Testing the tissue-specific mechanisms of Hoxa5 function in musculoskeletal patterning

摘要 软骨、骨骼、肌肉、肌腱和韧带的发育必须高度协调， 组织类型分化和形态发生以协调的方式发生，从而允许所产生的 组织一起发挥作用。轴向肌肉骨骼(脊柱和肋骨)由 体节，其中所有肌肉骨骼组织类型都是特定的，并协调发展。然而， 体节体衍生物的形态随体轴位置的不同而不同。这种变化由以下因素控制 HOX蛋白，保守的转录因子，形成大多数动物胚胎的体轴。在……里面 脊椎动物，HOX蛋白既赋予新生节段前后身份，又直接发挥作用 在发育后期的组织形态发生中的作用。然而，我们对他们的情况仍然知之甚少 作用机制，包括它们所作用的细胞和组织类型，它们的细胞行为 调控，并最终调控其转录靶标。Hoxa5非冗余型肌肉骨骼 颈-胸交界处的元素。为了了解它是如何起作用的，我们使用了基因谱系 命运标记定位Hoxa5表达细胞的后代。我们发现Hoxa5的后代 有助于有限数量的组织类型，如软骨和软骨膜，但仅有助于 很少是肌腱，从来不是肌肉。这种对Hoxa5后代命运的限制可能反映了一个重要的 它的功能的一个方面。在这里，我们建议研究Hoxa5模式的机制 小鼠的颈胸过渡肌肉骨骼，使用遗传谱系标记的组合， 条件性和功能丧失分析以及高通量测序。我们的具体目标是 设计的目的是：(1)确定Hoxa5在构建中轴骨骼模型中的组织特异性(2) 识别由Hoxa5自主调控的基因和基因网络；(3)直接识别 体节中Hoxa5的转录靶点。这项工程的顺利完成将会给我们带来光明 轴向花纹由HOX基因调控的机制，也可以应用于 一般了解Hoxa5在其他组织类型和癌症中的作用机制。更广泛地说， 通过应用工作来理解肌肉骨骼组织，工作与人类健康相关 正常发育和疾病过程中的图案化和分化。最后，这项工作将是 对巴纳德学院的本科生进行了调查，巴纳德学院是一所面向女性的文科学院。它将被执行 由PI实验室的研究学生提供，并将被引入一个新开发的全年实验室 在巴纳德的课程。基于课程的研究已被证明增加了学生的参与度， 包括那些来自STEM的代表不足的群体的学生，并增加对研究生的追求 干式训练。本课程将让本科生从事实质性的原创性研究，同时提供 他们接受了当前分子遗传学方法的培训。

Abstract Development of cartilage, bones, muscles, tendons and ligaments must be highly coordinated, with tissue type differentiation and morphogenesis occuring in a concerted way that allows the resulting tissues to function together. The axial musculoskeleton (vertebral column and ribs) develops from the somites, in which all musculoskeletal tissue types are specified and develop coordinately. However, the morphology of somite derivatives varies with position along the body axis. This variation is controlled by Hox proteins, conserved transcription factors that pattern the body axis of most animal embryos. In vertebrates, Hox proteins both confer anterior-posterior identity on nascent segments and play direct roles in tissue morphogenesis later in development. However, we still know relatively little about their mechanisms of action, including the cell and tissue types in which they act, the cellular behaviors they regulate, and ultimately their transcriptional targets. Hoxa5 non-redundantly patterns musculoskeletal elements at the cervical-thoracic transition. In order to understand how it acts, we used genetic lineage labeling to fate map the descendants of Hoxa5 expressing cells. We found that Hoxa5 descendants contribute to a restricted number tissue types, such as cartilage and perichondrium, but only contribute rarely to tendon and never to muscle. This restriction of Hoxa5 descendant fate may reflect an important aspect of its function. Here, we propose to investigate the mechanism through which Hoxa5 patterns the cervical-thoracic transition musculoskeleton of mice, using a combination of genetic lineage labeling, conditional and loss-of-function analysis, and high throughput sequencing. Our specific aims are designed to: (1) Identify the tissue specificity of Hoxa5 action in patterning the axial skeleton (2) Identify genes and gene networks regulated cell-autonomously by Hoxa5 and (3) Identify direct transcriptional targets of Hoxa5 in the somites. Successful completion of this project will shed light on the mechanisms through which axial patterning is regulated by Hox genes, and can also be applied to a general understanding the mechanisms of Hoxa5 in other tissue types and cancers. More generally, the work is relevant to human health through its application to understanding musculoskeletal tissue patterning and differentiation during normal development and disease. Finally, this work will be conducted with undergraduates at Barnard College, a liberal arts college for women. It will be performed by research students in the PI’s lab, and will be introduced into a newly developed, full year laboratory course at Barnard. Course-based research has been shown to increase participation of students, including those from underrepresented groups, in STEM and to increase the pursuit of postgraduate STEM training. This course will engage undergraduates in substantive original research while providing them with training in current molecular genetics approaches.