Antagonistic regulation of homeologous uncx.L and uncx.S genes orchestrates myotome and sclerotome differentiation in the evolutionarily divergent vertebral column of Xenopus laevis

In anurans, the vertebral column diverges widely from that of other tetrapods; yet the molecular mechanisms underlying its morphogenesis remain largely unexplored. In this study, we investigate the role of the homeologous uncx.L and uncx.S genes in the vertebral column morphogenesis of the allotetraploid frog Xenopus laevis. We initiated our study by cloning the uncx orthologous genes in the anuran Xenopus and determining their spatial expression patterns using in situ hybridization. Additionally, we employed gain-of-function and loss-of-function approaches through dexamethasone-inducible uncx constructs and antisense morpholino oligonucleotides, respectively. Comparative analysis of the messenger RNA sequences of homeologous uncx genes revealed that the uncx.L variant lacks the eh1-like repressor domain. Our spatial expression analysis indicated that in the presomitic mesoderm and somites, the transcripts of uncx.L and uncx.S are located in overlapping domains. Alterations in the function of uncx genes significantly impact the development and differentiation of the sclerotome and myotome, resulting in axial skeleton malformations. Our findings suggest a scenario where the homeologous genes uncx.L and uncx.S exhibit antagonistic functions during somitogenesis. Specifically, uncx.S appears to be crucial for sclerotome development and differentiation, while uncx.L primarily influences myotome development. Postallotetraploidization, the uncx.L gene in X. laevis evolved to lose its eh1-like repressor domain, transforming into a “native dominant negative” variant that potentially competes with uncx.S for the same target genes. Finally, the histological analysis revealed that uncx.S expression is necessary for the correct formation of pedicles and neural arch of the vertebrae, and uncx.L is required for trunk muscle development.