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Personal genome screening (PGenS) is increasingly being offered as a screen for future health management, and to identify carrier status pertinent to reproductive decision-making. The aim of this study was therefore to explore the experience of individuals who undertook PGenS through the 2014 Sydney “Understand Your Genome (UYG)” event and a 2015 offer of PGenS by Australian biotechnology company Life Letters (LL). Eligible individuals were invited to participate by their clinical geneticist (UYG), or email from Director of LL. Semi-structured telephone interviews with 17 individuals were audio-recorded, transcribed, de-identified and analyzed by two coders using thematic analysis with an inductive approach. Nine participants had genetic/genomics expertise and eight were well-informed health and business professionals. Individual participant PGenS results included: an autosomal dominant condition not previously clinically identified (n = 1); carrier status for recessive condition(s) (n = 8); a number of disease-causing variants associated with an increased susceptibility to an inherited disorder (n = 7); variants of uncertain significance (n = 5); and a few pharmacogenomically-relevant variants (n = 4). The majority of participants described the importance of pre-test genetic counseling, information and/or consent (n = 12). Some barriers to uptake were identified, including scepticism by GPs (n = 6), colleagues (n = 3), and family members (n = 2), as well as privacy concerns (n = 4). Those without genetic/genomics expertise were mostly motivated to have testing by curiosity or interest in personal health (6/8), one seeking a diagnosis for an inherited medical condition and another for future health management. For many with genetic/genomics experience, the motivation was professional interest (8/9) and/or curiosity (5/9), without concern for personal health risk (4/9). On reflection, despite this initial motivation by the latter, the test result had unanticipated personal impact for some of this group, which changed over time (4/5). Several later recognized this, as health problems developed or family history was interrogated more closely. For all participants, disclosure of results to extended family members was limited. Most participants felt personal and family implications and communication (5/17) and/or expectations (3/17) should be addressed at the pre-test session, including more emphasis on residual risk and changes in interpretation with developing phenotypes. Those without genetics/genomics expertise highlighted the need for easy to understand pre-test information and/or an example report to be provided (7/8). These results are consistent with a need to develop more accessible resources, and more personalized counseling approaches to address expectations, dissemination of results, and preparedness for unexpected findings.

High-throughput screening of a small-molecule library identified a 5-triazolo-2-arylpyridazinone as a novel inhibitor of the important glycolytic enzyme 6-phosphofructo-2-kinase/2,6-bisphosphatase 3 (PFKFB3). Such inhibitors are of interest due to PFKFB3's control of the important glycolytic pathway used by cancer cells to generate ATP. A series of analogues was synthesized to study structure-activity relationships key to enzyme inhibition. Changes to the triazolo or pyridazinone rings were not favoured, but limited-size substitutions on the aryl ring provided modest increases in potency against the enzyme. Selected analogues and literature-described inhibitors were evaluated for their ability to suppress the glycolytic pathway, as detected by a decrease in lactate production, but none of these compounds demonstrated such suppression at non-cytotoxic concentrations.

Two important lines of research have enhanced our understanding of the molecular role of nutrition in influencing behavior. First, exposure to an adverse environment during early life can influence the long-term behavior of the offspring. Second, regulation of the nervous system development and functioning appears to involve epigenetic mechanisms that require a continuous supply of methyl group donors in food. We hypothesized that a maternal diet during pregnancy deficient in methyl donors (MDD) may lead to altered behavior in offspring through permanent changes in hippocampal DNA methylation. We used a rat model of prenatal dietary MDD to test this hypothesis in female offspring as they aged. Prenatal MDD reduced birth weight, litter size, and newborn viability. Aged female offspring of MDD mothers showed increased anxiety and increased learning ability in comparison with control diet group offspring. To explore the role of MDD on epigenetic mechanisms in the brain of adult offspring, we studied expression and methylation of 4 selected genes coding for glucocorticoid receptor, hydroxysteroid dehydrogenase 11 type 2, neuronatin, and reelin proteins in the hippocampus. No major group differences in methylation or expression of the studied genes were detected, except for a significant down-regulation of the reelin gene in the MDD female offspring. The prenatal MDD diet caused intrauterine growth restriction, associated with long-term effects on the behavior of the offspring. However, the observed behavioral differences between the MDD and control diet offspring cannot be explained by epigenetic regulation of the specific genes investigated in this study.

Calcium (Ca(2+)) influx through the plasma membrane store-operated Ca(2+) channel ORAI1 is controlled by Ca(2+) sensors of the stromal interaction molecule (STIM) family. STIM1 responds to endoplasmic reticulum (ER) Ca(2+) store depletion by redistributing and activating ORAI1 from regions of the ER juxtaposed to the plasma membrane. Unlike STIM1, STIM2 can regulate ORAI1 in a store-dependent and store-independent manner, but the mechanism by which this is achieved is unknown. Here we find that STIM2 is translated from a highly conserved methionine residue and is directed to the ER by an incredibly long 101-amino acid signal peptide. We find that although the majority of the total STIM2 population resides on the ER membrane, a second population escapes ER targeting to accumulate as a full-length preprotein in the cytosol, signal peptide intact. Unlike STIM2, preSTIM2 localizes to the inner leaflet of the plasma membrane where it interacts with ORAI1 to regulate basal Ca(2+) concentration and Ca(2+)-dependent gene transcription in a store-independent manner. Furthermore, a third protein comprising a fragment of the STIM2 signal peptide is released from the ER membrane into the cytosol where it regulates gene transcription in a Ca(2+)-independent manner. This study establishes a new model for STIM2-mediated regulation of ORAI1 in which two distinct proteins, STIM2 and preSTIM2, control store-dependent and store-independent modes of ORAI1 activation.

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.

In all cells Ca2+ signals are key to controlling a spectrum of cellular responses. Ca2+ signals activated by phospholipase C-coupled receptors have two components—rapid Ca2+ release from ER stores followed by slower Ca2+ entry from outside the cell. The coupling process between ER and PM to mediate this “store-operated” Ca2+ entry process has remained a molecular and mechanistic mystery. Through a combination of high throughput screening and molecular physiological approaches, the machinery and mechanism of this process have been elucidated. Two proteins are key to the coupling process. STIM1, a single spanning membrane protein with an unpaired Ca2+ binding EF-hand functions as the sensor of ER luminal Ca2+ and through redistribution in the ER transduces information directly to the PM. Orai1, a tetra-spanning PM protein, functions as the highly Ca2+ selective channel in the PM that is gated through interactions with the store-activated ER Ca2+ sensor. This molecular pas-de-deux between ER and PM components represents not only a crucial signaling pathway, but also a new paradigm in inter-organelle communication.

Summary The coupling mechanism between endoplasmic reticulum (ER) Ca 2+ stores and plasma membrane (PM) store-operated channels (SOCs) remains elusive [1–3]. STIM1 was shown to play a crucial role in this coupling process [4–7]; however, the role of the closely related STIM2 protein remains undetermined. We reveal that STIM2 is a powerful SOC inhibitor when expressed in HEK293, PC12, A7r5, and Jurkat T cells. This contrasts with gain of SOC function in STIM1-expressing cells. While STIM1 is expressed in both the ER and plasma membrane, STIM2 is expressed only intracellularly. Store depletion induces redistribution of STIM1 into distinct "puncta." STIM2 translocates into puncta upon store depletion only when coexpressed with STIM1. Double labeling shows coincidence of STIM1 and STIM2 within puncta, and immunoprecipitation reveals direct interactions between STIM1 and STIM2. Independent of store depletion, STIM2 colocalizes with and blocks the function of a STIM1 EF-hand mutant that preexists in puncta and is constitutively coupled to activate SOCs. Thus, whereas STIM1 is a required mediator of SOC activation, STIM2 is a powerful inhibitor of this process, interfering with STIM1-mediated SOC activation at a point downstream of puncta formation. The opposing functions of STIM1 and STIM2 suggest they may play a coordinated role in controlling SOC-mediated Ca 2+ entry signals.

Receptor-induced Ca 2+ signals are key to the function of all cells and involve release of Ca 2+ from endoplasmic reticulum (ER) stores, triggering Ca 2+ entry through plasma membrane (PM) “store-operated channels” (SOCs). The identity of SOCs and their coupling to store depletion remain molecular and mechanistic mysteries. The single transmembrane-spanning Ca 2+ -binding protein, STIM1, is necessary in this coupling process and is proposed to function as an ER Ca 2+ sensor to provide the trigger for SOC activation. Here we reveal that, in addition to being an ER Ca 2+ sensor, STIM1 functions within the PM to control operation of the Ca 2+ entry channel itself. Increased expression levels of STIM1 correlate with a gain in function of Ca 2+ release-activated Ca 2+ (CRAC) channel activity. Point mutation of the N-terminal EF hand transforms the CRAC channel current ( I CRAC ) into a constitutively active, Ca 2+ store-independent mode. Mutants in the EF hand and cytoplasmic C terminus of STIM1 alter operational parameters of CRAC channels, including pharmacological profile and inactivation properties. Last, Ab externally applied to the STIM1 N-terminal EF hand blocks both I CRAC in hematopoietic cells and SOC-mediated Ca 2+ entry in HEK293 cells, revealing that STIM1 has an important functional presence within the PM. The results reveal that, in addition to being an ER Ca 2+ sensor, STIM1 functions within the PM to exert control over the operation of SOCs. As a cell surface signaling protein, STIM1 represents a key pharmacological target to control fundamental Ca 2+ -regulated processes including secretion, contraction, metabolism, cell division, and apoptosis.

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.

The anti-adhesive extracellular matrix protein SPARC (secreted protein and rich in cysteine; osteonectin or BM-40) has been implicated in the regulation of matrix turnover, cell migration, and proliferation. The present study sought to examine whether modulation in the expression of this protein may play a role in diabetes-associated vascular remodeling. SPARC mRNA and protein were measured in mesenteric vessels of diabetic rats and controls. Hypertrophy of mesenteric vessels was noted after 3 and 32 weeks of diabetes as revealed by the increase in mesenteric vessel wet weight and an increased wall/lumen ratio. SPARC mRNA was sparsely present in intima and adventitia of control vessels. There was a marked increase in SPARC gene expression in the intima and adventitia of mesenteric vessels after 1, 3, and 32 weeks of diabetes. SPARC protein was demonstrated in the vessel wall in control animals and was increased in the mesenteric vessels of diabetic rats after 1 and 32 weeks of diabetes. Administration of the inhibitor of advanced glycation end-product formation, aminoguanidine, to diabetic rats attenuated both the hypertrophic response in mesenteric vessels and the overexpression of SPARC mRNA and protein without affecting glycemic control or food intake. In summary, diabetes-related mesenteric vascular hypertrophy is associated with an increase in SPARC expression in the vessel wall. The modulation of SPARC expression in mesenteric vessels of diabetic rats might be of pathogenetic significance in the development of vascular remodeling in diabetes.

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.

To characterize developmental changes in the extracellular matrix of the postnatal rat testis in relationship to the timing of germ cell maturation, we immunolocalized fibulin-1, fibulin-2, and other matrix components in the testes of normal and propylthiouracil (PTU)-induced hypothyroid animals. Unlike laminin, nidogen, and perlecan, which were present in the seminiferous tubule basement membrane (BM) throughout postnatal development, fibulins were found to disappear from the postnatal tubule BM. Fibulin-1 was no longer detected after Day 5 whereas fibulin-2 became localized in a segmental manner within the BM of each seminiferous tubule on Days 10 and 15 and disappeared by Day 20. Fibronectin showed a segmental pattern in the level of immunostaining of the tubule BM on Days 10 and 15, with a more uniform staining seen at earlier and later ages. Collagen VI was initially confined to the interstitial matrix in the Day 5 testis and became progressively more closely associated with the seminiferous tubule BM at later stages. The disappearance of fibulin-2 and the BM-association of collagen VI were both delayed in the PTU-treated testes. The developmental changes in the staining patterns for fibulin-2 and fibronectin coincide with the adhesion and alignment of peritubular cells on the inner seminiferous tubule BM. The delay in maturation of the seminiferous tubule BM in the testes of PTU-treated rats demonstrates a correlation between changes in the composition of the tubule BM and cellular development of the testis.

Collagen VI is a microfibrillar protein found in the extracellular matrix of virtually all connective tissues. Three genetically distinct subunits, the alpha1(VI), alpha2(VI), and alpha3(VI) chains, associate intracellularly to form triple-helical monomers, which then assemble into disulfide-bonded dimers and tetramers before secretion. Although sequence considerations suggest that collagen VI monomers composed of all three chains are the most stable isoform, the precise chain composition of collagen VI remains controversial and alternative assemblies containing only alpha1(VI) and alpha2(VI) chains have also been proposed. To address this question directly and study the role of the alpha3(VI) chain in assembly, we have characterized collagen VI biosynthesis and in vitro matrix formation by a human osteosarcoma cell line (SaOS-2) that is deficient in alpha3(VI) production. Northern analysis showed an abundance of alpha1(VI) and alpha2(VI) mRNAs, but no detectable alpha3(VI) mRNA was apparent in SaOS-2 cells. By day 30 of culture, however, small amounts of alpha3(VI) mRNA were detected, although the level of expression was still much less than alpha1(VI) and alpha2(VI). Collagen VI protein was not detected in SaOS-2 medium or cell layer samples until day 30 of culture, demonstrating that despite the abundant synthesis of alpha1(VI) and alpha2(VI), no stable collagen VI protein was produced without expression of alpha3(VI). The alpha1(VI) and alpha2(VI) chains produced in the absence of alpha3(VI) were non-helical and were largely retained intracellularly and degraded. The critical role of the alpha3(VI) chain in collagen VI assembly was directly demonstrated after stable transfection of SaOS-2 cells with an alpha3(VI) cDNA expression construct that lacked 4 of the 10 N-terminal type A subdomains. The transfected alpha3(VI) N6-C5 chains associated with endogenous alpha1(VI) and alpha2(VI) and formed collagen VI dimers and tetramers, which were secreted and deposited into an extensive network in the extracellular matrix. These data demonstrated that alpha3(VI) is essential for the formation of stable collagen VI molecules and subdomains N10-N7 are not required for molecular assembly.

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.

Secreted protein acidic and rich in cysteine (SPARC) is an extracellular matrix-associated protein with antiadhesive, antiproliferative, and matrix remodeling properties. SPARC gene and protein expression were investigated after subtotal nephrectomy (STNx), a model of noninflammatory progressive renal injury. In addition, the effect of blockade of the renin- angiotensin system by the angiotensin-converting enzyme in- hibitor ramipril or by the angiotensin II receptor antagonist valsartan was examined. The STNx rats developed hyperten- sion, proteinuria, and renal impairment. These changes were associated with a 2.4-fold increase in SPARC gene expression in STNx compared with SHAM kidneys (P < 0.001). In situ hybridization revealed increased SPARC mRNA in glomeruli and interstitial cells, as well as de novo expression by tubular epithelial cells at sites of renal injury. Immunofluorescence studies confirmed concordant changes in SPARC protein. Both ramipril and valsartan ameliorated renal injury and signifi- cantly reduced SPARC overexpression in the STNx animals. The findings of the present study suggest that SPARC, in the context of its known biological actions, may influence some of the pathological features associated with significant renal mass reduction. (J Am Soc Nephrol 8: 1373-1382, 1997)

Renal enlargement is a characteristic feature of human and experimental diabetes mellitus that may be predictive of subsequent nephropathy. In the streptozotocin diabetic rat kidney growth rapidly follows the induction of experimental diabetes but the mechanisms responsible for this growth are poorly understood. Epidermal growth factor (EGF) is a potent mitogen for renal tubular cells. Thirty one male Sprague-Dawley rats aged 13 weeks were randomised to receive either streptozotocin (diabetic, n = 20) or buffer (control, n = 11). Animals were studied on days 1, 3, 5 and 7 following streptozotocin. Diabetes was associated with a 3-fold increase in urinary EGF excretion (223 ± 15 vs 59 ± 5 ng/day, mean ± SEM, diabetic vs control, p < 0.0001) and 3–6 fold increase in renal EGF mRNA relative to controls (p < 0.001). A transient rise in kidney EGF protein was noted on day 1. There was no difference between diabetic and control animals with regard to intrarenal sites of EGF expression or in plasma EGF. These data suggest that the increased urinary EGF excretion in diabetic animals is the result of enhanced local production and that EGF is not stored for a prolonged period within renal tubular cells but is released following its synthesis. In the context of the known intrarenal actions of EGF this growth factor may play a role in the pathogenesis of diabetes related kidney growth. [Diabetologia (1997) 40: 778–785]

Transforming growth factor β1 and renal injury following subtotal nephrectomy in the rat: Role of the renin-angiotensin system. Transforming growth factor-β (TGF-β) and the renin-angiotensin system (RAS) have both been implicated in the pathogenesis of chronic renal disease. The present experiment investigated the chronology of TGF-β1 gene expression following subtotal nephrectomy (STNx) in the rat and the effect of blocking the RAS by angiotensin converting enzyme (ACE) inhibition or by angiotensin II receptor (AT1) antagonism. Rats that had undergone subtotal nephrectomy developed hypertension, proteinuria, renal impairment, glomerulosclerosis, tubulointerstitial fibrosis and mononuclear cell infiltration. These changes were associated with a 2.5-fold increase in TGF-β1 gene expression during a 16-week time course. In situ hybridization localized TGF-β1 mRNA to sclerotic glomeruli, areas of tubulointerstitial injury and sites of mononuclear cell infiltration. Administration of the ACE inhibitor ramipril and the AT1 receptor blocker valsartan blunted the increase in TGF-β1 mRNA, and attenuated the structural and functional manifestations of injury. These data suggest an interaction between the intrarenal RAS and TGF-β in the pathogenesis of the glomerular and tubulointerstitial fibrosis that follow a major reduction in renal mass.

p57KIP2 is a cyclin-dependent kinase inhibitor that maps to human chromosome band 11p15.5, placing it in a genomically imprinted region that has been implicated in the etiology of Wilms' tumor and in the Beckwith-Wiedemann syndrome. Recent analysis of p57KIP2 expression in the mouse has determined that this gene is exclusively expressed from the maternal allele. It has been suggested that p57KIP2 is the WT2 tumor suppressor gene in the 11p15.5 region. We have used reverse transcriptase PCR to determine whether loss of p57KIP2 expression occurs in Wilms' tumor samples that have undergone maternal loss of heterozygosity of 11p15.5. p57KIP2 mRNA was amplified in both the Wilms' tumor tissue and in normal kidney tissue of all five patients analyzed. Semi-quantitative PCR analyses demonstrated that the relative level of p57KIP2 expression in tumor tissue is not markedly different from that in normal kidney. Our data indicate that if the p57KIP2 gene is imprinted in humans and expressed exclusively from the maternal allele, reactivation of the paternal allele has occurred in all five Wilms' tumor samples analyzed in this study. Sequence analysis of the p57KIP2 Cdk inhibitory domain in genomic DNA from primary and secondary tumors from two patients showed only a single base change in one secondary WT, resulting in a predicted methionine to isoleucine substitution at amino acid position 70. These studies suggest that p57KIP2 may not be the WT2 gene.

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.

SPARC gene expression is reduced in early diabetes-related kidney growth. Renal enlargement is a characteristic feature of diabetes in humans and experimental animals that may predict subsequent renal disease. The biological processes involved in diabetes-related kidney growth are complex and involve changes in extracellular matrix, cell hypertrophy and hyperplasia. Secreted protein acidic and rich in cysteine (SPARC) is an extracellular matrix protein with anti-adhesive, antiproliferative and matrix remodeling properties. We examined kidney SPARC gene expression and protein content in early experimental diabetes. By Northern blot analysis, kidney SPARC mRNA fell in diabetic animals at day 1 to 40 ± 15% of controls levels (mean ± SEM, P < 0.01), to 42% ± 11% on day 3 (P < 0.01) with a further decrease at day 7 to 29 ± 7% (P < 0.001). In situ hybridization demonstrated SPARC mRNA within glomeruli, renal interstitial cells and in blood vessels but not in tubular epithelial cells. SPARC mRNA was decreased in diabetic rats without a change in the pattern of distribution. By immunofluorescence, SPARC protein was detected in glomeruli and tubular basement membrane. Diabetes was associated with a decrease in SPARC protein at both sites. These data demonstrate that the onset of diabetes-related kidney growth is associated with a reduction in SPARC mRNA and protein. In the context of the known biological actions of SPARC, the findings in the present study implicate this matrix protein in the pathogenesis of diabetes related kidney growth.

The alpha 1, alpha 2, and alpha 3 chains of collagen VI and mRNAs for these chains were localized in the female mouse reproductive tract by immunofluorescence and in situ hybridization. High levels of collagen VI protein and mRNAs were present in the endometrium and myometrium of the uterus up to Day 4.5 of pregnancy. After embryo implantation, reduction in collagen VI protein within the decidualizing endometrium correlated with significantly reduced steady-state levels of alpha 1(VI), alpha 2(VI), and alpha 3(VI) mRNAs, indicating either transcriptional down-regulation of collagen VI gene expression or decreased stability of transcripts. High levels of alpha 1(V1) and alpha 2(VI) mRNAs, but not alpha 3(VI) mRNA, in cells surrounding the uterine epithelium in the mesometrial region did not correlate with deposition of collagen VI protein in this region. These data are consistent with an important role of alpha 3(VI) in assembly of collagen VI heterotrimers. However, distinct immunostaining with antiserum to alpha 2(VI) chains in the extracellular matrix immediately beneath the uterine epithelium may indicate that alpha 2(VI) chains are deposited without the alpha 1(VI) or alpha 3(VI) collagen chains. No collagen VI protein or mRNAs were detected in any tissue layers of the embryo on Days 5.5 or 6.5 of gestation.

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.

Background/Aims: Early-life methyl-donor deficiency is implicated in growth restriction and later-life development of type 2 diabetes mellitus. We ascertained whether dietary methyl-donor deficiency in the mother during pregnancy or during postweaning growth in the rat would impair glucose homeostasis, insulin secretion and pancreatic endocrine development in young adults. Methods: Effects of maternal methyl deficiency (90% deficiency in methionine, folate and choline) were compared with those of postweaning methyl deficiency and with control diets for effects on growth, impaired glucose tolerance, insulin secretion and pancreas development in offspring. Studies focussed on male offspring, which have been shown more susceptible to early-life influences on later disease development. Results: Prenatal methyl deficiency delayed delivery, restricted birthweight by 22%, reduced litter size by 33% and increased offspring mortality to 23% shortly after birth. It reduced relative endocrine pancreatic mass in adult male offspring to 46% of endocrine mass in controls, but only mildly impaired their glucose tolerance and insulin secretion. In contrast, postweaning methyl deficiency restricted growth of male rats and reduced relative pancreatic endocrine mass (–40%), but improved their glucose tolerance, despite decreased insulin secretion. Conclusion: It is clear that the global undernutrition (UN) during pregnancy in rodents alters glucose metabolism in adult offspring. It has been hypothesised that alterations in epigenetic mechanisms may underlie this phenotype. However, removing all methyl donors during pregnancy, which are essential for epigenetic processes in development, did not cause any alteration in glucose metabolism in offspring as seen in the global UN model.

The expression patterns of laminin A, B1, B2, and nidogen genes were identified by in situ hybridization in postimplantation mouse extraembryonic tissues and maternal decidua during the period when the chorioallantoic placenta is established. Laminin and nidogen genes were not coordinately expressed either in the decidua or in trophoblast cells, indicating that these genes are regulated independently in these cell types during the establishment of the placenta. Laminin A mRNA was absent from the decidua except in the outer layer of cells adjacent to the myometrium and in the central decidual zone adjacent to the remnant of the uterine epithelium on Day 9. At this stage laminin B1, B2, and nidogen genes were strongly expressed in these cells and also in other regions of the decidua. Laminin B1 mRNA was present at higher levels in the decidua capsularis than in the decidua basalis, while nidogen mRNA showed highest expression in the decidua basalis. Laminin B2 mRNA was produced uniformly throughout the decidua at very high levels, suggesting that laminin B2 chains may be an important component of the decidual matrix. By Day 11, the nidogen gene was expressed only in endothelial cells lining the maternal blood spaces within the decidua. Laminin B1 and nidogen mRNAs were found at high levels within trophoblast giant cells at all stages, while laminin A mRNA was detected in trophoblast giant cells at later stages and laminin B2 mRNA was not produced in high levels by these cells. The patterns of gene expression show a very high degree of regional specialization, suggesting that the extracellular matrices in different regions of the decidua and extraembryonic membranes are likely to be composed of quite different ratios of laminin and nidogen polypeptides.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Major components of basement membranes, including collagen IV, laminin, heparan sulphate proteoglycan and nidogen, were isolated from the matrix of the EHS sarcoma. The purified components were analysed for their domain structure and for the participation of distinct domains in molecular interactions and cell binding. Collagen IV consists of four domains which have triple helical or non-collagenous structures. Self-assembly of the protein into a network-like organization occurs by specific interactions between N-terminal triple helical segments and between the C-terminal globules. Cell binding requires a central triple helical segment. Laminin has the shape of an asymmetrical cross; different globular domains within this structure mediate binding to proteoglycan and to cells. The proteoglycan consists of four heparan sulphate chains attached to a small protein core. These chains have the potential to bind laminin, fibronectin and collagen IV. Nidogen was isolated in several molecular forms which showed either self-aggregation or binding to laminin.

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.

Basement membranes contain at least three different proteoglycans. These are a large, low buoyant density heparan sulphate proteoglycan and two smaller, high density proteoglycans with either heparan sulphate or chondroitin sulphate side-chains. The large (Mr 400K-600K and small (Mr 130K) heparan sulphate proteoglycans were purified from the mouse EHS tumour. These proteoglycans are immunologically related by sharing some protein core antigenic determinants (epitopes) but do not cross-react with cell-surface heparan sulphate proteoglycans or with proteoglycans from interstitial connective tissue. This indicates that they belong to a distinct family of proteoglycans. Structural models were developed, based on electron microscopy and analytical ultracentrifugation, demonstrating that the small proteoglycan contains on average four heparan sulphate chains of about 30 nm in length, while the large proteoglycan consists of three long (about 90 nm) heparan sulphate chains connected to one end of a large core protein. Single heparan sulphate chains were isolated from the EHS tumour proteoglycans and from the corresponding proteoglycans from Reichert's membrane of the mouse embryo. The heparan sulphate from Reichert's membrane bound to antithrombin with high affinity and was found to contain the unique 3-O-sulphated glucosamine residue previously identified in the antithrombin-binding region of heparin. The EHS tumour heparan sulphate showed a higher N-/O-sulphate ratio and a lower affinity for antithrombin.

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.

Stromal interaction molecule 1 (STIM1) is a cell surface transmembrane glycoprotein implicated in tumour growth control and stromal-haematopoietic cell interactions. A single sterile alpha motif (SAM) protein-protein interaction domain is modelled within its extracellular region, a subcellular localisation not previously described for other SAM domain-containing proteins. We have defined the transmembrane topology of STIM1 by determining the sites of N-linked glycosylation. We have confirmed that STIM1 is modified by N-linked glycosylation at two sites within the SAM domain itself, deduced as asparagine residues N131 and N171, demonstrating that STIM1 is translocated across the membrane of the endoplasmic reticulum such that the SAM domain resides within the endoplasmic reticulum (ER) lumen. Both N-linked oligosaccharides remain endoglycosidase H-sensitive, indicating absence of full processing within the ER and Golgi. This immature modification is nevertheless sufficient and critical for cell surface expression of STIM1. We show that STIM1-STIM1 homotypic interactions are mediated via the cytoplasmic rather than the extracellular region of STIM1, excluding an essential role for the SAM domain in these protein interactions. These studies provide the first evidence for an extracellular localisation of a SAM domain within any protein, and the first example of a SAM domain modified by N-linked glycosylation.

STIM1 is a novel candidate growth suppressor gene mapping to the human chromosome region 11p15.5 that is associated with several malignancies. STIM1 overexpression studies in G401 rhabdoid tumour, rhabdomyosarcoma and rodent myoblast cell lines causes growth arrest, consistent with a potential role as a tumour growth suppressor. We used highly specific antibodies to show by immunofluorescence and cell surface biotinylation studies that STIM1 is located at the cell surface of K562 cells. Western blot analysis revealed that the 90-kDa STIM1 protein is ubiquitously expressed in various human primary cells and tumour cell lines. STIM1 is not secreted from cells and does not appear to undergo proteolytic processing. We show evidence of post-translational modification of STIM1, namely phosphorylation and N-linked glycosylation. Phosphorylation of STIM1 in vivo occurs predominantly on serine residues. Thus, STIM1, the putative tumour growth suppressor gene is ubiquitously expressed and has features of a regulatory cell-surface phosphoprotein.

Transthyretin, an extracellular thyroid-hormone-binding protein (THBP) in higher vertebrates, is synthesized and secreted by the choroid plexus of all classes of vertebrates, except fish and amphibians, and synthesized in the liver of endothermic animals. Here, we report the nucleotide sequence of the cDNA for a THBP found in plasma of bullfrog (Rana catesbeiana) tadpoles before the climax of metamorphosis. The amino acid sequence clearly shows this protein to be an amphibian transthyretin. The three-dimensional structure of bullfrog transthyretin was derived using homology modeling. Compared with transthyretins from other vertebrate species, bullfrog transthyretin is highly conserved at the thyroid hormone-binding sites and other important structural regions of the subunits. Bullfrog transthyretin mRNA was found in tadpole liver, but not in tadpole choroid plexus. Thus, during evolution, synthesis of transthyretin in the liver of metamorphosing amphibians preceded that in the choroid plexus of reptiles, birds and mammals. It was previously observed that the protein most abundantly synthesized and secreted by the choroid plexus in adult amphibians is a lipocalin [Achen, M. G., Harms, P. J., Thomas, T., Richardson, S. J., Wettenhall, R. E. H. & Schreiber, G. (1992) J. Biol. Chem. 267, 23 170−23 174], in contrast to transthyretin being the most abundantly synthesized and secreted protein in the choroid plexus of mammals, birds and reptiles. Lipocalin mRNA was found in large amounts in tadpole choroid plexus, but not livers.

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.

Laminin and nidogen (entactin) are major glycoprotein components of basement membranes. At least seven different isoforms of laminin have been identified. Laminin and nidogen form high affinity complexes in basement membranes by specific binding between the laminin gamma 1 chain and the G3 globule of nidogen. Additional interactions between nidogen and collagen IV, perlecan and other basement membrane components result in the formation of ternary complexes between these matrix components. Nidogen is highly susceptible to proteolytic cleavage, and binding to laminin protects nidogen from degradation. Nidogen is considered to have a crucial role as a link protein in the assembly of basement membranes. Basement membrane components are synthesized at high levels during tissue growth and development, and sites of morphogenesis correlate with localized remodelling of basement membranes. The formation of distinct basement membrane matrices in the developing embryo is influenced by the laminin isoforms produced and by whether laminin and nidogen are co-expressed and secreted as a complex or are produced by cooperation between two cell layers. The potential roles of laminin-nidogen complexes, cell-matrix interactions, and other intermolecular interactions within the matrix in basement membrane assembly and stability are discussed in this review.

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.

MicroRNAs (miRNAs) are short, single-stranded, non-coding RNAs that regulate translation, by interacting with complementary sites in the 3′UTRs of target mRNAs. Each can target multiple mRNAs and each mRNA can be targeted by many different miRNAs to form regulatory networks. Placental development is characterised by dynamic, co-ordinated changes in expression of regulatory and functional genes that drive invasion, differentiation and growth. These changes may involve miRNAs, but placental miRNA expression during gestation has been little studied in any species to date. MiRNA expression was examined (by microarray) in the rat in mid (day 16) and late (day 20) gestation (term ~22 days) and the two major zones of the placenta, the labyrinth and junctional zones, at and between each gestational age. At day 16, 107 miRNAs were differentially expressed between the labyrinth and junctional zone (P < 0.05); 47% being more highly expressed and the remainder less expressed in the labyrinth compared to the junctional zone. At day 20, 78 miRNAs were differentially expressed between the two zones (P < 0.05); 53% being higher and the remainder lower in the labyrinth compared to the junctional zone. 216 miRNAs were differentially expressed in the labyrinth zone between mid and late gestation; 73% of the differentially expressed labyrinth zone miRNAs were upregulated at day 20, many from a cluster on chromosome 6, a feature of the late gestation mouse placenta as well (Kong et al., unpublished). In the mouse, these same clustered miRNAs are located on chromosome 12 in an imprinted region, with the miRNAs expressed from the maternal allele. Disruption of their expression impairs placental vascular, labyrinth and junctional zone development [1]. Together these findings imply that these clustered miRNAs are likely to play a role in labyrinth zone development and further study is warranted into which placental transcripts are targeted by these imprinted miRNAs. (1) Georgiades P, et al., Parental origin-specific developmental defects in mice with uniparental disomy for chromosome 12. Development, 2000. 127(21): 4719–28.