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

Abstract

The correct positioning of joints in the vertebrate skeleton is not well understood. Mutations in connexin43 (cx43) cause the short segment phenotype of the zebrafish short fin (sof b123) mutant. We have shown that Cx43 suppresses evx1 expression, a transcription factor required for joint formation. Here, we provide novel insights into how Cx43 influences evx1 transcription. First, we find that Simplet (Smp) knockdown recapitulates the sof b123 phenotypes of reduced regenerate length and reduced segment length, and we find evidence for synergy between cx43 and smp. Moreover, knockdown of Smp increases the evx1 expression, similar to cx43 knockdown. Previous studies have shown that Smp is required for the nuclear localization of β-catenin. Indeed, β-catenin activity is required for segment length, and is reduced in both sof b123 mutants and following Smp knockdown in regenerating fins. We further show that blocking canonical Wnt signaling results in a synergistic reduction in segment length in sof b123/+ heterozygotes. Together, our findings suggest that both Smp and β-catenin function in a common molecular pathway with cx43 to influence both evx1 expression and joint location.

Connexin43 gap junctional intercellular communication inhibits evx1 expression and joint formation in regenerating fins

“…To minimize fin to fin variation, we used the percentage similarity method to determine whether the peptide had an effect (i.e. Bhattacharya et al, 2018;Govindan et al, 2016). We measured the ratio of the injected side to the uninjected side reported as a percentage.…”

“…Previously, we identified both Smp and Sema3d pathways acting downstream of Cx43 during joint formation (Bhattacharya et al, 2018;Ton and Iovine, 2012). To address whether Smp and Sema3d function in a common pathway downstream of Cx43, we tested for evidence of synergy by co-injecting subthreshold doses of both gene-targeting morpholinos (MOs).…”

“…Together, these studies indicate that Cx43 suppresses joint formation, and that the short segment phenotype of sof b123 is the result of premature joint formation. We have suggested that the gap junction protein Connexin 43 (Cx43) influences the specification of cell fate in SPCs by regulating the expression of evx1 (Dardis et al, 2017;Bhattacharya et al, 2018). Thus, high Cx43 favors the differentiation of SPCs into osteoblasts (and evx1 is reduced), whereas lower Cx43 favors the differentiation of SPCs into joint-forming cells (and evx1 is increased) (Dardis et al, 2017).…”

“…Sema3d is a secreted growth factor; in our previous study we suggested that Sema3d interacts with the plexin A3 receptor to influence evx1 expression in SPCs. More recently, we showed that simplet (smp) is molecularly and functionally downstream of cx43 (Bhattacharya et al, 2018). Smp protein brings β-catenin into the nucleus to regulate gene expression (Kizil et al, 2014).…”

“…Smp protein brings β-catenin into the nucleus to regulate gene expression (Kizil et al, 2014). Indeed, β-catenin signaling is also downstream of cx43 and contributes to determining segment length (Bhattacharya et al, 2018). Although cx43 acts upstream of both sema3d and smp, it is notable that these genes are not expressed in the same cellular compartments.…”