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

Abstract

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.

Simplet-dependent regulation of β-catenin signaling influences skeletal patterning downstream of Cx43

“…We use the percentage similarity method to determine whether the gene-targeting MO has an effect (i.e. Banerji et al, 2017;Govindan et al, 2016). With this method, we take the ratio of the injected side over the uninjected side and multiply by 100, and we compare these values for the Smp-MOand for the STD-MO-treated fins (values close to 100% indicate little effect of the MO; values with low similarity indicate that the MO had an effect).…”

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

“…Using both MOs we carried out microinjection and electroporation as previously described ( Govindan et al, 2016 ; Banerji et al, 2016 ) ( Fig. 2 D).…”

“…To reduce the effect of fin-to-fin variation, we utilized the percent similarity method in which values close to 100% indicates no difference between injected and non-injected sides of the same fin. Values less than 100% indicate reduced growth of the injected fin side compared to the non-injected side of the same fin, whereas values greater than 100% indicate increased growth of the injected fin side compared to the non-injected side ( Govindan et al, 2016 ; Bhadra and Iovine, 2015 ; Banerji et al, 2016 ). Quantification of regenerate length was based on the distance from the plane of amputation to the distal end of the 3rd fin ray.…”

Connexin Communication Compartments and Wound Repair in Epithelial Tissue

“…In contrast, when Cx43 function is suppressed, cell proliferation is also suppressed because Neurpilin2a is activated, and joint formation is activated by negative regulation of PlexinA3. Furthermore, with regard to cell proliferation, Hela1n1a protein is involved in the stabilisation of Sema3d function [ 152 , 153 ].…”