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一个透明器官，对视力至关重要。角膜基质和内皮层来源于视游走流的神经脊细胞(NCC)。NCC在其靶组织中的命运受其沿神经管嘴尾轴的位置、离开神经管后在迁移流中的位置以及其靶组织微环境中各种信号的诱导等因素的影响。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整合。实时解剖塑造角膜的细胞事件将提供巨大的洞察力，了解角膜是如何雕刻成一个功能正常的视觉器官的。我将使用4D共焦成像和光可转换荧光蛋白技术来观察这些事件，这将提供更完整的角膜发育画面。为了追踪和观察NCC的亚群，我将用KikGR光可转换荧光蛋白标记光学迁移流。使用这项技术，我将清楚地标记眼周区域的视流或其衍生品的亚群，并在它们侵入角膜时追踪它们的命运。此外，我将确定每个亚群对细胞引导分子Semaphorin3A(Sema3A)的反应，该分子已被证明影响NCC的眼睛命运。这项提议将实现以下目标：1：在视游走流和眼周区域内确定NCC的亚群。2：通过在体时间推移视频显微镜研究角膜发育过程中NCC来源的周眼细胞对晶状体来源的信号的细胞反应。3：确定晶状体近端和晶状体远端NCC来源的眼周亚群在体外对Sema3A信号的反应是否表现出不同的迁移行为。这些目标将填补我们在视觉迁移流细胞如何获得其位置识别和发育程序以形成角膜组织方面的知识空白。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，这些输入驱动其他系统中的器官发生。这些目标相互关联但又相互独立，每个目标都涉及眼球NCC走向角膜命运的一个方面。这项研究的结果将有助于阐明多种输入和细胞行为在NCC群体分化过程中的整合，并将对我们理解先天性角膜缺陷具有重要影响。此外，这项研究的结果可能会对其他NCC群体的迁移和侵袭细胞行为产生进一步的影响，导致关于这些过程的失调的新信息，这些过程可能导致头面部出生缺陷和一些癌症。

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.