Cellular Biomechanics of Heat-Shock Induced Defects in Drosophila Embryogenesis

描述(由申请人提供)：胚胎发育早期的非特定环境应激，如热休克、缺氧或化学暴露，可导致显著特定的发育缺陷。例如，在人类和其他哺乳动物中，暴露在极端高温下会导致神经管缺陷，暴露在酒精中可能会导致一系列特有的缺陷，在最严重的情况下表现为胎儿酒精综合征。该项目专注于在果蝇模型系统中的类似效应，在该模型系统中，发育缺陷被称为物候学，因为它们有能力模仿特定基因突变的表型。这些表型缺陷中的许多是由于环境应激对整个胚胎中细胞命运规范的模式的影响而产生的；然而，其他的原因是在细胞致力于特定的细胞命运之后发生的应激。在后一种情况下，缺陷是由于承诺细胞通过细胞和组织水平的生物力学的时空调节来驱动形态发生的过程中的错误造成的。本项目将侧重于这种效应的一个例子--胚胎发育原肠形成阶段的热休克，它会在大约4-5小时后导致生殖带回缩阶段的发育缺陷。该项目试图解释热休克引起的异常细胞行为和细胞分裂模式是如何导致组织生物力学改变，从而导致发育缺陷的。使用的方法包括对GFP标记的果蝇胚胎进行时移共聚焦成像，激光显微外科手术探测体内生物力学，激光诱导胚胎内部局部区域的热休克，以及一种名为视频力显微镜(VFM)的先进计算方法，该方法使用逆有限元方法从运动/变形组织的时移图像中推断细胞力。这个项目的四个具体目标是：1.评估羊膜浆膜中额外的第14个有丝分裂--一种在胚胎发育中起关键机械作用的组织--与随后的生殖带收缩缺陷之间的相关性2.使用仅传递到假定的羊膜浆膜细胞的局部激光诱导的热休克来证实这种相关性的特异性3.使用视频力显微镜(VFM)评估细胞和组织水平的力被改变，以及这些改变的力是如何导致生殖带收缩缺陷的方法使用视频力显微镜(VFM)4.为了验证VFM获得的动态力图，并在完成后用激光显微手术挑战我们的机械假说，这四个目标的结果应该描绘出从早期胚胎发育的环境压力到后来的发育缺陷的生物力学链的最完整图景。 与公共卫生相关：在胚胎发育早期，广泛的环境压力--例如过热、氧气不足和酒精摄入--可能会产生有害影响，导致严重的发育异常。本项目的目标是研究黑腹果蝇模型系统中的热休克，以便从根本上了解早期胚胎发育过程中短暂的环境应激如何影响胚胎内关键的细胞和组织水平的力量，并导致畸形缺陷。

DESCRIPTION (provided by applicant): Non-specific environmental stress in early embryogenesis, e.g. heat shock, anoxia or chemical exposure, can lead to remarkably specific developmental defects. For example, in humans and other mammals, exposure to extreme heat can lead to neural tube defects, and exposure to alcohol can lead to a characteristic spectrum of defects, in the most severe cases characterized as fetal alcohol syndrome. The project focuses on similar effects in the model system of Drosophila melanogaster in which the developmental defects have been termed phenocopies for their ability to mimic the phenotypes of specific genetic mutations. Many of these phenocopy defects arise due to the effects of environmental stress on the patterning of cell fate specifications across the embryo; however, others arise from stresses that occur after cells have committed to specific cell fates. In the latter case, the defect is caused by errors in the process by which committed cells drive morphogenesis through spatiotemporal regulation of cell- and tissue-level biomechanics. This project will focus on one example of this effect - heat shocks during the gastrulation stage of embryogenesis that lead to developmental defects some 4-5 hours later during the germband-retraction stage. The project seeks to explain how aberrant cell behaviors and cell division patterns induced by the heat shock later lead to altered tissue biomechanics and hence a developmental defect. Methods to be used include time-lapse confocal imaging of GFP-labeled Drosophila embryos, laser-microsurgery to probe the biomechanics in vivo, laser-induced heat shocks of local regions within the embryos, and an advanced computational method called video force microscopy (VFM) that uses an inverse finite element method to infer cellular forces from time-lapse images of moving/deforming tissues. The four specific aims of this project are: 1. To evaluate the correlation between an extra 14th mitosis in the amnioserosa - a tissue which plays a critical mechanical role in embryogenesis - and subsequent defects in germband retraction 2. To confirm the specificity of this correlation using local, laser-induced heat shocks delivered only to cells of the presumptive amnioserosa 3. To evaluate the cell- and tissue-level forces that are altered by heat-shock and the means by which these altered forces lead to defects in germband retraction using video force microscopy (VFM) 4. To validate the dynamic force maps obtained by VFM and challenge our mechanical hypothesis with laser-microsurgery Once complete, the results from these four aims should paint the most complete picture available for the biomechanical chain of events that leads from an environmental stress in early embryogenesis to a later developmental defect. PUBLIC HEALTH RELEVANCE: During early embryonic development, a wide range of environmental stresses - e.g., excess heat, insufficient oxygen, and alcohol ingestion - can have deleterious effects that lead to severe developmental abnormalities. The goal of our project is to investigate heat shocks in the model system of Drosophila melanogaster so as gain a fundamental understanding of how a short exposure to environmental stress in early embryogenesis affects critical cell- and tissue-level forces within the embryo and causes a malformation defect.