Integrating physiological and genetic mechanisms of body size regulation

描述（由申请人提供）：身体大小深刻地影响动物生物学的许多方面，但它仍然是发育生物学的基本未解决的问题之一。全变态昆虫是研究动物体型调节的主要模型，它们不像成虫那样生长，因此幼虫开始变态的大小决定了它们成虫的大小。在全变态昆虫幼虫中，停止生长和变态的决定是在特定的重量下实现的，称为临界重量。达到临界体重启动激素级联反应，最终导致蜕皮激素的合成和释放，蜕皮激素是协调幼虫蜕皮和变形所需的发育事件的激素。临界体重的现象已经被观察了几十年，最近的研究已经阐明了调节蜕皮类固醇合成的信号通路。然而，幼虫用来感知其大小并激活这些信号通路的机制在很大程度上是未知的。其结果是我们对调节身体大小的机制的理解存在明显的差距。 我假设，随着幼虫在一个龄期中的生长，组织相对于供给结构的生长产生了内部缺氧，而内部缺氧是一种生理线索 引发蜕皮和变态的激素级联。此外，我推测，氧气对临界体重的影响是由缺氧与已知的调节蜕皮激素分泌的途径（胰岛素/IGF，TOR和Ras/Raf/MAPK）的相互作用介导的。我将用生理学、形态学和分子遗传学的方法来研究这个问题。首先，我将测试在缺氧条件下临界体重的降低是否反映了蜕皮激素信号传导的潜在生理学变化。我将测试幼虫是否成为缺氧后，在龄期通过测量HIF-1（缺氧诱导因子）蛋白使用蛋白质印迹法对整个苍蝇，并通过评估HIF-1信号在不同的细胞类型使用苍蝇GFP报告结构。我将探讨HIF-1信号在调节临界体重在常氧和缺氧条件下的作用，使用苍蝇，其中HIF-1信号是组成性活跃或缺席。 总之，这些实验将测试内部缺氧是否是在果蝇的大小感应和变态启动的关键线索。这项研究有可能解释为什么包括人类在内的许多物种在高海拔（低氧条件）下体型较小等现象。通过阐明缺氧在生长和大小的正常调节中的作用，本研究还将阐明缺氧调节癌症肿瘤的病理生长和大小的机制。

DESCRIPTION (provided by applicant): Body size profoundly affects many aspects of animal biology, yet it remains one of the fundamental unsolved problems of developmental biology. Holometabolous insects - the primary model for the study of size regulation in animals - do not grow as adults, so the size at which larvae initiate metamorphosis determines their adult size. In holometabolous insect larvae, the decision to stop growing and metamorphose is attained at a particular weight, called the critical weight. Attainment of critical weight initiates a hormonal cascade that ultimately results in the synthesis and release of ecdysone, the hormone that coordinates the developmental events necessary for a larva to molt and metamorphose. The phenomenon of the critical weight has been observed for decades, and more recent research has elucidated the signaling pathways that regulate the synthesis of ecdysteroids. However, the mechanisms that a larva uses to sense its size and activate these signaling pathways are largely unknown. The result is a conspicuous gap in our understanding of the mechanisms that regulate body size. I hypothesize that, as larvae grow through an instar, the growth of tissues relative to supply structures creates internal hypoxia, and internal hypoxia is a physiological cue that initiates the hormonal cascade for molting and metamorphosis. Further, I hypothesize that oxygen effects on critical weight are mediated by hypoxia's interaction with the known pathways (insulin/IGF, TOR and Ras/Raf/MAPK) that regulate ecdysone secretion. I will investigate this question using physiological, morphological and molecular-genetic methods. First I will test whether the reduction in the critical weight in hypoxic conditions reflects a shift in the underlyig physiology of ecdysone signaling. I will test whether larvae become hypoxic later in the instar by measuring HIF-1 (hypoxia inducible factor) proteins using western blotting on whole flies, and by assessing HIF-1 signaling in different cell types using flies with GFP-reporter constructs. I will explore the role of HIF-1 signaling in regulating critical weight in normoxic and hypoxic conditions, using flies in which HIF-1 signaling is constitutively active or absent. Together, these experiments will test whether internal hypoxia is a critical cue in size-sensing and metamorphosis initiation in Drosophila. This study has the potential to explain phenomena such as why many species, including humans, have smaller body sizes at high altitude (low oxygen conditions). By elucidating the role of hypoxia in the normal regulation of growth and size, this study will also shed light on the mechanisms by which hypoxia regulates the pathological growth and size of cancer tumors.