Molecular Mechanisms of Cerebral Cortical Patterning - Resubmission 01

描述（由申请人提供）：拟议研究的长期目标是了解启动哺乳动物大脑皮层发育的机制，并控制新皮层区域图的形成。研究结果应该与一系列源于大脑皮层发育缺陷的人类疾病有关。这一提议有三个目的，它基于信号分子成纤维细胞生长因子（FGF） 8在胚胎小鼠新皮质原基（NP）中作为渐变形态原，从而启动基本NP模式的证据。引人注目的是，NP中的异位FGF8可以诱导重复区域，甚至复杂的地图。目的1验证了FGF8启动一系列指定区域图的基因调控事件的假设。系统搜索FGF8下游在皮层区域化中起作用的基因将使用下一代深度测序（RNA-Seq）来比较暴露于不同浓度FGF8的NP组织的转录组，模拟FGF8的自然梯度，或新解剖的NP部分，代表不同的推定新皮层类型。二次筛选将寻找具有区域性NP表达的候选人。目的2将研究FGF8如何与FGF受体（fgfr）和潜在的内源性抑制剂相互作用，以控制正常区域模式的发展。野生型或突变型fgfr将通过子宫内电穿孔引入胚胎NP的不同区域。在适当的存活时间后，将检查大脑对FGF8扩散和信号传导以及成熟区域图组织的影响。一些前额叶区域来源于FGF8来源内的祖细胞，而且，最有可能的是，来自一个更大的FGF17来源，即FGF8家族成员。命运映射实验将研究FGF8和FGF17的巢状来源是否会产生前额皮质的巢状区域。Aim 3建立在一个新发现的基础上，即皮层投射神经元的主要类别，嗅球（OB）的二尖瓣细胞，也由FGF8/17源内的祖细胞产生。实验将确定FGF8/17信号传导是否诱导OB原基（一个有边界的皮质区域），证实FGF8/17驱动二尖瓣细胞发育，并鉴定FGF8/17下游特异性二尖瓣细胞的转录因子。

DESCRIPTION (provided by applicant): Longterm goals of the proposed research are to understand the mechanisms that initiate development of mammalian cerebral cortex, and control formation of the neocortical area map. Findings should be relevant to a range of human disorders that stem from developmental defects in cerebral cortex. This proposal, which has three aims, is based on evidence that the signaling molecule Fibroblast Growth Factor (FGF) 8 acts as a graded morphogen in the embryonic mouse neocortical primordium (NP), thereby initiating basic NP patterning. Strikingly, ectopic FGF8 in the NP can induce duplicate areas, and even complex maps. Aim 1 tests the hypothesis that FGF8 initiates a series of gene regulatory events that specifies the area map. A systematic search for genes downstream of FGF8 that have a role in cortical regionalization will use next generation deep sequencing (RNA-Seq) to compare transcriptomes of NP tissue exposed to different concentrations of FGF8 in vitro, mimicking the natural gradient of FGF8, or freshly dissected parts of the NP, representing different presumptive types of neocortex. Secondary screening will look for candidates with regional NP expression. Aim 2 will investigate how FGF8 interacts with FGF receptors (FGFRs), and a potential endogenous inhibitor, to control development of the normal area pattern. Wild type or mutant FGFRs will be introduced into different regions of the embryonic NP with in utero electroporation. After appropriate survival times, brains will be examined for effects on FGF8 diffusion and signaling, and mature area map organization. Some prefrontal areas derive from progenitors inside the source of FGF8, and, most likely, inside a larger source of FGF17, an FGF8 family member. Fate-mapping experiments will investigate if the nested sources of FGF8 and FGF17 give rise to nested areas of prefrontal cortex. Aim 3 builds on a new finding, namely that a major class of cortical projection neurons, mitral cells of the olfactory bulb (OB), is also generated by progenitors inside the FGF8/17 source. Experiments are proposed to determine if FGF8/17 signaling induces the OB primordium, a bounded cortical region, confirm that FGF8/17 drive mitral cell development, and identify transcription factors downstream of FGF8/17 that specify mitral cells.