A minimal tooth enhancer regulates dlx2b expression during zebrafish tooth formation: insights into cis -regulatory logic in organogenesis

Despite growing recognition of the importance of cis -regulatory elements in vertebrate development, the mechanisms by which enhancers control gene expression during organogenesis remain incompletely understood. To address this gap, we investigated the regulation of the transcription factor dlx2b during zebrafish larval tooth formation. Using CRISPR/Cas9-mediated genome editing, we generated a GFP knock-in line that recapitulates dlx2b expression in developing tooth germs. Through targeted manipulation of enhancer sequences, we identified a minimal tooth enhancer (MTE), which is sufficient to drive most of the endogenous dlx2b tooth germ expression pattern in vivo . Functional dissection of the MTE revealed that four evolutionarily conserved transcription factor binding sites are essential for enhancer activity. Mutating these sites within a transgenic reporter abolishes enhancer-driven expression, while deletion of the same sequences at the endogenous dlx2b locus causes a dramatic shift in the gene’s expression pattern. These findings suggest that loss of MTE function permits alternative cis -regulatory elements to gain control of the promoter, highlighting the dynamic nature of enhancer-promoter interactions during development. Together, these results uncover fundamental principles of enhancer function during vertebrate organogenesis and demonstrate the power of empirical dissection in decoding cis -regulatory architecture.