Search

Ca(2+) plays a complex role in the differentiation of committed pre-adipocytes into mature, fat laden adipocytes. Stim1 is a single pass transmembrane protein that has an essential role in regulating the influx of Ca(2+) ions through specific plasma membrane store-operated Ca(2+) channels. Stim1 is a sensor of endoplasmic reticulum Ca(2+) store content and when these stores are depleted ER-localized Stim1 interacts with molecular components of store-operated Ca(2+) channels in the plasma membrane to activate these channels and induce Ca(2+) influx. To investigate the potential role of Stim1 in Ca(2+)-mediated adipogenesis, we investigated the expression of Stim1 during adipocyte differentiation and the effects of altering Stim1 expression on the differentiation process. Western blotting revealed that Stim1 was expressed at low levels in 3T3-L1 pre-adipocytes and was upregulated 4 days following induction of differentiation. However, overexpression of Stim1 potently inhibited their ability to differentiate and accumulate lipid, and reduced the expression of C/EBP alpha and adiponectin. Stim1-mediated differentiation was shown to be dependent on store-operated Ca(2+) entry, which was increased upon overexpression of Stim1. Overexpression of Stim1 did not disrupt cell proliferation, mitotic clonal expansion or subsequent growth arrest. siRNA-mediated knockdown of endogenous Stim1 had the opposite effect, with increased 3T3-L1 differentiation and increased expression of C/EBP alpha and adiponectin. We thus demonstrate for the first time the presence of store-operated Ca(2+) entry in 3T3-L1 adipocytes, and that Stim1-mediated Ca(2+) entry negatively regulates adipocyte differentiation. We suggest that increased expression of Stim1 during 3T3-L1 differentiation may act, through its ability to modify the level of Ca(2+) influx through store-operated channels, to balance the level of differentiation in these cells in vitro.

Three distinct alpha chains form the collagen VI monomer, the alpha 3(VI) chain being much larger than the alpha 1(VI) and alpha 2(VI) chains. The alpha 3(VI) chain has 10 von Willebrand Factor type A domains of approximately 200 amino acids at the N-terminus (N1-N10) compared with only one such domain in the alpha 1(VI) and alpha 2(VI) chains. Domains N10, N9, N7 and N3 of the alpha 3(VI) chain are subject to alternative splicing in chick and/or human tissues, indicating the possibility of isoforms that have different functions depending on which N-terminal domains are included or excluded. In this study we have PCR amplified and sequenced mouse alpha 3(VI) cDNA encoding the N2-N10 domains. By reverse transcription-PCR using oligonucleotides spanning different regions of the cDNA we have undertaken a comprehensive analysis of alternative splicing of the alpha 3(VI) mRNA in embryonic and adult mouse tissues. We demonstrate that domains N10, N9 and N7 are also subject to alternative splicing in mouse tissues and in addition identify an abundant novel variant transcript that lacks all four N-terminal domains (N7-N10) in mouse tissues and human cells. We also identify less abundant transcripts that lack a large part of the N3 domain, and transcripts lacking the entire N5 domain. Using specific RNase protection assays we show that the shorter transcripts containing domains (N8+N7+N6), (N8+N6) and N6 are present at higher levels than transcripts containing the N10 and/or N9 domains, with tissue-specific variation in the levels of variant transcripts. These studies demonstrate a larger range of collagen VI protein variants than previously described.

Lung epithelial branching morphogenesis results from a repetitive series of cleft and bud formation, a process dependent upon a complex interaction with the surrounding mesenchyme. The present study describes these cleft- and bud-forming regions as autonomous morphogenetic compartments within the embryonic day 11.5 (E11.5) mouse lung and directly correlates their identity with differences in epithelial proliferation rates and the localization pattern of specific basement membrane components. Lung buds were cultured in vitro, in two-dimensional planes, and labeled with a series of 5-bromo-2'-deoxyuridine (BrdU) pulses. Collectively, epithelial cells within actively budding regions of the bronchiolar tree demonstrated an at least 2.5-fold greater proliferation rate than those situated in the adjacent cleft-forming regions. Epithelial proliferation rates showed an inverse relationship with the degree of immunoreactivity of nidogen, laminin-1, fibronectin, and collagen IV within the underlying basement membrane. Epithelial cells dissected free from mesenchyme demonstrated cell-cell contact-dependent proliferation, thus revealing a hierarchy between mesenchymal signaling and direct epithelial cell-cell communication during branch formation. Dissection of the E11.5 bronchiolar tree into specific distalbud and interbud regions and their in vitro culture demonstrated differences in their autonomous morphogenetic potential. Tissue dissected from the distal tips of the lung continued to branch, whereas tissue dissected from immediately adjacent cleft regions seldom branched. Isolated distalbud tissue also continued to correlate regional differences in epithelial proliferation rates and immunolocalization patterns of nidogen, laminin-1, fibronectin, and collagen IV with branch formation. These results support the basement membrane remodeling hypothesis, thus connecting nidogen, collagen type IV, fibronectin, and laminin-1 localization with the molecular processes directing epithelial proliferation and supporting bud outgrowth and cleft formation/stabilization during lung morphogenesis.

Igf2 and H19 are closely linked imprinted genes in both mice and humans that are expressed from opposite parental alleles. In this study we demonstrate that these two genes are also closely linked in the rat, with the H19 gene mapping to within 145 kb of Igf2 on rat chromosome 1. We identified polymorphisms in H19 and Igf2 transcripts in two inbred rat strains and determined the expression of the parental alleles in F1 offspring. The H19 gene was shown to be expressed exclusively from the maternal allele in all fetal and neonatal tissues. Monoallelic expression of Igf2 from the paternal allele was found in all tissues except the leptomeninges and choroid plexus. Igf2 in the choroid plexus was monoallelic at days 13.5 and 15.5 of gestation with a switch to biallelic expression by day 18.5, demonstrating a loss of imprinting after the choroid plexus has differentiated. Biallelic expression of Igf2 was observed in the leptomeninges at all fetal and neonatal stages analyzed. These studies demonstrate conservation of imprinting of two closely linked genes transcribed from opposite parental alleles in a species other than human or mouse. A comparative approach between different species will be important in defining the mechanisms that regulate the tissue-specific expression of imprinted genes.

Collagen VI is a microfibrillar component of the extracellular matrix that is predicted to have an important structural role in matrix organization and a biological function in mediating cell-matrix interactions. Secreted collagen VI molecules are composed of three distinct subunits, the alpha 1(VI), alpha 2(VI), and alpha 3(VI) chains. To determine when, and in which tissues, collagen VI is likely to have a role in embryonic processes, we have analyzed the expression patterns of the three subunit chains during postimplantation mouse development by reverse transcriptase-PCR (RT-PCR), in situ hybridization, and immunofluorescence. No collagen VI protein could be detected in the mouse embryo until Day 11.5 of gestation, when low levels were localized within the mesoderm layer of the visceral yolk sac, the subepidermal matrix of branchial arches, and the vessel wall of the dorsal aorta. Levels of collagen VI mRNA and protein increased during the period from Days 12.5 to 14.5 in the visceral yolk sac, subepidermal mesenchyme, lung, gut, meninges, muscle, perichondrium, and vertebral column. The cartilage matrix of ribs and developing long bones was not stained with collagen VI antisera, but pericellular staining of chondrocytes was seen in both tissues. Low levels of collagen VI mRNA and protein were seen in the fetal liver except for the connective tissue of the liver capsule, which was highly stained. Collagen VI was first detected at significant levels in the developing heart on Day 14.5. These data demonstrate a tissue-specific onset of collagen VI synthesis and deposition in the extracellular matrix of developing mouse embryos at a much later stage of development than that reported for fibronectin or collagen I. Sensitive RT-PCR assays showed that alpha 1(VI) and alpha 2(VI) mRNAs were amplified from extracts of embryonic tissues as early as Day 7.5, while alpha 3(VI) mRNA was not detected until Day 10.5. Expression of the alpha 3(VI) gene immediately preceded the appearance of collagen VI protein in embryonic tissues. This correlation is consistent with the proposal that expression of alpha 3(VI) chains regulates the formation and secretion of collagen VI trimers and collagen VI matrix deposition during development.

The Hba(th-J) mouse mutation is a deletion on chromosome 11 that spans the alpha-globin complex and causes alpha-thalassemia in heterozygous animals and in utero death of embryos homozygous for the deletion. We hypothesised that one or more genes closely linked to the Hba locus are also deleted in these mutant mice and that deletion of these additional genes is responsible for the embryo lethality. We have analysed the developmental profile of mutant embryos using a PCR assay to distinguish homozygous embryos from wild-type and heterozygous embryos. No homozygous embryos are detectable on Day 6.5 of gestation and morphological analysis of embryos on Day 5.5 shows that both the embryonic and extraembryonic ectoderm of the egg cylinder are reduced in size and contain degenerate cells. Preimplantation homozygous embryos are morphologically normal with the same proportion developing to the blastocyst stage as control embryos. However, the cell number of homozygous embryos on Day 4.5 is significantly reduced due predominantly to a decrease in the cell number of the trophectoderm and not the inner cell mass. When homozygous blastocysts are plated in vitro, outgrowth of giant trophectoderm cells appears similar to that of wild-type embryos but outgrowth of the inner cell mass is affected. Cells from the inner cell mass of homozygous embryos appear to undergo necrosis and dissociate from the trophectoderm outgrowth after 3 to 4 days in culture. These studies demonstrate that the development of both the inner cell mass and the trophectoderm of embryos homozygous for the Hbath-J deletion is affected by the mutation. We have used quantitative Southern blotting to show that 3-methyladenine glycosylase (mpg) and dist1, two genes closely linked to the Hba locus on chromosome 11, are also deleted in this mutation. Reverse transcriptase-polymerase chain reaction analyses demonstrate that mpg and dist1 are normally expressed by preimplantation and early postimplantation embryos, whereas alpha-globin transcripts from the Hba locus are not detected until Day 7.5 of gestation. These studies demonstrate that deletion of the mpg or dist1 genes is likely to be responsible for the homozygote embryo lethality and the potential roles of these gene products in early embryogenesis are discussed.

The patterns of laminin A, B1, B2, nidogen and collagen alpha 1(IV) gene expression in the embryonic mouse lung were determined using in situ hybridization histochemistry at a stage when branching morphogenesis is taking place. Collagen alpha 1(IV), laminin B1 and B2 genes were expressed throughout the mesenchyme and epithelium. Nidogen gene expression was uniform throughout the mesenchyme but was not detected in epithelial cells. Laminin A mRNA was localized to cells closely associated with a basement membrane at the epithelial-mesenchymal interface. However, expression of the laminin A gene was limited to the mesenchymal cells in bronchial regions and to epithelial cells in distal terminal lobules. We propose that the pattern of laminin A gene expression in different regions of the developing lung will influence the structure of the basement membrane at the epithelial-mesenchymal interface and thus have a role in branching morphogenesis.

The pattern of laminin A, B1, B2, nidogen, and collagen alpha 1 (IV) gene expression in the 12.5-day mouse embryo was determined by in situ hybridization. Laminin B1, B2, and collagen alpha 1 (IV) mRNAs were present in many epithelial and mesenchymal compartments. Laminin A mRNA had a more restricted distribution, being present in cells closely associated with basement membranes and also in the ependymal layer of the neural tube. Nidogen was not produced by any epithelium, but was abundant in mesenchymal and endothelial cells. These results demonstrate that mesenchymal cells contribute significantly to basement membrane production, and that many cells not associated with typical basement membranes produced high levels of mRNAs coding for basement membrane components. Very few cell types produced all five gene products, and some tissues preferentially expressed only one or two of the five genes. This study shows that basement membranes at the epithelial-mesenchymal interface in the majority of mouse embryonic tissues are assembled from components derived from both cell types, and that heterogeneous matrix structures containing different laminin subunits and/or nidogen are likely to be present in the central nervous system and other tissues of the midgestation mouse embryo.

The stromal interaction molecules STIM1 and STIM2 are endoplasmic reticulum Ca(2+) sensors, serving to detect changes in receptor-mediated ER Ca(2+) store depletion and to relay this information to plasma membrane localized proteins, including the store-operated Ca(2+) channels of the ORAI family. The resulting Ca(2+) influx sustains the high cytosolic Ca(2+) levels required for activation of many intracellular signal transducers such as the NFAT family of transcription factors. Models of STIM protein deficiency in mice, Drosophila melanogaster and Caenorhabditis elegans, in addition to the phenotype of patients bearing mutations in STIM1 have provided great insight into the role of these proteins in cell physiology and pathology. It is now becoming clear that STIM1 and STIM2 are critical for the development and functioning of many cell types, including lymphocytes, skeletal and smooth muscle myoblasts, adipocytes and neurons, and can interact with a variety of signalling proteins and pathways in a cell- and tissue-type specific manner. This review focuses on the role of STIM proteins in development, differentiation and disease, in particular highlighting the functional differences between STIM1 and STIM2.

We have developed a primary culture system for fetal mouse choroid plexus epithelial cells which maintains their differentiated phenotype. When grown on a reconstituted basement membrane substrate (Matrigel) epithelial cells formed aggregates which became embedded in the matrix and developed into characteristic and highly reproducible multicellular vesicular structures. These vesicles consisted of a squamous layer of epithelial cells with extensive attachment to the matrix substrate, surrounding a fluid-filled lumen. Electron microscopy showed that cells comprising these vesicles had a high degree of membrane specialization and polarized morphology which in many respects mimicked the in vivo morphology. Biochemical analyses demonstrated that under these culture conditions the tissue-specific pattern of gene expression of fetal choroid plexus epithelium was maintained. After 6 days in culture these cells contained approximately the same amount of transthyretin mRNA as the 12.5-day choroid plexus in vivo, and the level of total RNA per cell, which is proportional to the protein synthetic capability of the cells, was also maintained. The pattern of protein secretion was also very similar to that generated by fetal mouse choroid plexus cells in vivo. In contrast choroid plexus epithelial cells attached poorly to collagen I gels. Heterogeneous aggregates were formed in which cell-cell interactions were more extensive than cell-substrate interactions, and in no cases was a central lumen observed. Cells on the surface of large aggregates showed some evidence of membrane polarization, while the majority of cells in the cultures exhibited little evidence of polarized morphology. Despite the striking difference in morphology and multicellular organization these cells still expressed high levels of transthyretin mRNA and maintained the same pattern of protein synthesis as cells cultured on Matrigel. These results indicate that the basement membrane is important for the organization of choroid plexus epithelial cells into a functional epithelium in vitro and thus presumably the maintenance of the integrity of the blood-brain barrier in vivo. In contrast to several other epithelial systems which have been studied, the type of extracellular matrix does not appear to directly influence tissue-specific gene expression by choroid plexus epithelial cells. Thus the level of gene expression is not dependent on the cytoarchitecture and multicellular organization of this cell type.

We report the isolation of eight independent cell lines from preimplantation mouse embryos, which have a parietal endoderm phenotype. When grown as aggregates, these cell lines produce large amounts of a basement membrane matrix, that contains laminin, nidogen, heparan sulfate proteoglycan, collagen IV, and BM-40. The biosynthetic profiles of all eight cell lines are very similar to parietal endoderm cells in vivo which synthesize Reichert's membrane. The structure of the matrix produced by the parietal endoderm cell lines (PEC lines) resembles more closely Reichert's membrane than the Engelbreth—Holm—Swarm (EHS) tumor in susceptibility to proteolytic degradation. Since these cell lines produce large quantities of basement membrane they will be useful for structural and functional comparison of a Reichert's membrane matrix with the basement membrane produced by the EHS tumor.

The protein BM-90 was solubilized from the mouse Engelbreth-Holm-Swarm tumor with neutral buffers in molar yields lower (15-30%) than found for other basement membrane proteins (e.g. laminin, BM-40). The purified protein was shown to be rich in cysteine (5 mol%) and to change in SDS electrophoresis from an 84-kDa position to a 95-kDa one upon reduction. BM-90 was also shown to be a calcium-binding protein. The N-terminal sequence of BM-90, as well as those of several internal peptides, showed no identity with any known protein sequences, indicating that it is a new protein. Specific radioimmunoassays showed no or only minor cross-reactions with other known basement membrane proteins. Immunological assays demonstrated BM-90 to be present in neutral salt extracts from mouse heart and kidney, in serum (20-40 micrograms/ml) and in the medium of various cultured cells (0.1-1 microgram/ml). The protein in these samples was identical in size to BM-90 purified from the tumor, indicating that negligible degradation occurs during purification. An extracellular matrix localization of BM-90 was shown by immunofluorescence for Reichert's membrane, lens capsules and other basement membranes. Thus, BM-90 appears to be a novel basement membrane protein whose functions remain to be studied.

Genetic analysis has revealed an important function in myogenesis for Myostatin, a member of the TGF-β superfamily. However, the cascade of genes that responds to Myostatin signalling to regulate myogenesis is not well understood. Thus, a suppressive subtraction hybridization to identify such genes was undertaken and here we report the cloning and characterization of a novel gene, Mighty. Mighty is expressed in a variety of different tissues but appears to be specifically regulated by Myostatin in skeletal muscle. Overexpression of Mighty in C2C12 cells results in early withdrawal of myoblasts from the cell cycle, enhanced and accelerated differentiation and hypertrophy of myotubes. Most importantly, Mighty overexpression leads to increased and earlier expression of MyoD and increased secretion of another known differentiation inducing factor, IGF-II. Furthermore, viral expression of Mighty in mdx mice resulted in an increase in the number of larger healthy muscle fibers. Given its role in myogenesis, we propose that Mighty is a critical promyogenic factor which plays a key role in the signalling pathway downstream of Myostatin.

Human and murine STIM1 were originally discovered as candidate growth regulators in tumours and in the bone marrow stroma, and the structurally related vertebrate family members, STIM2 and the Drosophila homologue D-Stim, were subsequently identified. STIM proteins are ubiquitously expressed type I single-pass transmembrane proteins which have a unique combination of structural motifs within their polypeptide sequences. The extracellular regions contain an N-terminal unpaired EF-hand Ca(2+) binding motif adjacent to an unconventional glycosylated SAM domain, while the cytoplasmic regions contain alpha-helical coiled-coil domains within a region having homology to ERM domains adjacent to the transmembrane region, and phosphorylated proline-rich domains near the C-terminus. STIM1, STIM2 and D-Stim diverge significantly only in their structure C-terminal to the coiled-coil/ERM domains. The STIM structural domains were predicted to function in Ca(2+) binding as well as in mediating interactions between STIM proteins and other proteins, and homotypic STIM1-STIM1 and heterotypic STIM1-STIM2 interactions were demonstrated biochemically. However, the functional significance of the cellular localisation of STIM1 and its domain structure only became evident after recent breakthrough research identified STIM1 as a key regulator of store-operated calcium (SOC) entry into cells. It is now clear that STIM1 is both a sensor of Ca(2+) depletion in the endoplasmic reticulum (ER) lumen and an activator of Orai1-containing SOC channels in the plasma membrane. On the basis of recent functional studies a model can be proposed to explain how the biochemical properties of STIM1 contribute to its precise membrane localisation and its function in regulating SOC entry.

STIM1 (GOK) maps to a region of human Chromosome (Chr) 11p15.5 that is implicated in several embryonal tumors, and some evidence indicates that STIM1 may have a growth suppressor role in rhabdomyosarcoma. In this study we have mapped the murine homolog, Stim1, to the same position as Hbb on distal mouse Chr 7. This region is separated by 20 cM from the region of distal Chr 7 that contains Igf2, H19, and other imprinted genes. Using strain-specific polymorphisms, we have shown that Stim1 is expressed from both parental alleles in fetal and neonatal mouse tissues. Similar analyses of human Wilms' tumor and normal kidney tissues demonstrated biallelic expression of STIM1 in the majority of samples. These data demonstrate that Stim1 expression is not regulated by genomic imprinting in either mouse or human tissues. Thus, if STIM1 is a tumor suppressor at 11p15.5, loss of expression is not due to imprinting effects.

We have previously shown that isolated mouse fetal choroid plexus epithelial (CPE) cells penetrate a basement membrane matrix (Matrigel) substratein vitroto form single-layered epithelial vesicles embedded within the matrix. To determine which properties of the matrix are important for inducing or permitting cells to penetrate the substrate and organize into multicellular vesicles we have made quantitative changes to the basement membrane components and growth factors in cell cultures. Matrigel diluted to 33 or 10% with a collagen I gel was not permissive to cell invasion, and CPE cells formed a polarized epithelial monolayer on the substrate surface which had ultrastructural characteristics similar to those of CPE vesicles. Cells in these monolayers proliferated more rapidly than cells in epithelial vesicles. When deliberately embedded within a 33 or 10% Matrigel matrix, CPE cells were able to form vesicles, indicating that a dilute matrix is nonpermissive to cell invasion but promotes epithelial polarization and organization into vesicles. Cells embedded within a 100% collagen I matrix did not proliferate or form epithelial vesicles and the majority of cells did not remain viable. Addition of laminin to the collagen I gel promoted cell adhesion and cell survival, but did not promote the formation of extensive monolayers on the substrate nor the formation of epithelial vesicles within the matrix. Cell invasion into the 33% Matrigel matrix was induced by addition of laminin, nidogen, or a laminin–nidogen complex to the substrate or by addition of TGFβ2 to the culture medium, but not TGFβ1 or PDGF. These studies show that CPE cells are sensitive to quantitative changes in matrix composition, which influences their survival and proliferation and also their ability to penetrate the matrix and organize into multicellular epithelial vesicles.

Genomic imprinting is a process that results in the differential expression of genes according to their parental inheritance. Two imprinted genes, insulin-like growth factor 2 (Igf2) and H19 are closely linked on mouse chromosome 7, and are expressed from the paternal and maternal alleles, respectively. The genes show striking similarity in their tissue-specific expression patterns, which led to the proposal that their transcription is controlled by a common regulatory domain that enables only one gene to be active from each chromosome. Evidence is accumulating, however, that the expression of H19 and Igf2 genes is not always from their respective maternal and paternal alleles. This suggests that their expression is regulated independently of imprinting in some tissues and teratomas. We have analysed the extent of non-imprinted expression of H19 and Igf2 in uniparental mouse embryonic stem (ES) cells during in vitro differentiation, and differentiation in teratomas using Northern blot and in situ hybridisation analysis. The expression patterns observed indicate that both imprinting and non-imprinting mechanisms regulate transcription of these genes. Expression of one or the other gene was observed in certain cell types in differentiated cultures and in teratomas, suggesting that imprinting regulates the expression of H19 and Igf2 genes in some differentiating cell lineages. At the same time, in other subpopulations of cells, co-expression of both genes was observed, demonstrating that the expression of these genes is not always regulated by genomic imprinting.

The distribution of laminin A, B1, B2, nidogen and collagen alpha 1(IV) mRNA was studied in the 12.5-day mouse placenta and uterus. This was compared to the pattern of laminin, nidogen and collagen IV immunoreactivity in the placenta at this time. High levels of B2 mRNA were distributed throughout the decidual layer, compared to lower levels of laminin B1, nidogen and collagen IV. In contrast, laminin B1 and nidogen mRNA were found at very high levels in the trophoblast giant cells and cytotrophoblast. Laminin B2 was much lower in trophoblast cells than in the decidua. Nidogen mRNA levels were low in the decidual cells, but high in endothelial cells lining the placental blood spaces in the decidual layer. Immunofluorescence staining of the placenta showed colocalization of laminin, nidogen and collagen IV in fetal and maternal layers of the placenta. In the antimesometrial area where the uterine epithelium was reforming laminin B1 and B2 mRNA were uniformly distributed between the epithelium and stroma, whereas nidogen and collagen alpha 1(IV) mRNA were only produced by the stromal cells. In all cell types, apart from parietal endoderm cells forming Reichert's membrane, laminin A chain mRNA was very low or absent. These results demonstrate that laminin and nidogen genes are not coordinately expressed in the midgestation mouse placenta. Comparison of the distribution of these mRNAs with collagen alpha 1(IV) mRNA suggests that different regions of the placenta produce specialized extracellular matrices which may contain different ratios of these polypeptides.

Clomiphene citrate was administered to female mice at different doses and different times prior to ovulation, in the preimplantation period after ovulation, and after implantation. Pregnancy outcome was determined on day 15 of gestation, when the number of implantations and resorptions were calculated relative to the number of ovulations, and fetuses were assessed for size and stage of development and morphological abnormalities. Preovulatory administration of clomiphene citrate caused decreased implantation rates and growth retardation of surviving fetuses, the degree of the effect being dependent on the dose and the time of drug injection relative to ovulation. The implantation rate was lowest, and the degree of fetal growth retardation highest, when clomiphene citrate was administered immediately before ovulation. An increased incidence of exencephaly was found in the fetuses of females injected with clomiphene citrate prior to ovulation. Transfer of blastocysts from treated mice to untreated fosters showed the effect of clomiphene citrate on implantation and fetal growth to be predominantly mediated through the female reproductive tract, rather than a direct effect on the embryo itself. Administration of clomiphene citrate in the preimplantational period resulted in complete inhibition of implantation, while the only effect when administration was after implantation was a slight reduction in fetal weight. These results indicate that preovulatory clomiphene citrate impairs uterine function, which has an indirect effect on the growth and development of the postimplantation embryo. © 1993 Wiley-Liss, Inc.

Mouse embryos were mechanically bisected at the morula, early blastocyst or expanded blastocyst stages of development and cultured in vitro to the expanded blastocyst stage. Their capacity for postimplantation development was assessed after transfer to pseudopregnant foster mice. Embryos bisected at blastocyst stages had a higher survival rate in vitro than those bisected at the morula stage. Half-embryos had approximately half the number of cells at the blastocyst stage as control embryos, but the proportion of cells in the inner cell mass (ICM) was unaltered. The implantation rate of blastocysts derived from bisected embryos was only slightly lower than that of control embryos, but bisected embryos had a significantly reduced capacity to form fetuses. Histological analyses showed that failure to form a fetus is due to the absence of egg cylinder development, which correlates with the reduced number of cells in the ICM of bisected embryos. Postimplantation viability of half-embryos was significantly higher when blastocysts were transferred to Day-3 rather than Day-4 pseudopregnant recipients, presumably because of an increase in cell number in vivo prior to implantation.

A monoclonal antibody was produced against purified nidogen extracted from a mouse basement-membrane-producing tumor. This antibody reacted with a determinant on Nd-40, a rod which separates the globular domains of nidogen. Antigenicity depends on intrachain disulfide bonds within this rod. The monoclonal antibody was used to detect nidogen fragments after proteolytic cleavage of isolated nidogen, and nidogen complexed to laminin. The data indicate that thrombin and thermolysin generated very different patterns of degradation, but in both cases no differences were found between isolated and complexed nidogen. In contrast, nidogen in the laminin-nidogen complex was much less degraded by trypsin than isolated nidogen, indicating that an interaction between these basement membrane components reduces the susceptibility of nidogen to trypsin digestion. Immunofluorescent studies, using the monoclonal antibody on sections of the EHS tumor after proteolytic digestion, showed that the retention or disappearance of the Nd-40 determinant correlated with the in vitro digestion pattern of the laminin-nidogen complex.

Interstitial collagens are thought to mediate epithelial-mesenchymal interactions during organogenesis. We have used the collagen I-deficient mouse mutant Mov13 to directly investigate the role of this major representative of the interstitial collagens in epithelial branching morphogenesis. Since homozygous embryos die at midgestation, we have studied the development of organ rudiments from Mov13 homozygous (i.e., collagen I-deficient), heterozygous, and wild-type embryos in culture. Development of all explants, including lung, kidney, salivary glands, pancreas, and skin, was normal by light and electron microscopic criteria and was independent of the genotype of the donor embryo. Metabolic labeling and immune staining verified the complete absence of collagen I in homozygous explants while revealing substantial production of collagens III and V in explants of all three genotypes. These results indicate either that collagen I has no role in the morphogenesis of these organs, or that its function is shared, or can be substituted for, by other fibrillar collagens.

Extraction of a basement-membrane-producing mouse tumor with 6 M guanidine/HCl in the presence of protease inhibitors allowed the purification of the genuine form of the matrix protein nidogen (Mr = 150,000) and, in addition, two defined fragments (Mr = 130,000 and 100,000). Smaller fragments (Mr = 80,000 and 40,000) were obtained under conditions with less stringent control of endogenous proteolysis. Intact nidogen and the larger fragments were similar in amino acid and carbohydrate (about 5%) composition, the presence of a single polypeptide chain, conformational features as revealed by CD spectroscopy and all shared major epitopes located on the Mr = 80,000 fragment. Additional epitopes were found on intact nidogen and the Mr = 130,000 fragment. Nidogen and the various fragments possess different N-terminal amino acid sequences indicating a stepwise degradation from the N-terminal end of the molecule. Electron microscopical and hydrodynamic studies of the Mr = 80,000 fragment demonstrated a structure consisting of a globular head connected to a thin tail. Intact nidogen appears to contain a somewhat larger globule but the same tail, which is terminated at its opposite end by a second, smaller globular structure. The data suggest a multidomain structure for nidogen containing sites highly susceptible to proteolytic cleavage.

A 55-kilodalton (kDal) protein was detected recently in primary cultures of day 12 mouse embryos by immunoprecipitation with serum from simian virus 40 (SV40) tumor-bearing hamsters (T serum), Preliminary evidence suggested that this protein was similar to a cellular 55-kDal protein induced after SV40 transformation of mouse cells. We now show that specific approximately 55-kDal [35S]methionine-labeled proteins precipitate from primary cultures of midgestation mouse, rat, and hamster embryos on addition of T serum or monoclonal antiserum prepared against the SV40-induced mouse 55-kDal proteins. The two-dimensional maps of the [35S]methionine-labeled tryptic peptides of the mouse, hamster, and rat embryo proteins are similar to the maps of the corresponding proteins from SV40-transformed cells. Primary cells from midgestation mouse, hamster, or rat embryos contain one-third to one-half as much 55-kDal protein as a SV40-transformed mouse fibroblast cell and nearly the same amount as F9 mouse embryonal carcinoma cells. The amount of 55-kDal protein is greatly reduced on replating the mouse, rat, or hamster embryo primary cells. The amount of this protein in mouse embryos is dependent on the stage of the embryo. The embryo proteins are phosphoproteins.

A novel acidic glycoprotein, BM-40, with Mr = 40,000, was purified from the basement-membrane-producing mouse EHS tumor and characterized with regard to its unique chemical and antigenic properties. It was obtained from the tumor in a neutral salt-soluble form or as a component requiring extraction with 6M guanidine X HCl. This protein could also be identified in many other tissue extracts and cell and tissue cultures. The most intact form of BM-40 consists of a single polypeptide chain which undergoes limited proteolysis during extraction and purification. BM-40 exists in most tissues in stoichiometric amounts compared to other basement membrane proteins (laminin, nidogen) and is secreted by various teratocarcinoma and epithelial cells. It can be visualized by immunofluorescence in the extracellular matrix of the EHS tumor and Reichert's membrane. Other tissues which contain extractable BM-40 were negative in immunofluorescence.

We have studied susceptibility of basement membranes in a variety of tissues to solubility in guanidine hydrochloride and to proteolytic degradation by trypsin and thermolysin. Unfixed sections from embryonic and adult mouse tissues and the EHS tumor were subjected to sol vent buffers or digested with enzymes. The retention or disappearance of the basement-membrane components nidogen, laminin, collagen IV, and heparan sulfate proteoglycan was subsequently assayed by immunofluorescence. Our data show ed that in all tissues nidogen was the most readily solubilized component and the most susceptible to proteolytic degradation. With few exceptions, nidogen in embryonic tissues was more susceptible to degradation than that in adult tissues, and this correlated well with the susceptibility of the other basement-membrane components to be degraded. We conclude that basement membranes differ quite markedly in their solubility and their susceptibility to proteolytic degradation and that these properties reflect differences in their molecular structure.

Nidogen was purified from a mouse tumor basement membrane where it accounted for 2-3% of the total proteins. It was isolated as two forms (A and B) of a monomer (Mr = 80000) each consisting of a single polypeptide chain folded into a globular head connected to a small tail. The B form of the monomer was shown to be capable of aggregating into a nest-like structure (Mr greater than 250000). A smaller form (Mr = 45000) was observed in some of the extracts. The amino acid composition of nidogen was different to that of other basement membrane proteins. It contained about 10% carbohydrate, with N-linked and O-linked oligosaccharide chains in similar proportions. Isoelectrofocussing demonstrated a limited heterogeneity of nidogen with pI in the range 6.5 - 7. Monomeric nidogen failed to interact with other basement membrane components and heparin. Aggregation could be induced by limited proteolysis and was reversed by detergents or high salt concentrations. Together with the observation that most of the nidogen could be solubilized only after destroying the collagenous matrix, the data indicate that aggregation of nidogen reflects an activity involved in matrix assembly. Specific antibodies raised against nidogen did not distinguish between the monomeric and aggregated form of the protein but showed that the fragment was antigenically deficient. These antibodies did not cross-react with collagen type IV, laminin, entactin and heparansulfate proteoglycan. Immunofluorescence staining and absorption studies demonstrated that nidogen is a common component of authentic basement membranes. Larger forms of nidogen (Mr about 100000 and 150000) were found in organ cultures of Reichert's membrane suggesting that it is synthesized in precursor forms.

Seven different sulphated macromolecules were detected in 6 M-guanidinium chloride extracts of metabolically [35S]sulphate-labelled mouse Reichert's membrane and were partially separated. Polypeptide bands of apparent Mr 50 000, 150 000 (tentatively identified as entactin) and 170 000 contained essentially tyrosine O-sulphate as the labelled component. Most of the radioactive sulphate was incorporated into three different proteoglycans, which could be separated by chromatography and density-gradient centrifugation before and after enzymic degradation. Enzymic analysis of glycosaminoglycans and of protein cores by immunoassays identified these components as low-density and high-density forms of heparan sulphate proteoglycan and a high-density form of chondroitin sulphate or dermatan sulphate proteoglycan.

Inner cell masses (ICMs) were isolated by immunosurgery from giant blastocysts formed by the aggregation of three morulae. A layer of endoderm cells formed on the outer surface of these primary ICMs in vitro. When this layer was removed by immunosurgery, a secondary endoderm layer formed. Alphafetoprotein (AFP) was used as a biochemical marker to characterize visceral endoderm formation in these cultured ICMs. The immunoperoxidase reaction on sections of ICMs cultured for intervals up to 120 h in vitro showed that some primary endoderm cells contained AFP, but these were always in the minority. The secondary endoderm layer, on the other hand, was composed of predominantly AFP-positive cells. It is concluded that the primary endoderm contains mainly parietal endoderm cells, while the secondary layer contains visceral endoderm cells. A model is proposed for the consecutive differentiation of parietal and visceral endoderm cell types from the ICM of mouse blasto-cysts.

The Mov-13 mouse strain carries a retroviral insertion in the pro alpha 1(I) collagen gene that prevents transcription of the gene. Cell lines derived from homozygous embryos do not express type I collagen although normal amounts of pro alpha 2 mRNA are synthesized. We have introduced genomic clones of either the human or mouse pro alpha 1(I) collagen gene into homozygous cell lines to assess whether the human or mouse pro alpha 1(I) chains can associate with the endogenous mouse pro alpha 2(I) chain to form stable type I collagen. The human gene under control of the simian virus 40 promoter was efficiently transcribed in the transfected cells. Protein analyses revealed that stable heterotrimers consisting of two human alpha 1 chains and one mouse alpha 2 chain were formed and that type I collagen was secreted by the transfected cells at normal rates. However, the electrophoretic migration of both alpha 1(I) and alpha 2(I) chains in the human-mouse hybrid molecules were retarded, compared to the alpha (I) chains in control mouse cells. Inhibition of the posttranslational hydroxylation of lysine and proline resulted in comigration of human and mouse alpha 1 and alpha 2 chains, suggesting that increased posttranslational modification caused the altered electrophoretic migration in the human-mouse hybrid molecules. Amino acid sequence differences between the mouse and human alpha chains may interfere with the normal rate of helix formation and increase the degree of posttranslational modifications similar to those observed in patients with lethal perinatal osteogenesis imperfecta. The Mov-13 mouse system should allow us to study the effect specific mutations introduced in transfected pro alpha 1(I) genes have on the synthesis, assembly, and function of collagen I.

Mouse embryos homozygous for the Mov-13 mutation produce no collagen I, owing to transcriptional blockage of the collagen alpha 1(I) gene by a retroviral insert. Fibroblast-like cell lines derived from these embryos were compared with similar lines derived from heterozygous and wild-type embryos with respect to the total amounts, and types, of collagen synthesized. Total collagen synthesized by either cloned or uncloned cell lines correlated with their genotype, demonstrating no compensation for absence of collagen I production by an increase in synthesis of other collagen types. Procollagen alpha 2(I) chains were not detected in the homozygous cell lines, demonstrating that these chains do not form homotrimers, nor do they form heterotrimers with alpha-chains of other collagen types. Procollagen III levels were quantified by radioimmunoassay and found to be similar in all cell lines.

Mouse Schwann cells and rat RN22 schwannoma cells cultured in the absence of neurons and fibroblasts produce typical basement membrane proteins. Heparan sulfate proteoglycan (low density form), nidogen, and protein BM-40 were identified by radioimmunoassays, immunoblotting, and by immunoprecipitation after metabolic labeling. The cells also produce a laminin-like protein that differs from authentic laminin by a reduced A chain content and lack of antigenic determinants located in the long arm of laminin. Laminin possessing A and B chains is, however, produced by PYS-2 teratocarcinoma cells grown under the same conditions. Laminin from Schwann cell culture medium promotes neurite outgrowth, and this activity could be immunoprecipitated but not blocked by various antibodies against authentic laminin. In addition, Schwann cell laminin is found complexed noncovalently with nidogen. Sulfate incorporation revealed the synthesis of proteoglycans and entactin. A similar set of proteins and in addition collagen IV could be demonstrated in neurofibroma tissue by immunohistology, and were localized to the laminae densae of the multilayered basement membranes around Schwann cells and capillaries. Laminin purified from 0.5 M NaCl neurofibroma tissue extracts possessed both A and B chains. Nidogen was identified in a partially degraded form.

While bone morphogenetic proteins (BMPs) 2, 4 and 7 have recently been implicated in aspects of metanephric development, and expression patterns of these ligands have been described in the developing metanephros, the distribution of BMP receptors in developing metanephroi remains unknown. In the present study, in situ hybridisation histochemistry was used to localise mRNAs for BMP type-I receptors (BMPR-IA and BMPR-IB) and the BMP type-II receptor (BMPR-II) in developing mouse metanephroi. At embryonic day 12.5 (E12.5) and E14.5 transcripts for BMP type-I receptors were localised to the tips and body of the branching ureter as well as mesenchymal condensates, developing vesicles and comma-shaped bodies. Localisation of BMPR-II transcripts was similar although expression was not observed in the body of the ureter. At E17.5, transcripts for all three receptors were localised in the nephrogenic zone including ureteric tips, vesicles, comma- and S-shaped bodies as well the body of the ureter and in tubules. BMP type-I and type-II receptor transcripts co-localised with each other, in agreement with the well-documented evidence that BMPs signal via heterotetrameric complexes of type-I and type-II receptors and with the previously reported metanephric expression pattern of BMPs. These patterns of receptor expression suggest that these molecules are important regulators of epithelial-mesenchymal interactions, nephron development and ureteric branching morphogenesis.