Gene Expression At The Beginning Of Mammalian Developmen

哺乳动物发育初期的基因表达

• 批准号: 7333867
• 负责人: Melvin DePamphilis
• 金额: --
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7333867
• 关键词: Gene; Expression; Beginning; Mammalian; Developmen

Human development begins when an egg is fertilized by a sperm. This event activates the first round of genome duplication, after which the resulting 2-cell embryo activates the expression of genes in the newly formed zygote. Five more rounds of cell division and a blastocyst appears, marking the beginning of cell differentiation: trophoblasts stem (TS) cells on the spherical surface of the blastocyst will give rise to the placental, and embryonic stem (ES) cells inside the blastocyst will give rise to the embryo. During this process, a remarkable and very rare event occurs in which TS cells differentiate into trophoblast giant (TG) cells that are required for implantation of the embryo into the uterine lining. During all of the trillions of cell divisions required to make an adult human, genome duplication is restricted to once per cell division, but in TG cells and in megakaryocytes, endoduplication occurs, a process whereby the genome is duplicated multiple times without undergoing cell division. We are taking advantage of our background in DNA replication to focus on three questions concerning preimplantation mammalian development: How is DNA replication activated in fertilized eggs? What genes are involved in the production and subsequent differentiation of TS and ES cells? What triggers endoreduplication during differentiation of TS to TG cells? During the past year, we have made the following discoveries: 1) Dickkopf-like 1 (DkkL1) is a gene that is related to the Dickkopf gene family which is a group of proteins that are characterized as secreted antagonists of Wingless signal transduction proteins. DkkL1 mRNA is found in preimplantation mouse embryos and in developing neural tissue, but in adults it is found primarily in the testes. In an effort to elucidate its function, the distribution of Dickkopf-like 1 protein (DkkL1) in mouse testis and mature sperm was analyzed by immuno-histochemistry and immuno-blotting techniques. DkkL1 first appeared in the developing spermatocytes in seminiferous tubules as early as Stage XII, coincident with the appearance of DkkL1 mRNA. Surprisingly, however, DkkL1 localized to the developing acrosome in spermatocytes and spermatids and to the acrosome in mature sperm. Furthermore, DkkL1 was N-glycosylated in the testis, but it did not appear to be excreted, and the DkkL1 in mature sperm was no longer N-glycosylated, suggesting that additional post-translational modifications occurred during the final stages of spermatogenesis. These results identify a member of the Dickkopf family as a novel acrosomal protein that may be involved in acrosome assembly or function, a unique role for a secreted signaling molecule. 2) The La protein is a target of autoantibodies in patients suffering from Sjogren's syndrome, systemic lupus erythematosus, and neonatal lupus. Ubiquitous in eukaryotes, La functions as a RNA-binding protein that promotes the maturation of tRNA precursors and other nascent transcripts synthesized by RNA polymerase III as well as other noncoding RNAs. La also associates with a class of mRNAs that encode ribosome subunits and precursors to snoRNAs involved in ribosome biogenesis. Thus, it was surprising that La is dispensable in the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, the organisms from which it has been characterized most extensively. To determine whether La is essential in mammals and if so, at which developmental stage it is required, mice were created with a disrupted La gene, and the offspring from La+/-intercrosses were analyzed. La-/- offspring were detected at the expected frequency among blastocysts prior to implantation, whereas no nullizygotes were detected after implantation, indicating that La is required early in development. Blastocysts derived from La+/- intercrosses yielded 38 La+/+ and La+/- embryonic stem (ES) cell lines but no La-/- ES cell lines, suggesting that La contributes a critical function toward the establishment or survival of ES cells. Consistent with this, La-/- blastocyst outgrowths revealed loss of the inner cell mass (ICM). The results indicate that in contrast to the situation in yeasts, La is essential in mammals and is one of a limited number of genes required as early as the development of the ICM. For additional information, visit our web site at (http://depamphilislab.nichd.nih.gov/), and read our three recent publications. Kohn, M.J., K.J. Kaneko and M.L. DePamphilis (2005) DkkL1(Soggy), A Dickkopf Family Member, Localizes To The Acrosome During Mammalian Spermatogenesis, Mol. Reprod. Dev. 71:516-522. Park, J-M., M. J. Kohn, M. Bruinsma, C. Vech, R.V. Intine, S. Fuhrmann, A. Grinberg, I. Mukherjee, P.E. Love, M.S. Ko, M.L. DePamphilis and R.J. Maraia. (2006) The multifunctional RNA-binding protein La is required for mouse development and for the establishment of embryonic stem cells., Mol. Cell. Biol. 26:1445-51. Kohn, M.J., Kaneko, K.J., Yagi, R., Litscher, E.S., Wassarman, P.M., DePamphilis, M.L. (2006) Dickkopf-like 1- a Protein Unique to Mammals that is Associated Both With Formation of Trophoblast Stem Cells and With Spermatogenesis. In ?Y Chromosome and Male Germ Cell Biology? (Lau, Y.-F.C. and Chan, W.Y., eds), World Scientific Publishers, Hackensack, NJ, in press.

当卵子与精子受精时，人类的发育就开始了。该事件激活第一轮基因组复制，之后产生的 2 细胞胚胎激活新形成的受精卵中的基因表达。再经过五轮细胞分裂，囊胚出现，标志着细胞分化的开始：囊胚球形表面的滋养层干细胞（TS）将产生胎盘，囊胚内的胚胎干细胞（ES）将产生胎盘。上升到胚胎。在此过程中，会发生一个显着且非常罕见的事件，即 TS 细胞分化为胚胎植入子宫内膜所需的滋养层巨细胞 (TG)。在形成成年人所需的所有数万亿次细胞分裂期间，基因组复制仅限于每次细胞分裂一次，但在TG细胞和巨核细胞中，会发生内复制，这是基因组在不经历细胞分裂的情况下多次复制的过程。我们利用 DNA 复制方面的背景，重点研究有关植入前哺乳动物发育的三个问题：受精卵中的 DNA 复制是如何激活的？哪些基因参与 TS 和 ES 细胞的产生和随后的分化？在 TS 细胞分化为 TG 细胞的过程中，什么触发了核内复制？ 在过去的一年里，我们有以下发现： 1) Dickkopf-like 1 (DkkL1) 是与 Dickkopf 基因家族相关的基因，该家族是一组以 Wingless 信号转导蛋白的分泌拮抗剂为特征的蛋白质。 DkkL1 mRNA 存在于植入前小鼠胚胎和发育中的神经组织中，但在成人中，它主要存在于睾丸中。为了阐明其功能，通过免疫组织化学和免疫印迹技术分析了 Dickkopf 样 1 蛋白 (DkkL1) 在小鼠睾丸和成熟精子中的分布。 DkkL1 早在第 XII 阶段就首次出现在生精小管中正在发育的精母细胞中，与 DkkL1 mRNA 的出现一致。然而，令人惊讶的是，DkkL1 定位于精母细胞和精子细胞中正在发育的顶体以及成熟精子中的顶体。此外，DkkL1在睾丸中被N-糖基化，但似乎没有被排出体外，并且成熟精子中的DkkL1不再被N-糖基化，这表明在精子发生的最后阶段发生了额外的翻译后修饰。这些结果将 Dickkopf 家族的成员鉴定为一种新型顶体蛋白，可能参与顶体组装或功能，这是分泌信号分子的独特作用。 2) La蛋白是干燥综合征、系统性红斑狼疮和新生儿狼疮患者自身抗体的靶标。 La 在真核生物中普遍存在，作为一种 RNA 结合蛋白，促进 tRNA 前体和 RNA 聚合酶 III 以及其他非编码 RNA 合成的其他新生转录物的成熟。 La 还与一类编码核糖体亚基和参与核糖体生物合成的 snoRNA 前体的 mRNA 相关。因此，令人惊讶的是，La在酿酒酵母和粟酒裂殖酵母中是可有可无的，这两种生物体中La的特征最为广泛。为了确定 La 是否对哺乳动物来说是必需的，如果是的话，在哪个发育阶段需要它，用 La 基因被破坏的小鼠被创建，并对 La+/- 杂交的后代进行了分析。植入前在囊胚中以预期频率检测到 La-/- 后代，而植入后未检测到无效合子，这表明 La 在发育早期是必需的。 La+/- 杂交产生的囊胚产生了 38 个 La+/+ 和 La+/- 胚胎干 (ES) 细胞系，但没有 La-/- ES 细胞系，这表明 La 对 ES 细胞的建立或存活发挥着关键作用。与此一致的是，La-/-囊胚的生长显示出内细胞团（ICM）的损失。结果表明，与酵母中的情况相反，La在哺乳动物中是必需的，并且是早在ICM发展时所需的有限数量的基因之一。 如需更多信息，请访问我们的网站 (http://depamphilislab.nichd.nih.gov/)，并阅读我们最近的三份出版物。 科恩，M.J.，K.J.金子和 M.L. DePamphilis (2005) DkkL1(Soggy)，迪克科普夫家族成员，在哺乳动物精子发生过程中定位于顶体，分子生物学。重现。开发。 71：516-522。 Park, J-M.、M. J. Kohn、M. Bruinsma、C. Vech、R.V. Intine、S. Fuhrmann、A. Grinberg、I. Mukherjee、P.E.爱，M.S.高，M.L.德潘菲利斯和 R.J.玛拉亚。 (2006) 多功能 RNA 结合蛋白 La 是小鼠发育和胚胎干细胞建立所必需的。, Mol.细胞。生物。 26:1445-51。 Kohn, M.J.、Kaneko, K.J.、Yagi, R.、Litscher, E.S.、Wassarman, P.M.、DePamphilis, M.L. (2006) Dickkopf-like 1 - 一种哺乳动物特有的蛋白质，与滋养层干细胞的形成和精子发生有关。在“Y染色体和男性生殖细胞生物学”中？ （Lau, Y.-F.C. 和 Chan, W.Y. 编辑），世界科学出版社，新泽西州哈肯萨克，印刷中。