Mice with conditional inactivation of fibroblast growth factor receptor-2 signaling in oligodendrocytes have normal myelin but display dramatic hyperactivity when combined with Cnp1 inactivation.

Fibroblast growth factor receptors (Fgfr) comprise a widely expressed family of developmental regulators implicated in oligodendrocyte (OL) maturation of the CNS. Fgfr2 is expressed by OLs in myelinated fiber tracks. In vitro, Fgfr2 is highly upregulated during OL terminal differentiation, and its activation leads to enhanced growth of OL processes and the formation of myelin-like membranes. To investigate the in vivo function of Fgfr2 signaling by myelinating glial cells, we inactivated the floxed Fgfr2 gene in mice that coexpress Cre recombinase (cre) as a knock-in gene into the OL-specific 2',3'-cyclic nucleotide phosphodiesterase (Cnp1) locus. Surprisingly, no obvious defects were detected in brain development of these conditional mutants, including the number of OLs, the onset and extent of myelination, the ultrastructure of myelin, and the expression level of myelin proteins. However, unexpectedly, a subset of these conditional Fgfr2 knock-out mice that are homozygous for cre and therefore are also Cnp1 null, displayed a dramatic hyperactive behavior starting at approximately 2 weeks of age. This hyperactivity was abolished by treatment with dopamine receptor antagonists or catecholamine biosynthesis inhibitors, suggesting that the symptoms involve a dysregulation of the dopaminergic system. Although the molecular mechanisms are presently unknown, this novel mouse model of hyperactivity demonstrates the potential involvement of OLs in neuropsychiatric disorders, as well as the nonpredictable role of genetic interactions in the behavioral phenotype of mice.

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.

Development and maintenance of otoconia: biochemical considerations.

The first part of this review deals with recent advances in the understanding of biochemical mechanisms of otoconial morphogenesis. Most important in this regard is the molecular characterization of otoconin 90, the principal matrix protein of mammalian calcitic otoconia, which was found to be a homologue of the phospholytic enzyme PLA2. The unique and unexpected expression pattern of this protein required radical rethinking of traditional concepts. The new data, when integrated with existing information, provide a rational basis for an explanation of the mechanisms leading to crystal nucleation and growth. Based on this information, a hypothetical model is presented that posits interaction of otoconin 90 with microvesicles derived from the supporting cells as a key event in the formation of otoconia. The second part of the review is directed at the controversial subject of maintenance of mature otoconia and systematically analyzes the available indirect information on this topic. A synthesis of these theoretical considerations is viewed in relation to the pathogenesis of the important otoneurologic entities of BPPN and senile otoconial degeneration. The last part of the review deals with several animal models that promise to help elucidate normal and abnormal mechanisms of otoconial morphogenesis, including mineral deficiencies, mutations with selective otoconial agenesis, as well as targeted disruption of essential genes.

Physical mapping of the mouse tilted locus identifies an association between human deafness loci DFNA6/14 and vestibular system development.

The tilted (tlt) mouse carries a recessive mutation causing vestibular dysfunction. The defect in tlt homozygous mice is limited to the utricle and saccule of the inner ear, which completely lack otoconia. Genetic mapping of tlt placed it in a region orthologous with human 4p16.3-p15 that contains two loci, DFNA6 and DFNA14, responsible for autosomal dominant, nonsyndromic hereditary hearing impairment. To identify a possible relationship between tlt in mice and DFNA6 and DFNA14 in humans, we have refined the mouse genetic map, assembled a BAC contig spanning the tlt locus, and developed a comprehensive comparative map between mouse and human. We have determined the position of tlt relative to 17 mouse chromosome 5 genes with orthologous loci in the human 4p16.3-p15 region. This analysis identified an inversion between the mouse and human genomes that places tlt and DFNA6/14 in close proximity.

Differential regulation of endochondral bone growth and joint development by FGFR1 and FGFR3 tyrosine kinase domains.

Fibroblast growth factor receptors (FGFR) 1 and 3 have distinct mitogenic activities in vitro. In several cultured cell lines, FGFR1 transmits a potent mitogenic signal, whereas FGFR3 has little or no mitogenic activity. However, in other in vitro assays the FGFR3 intracellular domain is comparable with that of FGFR1. In vivo, FGFR3 negatively regulates chondrocyte proliferation and differentiation, and activating mutations are the molecular etiology of achondroplasia. By contrast, FGFR1 transmits a proliferative signal in various cell types in vivo. These observations suggest that inhibition of the proliferating chondrocyte could be a unique property of FGFR3 or, alternatively, a unique property of the proliferating chondrocyte. To test this hypothesis, FGFR1 signaling was activated in the growth plate in cells that normally express FGFR3. Comparison of transgenic mice with an activated FGFR1 signaling pathway with an achondroplasia-like mouse that expresses a similarly activated FGFR3 signaling pathway demonstrated that both transgenes result in a similar achondroplasia-like dwarfism. These data demonstrate that suppression of mitogenic activity by FGFR signaling is a property that is unique to growth plate chondrocytes. Surprisingly, we observed that in transgenic mice expressing an activated FGFR, some synovial joints failed to develop and were replaced by cartilage. The defects in the digit joints phenocopied the symphalangism that occurs in Apert syndrome and the number of affected joints was dependent on transgene dose. In contrast to the phenotype in the growth plate, the joint phenotype was more severe in transgenic mice with an activated FGFR1 signaling pathway. The failure of joint development resulted from expanded chondrification in the presumptive joint space, suggesting a crucial role for FGF signaling in regulating the transition of condensed mesenchyme to cartilage and in defining the boundary of skeletal elements.

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.

Structural basis for fibroblast growth factor receptor 2 activation in Apert syndrome.

Apert syndrome (AS) is characterized by craniosynostosis (premature fusion of cranial sutures) and severe syndactyly of the hands and feet. Two activating mutations, Ser-252 --> Trp and Pro-253 --> Arg, in fibroblast growth factor receptor 2 (FGFR2) account for nearly all known cases of AS. To elucidate the mechanism by which these substitutions cause AS, we determined the crystal structures of these two FGFR2 mutants in complex with fibroblast growth factor 2 (FGF2). These structures demonstrate that both mutations introduce additional interactions between FGFR2 and FGF2, thereby augmenting FGFR2-FGF2 affinity. Moreover, based on these structures and sequence alignment of the FGF family, we propose that the Pro-253 --> Arg mutation will indiscriminately increase the affinity of FGFR2 toward any FGF. In contrast, the Ser-252 --> Trp mutation will selectively enhance the affinity of FGFR2 toward a limited subset of FGFs. These predictions are consistent with previous biochemical data describing the effects of AS mutations on FGF binding. Alterations in FGFR2 ligand affinity and specificity may allow inappropriate autocrine or paracrine activation of FGFR2. Furthermore, the distinct gain-of-function interactions observed in each crystal structure provide a model to explain the phenotypic variability among AS patients.

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.

Regulation of chondrocyte growth and differentiation by fibroblast growth factor receptor 3.

Both gain-of-function and loss-of-function mutations in fibroblast growth factor receptor 3 (Fgfr3) have revealed unique roles for this receptor during skeletal development. Loss-of-function alleles of Fgfr3 lead to an increase in the size of the hypertrophic zone, delayed closure of the growth plate and the subsequent overgrowth of long bones. Gain-of-function mutations in Fgfr3 have been genetically linked to autosomal dominant dwarfing chondrodysplasia syndromes where both the size and architecture of the epiphyseal growth plate are altered. Analysis of these phenotypes and the biochemical consequences of the mutations in FGFR3 demonstrate that FGFR3-mediated signalling is an essential negative regulator of endochondral ossification.

Fibroblast growth factors.

SUMMARY: Fibroblast growth factors (FGFs) make up a large family of polypeptide growth factors that are found in organisms ranging from nematodes to humans. In vertebrates, the 22 members of the FGF family range in molecular mass from 17 to 34 kDa and share 13-71% amino acid identity. Between vertebrate species, FGFs are highly conserved in both gene structure and amino-acid sequence. FGFs have a high affinity for heparan sulfate proteoglycans and require heparan sulfate to activate one of four cell-surface FGF receptors. During embryonic development, FGFs have diverse roles in regulating cell proliferation, migration and differentiation. In the adult organism, FGFs are homeostatic factors and function in tissue repair and response to injury. When inappropriately expressed, some FGFs can contribute to the pathogenesis of cancer. A subset of the FGF family, expressed in adult tissue, is important for neuronal signal transduction in the central and peripheral nervous systems.

Loss of fibroblast growth factor receptor 2 ligand-binding specificity in Apert syndrome.

Craniosynostosis syndromes are autosomal dominant human skeletal diseases that result from various mutations in fibroblast growth factor receptor genes (Fgfrs). Apert syndrome (AS) is one of the most severe craniosynostosis syndromes and is associated with severe syndactyly of the hands and feet and with central nervous system malformations. AS is caused by specific missense mutations in one of two adjacent amino acid residues (S252W or P253R) in the highly conserved region linking Ig-like domains II and III of FGFR2. Here we demonstrate that these mutations break one of the cardinal rules governing ligand specificity of FGFR2. We show that the S252W mutation allows the mesenchymal splice form of FGFR2 (FGFR2c) to bind and to be activated by the mesenchymally expressed ligands FGF7 or FGF10 and the epithelial splice form of FGFR2 (FGFR2b) to be activated by FGF2, FGF6, and FGF9. These data demonstrate loss of ligand specificity of FGFR2 with retained ligand dependence for receptor activation. These data suggest that the severe phenotypes of AS likely result from ectopic ligand-dependent activation of FGFR2.

A high-resolution radiation hybrid map of the proximal portion of mouse chromosome 5.

Radiation hybrid (RH) mapping of the mouse genome provides a useful tool in the integration of existing genetic and physical maps, as well as in the ongoing effort to generate a dense map of expressed sequence tags. To facilitate functional analysis of mouse Chromosome 5, we have constructed a high-resolution RH map spanning 75 cM of the chromosome. During the course of these studies, we have developed RHBase, an RH data management program that provides data storage and an interface to several RH mapping programs and databases. We have typed 95 markers on the T31 RH panel and generated an integrated map, pooling data from several sources. The integrated RH map ranges from the most proximal marker, D5Mit331 (Chromosome Committee offset, 3 cM), to D5Mit326, 74.5 cM distal on our genetic map (Chromosome Committee offset, 80 cM), and consists of 138 markers, including 89 simple sequence length polymorphic markers, 11 sequence-tagged sites generated from BAC end sequence, and 38 gene loci, and represents average coverage of approximately one locus per 0.5 cM with some regions more densely mapped. In addition to the RH mapping of markers and genes previously localized on mouse Chromosome 5, this RH map places the alpha-4 GABA(A) receptor subunit gene (Gabra4) in the central portion of the chromosome, in the vicinity of the cluster of three other GABA(A) receptor subunit genes (Gabrg1-Gabra2-Gabrb1). Our mapping effort has also defined a new cluster of four genes in the semaphorin gene family (Sema3a, Sema3c, Sema3d, and Sema3e) and the Wolfram syndrome gene (Wfs1) in this region of the chromosome.

Temporal and spatial gradients of Fgf8 and Fgf17 regulate proliferation and differentiation of midline cerebellar structures.

The midbrain-hindbrain (MHB) junction has the properties of an organizer that patterns the MHB region early in vertebrate development. Fgf8 is thought to mediate this organizer function. In addition to Fgf8, Fgf17 and Fgf18 are also expressed in the MHB junction. Fgf17 is expressed later and broader than either Fgf8 or Fgf18. Disrupting the Fgf17 gene in the mouse decreased precursor cell proliferation in the medial cerebellar (vermis) anlage after E11.5. Loss of an additional copy of Fgf8 enhanced the phenotype and accelerated its onset, demonstrating that both molecules cooperate to regulate the size of the precursor pool of cells that develop into the cerebellar vermis. However, expression patterns of Wnt1, En2, Pax5 and Otx2 were not altered suggesting that specification and patterning of MHB tissue was not perturbed and that these FGFs are not required to pattern the vermis at this stage of development. The consequence of this developmental defect is a progressive, dose-dependent loss of the most anterior lobe of the vermis in mice lacking Fgf17 and in mice lacking Fgf17 and one copy of Fgf8. Significantly, the differentiation of anterior vermis neuroepithelium was shifted rostrally and medially demonstrating that FGF also regulates the polarized progression of differentiation in the vermis anlage. Finally, this developmental defect results in an ataxic gait in some mice.

FGFs, heparan sulfate and FGFRs: complex interactions essential for development.

Fibroblast growth factors (FGFs) comprise a large family of developmental and physiological signaling molecules. All FGFs have a high affinity for the glycosaminoglycan heparin and for cell surface heparan sulfate proteoglycans. A large body of biochemical and cellular evidence points to a direct role for heparin/heparan sulfate in the formation of an active FGF/FGF receptor signaling complex. However, until recently there has been no direct demonstration that heparan is required for the biological activity of FGF in a developmental system in vivo. A recent paper by Lin et al.(1) has broken through this barrier to demonstrate that heparan sulfate is essential for FGF function during Drosophila development. The establishment of a role for heparan sulfate in FGFR activation in vivo suggests that tissue-specific differences in the structure of heparan may modulate the activity of FGF. BioEssays 22:108-112, 2000.

Expression of FGFR3 with the G380R achondroplasia mutation inhibits proliferation and maturation of CFK2 chondrocytic cells.

A G380R substitution in the transmembrane-spanning region of FGFR3 (FGFR3Ach) results in constitutive receptor kinase activity and is the most common cause of achondroplastic dwarfism in humans. The epiphyseal growth plates of affected individuals are disorganized and hypocellular and show aberrant chondrocyte maturation. To examine the molecular basis of these abnormalities, we used a chondrocytic cell line, CFK2, to stably express the b variant of wild-type FGFR3 or the the constitutively active FGFR3Ach. Overexpression of FGFR3 had minimal effects on CFK2 proliferation and maturation compared with the severe growth retardation found in cells expressing FGFR3Ach. Cells expressing the mutant receptor also showed an abnormal apoptotic response to serum deprivation and failed to undergo differentiation under appropriate culture conditions. These changes were associated with altered expression of integrin subunits, which effectively led to a switch in substrate preference of the immature cell from fibronectin to type II collagen. These in vitro observations support those from in vivo studies indicating that FGFR3 mediates an inhibitory influence on chondrocyte proliferation. We now suggest that the mechanism is related to altered integrin expression.

Subcellular and developmental expression of alternatively spliced forms of fibroblast growth factor 14.

Fibroblast growth factors (FGFs) 11-14 comprise a subfamily of FGFs with poorly defined biological function. Here we characterize two isoforms of FGF14 (FGF14-1a and FGF14-1b) that result from the alternative usage of two different first exons. We demonstrate that these isoforms have differential subcellular localization and that they are differentially expressed in various adult tissues. Using in situ hybridization we show that Fgf14 is widely expressed in brain, spinal cord, major arteries and thymus between 12.5 and 14.5 days of mouse embryonic development. We also show that during cerebellar development, Fgf14 is first observed at postnatal day 1 in post mitotic granule cells, and later in development, in migrating and post migratory granule cells. The developmental expression pattern of Fgf14 in the cerebellum is complementary to that of Math1, a marker for proliferating granule cells in the external germinal layer.

Mapping ligand binding domains in chimeric fibroblast growth factor receptor molecules. Multiple regions determine ligand binding specificity.

Fibroblast growth factors (FGFs) mediate essential cellular functions by activating one of four alternatively spliced FGF receptors (FGFRs). To determine the mechanism regulating ligand binding affinity and specificity, soluble FGFR1 and FGFR3 binding domains were compared for activity. FGFR1 bound well to FGF2 but poorly to FGF8 and FGF9. In contrast, FGFR3 bound well to FGF8 and FGF9 but poorly to FGF2. The differential ligand binding specificity of these two receptors was exploited to map specific ligand binding regions in mutant and chimeric receptor molecules. Deletion of immunoglobulin-like (Ig) domain I did not effect ligand binding, thus localizing the binding region(s) to the distal two Ig domains. Mapping studies identified two regions that contribute to FGF binding. Additionally, FGF2 binding showed positive cooperativity, suggesting the presence of two binding sites on a single FGFR or two interacting sites on an FGFR dimer. Analysis of FGF8 and FGF9 binding to chimeric receptors showed that a broad region spanning Ig domain II and sequences further N-terminal determines binding specificity for these ligands. These data demonstrate that multiple regions of the FGFR regulate ligand binding specificity and that these regions are distinct with respect to different members of the FGF family.

Fibroblast growth factor receptor 3 gene transcription is suppressed by cyclic adenosine 3',5'-monophosphate. Identification of a chondrocytic regulatory element.

Signaling through fibroblast growth factor receptors (FGFRs) is critical for the development and patterning of the vertebrate skeleton. Gain-of-function alleles of fgfr2 and fgfr3 have been linked to several dominant skeletal disorders in humans, while null mutations in fgfr3 result in the overgrowth of long bones in a mouse model system. Interestingly, the expression pattern of fgfr3 in growth plate chondrocytes overlaps that of the parathyroid hormone (PTH)-related peptide (PTHrP) receptor, a signaling molecule that also regulates endochondral ossification. The coincident expression of these two receptors suggests that their signaling pathways may also interact. To gain insight into the regulatory mechanism(s) that govern the expression of the fgfr3 gene in chondrocytes, we have identified a cell-specific transcriptional regulatory element (CSRh) by measuring the activity of various promoter fragments in FGFR3-expressing (CFK2) and nonexpressing (RCJ) chondrocyte-like cell lines. Furthermore, we demonstrate that activation of PTH/PTHrP receptors, either by stimulation with PTH or through the introduction of activating mutations, represses CSRh-mediated transcriptional activity. Finally, the transcriptional repression of the CSRh element was mimicked by treatment with forskolin, 8-bromo-cAMP, and 3-isobutyl-1-methylxanthine or by overexpression of the catalytic subunit of protein kinase A. Together, these data suggest that protein kinase A activity is a critical factor that regulates fgfr3 gene expression in the proliferative or prehypertrophic compartment of the epiphyseal growth plate. Furthermore, these results provide a possible link between PTHrP signaling and fgfr3 gene expression during the process of endochondral ossification.

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.