Molecular Mechanisms of Cerebral Cortical Patterning - Resubmission 01

描述(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，并控制新皮质区域图的形成。这些发现应该与一系列人类疾病相关，这些疾病源于大脑皮层发育缺陷。这一建议有三个目的，基于信号分子成纤维细胞生长因子8在胚胎小鼠新皮质原基(NP)中作为分级形态因子，从而启动基本NP模式的证据。值得注意的是，NP中的异位FGF8可以诱导重复的区域，甚至是复杂的地图。AIM 1验证了这样的假设，即FGF8启动了一系列指定区域地图的基因调控事件。对FGF8下游在皮质区域化中起作用的基因的系统搜索将使用下一代深度测序(RNA-Seq)来比较体外暴露于不同浓度FGF8的NP组织的转录，模仿FGF8的自然梯度，或新解剖的NP部分，代表不同类型的新皮质。二次筛查将寻找具有地区性NP表达的候选人。目的2研究成纤维细胞生长因子8(FGF8)如何与成纤维细胞生长因子受体(FGFRs)和潜在的内源性抑制物相互作用，以控制正常区域模式的发展。野生型或突变型FGFRs将通过宫内电穿孔被引入胚胎NP的不同区域。在适当的存活时间后，将检查大脑对FGF8扩散和信号传递以及成熟区域地图组织的影响。一些前额叶区域来自FGF8来源内的祖细胞，很可能是在FGF17的更大来源内，FGF17是FGF8家族成员。命运图实验将调查FGF8和FGF17的嵌套来源是否会导致前额叶皮质的嵌套区。AIM 3建立在一项新发现的基础上，即一类主要的皮质投射神经元，即嗅球(OB)的二尖瓣细胞，也是由FGF8/17来源内的前体细胞产生的。实验旨在确定FGF8/17信号是否诱导OB原基(一个有界的皮质区域)，确认FGF8/17驱动二尖瓣细胞的发育，并确定FGF8/17下游指定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.