Live Imaging of Neural Crest Contribution to the Developing Cornea

描述（申请人提供）：角膜是位于眼睛前部的透明器官，对视力至关重要。角膜基质和内皮层来源于视神经迁移流的神经嵴细胞（NCCs）。NCCs在靶组织中的命运受到以下因素的影响：它们沿神经管上尾轴的位置、离开神经管后在迁移流中的位置以及靶组织微环境中各种信号的诱导。许多这些输入在时间上是重叠的；因此，NCC规范可能依赖于这些影响的集成。解剖实时形成角膜的细胞事件将为了解角膜如何被塑造成一个功能性视觉器官提供巨大的见解。我将使用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图像。为了跟踪和观察NCCs亚群，我将用KikGR光转换荧光蛋白标记光迁移流。使用这项技术，我将在眼周区域清楚地标记视光流亚群或其衍生物，并在它们侵入角膜时跟踪它们的命运。此外，我将确定每个亚群对细胞引导分子Semaphorin3A （Sema3A）的反应，该分子已被证明会影响ncc的眼部命运。该提案将实现以下目标：1：确定光学迁移流和眼周区域内的ncc亚群。2：通过体内延时视频显微镜确定角膜发育过程中nc源性眼周细胞对晶状体源性信号的细胞反应。3：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对Sema3A信号的响应中表现出不同的迁移行为。这些目标将填补我们对来自光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。这些发现将为理解单个细胞的多种输入的整合提供一个起点，这些输入驱动了其他系统中的器官发生。这些目标是相互关联但又独立的，每个目标都涉及角膜NCC进展的一个方面。本研究结果将揭示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，本研究的结果可能对其他NCC群体的迁移和侵袭性细胞行为有进一步的影响，从而为这些过程的失调提供新的信息，这些过程可能导致颅面出生缺陷和某些癌症。

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.