Mechanism of apicosome-driven lumen formation during human and mouse embryogenesis

人类和小鼠胚胎发生过程中顶端体驱动的管腔形成机制

• 批准号: 10424552
• 负责人: Kenichiro Taniguchi
• 金额: $ 38.22万
• 依托单位: MEDICAL COLLEGE OF WISCONSIN
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-01 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10424552
• 关键词: ATP phosphohydrolase; Actins; Amino Acid Transport System L; Amino Acid Transporter; Amino Acids; Apical; Automobile Driving; Basic Amino Acid Transport Systems; Biogenesis; Biological Models; Biology; Biotinylation; CDC42 gene; CDK6-associated protein p18; Carrier Proteins; Cell Polarity; Cells; Charge; Cilia; Coupled; Cysteine; Cytoskeletal Modeling; Data; Development; Embryo; Embryonic Development; Endoderm; Engineering; Epiblast; Epigenetic Process; Ethics; Event; FRAP1 gene; Fertility; Glutamate Transporter; Goals; Human; Human Development; Image; Implant; In Vitro; Investigation; Leukocytes; Link; Mass Spectrum Analysis; Membrane; Modeling; Molecular; Monomeric GTP-Binding Proteins; Mus; Names; Nuclear; Organelles; Peroxidases; Process; Property; Proteins; Proteome; Proteomics; Protons; Radial; Reproduction; Rosaniline Dyes; Signal Transduction; Structure; Subcellular structure; Surface; System; Tissues; Uterus; Vesicle; Work; ascorbate; base; cellular microvillus; cilium biogenesis; experimental study; extracellular; human model; human pluripotent stem cell; implantation; in vivo; locus ceruleus structure; mouse model; natural Blastocyst Implantation; novel; polarized cell; tool; trafficking; transcriptomics; trophoblast

PROJECT SUMMARY Epiblast cavity formation occurs as the embryo implants into the uterine wall, making this step ethically inaccessible to experimental study in humans. While mouse embryos do provide a genetically tractable tool for exploring mechanisms associated with epiblast cavity formation, such investigations are restricted to embryo size and number, and live imaging of peri-implantation events is still limited. Thus, there is a critical need for an in vitro platform to model, manipulate and directly study key steps involved. Recently, we showed that aggregates of human pluripotent stem cells (hPSC) recapitulate several of these embryogenic events: they readily polarize and self-organize into radial structures, forming spheroids with a central lumen (hPSC- spheroid). This lumenal spheroid forming property, combined with the transcriptomic and epigenetic similarity of hPSC to epiblast cells in vivo, makes this hPSC-based system an attractive model system for investigation of the cellular and molecular mechanisms underlying epiblast cavity formation. Strikingly, apical polarization, radial organization and lumenogenesis in this system are driven by formation and membrane integration of an apicosome, an apically polarized membranous organelle with extracellular-like features (i.e. microvilli, primary cilium and accumulated Ca2+). To further expand the mechanistic understanding of apicosome biology, we examined the comprehensive proteome of the apicosome territory using an APEX2 (engineered ascorbate peroxidase 2)-based proximity biotinylation system, coupled with quantitative mass spectrometry. We discovered several proteins that are enriched in the apicosome territory, including proteins with known functions in vesicular trafficking and actin cytoskeletal organization (RAB35 and CDC42) as well as mTORC1 signaling (LAMTOR1/p18 and V-type proton ATPases). Our preliminary results show that these proteins are localized to the apicosome and apicosome precursor vesicles, and that the cellular and signaling processes that are governed by these proteins are involved in apicosome formation. To further investigate this, we will: 1) Explore how the small GTPase RAB35 regulates the formation and trafficking of the apicosome and establish CDC42 as a downstream effector of RAB35; 2) Examine the requirement of mTORC1 signaling in apicosome formation; 3) Determine mTORC1 function during ciliogenesis in the apicosome. Establishment of primary cilia and apicobasal cell polarity are tightly linked. Proteomic analysis reveals that SLC7 amino acid transporter proteins, including SLC7A3 (cationic amino acid transporter 3), SLC7A8 (large neutral amino acids transporter small subunit 2) and SLC7A11 (cysteine/glutamate transporter), are enriched in the apicosome territory. mTORC1 signaling was recently shown to regulate primary cilium formation downstream of SLC7A8. The work proposed here will greatly accelerate the pace of discovery regarding these essential but previously inaccessible peri-implantation events, and will have enormous implications for understanding early process that impact embryonic development and human fertility.

项目摘要 当胚胎植入子宫壁时，epiblast腔形成发生 无法在人类中实验研究。小鼠胚胎确实为遗传处理工具提供了 探索与层状腔形成相关的机制，此类研究仅限于胚胎 大小和数字以及植入周期事件的实时成像仍然有限。因此，迫切需要 在体外平台建模，操纵和直接研究涉及的关键步骤。最近，我们表明 人多能干细胞（HPSC）的聚集体概括了以下几个胚胎事件：它们 很容易地将极化并自组织成径向结构，并用中央管腔形成球体（HPSC- 球体）。这种腔体球形形成特性，结合了转录组和表观遗传相似性 HPSC到体内的epiblast细胞，使该基于HPSC的系统成为研究的有吸引力的模型系统 细胞和分子机制的基础型腔形成。令人惊讶的是，顶极化， 该系统中的径向组织和腔内发生是由形成和膜整合的驱动 Apicosom，一种具有细胞外特征的顶极化的膜细胞器（即微绒毛） 纤毛和累积的Ca2+）。为了进一步扩大对尖体生物学的机械理解，我们 使用Apex2（工程抗坏血酸盐）检查了Apicosom区域的全面蛋白质组 基于过氧化物酶2）基于定量质谱法结合的近端生物素化系统。我们 发现了几种富集在Apicosom体积的蛋白质，包括已知的蛋白质 囊泡运输和肌动蛋白细胞骨架组织（RAB35和CDC42）以及MTORC1的功能 信号传导（LAMTOR1/P18和V型质子ATPase）。我们的初步结果表明这些蛋白质是 位于尖体和尖体前体囊泡上，细胞和信号过程 由这些蛋白质控制的蛋白质与尖体形成有关。为了进一步调查，我们将：1） 探索小型GTPase Rab35如何调节apicosom的形成和贩运 Cdc42是Rab35的下游效应子； 2）检查Apicosom中MTORC1信号的需求 形成； 3）确定Apicosom中纤毛发生过程中的MTORC1功能。建立主要纤毛 和apicobasal细胞极性紧密相连。蛋白质组学分析表明SLC7氨基酸转运蛋白 蛋白质，包括SLC7A3（阳离子氨基酸转运蛋白3），SLC7A8（大中性氨基酸转运蛋白 小亚基2）和SLC7A11（半胱氨酸/谷氨酸转运蛋白）富含在Apicosom的区域中。 最近显示MTORC1信号传导调节SLC7A8下游的原发性纤毛形成。工作 在这里提出的建议将大大加速有关这些基本但以前的发现的速度 无法访问的植入周期事件，并将对理解早期过程产生巨大影响 这会影响胚胎发育和人类生育能力。