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.     

Co-expression of the proto-oncogene fos (c-fos) and an embryonic interferon (ovine trophoblastin) by sheep conceptuses during implantation.

Expression of the c-fos proto-oncogene by ovine conceptuses was analyzed by Northern and slot blots and indirect immunohistofluorescence in relation to the expression of the embryonic interferon-alpha (oTP) during implantation. c-fos was expressed initially in the trophoblast, and then in the allantois, when this tissue began to develop (day 17). In the embryonic tissues, the c-fos proto-oncogene was weakly expressed up to day 22 and increased thereafter. In the trophoblast, the expression of c-fos proto-oncogene was transient, occurring when the oTP gene was transcribed at a maximal level at the beginning of implantation (days 14-15), and decreased thereafter, following the pattern of oTP gene expression. This decline is due essentially to the arrest of c-fos and oTP gene expression by the trophoblastic cells which established cellular contacts with the uterine epithelium during the implantation process.     

Characterization of a prolactin-regulated gene in reproductive tissues using the prolactin receptor knockout mouse model

Prolactin (PRL) exerts pleiotropic physiological effects in various cells and tissues, although it is mainly considered as a regulator of reproduction and cell growth. Null mutation of the prolactin receptor (PRLR) gene leads to female sterility due to a failure of embryo implantation. Using this mouse model and the method of mRNA differential display, we identified PRL target genes that are regulated during the peri-implantation period. We characterized 1 among the 45 isolated genes, UA-3, which is regulated in the uterus as well as in the ovary during early pregnancy. This gene corresponds to a P311 mouse cDNA that was originally identified for its high expression in late-stage embryonic brain and adult cerebellum. We report here that UA-3 is present in numerous tissues as well as in ovary and uterus at the site of blastocyst apposition, and that its expression is hormonally regulated. Moreover, in situ hybridization reveals high expression in ovarian granulosa cells and in uterine epithelium. Recently, it has been suggested that P311 expression is tightly regulated at several levels by mechanisms that control cellular growth, transformation, motility, or a combination of these. Taken together, these results suggest that P311 could be involved in these processes during pregnancy, although its function remains to be clearly established.     

Subcloning, localization, and expression of the rat intestinal sodium-hydrogen exchanger isoform 8

Apically expressed intestinal and renal sodium-hydrogen exchangers (NHEs) play a major role in Na(+) absorption. Our previous studies on NHE ontogeny have shown that NHE-2 and NHE-3 are expressed at very low levels in young animals. Furthermore, single and/or double NHE-2 and NHE-3 knockout mice display no obvious abnormalities before weaning. These observations suggest that other transporter(s) may be involved in intestinal Na+ absorption during early life. The present studies were designed to clone the novel rat intestinal NHE-8 cDNA and to decipher the NHE-8 protein localization and gene expression pattern during different developmental stages. The rat NHE-8 cDNA has 2,160 bp and encodes a 575-amino acid protein. An antibody against NHE-8 protein was developed. Immunohistochemistry staining indicated apical localization of NHE-8 protein in rat intestinal epithelial cells. The apical localization of NHE-8 was also confirmed by its presence in brush-border membrane and its absence in basolateral membrane preparations. Northern blotting utilizing a NHE-8-specific probe demonstrated higher NHE-8 mRNA expression in young animals compared with adult animals. Western blot analysis revealed a similar pattern. Tissue distribution with multiple human tissue RNA blot showed that NHE-8 was expressed in multiple tissues including the gastrointestinal tract. In conclusion, we have cloned the full-length NHE-8 cDNA from rat intestine and further showed its apical localization in intestinal epithelial cells. We have also shown that NHE-8 gene expression and protein expression were regulated during ontogeny. Our data suggests that NHE-8 may play an important role in intestinal Na+ absorption during early life.     

Embryonic mouse blood flow and oxygen correlate with early pancreatic differentiation

The mammalian embryo represents a fundamental paradox in biology. Its location within the uterus, especially early during development when embryonic cardiovascular development and placental blood flow are not well-established, leads to an obligate hypoxic environment. Despite this hypoxia, the embryonic cells are able to undergo remarkable growth, morphogenesis, and differentiation. Recent evidence suggests that embryonic organ differentiation, including pancreatic β-cells, is tightly regulated by oxygen levels. Since a major determinant of oxygen tension in mammalian embryos after implantation is embryonic blood flow, here we used a novel survivable in utero intracardiac injection technique to deliver a vascular tracer to living mouse embryos. Once injected, the embryonic heart could be visualized to continue contracting normally, thereby distributing the tracer specifically only to those regions where embryonic blood was flowing. We found that the embryonic pancreas early in development shows a remarkable paucity of blood flow and that the presence of blood flow correlates with the differentiation state of the developing pancreatic epithelial cells in the region of the blood flow.     

Differentiation of murine embryonic stem cells induces progesterone receptor gene expression

The role of steroid hormone receptors in very early embryonic development remains unknown. Clearly, expression during organogenesis is important for tissue-specific development. However, progesterone receptor (PR) and estrogen receptors (ERalpha, ERbeta) are expressed during early development through the blastocyst stage in mice and other species, and yet are not essential for embryonic viability. We have utilized the mouse embryonic stem (mES) cell model to investigate the regulated expression of these receptors during differentiation. Surprisingly, one of the earliest changes in gene expression in response to a differentiation signal observed is PR gene induction. It parallels the time course of expression for the patterning genes Hoxb1 and Hoxa5. Unexpectedly, PR gene expression is not regulated in an estrogen-dependent manner by endogenous ERs or by transiently overexpressed ERalpha. Our results suggest a potentially novel mechanism of PR gene regulation within mES cells compared to adult tissues and the possibility of unique targets of PR action during early mES cell differentiation.     

人胚胎干细胞向滋养层分化的研究进展

哺乳动物胚胎发育产生的第一个细胞系的分离是内细胞团和滋养层的分离,不同哺乳动物之间胚胎干细胞向滋养层细胞分化不同,滋养层细胞对胚胎的植入、促进胚胎在子宫内的生存和生长至关重要.人胚胎干细胞为研究人类胚胎发育及向滋养层分化提供了一个独特的模型.人胚胎干细胞可以在实验室条件下保持无限期稳定的培养,用于最初胚胎和滋养外胚层发生的机制研究.目前人胚胎干细胞分化为滋养层细胞在体外可以通过自发分化、基因敲除、分离EB小体和BMP4诱导等几种途径实现.不同哺乳动物之间胚胎干细胞向滋养层分化机制,主要通过信号通路如BMP4,LIF等以及某些标志基因如OCT4,CDX2,Eomes等的变化调节.人胚胎干细胞向滋养层分化的研究为临床应用提供了一定的基础.     

Cloning of the porcine interferon-γ receptor and its foeto-endometrial expression in early pregnancy

In early gestation, trophoblastic cells of porcine preimplanting conceptuses transiently and massively secrete two distinct interferons (IFNs), one of which is IFN-γ. In order to localize possible cellular target(s) for this IFN-γ, the expression of the porcine IFN-γ receptor and its developmental regulation have been investigated on the maternal endometrium and on the embryonic tissues. A cDNA encoding the porcine IFN-γ binding-chain (pIFNGR1) was isolated. When expressed in COS-7 cells, it displayed a specific binding to radiolabelled pIFN-γ and was shown to be a glycosylated membrane protein with an apparent molecular mass of 92 kDa. Porcine IFNGR1 mRNA was detected by RT-PCR not only in uterine epithelial cells but also in embryonic tissues from at least as early as day 10 of gestation. Moreover, membrane expression of the pIFN-γ receptor quantified by binding and crosslinking of ³²P-pIFN-γ was demonstrated in uterine epithelium and in the trophoblast. In the trophoblast, expression of the receptor was found to be developmentally regulated: although expression was weak on days 12 and 15 of gestation, it reached a level similar to that found on some IFN-γ–sensitive cells on day 16. This study shows that maternal endometrium is not the only possible target for trophoblastic IFN-γ: the induction of pIFN-γ receptor expression in the trophoblast around day 16 of gestation could suggest the appearance of responsiveness to pIFN-γ in this implanted tissue and therefore a possible delayed autocrine effect of trophoblastic pIFN-γ. Mol. Reprod. Dev. 51:225–234, 1998.© 1998 Wiley-Liss, Inc.     

Molecular cloning and functional analysis of a human cDNA encoding an Escherichia coli AlkB homolog, a protein involved in DNA alkylation damage repair.

The Escherichia coli AlkB protein is involved in protecting cells against mutation and cell death induced specifically by SN2-type alkylating agents such as methyl methanesulfonate (MMS). A human cDNA encoding a polypeptide homologous to E.coli AlkB was discovered by searching a database of expressed sequence tags (ESTs) derived from high throughput cDNA sequencing. The full-length human AlkB homolog (hABH) cDNA clone contains a 924 bp open reading frame encoding a 34 kDa protein which is 52% similar and 23% identical to E.coli AlkB. The hABH gene, which maps to chromosome 14q24, was ubiquitously expressed in 16 human tissues examined. When hABH was expressed in E.coli alkB mutant cells partial rescue of the cells from MMS-induced cell death occurred. Under the conditions used expression of hABH in skin fibroblasts was not regulated by treatment with MMS. Our findings show that the AlkB protein is structurally and functionally conserved from bacteria to human, but its regulation may have diverged during evolution.     

ASB4 is a hydroxylation substrate of FIH and promotes vascular differentiation via an oxygen-dependent mechanism

The molecular mechanisms of endothelial differentiation into a functional vascular network are incompletely understood. To identify novel factors in endothelial development, we used a microarray screen with differentiating embryonic stem (ES) cells that identified the gene for ankyrin repeat and SOCS box protein 4 (ASB4) as the most highly differentially expressed gene in the vascular lineage during early differentiation. Like other SOCS box-containing proteins, ASB4 is the substrate recognition molecule of an elongin B/elongin C/cullin/Roc ubiquitin ligase complex that mediates the ubiquitination and degradation of substrate protein(s). High levels of ASB4 expression in the embryonic vasculature coincide with drastic increases in oxygen tension as placental blood flow is initiated. However, as vessels mature and oxygen levels stabilize, ASB4 expression is quickly downregulated, suggesting that ASB4 may function to modulate an endothelium-specific response to increasing oxygen tension. Consistent with the hypothesis that ASB4 function is regulated by oxygen concentration, ASB4 interacts with the factor inhibiting HIF1alpha (FIH) and is a substrate for FIH-mediated hydroxylation via an oxygen-dependent mechanism. Additionally, overexpression of ASB4 in ES cells promotes differentiation into the vascular lineage in an oxygen-dependent manner. We postulate that hydroxylation of ASB4 in normoxia promotes binding to and degradation of substrate protein(s) to modulate vascular differentiation.     

Spatiotemporal patterns of macrophage migration inhibitory factor (Mif) expression in the mouse placenta

Background  

Macrophage migration inhibitory factor (MIF) has special pro-inflammatory roles, affecting the functions of macrophages and lymphocytes and counter-regulating the effects of glucocorticoids on the immune response. The conspicuous expression of MIF during human implantation and early embryonic development also suggests this factor acts in reproductive functions. The overall goal of this study was to evaluate Mif expression by trophoblast and embryo placental cells during mouse pregnancy.