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

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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.