Transcriptional autoregulation of zebrafish tbx6 is required for somite segmentation.

The presumptive somite boundary in the presomitic mesoderm (PSM) is defined by the anterior border of the expression domain of Tbx6 protein. During somite segmentation, the expression domain of Tbx6 is regressed by Ripply-meditated degradation of Tbx6 protein. Although the expression of zebrafish tbx6 remains restricted to the PSM, the transcriptional regulation of tbx6 remains poorly understood. Here, we show that the expression of zebrafish tbx6 is maintained by transcriptional autoregulation. We find that a proximal-located cis-regulatory module, TR1, which contains two putative T-box sites, is required for somite segmentation in the intermediate body and for proper expression of segmentation genes. Embryos with deletion of TR1 exhibit significant reduction of tbx6 expression at the 12-somite stage, although its expression is initially observed. Additionally, Tbx6 is associated with TR1 and activates its own expression in the anterior PSM. Furthermore, the anterior expansion of tbx6 expression in ripply gene mutants is suppressed in a TR1-dependent manner. The results suggest that the autoregulatory loop of zebrafish tbx6 facilitates immediate removal of Tbx6 protein through termination of its own transcription at the anterior PSM.

Functional roles of the Ripply-mediated suppression of segmentation gene expression at the anterior presomitic mesoderm in zebrafish.

Somites sequentially form with a regular interval by the segmentation from the anterior region of the presomitic mesoderm (PSM). The expression of several genes involved in the somite segmentation is switched off at the transition from the anterior PSM to somites. Zebrafish Ripply1, which down-regulates a T-box transcription factor Tbx6, is required for the suppression of segmentation gene expression. However, the functional roles of the Ripply-mediated suppression of segmentation gene expression at the anterior PSM remain elusive. In this study, we generated ripply1 mutants and examined genetic interaction between ripply1/2 and tbx6. Zebrafish ripply1-/- embryos failed to form the somite boundaries as was observed in knockdown embryos. We found that somite segmentation defects in ripply1 mutants were suppressed by heterozygous mutation of tbx6 or partial translational inhibition of tbx6 by antisense morpholino. We further showed that somite boundaries that were recovered in tbx6+/-; ripply1-/- embryos were dependent on the function of ripply2, indicating that relative gene dosage between ripply1/2 and tbx6 plays a critical role in the somite formation. Interestingly, the expression of segmentation genes such mesp as was still not fully suppressed at the anterior PSM of tbx6+/-; ripply1-/- embryos although the somite formation and rostral-caudal polarity of somites were properly established. Furthermore, impaired myogenesis was observed in the segmented somites in tbx6+/-; ripply1-/- embryos. These results revealed that partial suppression of the segmentation gene expression by Ripply is sufficient to establish the rostral-caudal polarity of somites, and that stronger suppression of the segmentation gene expression by Ripply is required for proper myogenesis in zebrafish embryos.