Intracellular pH Dynamics in Zebrafish Cranial Neural Crest Development

项目摘要/摘要 细胞内pH(Phi)的动态变化是细胞命运变化的重要调节因素。我们的实验室和其他人展示了 PHI的增加对于多种细胞类型的分化和谱系规范是必要的， 包括小鼠胚胎干细胞，果蝇卵泡干细胞，小鼠肠道干细胞，以及 鸡旁轴中胚层。我们的实验室还展示了phi动力学如何调节细胞黏附和迁移。 通过改变pH敏感蛋白的质子化状态的行为，包括-连环蛋白、粘连蛋白、Talin和焦点 黏附蛋白激酶。然而，phi调控细胞命运变化的潜在机制仍未解决。 此外，我们对体内胚胎发育过程中的phi动态的了解是有限的，这主要是由于 缺乏合适的模型。为了解决这个问题，我生成了一个新的系统来审问 遗传易感性斑马鱼体内胚胎发育过程中PHI动态变化的意义 适合活体内胚胎细胞成像的有机体。使用这个新模型，我将确定Phi的角色 高度保守的脊椎动物胚胎细胞--颅神经脊发育的动力学研究 产生多种细胞类型的群体，包括软骨细胞、骨细胞和成牙本质细胞。许多 参与颅骨NC发育的细胞行为在其他类型的细胞中受到phi动力学的调节，包括 谱系指定，细胞迁移，上皮细胞向间充质细胞转变。因此，颅骨NC代表一种 理想的模型来解决我们在胚胎发育过程中对PHI动态的理解上的差距 活着。通过跟踪早期NC细胞通过颅骨谱系的分化，我将测试中央 在分层阶段，PHI动力学调节颅骨NC发育的假说， 迁徙，或世系规范。在目标1中，我将解析空间和时间Phi动力学 活体细胞成像技术研究斑马鱼颅骨发育。我的初步数据显示， 与迁移前相比，迁移的颅骨NC细胞。在目标2中，我将在实验中干扰斑马鱼的phi NC细胞通过药理和遗传调控质膜离子转运蛋白并确定 对颅骨NC细胞行为和转录的影响，从而确立了Phi的功能意义 斑马鱼颅骨NC发育过程中的动力学。我的发现有望揭示出关于 控制颅面发育的细胞和分子因素及其对人类的重要意义 先天性疾病和组织修复。

PROJECT SUMMARY/ABSTRACT Intracellular pH (pHi) dynamics are a critical regulator of cell fate changes. Our lab and others demonstrated that an increase in pHi is necessary for the differentiation and lineage specification of multiple cell types, including mouse embryonic stem cells, Drosophila ovarian follicle stem cells, mouse intestinal stem cells, and chick paraxial mesoderm. Our lab has also shown how pHi dynamics regulate cell adhesion and migration behaviors by modifying the protonation state of pH-sensing proteins, including -catenin, cofilin, talin, and focal adhesion kinase. However, the mechanisms underlying pHi regulation of cell fate changes remain unresolved. Furthermore, our understanding of pHi dynamics during embryonic development in vivo is limited, largely due to lack of appropriate models. To address this, I generated a novel system for interrogating the functional significance of pHi dynamics during embryonic development in vivo using zebrafish, a genetically tractable organism suited for in vivo live cell imaging of embryos. Using this new model, I will determine the role of pHi dynamics in the development of cranial neural crest (NC), a highly conserved vertebrate embryonic cell population that gives rise to diverse cell types, including chondrocytes, osteocytes, and odontoblasts. Many cell behaviors involved in cranial NC development are regulated by pHi dynamics in other cell types, including lineage specification, cell migration, and epithelial to mesenchymal transition. Thus, cranial NC represents an ideal model for addressing the gaps in our understanding of pHi dynamics during embryonic development in vivo. By following early NC cells through the differentiation of cranial lineages, I will test the central hypothesis that pHi dynamics regulate cranial NC development, at the stage of delamination, migration, or lineage specification. In Aim 1, I will resolve spatial and temporal pHi dynamics during zebrafish cranial NC development by in vivo live cell imaging. My preliminary data indicate a higher pHi in migratory compared with premigratory cranial NC cells. In Aim 2, I will experimentally perturb pHi in zebrafish NC cells through pharmacologic and genetic modulation of plasma membrane ion transporters and determine the effect on cranial NC cell behaviors and transcription, thus establishing the functional significance of pHi dynamics during zebrafish cranial NC development. My findings have promise to reveal new insight on the cellular and molecular factors controlling craniofacial development, with important implications for human congenital diseases and tissue repair.