Activating Fgfr3 Y367C mutation causes hearing loss and inner ear defect in a mouse model of chondrodysplasia

Fibroblast growth factor receptor 3 (FGFR3) is a key regulator of skeletal development and activating mutations in FGFR3 cause skeletal dysplasias, including hypochondroplasia, achondroplasia and thanatophoric dysplasia. The introduction of the Y367C mutation corresponding to the human Y373C thanatophoric dysplasia type I (TDI) mutation into the mouse genome, resulted in dwarfism with a skeletal phenotype remarkably similar to that of human chondrodysplasia. To investigate the role of the activating Fgfr3 Y367C mutation in auditory function, the middle and inner ear of the heterozygous mutant Fgfr3^Y367C/+ mice were examined. The mutant Fgfr3^Y367C/+ mice exhibit fully penetrant deafness with a significantly elevated auditory brainstem response threshold for all frequencies tested. The inner ear defect is mainly associated with an increased number of pillar cells or modified supporting cells in the organ of Corti. Hearing loss in the Fgfr3^Y367C/+ mouse model demonstrates the crucial role of Fgfr3 in the development of the inner ear and provides novel insight on the biological consequences of FGFR3 mutations in chondrodysplasia.     

Postnatal male germ‐cell expression of cre recombinase in Tex101‐iCre transgenic mice

We have generated a transgenic mouse line that expresses improved Cre recombinase (iCre) under the control of the testis‐expressed gene 101 (Tex101) promoter. This transgenic mouse line was named Tex101‐iCre. Using the floxed ROSA reporter mice, we found that robust Cre recombinase activity was detected in postnatal testes with weak or no activity in other tissues. Within the testis, Cre recombinase was active in spermatogenic cells as early as the prospermatogonia stage at day 1 after birth. In 30‐ and 60‐day‐old mice, positive Cre recombinase activity was detected not only in prospermatogonia but also in spermatogenic cells at later stages of spermatogenesis. There was little or no Cre activity in interstitial cells. Breeding wild‐type females with homozygous floxed fibroblast growth factor receptor 2 (Fgfr2) males carrying the Tex101‐iCre transgene did not produce any progeny with the floxed Fgfr2 allele. All the progeny inherited a recombined Fgfr2 allele, indicating that complete deletion of the floxed Fgfr2 allele by Tex101‐iCre can be achieved in the male germline. Furthermore, FGFR2 protein was not detected in spermatocytes and spermatids of adult Fgfr2^fl/fl;Tex101‐iCre mice. Taken together, our results suggest that the Tex101‐iCre mouse line allows the inactivation of a floxed gene in spermatogenic cells in adult mice, which will facilitate the functional characterization of genes in normal spermatogenesis and male fertility. genesis 48:717–722, 2010. © 2010 Wiley‐Liss, Inc.     

Activation of p38 MAPK pathway in the skull abnormalities of Apert syndrome Fgfr2 +P253R mice

Background

Apert syndrome is characterized by craniosynostosis and limb abnormalities and is primarily caused by FGFR2 +/P253R and +/S252W mutations. The former mutation is present in approximately one third whereas the latter mutation is present in two-thirds of the patients with this condition. We previously reported an inbred transgenic mouse model with the Fgfr2 +/S252W mutation on the C57BL/6J background for Apert syndrome. Here we present a mouse model for the Fgfr2+/P253R mutation.

Results

We generated inbred Fgfr2 ^+/P253R mice on the same C56BL/6J genetic background and analyzed their skeletal abnormalities. 3D micro-CT scans of the skulls of the Fgfr2 ^+/P253R mice revealed that the skull length was shortened with the length of the anterior cranial base significantly shorter than that of the Fgfr2 ^+/S252W mice at P0. The Fgfr2 ^+/P253R mice presented with synostosis of the coronal suture and proximate fronts with disorganized cellularity in sagittal and lambdoid sutures. Abnormal osteogenesis and proliferation were observed at the developing coronal suture and long bones of the Fgfr2 ^+/P253R mice as in the Fgfr2 ^+/S252W mice. Activation of mitogen-activated protein kinases (MAPK) was observed in the Fgfr2 ^+/P253R neurocranium with an increase in phosphorylated p38 as well as ERK1/2, whereas phosphorylated AKT and PKCα were not obviously changed as compared to those of wild-type controls. There were localized phenotypic and molecular variations among individual embryos with different mutations and among those with the same mutation.

Conclusions

Our in vivo studies demonstrated that the Fgfr2 +/P253R mutation resulted in mice with cranial features that resemble those of the Fgfr2 ^+/S252W mice and human Apert syndrome. Activated p38 in addition to the ERK1/2 signaling pathways may mediate the mutant neurocranial phenotype. Though Apert syndrome is traditionally thought to be a consistent phenotype, our results suggest localized and regional variations in the phenotypes that characterize Apert syndrome.     

High-resolution mapping of tlt, a mouse mutant lacking otoconia

The ability to sense gravity is enhanced by an extracellular structure that overlies the macular sensory epithelium. This complex consists of high density particles, otoconia, embedded within a gelatinous membrane. The tilted mouse specifically lacks otoconia, yet has no other detectable anatomic lesions. Furthermore, the penetrance of the tilted phenotype is nearly 100%. This mouse provides a model to identify genes that are involved in the development and function of vestibular otoconia. Using SSLP markers, we have mapped the tilted (tlt) gene on mouse Chromosome (Chr) 5 between D5Mit421 and D5Mit353/D5Mit128/D5Mit266/D5Mit267 by analysis of the progeny of an intersubspecific F₂ intercross. We also mapped the fibroblast growth factor receptor 3 (Fgfr3) gene, a potential candidate for tlt, and the Huntington's disease homolog (Hdh) gene to D5Mit268, approximately 4.3 centiMorgans (cM) from the tilted locus. This study excludes both Fgfr3 and Hdh as candidate genes for tlt and identifies closely linked microsatellite markers that will be useful for the positional cloning of tlt. Received: 17 November 1998 / Accepted: 1 February 1999     

Genetic mosaic analysis reveals FGF receptor 2 function in terminal end buds during mammary gland branching morphogenesis

FGF signaling is associated with breast cancer and is required for mammary placode formation in the mouse. In this study, we employed a genetic mosaic analysis based on Cre-mediated recombination to investigate FGF receptor 2 (Fgfr2) function in the postnatal mammary gland. Mosaic inactivation of Fgfr2 by the MMTV-Cre transgene enabled us to compare the behavior of Fgfr2 null and Fgfr2 heterozygous cells in the same gland. Fgfr2 null cells were at a competitive disadvantage to their Fgfr2 heterozygous neighbors in the highly proliferative terminal end buds (TEBs) at the invasion front, owing to a negative effect of loss of Fgfr2 function on cell proliferation. However, Fgfr2 null cells were tolerated in mature ducts. In these genetic mosaic mammary glands, the epithelial network is apparently built by TEBs that over time are composed of a progressively larger proportion of Fgfr2-positive cells. However, subsequently, most cells lose Fgfr2 function, presumably due to additional rounds of Cre-mediated recombination. Using an independent strategy to create mosaic mammary glands, which employed an adenovirus-Cre that acts only once, we confirmed that Fgfr2 null cells were out-competed by neighboring Fgfr2 heterozygous cells. Together, our data demonstrate that Fgfr2 functions in the proliferating and invading TEBs, but it is not required in the mature ducts of the pubertal mammary gland.     

Isolation and Chromosomal Mapping of a Mouse Homolog of the Batten Disease GeneCLN3

We describe the isolation and chromosomal mapping of a mouse homolog of the Batten disease gene,CLN3.Like its human counterpart, the mouse cDNA contains an open reading frame of 1314 bp encoding a predicted protein product of 438 amino acids. The mouse and human coding regions are 82 and 85% identical at the nucleic acid and amino acid levels, respectively. The mouse gene maps to distal Chromosome 7, in a region containing genes whose homologs are on human chromosome 16p12, whereCLN3maps. Isolation of a mouseCLN3homolog will facilitate the creation of a mouse model of Batten disease.     

Mapping of adenylyl cyclase genes type I,II, III,IV, V,and VI in mouse

The adenylyl cyclases (AC) act as second messengers in regulatory processes in the central nervous system. They might be involved in the pathophysiology of diseases, but their biological function is unknown, except for AC type I, which has been implicated in learning and memory. We previously mapped the gene encoding AC I to human Chromosome (Chr) 7p12. In this study we report the mapping of the adenylyl cyclase genes type I–VI to mouse chromosomes by fluorescence in situ hybridization (FISH): Adcy1 to Chr 11A2, Adcy2 to 13C1, Adcy3 to 12A-B, Adcy4 to 14D3, Adcy5 to 16B5, and Adcy6 to 15F. We also confirmed previously reported mapping results of the corresponding human loci ADCY2, ADCY3, ADCY5, and ADCY6 to human chromosomes and, in addition, determined the chromosomal location of ADCY4 to human Chr 14q11.2. The mapping data confirm known areas of conservation between mouse and human chromosomes.     

FGF10/FGFR2b signaling plays essential roles during in vivo embryonic submandibular salivary gland morphogenesis

Background  

Analyses of Fgf10 and Fgfr2b mutant mice, as well as human studies, suggest that FGF10/FGFR2b signaling may play an essential, nonredundant role during embryonic SMG development. To address this question, we have analyzed the SMG phenotype in Fgf10 and Fgfr2b heterozygous and null mutant mice. In addition, although previous studies suggest that the FGF10/FGFR2b and FGF8/FGFR2c signaling pathways are functionally interrelated, little is known about the functional relationship between these two pathways during SMG development. We have designed in vivo and in vitro experiments to address this question.     

Tissue-Specific Expression and Mapping of theCox7ahGene in Mouse

We have isolated and examined the gene for the heart isoform of cytochromecoxidase subunit VIIa (COX VIIa-H) in mouse, an isoform gene previously thought to be lacking in rodents. Interspecies amino acid comparisons indicate that mouse COX VIIa-H protein displays 82.5 and 70.9% identity with the bovine and human heart isoforms of COX VIIa, but only 53.7% identity with the paralogous mouse liver isoform (COX VIIa-L). Expression in adult mouse tissues is limited to heart and skeletal muscle, as found in other species. In the early mouse embryo,Cox7alwas the exclusive isoform expressed andCox7ahmRNA was not detectable until day 17postcoitum.That the mouseCox7ahgene characterized in this study is orthologous to the humanCOX7AHgene was also suggested by its mapping to mouse chromosome 7, to a conserved region syntenic with the human chromosome location ofCOX7AH,19q13.1. As a result, all three COX heart isoform genes in mouse group to chromosome 7. Interestingly, mapping of the mouseCox7alto chromosome 9 suggests a new syntenic region between the mouse and the human genomes.     

Human fibroblast growth factor receptor 3 gene (FGFR3): genomic sequence and primer set information for gene analysis

We have determined the nucleotide sequence of the human fibroblast growth factor receptor 3 (FGFR3) gene, including 800 bp of the 5′-flanking region and compared the sequence with the previously published murine Fgfr3 gene. The organization of the gene is highly conserved between man and mouse. We used the intron sequences to design a set of primers that allow amplification of the 17 exons (2–18) that encode the complete open reading frame. Using these primers the FGFR3 gene can be amplified at the genomic level, which significantly facilitates mutational screening. Received: 27 December 1996 / Accepted: 6 March 1997     

Wnt11/Fgfr1b cross-talk modulates the fate of cells in palate development

Various cellular and molecular events underlie the elevation and fusion of the developing palate that occurs during embryonic development. This includes convergent extension, where the medial edge epithelium is intercalated into the midline epithelial seam. We examined the expression patterns of Wnt11 and Fgfr1b - which are believed to be key factors in convergent extension - in mouse palate development. Wnt-11 overexpression and beads soaked in SU5402 (an Fgfr1 inhibitor) were employed in in vitro organ cultures. The results suggested that interactions between Wnt11 and Fgfr1b are important in modulating cellular events such as cell proliferation for growth and apoptosis for fusion. Moreover, the Wnt11 siRNA results showed that Wnt11-induced apoptosis was necessary for palatal fusion. In summary, Fgfr1b induces cell proliferation in the developing palate mesenchyme so that the palate grows and contacts each palatal shelf, with negative feedback of Fgfs triggered by excessive cell proliferation then inhibiting the expression of Fgfr1b and activating the expression of Wnt11 to fuse each palate by activating apoptosis.     

A Physical Map of the Mouseshaker-2Region Contains Many of the Genes Commonly Deleted in Smith–Magenis Syndrome (del17p11.2p11.2)

We report the construction of a physical map of the region of mouse chromosome 11 that encompassesshaker-2(sh2), a model for the human nonsyndromic deafnessDFNB3. DFNB3maps within the common deletion region of Smith–Magenis syndrome (SMS), del(17)(p11.2p11.2). Eleven of the genes mapping within the SMS common deletion region have murine homologs on thesh2physical map. The gene order in this region is not perfectly conserved between mouse and human, a finding to be considered as we engineer a mouse model of Smith–Magenis syndrome.     

Dlx2 (Tes1), a homeobox gene of the Distal-less family, assigned to conserved regions on human and mouse chromosomes 2

Dlx-2 (also called Tes-1), a mammalian member of the Distal-less family of homeobox genes, is expressed during murine fetal development in spatially restricted domains of the forebrain. Searching for a candidate neurological mutation that might involve this gene, we have assigned the human and mouse loci to regions of conserved synteny on human chromosome 2, region cen-q33, and mouse chromosome 2 by Southern analysis of somatic cell hybrid lines. An EcoRI dimorphism, discovered in common inbred laboratory strains, was used for recombinant inbred strain mapping. The results place Dlx-2/Tes-1 near the Hox-4 cluster on mouse chromosome 2.     

MappingTNNC1, the Gene That Encodes Cardiac Troponin I in the Human and the Mouse

We have mapped the TNNC1 gene, whose protein product is the cardiac TnI protein. TnI is one of the proteins that makes up the troponin complex, which mediates the response of muscle to calcium ions. The human TNNC1 locus had been assigned to a large region of chromosome 19, and we have refined the mapping position to the distal end of the chromosome by amplification of DNAs from a chromosome 19 mapping panel. We have also mapped the mouse Tnnc1 locus, by following the segregation of an intron sequence through DNAs from the European Interspecific Backcross. Tnnc1 maps close to the centromere on mouse chromosome 7.     

Mapping of the structural gene for S-adenosyl homocysteine hydrolase to mouse Chromosome 2, and related sequences to Chromosomes 8 and X

Comparative mapping studies in human and mouse have shown that, to date, human Chromosome (Chr) 20 is completely syntenic with distal mouse Chr 2. The structural locus for S-adenosyl-l-homocysteine hydrolase (EC 3.3.1.1) in human, AHCY, maps to 20 qterq13.1, and we report here that the homologous locus in the mouse, Ahcy, maps to distal mouse Chr 2 with gene order Pcna-Ahcy-Ada. Analysis of 123 progeny of an interspecific backcross between a laboratory stock, AN, and Mus spretus using a rat cDNA probe revealed the presence of at least two other Ahcy-related sequences segregating independently in the mouse genome. One, Ahcy-rs1, was mapped to Chr 8 in the BXH recombinant inbred strains, and the other, Ahcy-rs2, shows a pattern of inheritance consistent with X-linkage.     

Assignment of the Y4Receptor Gene (PPYR1) to Human Chromosome 10q11.2 and Mouse Chromosome 14

The human and mouse genes for the neuropeptide Y₄receptor have been isolated, sequenced, and shown to contain no introns within the coding region of the gene. Nonisotopicin situhybridization and interspecific mouse backcross mapping have localized the genes to human chromosome 10q11.2 and mouse chromosome 14. Five nucleotide variants, which do not alter the protein sequence, have been identified within the coding region of the human receptor gene. The human Y₄subtype is most closely related to the Y₁-receptor subtype (42%), suggesting that it evolved from an ancestral Y₁-like receptor via an RNA-mediated transpositional event.