of a tracheal bud from the foregut endoderm. The tracheal bud elongates until it penetrates the splanchnic mesoderm where it is induced to branch repeatedly, resulting in the formation of the mature bronchial tree. Throughout tracheal development (9.5–16.5 days p.c.), FGFR2 was expressed in tracheal epithelium (Figs 6C, F and 5C), while FGFR1 was expressed in tracheal mesenchyme (Figs 6B, E and 5B). Similarly, in early lung development (12.5 days p.c.), FGFR2 was expressed in lung bud epithelium (Fig. 4C), but FGFR1 was expressed predominantly in the mesenchyme (Fig. 4B). However, later in lung development (16.5 days p.c.), FGFR2 was expressed in terminal bronchioles and not in the more proximal airways (Fig. 4F), and FGFR1 expression in lung bud mesenchyme was substantially decreased (Fig. 4E).

Fig. 4.

Expression of FGF receptors in developing lung. Sections of 12.5 (A-C) and 16.5 (D-F) day embryos probed with antisense transcripts for either FGFR1 (B,E) or FGFR2 (C,F). b, lung bud bronchiole; m, lung bud mesenchyme; small arrowheads indicate terminal airways; large arrowheads indicate larger secondary airways. Magnification (A-C), 200×; (D-C), 100×.

Fig. 4.

Expression of FGF receptors in developing lung. Sections of 12.5 (A-C) and 16.5 (D-F) day embryos probed with antisense transcripts for either FGFR1 (B,E) or FGFR2 (C,F). b, lung bud bronchiole; m, lung bud mesenchyme; small arrowheads indicate terminal airways; large arrowheads indicate larger secondary airways. Magnification (A-C), 200×; (D-C), 100×.

Glazer and Shilo have cloned a Drosophila FGF receptor homologue and provided evidence that it represents the sole Drosophila FGF receptor gene (Glazer and Shilo, 1991). In Drosophila embryos, this receptor was expressed in the developing tracheal tree. In embryos homozygous for a multigene chromosomal deletion that included the FGF receptor gene, tracheal outgrowth was irregular and incomplete. These findings imply that, in Drosophila, the FGF receptor is required for normal epithelial proliferation and/or migration. Extended to mammalian development, these findings suggest that FGFR2 might be important in the development of the lung and other branched organs such as the kidney (see below).

In the developing esophagus and stomach, as in the lung, FGFR2 was expressed predominantly in the epithelium (Figs 5C, F, I and 6C, F) and FGFR1 was expressed in the surrounding mesenchyme (Figs 5B, E, H and 6B, E). In the intestine, FGFR1 and FGFR2 were coexpressed in the mesenchyme at modest levels (data not shown). FGFR1 was expressed in the gut mesenchyme and in the mesocolon, but FGFR2 was expressed only in gut mesenchyme.

Fig. 5.

FGF receptor expression in the developing digestive tract. Embryo sections were hybridized with antisense transcripts for FGFR1 (B, E, H) or FGFR2 (C, F, I). In A-C the esophagus (e) and trachea (t) are shown at 16.5 days p.c., and in D-I the stomach (s) is shown at 12.5 days (D-F) and 16.5 days (G-I) p.c. 1, liver; c, tracheal cartilage. Magnification (A-C, G-I), 100×; (D-F) 200×.

Fig. 5.

FGF receptor expression in the developing digestive tract. Embryo sections were hybridized with antisense transcripts for FGFR1 (B, E, H) or FGFR2 (C, F, I). In A-C the esophagus (e) and trachea (t) are shown at 16.5 days p.c., and in D-I the stomach (s) is shown at 12.5 days (D-F) and 16.5 days (G-I) p.c. 1, liver; c, tracheal cartilage. Magnification (A-C, G-I), 100×; (D-F) 200×.

Fig. 6.

FGF receptor expression in differentiating somites and in the developing esophagus and trachea. Serial sections of mouse embryos at 10.5 (A-C) and 12.5 (D-F) days p.c. were hybridized with antisense transcripts for FGFR1 (B, E) or FGFR2 (C, F). Transverse (A-C) and sagittal (D-F) sections through somites and the trachea and esophagus are depicted, s, sclerotome; m, myotome; d, dermatome; r, rostral sclerotome; c, caudal sclerotome; large arrowhead, trachea; small arrowhead, esophagus. Magnification (A-F), 100 ×.

Fig. 6.

FGF receptor expression in differentiating somites and in the developing esophagus and trachea. Serial sections of mouse embryos at 10.5 (A-C) and 12.5 (D-F) days p.c. were hybridized with antisense transcripts for FGFR1 (B, E) or FGFR2 (C, F). Transverse (A-C) and sagittal (D-F) sections through somites and the trachea and esophagus are depicted, s, sclerotome; m, myotome; d, dermatome; r, rostral sclerotome; c, caudal sclerotome; large arrowhead, trachea; small arrowhead, esophagus. Magnification (A-F), 100 ×.

FGF receptor expression in the embryonic kidney

Kidney development is governed by reciprocal epithelial-mesenchymal inductive events involving the metanephric duct epithelium and the surrounding mesenchyme (Ekblom, 1989). Nephrogenic mesenchyme induces the branching and elongation of the metanephric duct epithelium, and the terminal portions of the metanephric ducts, in turn, induce the formation of renal tubule and Bowman’s capsule epithelium from the mesenchyme. During kidney development, FGFR2 was expressed in the epithelium of the metanephric collecting ducts (Fig. 7C). FGFR1 hybridization,however, was expressed at higher levels in developing renal tubule and Bowman’s capsule epithelium (Fig. 7B). Interestingly, FGFR1 was also expressed in the cortical mesenchyme from which the renal tubule system originates.

Fig. 7.

FGF receptor expression during kidney development. Photomicrographs showing FGFR1 (B) and FGFR2 (C) expression in the renal cortex of a 16.5 day p.c, mouse embryo, m, cortex mesenchyme; large arrowheads indicate Bowman’s capsule/renal tubule complexes; small arrowheads indicate collecting ducts. Magnification (A-C), 200×.

Fig. 7.

FGF receptor expression during kidney development. Photomicrographs showing FGFR1 (B) and FGFR2 (C) expression in the renal cortex of a 16.5 day p.c, mouse embryo, m, cortex mesenchyme; large arrowheads indicate Bowman’s capsule/renal tubule complexes; small arrowheads indicate collecting ducts. Magnification (A-C), 200×.

FGF receptor expression in somite differentiation

At 9.5–10.5 days p.c., when de-epithelialization of the sclerotome is well underway, FGFR1 was expressed in both the dermatome and the sclerotome (Fig. 6B). The highest level of FGFR2, however, was in the perichordal sclerotome (Fig. 6C). Neither FGF receptor was detected in the myotome. By 12.5 days, the sclerotome becomes segmented into rostral and caudal “half somites” that can be identified on the basis of various anatomical and histochemical criteria (Stem and Keynes, 1986). FGFR1 was preferentially expressed in the caudal half (Fig. 6E), while FGFR2 showed the highest level of expression in the rostral half of the sclerotome (Fig. 6F).

FGF receptor expression in developing bone and tooth

FGF receptors were expressed in all phases of endochondral ossification. Both FGFR1 and FGFR2 were expressed in the precartilage blastema of the vertebrae, long bones and mandible (Figs 2 and 6 and data not shown). Later, FGFR1 was expressed prominently in hypertrophied cartilage, osteocytes and osteoblasts (Fig. 8B). FGFR2 expression, however, was concentrated in the periostium and perichondrium, and in cells within the degenerative matrix (presumptive marrow) between the hypertrophied cartilage and the newly forming bone (Fig. 8C).

Fig. 8.

Expression of FGF receptors in developing bone and tooth. Serial sections from a 16.5 day p.c. embryo were hybridized with antisense probes for FGFR1 (B. E) and FGFR2 (C, F). Panels A-C show developing longbone and panels D-F show an incisor. In A-C: s, skeletal muscle; b, developing bone; m, presumptive marrow; h, hypertrophic cartilage; c, cartilage; p, perichondrium/periostium; arrowheads indicate location of osteoblasts. In D-F: e, epidermal island; a, ameloblast layer; o, odontoblast layer; d, dental papilla. Magnification (A-F), 200×.

Fig. 8.

Expression of FGF receptors in developing bone and tooth. Serial sections from a 16.5 day p.c. embryo were hybridized with antisense probes for FGFR1 (B. E) and FGFR2 (C, F). Panels A-C show developing longbone and panels D-F show an incisor. In A-C: s, skeletal muscle; b, developing bone; m, presumptive marrow; h, hypertrophic cartilage; c, cartilage; p, perichondrium/periostium; arrowheads indicate location of osteoblasts. In D-F: e, epidermal island; a, ameloblast layer; o, odontoblast layer; d, dental papilla. Magnification (A-F), 200×.

Members of the FGF family are mitogens and morphogens for chondrocytes and osteocytes (McCarthy et al., 1989; Globus et al., 1988; Kasperk et al., 1990; Kato et al., 1987; Too et al., 1987; Kato and Gospodarowicz, 1985a, b), and can be isolated from cartilage matrix and osseous tissue (Hauschka et al., 1986; Lobb et al., 1986). Nicoll and his colleagues have shown that antibodies to basic FGF impair cartilage and bone development in embryonic rats (Liu and Nicoll, 1988). These data, and the present data demonstrating the presence of FGF receptors, suggest that FGFs participate in the development of cartilage and bone.

In mouse embryos at 16.5 days p.c., both the enamel organ and the dental papilla of the inscisor are well formed (Fig. 8D). In the enamel organ, at this stage, both FGFR1 and FGFR2 were highly expressed in the ameloblast layer (Fig. 8E, F). In the dental papilla, FGFR1 was expressed abundantly in the odontoblast layer and in the underlying mesenchyme (Fig. 8E), but FGFR2 was not detected (Fig. 8F).

FGF receptor expression during skeletal muscle differentiation

The myotomes are the earliest skeletal muscles to differentiate, giving rise to the vertebral and intercostal muscles. As shown in Fig. 6, neither FGFR1 nor FGFR2 were detected in the myotome. FGFR1, however, is expressed in premuscle mesenchyme of the limb buds from 9.5–12.5 days p.c (Fig. 2B, E). At 16.5 days, FGFR1 expression can be detected in differentiated skeletal muscle, but at a much lower level compared with expression in cartilage, bone and skin (Fig. 8B).

FGF receptor expression in the developing cardiovascular system

In the developing heart the most prominent expression of both FGF receptors was in the endocardial cushions. From 9.5 to 12.5 days p.c. expression of FGFR2 in the heart was limited to the endocardial cushions and the endothelium overlying them (Fig. 9C). FGFR1 expression, however, was more generalized; expression was detected at moderate levels in the endocardial cushions and at lower levels in the myocardium (Fig. 9B). Similarly, at 16.5 days p.c., FGFR2 expression was detected exclusively in the developing cardiac skeleton and in the atrioventricular (AV) and semilunar valves (Fig. 9F), whereas FGFR1 expression was detected at low levels in endocardial cushion derivatives and diffusely in myocardium (Fig. 9E). The localization of FGF receptors, particularly FGFR2, in the endocardial cushions and in the valve primordia suggests that FGFs play some role in cardiac septation and valve formation.

Fig. 9.

FGF receptor expression in the developing heart and aorta. Sections from 12.5 (A-C) and 16.5 (D-F) day p.c. embryos were hybridized with antisense transcripts for FGFR1 (B, E) or FGFR2 (C, F). e, endocardial cushions; m, myocardium; a, aortic outflow tract. Arrowheads indicate the developing mitral and aortic valve apparatus. Asterisks indicate blood columns in the heart and great vessels. Magnification (A-C), 200×; (D-F), 100×.

Fig. 9.

FGF receptor expression in the developing heart and aorta. Sections from 12.5 (A-C) and 16.5 (D-F) day p.c. embryos were hybridized with antisense transcripts for FGFR1 (B, E) or FGFR2 (C, F). e, endocardial cushions; m, myocardium; a, aortic outflow tract. Arrowheads indicate the developing mitral and aortic valve apparatus. Asterisks indicate blood columns in the heart and great vessels. Magnification (A-C), 200×; (D-F), 100×.

Although FGF is a potent angiogenic factor, neither FGF receptor was clearly detected in small blood vessels. In the aorta, however, FGFR1 was detected in the endothelium and smooth muscle at 12.5 days p.c. and at apparently lower levels at 16.5 days p.c. (Fig. 9B, E). FGFR2 was rarely detected in blood vessels but was detected, in smooth muscle, at branch points of the dorsal aorta (data not shown). This distribution of FGF receptors in large vessels suggests a role in the growth of pre-existing vessels.

FGF receptor expression in the embryonic nervous system

Early in brain development (9.5–10.5 days p.c.), both FGFR1 and FGFR2 were detected in the ventricular layer, the germinal cell layer from which both neurons and glia are derived (Fig. 10B, C). FGFR1 was expressed uniformly in the ventricular layer throughout the developing brain; whereas, FGFR2 was expressed more discretely with highest levels detected in the mesencephalon and the myelencephalon. In the developing spinal cord, FGFR1 was detected at relatively high levels in the developing parenchyma of the ventral horns and at lower levels in the ventricular layer (Figs 2B and 6B). FGFR2, however, was expressed at relatively high levels in the ventricular layer of the spinal cord and was not detected in the ventral horns (Figs 2C and 6C).

Fig. 10.

Expression of FGF receptors in the developing nervous system. Sagittal sections of embryos at 9.5 (A-C) and 16.5 days (D-L) were probed with radiolabeled transcripts for FGFR1 (B, E, H, K) or FGFR2 (C, F, I, L). In A-C my, myelencephalon; me, mesencephalon; d, diencephalon; t, telencephalon. In D-L v, ventricular layer; h, dentate gyruhippocampus; ce, cerebellum; cp, choroid plexus; b, brainstem; tg, trigeminal ganglion. Arrowhead in panel A indicates Rathke’s pouch and the infundibulum. Magnification (A-L), 200×.

Fig. 10.

Expression of FGF receptors in the developing nervous system. Sagittal sections of embryos at 9.5 (A-C) and 16.5 days (D-L) were probed with radiolabeled transcripts for FGFR1 (B, E, H, K) or FGFR2 (C, F, I, L). In A-C my, myelencephalon; me, mesencephalon; d, diencephalon; t, telencephalon. In D-L v, ventricular layer; h, dentate gyruhippocampus; ce, cerebellum; cp, choroid plexus; b, brainstem; tg, trigeminal ganglion. Arrowhead in panel A indicates Rathke’s pouch and the infundibulum. Magnification (A-L), 200×.

Later in brain development (16.5 days), both receptors were still detected in the ventricular layer (Fig. 10E, F). FGFR1 was also expressed in maturing neuronal populations in the dentate gyrus, hippocampus, brainstem, cerebellum and trigeminal ganglia (Fig. 10E, H, K). FGFR2 was expressed diffusely at relatively low levels outside the ventricular layer with the exception of the relatively high level of expression in the choroid plexus (Fig. 10F, I, L). These findings are consistent with expression in the adult mouse CNS where FGFR1 was expressed preferentially in neuronal populations but FGFR2 localization correlated best with expression in glia (K.P. unpublished data). Based on these findings, it is conceivable that FGFR1 and FGFR2 could provide lineage markers for specific populations of neurons and glia and that FGFs might participate in the growth and differentiation of both neurons and glia.

Conclusions

Consistent with the pleiotrophic effects of FGFs, FGFR1 and FGFR2 were detected in embryonic cells of every lineage; neuroectodermal, mesenchymal, ectodermal and endodermal. The most dramatic finding of this study was that FGFR2 was expressed predominantly in epithelium, whereas FGFR1 was expressed in mesenchyme. This pattern of mesenchymal/epithelial expression of FGFR1 and FGFR2 was observed in a variety of embryonic tissues including skin, limb, gut and respiratory tract. The functional significance of the differential distribution of these receptors is not known. It will be important to determine whether FGFR1 and FGFR2 elicit different responses at the cellular level, and whether there are stimuli, such as tissue injury, that regulate their expression differently.

FGF activity in embryonic tissues depends on both the presence of FGF responsive cells and the availability of the appropriate FGF ligand. Most published data suggest that FGFR1 and FGFR2 bind basic and acidic FGF with approximately equal affinities (Dionne et al., 1990). Recently, however, we and others have shown that FGF receptor isoforms that arise as the result of alternative mRNA splicing in the extracellular region, in particular the third Ig-like domain, display different binding characteristics from the originally isolated forms of the receptor (Johnson et al., 1991 and Eisemann et al., 1991). One such isoform of FGFR2 binds KGF (keratinocyte growth factor) and acidic FGF with fifteen-fold higher affinity than basic FGF (Miki et al., 1991). We have recently characterized a similar isoform of FGFR1 that binds acidic FGF with much higher affinity than basic FGF (S. W. in press MCB). Since the probes used in this study do not differentiate between these two isoforms of FGFR1 and FGFR2, their relative distribution is not known. In future experiments it will be important to determine if the specific receptor isoforms are expressed in the same tissues as the ligands for which they have the highest affinity.

The authors wish to thank Melanie Bedolli for expert technical advice and assistance. We also thank Lee Niswander, Marc Tessier-Lavigne, Tony Muslin and Jenny Lavail for their critical reading of the manuscript and Mike Solursh, Margaret Kirby and Ray Runyon for helpful discussions. K. P. is supported by a Physician Scientist Award from the NIH. S. W. is supported by an Otto-Hahn fellowship of the Max-Planck Gesellschaft, Munich, Germany.

Burgess
,
W.
and
Maciag
,
T.
(
1989
).
The heparin-binding (fibroblast) growth factor family of proteins
.
Annu. Rev. Biochem
.
58
,
575
606
.
Caday
,
C.
,
Klagsbrun
,
M.
,
Fanning
,
P.
,
Mirzabegian
,
A.
and
Finkelstein
,
S.
(
1990
).
Fibroblast growth factor (FGF) levels in the developing rat brain
.
Dev. Brain Res
.
52
,
241
246
.
Chirgwin
,
J.
,
Przybyla
,
A.
,
Macdonald
,
R.
and
Rutter
,
W.
(
1979
).
Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease
.
Biochemistry
18
,
5294
.
Dionne
,
C.
,
Crumley
,
G.
,
Bellot
,
F.
,
Kaplow
,
J.
,
Searfoss
,
G.
,
Ruta
,
M.
,
Burgess
,
W.
,
Jaye
,
M.
and
Schlessinger
,
J.
(
1990
).
Cloning and expression of two distinct high-affinity receptors cross-reacting with acidic and basic fibroblast growth factors
.
EMBO J
.
9
,
2685
2692
.
Eisemann
,
A.
,
Ahn
,
J.
,
Graziani
,
G.
,
Tronick
,
S.
and
Ron
,
D.
(
1991
).
Alternative splicing generates at least five different isoforms of the human basic-FGF receptor
.
Oncogene
6
,
1195
1202
.
Ekblom
,
P.
(
1989
).
Developmentally regulated conversion of mesenchyme to epithelium
.
FASEB J
.
3
,
2141
2150
.
Finch
,
P.
,
Rubin
,
J.
,
Mikl
,
T.
,
Ron
,
D.
and
Aaronson
,
S.
(
1989
).
Human KGF is FGF-related with properties of a paracrine effector of epithelial cell growth
.
Science
245
,
752
755
.
Frohman
,
M.
,
Boyle
,
M.
and
Martin
,
G.
(
1990
).
Isolation of the mouse Hox-2.9 gene; analysis of embryonic expression suggests that positional information along the anterior-posterior axis is specified by mesoderm
.
Development
110
,
589
-
607
. ’
Fu
,
Y-M.
,
Spirito
,
P.
,
Yu
,
Z-X.
,
Biro
,
S.
,
Sasse
,
J.
,
Lei
,
J.
,
Ferrans
,
V.
,
Epstein
,
S.
and
Casscells
,
VV
. (
1991
).
Acidic fibroblast growth factor in the developing rat embryo
.
J. Cell Biol
.
114
,
1261
1273
.
Glazer
,
L.
and
Shilo
,
B.
(
1991
).
The Drosophila FGF-R homolog is expressed in the embryonic tracheal system and appears to be required for directed tracheal cell extension
.
Genes Dev
.
5
,
697705
.
Globus
,
R.
,
Patterson-Buckendahl
,
P.
and
Gospodarowicz
,
D.
(
1988
).
Regulation of bovine bone cell proliferation by fibroblast growth factor and transforming growth factor-β
.
Endocrinology
123
,
98105
.
Gonzalez
,
A.
,
Buscaglia
,
M.
,
Ong
,
M.
and
Baird
,
A.
(
1990
).
Distribution of basic fibroblast growth factor in the 18-day rat fetus: Localization in the basement membranes of diverse tissues
.
J. Cell Biol
.
110
,
753
765
.
Gospodarowicz
,
D.
(
1990
).
Fibroblast growth factor and its involvement in developmental processes
.
Current Topics in Developmental Biol. 1A, 57-93
.
Hattori
,
Y.
,
Odagiri
,
H.
,
Nakatani
,
H.
,
Miyagawa
,
K.
,
Naito
,
K.
,
Sakamoto
,
H.
,
Katoh
,
O.
,
Yoshida
,
Y.
,
Suglmura
,
T.
and
Terada
,
M.
(
1990
).
K-sam, an amplified gene in stomach cancer, is a member of the heparin-binding growth factor receptor genes
.
Proc. Natl. Acad. Sci. USA
87
,
5983
5987
.
Haub
,
O.
and
Goldfarb
,
M.
(
1991
).
Expression of the fibroblast growth factor-5 gene in the mouse embryo
.
Development
112
,
397406
.
Hauschka
,
P.
,
Mavrakos
,
A.
,
Lafrati
,
M.
,
Doleman
,
S.
and
Klagsbrun
,
M.
(
1986
).
Growth factors in bone matrix: Isolation of multiple types by affinity chromatography on heparin sepharose
.
J. Biol. Chem
.
261
,
12665
12674
.
Hebert
,
J.
,
Basifico
,
C.
,
Goldfarb
,
M.
,
Haub
,
O.
and
Martin
,
G.
(
1990
).
Isolation of cDNA’s encoding four mouse FGF family members and characterization of their expression patterns during embryogenesis
.
Developmental Biol
.
138
,
454
463
.
Heuer
,
J.
,
Von Bartheld
,
C.
,
Kinoshi Ta
,
Y.
,
Evers
,
P.
and
Bothwell
,
M.
(
1990
).
Alternating Phases of FGF Receptor and NGF Receptor Expression in the Developing Chicken Nervous System
.
Neuron
5
,
283
296
.
Houssaint
,
E.
,
Blanquet
,
P.
,
Champion-Arnaud
,
P.
,
Gesnel
,
M.
,
Torriglla
,
A.
,
Courtois
,
Y.
and
Breathnach
,
R.
(
1990
).
Related fibroblast growth factor receptor genes exist in the human genome
.
Proc. Nad. Acad. Sci. USA
87
,
8189
8184
.
Johnson
,
D.
,
Lu
,
J.
,
Chen
,
H.
,
Werner
,
S.
and
Williams
,
L.
(
1991
).
The human fibroblast growth factor receptor genes: a common structural arrangement underlies the mechanisms for generating receptor forms that differ in their third immunoglobulin domain
.
Mol. Cell. Biol
.
11
,
4627
4634
.
Joseph-Silverstein
,
J.
,
Conslgli
,
S.
,
Lyser
,
K.
and
Ver Pault
,
C.
(
1989
).
Basic fibroblast growth factor in the chick embryo: Immunolocalization to striated muscle cells and their precursors
.
J. Cell Biol
.
108
,
2459
2466
.
Kasperk
,
C.
,
Wegedal
,
J.
,
Mohan
,
S.
,
Long
,
D.
,
Lau
,
K.
and
Baylink
,
D.
(
1990
).
Interactions of growth factors present in bone matrix with bone cells: Effects on DNA synthesis and alkaline phosphatase
.
Growth Factors
3
,
147
158
.
Kato
,
Y.
and
Gospodarowitcz
,
D.
(
1985a
).
Sulfated proteoglycan synthesis by confluent cultures of rabbit costal chondrocytes grown in the presence of fibroblast growth factor
.
J. Cell Biol
.
100
,
477485
.
Kato
,
Y.
and
Gospodarowicz
,
D.
(
1985b
).
Effect of exogenous extracellular matrices on proteoglycan synthesis by cultured rabbit costal chondrocytes
.
J. Cell Biol
.
100
,
486
495
.
Kato
,
Y.
,
Iwamoto
,
M.
and
Koike
,
T.
(
1987
).
Fibroblast growth factor stimulates colony formation of differentiated chondrocytes in soft agar
.
J. Cell. Physiol
.
133
,
491
498
.
Keegan
,
K.
,
Johnson
,
D.
,
Williams
,
L.
and
Hayman
,
M.
(
1991
).
Isolation of an additional member of the fibroblast growth factor receptor family, FGFR-3
.
Proc. Natl. Acad. Sci. USA
88
,
10951099
.
Kimelman
,
D.
and
Kirschner
,
M.
(
1987
).
Synergistic induction of mesoderm by FGF and TGF-beta and the identification of an mRNA coding for FGF in the early Xenopus embryo
.
Cell
51
,
869877
.
Kombluth
,
S.
,
Paulson
,
E.
and
Hanafusa
,
H.
(
1988
).
Novel tyrosine kinase identified by phosphotyrosine antibody screening of cDNA libraries
.
Mol. Cell. Biol
.
8
,
5541
5544
.
Lee
,
P.
,
Johnson
,
D.
,
Cousens
,
L.
,
Fried
,
V.
and
Williams
,
L.
(
1989
).
Purification and complementary DNA cloning of a receptor for basic fibroblast growth factor
.
Science
245
,
57
60
.
Liu
,
L.
and
Nicoll
,
C.
(
1988
).
Evidence for a role of basic fibroblast growth factor in rat embryonic growth and differentiation
.
Endocrinology
123
,
2027
2031
.
Lobb
,
R.
,
Sasse
,
J.
,
Sullivan
,
R.
,
Shing
,
Y.
,
D’amore
,
P.
,
Jacobs
,
J.
and
Klagsbrun
,
M.
(
1986
).
Purification and characterization of heparin-binding endothelial cell growth factors
.
J. Biol. Chem
.
261
,
1924
1928
.
Mccarthy
,
T.
,
Centrella
,
M.
and
Canalls
,
E.
(
1989
).
Effects of fibroblast growth factors on deoxyribonucleic acid and collagen synthesis in rat parietal bone cells
.
Endocrinology
125
,
2118
2126
.
Melton
,
D.
,
Krieg
,
P.
,
Regabliati
,
T.
,
Maniatis
,
K.
,
Zinn
,
K.
and
Green
,
M.
(
1984
).
Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter
.
Nucleic Acids Res
.
12
,
7035
7056
.
Miki
,
T.
,
Fleming
,
T.
,
Bottaro
,
D.
,
Rubin
,
J.
,
Ron
,
D.
and
Aaronson
,
S.
(
1991
).
Expression cDNA cloning of the KGF receptor by creation of a transforming autocrine loop
.
Science
251
,
72
75
.
Olwln
,
B.
and
Hauschka
,
S.
(
1990
).
Fibroblast growth factor receptor levels decrease during chick embryogenesis
.
J. Cell Biol
.
110
,
503509
.
Partanen
,
J.
,
Makela
,
T.
,
Eerola
,
E.
,
Korhonen
,
J.
,
Hlrvonen
,
H.
,
Claesson-Welsh
,
L.
and
Alitalo
,
K.
(
1991
).
FGFR-4, a novel acidic fibroblast growth factor receptor with a distinct expression pattern
.
EMBO J
.
10
,
1347
1354
.
Pasquale
,
E.
(
1990
).
A distinctive family of embryonic proteintyrosine kinase receptors
.
Proc. Natl. Acad. Sci. USA
87
,
58125816
.
Reid
,
H.
,
Wilks
,
A.
and
Bernard
,
O.
(
1990
).
Two forms of the basic fibroblast growth factor receptor-like mRNA are expressed in the developing mouse brain
.
Proc. Natl. Acad. Sci. USA
87
,
1596
1600
.
Rifkin
,
D.
and
Moscatelli
,
B.
(
1989
).
Recent developments in the cell biology of basic fibroblast growth factor
.
J. Cell Biol
.
109
,
1
6
.
Ruta
,
T.
,
Howk
,
R.
,
Ricca
,
G.
,
Drohan
,
W.
,
Zabelshansky
,
M.
,
Laureys
,
G.
,
Barton
,
D.
,
Franke
,
U.
,
Schlessinger
,
J.
and
Givol
,
D.
(
1988
).
A novel protein tyrosine kinase gene whose expression is modulated during endothelial cell differentiation
.
Oncogene
3
,
915
.
Safran
,
A.
,
Avivi
,
A.
,
Orr-Urtereger
,
A.
,
Neufeld
,
G.
,
Lonai
,
P.
,
Givol
,
D.
and
Yarden
,
Y.
(
1990
).
The murine flg gene encodes a receptor for fibroblast growth factor
.
Oncogene
5
,
635
645
.
Schnurch
,
H.
and
Risau
,
W.
(
1991
).
Differentiating and mature neurons express the acidic fibroblast growth factor gene during chick neural development
.
Development
111
,
1143
1154
.
Slack
,
J.
,
Darlington
,
B.
,
Heath
,
J.
and
Godsave
,
S.
(
1987
).
Mesoderm induction in early Xenopus embryos by heparin-binding growth factors
.
Nature
326
,
197
203
.
Stem
,
C.
and
Keynes
,
R.
(
1986
).
Cell lineage and the formation and maintenance of half somites
. In
Somites in Developing Embryos
(
Bellairs
,
R.
,
Ede
,
D.
and
Lash
,
J. eds.
), pp.
147
159
.
Too
,
C.
,
Murphy
,
P.
,
Hamel
,
A-M.
and
Friesen
,
H.
(
1987
).
Further purification of human pituitary-derived chondrocyte growth factor: Heparin-binding and cross-reactivity with antiserum to basic FGF
.
Biochem. Biophys. Res. Com
.
144
,
1128
1134
.
Wanaka
,
A.
,
Milbrandt
,
J.
and
Johnson
,
E.
(
1991
).
Expression of FGF receptor gene in rat development
.
Development
111
,
455
468
.
Wilkinson
,
D.
,
Bailes
,
J.
,
Champion
,
J.
and
Mcmahon
,
A.
(
1987
).
A molecular analysis of mouse development from 8 to 10 days post coitum detects changes only in embryonic globin expression
.
Development
99
,
493
500
.
Wilkinson
,
D.
,
Bhatt
,
S.
and
Mcmahon
,
A.
(
1989
).
Expression of the FGF-related proto-oncogene int-2 suggests multiple roles in fetal development
.
Development
105
,
131
136
.