Live Imaging of Neural Crest Contribution to the Developing Cornea

描述（由申请人提供）：角膜是眼睛前部的透明器官，对视力至关重要。角膜基质层和内皮层来源于视神经迁移流的神经嵴细胞（NCC）。NCC在其靶组织中的命运受其沿神经管的头尾轴的沿着位置、一旦它们离开神经管它们在迁移流内的位置以及来自其靶组织的微环境内的各种信号的诱导的影响。这些输入中有许多在时间上重叠;因此，NCC规格可能依赖于这些影响的整合。在真实的时间内解剖塑造角膜的细胞事件将为角膜如何塑造成功能性视觉器官提供巨大的洞察力。我将采用4D共聚焦成像和光转换荧光蛋白技术来观察这些事件，这将提供一个更完整的角膜发育的图片。为了追踪和观察NCC的亚群，我将用KikGR光转换荧光蛋白标记光学迁移流。利用这项技术，我将清楚地标记眼周区域的视流或其衍生物的亚群，并跟踪它们侵入角膜时的命运。此外，我将确定每个亚群对细胞导向分子Semaphorin 3A（Sema 3A）的反应，Semaphorin 3A已被证明会影响NCC的眼部命运。该提案将承担以下目标：1：定义在视神经迁移流和眼周区域内的NCC亚群。第二章：通过体内延时视频显微镜确定角膜发育过程中NCC衍生的眼周细胞对晶状体衍生信号的细胞反应。第三章：确定晶状体近端和晶状体远端NCC衍生的眼周亚群是否在体外响应Sema 3A信号传导时表现出差异迁移行为。这些目标将填补我们对来自视神经迁移流的细胞如何获得其位置身份和发育程序以产生角膜组织的知识空白。这些发现将为理解多个输入到单个细胞的整合提供一个起点，这些细胞驱动其他系统中的器官发生。这些目标是相关但独立的，并且每个目标都解决了光学NCC向角膜命运进展的一个方面。本研究的结果将揭示NCC群体分化过程中多种输入和细胞行为的整合，并将对我们对先天性角膜缺陷的理解产生重要影响。此外，这项研究的结果可能对其他NCC群体的迁移和侵入性细胞行为产生进一步的影响，从而获得有关这些过程失调的新信息，这些过程可能导致颅面出生缺陷和某些癌症。 公共卫生相关性：角膜是一种透明的、无血管的、高度神经支配的组织，位于眼睛的前部;该器官的正常发育对视力至关重要，异常发育可导致视力丧失或失明。角膜基质和内皮层由源自神经嵴（NCC）的细胞组成，神经嵴是起源于神经管并产生包括角膜在内的许多器官的短暂迁移群体。已知NCC的光流对角膜有贡献，但尚不清楚NCC如何或何时被指定为它们的最终命运，这是角膜发育中的关键事件。在这里，我建议使用真实的时间4D共聚焦成像和光转换荧光蛋白来弥合这一知识差距，以确定NCC及其衍生物的亚群，因为它们与角膜的命运。

DESCRIPTION (provided by applicant): The cornea is a transparent organ at the anterior of the eye which is critical to visual acuity. Corneal stroma and endothelia layers are derived from neural crest cells (NCCs) of the optic migratory stream. The fate of NCCs in their target tissue is influenced by their position along the rostro-caudal axis of the neural tube, their position within the migratory stream once they leave the neural tube, and induction from various signals within the microenvironments of their target tissues. Many of these inputs overlap in time; as such NCC specification likely relies on the integration of these influences. Dissecting the cellular events that shape the cornea in real time will provide tremendous insight into how the cornea is sculpted into a functional visual organ. I will employ 4D confocal imaging and photoconvertible fluorescent protein technology to observe these events, which will provide a more complete picture of corneal development. In order to track and observe subpopulations of NCCs, I will label the optic migratory stream with the KikGR photoconvertible fluorescent protein. Using this technology I will distinctly label subpopulations of the optic stream or their derivatives in the periocular region and track their fate as they invade the cornea. In addition, I will determine the response of each subpopulation to the cell guidance molecule Semaphorin3A (Sema3A) that has been shown to influence the ocular fate of NCCs. This proposal will undertake the following aims: 1: Define sub-populations of NCCs within the optic migratory stream and periocular region. 2: Define the cellular response of NCC-derived periocular cells to lens-derived signaling during cornea development by in vivo time-lapse video microscopy. 3: Determine whether lens-proximal and lens-distal NCC-derived periocular subpopulations exhibit differential migratory behavior in response to Sema3A signaling in vitro. These aims will fill gaps in our knowledge of how cells from the optic migratory stream acquire their positional identity and developmental program to give rise to corneal tissues. These findings will provide a jumping-off point for understanding the integration of multiple inputs to individual cells, which drive organogenesis in other systems. These aims are related but independent and each address an aspect of optic NCC progression toward a corneal fate. Findings from this study will shed light on the integration of multiple inputs and cellular behaviors during differentiation of NCC populations and will have important impacts on our understanding of congenital corneal defects. In addition, results from this study may have further reaching implications for migration of other NCC populations and invasive cell behaviors, leading to new information about dysregulation of these processes which can lead to craniofacial birth defects and some cancers. PUBLIC HEALTH RELEVANCE: The cornea is a transparent, avascular, and highly innervated tissue occupying the anterior position of the eye; proper development of this organ is critical to vision, and aberrant development can result in loss of visual acuity or blindness. The corneal stroma and endothelial layers are comprised of cells derived from the neural crest (NCCs), a transient migratory population that originates in the neural tube and gives rise to many organs, including the cornea. The optic stream of NCCs is known to contribute to the cornea, but it is not clear how or when the NCCs are specified toward their ultimate fate, a key event in cornea development. Here I propose to bridge this knowledge gap using real time 4D confocal imaging and photoconvertible fluorescent proteins to identify subpopulations of NCCs and their derivatives as they relate to corneal fate.