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In terrestrial vertebrates, spermatozoa generated in the testis are transported through the reproductive tract toward outside the body. In addition to as the pathway of sperm transport, the male reproductive tract also functions as the site of post-testicular sperm maturation and the epididymis, which constitutes the majority of male reproductive tract, and plays central roles in such a sperm maturation. Recent studies with gene-modified animals have been unveiling not only the molecular mechanisms of sperm maturation in the epididymis but also the regulatory system by which the epididymis acquires and executes sperm-maturing functions. In this review, the mechanisms of mammalian sperm maturation will be summarized, based on recent findings, including the lumicrine regulation of sperm maturation.

Local control of sperm maturation Newly produced spermatozoa within the testis do not have fertilizing ability but become fully functional when they mature in the epididymis. The development of the epididymis itself is dependent on testicular factors arriving via luminal flow. Improper signaling between the testis and epididymis is hypothesized to result in male infertility. Kiyozumi et al. identified NELL2 as a testicular luminal protein that binds to its receptor, ROS1, on the luminal epididymis surface and induces epididymal differentiation (see the Perspective by Lord and Oatley). In turn, differentiated epididymis secretes a fertility-essential protease, ovochymase-2, to make spermatozoa fully mature and functional. Thus, testis-epididymis interorgan communication by this “lumicrine” regulation ensures mammalian reproduction. Science , this issue p. 1132 ; see also p. 1053

QBRICK, FRAS1, and FREM2 compose a family of extracellular matrix proteins characterized by twelve consecutive CSPG repeats and single or multiple Calx-β motifs. Dysfunction of these proteins have been associated with Fraser syndrome, which is characterized by malformation of skin, eyes, digits, and kidneys. FREM3 is another member of the 12-CSPG protein family. However, it remains unknown whether genetic dysfunction of FREM3 also causes Fraser syndrome or another developmental disorder. Here we investigated a Frem3 mutant mouse line generated by CRISPR/Cas9-mediated genome editing. The FREM3 mutant homozygotes were born at the expected Mendelian ratio and did not possess any defects characteristic of Fraser syndrome. These results indicate that the dysfunction of FREM3 is not associated with Fraser syndrome.

Preeclampsia is a systemic disease caused by abnormal placentation that affects both mother and fetus. It was reported that Laeverin (LVRN, also known as Aminopeptidase Q) was up-regulated in the placenta of preeclamptic patients. However, physiological and pathological functions of LVRN remained to be unknown. Here we characterized Lvrn function during placentation in mice. RT-PCR showed that Lvrn is expressed in both fetus and placenta during embryogenesis, and several adult tissues. When we overexpressed Lvrn in a placenta-specific manner using lentiviral vectors, we did not see any defects in both placentae and fetuses. The mice carrying Lvrn overexpressing placentas did not show any preeclampsia-like symptoms such as maternal high blood pressure and fetal growth restriction. We next ablated Lvrn by CRISPR/Cas9-mediated genome editing to see physiological function. In Lvrn ablated mice, maternal blood pressure during pregnancy was not affected, and both placentas and fetuses grew normally. Collectively, these results suggest that, LVRN is irrelevant to preeclampsia and dispensable for normal placentation and embryonic development in mice.

Yuya Sato, Daiji Kiyozumi, Sugiko Futaki, Itsuko Nakano, Chisei Shimono, Naoko Kaneko, Masahito Ikawa, Masaru Okabe, Kazunobu Sawamoto, and Kiyotoshi Sekiguchi, "Ventricular–subventricular zone fractones are speckled basement membranes that function as a neural stem cell niche", Molecular Biology of the Cell, Vol. 30, No.1, 56-68, American Society for Cell Biology, 2018, Neural stem cells (NSCs) are retained in the adult ventricular–subventricular zone (V-SVZ), a specialized neurogenic niche with a unique cellular architecture. It currently remains unclear whether or how NSCs utilize basement membranes (BMs) in this niche. Here, we examine the molecular compositions and functions of BMs in the adult mouse V-SVZ. Whole-mount V-SVZ immunostaining revealed that fractones, which are fingerlike processes of extravascular BMs, are speckled BMs unconnected to the vasculature, and differ in their molecular composition from vascular BMs. Glial fibrillary acidic protein (GFAP)-positive astrocytes and NSCs produce and adhere to speckled BMs. Furthermore, Gfap-Cre-mediated Lamc1flox(E1605Q) knockin mice, in which integrin-binding activities of laminins are specifically nullified in GFAP-positive cells, exhibit a decreased number and size of speckled BMs and reduced in vitro neurosphere-forming activity. Our results reveal niche activities of fractones/speckled BMs for NSCs and provide molecular insights into how laminin–integrin interactions regulate NSCs in vivo.

There are over 200 genes that are predicted to be solely expressed in the oocyte and ovary, and thousands more that have expression patterns in the female reproductive tract. Unfortunately, many of their physiological functions, such as their roles in oogenesis or fertilization, have yet to be elucidated. Previous knockout (KO) mice studies have proven that many of the genes that were once thought to be essential for fertility are dispensable in vivo. Therefore, it is extremely important to confirm the roles of all genes before spending immense time studying them in vitro. To do this, our laboratory analyzes the functions of ovary and oocyte-enriched genes in vivo through generating CRISPR/Cas9 KO mice and examining their fertility. In this study, we have knocked out three Oosp family genes (Oosp1, Oosp2, and Oosp3) that have expression patterns linked to the female reproductive system and found that the triple KO (TKO) mutant mice generated exhibited decreased prolificacy but were not infertile, thus, these genes may potentially be dispensable for fertility. We also generated Cd160 and Egfl6 KO mice and found these genes are individually dispensable for female fertility. KO mice with no phenotypic data are seldom published, but we believe that this information must be shared to prevent unnecessary experimentation by other laboratories.

Laminin functions as an ECM niche factor for trophoblast stem cells and secures trophoblast stem cell expansion through its interactions with integrin., The niche is a specialized microenvironment for tissue stem cells in vivo. It has long been emphasized that niche ECM molecules act on tissue stem cells to regulate their behavior, but the molecular entities of these interactions remain to be fully elucidated. Here, we report that laminin forms the in vivo ECM niche for trophoblast stem cells (TSCs), the tissue stem cells of the placenta. TSCs expressed fibronectin-binding, vitronectin-binding, and laminin-binding integrins, whereas the integrin ligands present in the TSC niche were collagen and laminin. Therefore, the only niche integrin ligand available for TSCs in vivo was laminin. Laminin promoted TSC adhesion and proliferation in vitro in an integrin binding–dependent manner. Importantly, when the integrin-binding ability of laminin was genetically ablated in mice, the size of the TSC population was significantly reduced compared with that in control mice. The present findings underscore an ECM niche function of laminin to support tissue stem cell maintenance in vivo.

The placenta is an essential organ for embryo development in the uterus of eutherian mammals. Large contributions in unveiling molecular mechanisms and physiological functions underlying placental formation were made by analyzing mutant and transgenic animals. However, it had been difficult to elucidate whether the placental defects observed in such animals originate from the placenta itself or from the fetus, as both placental and fetal genomes are modified. Therefore strategies to modify the placental genome without affecting the "fetal genome" had been needed. Through the ingenious use of lentiviral (LV) vectors, placenta-specific modification is now possible. Lentivirus is a genus of retroviruses that use reverse-transcriptase to convert its single-strand RNA genome to double-strand DNA and integrate into the host genome. Previous studies showed that when LV vectors were used to transduce embryos at the 2-cell stage, the viral genome is systemically introduced into host genome. Interestingly, by delaying the timing of transduction to the blastocyst stage, the transgene is expressed specifically in the placenta as a consequence of trophectoderm-specific viral transduction. This review summarizes the development of the LV vector-mediated placenta-specific gene manipulation technology and its application in placental research over the past decade. A perspective for future application of LV vectors to further placenta research, especially in combination with next generation genome editing technologies, is also presented.

QBRICK, FRAS1, and FREM2 compose a family of extracellular matrix proteins characterized by twelve consecutive CSPG repeats and single or multiple Calx-β motifs. Dysfunction of these proteins have been associated with Fraser syndrome, which is characterized by malformation of skin, eyes, digits, and kidneys. FREM3 is another member of the 12-CSPG protein family. However, it remains unknown whether genetic dysfunction of FREM3 also causes Fraser syndrome or another developmental disorder. Here we investigated a Frem3 mutant mouse line generated by CRISPR/Cas9-mediated genome editing. The FREM3 mutant homozygotes were born at the expected Mendelian ratio and did not possess any defects characteristic of Fraser syndrome. These results indicate that the dysfunction of FREM3 is not associated with Fraser syndrome.

More than 1000 genes are predicted to be predominantly expressed in mouse testis, yet many of them remain unstudied in terms of their roles in spermatogenesis and sperm function and their essentiality in male reproduction. Since individually indispensable factors can provide important implications for the diagnosis of genetically related idiopathic male infertility and may serve as candidate targets for the development of nonhormonal male contraceptives, our laboratories continuously analyze the functions of testis-enriched genes in vivo by generating knockout mouse lines using the CRISPR/Cas9 system. The dispensability of genes in male reproduction is easily determined by examining the fecundity of knockout males. During our large-scale screening of essential factors, we knocked out 30 genes that have a strong bias of expression in the testis and are mostly conserved in mammalian species including human. Fertility tests reveal that the mutant males exhibited normal fecundity, suggesting these genes are individually dispensable for male reproduction. Since such functionally redundant genes are of diminished biological and clinical significance, we believe that it is crucial to disseminate this list of genes, along with their phenotypic information, to the scientific community to avoid unnecessary expenditure of time and research funds and duplication of efforts by other laboratories., Thirty testis-enriched genes are dispensable for male fertility based on phenotypic analyses of knockout mice produced by the CRISPR/Cas9 system.

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.

Masanaga Muto, Yoshitaka Fujihara, Tomohiro Tobita, Daiji Kiyozumi, Masahito Ikawa, Lentiviral Vector-Mediated Complementation Restored Fetal Viability but Not Placental Hyperplasia in Plac1-Deficient Mice, Biology of Reproduction, Volume 94, Issue 1, 1 January 2016, 6, 1–9, https://doi.org/10.1095/biolreprod.115.133454, 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.

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.

QBRICK facilitates the integrin α8β1–dependent interactions of cells with basement membranes by regulating the basement membrane assembly of nephronectin., Dysfunction of the basement membrane protein QBRICK provokes Fraser syndrome, which results in renal dysmorphogenesis, cryptophthalmos, syndactyly, and dystrophic epidermolysis bullosa through unknown mechanisms. Here, we show that integrin α8β1 binding to basement membranes was significantly impaired in Qbrick-null mice. This impaired integrin α8β1 binding was not a direct consequence of the loss of QBRICK, which itself is a ligand of integrin α8β1, because knock-in mice with a mutation in the integrin-binding site of QBRICK developed normally and do not exhibit any defects in integrin α8β1 binding. Instead, the loss of QBRICK significantly diminished the expression of nephronectin, an integrin α8β1 ligand necessary for renal development. In vivo, nephronectin associated with QBRICK and localized at the sublamina densa region, where QBRICK was also located. Collectively, these findings indicate that QBRICK facilitates the integrin α8β1–dependent interactions of cells with basement membranes by regulating the basement membrane assembly of nephronectin and explain why renal defects occur in Fraser syndrome.

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.

A novel protein family characterized by the presence of 12 CSPG repeats and Calx-β domains includes Fras1, QBRICK/Frem1, Frem2 and Frem3. Although Fras1, QBRICK/Frem1 and Frem2 have been shown to localize at the basement membrane through reciprocal stabilization, it remains unclear whether Frem3 is also deposited at the basement membrane in a similar manner. Here we show that Frem3 localizes at the basement membrane with tissue distribution patterns clearly distinct from those of other 12 CSPG repeats-containing proteins (12-CSPG proteins). In adult mice, Frem3 was present at the basement membrane underlying ductal cells of the salivary gland, retinal ganglion cells, basal cells of epidermis and hair follicles, where other 12-CSPG proteins were barely expressed. In embryos and neonates, the expression of Frem3 transcripts was significantly lower than that of the other 12-CSPG proteins, although Frem3 protein was coexpressed with other 12-CSPG proteins at the basement membranes of retina, renal epithelia and epidermis. Interestingly, Frem3 deposition at the epidermal basement membrane was not severely compromised in blebbing mutant embryos, in which the basement membrane deposition of other 12-CSPG proteins was dramatically reduced due to the breakdown of their reciprocal stabilization. These results indicate that Frem3 is a basement membrane protein that is distinct from other 12-CSPG proteins in its tissue distribution and competence to assemble into the basement membrane.

Integrins are cell surface receptors for cell adhesion. Integrin-mediated cell adhesion regulates various cellular processes, including cell survival, migration, proliferation, and differentiation. In vivo, ligands for integrins are immobilized within extracellular matrices, insoluble sheet-like or fibrous supramolecular complexes that associate with or surround cells. To better understand the molecular basis of integrin-mediated regulation of cellular behavior in vivo, it is of critical importance to collect information regarding the activities as well as spatial distributions of integrin ligands in situ. This unit describes a protocol for detecting the spatial distribution of the complement of integrin ligands in situ by overlaying soluble recombinant integrins.

We screened for genes specifically expressed in the mesenchymes of developing hair follicles using representational differential analysis; one gene identified was MAEG, which encodes a protein consisting of five EGF-like repeats, a linker segment containing a cell-adhesive Arg-Gly-Asp (RGD) motif, and a MAM domain. Immunohistochemistry showed that MAEG protein was localized at the basement membrane of embryonic skin and developing hair follicles, while MAEG expression diminished at the tip of the hair bud. A recombinant MAEG fragment containing the RGD motif was active in mediating adhesion of keratinocytes to the substratum in an RGD-dependent manner. One of the adhesion receptors recognizing the RGD motif was found to be the alpha8beta1 integrin, the expression of which was detected in the placode close to MAEG-positive mesenchymal cells, but later became restricted to the tip of the developing hair bud. Given its localized expression at the basement membrane in developing hair follicles and the RGD-dependent cell-adhesive activity, MAEG may play a role as a mediator regulating epithelial-mesenchymal interaction through binding to RGD-binding integrins including alpha8beta1 during hair follicle development.

A polymorphic protein well-correlated to the diploid S genotypes of the pollen parent was detected by two-dimensional gel electrophoresis in Pyrus pyrifolia (Japanese pear). Its molecular weight was about 50 kDa, and it was expressed primarily in pollen. Partial amino acid sequences of the polymorphic protein from 'Nijisseiki' (S2S4), a cultivar of P. pyrifolia, were determined. Based on these sequences, two cDNA sequences associated with the S2 and S4 genotypes were identified by PCR-based methods. Both encode a protein of 458 amino acids whose sequence has high similarity to eukaryotic UDP-glucose pyrophosphorylases (UGPases) (EC 2.7.7.9), so they were named UGPases PA and PC. As there are only three amino acid substitutions between UGPases PA and PC, it is unlikely that they are pollen factors that recognize self and non-self S-RNases. Although this UGPase had more than 75% sequence identity to the known plant UGPases, its C-terminal sequences differed markedly. This unique C-terminal region of UGPases PA and PC may act in their subcellular localization in the pollen or interact with some other factor(s).

Jeong S-J, Luo R, Singer K, Giera S, Kreidberg J, Kiyozumi D, et al. (2013) GPR56 Functions Together with α3β1 Integrin in Regulating Cerebral Cortical Development. PLoS ONE 8(7): e68781. https://doi.org/10.1371/journal.pone.0068781, 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.

This research was originally published in the Journal of Biological Chemistry. Ryoko Sato-Nishiuchi, Itsuko Nakano, Akio Ozawa, Yuya Sato, Makiko Takeichi, Daiji Kiyozumi, Kiyoshi Yamazaki, Teruo Yasunaga, Sugiko Futaki and Kiyotoshi Sekiguchi. Polydom/SVEP1 Is a Ligand for Integrin α9β1. J. Biol. Chem. 2012; 287: 25615-25630 © the American Society for Biochemistry and Molecular Biology, 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.

Fused pulmonary lobes (fpl) is a mutant gene that is inherited in an autosomal recessive manner and causes various developmental defects, including fusion of pulmonary lobes, and eyelid and digit anomalies in rats. Since these developmental defects closely resemble those observed in patients with Fraser syndrome, a recessive multiorgan disorder, and its model animals, we investigated whether the abnormal phenotypes observed in fpl/fpl mutant rats are attributable to a genetic disorder similar to Fraser syndrome. At the epidermal basement membrane in fpl/fpl mutant neonates, the expression of QBRICK, a basement membrane protein whose expression is attenuated in Fraser syndrome model mice, was greatly diminished compared with control littermates. Quantitative RT-PCR analyses of Fraser syndrome-related genes revealed that Frem2 transcripts were markedly diminished in QBRICK-negative embryos. Genomic DNA sequencing of the fpl/fpl mutant identified a nonsense mutation that introduced a stop codon at serine 2005 in Frem2. These findings indicate that the fpl mutant is a rat model of human Fraser syndrome.

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.

Carney TJ, Feitosa NM, Sonntag C, Slanchev K, Kluger J, Kiyozumi D, et al. (2010) Genetic Analysis of Fin Development in Zebrafish Identifies Furin and Hemicentin1 as Potential Novel Fraser Syndrome Disease Genes. PLoS Genet 6(4): e1000907. https://doi.org/10.1371/journal.pgen.1000907, 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.

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.

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.

We used PCR-based cDNA subtraction to screen for genes up-regulated during mouse hair morphogenesis. One gene selected was predominantly expressed at the tip of developing hair follicles and encoded a protein characterized by the presence of twelve tandem repeats of approximately 120 amino acids and a novel N-terminal domain containing an Arg-Gly-Asp cell-adhesive motif. Immunohistochemistry demonstrated that the protein encoded by this gene, named QBRICK, was localized at the basement membrane zone of embryonic epidermis and hair follicles, in which it was more enriched at the tip rather than the stalk region. Cell adhesion assays showed that QBRICK was active in mediating cell-substratum adhesion through integrins containing alphav or alpha8 chain, but not integrin alpha5beta1. Immunohistochemistry showed that QBRICK colocalized with alphav-containing integrins in the interfollicular region, but with the alpha8-containing integrin at the tip region of developing hair follicles. These results, together, indicate that QBRICK is an adhesive ligand of basement membrane distinctively recognized by cells in the embryonic skin and hair follicles through different types of integrins directed to the Arg-Gly-Asp motif.