Using forward genetics, we have identified the genes mutated in two classes of zebrafish fin mutants. The mutants of the first class are characterized by defects in embryonic fin morphogenesis, which are due to mutations in a Laminin subunit or an Integrin alpha receptor, respectively. The mutants of the second class display characteristic blistering underneath the basement membrane of the fin epidermis. Three of them are due to mutations in zebrafish orthologues of FRAS1, FREM1, or FREM2, large basement membrane protein encoding genes that are mutated in mouse bleb mutants and in human patients suffering from Fraser Syndrome, a rare congenital condition characterized by syndactyly and cryptophthalmos. Fin blistering in a fourth group of zebrafish mutants is caused by mutations in Hemicentin1 (Hmcn1), another large extracellular matrix protein the function of which in vertebrates was hitherto unknown. Our mutant and dose-dependent interaction data suggest a potential involvement of Hmcn1 in Fraser complex-dependent basement membrane anchorage. Furthermore, we present biochemical and genetic data suggesting a role for the proprotein convertase FurinA in zebrafish fin development and cell surface shedding of Fras1 and Frem2, thereby allowing proper localization of the proteins within the basement membrane of forming fins. Finally, we identify the extracellular matrix protein Fibrillin2 as an indispensable interaction partner of Hmcn1. Thus we have defined a series of zebrafish mutants modelling Fraser Syndrome and have identified several implicated novel genes that might help to further elucidate the mechanisms of basement membrane anchorage and of the disease's aetiology. In addition, the novel genes might prove helpful to unravel the molecular nature of thus far unresolved cases of the human disease.

An emerging family of extracellular matrix proteins characterized by 12 consecutive CSPG repeats and the presence of Calx-␤ motif(s) includes Fras1, QBRICK͞Frem1, and Frem2. Mutations in the genes encoding these proteins have been associated with mouse models of Fraser syndrome, which is characterized by subepidermal blistering, cryptophthalmos, syndactyly, and renal dysmorphogenesis. Here, we report that all of these proteins are localized to the basement membrane, and that their basement membrane localization is simultaneously impaired in Fraser syndrome model mice. In Frem2 mutant mice, not only Frem2 but Fras1 and QBRICK͞Frem1 were depleted from the basement membrane zone. This coordinated reduction in basement membrane deposition was also observed in another Fraser syndrome model mouse, in which GRIP1, a Fras1-and Frem2-interacting adaptor protein, is primarily affected. Targeted disruption of Qbrick͞Frem1 also resulted in diminished expression of Fras1 and Frem2 at the epidermal basement membrane, confirming the reciprocal stabilization of QBRICK͞ Frem1, Fras1, and Frem2 in this location. When expressed and secreted by transfected cells, these proteins formed a ternary complex, raising the possibility that their reciprocal stabilization at the basement membrane is due to complex formation. Given the close association of Fraser syndrome phenotypes with defective epidermal-dermal interactions, the coordinated assembly of three Fraser syndrome-associated proteins at the basement membrane appears to be instrumental in epidermal-dermal interactions during morphogenetic processes.epithelial-mesenchymal interaction ͉ gene targeting ͉ morphogenesis T he extracellular matrix (ECM) is an insoluble supramolecular complex surrounding metazoan cells that is often fibrous or sheet-like. The ECM functions in the control of cellular behaviors, including migration, proliferation, and differentiation, and mediates intercellular communication, as in epithelialmesenchymal interactions; both of these functions are critical during development. Because individual ECM components often function in combination with other ECM components and soluble factors, genetic disorders of the ECM are often linked to severe developmental abnormalities.

Gene-expression analysis studies from Schultz et al. estimate that more than 2,300 genes in the mouse genome are expressed predominantly in the male germ line. As of their 2003 publication [Schultz N, Hamra FK, Garbers DL (2003) Proc Natl Acad Sci USA 100(21):12201–12206], the functions of the majority of these testis-enriched genes during spermatogenesis and fertilization were largely unknown. Since the study by Schultz et al., functional analysis of hundreds of reproductive-tract–enriched genes have been performed, but there remain many testis-enriched genes for which their relevance to reproduction remain unexplored or unreported. Historically, a gene knockout is the “gold standard” to determine whether a gene’s function is essential in vivo. Although knockout mice without apparent phenotypes are rarely published, these knockout mouse lines and their phenotypic information need to be shared to prevent redundant experiments. Herein, we used bioinformatic and experimental approaches to uncover mouse testis-enriched genes that are evolutionarily conserved in humans. We then used gene-disruption approaches, including Knockout Mouse Project resources (targeting vectors and mice) and CRISPR/Cas9, to mutate and quickly analyze the fertility of these mutant mice. We discovered that 54 mutant mouse lines were fertile. Thus, despite evolutionary conservation of these genes in vertebrates and in some cases in all eukaryotes, our results indicate that these genes are not individually essential for male mouse fertility. Our phenotypic data are highly relevant in this fiscally tight funding period and postgenomic age when large numbers of genomes are being analyzed for disease association, and will prevent unnecessary expenditures and duplications of effort by others.

Extracellular matrix (ECM), which provides critical scaffolds for all adhesive cells, regulates proliferation, differentiation, and apoptosis. Different cell types employ customized ECMs, which are thought to play important roles in the generation of so-called niches that contribute to cell-specific functions. The molecular entities of these customized ECMs, however, have not been elucidated. Here, we describe a strategy for transcriptome-wide identification of ECM proteins based on computational screening of >60,000 full-length mouse cDNAs for secreted proteins, followed by in vitro functional assays. These assays screened the candidate proteins for ECM-assembling activities, interactions with other ECM molecules, modifications with glycosaminoglycans, and cell-adhesive activities, and were then complemented with immunohistochemical analysis. We identified 16 ECM proteins, of which seven were localized in basement membrane (BM) zones. The identification of these previously unknown BM proteins allowed us to construct a body map of BM proteins, which represents the comprehensive immunohistochemistry-based expression profiles of the tissue-specific customization of BMs. basement membrane ͉ body map ͉ niche ͉ cell adhesion ͉ glycosaminoglycan

The lumicrine system is a postulated signaling system in which testis-derived (upstream) secreted factors enter the male reproductive tract to regulate epididymal (downstream) pathways required for sperm maturation. Until now, no lumicrine factors have been identified. We demonstrate that a testicular germ-cell–secreted epidermal growth factor–like protein, neural epidermal growth factor–like–like 2 (NELL2), specifically binds to an orphan receptor tyrosine kinase, c-ros oncogene 1 (ROS1), and mediates the differentiation of the initial segment (IS) of the caput epididymis. Male mice in which Nell2 had been knocked out were infertile. The IS-specific secreted proteases, ovochymase 2 (OVCH2) and A disintegrin and metallopeptidase 28 (ADAM28), were expressed upon IS maturation, and OVCH2 was required for processing of the sperm surface protein ADAM3, which is required for sperm fertilizing ability. This work identifies a lumicrine system essential for testis-epididymis-spermatozoa (NELL2-ROS1-OVCH2-ADAM3) signaling and male fertility.

Background: Polydom/SVEP1 is a putative extracellular matrix protein of unknown function. Results: Polydom/SVEP1 is a potent ligand for integrin ␣9␤1 and colocalizes with the integrin in vivo. Conclusion: Polydom/SVEP1 is a hitherto unknown high affinity ligand for integrin ␣9␤1. Significance: The identification of this high affinity ligand offers important clues toward better understanding of the consequences of integrin ␣9␤1-mediated cell-extracellular matrix interactions.

QBRICK facilitates the integrin α8β1–dependent interactions of cells with basement membranes by regulating the basement membrane assembly of nephronectin.

The premature fusion of the paired frontal bones results in metopic craniosynostosis (MC) and gives rise to the clinical phenotype of trigonocephaly. Deletions of chromosome 9p22.3 are well described as a cause of MC with variably penetrant midface hypoplasia. In order to identify the gene responsible for the trigonocephaly component of the 9p22.3 syndrome, a cohort of 109 patients were assessed by high-resolution arrays and MLPA for copy number variations (CNVs) involving 9p22. Five CNVs involving FREM1, all of which were de novo variants, were identified by array-based analyses. The remaining 104 patients with MC were then subjected to targeted FREM1 gene re-sequencing, which identified 3 further mutant alleles, one of which was de novo. Consistent with a pathogenic role, mouse Frem1 mRNA and protein expression was demonstrated in the metopic suture as well as in the pericranium and dura mater. Micro-computed tomography based analyses of the mouse posterior frontal (PF) suture, the human metopic suture equivalent, revealed advanced fusion in all mice homozygous for either of two different Frem1 mutant alleles, while heterozygotes exhibited variably penetrant PF suture anomalies. Gene dosage-related penetrance of midfacial hypoplasia was also evident in the Frem1 mutants. These data suggest that CNVs and mutations involving FREM1 can be identified in a significant percentage of people with MC with or without midface hypoplasia. Furthermore, we present Frem1 mutant mice as the first bona fide mouse model of human metopic craniosynostosis and a new model for midfacial hypoplasia.