The role of arginine vasopressin in human labour: functional studies, fetal production and localisation of V1a receptor mRNA.

OBJECTIVE: To investigate labour-associated changes in: 1. the myometrial contractile response to arginine vasopressin compared with oxytocin in vitro 2. fetal production of arginine vasopressin and 3. myometrial vasopressin V1a receptor mRNA. DESIGN: The contractile response to vasopressin (compared with oxytocin) was investigated in paired myometrial strips in vitro. Blood was taken from the umbilical artery and vein at delivery and arginine vasopressin measured by radio-immunoassay. V1a receptor mRNA was determined by in situ hybridisation. RESULTS: Myometrium was more sensitive to arginine vasopressin than oxytocin (P<0.05 for frequency, amplitude and activity integral in paired strips) after, but not before labour. There was a marked umbilical arteriovenous difference in arginine vasopressin concentration at delivery suggesting fetal production which was not influenced by labour. Myometrial vasopressin V1a receptor mRNA was not increased after the onset of labour. CONCLUSIONS: The human uterus is extremely sensitive to arginine vasopressin in vitro. Arginine vasopressin is produced by the fetus but fetal formation is not increased during labour.

Evolutionary conservation, developmental expression, and genomic mapping of mammalian Twisted gastrulation.

The twisted gastrulation gene (tsg) encodes a secreted protein required for the correct specification of dorsal midline cell fate during gastrulation in Drosophila. We report that tsg homologs from human, mouse, zebrafish, and Xenopus share 72-98% identity at the amino acid level and retain all 24 cysteine residues from Drosophila. In contrast to Drosophila where tsg expression is limited to early embryos, expression is found throughout mouse and human development. In Drosophila, tsg acts in synergy with decapentaplegic (dpp), a member of the TGF-beta family of secreted proteins. The vertebrate orthologs of dpp, BMP-2 and -4, are crucial for gastrulation and neural induction, and aberrant signaling by BMPs and other TGF-beta family members results in developmental defects including holoprosencephaly (HPE). Interestingly, human TSG maps to the HPE4 locus on Chromosome 18p11.3, and our analysis places the gene within 5 Mbp of TG-interacting factor (TGIF).

Induction of the mammalian node requires Arkadia function in the extraembryonic lineages.

The early mammalian embryo is patterned by signals emanating from extraembryonic and embryonic signalling centres, most notably the anterior visceral endoderm (AVE) and the node, respectively. The AVE is responsible for anterior development, whereas further axis specification depends on the node, the equivalent of Spemann's organizer. Formation of the node, at the anterior primitive streak, depends on expression of the transcription factor HNF3beta (ref. 4). However, both the source and the nature of the signals responsible for inducing the node have been unknown. Here we describe a recessive lethal mutation, arkadia, generated using gene-trap mutagenesis. Mutant embryos establish an AVE but fail to maintain anterior embryonic structures and lack a node. The mutation has disrupted the Arkadia gene, which encodes a putative intracellular protein containing a RING domain. Arkadia is essential for HNF3beta expression in the anterior primitive streak. Analysis with chimaeras, however, shows that Arkadia functions within extraembryonic tissues, revealing that these are required to induce the node. Furthermore, our experiments show that Arkadia interacts genetically with the transforming growth factor (TGF)beta-like factor Nodal, implying that Nodal mediates the function of Arkadia in node induction.

Expression of galectin-7 during epithelial development coincides with the onset of stratification.

Galectin-7 is a 14 KDa member of the galectin family that we have cloned from human, rat and mouse. Our previous studies have shown that in the adult, galectin-7 is expressed in all cell layers of epidermis and of other stratified epithelia such asthe cornea and the lining of the oesophagus. This suggested that galectin-7 expression might be induced at a particular stage in the embryonic development of stratified epithelia. In the present study we have investigated this hypothesis by in situ hybridization of galectin-7 mRNA in mouse embryos. Starting from E13.5, weak expression of galectin-7 was detected in bilayered ectoderm, and stronger expression was found in areas of embryonic epidermis where stratification was more advanced. Galectin-7 expression was maintained in all living layers after epidermal development was completed. Galectin-7 was also strongly and specifically expressed in stratified regions of ectodermally-derived non-epidermal epithelia such as the lining of the buccal cavity, the oesophagus and the ano-rectal region. In contrast, no expression of galectin-7 was found in epithelia derived from endoderm, such as lining of the intestine, kidney and lung. Our results demonstrate that galectin-7 is expressed in all stratified epithelia examined so far, and that the onset of its expression coincides with the first visible signs of stratification. These results establish galectin-7 as the first region-independent marker of epithelial stratification.

Oxytocin receptor expression in human term and preterm gestational tissues prior to and following the onset of labour.

Oxytocin receptor (OTR) mRNA expression has previously been demonstrated in human myometrium, decidua, chorion and amnion but the effect of gestational age and the onset of labour has not been determined in these individual tissues. Spatial OTR mRNA expression was examined by in situ hybridization and ligand binding was confirmed using autoradiography with the iodinated oxytocin antagonist d(CH2)5[Tyr(Me)2,Thr4,Tyr-NH29]-vasotocin (125I-OTA). Tissue was collected at term (>37 weeks of gestation) or preterm (24-36 weeks of gestation) caesarean section and classified as labour (contractions every 5 min associated with cervical dilatation) or non-labour. OTR mRNA expression was measured as optical density units from autoradiographs. There was a highly significant (P<0.001) effect of tissue type on expression of OTR mRNA with expression greatest in myometrium, low in decidua and chorion and not detected in placenta. Similar results were obtained with the 125I-OTA-binding studies, indicating that the message was translated. Amnion had an apparently high level of both hybridization and 125I-OTA binding in some samples, but a lack of specificity prevented quantification of the signal in this tissue type. Term myometrium (labour and non-labour) had significantly higher (P<0.01) OTR mRNA expression than preterm myometrium, but there was no further increase in mRNA concentration associated with labour onset. In contrast, 125I-OTA binding in myometrium was already high at 33 weeks and did not increase further either later in pregnancy or with labour. In decidua there was no effect of gestational age or labour onset on OTR mRNA expression or 125I-OTA binding. In summary, OTR mRNA expression in the myometrium increased in late pregnancy whereas decidual expression was much lower and did not rise at term.

Expression and function of Pax 1 during development of the pectoral girdle.

Pax 1 is a member of the paired-box containing gene family. Expression has previously been observed in the developing sclerotomes and later in the anlagen of the intervertebral discs. Analysis of Pax 1-deficient undulated mice revealed an important role for this gene in the development of the axial skeleton, in which Pax 1 apparently functions as a mediator of notochordal signals during sclerotome differentiation. Here we demonstrate that Pax 1 is also transiently expressed in the developing limb buds. A comparative phenotypic analysis of different undulated alleles shows that this expression is of functional significance. In mice that are mutant for the Pax 1 gene severe developmental abnormalities are found in the pectoral girdle. These include fusions of skeletal elements which would normally remain separate, and failures in the differentiation of blastemas into cartilaginous structures. Although Pax 1 is also expressed in the developing hindlimb buds and Wolffian ridge, no malformations could be detected in the corresponding regions of Pax 1 mutant mice. These findings show that, in addition to its role in the developing vertebral column, Pax 1 has an important function in the development of parts of the appendicular skeleton.

The gene encoding the calcium binding protein calcyclin is expressed at sites of exocytosis in the mouse.

Calcyclin is a member of the S100 family of calcium binding proteins. We have found by in situ hybridization that calcyclin transcripts are restricted to specific cell types within a limited number of mouse organs. High levels of expression in the epithelia lining the gastrointestinal, respiratory and urinary tracts, and specific localization of the transcripts to the goblet cells in the small intestine, lead us to suggest a role for calcyclin in the process of mucus secretion. In addition, calcyclin expression was detected in the corpus luteum, placenta and nerves within the gut wall, which are all sites of regulated exocytosis. We propose that this S100-like protein may be part of a calcium signalling pathway utilized in the secretion of various products by different cell types.

Expression of the L14 lectin during mouse embryogenesis suggests multiple roles during pre- and post-implantation development.

A cDNA encoding L14, the lactose-binding, soluble lectin of relative molecular mass 14 x 10(3), has been isolated in a differential screen designed to identify genes that are regulated during the differentiation of murine embryonic stem cells in vitro. The expression patterns of the gene and of the encoded protein during mouse embryogenesis are consistent with the lectin playing a role at several stages of development. Firstly, it is initially synthesised in the trophectoderm of expanded blastocysts immediately prior to implantation, suggesting that it may be involved in the attachment of the embryo to the uterine epithelium. Secondly, in the postimplantation embryo, the lectin is abundantly expressed in the myotomes of the somites. This observation, when taken together with data indicating a role for the lectin in myoblast differentiation in culture, suggests that the protein is important in muscle cell differentiation. Finally, within the nervous system expression of this gene is activated early during the differentiation of a particular subset of neurones.

Activation of endogenous retroviral transcription in SV40-transformed mouse cells.

We have previously isolated a number of cDNA clones that correspond to mRNAs present at higher levels in SV40-transformed cells than in the untransformed parental cells (Scott, M.R.D., Westphal, K.-H. and Rigby, P.W.J. (1983) Cell 34, 557-567). We have now determined the nucleotide sequence of the archetypal Set 2 clone, pAG59, and can thus identify it as corresponding to the env gene of the endogenous, ecotropic C-type retrovirus of Balb/c mice, Emv-1. We have shown that in the subset of SV40-transformed cells that express the provirus both of the proteins encoded by env, gp70 and p15E, are synthesised and that the former is displayed on the cell surface. We discuss the significance of these observations for the biology of SV40 transformation.

The murine H19 gene is activated during embryonic stem cell differentiation in vitro and at the time of implantation in the developing embryo.

The differentiation in vitro of murine embryonic stem cells to embryoid bodies mimics events that occur in vivo shortly before and after embryonic implantation. We have used this system, together with differential cDNA cloning, to identify genes the expression of which is regulated during early embryogenesis. Here we describe the isolation of several such cDNA clones, one of which corresponds to the gene H19. This gene is activated in extraembryonic cell types at the time of implantation, suggesting that it may play a role at this stage of development, and is subsequently expressed in all of the cells of the mid-gestation embryo with the striking exception of most of those of the developing central and peripheral nervous systems. After birth, expression of this gene ceases or is dramatically reduced in all tissues.

Transcription factor AP-2 is expressed in neural crest cell lineages during mouse embryogenesis.

We have analyzed the expression pattern of transcription factor AP-2 in mouse embryos to evaluate the potential of AP-2 as a regulator during vertebrate development. A partial cDNA encoding AP-2 was isolated from a mouse embryo cDNA library and used to prepare probes to measure AP-2 mRNA levels by RNase protection and RNA in situ hybridization. Between 10.5 and 15.5 days of embryogenesis, the relative abundance of AP-2 mRNA is greatest at 11.5 days and declines steadily thereafter. RNA in situ hybridization analysis of embryos between 8.5 and 12.5 days of gestation identified a novel expression pattern for AP-2. The principle part of this expression occurs in neural crest cells and their major derivatives, including cranial and spinal sensory ganglia and facial mesenchyme. AP-2 is also expressed in surface ectoderm and in a longitudinal column of the spinal cord and hindbrain that is contacted by neural crest-derived sensory ganglia. Additional expression of AP-2 occurs in limb bud mesenchyme and in meso-metanephric regions. This embryonic expression pattern is spatially and temporally consistent with a role for AP-2 in regulating transcription of genes involved in the morphogenesis of the peripheral nervous system, face, limbs, skin, and nephric tissues.

A POU-domain transcription factor in early stem cells and germ cells of the mammalian embryo.

The murine oct-3 gene encodes a transcription factor containing a POU-specific domain and a homeodomain. In marked contrast to other homeodomain-encoding genes, oct-3 is expressed in the totipotent and pluripotent stem cells of the pregastrulation embryo and is down-regulated during differentiation to endoderm and mesoderm, suggesting that it has a role in early development. The oct-3 gene is also expressed in primordial germ cells and in the female germ line.

Developmental control of transduced dopa decarboxylase genes in D. melanogaster.

Seventeen new euchromatic integration sites of the dopa-decarboxylase gene (Ddc) have been generated using p-mediated transduction. The developmental expression of the integrated genes was examined by monitoring the embryonic induction of dopa decarboxylase enzyme activity (DDC) and by monitoring the developmental pattern of DDC activity from late third instar to eclosion. The majority of inserts are regulated correctly within about 30% of controls. Several cases of multiple insertion events were recovered and these show correspondingly elevated levels of activity and are regulated normally. The pattern of expression of one insert (15C) falls outside the normal range. Multiple copies of transduced Ddc genes are used to test for effects of elevated gene dose on levels of expression. One insert on the X chromosome shows little or no dosage compensation. Possible reasons for the differences between the regulation of transduced genes in Drosophila and the regulation of transformed genes in mammalian systems are discussed.