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

点击翻译按钮获取中文摘要

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.