Cellular Biomechanics of Heat-Shock Induced Defects in Drosophila Embryogenesis

描述（由申请人提供）：胚胎发生早期的非特异性环境胁迫，如热休克、缺氧或化学物质暴露，可导致非常特异性的发育缺陷。例如，在人类和其他哺乳动物中，暴露于极端高温可导致神经管缺陷，暴露于酒精可导致一系列特征性缺陷，在最严重的情况下表现为胎儿酒精综合症。该项目的重点是在黑胃果蝇模型系统中的类似效应，其中发育缺陷被称为表型，因为它们能够模仿特定基因突变的表型。许多这些表型缺陷是由于环境压力对胚胎细胞命运规范模式的影响而产生的；然而，另一些则是由于细胞已承诺特定的细胞命运后发生的压力。在后一种情况下，缺陷是由细胞通过细胞和组织水平生物力学的时空调节驱动形态发生的过程中的错误引起的。本项目将重点研究这种影响的一个例子——胚胎发生的原肠胚期的热冲击，在大约4-5小时后的德国收缩期导致发育缺陷。该项目试图解释由热休克引起的异常细胞行为和细胞分裂模式如何导致组织生物力学的改变，从而导致发育缺陷。使用的方法包括对gfp标记的果蝇胚胎进行延时共聚焦成像，激光显微手术探测体内生物力学，激光诱导胚胎局部区域的热冲击，以及一种称为视频力显微镜（VFM）的先进计算方法，该方法使用逆有限元方法从运动/变形组织的延时图像中推断细胞力。该项目的四个具体目标是：1。评估羊膜（在胚胎发生中起关键机械作用的组织）中额外的第14次有丝分裂与随后的胚带收缩缺陷之间的关系2。为了确认这种相关性的特异性，使用局部激光诱导的热冲击，仅传递给假定的羊膜3细胞。利用视频力显微镜（VFM）评估热冲击改变的细胞和组织水平的力，以及这些改变的力导致胚带收缩缺陷的方法4。为了验证VFM获得的动态力图，并通过激光显微手术挑战我们的力学假说，一旦完成，这四个目标的结果将描绘出最完整的生物力学事件链，从早期胚胎发生的环境压力到后来的发育缺陷。

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.