Sox, Fox, and Lmx1b binding sites differentially regulate a Gdf5-Associated regulatory region during elbow development

• Published in: Frontiers in cell and developmental biology Vol. 11; p. 1215406
• Main Authors: Yeboah, Ruth-Love, Pira, Charmaine U, Shankel, Matthew, Cooper, Allen M, Haro, Endika, Ly, Van-Dai, Wysong, Kenrick, Zhang, Michael, Sandoval, Nicole, Oberg, Kerby C
• Format: Journal Article
• Language: English
• Published: Switzerland Frontiers Media S.A 10.07.2023
• Subjects: Cell and Developmental Biology; cis-regulatory module (CRM); FOX transcription factor family; GDF5 gene; joint development; LMX1b transcription factor; SOX transcription factors; osteoarthritis; cis-regulatory module (CRM); joint development; FOX transcription factor family; SOX transcription factors; GDF5 gene; LMX1b transcription factor
• ISSN: 2296-634X; 2296-634X
• DOI: 10.3389/fcell.2023.1215406

The articulating ends of limb bones have precise morphology and asymmetry that ensures proper joint function. Growth differentiation factor 5 (Gdf5) is a secreted morphogen involved in cartilage and bone development that contributes to the architecture of developing joints. Dysregulation of Gdf5 results in joint dysmorphogenesis often leading to progressive joint degeneration or osteoarthritis (OA). The transcription factors and -regulatory modules (CRMs) that regulate expression are not well characterized. We previously identified a Gdf5-associated regulatory region ( ) that contains predicted binding sites for Lmx1b, Osr2, Fox, and the Sox transcription factors. These transcription factors are recognized factors involved in joint morphogenesis and skeletal development. We used hybridization to , , and the transcription factors of interest in developing chicken limbs to determine potential overlap in expression. We further analyzed scRNA-seq data derived from limbs and knees in published mouse and chicken datasets, identifying cells with coexpression of and the transcription factors of interest. We also performed site-directed mutatgenesis of the predicted transcription factor binding sites in a -reporter construct and determined any change in activity using targeted regional electroporation (TREP) in micromass and embryonic chicken wing bioassays. expression overlapped the expression of these transcription factors during joint development both by hybridization (ISH) and scRNA-seq analyses. Within the CRM, mutation of two binding sites common to Fox and Sox transcripstion factors reduced enhancer activity to background levels in micromass cultures and embryonic chicken wing bioassays, whereas mutation of two Sox-only binding sites caused a significant increase in activity. These results indicate that the Fox/Sox binding sites are required for activity, while the Sox-only sites are involved in repression of activity. Mutation of Lmx1b binding sites in caused an overall reduction in enhancer activity and a dorsal reduction . Despite a recognized role for Osr2 in joint development, disruption of the predicted Osr2 site did not alter activity. Taken together, our data indicates that integrates positive, repressive, and asymmetrical inputs to fine-tune the expression of during elbow joint development.