Mechanisms underlying asymmetric rotation and vascular development of the midgut

中肠不对称旋转和血管发育的机制

• 批准号: 8297300
• 负责人: Natasza A Kurpios
• 金额: $ 33.11万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-04-01 至 2017-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8297300
• 关键词: Actins; Address; Affect; Angioblast; Architecture; Automobile Driving; Behavior; Biological Assay; Biological Models; Birds; Blood; Blood Vessels; Cataloging; Catalogs; Cell Polarity; Cell Shape; Cells; Chickens; Childhood; Congenital Abnormality; Coupled; Cues; Cytoskeleton; Data; Development; Diagnosis; Dorsal; ES Cell Line; Embryo; Embryology; Enzymes; Exhibits; Failure; GTP-Binding Proteins; Gastrointestinal tract structure; Gel; Gene Expression; Gene Expression Regulation; Genes; Genetic; Goals; Heparan Sulfate Proteoglycan; Heparitin Sulfate; Human; Intestinal Volvulus; Intestines; Knockout Mice; Lasers; Lead; Learning; Left; Ligands; Link; Live Birth; Mediator of activation protein; Mesenchymal; Mesenteric Arteries; Mesentery; Microarray Analysis; Midgut; Modification; Molecular; Morphogenesis; Morphology; Mus; Mutant Strains Mice; Neoplasm Metastasis; Organ; Pathway interactions; Patients; Pattern; Physical condensation; Play; Positioning Attribute; Process; Property; Proteins; Publishing; Quail; Randomized; Research; Role; Rotation; Side; Signal Transduction; System; Technology; Testing; Time; Transplantation; Tube; Tubular formation; Vascular blood supply; Vascularization; angiogenesis; base; cell behavior; cellular targeting; chemokine receptor; chromatin immunoprecipitation; egg; extracellular; gene function; genetic manipulation; glypican 3; improved; in vivo; laser capture microdissection; member; molecular asymmetry; mouse model; neonate; novel; research study; rho; tissue repair; tool; transcription factor; tumor progression; vasculogenesis

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.

描述（由申请人提供）：在发育早期，中肠必须旋转，使其腹侧边缘向左移动；否则会导致旋转不良，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管子本身固有的。然而，我的研究表明，旋转是由悬置肠道的背侧肠系膜内的不对称细胞变化决定的。肠系膜有四个并置但不同的细胞区室，沿其左右轴分布，每个区室的变化都是正确的肠道旋转所必需的。结合鸡蛋独特的可及性，这种细胞结构将背侧肠系膜建立为一个强大的模型系统，在体内定义基本的遗传和细胞机制，器官通过这些机制获得空间组织，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左右对称性破坏的转录因子 Pitx2。在小鼠和鸟类中，Pitx2 对于产生向左倾斜是必要且充分的，并且这种旋转在缺乏 Pitx2 活性的胚胎中是随机的。然而，这种转录因子引导引起肠道旋转所需的下游细胞变化的机制仍然未知。为了识别四个区室中每个区室中 Pitx2 的细胞靶标，我们采用激光捕获显微切割来分离并编录向左倾斜时每个细胞区室中表达的基因。利用这些数据，第一个目标是追求涉及对信号传导、识别细胞外信号和重塑细胞骨架结构至关重要的基因子集的级联。将通过将功能增强或减弱的基因构建体引入每个区室来评估关键参与者的作用。在我们的第二个目标中，我们使用类似于第一个目标的实验方法来解决将血液输送到肠道的肠系膜内动脉形成过程中以前未知的不对称性 目的只是测定血管生成的正调节因子和负调节因子。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扩展了我们的研究，并使用体内染色质免疫沉淀来识别真正的 Pitx2 转录靶标。从这些实验中吸取的教训将影响肠道其他区域和一般管状器官的研究，其中一些器官与脊椎动物中肠在形态发生和遗传模式方面具有惊人相似的特征。 公众健康相关性：肠道旋转不良是一种肠道旋转异常的先天缺陷，大约 500 名活产婴儿中就会出现一次。虽然肠旋转不良会使受影响的婴儿容易发生肠扭转，这是一种灾难性的肠道及其血液供应绞窄，但这种异常的起源仍然完全未知。我们开发了遗传工具来回答有关正确肠道旋转的基本问题，利用鸡蛋和遗传小鼠模型的可及性，最终改善新生儿的诊断。