Fibroblast growth factors (FGFs) and sonic hedgehog (SHH) are mitogens known to affect limb growth and patterning during embryonic development. While FGFs establish the proximal‐distal axis from the apical ectodermal ridge (AER), SHH directs anterior‐posterior patterning from the zone of polarizing activity (ZPA). In addition, FGFs and SHH act in an autoregulatory loop by maintaining each other’s expression to coordinate limb patterning during limb outgrowth. The LIM Homeobox 2 (LHX2) transcription factor, which acts in the progress zone (PZ), was identified as an intermediary in the FGF to SHH arm of the maintenance loop. FGF mediates its action on cells through multiple intracellular signaling pathways including the JAK‐STAT self‐renewal pathway, PKC cell motility pathway, PDK1‐AKT cell survival pathway, and RAS‐associated cell proliferation pathway. We wondered whether one or more of these pathways directed FGF‐mediated upregulation of LHX2 in the FGF‐SHH regulatory loop. To evaluate a specific FGF signaling pathway, we implanted a bead soaked in a selective pathway‐specific inhibitor adjacent to an FGF2‐soaked bead in the posterior margin of Hamburger‐Hamilton stage 23–24 (HH23‐HH24) chicken limbs, a region known to induce LHX2 expression in response to FGF. The embryos were harvested after 4 hours and assayed for LHX2 and SHH expression by whole mount in situ hybridization. Ectopic LHX2 and SHH expression levels were reduced in the presence of the RAS and MEK inhibitors, indicating that FGF requires functional RAS‐associated pathways that utilize MEK to upregulate LHX2 expression.
The direct molecular mechanism underlying joint formation and remodeling has been studied for decades yet still is not fully understood. Growth differentiation factor 5 (GDF5), a critical joint‐associated transcription factor linked to accelerated osteoarthritis, has been shown to be one of the intermediate markers for precursors of joint associated cells. Understanding the mechanisms regulating GDF5 expression will further illuminate some of the key steps of joint development. Previously, we identified a GDF5 Associated Regulatory Region (GARR) with putative Sox binding sites; the SOX11 transcription factor has been shown to upregulate GDF5. Hence, we hypothesize that SOX11 (SOXC family) is necessary for GARR‐mediated expression of GDF5 during joint development and maintenance. We previously demonstrated that site‐directed mutagenesis of the putative Sox binding sites in a GARR reporter construct markedly reduced activity. To confirm the role of SOX11 we performed over expression experiments in transformed chicken fibroblast (DF1) cells, a primary limb bud micromass culture, and in ovo in the developing chicken wing. Co‐transfection studies demonstrated increased GARR activity both in vivo and in vitro with over expression of SOX11. Taken together, these data suggest that SOX11 may play a role in the regulation of GDF5 through GARR.
Growth differentiation factor 5 (GDF5) is associated with the formation and maintenance of joints. Disruption of GDF5 expression is also linked to accelerated osteoarthritis. We have identified a GDF5 Associated Regulatory Region (GARR) that contains several Sox binding sites. The SOX11 transcription factor has been shown to upregulate GDF5 and we hypothesized that SOX11 is necessary for GARR mediated expression of GDF5 during joint development and maintenance. To test this hypothesis, we compared the expression of SOX11 and GDF5 in chicken limbs during development using in‐situ hybridization, using MYOD, TNMD, and Col1A2 as muscle and tendon markers. We also generated a GARR reporter construct and mutated the Sox binding sites. The native and mutated GARR reporters were transfected into developing chick wings. Our results showed that in chicken, SOX11 expression primarily surrounds GDF5 expression in joint spaces at Hamburger‐Hamilton stages (HH) 25‐27. At HH 29, the expression of SOX11 and GDF5 colocalized in joint spaces. SOX11 expression was not confined to joints, but also found in muscles (MYOD) and tendons (TNMD, Col1A2). Disruption of the Sox binding sites greatly reduced GARR activity within joint spaces. Colocalization of SOX11 and GDF5 in joints shows that SOX11 may play a role in the regulation of GDF5 through GARR. Additionally, it may be a competency factor for the formation and organization of joint‐related tissues. Decreased activity with mutant GARR suggests it is required for GARR activity. Further experiments are needed to clarify the role of SOX11 in regulating joints and joint associated tissues.
SOX11 has been implicated in several aspects of development including epithelial to mesenchymal interactions, neuronal migration, skeletogenesis, joint maintenance, and hedgehog signaling. As a first step in evaluating its role in limb development, we determined the expression pattern for SOX11 between Hamilton‐Hamburger (HH) stages 19–35 chicken embryos. Initally (HH19), SOX11 expression is detected in the the emerging limb bud, with strong ventral expression centrally. At HH22, dorsal anterior and posterior proximal expression is evident. As the limb develops (HH24), SOX11 expression shifts distally and wanes proximally. At HH25, expression surrounds condensing cartilage, and by HH29, SOX11 expression surrounds and accentuates the elbow and distal phalanges. By HH35, SOX11 has clearly shifted to selected muscles and tendons but is poorly expressed in joint spaces. This is the first report to implicate SOX11 in myogenesis. These findings suggest that SOX11 may play multiple roles during skeletogenesis, including the regulation of selected muscle tracts. Further work is needed to determine whether there is a common role for SOX11 during these various components of musculoskeletal development or whether SOX11 adapts to multiple pleiotropic roles that occur during limb development.
Sox transcription factors are involved in a number of developmental processes including joint formation. Sox9, Sox5, and Sox6 (the Sox‐trio) form a complex that promotes chondrogenesis. In joint‐associated cells, Sox9 may give rise to a subpopulation of cells expressing growth differentiation factor 5 (Gdf5). Gdf5, a secreted morphogen critical to joint development, regulates cartilage growth and tissue differentiation. Osteoarthritis (OA) is condition characterized by articular cartilage degradation and is a leading cause of disability worldwide. Gdf5 dysregulation has been identified as a risk factor for OA, and in the joints of OA patients, the Sox‐trio are downregulated. The link between Sox, Gdf5, and joint development is not fully understood. We previously identified an enhancer for Gdf5, known as Gdf5‐Associated Regulatory Region (GARR), and have identified possible Sox9 and 5/6 binding sites within GARR by in silico analysis. Thus, we hypothesized that Sox9 is required for Gdf5 upregulation through GARR, while other Sox transcription factors (Sox5/6) enhance or stabilize this interaction. To test this hypothesis, we analyzed publicly available single cell RNA sequencing (scRNA‐seq) data from mouse embryo (E12‐5‐E15.5) knee joints. Analysis revealed a strong correlation between Gdf5 and Sox9. Sox9 was upregulated in Gdf5 positive cells. Sox5 and 6 were upregulated in Gdf5 positive cells but had a lower correlation to Gdf5 expression indicating a weaker or indirect/secondary role in Gdf5 regulation. We also compared the activity of the wildtype GARR enhancer‐reporter construct to constructs with mutated Sox binding sites in developing chicken limbs to determine the role of these sites on enhancer activity. We found that in developing chicken embryo limbs, constructs with mutated Sox9 binding sites had a marked decrease in enhancer activity in the elbow but not in the autopod. Mutation of predicted Sox5/6 binding sites did not seem to affect activity relative to the non‐mutated control in the forelimb joints. Our findings suggest that Sox9 is a key regulator of elbow development through GARR‐mediated expression of Gdf5. The correlations identified in the transcriptomic data may be relevant only to the elbow and knee joints. Alternatively, the Sox transcription factors may be acting though other Gdf5‐associated enhancers in the autopod joints. Further investigation of enhancer activity in the presence of various combinations of Sox5/6/9 will throw more light on how they collectively affect Gdf5 expression as well as joint development and OA progression.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.