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

项目摘要 胚胎植入子宫壁时会形成上胚层腔，这一步骤在伦理上 无法进行人体实验研究。虽然小鼠胚胎确实提供了一种遗传上易于处理的工具， 探索与上胚层腔形成相关的机制，这种研究仅限于胚胎 但是，其尺寸和数量以及植入前后事件的实时成像仍然有限。因此，迫切需要一个 体外平台来建模、操作和直接研究所涉及的关键步骤。最近，我们发现， 人多能干细胞（hPSC）的聚集体概括了这些胚胎发生事件中的几个：它们 容易折叠和自组织成放射状结构，形成具有中心腔的球状体（hPSC-1）。 球体）。这种内腔球体形成特性，结合转录组学和表观遗传学的相似性， 的hPSC的外胚层细胞在体内，使这种基于hPSC的系统有吸引力的模型系统的研究 上胚层腔形成的细胞和分子机制。引人注目的是，顶端极化， 该系统中的放射状组织和管腔形成是由一个细胞的形成和膜整合驱动的， 顶体，一种顶端极化的膜性细胞器，具有细胞外样特征（即微绒毛，初级 纤毛和积累的Ca ~（2+）。为了进一步扩展对顶体生物学的机制理解，我们 使用APEX 2（工程抗坏血酸）检测了顶体区域的全面蛋白质组 基于过氧化物酶2）的邻近生物素化系统，结合定量质谱法。我们 他发现了几种富含顶核体区域的蛋白质，包括已知的 在囊泡运输和肌动蛋白细胞骨架组织（RAB 35和CDC 42）以及mTORC 1中起作用 信号传导（LAMTOR 1/p18和V型质子ATP酶）。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囊泡，并且细胞和信号过程 与顶粒体的形成有关。为了进一步研究这一点，我们将：1） 探索小GTTRAB 35如何调节顶体的形成和运输，并建立 CDC 42作为RAB 35的下游效应子; 2）检查顶粒体中mTORC 1信号传导的需要 3）确定顶体中纤毛发生过程中mTORC 1的功能。初级纤毛的建立 和顶基细胞极性紧密相连。蛋白质组学分析表明，SLC 7氨基酸转运蛋白 蛋白质，包括SLC 7A 3（阳离子氨基酸转运蛋白3）、SLC 7A 8（大中性氨基酸转运蛋白 小亚基2）和SLC 7A 11（半胱氨酸/谷氨酸转运蛋白），富集在顶粒领域。 mTORC 1信号传导最近显示调节SLC 7A 8下游的初级纤毛形成。工作 这里提出的将大大加快发现这些基本的，但以前的步伐， 难以接近的围植入期事件，并将对理解早期过程产生巨大影响 影响胚胎发育和人类生育能力的因素。

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.