Integrating physiological and genetic mechanisms of body size regulation

描述(申请人提供)：身体大小深刻影响着动物生物学的许多方面，但它仍然是发育生物学中尚未解决的基本问题之一。全变态昆虫--研究动物大小调节的主要模型--不会作为成虫生长，因此幼虫开始变态的大小决定了它们的成虫大小。在全变态昆虫幼虫中，停止生长和变态的决定是在一个特定的重量上实现的，称为临界重量。达到临界体重会引发荷尔蒙的级联反应，最终导致蜕皮激素的合成和释放，蜕皮激素是一种协调幼虫蜕皮和变态所需的发育事件的激素。临界体重的现象已经观察了几十年，最近的研究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出现了明显的差距。我假设，当幼虫在幼虫发育到一龄时，相对于供应结构的组织生长会产生内部缺氧，而内部缺氧是一种生理信号。 这启动了蜕皮和变态的荷尔蒙级联反应。此外，我假设氧对临界体重的影响是通过低氧与调节蜕皮激素分泌的已知途径(胰岛素/IGF、TOR和Ras/Raf/MAPK)的相互作用来调节的。我将使用生理学、形态学和分子遗传学的方法来研究这个问题。首先，我将测试在低氧条件下临界体重的减少是否反映了蜕皮激素信号的基础生理学的转变。我将通过在整个果蝇上使用Western blotting来测量HIF-1(缺氧诱导因子)蛋白，并通过使用GFP-Report构建的果蝇来评估不同细胞类型的HIF-1信号，来测试幼虫是否在幼虫后期变得缺氧。我将使用HIF-1信号正常或不活跃的果蝇，探索HIF-1信号在常氧和低氧条件下调节临界体重中的作用。总之，这些实验将测试内部缺氧是否是果蝇大小感知和变态启动的关键线索。这项研究有可能解释一些现象，比如为什么包括人类在内的许多物种在高海拔(低氧条件)下身体较小。通过阐明低氧在正常生长和大小调节中的作用，这项研究也将阐明低氧调节癌症肿瘤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.