Daiji Kiyozumi

Transcriptome-based systematic identification of extracellular matrix proteins

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.

Breakdown of the reciprocal stabilization of QBRICK/Frem1, Fras1, and Frem2 at the basement membrane provokes Fraser syndrome-like defects

An emerging family of extracellular matrix proteins characterized by 12 consecutive CSPG repeats and the presence of Calx-β motif(s) includes Fras1, QBRICK/Frem1, 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 QBRICK/Frem1 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 Qbrick/Frem1 also resulted in diminished expression of Fras1 and Frem2 at the epidermal basement membrane, confirming the reciprocal stabilization of QBRICK/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.

Genetic Analysis of Fin Development in Zebrafish Identifies Furin and Hemicentin1 as Potential Novel Fraser Syndrome Disease Genes

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 diseases aetiology. In addition, the novel genes might prove helpful to unravel the molecular nature of thus far unresolved cases of the human disease.

Heterozygous mutations of FREM1 are associated with an increased risk of isolated metopic craniosynostosis in humans and mice

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.

Polydom/SVEP1 Is a Ligand for Integrin α9β1

Background: Polydom/SVEP1 is a putative extracellular matrix protein of unknown function. Results: Polydom/SVEP1 is a potent ligand for integrin α9β1 and colocalizes with the integrin in vivo. Conclusion: Polydom/SVEP1 is a hitherto unknown high affinity ligand for integrin α9β1. Significance: The identification of this high affinity ligand offers important clues toward better understanding of the consequences of integrin α9β1-mediated cell-extracellular matrix interactions. A variety of proteins, including tenascin-C and osteopontin, have been identified as ligands for integrin α9β1. However, their affinities for integrin α9β1 are apparently much lower than those of other integrins (e.g. α3β1, α5β1, and α8β1) for their specific ligands, leaving the possibility that physiological ligands for integrin α9β1 still remain unidentified. In this study, we found that polydom (also named SVEP1) mediates cell adhesion in an integrin α9β1-dependent manner and binds directly to recombinant integrin α9β1 with an affinity that far exceeds those of the known ligands. Using a series of recombinant polydom proteins with N-terminal deletions, we mapped the integrin-binding site to the 21st complement control protein domain. Alanine-scanning mutagenesis revealed that the EDDMMEVPY sequence (amino acids 2636–2644) in the 21st complement control protein domain was involved in the binding to integrin α9β1 and that Glu2641 was the critical acidic residue for the integrin binding. The importance of this sequence was further confirmed by integrin binding inhibition assays using synthetic peptides. Immunohistochemical analyses of mouse embryonic tissues showed that polydom colocalized with integrin α9 in the stomach, intestine, and other organs. Furthermore, in situ integrin α9β1 binding assays using frozen mouse tissues showed that polydom accounts for most, but not all, of the integrin α9β1 ligands in tissues. Taken together, the present findings indicate that polydom is a hitherto unknown ligand for integrin α9β1 that functions as a physiological ligand in vivo.

Basement membrane assembly of the integrin α8β1 ligand nephronectin requires Fraser syndrome–associated proteins

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

Genome engineering uncovers 54 evolutionarily conserved and testis-enriched genes that are not required for male fertility in mice.

Significance In the mouse genome, thousands of genes are predominantly expressed in the testis, where these genes are thought to play important roles in spermatogenesis and fertilization. However, in this study, we report that 54 evolutionarily conserved and testis-enriched genes are not essential individually for male mouse fertility. Because the recent development of the CRISPR/Cas9 system has made it faster and easier to produce knockout mice, our results suggest that one should determine whether a gene of interest is essential for male fertility in vivo before spending significant effort to analyze the molecular function of the gene in vitro. 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.

GPR56 Functions Together with α3β1 Integrin in Regulating Cerebral Cortical Development

Loss of function mutations in GPR56, which encodes a G protein-coupled receptor, cause a specific human brain malformation called bilateral frontoparietal polymicrogyria (BFPP). Studies from BFPP postmortem brain tissue and Gpr56 knockout mice have previously showed that GPR56 deletion leads to breaches in the pial basement membrane (BM) and neuronal ectopias during cerebral cortical development. Since α3β1 integrin also plays a role in pial BM assembly and maintenance, we evaluated whether it functions together with GPR56 in regulating the same developmental process. We reveal that loss of α3 integrin enhances the cortical phenotype associated with Gpr56 deletion, and that neuronal overmigration through a breached pial BM occurs earlier in double knockout than in Gpr56 single knockout mice. These observations provide compelling evidence of the synergism of GPR56 and α3β1 integrin in regulating the development of cerebral cortex.

Lentiviral Vector Mediated Complementation Restored Fetal Viability but Not Placental hyperplasia in Plac1-Deficient Mice

ABSTRACT The X-linked Plac1 gene is maternally expressed in trophoblast cells during placentation, and its disruption causes placental hyperplasia and intrauterine growth restriction. In contrast, Plac1 is also reported to be one of the upregulated genes in the hyperplastic placenta generated by nuclear transfer. However, the effect of overexpressed Plac1 on placental formation and function remained unaddressed. We complemented the Plac1 knockout placental dysfunction by lentiviral vector-mediated, placenta-specific Plac1 transgene expression. Whereas fetal development and the morphology of maternal blood sinuses in the labyrinth zone improved, placental hyperplasia remained, with an expanded the junctional zone that migrated and encroached into the labyrinth zone. Further experiments revealed that wild-type placenta with transgenically expressed Plac1 resulted in placental hyperplasia without the encroaching of the junctional zone. Our findings suggest that Plac1 is involved in trophoblast cell proliferation, differentiation, and migration. Its proper expression is required for normal placentation and fetal development.

Identification of genes expressed during hair follicle induction

The hair follicle is one of the skin appendages that develops through reciprocal epithelial‐mesenchymal interactions. Although a large number of studies have been made on the mechanisms of hair follicle development, the whole molecular mechanism that governs hair follicle development remains poorly defined. To further understand the molecular basis of hair follicle development, it is necessary to identify genes that drive hair morphogenesis. As an initial approach, we attempted to identify gene products associated with mouse hair follicle development. Genes upregulated in the vibrissal hair placodes were screened by polymerase chain reaction (PCR)‐based cDNA subtraction. The genes thus isolated were evaluated for their hair development‐associated induction and spatiotemporal expression by quantitative reverse‐transcription‐PCR analysis and whole‐mount in situ hybridization, respectively. Finally, we identified four genes whose upregulation and spatiotemporal expression in developing hair follicles were confirmed. Successful identification of novel hair development‐associated genes will be informative as clues for further characterization of hair follicle development at the molecular level.