Gene regulatory mechanisms required for neuronal chemosensitivity

项目摘要/摘要： 哺乳动物脑干的化学敏感区调节呼吸，并受 动脉血中的二氧化碳(CO2)水平。这种化疗敏感性的遗传和分子基础很差。 明白了。线虫是探索这一问题的极好的遗传模型，因为它对二氧化碳敏感。 神经元--袋状神经元--调节刻板行为，其细胞生理学可以在体内进行研究。 BAG细胞的命运由Ets结构域转录因子Ets-5决定。令人惊讶的是，哺乳动物的同源物 在Ets-5中，Pet1是二氧化碳化学敏感脑区发育所必需的，而Pet1突变体有 应对二氧化碳挑战时调节呼吸的缺陷。在C.中寻找二氧化碳传感中重要的分子。 在线虫和脊椎动物中，我使用芯片序列分析了ETS-5的直接转录靶标。然后，到 在BAG中阐明这些靶点中哪些是由ETS-5直接调控的，我在野生型上进行了mrna-seq 与Ets-5突变型BAG神经元的对比。要确定这些目标中哪些对二氧化碳具有重要的功能 感觉到了这一点，我进行了一次行为筛查，以确定是否存在二氧化碳规避缺陷。RGS-6是一种G蛋白激活蛋白，是一种 在Ets-5突变背景中似乎下调的直接Ets-5转录靶点，以及RGS-6 突变也会导致回避缺陷。为了更好地了解RGS-6在二氧化碳传感中的作用，我将(1) 确定RGS-6在二氧化碳传感中的表达模式、作用部位和作用时间，(2)将RGS-6放入GPCR中 以及(3)确定RGS-6在二氧化碳感应中的生理作用。拟议的研究将整合 分子遗传学、基因组学和体内功能成像阐明一种新的RGS蛋白在二氧化碳中的作用 感官。由于类Pet1因子在线虫和人类之间是保守的，我们的研究可能会澄清 大脑中化学敏感神经元功能所需的机制，这些机制在调节 呼吸功能障碍与致命的呼吸暂停有关。

PROJECT SUMMARY / ABSTRACT: Chemosensitive areas of the mammalian brainstem regulate breathing and are stimulated by small increases in carbon dioxide (CO2) levels in arterial blood. The genetic and molecular bases for this chemosensitivity are poorly understood. C. elegans is an excellent genetic model for probing this question because it possesses CO2-sensitive neurons - the BAG neurons - that mediate stereotyped behavior and whose cell physiology can be studied in vivo. BAG cell fate is determined by ETS-5, an ETS-domain transcription factor. Strikingly, the mammalian homolog of ETS-5, Pet1, is required for the development of CO2-chemosensitive brain regions, and Pet1 mutants have defects regulating breathing in response to CO2 challenges. To find molecules important in CO2 sensing in C. elegans and perhaps in vertebrates, I analyzed the direct transcriptional targets of ETS-5 using ChIP-seq. Then, to clarify which of these targets are being directly regulated by ETS-5 in BAG, I performed mRNA-seq on wild type versus ets-5 mutant BAG neurons. To determine which of these targets are functionally important for CO2 sensing, I performed a behavioral screen for CO2 avoidance defects. RGS-6 is a G-protein activating protein that is a direct ETS-5 transcriptional target that appears to be down-regulated in an ets-5 mutant background, and rgs-6 mutation also results in an avoidance defect. To better understand the role of RGS-6 in CO2 sensing, I will (1) determine the expression pattern, site, and time of action of rgs-6 in CO2 sensing, (2) place RGS-6 in a GPCR pathway, and (3) determine the physiological role of RGS-6 in CO2 sensing. The proposed studies will integrate molecular genetics, genomics, and in vivo functional imaging to elucidate the role of a novel RGS protein in CO2 sensing. Because Pet1-like factors are conserved between C. elegans and humans, our studies will likely elucidate mechanisms required for the function of chemosensitive neurons in the brain, which play critical roles in regulating breathing and whose dysfunction is linked to fatal apneas.