Novel Modulators of Limb Development and Chondrocyte Maturation

Project Summary Disorders of growth plate chondrocyte maturation impact the growth of the skeleton, resulting in a spectrum of diseases from skeletal dysplasia to extreme short stature. These diverse conditions underscore the importance of tight regulation to normal epiphysial physiology, yet the genetic pathways directing chondrocyte maturation are poorly understood. The current proposal leverages an in vitro model of the growth plate to (1) conduct high- throughput, genome-wide functional KO screening of chondrocyte maturation, (2) prioritize screening hits with orthologues linked to human skeletal growth through genome-wide association studies (GWAS), and (3) investigate the mechanisms by which top screening targets act to affect chondrocyte maturation. During the course of my K08 award thus far, I have conducted a screening assay in which a lentiviral library of 80,000 unique single-guide RNAs (sgRNAs) is transduced into Cas9+ chondrocytes (300 million cells from the MLB13 cell line) to simultaneously KO 20,000 genes in replicate. After four days of maturation, a time at which 95% of cells are usually immature, KO chondrocytes are FACS sorted for the maturation marker CD-200; KOs driving early maturation (CD-200 high) are compared to immature internal controls (CD-200 low). This screening assay can robustly detect genetic determinants of chondrocyte maturation and has already identified genes highly relevant to skeletal biology, including members of the Indian hedgehog signaling family. Interestingly, I found that loss of Protein Inhibitor of Activated STAT1 (PIAS1) results in significantly premature chondrocyte maturation. Not yet known to function in the growth plate, PIAS1 is a highly plausible candidate as it has been shown to modulate the activity of transcription factors in other cell types that also direct growth plate elongation and maturation. In the present application, I intend to investigate mechanisms by which PIAS1 acts to delay growth plate chondrocyte maturation in vivo (Aim 1) and probe a role for PIAS1 in murine limb development (Aim 2). Securing this award will be instrumental in providing crucial support for generating preliminary data essential for a subsequent R01-level grant. This subsequent grant will enable me to conduct an in-depth investigation into the mechanistic role of PIAS1, positioning it as a promising and innovative target in both limb development and growth plate maturation.