Factors Regulating Development of Appendicular Skeletal Progenitors - PROJECT SUMMARY Appendicular skeletal progenitors are generated when trunk lateral plate mesoderm undergoes an epithelial-to-mesenchymal transition to generate mesenchymal skeletal progenitors that migrate away from the body axis to form the limb buds. Deciphering the mechanism that stimulates the generation of limb skeletal progenitors is important for understanding and treating appendicular skeletal defects. Recent studies have shown that retinoic acid (RA), an active metabolite of vitamin A, is required specifically for generation of forelimb buds which give rise primarily to forelimb skeletal progenitors; studies on RA-deficient mutant mouse embryos demonstrated a blockage of forelimb but not hindlimb budding. RA signaling is controlled by RA-synthesizing enzymes, including retinol dehydrogenase 10 (RDH10) and retinaldehyde dehydrogenase 2 (ALDH1A2; RALDH2). Interestingly, a human forelimb-specific role for RA was shown by a genome-wide association study showing that severe osteoarthritis of the hand, but not hip or knee osteoarthritis, is associated with human ALDH1A2 gene variants that exhibit lower expression in articular cartilage. RA directly regulates gene transcription by functioning as a ligand for nuclear RA receptors that bind RA response elements (RAREs) near target genes. Binding of RA controls recruitment of transcriptional coregulators including nuclear receptor coactivator (NCOA) that recruits the histone acetytransferase p300, and nuclear receptor corepressor (NCOR) that recruits the histone methyltransferase PRC2. Aldh1a2-/- embryos (that completely lack RA activity) fail to induce expression of Tbx5, the earliest known marker of forelimb development, whereas Rdh10-/- embryos (with greatly reduced RA activity) exhibit delayed/reduced Tbx5 expression and stunted forelimbs. We recently reported that Fgf8 needs to be directly repressed by RA and NCOR to allow Tbx5 gene activation during forelimb initiation. As identification of direct RA target genes is slow when proceeding one gene at a time, we performed a global analysis comparing ChIP-seq and RNA-seq for wild-type vs Aldh1a2-/- trunk (where forelimb buds arise), thus identifying RA-regulated genes that also have nearby RA-regulated deposition of H3K27ac (activation mark) or H3K27me3 (repression mark) near RAREs which is expected if a gene is a direct target of RA. We identified new candidates for direct RA target genes; CRISPR knockout of two new candidate RA target genes (Meis1 and Meis2) demonstrated they are needed for forelimb initiation. Here, we will investigate Meis1/Meis2 gene function during forelimb initiation, identify a Tbx5 forelimb enhancer, and initiate studies on transcriptional coregulators to uncover the mechanisms through which RA stimulates forelimb initiation through regulation of Meis1/Meis2, Tbx5, Fgf8, or other factors.
