Patterning the optic neuroepithelium by FGF signaling and Ras activation.

During vertebrate embryogenesis, the neuroectoderm differentiates into neural tissues and also into non-neural tissues such as the choroid plexus in the brain and the retinal pigment epithelium in the eye. The molecular mechanisms that pattern neural and non-neural tissues within the neuroectoderm remain unknown. We report that FGF9 is normally expressed in the distal region of the optic vesicle that is destined to become the neural retina, suggesting a role in neural patterning in the optic neuroepithelium. Ectopic expression of FGF9 in the proximal region of the optic vesicle extends neural differentiation into the presumptive retinal pigment epithelium, resulting in a duplicate neural retina in transgenic mice. Ectopic expression of constitutively active Ras is also sufficient to convert the retinal pigment epithelium to neural retina, suggesting that Ras-mediated signaling may be involved in neural differentiation in the immature optic vesicle. The original and the duplicate neural retinae differentiate and laminate with mirror-image polarity in the absence of an RPE, suggesting that the program of neuronal differentiation in the retina is autonomously regulated. In mouse embryos lacking FGF9, the retinal pigment epithelium extends into the presumptive neural retina, indicating a role of FGF9 in defining the boundary of the neural retina.

Lung hypoplasia and neonatal death in Fgf9-null mice identify this gene as an essential regulator of lung mesenchyme.

Mammalian lung develops as an evagination of ventral gut endoderm into the underlying mesenchyme. Iterative epithelial branching, regulated by the surrounding mesenchyme, generates an elaborate network of airways from the initial lung bud. Fibroblast growth factors (FGFs) often mediate epithelial-mesenchymal interactions and mesenchymal Fgf10 is essential for epithelial branching in the developing lung. However, no FGF has been shown to regulate lung mesenchyme. In embryonic lung, Fgf9 is detected in airway epithelium and visceral pleura at E10.5, but is restricted to the pleura by E12.5. We report that mice homozygous for a targeted disruption of Fgf9 exhibit lung hypoplasia and early postnatal death. Fgf9(-/-) lungs exhibit reduced mesenchyme and decreased branching of airways, but show significant distal airspace formation and pneumocyte differentiation. Our results suggest that Fgf9 affects lung size by stimulating mesenchymal proliferation. The reduction in the amount of mesenchyme in Fgf9(-/-) lungs limits expression of mesenchymal Fgf10. We suggest a model whereby FGF9 signaling from the epithelium and reciprocal FGF10 signaling from the mesenchyme coordinately regulate epithelial airway branching and organ size during lung embryogenesis.

Male-to-female sex reversal in mice lacking fibroblast growth factor 9.

Fgfs direct embryogenesis of several organs, including the lung, limb, and anterior pituitary. Here we report male-to-female sex reversal in mice lacking Fibroblast growth factor 9 (Fgf9), demonstrating a novel role for FGF signaling in testicular embryogenesis. Fgf9(-/-) mice also exhibit lung hypoplasia and die at birth. Reproductive system phenotypes range from testicular hypoplasia to complete sex reversal, with most Fgf9(-/-) XY reproductive systems appearing grossly female at birth. Fgf9 appears to act downstream of Sry to stimulate mesenchymal proliferation, mesonephric cell migration, and Sertoli cell differentiation in the embryonic testis. While Sry is found only in some mammals, Fgfs are highly conserved. Thus, Fgfs may function in sex determination and reproductive system development in many species.

Genomic organization and embryonic expression of the mouse fibroblast growth factor 9 gene.

Fibroblast growth factor 9 (FGF9), originally cloned as glial-activating factor from human glioma cells, is expressed in adult rat brain and kidney. Here we report the chromosomal localization, genomic organization, and embryonic expression pattern of the mouse Fgf9 gene. Fgf9 maps to chromosome 14 near the Ctla6 locus. The gene spans more than 34 kb and contains three exons and two introns. Translation initiation occurs in exon 1, and translation termination occurs in exon 3. Fgf9 RNA was detected during mouse embryogenesis in several tissues in which Fgf gene expression has not been previously described, including intermediate mesoderm of late-stage gastrulation, ventricular myocardium, lung pleura, skeletal myoblasts in the early limb bud, spinal cord motor neurons, olfactory bulb, and gut lumenal epithelium. Fgf9 is coexpressed with other Fgf genes in some skeletal myoblasts, in limb apical ectoderm, in craniofacial ectoderm, and in the retina, inner ear, and tooth bud. Dev Dyn 1999;216:72-88.

Repression of hedgehog signaling and BMP4 expression in growth plate cartilage by fibroblast growth factor receptor 3.

Fibroblast growth factor receptor 3 (FGFR3) is a key regulator of skeletal growth and activating mutations in Fgfr3 cause achondroplasia, the most common genetic form of dwarfism in humans. Little is known about the mechanism by which FGFR3 inhibits bone growth and how FGFR3 signaling interacts with other signaling pathways that regulate endochondral ossification. To understand these mechanisms, we targeted the expression of an activated FGFR3 to growth plate cartilage in mice using regulatory elements from the collagen II gene. As with humans carrying the achondroplasia mutation, the resulting transgenic mice are dwarfed, with axial, appendicular and craniofacial skeletal hypoplasia. We found that FGFR3 inhibited endochondral bone growth by markedly inhibiting chondrocyte proliferation and by slowing chondrocyte differentiation. Significantly, FGFR3 downregulated the Indian hedgehog (Ihh) signaling pathway and Bmp4 expression in both growth plate chondrocytes and in the perichondrium. Conversely, Bmp4 expression is upregulated in the perichondrium of Fgfr3-/- mice. These data support a model in which Fgfr3 is an upstream negative regulator of the hedgehog (Hh) signaling pathway. Additionally, Fgfr3 may coordinate the growth and differentiation of chondrocytes with the growth and differentiation of osteoprogenitor cells by simultaneously modulating Bmp4 and patched expression in both growth plate cartilage and in the perichondrium.

Receptor specificity of the fibroblast growth factor family.

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.

Skeletal overgrowth and deafness in mice lacking fibroblast growth factor receptor 3.

Fibroblast growth factor receptor 3 (Fgfr3) is a tyrosine kinase receptor expressed in developing bone, cochlea, brain and spinal cord. Achondroplasia, the most common genetic form of dwarfism, is caused by mutations in FGFR3. Here we show that mice homozygous for a targeted disruption of Fgfr3 exhibit skeletal and inner ear defects. Skeletal defects include kyphosis, scoliosis, crooked tails and curvature and overgrowth of long bones and vertebrae. Contrasts between the skeletal phenotype and achondroplasia suggest that activation of FGFR3 causes achondroplasia. Inner ear defects include failure of pillar cell differentiation and tunnel of Corti formation and result in profound deafness. Our results demonstrate that Fgfr3 is essential for normal endochondral ossification and inner ear development.

Expression and biological activity of mouse fibroblast growth factor-9.

Receptor specificity is an essential mechanism governing the activity of fibroblast growth factors (FGF). To begin to understand the developmental role of FGF-9/glial activating factor, we have cloned and sequenced the murine FGF-9 cDNA and expressed the protein in mammalian cells and in Escherichia coli. We demonstrate that the FGF-9 protein is highly conserved between mouse and human. Receptor specificity was determined by direct binding to soluble and cell surface forms of FGF receptor (FGFR) splice variants and by the mitogenic activity on cells, which express unique FGF receptor splice variants. Our data demonstrate that FGF-9 efficiently activates the "c" splice forms of FGFR2 and FGFR3, receptors expressed in potential target cells for FGF-9. Significantly, FGF-9 also binds to and activates the "b" splice form of FGFR3, thus becoming the first FGF ligand besides FGF-1 to activate this highly specific member of the FGF receptor family.

Inositol trisphosphate receptor localization in brain: variable stoichiometry with protein kinase C.

Many neurotransmitters, hormones and growth factors act at membrane receptors to stimulate the phosphodiesteratic hydrolysis of phosphatidyl-inositol 4,5-bisphosphate generating the comessengers inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) and diacylglycerol. Diacylglycerol stimulates protein kinase C3 while Ins(1,4,5)P3 is postulated to activate specific receptors leading to release of intracellular calcium, probably from the endoplasmic reticulum. In recent preliminary reports, Rubin and associates detected 32P-Ins(1,4,5)P3 binding to liver and adrenal microsomes and to permeabilized neutrophils and liver cells. We now report the biochemical and autoradiographic demonstration in brain of high affinity, selective binding sites for 3H- and 32P-labelled Ins(1,4,5)P3 at levels 100-300 times higher than those observed in peripheral tissues. The potencies of various myoinositol analogues at the Ins(1,4,5)P3 binding site correspond to their potencies in releasing calcium from microsomes, supporting the physiological relevance of this receptor. Brain autoradiograms demonstrate discrete, heterogeneous localization of Ins(1,4,5)P3 receptors. In some regions localizations of Ins(1,4,5)P3 receptors resemble those of protein kinase C14, while in others they differ markedly, suggesting a novel mechanism whereby the relative activity of the two limbs of the PI cycle can be differently regulated.