Mechanisms of growth plate patterning revealed by natural variation in mammalian ossification

骨骼生长发生在称为生长板的特定位置。这些细胞保存了大量的软骨细胞或软骨细胞，使骨骼能够快速生长。关于控制软骨细胞分化和增殖的基因和发育过程，我们知道得很多。然而，生长板是如何被诱导的，以及它们为什么出现在骨骼的特定位置，仍然是一个谜。例如，足部的跖骨仅在一端产生生长板，而梨状骨和跟骨是唯一形成生长板的腕骨和踝关节骨。通过将这些生长板与无法形成生长板的匹配骨骼位点进行比较，这些骨骼形成了理想的模型，可以分离出控制生长板形成方式和位置的基因表达和调控模式。鱼状生长板特别容易受到关键发育基因的干扰，这进一步使这些基因在生长板形成中的作用得以确定。该项目为本科生和研究生提供了实验室研究的机会，将发展生物学和比较基因组学的前沿方法应用于与人类发展和生长有关的基本问题。该研究项目支持科学家、学生和费城医师学院之间的合作，为缺乏此类机会的服务不足人群开展拓展工作。一个为天赋异禀但缺乏教育的费城高中学生开设的科学研讨会将提供与哺乳动物发育生物学和基因调控相关的课堂和研究经验。这项研究将采用一种新颖的方法来比较软骨内骨骼中形成和不形成生长板的等效区域。通过配对生长板形成组织和非形成组织的全转录组（RNA-seq）和染色质状态（ATAC-seq）测序，对梨状、跟骨和跖骨进行生长板形成的诱导因子鉴定。据推测，在生长板可见形成之前，软骨细胞群体中存在表达差异，这些因素也将与储备区和软骨膜细胞差异相关。此外，Hox基因在生长板模式中起作用，并且据信大多数腕骨和跗骨的结节性是由于Hox基因在其发育中的作用较有限。然而，Hoxa11和Hoxd11功能丧失导致近端腕骨和跗骨畸形，包括缩短的鹰嘴状骨。因此，鱼状骨和跟骨是确定晚期Hox11表达在生长板形成中的作用的天然测试案例。预测Hox11通过建立保留区和软骨膜软骨细胞群，直接参与梨形和跟骨生长板的诱导。此外，预测生长板形成位点将偏向于含有Hox11-13响应元件的活性增强子，并且增强子的活性模式将在不同的生长板中共享。

Skeletal growth occurs at specific locations called growth plates. These preserve pools of cartilage cells, or chondrocytes, that enable rapid bone growth. Much is known regarding the genes and developmental processes that control chondrocyte differentiation and proliferation. Yet, how growth plates are induced and why they occur at specific locations in the skeleton continues to be a mystery. For example, metatarsal bones in the feet produce a growth plate at only one end, and the pisiform and calcaneus are the only wrist and ankle bones to form a growth plate. By comparing these growth plates to the matched skeletal sites that fail to form them, these bones form ideal models to isolate the patterns of gene expression and regulation that control how and where growth plates are formed. The pisiform growth plate is particularly susceptible to the perturbation of key developmental genes that further allows the role of these genes in growth plate formation to be identified. This program expands laboratory research opportunities for a diverse body of undergraduate and graduate students in the application of cutting-edge methods in developmental biology and comparative genomics to fundamental questions related to human development and growth. This research project supports collaboration between scientists, students, and the College of Physicians of Philadelphia to develop outreach efforts to underserved populations lacking in such opportunities. A science workshop for gifted but underserved Philadelphia high school students will provide classroom and research experiences related to mammalian developmental biology and gene regulation.This research will employ a novel approach comparing otherwise equivalent regions of the endochondral skeleton that do and do not form growth plates. Through whole transcriptome (RNA-seq) and chromatin-state (ATAC-seq) sequencing of paired growth plate forming and non-forming tissues, the pisiform, calcaneus, and metatarsal will identify inductive factors of growth plate formation. It is hypothesized that expression differences exist in chondrocyte populations prior to the visible formation of growth plates and that these factors will also be differentially associated with reserve zone and perichondrial cells. In addition, Hox genes have a role in growth plate patterning, and it is believed that the nodular nature of most carpals and tarsals results from the more limited role for Hox genes in their development. However, Hoxa11 and Hoxd11 loss-of-function result in malformation of the proximal carpus and tarsus, including shortened pisiforms. Thus, the pisiform and calcaneus are natural test cases to determine the role that later stage Hox11 expression plays in growth plate formation. It is predicted that Hox11 has a direct role in pisiform and calcaneus growth plate induction by establishing reserve zone and perichondrial chondrocyte populations. Furthermore, it is predicted that growth plate forming sites will be biased for active enhancers containing Hox11-13 response elements and that enhancer activity patterns will be shared among different growth plates.