Mechanisms underlying asymmetric rotation and vascular development of the midgut

描述（由申请人提供）：在发育早期，中肠必须旋转，使其腹缘向左移动；如果不这样做，将导致旋转不良，并可能导致灾难性的中肠扭转。长期以来，人们一直认为肠道旋转是肠管本身固有的；然而，我的研究表明，旋转是由悬浮肠道的背肠系膜内不对称的细胞变化决定的。肠系膜沿其左右轴分布有四个并列但不同的细胞室，正确的肠旋转需要改变每个细胞室。结合鸡蛋独特的可接近性，这种细胞结构建立了背肠系膜作为一个强大的模型系统，在体内定义器官获得其空间组织的基本遗传和细胞机制，这是正常功能的先决条件。肠道旋转的起源可以追溯到早期的左右对称断裂转录因子Pitx2。在小鼠和鸟类中，Pitx2是产生向左倾斜的必要和充分条件，而这种旋转在缺乏Pitx2活性的胚胎中是随机的。然而，这种转录因子指导下游细胞变化的机制仍不清楚，这些细胞变化是引起肠道旋转所必需的。为了鉴定四个区室中每个区室中Pitx2的细胞靶点，我们采用激光捕获显微解剖方法分离并编目了每个细胞区室在向左倾斜时表达的基因。利用这些数据，第一个目标是寻找涉及基因亚群的级联反应，这些基因亚群对信号传导、识别细胞外信号和重塑细胞骨架结构至关重要。关键参与者的角色将通过引入功能基因结构的增加或减少来评估每个隔间。在我们的第二个目标中，我们使用与第一个类似的实验方法，解决了以前未知的肠系膜内动脉形成的不对称性，肠系膜内动脉将血液输送到肠道

DESCRIPTION (provided by applicant): Early in development, the midgut must rotate so that its ventral margin shifts to the left; failure to do so results in a malrotation and can lead to catastrophic midgut volvulus. It has long been assumed that gut rotation is intrinsic to the tube itself; however, my research has demonstrated that rotation is instead determined by asymmetric cellular changes within the dorsal mesentery that suspends the gut. This mesentery has four juxtaposed yet distinct cellular compartments distributed along its left-right axis, and changes in each are required for correct gut rotation. Combined with the unique accessibility of the chicken egg, this cellular architecture has established the dorsal mesentery as a powerful model system to define, in vivo, the fundamental genetic and cellular mechanisms through which organs acquire their spatial organization, which is a prerequisite for normal functioning. The genesis of gut rotation traces its origins to the early left-right symmetry-breaking transcription factor Pitx2. In mice and birds, Pitx2 is necessary and sufficient to produce the leftward tilt, and this rotation is randomized in embryos deficient for Pitx2 activity. However, th mechanisms by which this transcription factor directs downstream cellular changes necessary to cause gut rotation remain unknown. To identify cellular targets of Pitx2 in each of the four compartments, we employed laser capture microdissection to isolate then catalog the genes expressed in each cellular compartment at the time of the leftward tilt. Using these data, the firs aim pursues cascades involving subsets of genes that are critical for signaling, for recognizing extracellular cues, and for remodeling cytoskeletal architecture. The roles of key players will be assessed by introducing gain- or reduction- of function gene constructs into each compartment. In our second aim, we address previously unknown asymmetries in the formation of intra-mesenteric arteries that bring blood to the gut, using experimental approaches similar to the first aim but assaying for positive and negative regulators of vasculogenesis. In our third aim, we expand our studies using mouse models of asymmetric organ development and use chromatin immunoprecipitations in vivo to identify bona fide Pitx2 transcriptional targets. Lessons learned from these experiments will impact the study of other regions of the gut, and of tubular organs in general, some of which share strikingly similar features of morphogenesis and genetic patterning with the vertebrate midgut. PUBLIC HEALTH RELEVANCE: Gut malrotation is a birth defect of abnormal intestinal rotation that occurs once in approximately 500 live births. While malrotation predisposes affected babies to volvulus, a catastrophic strangulation of the intestine and its blood supply, th origin of this anomaly remains entirely unknown. We have developed the genetic tools to answer the fundamental questions surrounding proper gut rotation making use of the accessibility of the chicken egg and genetic mouse models to ultimately improve diagnosis in neonates.