Soluble dominant-negative receptor uncovers essential roles for fibroblast growth factors in multi-organ induction and patterning
1998; Springer Nature; Volume: 17; Issue: 6 Linguagem: Inglês
10.1093/emboj/17.6.1642
ISSN1460-2075
Autores Tópico(s)Genetic Syndromes and Imprinting
ResumoArticle16 March 1998free access Soluble dominant-negative receptor uncovers essential roles for fibroblast growth factors in multi-organ induction and patterning Giulia Celli Giulia Celli Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD, 20892 USA Search for more papers by this author William J. LaRochelle William J. LaRochelle Laboratory of Cellular and Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD, 20892 USA Search for more papers by this author Susan Mackem Susan Mackem Laboratory of Pathology, National Cancer Institute, National Institutes of Health, Bethesda, MD, 20892 USA Search for more papers by this author Richard Sharp Richard Sharp Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD, 20892 USA Search for more papers by this author Glenn Merlino Corresponding Author Glenn Merlino Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD, 20892 USA Search for more papers by this author Giulia Celli Giulia Celli Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD, 20892 USA Search for more papers by this author William J. LaRochelle William J. LaRochelle Laboratory of Cellular and Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD, 20892 USA Search for more papers by this author Susan Mackem Susan Mackem Laboratory of Pathology, National Cancer Institute, National Institutes of Health, Bethesda, MD, 20892 USA Search for more papers by this author Richard Sharp Richard Sharp Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD, 20892 USA Search for more papers by this author Glenn Merlino Corresponding Author Glenn Merlino Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD, 20892 USA Search for more papers by this author Author Information Giulia Celli1, William J. LaRochelle2, Susan Mackem3, Richard Sharp1 and Glenn Merlino 1 1Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD, 20892 USA 2Laboratory of Cellular and Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD, 20892 USA 3Laboratory of Pathology, National Cancer Institute, National Institutes of Health, Bethesda, MD, 20892 USA *Corresponding author. E-mail: [email protected] The EMBO Journal (1998)17:1642-1655https://doi.org/10.1093/emboj/17.6.1642 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Despite a wealth of experimental data implicating fibroblast growth factor (FGF) signaling in various developmental processes, genetic inactivation of individual genes encoding specific FGFs or their receptors (FGFRs) has generally failed to demonstrate their role in vertebrate organogenesis due to early embryonic lethality or functional redundancy. Here we show that broad mid-gestational expression of a novel secreted kinase-deficient receptor, specific for a defined subset of the FGF superfamily, caused agenesis or severe dysgenesis of kidney, lung, specific cutaneous structures, exocrine and endocrine glands, and craniofacial and limb abnormalities reminiscent of human skeletal disorders associated with FGFR mutations. Analysis of diagnostic molecular markers revealed that this soluble dominant-negative mutant disrupted early inductive signaling in affected tissues, indicating that FGF signaling is required for growth and patterning in a broad array of organs and in limbs. In contrast, transgenic mice expressing a membrane-tethered kinase-deficient FGFR were viable. Our results demonstrate that secreted FGFR mutants are uniquely effective as dominant-negative agents in vivo, and suggest that related soluble receptor isoforms expressed in wild-type mouse embryos may help regulate FGF activity during normal development. Introduction Fibroblast growth factors (FGFs) belong to a superfamily of signaling molecules thought to regulate cellular proliferation, migration and differentiation by binding to and activating members of a family of tyrosine kinase receptors (FGFRs). Mutations in FGFRs are associated with heritable human skeletal disorders involving craniofacial and limb anomalies, including Apert, Crouzon, Pfeiffer, Jackson–Weiss and Beare–Stevenson cutis gyrata syndromes, and achondroplasia (Johnson and Williams, 1993; Mason, 1994; Muenke and Schell, 1995; Przylepa et al., 1996). FGFR proteins, encoded by four genes, are characterized by three immunoglobulin (Ig)-like domains (designated either as loops I, II and III, or D1, D2 and D3) within the extracellular region, a single transmembrane region and a split cytoplasmic tyrosine kinase domain. Although both loops II and III are involved in ligand binding, ligand specificity is determined by the C-terminal portion of loop III (Givol and Yayon, 1992; Johnson and Williams, 1993; Cheon et al., 1994). For example, alternative splicing of FGFR2 results in the creation of the differentially responsive receptor isoforms designated FGFR2IIIb and FGFR2IIIc (here abbreviated FGFR2b and FGFR2c, respectively) (Miki et al., 1992). FGFR2b binds efficiently to aFGF, FGF3, FGF7 and FGF10 (A.Blunt and D.Ornitz, personal communication) in cultured BaF3 cells, while FGFR2c binds to aFGF, bFGF, FGF4, FGF6 and FGF9 (Ornitz et al., 1996, and references therein). Ligand binding induces receptor dimerization and intermolecular transphosphorylation of the receptor subunits, thereby initiating FGFR signaling. The regulation of FGFR signaling is intricate and complex because the multiple ligand and receptor isoforms that exist share overlapping recognition and redundant specificity (Givol and Yayon, 1992; Johnson and Williams, 1993). Further complexity is contributed by the generation of membrane-bound and secreted receptor isoforms from the same gene, and by interactions of FGF ligands with heparan sulfate proteoglycans on the cell surface and extracellular matrix (Givol and Yayon, 1992; Johnson and Williams, 1993). FGFs and their receptors have been implicated as regulators of vertebrate development based on analysis of embryonic expression patterns (Wilkinson et al., 1989; Orr-Urtreger et al., 1991, 1993; Stark et al., 1991; Niswander and Martin, 1992; Peters et al., 1993; Crossley and Martin, 1995; Yamasaki et al., 1996; Neubüser et al., 1997; Ohuchi et al., 1997). Gene targeting studies have provided compelling genetic evidence that FGFR signaling is involved in early developmental processes such as blastocyst growth, primary mesoderm induction and early pattern formation (Amaya et al., 1991; Deng et al., 1994; Yamaguchi et al., 1994; Feldman et al., 1995). A later developmental role in mediating mesenchymal–epithelial cell interactions during the formation of structures such as limb and lung has been suggested through use of FGF implants, antisense oligonucleotides and neutralizing antibodies (Represa et al., 1991; Niswander and Martin, 1993; Niswander et al., 1993; Cohn et al., 1995; Nogawa and Ito, 1995; Crossley et al., 1996; Post et al., 1996; Ohuchi et al., 1997). However, obtaining more definitive genetic data for a role in secondary inductive events and organogenesis from gene targeting approaches has been problematic due to early embryonic lethality prior to organ induction, as with null mutations in FGFR1, FGF4, FGF8 and FGFR2 (Deng et al., 1994; Yamaguchi et al., 1994; Feldman et al., 1995; C.Deng, personal communication; E.Meyers and G.Martin, personal communication), or conversely due to functional redundancy within both the FGF and FGFR families, yielding little or no embryonic phenotype. It has been shown that inactivation of FGF3 perturbs the development of the ear and tail (Mansour et al., 1993), loss of FGF5 or FGF7 function results in abnormal hair growth and development (Hébert et al., 1994; Guo et al., 1996), FGFR3 null mutants demonstrate prolonged enchondral bone growth and inner ear defects (Colvin et al., 1996; Deng et al., 1996), FGF6 inactivation perturbs adult skeletal muscle regeneration (Floss et al., 1997), and bFGF null mice show no overt phenotypes (S.Ortega and C.Basilico, personal communication). While chimeric embryos demonstrate a partial rescue and a delay in the early lethality associated with FGFR1 inactivation (Ciruna et al., 1997; Deng et al., 1997), their phenotypes are strongly influenced by generally unpredictable competitive interactions between wild-type and null embryonic cells. Dominant-negative transgenic strategies have been used with mixed success to overcome functional redundancies and early embryonic lethality. Truncated membrane-bound FGFRs that lack a functional tyrosine kinase domain have been shown in vitro to disrupt FGFR signaling of multiple receptor isoforms by competing for ligand binding and forming inactive heterodimers with endogenous FGFRs (Ueno et al., 1992). In vivo, this dominant-negative approach has been used to show that FGFs are required for Xenopus gastrulation (Amaya et al., 1991), and also for epidermal organization, differentiation and wound healing (Werner et al., 1993, 1994), branching morphogenesis of the lung (Peters et al., 1994), lens development (Robinson et al., 1995) and lobuloalveolar development of the mammary gland (Jackson et al., 1997). However, the efficacy of defective membrane-bound receptors is limited by the spatial and temporal expression properties of the transcriptional promoter directing expression of the transgene to specific cell types, and by the need to be greatly overexpressed in order to compete effectively for ligand binding with native receptors at the cell surface. We have overcome many of these limitations through the use of a secreted soluble dominant-negative receptor (DNR) mutant which can bind a specific subset of FGFs extracellularly and disrupt signaling of virtually all FGFR isoforms. By using a metallothionein (MT) promoter to express this soluble DNR mutant broadly in mouse embryos, we could demonstrate that FGFR signaling is required for the normal development of multiple organs, including kidney, lung and specific endocrine and exocrine glands, as well as of a variety of craniofacial and cutaneous structures and limbs. Results Efficacy of dominant-negative FGFR2 mutants in vitro The extracellular ligand-binding domain of the mouse FGFR2b cDNA, containing both D2 and D3 Ig-like domains, was used to generate two truncated DNR forms (Figure 1A). In one form, the transmembrane domain was retained, anchoring it to the cell membrane (dnFGFR-Tm), while in the other the transmembrane domain was replaced by the mouse Ig heavy chain hinge and Fc domains, thereby creating a stable chimeric protein (dnFGFR-HFc), secreted as a disulfide-linked dimer (Cheon et al., 1994) (Figure 1A). To test and compare their biological function in vitro, the two receptor mutants were overexpressed using the mouse MT I gene promoter (MMTneo vector) in Balb/MK cells, an epidermal cell line which expresses the native FGFR2b (Bottaro et al., 1990). Both dominant-negative receptor forms selectively reduced the mitogenic activity of aFGF and FGF7 (Figure 1B), but not that of epidermal growth factor (EGF), hepatocyte growth factor (HGF) or insulin-like growth factor-1 (IGF-1) (not shown), indicating that they were equally effective dominant-negative receptor forms in vitro. Figure 1.Structure and in vitro activity of dominant-negative FGFR2 mutants. (A) Schematic diagram of native and dominant-negative receptors, and transgene construct. D2 and D3, Ig-like loops 2 and 3 of FGFR2b extracellular ligand-binding domain; Tm, transmembrane region; TK, split tyrosine kinase domain; CH2 and CH3, constant regions 2 and 3 of Ig heavy chain; and the hinge region (zigzag line). The transgene consists of the mouse metallothionein (MTp) promoter and its flanking locus control regions (LCRs), the human growth hormone polyadenylation signal (hGHpA) and the cDNA of either truncated or chimeric FGFR2b. (B) Balb/MK cells were transfected with either dnFGFR-Tm or dnFGFR-HFc, and the activity of each mutant was measured as inhibition of the mitogenic response to aFGF and FGF7. (C) The ligand-binding specificity of the soluble mutant was determined by the ability of increasing amounts of purified dnFGFR-HFc to block the proliferative response of NIH 3T3 cells to distinct FGFs. (D) Starved NIH 3T3 cells were treated with aFGF or bFGF in the presence or absence of purified dnFGFR-HFc. Cellular extracts were immunoprecipitated (IP) and subsequently probed with either anti-PY or anti-FGFR1 antibodies. Mouse FGFR1 (arrowheads) had an apparent mol. wt of 130–145 kDa. Download figure Download PowerPoint The ligand-binding specificity of dnFGFR-HFc was tested in a mitogenic assay using serum-starved NIH 3T3 cells treated overnight with increasing concentrations of purified dnFGFR-HFc pre-incubated with 1 ng/ml aFGF, bFGF, FGF3 or FGF4. The mitogenic activity, measured as [3H]thymidine incorporation, is shown in Figure 1C. As expected (Ornitz et al., 1996), dnFGFR-HFc inhibited the mitogenic response to aFGF completely and to FGF3 less effectively, but did not affect bFGF or FGF4 activity. To determine that the dnFGFR-HFc mutant was actually blocking signaling through endogenous FGFRs, quiescent NIH 3T3 mouse fibroblasts were exposed to aFGF or bFGF pre-incubated with increasing amounts of the purified soluble DNR. Endogenous FGFR1 was immunoprecipitated with either anti-phosphotyrosine or anti-FGFR1 antibodies, and analyzed for phosphorylation levels by immunoblot analysis. The dnFGFR2-HFc mutant blocked phosphorylation of FGFR1 in response to aFGF but not bFGF, indicating that the soluble chimera functions as a dominant-negative inhibitor by interfering with specific FGFR signaling (Figure 1D). Soluble, but not tethered, dominant-negative FGFR2 mutant induces embryonic lethality To determine the in vivo consequences of perturbing FGFR signal transduction, the mouse MT promoter with flanking locus control regions (LCRs) was employed to achieve broad reproducible expression (Takayama et al., 1996) of either the dnFGFR-HFc or dnFGFR-Tm mutant forms in transgenic mice. Attempts to establish MT-dnFGFR-HFc transgenic lines were unsuccessful (0/89 potential founders screened), raising the possibility that expression of the soluble chimera was incompatible with normal mouse development. Therefore, mouse zygotes micro-injected with the MT-dnFGFR-HFc transgene were allowed to develop to 18.5 days post-coitum (d.p.c.) and harvested for analysis. Fifteen percent of viable fetuses were grossly abnormal, all of which harbored the MT-dnFGFR-HFc transgene (Table I). All abnormal transgenic fetuses were small and displayed limb truncations of varying degrees of severity, ranging from shortening of the most distal phalangeal elements to complete loss of the appendage (Figure 2A). Typically, limb phenotypes were accompanied by craniofacial anomalies, curly tails, thin featureless skin and open eyes with opacities. Transgenic embryos were also identified at 12.5 d.p.c., about half of which were smaller with related limb and head phenotypes (Figure 2B; Table I). Transgenic embryos at 9.5 d.p.c were unremarkable. Figure 2.Expression of a soluble FGFR2 mutant induces embryonic lethality. (A) E18.5 and (B) E12.5 transgenic (right) and control (left) mouse embryos. (C–F) Immunohistochemical detection of soluble FGFR2 mutant in transgenic (D and F) relative to control (C and E) E12.5 embryos using an anti-HFc antibody. Note the cytoplasmic and extracellular staining in transgenic muscle mass (D) and spinal cord (F). Magnification: 630×. (G) Immunoblot analysis of total protein lysates from wild-type (wt) and transgenic (tg) E12.5 embryos without (−) and with (+) an overt phenotype. The concentration of the purified FGFR2 chimera ranges from 5 to 100 ng per lane, while for each embryo, 5 and 100 μg were loaded per lane. The blot was probed with anti-mouse IgG (Fc). The chimeric protein (arrow) was 80 kDa. Download figure Download PowerPoint Table 1. Genotype and gross morphology of soluble FGFR2 mutant transgenic embryos Age (days) No. of viable embryos No. of TG embryos Overt phenotypesa 9.5 26 3 (12%) – 12.5 155 20 (13%) 9 (45%) 18.5 66 10 (15%) 9 (90%) aOf 10 E18.5 transgenic (TG) embryos, nine exhibited head, eye and limb abnormalities, and eight had a curly tail. Of 20 E12.5 embryos, nine had morphologically aberrant head and limbs. Variability in the penetrance and expressivity of the phenotypes demonstrated in these ‘transient’ transgenic embryos could be explained by spatial and/or temporal differences in transgene expression caused by genomic sequences near the site of integration. In situ hybridization revealed that the MT-dnFGFR-HFc transgene was broadly expressed (not shown), and the protein was found to be localized extracellularly throughout the embryo (Figure 2D and F). To determine if the variability in embryonic phenotype was dependent on the level of transgene expression achieved by mid-gestation, protein lysates were prepared from two transgenic mid-gestation embryos, one with and one without overt head and limb phenotypes, and quantified by Western blot analysis using an anti-mouse IgG (Fc) antibody. Figure 2G shows that the MT-dnFGFR-HFc transgene was highly expressed by 12.5 d.p.c. in the embryo displaying the severe phenotype, while the overtly normal embryo had little or no transgene expression at that time. To demonstrate that expression of native FGFRs was not affected by expression of the soluble chimera, the same blot was stripped and probed in turn with antibodies to FGFR1 and FGFR2. No detectable differences were observed in receptor protein levels between control and transgenic littermates (not shown), indicating that the soluble DNR mutant does not overtly alter native receptor physiology. In striking contrast to results using the MT-dnFGFR-HFc transgene, three viable founder lines carrying the MT-dnFGFR-Tm transgene were generated readily (3/22 potential founders screened). These mice were characterized by strong transgene expression in many adult tissues, including liver, kidney and gastrointestinal tract and, in two of three lines, by expression in mid-gestation embryos, as well as by relatively mild changes in the skin (not shown), resembling those reported previously in mice in which a K14 promoter was used to target expression of a similar membrane-bound FGFR2 kinase-deficient mutant (Werner et al., 1994). Therefore, the soluble chimera was far superior to the membrane-bound form at perturbing development, and much more effective as a dominant-negative agent. Soluble FGFR causes developmental abnormalities reminiscent of human skeletal disorders Recently, mutations in genes encoding FGFRs have been detected in a cluster of clinically related human autosomal dominant skeletal disorders involving craniofacial and limb anomalies (reviewed in Muenke and Schell, 1995; Mulvihill, 1995). Nine of 10 E18.5 MT-dnFGFR-HFc embryos displayed obvious anomalous limb and craniofacial features (Figure 3A and F). It was therefore of great interest to examine appendicular and craniofacial skeletal development in these transgenic embryos. Figure 3.Skeletal phenotypes in dnFGFR-HFc transgenic embryos. (A) Transgenic (TG) E18.5 embryos with increasingly severe limb phenotypes and a wild-type (WT) littermate. Note that the level of truncations is more proximal in hind than forelimb. (B) Normal forelimb skeleton stained with alizarin red and alcian blue compared with those of transgenic (C and D) embryos shown in (A). Inserts show normal carpal bones at the distal end of truncated limb compared with wild-type. Frontal view of normal (E) and transgenic (F) littermates. Note the open eyes, and the loss of toenails and the soft tissue syndactyly in the transgenic forelimb. Ventral (G and H) and side (I and J) views of skulls from control (G and I) and transgenic (H and J) embryos. Palantine bone (p), otic capsule (oc) and maxillary bone (m) are indicated. (L) Rudimentary pelvis from mutant embryo shown on the far right in (A), compared with that of a normal littermate (K). Download figure Download PowerPoint Limb bud outgrowth and determination of limb elements in a proximal to distal sequence (i.e. hip to toe) depend upon continuous FGF signals from the apical ectodermal ridge, or AER (Niswander et al., 1993; Fallon et al., 1994, and references therein). In transgenic embryos, truncations of forelimb and hindlimb skeleton occurred at different levels along the proximodistal limb axis (Figure 3A–D) within a phenotypic range that recapitulated the results of AER removal at varying times in the chick wing bud (Saunders, 1948), and presumably reflected the time of onset of transgene expression in a given embryo. Development of the normal mouse hindlimb is delayed by about half a day relative to the forelimb and, accordingly, truncations were always seen at a more proximal level in the hindlimb compared with the forelimb (Figure 3A). Notably, in the most severely affected E18.5 embryos, only a rudimentary partial pelvis was formed (Figure 3L), clearly establishing a requirement for FGF signaling in pelvic development. The least affected embryos displayed absent toenails and very mild soft tissue syndactyly in distal appendages (Figure 3F). Analysis of the skull of E18.5 transgenic fetuses revealed a widely cleft palate, reduced maxillary bone, absent otic capsule and rudimentary inner ear structures (Figure 3H and J). Therefore, the presence of the dnFGFR-HFc disrupted development of many skeletal tissues demonstrating abnormalities in FGFR-associated human syndromes. Soluble FGFR dominant-negative mutant disrupts the formation of specific organs Detailed histopathological analysis of E18.5 embryos expressing the soluble dnFGFR mutant revealed developmental anomalies in multiple organs (Table II), all of which demonstrate embryonic expression of FGFR1 and/or FGFR2 (Orr-Urtreger et al., 1991, 1993; Peters et al., 1992, 1993). In the most severely affected transgenic embryos, the kidneys failed to develop (Figure 4G). Occasionally a transgenic embryo demonstrated unilateral formation of a very small right kidney, which appeared disorganized and was characterized by small glomeruli and enlarged vacuolated tubules (Figure 4N). Although visceral and parietal pleura were present, the lungs routinely were absent (Figure 4B); the respiratory system consisted only of the trachea and rudimentary primary bronchi, which typically ended abruptly without further branching. However, in one of nine transgenic embryos analyzed, pulmonary branching morphogenesis had been initiated and then prematurely arrested, presumably due to a delay in transgene expression (Figure 4C). Figure 4.Agenesis or dysgenesis of multiple organs in E18.5 embryos expressing the soluble FGFR2 dominant-negative mutant. (A and B) Transverse section through the chest cavity of wild-type (A) and transgenic (B) embryos. Note the absence of lungs and the enlarged heart in (B). (C) Prematurely arrested bronchial branching morphogenesis found in one transgenic embryo. (D and E) Thyroid and salivary glands (arrowheads) in wild-type embryo (D) are absent in mutant embryo (E). (F–H) Transverse section through the abdominal cavity of wild-type (F) and two transgenic embryos (G and H). The arrowhead in (H) points to a unilateral kidney rudiment of a transgenic embryo (see N). (I and J) Transverse section through the inner ear of control (I) and transgenic (J) embryos. (K and L) Tooth buds (arrowheads) in wild-type (K) are absent in transgenic embryo (L). (M and N) Higher magnification of normal embryonic E18.5 kidney and adrenal gland (M) compared with the dysgenic kidney found in some transgenic embryos (N). (O and P) Thymus gland of control (O) and transgenic (P) embryo; note the absence of distinct medulla and cortex. (Q and R) The glandular stomach (arrowhead) of a wild-type embryo (Q) appears to be replaced by non-glandular epithelium in a transgenic littermate (R). (S and T) Liver and exocrine pancreas of a non-transgenic embryo (S) compared with the same tissues of a mutant embryo (T). In the latter, the pancreas is limited to the hypoplastic and morphologically atypical gland shown (T). a, adrenal gland; c, thymic cortex; h, heart; k, kidney; l, liver; lu, lung; m, thymic medulla; p, pancreas; s, stomach. Original magnification: (A, B and F–H) 25×; (C, I, J, M–P, S and T) 200×; (D, E, K and L) 50×; (Q and R) 100×. Download figure Download PowerPoint Table 2. Incidence of histological abnormalities in E18.5 embryos expressing the soluble FGFR2 mutant Organ No. of embryos examined Dysgenesisa Agenesisa Lung 8 – 8 Thyroid 5 – 5 Anterior pituitary 3 – 3 Tooth buds 6 – 6 Salivary glands 5 – 5 Kidney 8 3 5 Hair follicles 8 4 4 Inner ear 8 8 – Thymus 8 8 – Glandular stomach 8 8 – Pancreas 8 8 – aDysgenesis is defined as malformation or reduction in number or size, and agenesis as absence of organ. See text for details. Figure 4 also shows that in the head/neck region tooth buds did not form (Figure 4L), the thymus was small, hypoplastic and lacked a defined medulla and cortex (Figure 4P), the thyroid, salivary and pharyngeal serous glands were missing (Figure 4E, and not shown), the eyes were relatively small and never formed eyelids, and only a rudiment of the inner ear was present (Figure 4J). Furthermore, the central nervous system appeared hypocellular and disorganized, and the anterior pituitary was missing (not shown). In the abdomen, in addition to renal agenesis, the adrenal gland occasionally appeared hypoplastic and cytologically abnormal (Figure 4N). Of the gastrointestinal organs, the pancreas consisted of a greatly reduced number of morphologically aberrant acinar cells and no detectable islets (Figure 4T), the liver was smaller and exhibited increased red blood cell hematopoiesis (Figure 4G, H and T) and the glandular portion of the stomach was reduced or missing (Figure 4G and R). In contrast, the small and large intestine appeared overtly normal (Figure 4G and H), as did reproductive organs. The most striking change in the transgenic E18.5 skin, which clearly expressed the soluble DNR protein (Figure 5D), was in hair follicle development. In some embryos, hair follicles were reduced in number by 40–60%, while in the most severely affected embryos they were absent (Figure 5B and F; Table II). The transgenic epidermis was very thin relative to wild-type. Keratin K14 staining in the transgenic epidermis was unremarkable, as was staining for K1, loricrin and neural cell adhesion molecule (N-CAM) (Figure 5F and H, and data not shown). In contrast, K6 expression, which in wild-type E18.5 skin was undetectable, was high throughout the transgenic epidermis (Figure 5J). Relative to age-matched controls, cellular proliferation was reduced by 55% (2.2 versus 1.0 mitotic figures/mm) and 61% (3.6 versus 1.4 figures/mm) in the E18.5 transgenic epidermis and dermis, respectively. Moreover, in the epidermis of transgenic embryos lacking hair follicles, apoptotic cells were reduced by 71% relative to controls (21 versus 6 cells/mm) (Figure 5L). These results suggest that the normal program of differentiation had been significantly altered in embryonic skin expressing the soluble DNR. Figure 5.Expression of keratin K6 and reduced apoptosis in epidermis of dnFGFR-HFc transgenic embryos reflects an altered program of differentiation. Dorsal skin of E18.5 wild-type (A, C, E, G, I and K) and transgenic (B, D, F, H, J and L) embryos stained with H&E (A and B) or subjected to immunohistochemistry to detect the transgene protein (C and D), keratin markers K14 (E and F), K1 (G and H) and K6 (I and J) or apoptotic cells (K and L). Original magnification: 200×. Download figure Download PowerPoint Inhibition of FGFR signaling blocks inductive interactions and outgrowth To determine if dnFGFR-HFc expression disrupted mesenchymal–epithelial inductive interactions in affected appendages and organs, mid-gestational embryos were subjected to in situ hybridization using a variety of diagnostic molecular markers. In the normal developing limb, expression of the homeobox gene Msx-1, but not Msx-2, in the distal mesenchyme requires the presence of a functional AER (Wang and Sassoon, 1995, and references therein). The limb bud rudiment in a severely affected transgenic E12.5 embryo was identified by the presence of an anterior Msx-2 domain (Figure 6D). However, transcripts for Msx-1 were undetectable in mesenchyme or ectoderm (Figure 6B), suggesting that a functional as well as morphological AER was lost by E12.5, or had never formed. In another E12.5 transgenic embryo, in which limb buds clearly were initiated and then arrested, distal ‘progress zone’ mesenchymal proliferation was virtually absent (Figure 6F). In contrast, in the more proximal pre-cartilaginous condensations, proliferation was still active at this time. Figure 6.Diagnostic molecular markers demonstrate disruption of induction and outgrowth in embryos expressing the soluble FGFR2 chimera. Analysis of transcripts by in situ hybridization (A–D and G–J), or of BrdU incorporation by immunohistochemistry (E and F). Shown are wild-type (A, C, E, G and I) and transgenic (B, D,
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