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
Department of Pathology and Human Anatomy, Loma Linda University, Loma Linda, CA, United States.
Front Cell Dev Biol. 2023 Jul 10;11:1215406. doi: 10.3389/fcell.2023.1215406. eCollection 2023.
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.
四肢骨骼的关节末端具有精确的形态和不对称性，以确保关节功能正常。生长分化因子5（Gdf5）是一种分泌型形态发生素，参与软骨和骨骼发育，对发育中的关节结构有贡献。Gdf5失调会导致关节形态发生异常，常导致进行性关节退变或骨关节炎（OA）。调节其表达的转录因子和调控模块（CRMs）尚未得到充分表征。我们之前鉴定了一个与Gdf5相关的调控区域，该区域包含Lmx1b、Osr2、Fox和Sox转录因子的预测结合位点。这些转录因子是参与关节形态发生和骨骼发育的公认因子。我们使用原位杂交技术，对发育中的鸡四肢中的调控区域、以及感兴趣的转录因子进行检测，以确定表达上的潜在重叠。我们进一步分析了已发表的小鼠和鸡数据集中来自四肢和膝盖的单细胞RNA测序（scRNA-seq）数据，鉴定出同时表达调控区域和感兴趣转录因子的细胞。我们还对调控区域报告基因构建体中预测的转录因子结合位点进行了定点诱变，并在微团培养和胚胎鸡翅生物测定中使用靶向区域电穿孔（TREP）确定活性的任何变化。通过原位杂交（ISH）和scRNA-seq分析，在关节发育过程中，调控区域的表达与这些转录因子的表达重叠。在调控区域CRM内，Fox和Sox转录因子共有的两个结合位点发生突变，在微团培养和胚胎鸡翅生物测定中，增强子活性降低至背景水平，而仅Sox结合位点的两个突变导致活性显著增加。这些结果表明，Fox/Sox结合位点是活性所必需的，而仅Sox位点参与活性的抑制。调控区域中Lmx1b结合位点的突变导致增强子活性总体降低，背侧活性降低。尽管Osr2在关节发育中具有公认的作用，但预测的Osr2位点的破坏并未改变调控区域的活性。综上所述，我们的数据表明，调控区域整合了正向、抑制和不对称输入，以在肘关节发育过程中微调其表达。
