Mutations in different components of FGF signaling in LADD syndrome.

Lacrimo-auriculo-dento-digital (LADD) syndrome is characterized by lacrimal duct aplasia, malformed ears and deafness, small teeth and digital anomalies. We identified heterozygous mutations in the tyrosine kinase domains of the genes encoding fibroblast growth factor receptors 2 and 3 (FGFR2, FGFR3) in LADD families, and in one further LADD family, we detected a mutation in the gene encoding fibroblast growth factor 10 (FGF10), a known FGFR ligand. These findings increase the spectrum of anomalies associated with abnormal FGF signaling.

Independent roles of Fgfr2 and Frs2alpha in ureteric epithelium.

Mice with conditional deletion of fibroblast growth factor receptor 2 (Fgfr2) in the ureteric bud using a Hoxb7cre line (Fgfr2(UB-/-)) develop severe ureteric branching defects; however, ureteric deletion of fibroblast growth factor receptor substrate 2α (Frs2α), a key docking protein that transmits fibroblast growth factor receptor intracellular signaling (Frs2α(UB-/-)) leads to mild ureteric defects. Mice with point mutations in the Frs2α binding site of Fgfr2 (Fgfr2(LR/LR)) have normal kidneys. The aim of this study was to determine the relationship between Fgfr2 and Frs2α in the ureteric lineage. Mice with ureteric deletion of both Fgfr2 and Frs2α (Fgfr2/Frs2α(UB-/)) were compared with Frs2α(UB-/-) and Fgfr2(UB-/-) mice. To avoid potential rescue of Fgfr1 forming heterodimers with Fgfr2(LR) alleles to recruit Frs2α, compound mutant mice were generated with ureteric deletion of Fgfr1 and with Fgfr2(LR/LR) point mutations (Fgfr1(UB-/-)Fgfr2(LR/LR)). At E13.5, three-dimensional reconstructions and histological assessment showed that, whereas Fgfr2(UB-/-) kidneys had more severe ureteric branching defects than Frs2α(UB-/-), Fgfr2(UB-/-) kidneys were indistinguishable from Fgfr2/Frs2α(UB-/-). At later stages, however, Fgfr2/Frs2α(UB-/-) kidneys were more severely affected than either Fgfr2(UB-/-) or Frs2α(UB-/-) kidneys. Taken together, although Fgfr2 and Frs2α have crucial roles in the ureteric lineage, they appear to act separately and additively.

Ureteric morphogenesis requires Fgfr1 and Fgfr2/Frs2α signaling in the metanephric mesenchyme.

Conditional deletion of fibroblast growth factor receptors (Fgfrs) 1 and 2 in the metanephric mesenchyme (MM) of mice leads to a virtual absence of MM and unbranched ureteric buds that are occasionally duplex. Deletion of Fgfr2 in the MM leads to kidneys with cranially displaced ureteric buds along the Wolffian duct or duplex ureters. Mice with point mutations in Fgfr2's binding site for the docking protein Frs2α (Fgfr2(LR/LR)), however, have normal kidneys; the roles of the Fgfr2/Frs2α signaling axis in MM development and regulating the ureteric bud induction site are incompletely understood. Here, we generated mice with both Fgfr1 deleted in the MM and Fgfr2(LR/LR) point mutations (Fgfr1(Mes-/-)Fgfrf2(LR/LR)). Unlike mice lacking both Fgfr1 and Fgfr2 in the MM, these mice had no obvious MM defects but had cranially displaced or duplex ureteric buds, probably as a result of decreased Bmp4 expression. Fgfr1(Mes-/-)Fgfr2(LR/LR) mice also had subsequent defects in ureteric morphogenesis, including dilated, hyperproliferative tips and decreased branching. Ultimately, they developed progressive renal cystic dysplasia associated with abnormally oriented cell division. Furthermore, mutants had increased and ectopic expression of Ret and its downstream targets in ureteric trunks, and exhibited upregulation of Ret/Etv4/5 signaling effectors, including Met, Myb, Cxcr4, and Crlf1. These defects were associated with reduced expression of Bmp4 in mesenchymal cells near mutant ureteric bud tips. Taken together, these results demonstrate that Fgfr2/Frs2α signaling in the MM promotes Bmp4 expression, which represses Ret levels and signaling in the ureteric bud to ensure normal ureteric morphogenesis.

Fgfr1 and the IIIc isoform of Fgfr2 play critical roles in the metanephric mesenchyme mediating early inductive events in kidney development.

Fibroblast growth factor receptors (Fgfrs) have critical roles in kidney development. FgfrIIIb is thought to act in epithelium, while FgfrIIIc functions in mesenchyme. We aimed to determine roles of Fgfr2IIIc in kidney development. Mice with deletion of Fgfr2IIIc (Fgfr2IIIc-/-) had normal kidneys. Combination of Fgfr2IIIc-/- with conditional deletion of Fgfr1 in metanephric mesenchyme (MM) (Fgfr1(Mes-/-)Fgfr2IIIc-/-) had small but identifiable MM at embryonic day (E) 10.5, expressing mesenchymal markers including Eya1, Six2, Pax2, and Gdnf (unlike Fgfr1/2(Mes-/-) mice that have no obvious MM). E11.5 Fgfr1(Mes-/-)Fgfr2IIIc-/- mice had rudimentary MM expressing only Eya1. Control, Fgfr2IIIc-/-, and Fgfr1(Mes-/-)Fgfr2IIIc-/- kidney mesenchymal tissues also express Fgfr2IIIb. In ureteric lineages, E10.5 Fgfr1(Mes-/-)Fgfr2IIIc-/- embryos had ureteric outgrowth (sometimes multiple buds); however, by E11.5 Gdnf absence lead to no ureteric elongation or branching (similar to Fgfr1/2(Mes-/-) mice). Beyond E12.5, Fgfr1(Mes-/-)Fgfr2IIIc-/- mice had no renal tissue. In conclusion, Fgfr2IIIc and Fgfr1 in kidney mesenchyme (together) are critical for normal early renal development.

Lacrimo-auriculo-dento-digital syndrome is caused by reduced activity of the fibroblast growth factor 10 (FGF10)-FGF receptor 2 signaling pathway.

Lacrimo-auriculo-dento-digital (LADD) syndrome is characterized by abnormalities in lacrimal and salivary glands, in teeth, and in the distal limbs. Genetic studies have implicated heterozygous mutations in fibroblast growth factor 10 (FGF10) and in FGF receptor 2 (FGFR2) in LADD syndrome. However, it is not clear whether LADD syndrome mutations (LADD mutations) are gain- or loss-of-function mutations. In order to reveal the molecular mechanism underlying LADD syndrome, we have compared the biological properties of FGF10 LADD and FGFR2 LADD mutants to the activities of their normal counterparts. These experiments show that the biological activities of three different FGF10 LADD mutants are severely impaired by different mechanisms. Moreover, haploinsufficiency caused by defective FGF10 mutants leads to LADD syndrome. We also demonstrate that the tyrosine kinase activities of FGFR2 LADD mutants expressed in transfected cells are strongly compromised. Since tyrosine kinase activity is stimulated by ligand-induced receptor dimerization, FGFR2 LADD mutants may also exert a dominant inhibitory effect on signaling via wild-type FGFR2 expressed in the same cell. These experiments underscore the importance of signal strength in mediating biological responses and that relatively small changes in receptor signaling may influence the outcome of developmental processes in cells or organs that do not possess redundant signaling pathway.

Deletion of Frs2alpha from the ureteric epithelium causes renal hypoplasia.

Fibroblast growth factor receptor 2 (Fgfr2) signaling is critical in maintaining ureteric branching architecture and mesenchymal stromal morphogenesis in the kidney. Fibroblast growth factor receptor substrate 2alpha (Frs2alpha) is a major docking protein for Fgfr2 with downstream targets including Ets variant (Etv) 4 and Etv5 in other systems. Furthermore, global deletion of Frs2alpha causes early embryonic lethality. The purpose of the study was to determine the role of Frs2alpha in mediating Fgfr2 signaling in the ureteric epithelium. To that end, we generated mice with conditional deletion of Frs2alpha in the ureteric epithelium (Frs2alpha(UB-/-)) and mice with point mutations in the Frs2alpha binding site of Fgfr2 (Fgfr2(LR/LR)). Frs2alpha(UB-/-) mice developed mild renal hypoplasia characterized by decreased ureteric branching morphogenesis but maintained normal overall branching architecture and had normal mesenchymal stromal development. Reduced nephron endowment in postnatal mutant mice was observed, corresponding with the reduction in branching morphogenesis. Furthermore, there were no apparent renal abnormalities in Fgfr2(LR/LR) mice. Interestingly, Etv4 and Etv5 expression was unaltered in Frs2alpha(UB-/-) mice, as was Sprouty1, an antagonist of Frs2alpha signaling. However, Ret and Wnt11 (molecules critical for ureteric branching morphogenesis) mRNA levels were lower in mutants vs. controls. Taken together, these findings suggest that Fgfr2 signals through adapter molecules other than Frs2alpha in the ureteric epithelium. Furthermore, Frs2alpha may transmit signals through other receptor kinases present in ureteric epithelium. Finally, the renal hypoplasia observed in Frs2alpha(UB-/-) mice is likely secondary to decreased Ret and Wnt11 expression.

Fibroblast growth factor receptor 2 plays an essential role in telencephalic progenitors.

We used loss-of-function analysis to determine the role of fibroblast growth factor receptor 2 (FGFR2) in telencephalic progenitors, and also to examine interactions between FGFR and Notch signaling. While the telencephalon of FGFR2 mutants appears grossly normal, mutant telencephalic progenitors exhibit altered proliferative behavior in vivo and in vitro. Based upon our prior finding that Notch1 activation increased neurosphere frequency in FGF2, we tested whether this effect is mediated by FGFR1 or FGFR2. We found that Notch1 activation increased neurosphere frequency in cells mutant for either FGFR1 or FGFR2, but had no effect on the reduced size of neurospheres mutant for those receptors. Additional analyses revealed biochemical changes in the adult neocortex mutant for the IIIc isoform of FGFR2, and essential roles for FGFR2 in nasopharynx, eyelid, and cornea development.

Testis Determination Requires a Specific FGFR2 Isoform to Repress FOXL2.

Male sex determination in mammals relies on sex determining region Y-mediated upregulation of sex determining region-box 9 (SOX9) expression in XY gonads, whereas Wnt family member (WNT)/R-spondin 1 signaling and forkhead box L2 (FOXL2) drive female sex determination in XX gonads. Fibroblast growth factor (FGF) 9 signaling ensures sustained SOX9 expression through repression of one of the ovarian pathways (WNT signaling), whereas the significance of FGF-mediated repression of the FOXL2 pathway has not been studied. Previously, we demonstrated that FGFR2 is the receptor for FGF9 in the XY gonad. Whether a specific isoform (FGFR2b or FGFR2c) is required was puzzling. Here, we show that FGFR2c is required for male sex determination. Initially, in developing mouse embryos at 12.5 to 13.5 days postcoitum (dpc), XY Fgfr2c-/- gonads appear as ovotestes, with SOX9 and FOXL2 expression predominantly localized to the posterior and anterior gonadal poles, respectively. However, by 15.5 dpc, XY Fgfr2c-/- gonads show complete male-to-female sex reversal, evident by the lack of SOX9 and ectopic expression of FOXL2 throughout the gonads. Furthermore, ablation of the Foxl2 gene leads to partial or complete rescue of gonadal sex reversal in XY Fgfr2c-/- mice. Together with previous findings, our data suggest that testis determination involves FGFR2c-mediated repression of both the WNT4- and FOXL2-driven ovarian-determining pathways.

Regulation of external genitalia development by concerted actions of FGF ligands and FGF receptors.

Members of the fibroblast growth factor (FGF) family play diverse roles during the development and patterning of various organs. In human and mice, 22 FGFs and four receptors derived from several splice variants are present. Redundant expression and function of FGF genes in organogenesis have been reported, but their roles in embryonic external genitalia, genital tubercle (GT), development have not been studied in detail. To address the role of FGF during external genitalia development, we have analyzed the expression of FGF genes (Fgf8, 9, 10) and receptor genes (Fgfr1, r2IIIb, r2IIIc) in GT of mice. Furthermore, Fgf10 and Fgfr2IIIb mutant mice were analyzed to elucidate their roles in embryonic external genitalia development. Fgfr2IIIb was expressed in urethral plate epithelium during GT development. Fgfr2IIIb mutant mice display urethral dysmorphogenesis. Marker gene analysis for urethral plate and bilateral mesenchymal formation suggests the existence of epithelial-mesenchymal interaction during urethral morphogenesis. Therefore, FGF10/FGFR2IIIb signals seem to constitute a developmental cascade for such morphogenesis.

Combined KIT and FGFR2b signaling regulates epithelial progenitor expansion during organogenesis.

Organ formation and regeneration require epithelial progenitor expansion to engineer, maintain, and repair the branched tissue architecture. Identifying the mechanisms that control progenitor expansion will inform therapeutic organ (re)generation. Here, we discover that combined KIT and fibroblast growth factor receptor 2b (FGFR2b) signaling specifically increases distal progenitor expansion during salivary gland organogenesis. FGFR2b signaling upregulates the epithelial KIT pathway so that combined KIT/FGFR2b signaling, via separate AKT and mitogen-activated protein kinase (MAPK) pathways, amplifies FGFR2b-dependent transcription. Combined KIT/FGFR2b signaling selectively expands the number of KIT+K14+SOX10+ distal progenitors, and a genetic loss of KIT signaling depletes the distal progenitors but also unexpectedly depletes the K5+ proximal progenitors. This occurs because the distal progenitors produce neurotrophic factors that support gland innervation, which maintains the proximal progenitors. Furthermore, a rare population of KIT+FGFR2b+ cells is present in adult glands, in which KIT signaling also regulates epithelial-neuronal communication during homeostasis. Our findings provide a framework to direct regeneration of branched epithelial organs.

Skeletal overgrowth is mediated by deficiency in a specific isoform of fibroblast growth factor receptor 3.

Fibroblast growth factor receptor 3 (FGFR3) plays an important role in the control of chondrocyte proliferation and differentiation, a process critical for normal development of the skeleton. To reveal the contributions of the epithelial Fgfr3b isoform and the mesenchymal Fgfr3c isoform to skeletal overgrowth seen in mice, in which both isoforms have been inactivated (Fgf3c(-/-) mice), we have generated mice in which each of the two Fgfr3 isoforms has been selectively inactivated. Whereas no apparent phenotype was detected in Fgfr3b(-/-) mice, strong stimulation of chondrocyte proliferation in the growth plates of Fgf3c(-/-) mice caused dramatic skeletal overgrowth and other skeletal abnormalities resembling the phenotype of mice deficient in both Fgfr3 isoforms. In addition, Fgfr3c(-/-) mice exhibited decreased bone mineral density in the cortical and trabecular bone, whereas the bone mineral density of Fgfr3b(-/-) mice resembled that of WT mice. These experiments demonstrated that the mesenchymal Fgfr3c isoform is responsible for controlling chondrocyte proliferation and differentiation that mediate normal skeletal development, whereas the epithelial Fgfr3b isoform does not contribute toward this process.

Distinct target-derived signals organize formation, maturation, and maintenance of motor nerve terminals.

Target-derived factors organize synaptogenesis by promoting differentiation of nerve terminals at synaptic sites. Several candidate organizing molecules have been identified based on their bioactivities in vitro, but little is known about their roles in vivo. Here, we show that three sets of organizers act sequentially to pattern motor nerve terminals: FGFs, beta2 laminins, and collagen alpha(IV) chains. FGFs of the 7/10/22 subfamily and broadly distributed collagen IV chains (alpha1/2) promote clustering of synaptic vesicles as nerve terminals form. beta2 laminins concentrated at synaptic sites are dispensable for embryonic development of nerve terminals but are required for their postnatal maturation. Synapse-specific collagen IV chains (alpha3-6) accumulate only after synapses are mature and are required for synaptic maintenance. Thus, multiple target-derived signals permit discrete control of the formation, maturation, and maintenance of presynaptic specializations.

A gain-of-function mutation of Fgfr2c demonstrates the roles of this receptor variant in osteogenesis.

The b and c variants of fibroblast growth factor receptor 2 (FGFR2) differ in sequence, binding specificity, and localization. Fgfr2b, expressed in epithelia, is required for limb outgrowth and branching morphogenesis, whereas the mesenchymal Fgfr2c variant is required by the osteocyte lineage for normal skeletogenesis. Gain-of-function mutations in human FGFR2c are associated with craniosynostosis syndromes. To confirm and extend this evidence, we introduced a Cys342Tyr replacement into Fgfr2c to create a gain-of-function mutation equivalent to a mutation in human Crouzon and Pfeiffer syndromes. Fgfr2c(C342Y/)(+) heterozygote mice are viable and fertile with shortened face, protruding eyes, premature fusion of cranial sutures, and enhanced Spp1 expression in the calvaria. Homozygous mutants display multiple joint fusions, cleft palate, and trachea and lung defects, and die shortly after birth. They show enhanced Cbfa1/Runx2 expression without significant change in chondrocyte-specific Ihh, PTHrP, Sox9, Col2a, or Col10a gene expression. Histomorphometric analysis and bone marrow stromal cell culture showed a significant increase of osteoblast progenitors with no change in osteoclastogenic cells. Chondrocyte proliferation was decreased in the skull base at embryonic day 14.5 but not later. These results suggest that long-term aspects of the mutant phenotype, including craniosynostosis, are related to the Fgfr2c regulation of the osteoblast lineage. The effect on early chondrocyte proliferation but not gene expression suggests cooperation of Fgfr2c with Fgfr3 in the formation of the cartilage model for endochondral bone.