Signals and mechanical forces controlling radial gut morphogenesis

项目总结/摘要 中肠的特点是由一系列同心的，中胚层来源的平滑肌层 和围绕内胚层来源的上皮的粘膜。在成熟的肠道中， 指状突起伸入管腔，称为绒毛。在发育中的小鸡， 通过一系列的上皮弯曲事件逐步地发生。屈曲力是通过 当扩张受到邻近分化的限制时， 平滑肌上皮所经历的物理约束随着时间的推移而变化，因为不同层的上皮细胞在不同的时间段内会发生变化。 依次建立平滑肌。建立这些平滑肌层的位置 通过Shh和Bmp梯度的活性，它们分别对 平滑肌分化当它们形成时，每个肌肉层中纤维的方向取决于 肠的机械环境时，层经历分化。尽管这位将军 关于中肠结构是如何建立的，缺乏关于组织水平的信息。 其他肠段的构建。前肠和后肠都起源于相同的线性原肠 肠管与中肠相同，并具有相同的同心组织。然而，有重要的 不同肠段中平滑肌分化的厚度和时间的差异， 上皮衬里在三个节段中显著不同。在目标1中，负责不同 将阐明前肠和后肠中肌肉层的特征。已知的信号系统 负责确定中肠平滑肌的位置和厚度， 定性（通过原位杂交）以及定量（通过qPCR）的前肠和后肠片段， 确定它们与中肠的表达有何不同。这些信号将被操纵的选举， 通过在外植体培养中与激动剂和拮抗剂一起培养，并利用组织重组， 测试他们的功能。目标2中的实验将确定这些差异在多大程度上平滑 肌肉结构和动力学是负责不同的上皮形态的前，后 勇气将使用药物阻断平滑肌分化以测试它们的必要性。形态学和 生物物理参数将被测量并输入计算模型，以测试 在此基础上可以完全解释上皮形态学。最后，在目标3中，我们将评估转录如何 Hox和paraHox簇的因子，已知在肠道内指定区域身份，改变了分子生物学特性。 以及区分前肠、中肠和后肠的物理参数，从而将区域模式与 肠道的形态发生。为此，涉及区域身份的转录因子将被错误表达 利用逆转录病毒载体，以及生化和机械参数的变化将被检查。

PROJECT SUMMARY/ABSTRACT The midgut is characterized by a series of concentric, mesodermally-derived layers of smooth muscle and mucosa, surrounding an inner, endodermally-derived epithelium. In the mature gut, this epithelium forms finger-like projections extending into the lumen, called villi. In the developing chick, intestinal villi are generated in a step-wise manner, through a series of epithelial buckling events. Buckling forces are generated through the confined growth of the epithelium at a time when expansion is restricted by the adjacent differentiating smooth muscle. The physical constraint experienced by the epithelium changes over time as different layers of smooth muscle are established sequentially. The location of these smooth muscle layers is established through the activity of gradients of Shh and Bmp, which, respectively, have positive and negative effects on smooth muscle differentiation. As they form, the orientation of the fibers in each muscle layer depends upon the mechanical environment of the gut at the time the layer undergoes differentiation. In spite of this general outline of how the midgut architecture is established, there is a dearth of information regarding the tissue-level construction of the other gut segments. Both the foregut and the hindgut arise from the same linear primative gut tube as the midgut, and have the same general concentric organization. However, there are significant differences in the thickness and timing of smooth muscle differentiation in the different gut segments, and the epithelial lining differs dramatically in the three segments. In Aim 1, the mechanisms responsible for the distinct characteristics of the muscle layers in the fore- and hindgut will be elucidated. The signaling systems known to be responsible for defining the location and thickness of the smooth muscle in the midgut will be examined in the fore- and hindgut segments qualitatively (by in situ hybridization) as well as quantitatively (by qPCR) to determine how they differ in expression from the midgut. These signals will be manipulated by electoration in vivo and through culturing with agonists and antagonists in explant culture and utilizing tissue recombination to test their roles functionally. Experiments in Aim 2 will determine the extent to which these differences in smooth muscle architecture and dynamics are responsible for the distinct epithelial morphology of the fore-and hind guts. Drugs will be employed to block smooth muscle differentiation to test their necessity. Morphometric and biophysical parameters will be measured and entered into computational models to test the degree to which epithelial morphology can be entirely explained on this basis. Finally, in Aim 3 we will assess how transcription factors of the Hox and paraHox clusters, known to specify regional identity within the gut, alter the molecular and physical parameters that differentiate the fore-, mid- and hindgut, thereby connecting regional patterning to morphogenesis of the gut. To that end, transcription fctors involved in regional identity will be misexpressed ustilizing retroviral vectors, and changes in biochemical and mechanical parameters will be examined.