A combinatory microfluidic and in vivo modeling approach to evaluate collective migration during retinogenesis

细胞群的协调迁移是组织发育和神经系统干细胞修复的核心要素。在一个理想的模型中，干细胞样细胞（stlc）被引入到受损组织中，并作为一个单位集体迁移到精确的损伤部位，以重建神经元连接。事实上，来自细胞基因组成及其外部环境的线索对集体迁移的影响只得到了部分探索。发育中的视网膜提供了一个独特的机会来定量研究集体迁移，以支持视力的自然发展。由于不同物种之间引导视网膜发育的信号线索惊人地相似，常见的果蝇（Drosophila melanogaster）为研究这一现象提供了一个简单而优秀的模型。研究stlc如何自然迁移以启动或重新启动神经元与视网膜的连接将大大加深我们对视网膜发育的理解，并可以大大推进恢复视力的治疗。教育方面的努力将通过建立整合的课程模块，让学生团队参与设备原型设计、设计创新和基于细胞的实验室实验，为本科生提供教授和指导暑期高中生视网膜研究的机会。该项目将评估stlc的集体迁移，利用体内遗传学来调节黑腹果蝇（作为模型）的细胞内成纤维细胞生长因子受体（FGF- r）信号和微流体系统来控制外部FGF环境。这项研究的动机是，已知果蝇模型中的FGF信号通路介导通过视柄启动视觉所需的神经迁移，但尚不清楚FGF- r单独调节是否足够。该系统将以微米分辨率促进fgf - r介导的趋化性的遗传控制研究，同时定量研究维持可移动STLC集体的空间凝聚力所需的细胞间信号（通过innexin-1的细胞-细胞粘附）。研究计划分为三个目标。AIM 1将开发视网膜视柄（发育中的视网膜在果蝇脑叶和眼成像盘之间的部分）的微流体模型，以产生可控的外源性FGF场。实验将在果蝇视网膜的规模上建立一个体外系统的原型，并开发一个分析模型（有限元模拟）来描述其中的浓度梯度场。集体体外迁移的特征将通过有针对性的定量参数进行评估。AIM 2将检查表达FGF-R基因修饰元件的胶质细胞和神经元细胞的集体迁移，即具有和不具有功能受体的细胞。基因FGF-R的改变将用于实验确定FGF-R对微流体系统中运动集体的大小以及它们的神经组成和细胞-细胞粘附的贡献。AIM 3将使用转基因FGF-R胶质和神经元集体评估体内视网膜形成。在体内集体迁移过程中，FGF-R的具体作用将通过使用缺乏FGF-R功能的神经集体检查视网膜发生来研究。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

The coordinated migration of groups of cells is a central element of tissue development as well as of stem cell-based repair in the nervous system. In an idealized model, stem-like cells (STLCs) are introduced into a damaged tissue and migrate collectively, as one unit, toward precise injury sites to reestablish neuronal connectivity. In truth, the effects of cues from a cell's genetic makeup and its external environment on collective migration have been only partially explored. The developing retina provides a unique opportunity for quantitative study of collective migration to support the natural development of vision. As the signaling cues that guide retinal development are surprisingly similar among different species, the common fruit fly (Drosophila melanogaster) provides a simple yet excellent model to study this phenomenon. The study of how STLCs naturally migrate to initiate or re-initiate connectivity of the neurons with the retina will greatly deepen our understanding of retinal development and could greatly advance therapies to restore vision. Educational efforts will develop opportunities for undergraduates to teach and mentor summer high school students in retinal research by establishing integrated course modules that engage student teams in device prototyping, design innovation and cell-based laboratory experiments.This project will evaluate the collective migration of STLCs using in vivo genetics to regulate intracellular Fibroblast Growth Factor Receptor (FGF-R) signaling in Drosophila melanogaster (as a model) and microfluidic systems to control extrinsic FGF environments. The study is motived by knowledge that FGF signaling pathways in the Drosophila model are known to mediate the neural migration needed to initiate vision via the optic stalk, but it is not clear if FGF-R regulation alone is sufficient. The system will facilitate genetically-controlled study of FGF-R-mediated chemotaxis with micrometer resolution in tandem with quantitative study of the intercellular signaling needed to preserve spatial cohesion across motile STLC collectives (cell-cell adhesion via innexin-1). The Research Plan is organized under 3 aims. AIM 1 will develop a microfluidic model of the retinal optic stalk (the portion of the developing retina between the Drosophila Brain Lobe and the Eye Imaginal Disc) to generate controlled, extrinsic FGF fields. Experiments will prototype an in vitro system on the scale of the Drosophila retina and develop an analytical model (Finite Element simulation) to describe concentration gradient fields therein. Characteristics of collective in vitro migration will be evaluated by targeted, quantitative parameters. AIM 2 will examine the collective migration of glial and neuronal cells expressing genetically-modified elements of FGF-R, i.e., cells with and without functional receptors. Genetic FGF-R alteration will be used to experimentally determine contribution of FGF-R to the size of motile collectives as well as their neural composition and cell-cell adhesion within the microfluidic system. AIM 3 will evaluate in vivo retinal formation using genetically modified FGF-R glial and neuronal collectives. The specific role for FGF-R during collective migration in vivo will be studied by examining retinogenesis using neural collectives lacking FGF-R function.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.