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

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 (sofb123) 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 sofb123 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 sofb123 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 sofb123/+ 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.