Gene Expression At The Beginning Of Mammalian Development

哺乳动物发育初期的基因表达

基本信息

项目摘要

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. 顶体蛋白 Dickkopf-Like 1 (DKKL1) 对于生育能力不是必需的 目的:确定 Dkkl1 对小鼠发育、活力和生育能力的作用。设计:前瞻性实验研究。地点:政府研究机构。动物:C57BL/6和129X1/SvJ品系的小鼠以及C57BL/6和129X1/SvJ混合品系的转基因小鼠用于研究。干预措施:构建缺乏功能性 Dkkl1 基因的小鼠。主要结果指标:通过 DNA、RNA 和蛋白质分析证实基因删除;通过连续交配方案检查体内生育力。结果:之前的研究表明,Dkkl1 是哺乳动物特有的基因,主要(如果不是全部)在发育中的精母细胞中表达,并且 DKKL1 蛋白在成熟精子的顶体中积累。随后的研究(在随附文章中报道)表明 Dkkl1 也在滋养外胚层/胎盘谱系中表达。总而言之,这些结果强烈表明 DKKL1 蛋白是滋养层巨细胞或精子终末分化所必需的,两者都直接参与生育能力。为了挑战这一假设,使用条件定向诱变来消除小鼠中的 Dkkl1 基因。令人惊讶的是,Dkkl1 无效胚胎发育成可存活、可育的成体,尽管事实上它们未能产生 DKKL1 蛋白的任何部分。结论:DKKL1 是一种哺乳动物特异性顶体蛋白,对于发育或生育力都不是必需的。 2. 顶体蛋白 Dickkopf-Like 1 (DKKL1) 促进精子穿透透明带 目的:确定 Dkkl1 在小鼠发育、活力和生育力中的作用。设计:前瞻性实验研究。地点:政府研究机构。动物:C57BL/6、B6D2F1/J和129X1/SvJ品系的小鼠以及C57BL/6和129X1/SvJ混合品系的转基因小鼠用于研究。干预措施:在小鼠早期发育过程中对 Dkkl1 基因的表达进行了表征,并在体外和体内表征了 Dkkl1 消除对生殖和生育能力的影响。主要结果指标:Dkkl1 RNA 表达通过 Northern 印迹杂交以及定量逆转录酶-聚合酶链式反应测定来测定。体外受精试验用于评估缺乏功能性 Dkkl1 的雄性小鼠精子的生育能力。结果:Dkkl1 是哺乳动物特有的基因,主要在发育中的精母细胞中表达,其产物位于成熟精子的顶体中。在这里,我们表明 Dkkl1 也在滋养外胚层/胎盘谱系中表达。令人惊讶的是,缺乏 DKKL1 蛋白的胚胎发育成有活力、有生育能力的成体。然而,缺乏 DKKL1 蛋白的精子与野生型卵子受精的能力在体外受到严重损害。因为这种缺陷可以通过去除透明带或野生型精子的存在来克服,所以 Dkkl1 直接或间接地促进精子穿透透明带的能力。 Dkkl1(-)精子对透明带的穿透在体内和体外均被延迟,但体内的延迟在植入前发育过程中被其他因素补偿。因此,Dkkl1-/- 男性提供了一种体外受精模型,用于识别可能导致不育的因素。结论:DKKL1 是一种哺乳动物特异性顶体蛋白,它强烈影响体外受精,尽管这种影响在体内减弱。 3. 器官发生依赖于SoxC转录因子来维持神经和间充质祖细胞的存活 在器官发生过程中,神经和间充质祖细胞产生许多细胞谱系,但它们自我更新和谱系决定的分子要求尚不完全清楚。在这项研究中,我们表明它们的生存严重依赖于冗余作用的 SoxC 转录因子 Sox4、Sox11 和 Sox12。小鼠胚胎中删除的 SoxC 等位基因越多,器官发育不全就越严重和广泛。 SoxC 三无效胚胎在妊娠中期死亡,未发育成熟且很小,具有正常的模式和谱系规范,但神经和间充质祖细胞大量死亡。神经细胞和间充质细胞中 SoxC 基因的特异性失活会导致这些细胞的选择性凋亡,表明 SoxC 细胞具有自主作用。 Tead2 在胚胎发育过程中与 SoxC 基因发生功能性相互作用,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 Hippo 信号通路的转录介质发挥作用。 4. 检查点激酶-1 防止与终末细胞分化相关的细胞周期退出。 滋养层干 (TS) 细胞在成纤维细胞生长因子 4 存在的情况下增殖,但在缺乏成纤维细胞生长因子 4 的情况下,它们会分化为多倍体滋养层巨细胞 (TG),该细胞仍能存活但不增殖。分化与 CDK 特异性抑制剂 p21 和 p57 的表达一致;其中p57对于有丝分裂细胞周期向内周期的转换至关重要。在这里,我们发现,在没有诱导 DNA 损伤的情况下,检查点激酶 1 (CHK1)(一种防止 DNA 损伤导致有丝分裂所必需的酶)可作为有丝分裂原依赖性蛋白激酶,防止 TS 细胞过早分化为 TG通过抑制 p21 和 p57 的表达来抑制细胞的表达,但不抑制 p27(调节有丝分裂细胞周期的 CDK 抑制剂)。 CHK1 在特定位点磷酸化 p21 和 p57 蛋白,从而靶向它们被 26S 蛋白酶体降解。 TG细胞缺乏CHK1,恢复TG细胞中的CHK1活性会抑制p57的表达并恢复有丝分裂。因此,CHK1 是“G2 限制点”的一部分,可防止细胞周期过早退出被编程为终末分化的细胞,而 CHK2 无法发挥这一作用。

项目成果

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Melvin DePamphilis其他文献

Melvin DePamphilis的其他文献

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{{ truncateString('Melvin DePamphilis', 18)}}的其他基金

Initiation Of Dna Replication In Mammalian Chromosomes
哺乳动物染色体中 DNA 复制的起始
  • 批准号:
    6681730
  • 财政年份:
  • 资助金额:
    $ 103.25万
  • 项目类别:
Gene Expression At The Beginning Of Mammalian Developmen
哺乳动物发育初期的基因表达
  • 批准号:
    6681731
  • 财政年份:
  • 资助金额:
    $ 103.25万
  • 项目类别:
Initiation Of DNA Replication In Mammalian Chromosomes
哺乳动物染色体中 DNA 复制的起始
  • 批准号:
    8736813
  • 财政年份:
  • 资助金额:
    $ 103.25万
  • 项目类别:
Initiation Of DNA Replication In Mammalian Chromosomes
哺乳动物染色体中 DNA 复制的起始
  • 批准号:
    8351104
  • 财政年份:
  • 资助金额:
    $ 103.25万
  • 项目类别:
Gene Expression At The Beginning Of Mammalian Development
哺乳动物发育初期的基因表达
  • 批准号:
    9339944
  • 财政年份:
  • 资助金额:
    $ 103.25万
  • 项目类别:
Gene Expression At The Beginning Of Mammalian Development
哺乳动物发育初期的基因表达
  • 批准号:
    7594136
  • 财政年份:
  • 资助金额:
    $ 103.25万
  • 项目类别:
Gene Expression At Beginning Of Mammalian Development
哺乳动物发育初期的基因表达
  • 批准号:
    7201708
  • 财政年份:
  • 资助金额:
    $ 103.25万
  • 项目类别:
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
  • 财政年份:
  • 资助金额:
    $ 103.25万
  • 项目类别:
Gene Expression At The Beginning Of Mammalian Developmen
哺乳动物发育初期的基因表达
  • 批准号:
    6508740
  • 财政年份:
  • 资助金额:
    $ 103.25万
  • 项目类别:
Initiation Of DNA Replication In Mammalian Chromosomes
哺乳动物染色体中 DNA 复制的起始
  • 批准号:
    7594135
  • 财政年份:
  • 资助金额:
    $ 103.25万
  • 项目类别:

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精子功能期间的循环 AMP 作用
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    2010
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Cyclic AMP Action During Sperm Function
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Cyclic AMP Action During Sperm Function
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