Mechanisms underlying asymmetric rotation and vascular development of the midgut

描述(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，会导致旋转不良，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，旋转是由支撑肠道的背侧肠系膜内不对称的细胞变化决定的。这个肠系膜有四个平行但不同的细胞隔室，沿其左右轴分布，每个隔室的变化是正确的肠道旋转所必需的。结合鸡蛋独特的可及性，这种细胞结构建立了背侧肠系膜作为一个强大的模型系统，在体内定义器官获得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破坏转录因子Pitx2。在小鼠和鸟类中，Pitx2是产生向左倾斜的必要条件和充分条件，这种旋转是在缺乏Pitx2活性的胚胎中随机进行的。然而，这种转录因子引导引起肠道旋转所必需的下游细胞变化的机制仍不清楚。为了确定四个隔室中每个隔室中的Pitx2的细胞靶标，我们使用激光捕获显微解剖来分离并分类左倾斜时每个细胞隔室中表达的基因。利用这些数据，FIRS的目标是追求涉及对信号传递、识别细胞外信号和重塑细胞骨架结构至关重要的基因子集的级联。将通过将功能基因结构的增减引入每个隔间来评估关键角色的作用。在我们的第二个目标中，我们使用与第一个相似的实验方法，解决了以前未知的将血液输送到肠道的肠系膜内动脉形成的不对称性。 目的检测血管生成的正性和负性调节因子。在我们的第三个目标中，我们使用不对称器官发育的小鼠模型来扩大我们的研究，并在体内使用染色质免疫沉淀来识别真正的Pitx2转录靶标。从这些实验中学到的经验教训将影响对肠道其他区域和一般管状器官的研究，其中一些器官在形态发生和遗传模式方面与脊椎动物中肠有着惊人的相似特征。 与公共卫生相关：肠道旋转不良是一种肠道旋转异常的出生缺陷，大约每500名活产儿中就有一例发生这种情况。虽然旋转不良使受影响的婴儿容易发生扭转，这是一种灾难性的肠梗阻及其血液供应，但这种异常的来源仍然完全不清楚。我们已经开发了基因工具来回答围绕正确的肠道旋转的基本问题，利用鸡蛋和遗传小鼠模型的可及性，最终提高对新生儿的诊断。

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.