Mechanisms underlying asymmetric rotation and morphogenesis of the midgut

摘要 通过胚胎发生研究发现的机制对于理解 疾病我们使用经典的鸡胚胎学和复杂的小鼠遗传学来阐明基本的细胞 这些过程决定了器官的形状和功能。我们最着迷于左右（LR）器官不对称， 因为器官偏侧性的错误与危及生命的出生缺陷有关。肠道的逆时针旋转 是研究器官偏侧性的极好模型。这种旋转的一个关键方面是启动了一个新的倾斜 由LR不对称性的主调节器Pitx2指导。不这样做会导致肠道旋转不良， 小儿患者的灾难性肠扭转。虽然旋转力一直被认为是肠道固有的， 相反，我们发现肠道旋转是由相邻背侧的不对称变形驱动的。 肠系膜（DM），其悬浮肠道，并且其细胞结构在Pitx 2的下游。一个重要特性 DM的特点是其精致的二进制组织，具有不同的LR隔室， 基因操控每个隔室中的细胞和细胞外基质（ECM）变化导致DM 变形并倾斜所附的肠管。这种关键的偏见决定了肠道手性，并建立了一个模型， 解释LR基因表达如何最终导致细胞行为的变化， 器官发生DM也是为肠道服务的血液和淋巴管的唯一管道。我们 发现Pitx2依赖性机制指导肠道倾斜也对肠道模式至关重要 并提供一种机制来协调这两个过程。而大多数特定部位 器官发生依赖于Pitx2，下游的机制研究受到了一个混杂因素的阻碍。 Pitx2突变体中的“双右”异构现象。尽管所有脊椎动物中的Pitx2表达都被Nodal激活， 结节在不对称形态发生之前消失，留下未解决的问题，即Pitx2 指导器官形成。我们发现肠道中的Pitx 2表达并不是之前诱导的延伸 关于Nodal相反，我们证明肠道旋转需要Pitx2的“第二波”， 通过潜在的TGF β进行机械调节，将LR基因表达与强制翻译联系起来。在目标1中，我们确定 在肠道、血管和淋巴发育过程中Pitx2剂量反应机制， Pitx2的不同作用取决于其对BMP 4信号传导的抑制阈值。在目标2中，我们定义了 PITX2靶点PITX2引导间充质细胞极性的分子机制， 收缩的不对称性，从而引导力以避免倾斜。在目标3中，我们将联合收割机 用定量成像和计算建模测量组织特性，以阐明 刚度和力的各向异性通过机械反馈驱动肠旋转。这些研究将 显著推进我们对不对称肠道的转录和机械控制的理解， 血管形态发生是改善新生儿旋转不良诊断的关键一步。

ABSTRACT The mechanisms discovered through the study of embryogenesis have been fundamental to understanding disease. We use classic chicken embryology and sophisticated mouse genetics to elucidate how basic cellular processes define the shape and function of organs. We are most fascinated by left-right (LR) organ asymmetry, as errors of organ laterality are linked to life-threatening birth defects. The counterclockwise rotation of the gut is an excellent model to study organ laterality. A critical aspect of this rotation is initiation of a leftward tilt directed by the master regulator of LR asymmetry, Pitx2. Failure to do so leads to gut malrotation and catastrophic volvulus in pediatric patients. Whereas rotation forces had long been assumed intrinsic to the gut tube, we instead discovered that gut rotation is driven by asymmetric deformation of the adjacent dorsal mesentery (DM) that suspends the gut, and whose cellular architecture is downstream of Pitx2. A key property of the DM is its exquisite binary organization, with distinct LR compartments that are readily accessible to genetic manipulation. Cellular and extracellular matrix (ECM) changes in each compartment cause the DM to deform and tilt the attached gut tube leftward. This critical bias determines gut chirality and frames a model to explain how LR gene expression is ultimately responsible for changes in cell behavior that initiate asymmetric organogenesis. The DM is also the sole conduit for blood and lymphatic vessels that serve the gut. We discovered that Pitx2-dependent mechanisms directing gut tilting are also crucial for patterning the gut vasculature and provide a mechanism to coordinate these two processes. Whereas most situs-specific organogenesis depends on Pitx2, mechanistic studies downstream have been hampered by a confounding “double-right” isomerism in Pitx2 mutants. Whereas Pitx2 expression in all vertebrates is activated by Nodal, Nodal disappears before asymmetric morphogenesis, leaving unresolved the question of how Pitx2 directs organogenesis. We discovered that Pitx2 expression in the gut is not an extension of previous induction by Nodal. Instead, we demonstrate that gut rotation requires a “second wave” of Pitx2 that is subject to mechanoregulation by the latent TGFb, linking LR gene expression to force translation. In aim 1, we determine the mechanism of Pitx2 dose response during gut, vascular, and lymphatic development and identify two distinct roles for Pitx2 dependent on its repressive threshold on BMP4 signaling. In aim 2, we define the molecular mechanism by which the formin Daam2, a Pitx2 target, directs mesenchymal cell polarity, actomyosin contractile asymmetry, and thereby steers the forces to polarize tilting. In aim 3, we combine cutting-edge physical measurements of tissue properties with quantitative imaging and computational modeling, to elucidate how stiffness and force anisotropies drive gut rotation through mechanical feedback. Together, these studies will significantly advance our understanding of the transcriptional and mechanical control of asymmetric gut and vascular morphogenesis, a critical step toward improved malrotation diagnostics in newborns.