Clock-driven waves of Tbx6 expression prefigure somite boundaries
One of the major events of vertebrate embryonic development is the segmentation of the body axis into transient blocks of tissue, called somites, that ultimately give rise to vertebrae, ribs, and skeletal muscles of the adult body. Somites bud off from the presomitic mesoderm (PSM) by the sequential and rhythmic formation of new boundaries. The periodicity of the process is controlled by the segmentation clock, which manifests as spatiotemporal waves of Hes/Her gene expression travelling anteriorly in the PSM and reiterating during the formation of each somite. In zebrafish, the genes her1 and her7 form a the core of this genetic oscillator. We discovered that the protein expression of the gene Tbx6 oscillates, driven by the Her1;Her7 loop. Although Her binding to gene regulatory sequences has been tested in vitro, the binding of Her1 or Her7 to regulatory regions in the embryo remains unknown. To assess whether Her1 and Her7 directly bind to tbx6, we performed CUT&Tag using anti-GFP antibodies to address the genomic binding landscape of the fusion proteins Her1-YFP and Her7-YFP from their respective transgenic lines, as no antibodies against Her1 and Her7 currently exist. Overall design: We performed Cut&Tag on Tg(her1:her1-YFP) or Tg(her1:nls-mkate2,her7:her7-YFP) zebrafish embryos. Briefly, 120 bud stage embryos per replicate were dechorionated using 1mg/ml Pronase (Roche) and dissociated into single cells by pipetting up and down using a 200 µl pipette. For each transgenic line, we performed two replicates with two different antibodies.