Fibroblast growth factors (FGFs) are essential molecules for mammalian development. The nine known FGF ligands and the four signaling FGF receptors (and their alternatively spliced variants) are expressed in specific spatial and temporal patterns. The activity of this signaling pathway is regulated by ligand binding specificity, heparan sulfate proteoglycans, and the differential signaling capacity of individual FGF receptors. To determine potentially relevant ligand-receptor pairs we have engineered mitogenically responsive cell lines expressing the major splice variants of all the known FGF receptors. We have assayed the mitogenic activity of the nine known FGF ligands on these cell lines. These studies demonstrate that FGF 1 is the only FGF that can activate all FGF receptor splice variants. Using FGF 1 as an internal standard we have determined the relative activity of all the other members of the FGF family. These data should serve as a biochemical foundation for determining developmental, physiological, and pathophysiological processes that involve FGF signaling pathways. Fibroblast growth factor (FGF)1 was identified as an activity that stimulates the proliferation of NIH3T3 cells (1). Currently, FGFs comprise a family of nine structurally related proteins (FGF 1-9). FGFs are expressed in specific spatial and temporal patterns and are involved in developmental processes, angiogenesis, wound healing, and tumorigenesis (2-5).FGFs bind and activate high-affinity receptor tyrosine kinases. The cloning of FGF receptors (FGFRs) has identified four distinct genes (6 -13). These receptors bind members of the FGF family with varying affinity (13-16), and alternative mRNA splicing leads to isoforms of these receptors which have unique ligand binding properties (15,17,18). An additional mechanism regulating FGF activity involves heparin or heparan sulfate proteoglycans, molecules which facilitate ligandreceptor interactions (12,19,20). FGFRs contain an extracellular ligand binding domain, a single transmembrane domain, and an intracellular tyrosine kinase domain. The extracellular domain determines ligand binding specificity and mediates ligand-induced receptor dimerization. Dimerization in turn results in one or more trans-phosphorylation events and the subsequent activation of the receptor (21).The extracellular region of the FGFR contains three immunoglobulin-like (Ig-like) domains (6). Alternative mRNA splicing creates several forms of the FGF receptor which differ in their extracellular sequence and have unique ligand binding properties. One splicing event results in the skipping of exons encoding the amino-terminal Ig-like domain (domain I) resulting in a "short" two Ig-like domain form of the receptor (22). The ligand binding properties of the short (two Ig-like domain) and long (three Ig-like domain) FGFRs are similar.2 However, the short form of the receptor may have a higher affinity for some FGFs than the long form (23). Changes in this alternative splicing pattern may correlate with the progression of se...

We provide evidence that FGF8 serves as an endogenous inducer of chick limb formation and that its expression in the intermediate mesoderm at the appropriate time and place to trigger forelimb development is directly linked to the mechanism of embryonic kidney differentiation. One function of the limb inducer is to initiate Fgf8 gene expression in the ectoderm overlying the prospective limb-forming territories. FGF8 secreted by the ectoderm then appears to initiate limb bud formation by promoting outgrowth of and Sonic hedgehog expression in the underlying lateral plate mesoderm. FGF8 also maintains mesoderm outgrowth and Sonic hedgehog expression in the established limb bud. Our data thus point to FGF8 as a key regulator of limb development that not only induces and initiates the formation of a limb bud, but also sustains its subsequent development.

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Transgenic mice carrying the Wnt-1 protooncogene modified for expression in mammary epithelial cells exhibit hyperplastic mammar glands and stochastically develop mammary carcinomas, suggesting that additional events are necessary for tumorigenesis. To induce such events and to identify the genes involved, we have infected Wnt-1 transgenic mice with mouse mammary tumor virus (MMTV), intending to insertionally activate, and thereby molecularly tag, cooperating protooncogenes. Infection of breeding female Wnt-1 transgenics decreased the average age at which tumors appeared from =4 months to --2.5 months and increased the average number of primary tumors per mouse from 1-2 to >5. A smaller effect was observed in virgin females, and infection of transgenic males showed no ficant effect on tumor latency.More than half of the tumors from the infected breeding group contained one or more newly acquired MMTV proviruses in a pattern suggesting that most cells in tumors arose from a single infected cell. Analyses of provirus-containing tumors for induced or altered expression of int-2/Fgf-3, hst/Fgf-4, int-3, and Wnt-3 showed activation of int-2 in 39% of tumors, hst in 3%, and both int-2 and hst in 3%. DNA analyses with probes for protooncogenes and MMTV confirmed that the activations resulted from proviral insertions. There was no evidence for proviral insertions at the int-3, Wnt-3, or Wnt-1 loci. These findings provide further evidence that fibroblast growth factors Int-2 and Hst can cooperate with Wnt-1, another secreted factor, in mammary tumorigenesis, and they illustrate the capacity of this system to identify cooperating oncogenes.The oncogenic potential of Wnt-J (formerly int-i) was initially inferred from its frequent transcriptional activation by the nearby insertion of mouse mammary tumor virus (MMTV) proviruses in mammary tumors of infected mice (1). Expression of Wnt-1 in mouse mammary epithelial cell lines imparts some characteristics of transformation (2, 3); however, the generation of mice carrying a Wnt-J transgene under the influence of a MMTV enhancer was required to provide firm proofofthe oncogenic effects of Wnt-i. Both male and female transgenic mice develop mammary adenocarcinomas and, less frequently, salivary gland adenocarcinomas (4).The median latency of mammary tumor formation in female Wnt-J transgenics is -5 months of age, with >80%1o of mice developing tumors by the age of 7 months (4). Males develop tumors later in life and less frequently. Wnt-J transgenic mice of both sexes also display a marked hyperplasia of the mammary gland with extensive lobular alveolar development. This mammary hyperplasia, coupled with the delayed kinetics and sporadic nature of tumor development, argues that Wnt-J contributes to, but is not sufficient for, tumorigenesis in these mice. Other events, presumably genetic, are necessary for progression to neoplasia. In an attempt to identify genes that may be involved in multistep tumorigenesis in this model system, we have mutagenized Wnt-1 transgenic mice...

Fibroblast growth factors are essential molecules for development. Here we characterize Fgfl7, a new member of the fibroblast growth factor (FGF) family. The Fgfl7 gene maps to mouse chromosome 14 and is highly conserved between mouse and human (93% identity). It exhibits 60% amino acid identity with Fgf8 and 50% identity with Fgf8. Both Fgf8 and Fgf17 have a similar structure and a similar pattern of alternative splicing in the 5' coding region. When expressed in 3T3 fibroblasts, mouse FGF17 is transforming, indicating that it can activate the 'c' splice form of either FGF receptor (FGFR) one or two. During midgestation embryogenesis, in situ hybridization analysis localized Fgf17 expression to specific sites in the midline structures of the forebrain, the midbrain-hindbrain junction, the developing skeleton and in developing arteries. Comparison to Fgf8 revealed a striking similarity in expression patterns, especially in the central nervous system (CNS), suggesting that both genes may be important for CNS development, although Fgf17 is expressed somewhat later than Fgf8. In the developing skeleton, both genes are expressed in costal cartilage while Fgf8 is preferentially expressed in long bones. In the developing great vessels Fgfl7 is preferentially expressed, suggesting that it may have a more prominent role in vascular growth.

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FGF-8 is a member of the family of fibroblast growth factors and is expressed during vertebrate embryo development. Eight potential FGF-8 isoforms are generated by alternative splicing in mice, several of which are expressed during embryogenesis in epithelial locations. The significance of the multiple isoforms is currently unknown. In this report, we investigate the expression patterns and the specificity of the FGF-8 isoforms for known fibroblast growth factor (FGF) receptors. RNAs for seven of the eight potential isoforms are present at multiple sites of embryonic Fgf8 expression. None of the FGF-8 isoforms exhibited activity when assayed with BaF3 cells expressing the "b" splice forms of FGF receptors 1-3, which are mostly expressed in epithelial tissues. Mesenchymally expressed "c" splice forms of FGF receptors 2 and 3 and FGF receptor 4 were activated by several FGF-8 isoforms. These findings are consistent with the hypothesis that the multiple FGF-8 isoforms are functionally redundant and function to signal in paracrine (epithelial to mesenchymal) contexts.The mammalian fibroblast growth factor (FGF) 1 family currently consists of structurally related polypeptides encoded by 10 genes (FGF-1-10) (reviewed in Ref. 1;. Four distinct genes code for high affinity transmembrane receptor tyrosine kinases (FGFR1-4) that bind FGF ligands and display varying patterns of expression (reviewed in Ref. 5). Alternative mRNA splicing generates isoforms of receptors 1-3 that exhibit unique ligand binding characteristics (6 -9). FGF receptor activation involves ligand binding and receptor dimerization, followed by transphosphorylation of the receptor and transduction of the signal into a biological response (5). FGF signal transduction has been implicated in development, wound healing, angiogenesis, and tumorigenesis (reviewed in Ref. 1). Germ line mutations of FGFs and FGFRs in mice (10 -15) and humans (16 -21) demonstrate the importance of FGF signaling in the process of development.FGF-8 was first identified as an androgen-induced growth factor secreted by the mouse mammary tumor cell line SC-3 (2). Subsequently, Fgf8 was identified as a Wnt1-cooperating protooncogene in murine mammary tumorigenesis (22). Fgf8 expression has been detected during murine and chicken embryogenesis in regions of outgrowth and patterning, including the primitive epiblast, the apical ectodermal ridge of the limb bud, the primitive streak, the tail bud, the facial primordia, and the midbrain-hindbrain junction (22-29). The murine and human genes encoding FGF-8 have been localized to mouse chromosome 19 and human chromosome 10q24 -26 (25, 26, 30, 31). The murine Fgf8 gene is unusual in the FGF family in that there are four exons (exons 1A-1D) equivalent to the usual exon 1 in other FGF genes (22,25,32). Alternative splicing of the four alternatively spliced exons results in potentially eight protein isoforms that differ at their amino termini and share a common carboxyl terminus encoded by exons 1D, 2, and 3 (22,25,32). Human FGF8 is similar ...

Prior studies have identi®ed Fibroblast Growth Factor-8 (Fgf8) as a possible proto-oncogene in mouse mammary tumorigenesis. We now report on the generation of two types of Fgf8 transgenic mice that each utilize the mouse mammary tumor virus (MMTV) promoter. The ®rst transgene (MMTV-Fgf8b) results in the overexpression of the FGF8b isoform exclusively. Male and female MMTV-Fgf8b transgenic mice are viable and fertile. RNA for FGF8b is detected in mammary gland and salivary gland tissues of transgenic mice by Northern blot analysis. Nearly 85% of breeding transgenic female mice developed mammary lobular adenocarcinomas by 12 months of age, while no tumors developed in nontransgenic littermates. Salivary gland tumors occurred in some animals, always in association with mammary tumors. Several MMTV-Fgf8b transgenic mice had lung metastases at necropsy. The second transgene (MMTVFgf8) uses the entire Fgf8 gene and potentially encodes all FGF8 isoforms. Fgf8 is expressed by this transgene in several tissues in addition to those described above, notably the ovaries. The two MMTV-Fgf8 founders developed mammary ductal adenocarcinomas at ®ve and eight months of age, and both displayed ovarian stromal hyperplasia. The founders expressing either transgene did not successfully nurse their pups. These results demonstrate that production of FGF8b, and possibly other FGF8 isoforms, in the mammary and salivary glands contributes to oncogenesis, and that ovarian expression results in stromal hyperplasia.