Connexin43 regulates joint location in zebrafish fins

AbstractJoints are essential for skeletal form and function, yet their development remains poorly understood. In zebrafish fins, joints form between the bony fin ray segments providing essentially unlimited opportunities to evaluate joint morphogenesis. Mutations in cx43 cause the short segment phenotype of short fin (sofb123) mutants, suggesting that direct cell–cell communication may regulate joint location. Interestingly, increased cx43 expression in the another long fin (alfdty86) mutant appears to cause joint failure typical of that mutant. Indeed, knockdown of cx43 in alfdty86 mutant fins rescues joint formation. Together, these data reveal a correlation between the level of Cx43 expression in the fin ray mesenchyme and the location of joints. Cx43 was also observed laterally in cells associated with developing joints. Confocal microscopy revealed that the Cx43 protein initially surrounds the membranes of ZNS5-positive joint cells, but at later stages becomes polarized toward the underlying Cx43-positive mesenchymal cells. One possibility is that communication between the Cx43-positive mesenchyme and the overlying ZNS5-positive cells regulates joint location, and upregulation of Cx43 in joint-forming cells contributes to joint morphogenesis

Identification of an evx1-dependent joint-formation pathway during FIN regeneration.

Joints are essential for skeletal flexibly and form, yet the process underlying joint morphogenesis is poorly understood. Zebrafish caudal fins are comprised of numerous segmented bony fin rays, where growth occurs by the sequential addition of new segments and new joints. Here, we evaluate joint gene expression during fin regeneration. First, we identify three genes that influence joint formation, evx1, dlx5a, and mmp9. We place these genes in a common molecular pathway by evaluating both their expression patterns along the distal-proximal axis (i.e. where the youngest tissue is always the most distal), and by evaluating changes in gene expression following gene knockdown. Prior studies from our lab indicate that the gap junction protein Cx43 suppresses joint formation. Remarkably, changes in Cx43 activity alter the expression of joint markers. For example, the reduced levels of Cx43 in the sof (b123) mutant causes short fin ray segments/premature joints. We also find that the expression of evx1-dlx5a-mmp9 is shifted distally in sof (b123) , consistent with premature expression of these genes. In contrast, increased Cx43 in the alf (dty86) mutant leads to stochastic joint failure and stochastic loss of evx1 expression. Indeed, reducing the level of Cx43 in alf (dty86) rescues both the evx1 expression and joint formation. These results suggest that Cx43 influences the pattern of joint formation by influencing the timing of evx1 expression

Hapln1a Is Required for Connexin43-Dependent Growth and Patterning in the Regenerating Fin Skeleton

<div><p>Cell–cell communication, facilitating the exchange of small metabolites, ions and second messengers, takes place via aqueous proteinaceous channels called gap junctions. Connexins (cx) are the subunits of a gap junction channel. Mutations in zebrafish <i>cx43</i> produces the <i>short fin</i> (<i>sof ^b123</i>) phenotype and is characterized by short fins due to reduced segment length of the bony fin rays and reduced cell proliferation. Previously established results from our lab demonstrate that Cx43 plays a dual role regulating both cell proliferation (growth) and joint formation (patterning) during the process of skeletal morphogenesis. In this study, we show that Hapln1a (<u>H</u>y<u>a</u>luronan and <u>P</u>roteoglycan <u>L</u>i<u>n</u>k Protein 1a) functions downstream of <i>cx43</i>. Hapln1a belongs to the family of link proteins that play an important role in stabilizing the ECM by linking the aggregates of hyaluronan and proteoglycans. We validated that <i>hapln1a</i> is expressed downstream of <i>cx43</i> by in situ hybridization and quantitative RT-PCR methods. Moreover, in situ hybridization at different time points revealed that <i>hapln1a</i> expression peaks at 3 days post amputation. Expression of <i>hapln1a</i> is located in the medial mesenchyme and the in the lateral skeletal precursor cells. Furthermore, morpholino mediated knock-down of <i>hapln1a</i> resulted in reduced fin regenerate length, reduced bony segment length and reduced cell proliferation, recapitulating all the phenotypes of <i>cx43</i> knock-down. Moreover, Hyaluronic Acid (HA) levels are dramatically reduced in <i>hapln1a</i> knock-down fins, attesting the importance of Hapln1a in stabilizing the ECM. Attempts to place <i>hapln1a</i> in our previously defined <i>cx43</i>–<i>sema3d</i> pathway suggest that <i>hapln1a</i> functions in a parallel genetic pathway. Collectively, our data suggest that Cx43 mediates independent Sema3d and Hapln1a pathways in order to coordinate skeletal growth and patterning.</p></div

Cx43-Dependent Skeletal Phenotypes Are Mediated by Interactions between the Hapln1a-ECM and Sema3d during Fin Regeneration.

Skeletal development is a tightly regulated process and requires proper communication between the cells for efficient exchange of information. Analysis of fin length mutants has revealed that the gap junction protein Connexin43 (Cx43) coordinates cell proliferation (growth) and joint formation (patterning) during zebrafish caudal fin regeneration. Previous studies have shown that the extra cellular matrix (ECM) protein Hyaluronan and Proteoglycan Link Protein1a (Hapln1a) is molecularly and functionally downstream of Cx43, and that hapln1a knockdown leads to reduction of the glycosaminoglycan hyaluronan. Here we find that the proteoglycan aggrecan is similarly reduced following Hapln1a knockdown. Notably, we demonstrate that both hyaluronan and aggrecan are required for growth and patterning. Moreover, we provide evidence that the Hapln1a-ECM stabilizes the secreted growth factor Semaphorin3d (Sema3d), which has been independently shown to mediate Cx43 dependent phenotypes during regeneration. Double knockdown of hapln1a and sema3d reveal synergistic interactions. Further, hapln1a knockdown phenotypes were rescued by Sema3d overexpression. Therefore, Hapln1a maintains the composition of specific components of the ECM, which appears to be required for the stabilization of at least one growth factor, Sema3d. We propose that the Hapln1a dependent ECM provides the required conditions for Sema3d stabilization and function. Interactions between the ECM and signaling molecules are complex and our study demonstrates the requirement for components of the Hapln1a-ECM for Sema3d signal transduction

Quantitative RT-PCR confirms changes in gene expression.

<p>a. The ΔC_T value is determined by subtracting the average Actin C_T value from the average Gene C_T value. The standard deviation of the difference is calculated from the standard deviations of the gene and actin values using the Comparative Method.</p><p>b. The calculation of ΔΔC_T involves subtraction by the ΔC_T calibrator value. This is subtraction of an arbitrary constant, so the standard deviation of ΔΔC_T is the same as the standard deviation of the ΔC_T value.</p><p>c. The range given for gene relative to WT/MM is determined by evaluating the expression: 2∧ –ΔΔC_T with ΔΔC_T + s and ΔΔC_T – s, where s  =  the standard deviation of the ΔΔC_T value.</p

Cohesin mediates Esco2-dependent transcriptional regulation in a zebrafish regenerating fin model of Roberts Syndrome

Robert syndrome (RBS) and Cornelia de Lange syndrome (CdLS) are human developmental disorders characterized by craniofacial deformities, limb malformation and mental retardation. These birth defects are collectively termed cohesinopathies as both arise from mutations in cohesion genes. CdLS arises due to autosomal dominant mutations or haploinsufficiencies in cohesin subunits (SMC1A, SMC3 and RAD21) or cohesin auxiliary factors (NIPBL and HDAC8) that result in transcriptional dysregulation of developmental programs. RBS arises due to autosomal recessive mutations in cohesin auxiliary factor ESCO2, the gene that encodes an N-acetyltransferase which targets the SMC3 subunit of the cohesin complex. The mechanism that underlies RBS, however, remains unknown. A popular model states that RBS arises due to mitotic failure and loss of progenitor stem cells through apoptosis. Previous findings in the zebrafish regenerating fin, however, suggest that Esco2-knockdown results in transcription dysregulation, independent of apoptosis, similar to that observed in CdLS patients. Previously, we used the clinically relevant CX43 to demonstrate a transcriptional role for Esco2. CX43 is a gap junction gene conserved among all vertebrates that is required for direct cell-cell communication between adjacent cells such that cx43 mutations result in oculodentodigital dysplasia. Here, we show that morpholino-mediated knockdown of smc3 reduces cx43 expression and perturbs zebrafish bone and tissue regeneration similar to those previously reported for esco2 knockdown. Also similar to Esco2-dependent phenotypes, Smc3-dependent bone and tissue regeneration defects are rescued by transgenic Cx43 overexpression, suggesting that Smc3 and Esco2 cooperatively act to regulate cx43 transcription. In support of this model, chromatin immunoprecipitation assays reveal that Smc3 binds to a discrete region of the cx43 promoter, suggesting that Esco2 exerts transcriptional regulation of cx43 through modification of Smc3 bound to the cx43 promoter. These findings have the potential to unify RBS and CdLS as transcription-based mechanisms

Model depicting Cx43-Hapln1a mediated effect on skeletal patterning during fin regeneration.

<p>(A) Proposed pathway for Hapln1a mediated effects of Cx43. Hapln1a functions in a <i>cx43</i>-dependent, but <i>sema3d</i>-independent pathway, positively influencing cell proliferation and inhibiting joint formation. (B) Cartoon illustrating the compartments of gene in the regenerating fin: The <i>cx43</i> mRNA is expressed throughout the mesenchyme (red) accompanied by <i>cx43</i>-dependent <i>hapln1a</i> upregulation in the same compartment (red), but primarily in the distal blastema (blue-red) and to a lesser extent in the proximal skeletal precursor cells (blue). e, epithelium; m, mesenchyme; b, blastema; db, distal blastema; spc, skeletal precursor cells; ble, basal layer of epidermis.</p

<i>hapln1a</i> expression pattern at different time points in regenerating fin.

<p>Whole mount in situ hybridization for <i>hapln1a</i> on regenerating fins at different time points. The <i>hapln1a</i> gene is not expressed during normal fin growth (uncut). Expression of <i>hapln1a</i> initiates around 2 dpa and is maximally expressed at 3 dpa, followed by gradual reduction at 5 dpa and 8 dpa. Arrow identifies the distal end of the fin; the arrowhead identifies region of staining (or where staining would be observed in uncut fins). The amputation plane is indicated for 2 dpa, 3 dpa, and 5 dpa, and is out of the field of view for the 8 dpa image. Scale bar is 50 µm.</p

Primers and Morpholinos.

<p>The T7 RNA polymerase binding site is in bold in the reverse primers. F = Forward primer; RT7 = Reverse primer; MO = targeting morpholino; ATG MO = targets translation initiation; Splice MO = targets splicing event; 5MM = control morpholino with 5 mismatch pairs to target sequence. All primers and MOs are shown in the 5′-3′orientation.</p