Intracellular pH Dynamics in Zebrafish Cranial Neural Crest Development

项目总结/摘要 细胞内pH（pHi）动态是细胞命运变化的关键调节器。我们的实验室和其他人证明了 pHi的增加对于多种细胞类型的分化和谱系特化是必要的， 包括小鼠胚胎干细胞、果蝇卵泡干细胞、小鼠肠干细胞，以及 鸡近轴中胚层。我们的实验室还展示了pHi动力学如何调节细胞粘附和迁移 通过改变pH敏感蛋白的质子化状态，包括β-catenin，cofilin，talin和focal 粘附激酶然而，pHi调节细胞命运变化的机制仍然没有得到解决。 此外，我们对体内胚胎发育过程中pHi动态的理解是有限的，主要是由于 缺乏合适的模式。为了解决这个问题，我开发了一个新的系统， 斑马鱼胚胎发育过程中pHi动态的意义 适合于胚胎的体内活细胞成像的生物体。使用这个新模型，我将确定pHi的作用 颅神经嵴（NC）发育的动力学，一种高度保守的脊椎动物胚胎细胞 这是一个能产生多种细胞类型的细胞群，包括软骨细胞、骨细胞和成牙本质细胞。许多 参与颅NC发育的细胞行为受其他细胞类型中的pHi动力学调节，包括 谱系特化、细胞迁移和上皮向间充质转化。因此，颅骨NC代表 理想的模型，以解决我们在胚胎发育过程中的pHi动力学的理解差距， vivo.通过跟踪早期NC细胞的颅谱系分化，我将测试中枢神经系统。 假设pHi动态调节颅NC发育，在分层阶段， 迁移或血统规范。在目标1中，我将解决空间和时间pHi动态， 通过活体细胞成像的斑马鱼颅NC发育。我的初步数据显示， 与迁移前的颅NC细胞相比，迁移。在目标2中，我将通过实验干扰斑马鱼的pHi NC细胞通过药理学和遗传学调节质膜离子转运蛋白， 对颅NC细胞行为和转录的影响，从而确定pHi的功能意义 斑马鱼颅NC发育过程中的动力学。我的发现有望揭示新的见解， 控制颅面发育的细胞和分子因素，对人类具有重要意义 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.