Gene Expression At The Beginning Of Mammalian Development

哺乳动物发育初期的基因表达

• 批准号: 8736814
• 负责人: Melvin DePamphilis
• 金额: $ 103.25万
• 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/8736814
• 关键词: 26S proteasome; Ablation; Acrosome; Adult; Affect; Alleles; Anencephaly; Apoptosis; Attenuated; Biological Assay; C57BL/6 Mouse; CDKN1C gene; CHEK2 gene; Cell Cycle; Cell Differentiation process; Cell Lineage; Cell Proliferation; Cell Survival; Cells; Checkpoint kinase 1; Cleaved cell; Collaborations; Congenital Abnormality; Cyclin-Dependent Kinases; DNA; DNA Damage; DNA damage checkpoint; Defect; Development; Embryo; Embryonic Development; Endometrium; Ensure; Enzymes; Event; Excision; Fertility; Fertilization in Vitro; Folic Acid; Gene Deletion; Gene Expression; Genes; Genetic; Genetic Transcription; Genome; Giant Cells; Goals; Government; Human; Human Development; Implant; In Vitro; Infertility; Inner Cell Mass; Institution; Laboratories; Life; Link; Mammals; Mediator of activation protein; Mesenchymal; Mesenchymal Stem Cells; Mitogens; Mitosis; Mitotic Cell Cycle; Modeling; Molecular; Morula; Mus; Mutagenesis; Nervous system structure; Neural Tube Closure; Northern Blotting; Organ; Organism; Organogenesis; Partner in relationship; Pattern; Penetration; Placenta; Play; Polyploidy; Pre-Eclampsia; Premature Birth; Proliferating; Protein Kinase; Proteins; RNA; Reporting; Reproduction; Research; Reverse Transcriptase Polymerase Chain Reaction; Role; Scheme; Signal Pathway; Site; Specific qualifier value; Sperm Penetration; Spermatocytes; Staging; Stem cells; Transgenic Mice; Zona Pellucida; blastocyst; blastomere structure; cyclin-dependent kinase inhibitor 1B; egg; embryo cell; embryo stage 2; embryonic stem cell; in vivo; inhibitor/antagonist; male; monolayer; mouse development; natural Blastocyst Implantation; preimplantation; premature; prevent; progenitor; programs; prospective; proto-oncogene protein kfgf; relating to nervous system; research study; response; self-renewal; sperm cell; transcription factor; trophoblast; zygote

1. The Acrosomal Protein Dickkopf-Like 1 (DKKL1) Is Not Essential For Fertility OBJECTIVE: To determine the role of Dkkl1 on mouse development, viability, and fertility. DESIGN: Prospective experimental study. SETTING: Government research institution. ANIMAL(S): Mice of C57BL/6 and 129X1/SvJ strains, as well as transgenic mice of mixed C57BL/6 and 129X1/SvJ strains were used for the studies. INTERVENTION(S): Mice were constructed that lacked a functional Dkkl1 gene. MAIN OUTCOME MEASURE(S): Deletion of the gene was confirmed by DNA, RNA, and protein analyses; in vivo fertility was examined by continuous mating scheme. RESULT(S): Previous studies have shown that Dkkl1, a gene unique to mammals, is expressed predominantly, if not exclusively, in developing spermatocytes, and the DKKL1 protein accumulates in the acrosome of mature sperm. Subsequent studies (reported in the accompanying article) demonstrate that Dkkl1 also is expressed in the trophectoderm/placental lineage. Taken together, these results strongly suggested that DKKL1 protein is required for terminal differentiation either of trophoblast giant cells or of sperm, both of which are directly involved in fertility. To challenge this hypothesis, conditional targeted mutagenesis was used to ablate the Dkkl1 gene in mice. Surprisingly, Dkkl1 nullizygous embryos developed into viable, fertile adults, despite the fact that they failed to produce any portion of the DKKL1 protein. CONCLUSION(S): DKKL1 is a mammalian-specific acrosomal protein that is not essential either for development or fertility. 2. The Acrosomal Protein Dickkopf-Like 1 (DKKL1) Facilitates Sperm Penetration Of The Zona Pellucida OBJECTIVE: To determine the role of Dkkl1 in mouse development, viability, and fertility. DESIGN: Prospective experimental study. SETTING: Government research institution. ANIMAL(S): Mice of C57BL/6, B6D2F1/J, and 129X1/SvJ strains, as well as transgenic mice of mixed C57BL/6 and 129X1/SvJ strains were used for the studies. INTERVENTION(S): Expression of the Dkkl1 gene was characterized during early mouse development, and the effects of Dkkl1 ablation on reproduction and fertility were characterized in vitro and in vivo. MAIN OUTCOME MEASURE(S): Dkkl1 RNA expression was determined by Northern blotting hybridization as well as quantitative reverse transcriptase-polymerase chain reaction assays. In vitro fertilization assays were used to assess fertility of sperm from male mice lacking functional Dkkl1. RESULT(S): Dkkl1 is a gene unique to mammals that is expressed primarily in developing spermatocytes and its product localized in the acrosome of mature sperm. Here we show that Dkkl1 also is expressed in the trophectoderm/placental lineage. Surprisingly, embryos lacking DKKL1 protein developed into viable, fertile adults. Nevertheless, the ability of sperm that lacked DKKL1 protein to fertilize wild-type eggs was severely compromised in vitro. Because this defect could be overcome either by removal of the zona pellucida or by the presence of wild-type sperm, Dkkl1, either directly or indirectly, facilitates the ability of sperm to penetrate the zona pellucida. Penetration of the zona pellucida by Dkkl1(-) sperm was delayed in vivo as well as in vitro, but the delay in vivo was compensated by other factors during preimplantation development. Accordingly, Dkkl1-/- males offer an in vitro fertilization model for identifying factors that may contribute to infertility. CONCLUSION(S): DKKL1 is a mammalian-specific, acrosomal protein that strongly affects in vitro fertilization, although the effect is attenuated in vivo. 3. Organogenesis relies on SoxC transcription factors for the survival of neural and mesenchymal progenitors During organogenesis, neural and mesenchymal progenitor cells give rise to many cell lineages, but their molecular requirements for self-renewal and lineage decisions are incompletely understood. Inthis study, we show that their survival critically relies on the redundantly acting SoxC transcription factors Sox4, Sox11 and Sox12. The more SoxC alleles that are deleted in mouse embryos, the more severe and widespread organ hypoplasia is. SoxC triple-null embryos die at midgestation unturned and tiny, with normal patterning and lineage specification, but with massively dying neural and mesenchymal progenitor cells. Specific inactivation of SoxC genes in neural and mesenchymal cells leads to selective apoptosis of these cells, suggesting SoxC cell-autonomous roles. Tead2 functionally interacts with SoxC genes in embryonic development, and is a direct target of SoxC proteins. SoxC genes therefore ensure neural and mesenchymal progenitor cell survival, and function in part by activating this transcriptional mediator of the Hippo signalling pathway. 4. Checkpoint Kinase-1 Prevents Cell Cycle Exit Linked To Terminal Cell Differentiation. Trophoblast stem (TS) cells proliferate in the presence of fibroblast growth factor-4, but in its absence, they differentiate into polyploid trophoblast giant (TG) cells that remain viable but nonproliferative. Differentiation is coincident with expression of the CDK-specific inhibitors p21 and p57; of which p57 is essential for switching from mitotic cell cycles to endocycles. Here we show that, in the absence of induced DNA damage, checkpoint kinase-1 (CHK1), an enzyme essential for preventing mitosis in response to DNA damage, functions as a mitogen-dependent protein kinase that prevents premature differentiation of TS cells into TG cells by suppressing expression of p21 and p57, but not p27, the CDK-inhibitor that regulates mitotic cell cycles. CHK1 phosphorylates p21 and p57 proteins at specific sites, thereby targeting them for degradation by the 26S proteasome. TG cells lack CHK1, and restoring CHK1 activity in TG cells suppresses expression of p57 and restores mitosis. Thus, CHK1 is part of a 'G2 restriction point' that prevents premature cell cycle exit in cells programmed for terminal differentiation, a role that CHK2 cannot play.

1。粘体蛋白dickkopf样1（dkkl1）对于生育不可能 目的：确定DKKL1在小鼠发育，生存力和生育方面的作用。设计：前瞻性实验研究。环境：政府研究机构。动物：使用C57BL/6和129x1/SVJ菌株的小鼠，以及混合C57BL/6和129x1/SVJ菌株的转基因小鼠。干预：构造了缺乏功能性DKKL1基因的小鼠。主要结局指标：通过DNA，RNA和蛋白质分析证实了基因的缺失；通过连续交配方案检查体内生育能力。结果：先前的研究表明，DKKL1是哺乳动物独有的基因，主要（即使不是仅限于开发精子细胞）的表达，而DKKL1蛋白在成熟精子的高级体中积累。随后的研究（随附的文章中报道）表明，DKKL1在滋养剂/胎盘谱系中也表达。综上所述，这些结果强烈表明，DKKL1蛋白是终止分化的滋养细胞巨细胞或精子的终末分化所必需的，而这些细胞或精子都直接参与了生育能力。为了挑战这一假设，使用条件靶向诱变来消除小鼠中的DKKL1基因。令人惊讶的是，尽管他们未能产生DKKL1蛋白的任何一部分，但DKKL1无效的胚胎发展为可行，肥沃的成年人。结论：DKKL1是一种哺乳动物特异性的顶体蛋白，无论对发育还是生育不可能。 2。粘体蛋白dickkopf样1（DKKL1）促进了Zona pellucida的精子穿透 目的：确定DKKL1在小鼠发育，生存力和生育方面的作用。设计：前瞻性实验研究。环境：政府研究机构。动物：C57BL/6，B6D2F1/J和129x1/SVJ菌株的小鼠，以及混合C57BL/6和129x1/SVJ菌株的转基因小鼠。干预措施：在小鼠早期发育过程中表征了DKKL1基因的表达，并且在体外和体内表征了DKKL1消融对繁殖和生育能力的影响。主要结局度量：通过北面印迹杂交以及定量逆转录酶 - 聚合酶链链反应测定法测定DKKL1 RNA表达。体外受精测定法用于评估缺乏功能性DKKL1的雄性小鼠的生育能力。结果：DKKL1是哺乳动物所独有的基因，主要在发展精子细胞及其产物中局限于成熟精子的高素质体。在这里，我们表明DKKL1在滋养剂/胎盘谱系中也表达。令人惊讶的是，缺乏DKKL1蛋白的胚胎发展成为可行的肥沃成年人。然而，在体外严重损害了缺乏DKKL1蛋白施肥的精子施肥的精子。因为可以通过去除Zona pellucida或直接或间接地存在野生型精子DKKL1来克服这种缺陷，从而促进了精子穿透Zona Pellucida的能力。 DKKL1（ - ）精子在体内和体外延迟Zona pellucida的渗透，但体内延迟在植入前发育过程中被其他因素补偿。因此，DKKL1 - / - 男性为识别可能导致不育的因素提供了一种体外受精模型。结论：DKKL1是一种哺乳动物特异性的粘体蛋白，尽管在体内受到了影响，但强烈影响体外受精。 3。器官发生依靠SOXC转录因子来生存神经和间质祖细胞的生存 在器官发生过程中，神经和间充质祖细胞会产生许多细胞谱系，但是它们对自我更新和谱系决定的分子要求尚不完全了解。在这项研究中，我们表明它们的存活率非常依赖于冗余作用的SOXC转录因子Sox4，Sox11和Sox12。在小鼠胚胎中删除的SOXC等位基因越多，器官发育不全越严重。 SOXC三重无效的胚胎在中间孕期处于未弯曲和微小的状态，具有正常的图案和谱系规格，但具有巨大的神经和间充质祖细胞。 Soxc基因在神经和间充质细胞中的特异性失活导致这些细胞的选择性凋亡，这表明SOXC细胞自治作用。 TEAD2在胚胎发育中与SOXC基因的功能相互作用，并且是SOXC蛋白的直接靶标。因此，SOXC基因确保神经和间充质祖细胞的存活，并部分通过激活河马信号通路的转录介质来发挥作用。 4。检查点激酶1阻止与末端细胞分化相关的细胞周期出口。 滋养细胞（TS）细胞在成纤维细胞生长因子-4的情况下增殖，但在不存在的情况下，它们分化为多倍体滋养细胞巨型（TG）细胞，这些细胞（TG）细胞仍然可行但无增殖。分化与CDK特异性抑制剂p21和p57的表达一致。其中p57对于从有丝分裂细胞循环转换为内鼠是必不可少的。在这里，我们表明，在没有诱导DNA损伤的情况下，检查点激酶1（CHK1），这是一种预防有丝分裂响应DNA损伤所必需的酶，可作为一种有丝分裂原依赖性蛋白激酶，可通过抑制P21和p27的表达，但不抑制P27，但CDK-MIT cyk cyk in Cyk in Cycy cyk in Cycy cys cys cys cys cy cys cys cys cys依赖有丝分裂原依赖性蛋白激酶。 CHK1在特定位点磷酸化P21和P57蛋白，从而将它们靶向26S蛋白酶体降解。 TG细胞缺乏CHK1，并且在TG细胞中恢复CHK1活性抑制了p57的表达并恢复有丝分裂。因此，CHK1是“ G2限制点”的一部分，该点可以防止编程用于末端分化的细胞中的过早细胞周期退出，这是CHK2无法扮演的角色。