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

项目总结 当胚胎植入子宫壁时，就会形成上胚腔，这一步符合伦理道德 无法在人体上进行实验研究。虽然小鼠胚胎确实提供了一种遗传上易于处理的工具 探索与上胚腔形成相关的机制，此类研究仅限于胚胎。 植入围植入期事件的大小和数量以及实时成像仍然有限。因此，迫切需要一种 体外实验平台，用于建模、操作和直接研究所涉及的关键步骤。最近，我们展示了 人类多能干细胞聚集体(HPSC)概括了这些胚胎起源事件中的几个：它们 容易极化和自组织成放射状结构，形成具有中心管腔的球体(hPSC- 椭球体)。这种腔内球体的形成特性，结合转录和表观遗传的相似性 体内hPSC对上皮细胞的作用，使基于hPSC的系统成为一个有吸引力的研究模型系统 上胚腔形成的细胞和分子机制。令人惊讶的是，顶端极化， 该系统中的放射状组织和管腔形成是由An的形成和膜整合驱动的。 顶体，顶端极化的膜细胞器，具有细胞外特征(如微绒毛，初级 纤毛和积累的钙)。为了进一步扩大对顶体生物学的机理理解，我们 使用APEX2(工程抗坏血酸)检测了顶体区域的综合蛋白质组 基于过氧化物酶2)的邻近生物素化系统，与定量质谱联用。我们 发现了几种富含在顶体区域的蛋白质，包括已知的 囊泡运输和肌动蛋白细胞骨架组织的功能(RAB35和CDC42)以及mTORC1 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 定位于顶体和顶体前体小泡，细胞和信号过程 由这些蛋白质控制的蛋白质参与了顶体的形成。为进一步调查此事，我们将：1) 探索小GTP酶RAB35如何调节顶体的形成和运输，并建立 CDC42作为RAB35下游效应器；2)研究顶体对mTORC1信号的需求 3)测定顶体纤毛发生过程中mTORC1的功能。初生纤毛的建立 和顶基细胞的极性紧密相连。蛋白质组学分析表明SLC7氨基酸转运体 蛋白质，包括SLC7A3(阳离子氨基酸转运体3)、SLC7A8(大的中性氨基酸转运体 小亚基2)和SLC7A11(半胱氨酸/谷氨酸转运蛋白)在顶体区域富含。 最近发现mTORC1信号调节SLC7A8下游初级纤毛的形成。这项工作 在这里提出的建议将大大加快发现这些基本的但以前 难以接近的围植入期事件，这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。

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.