A gal4 insertion in the rx3 locus as a tool for visualization and manipulation of eye fated cells in zebrafish

The rx3 gene encodes a transcription factor essential for eye development in zebrafish. Despite its importance, the spatiotemporal dynamics of gene expression during embryogenesis are not fully understood. This knowledge gap, coupled with the limitations of existing genetic tools, highlights the need for novel and more faithful reporters. In this study, we characterized a new transgenic zebrafish line with an insertion of gal4 transcriptional activator in the second exon of the rx3 gene, named tg(gSAIzGFFD2459B), to visualize and analyse gene function. In heterozygous embryos, the gal4 insertion drives gene expression to label eye-fated progenitor cells, providing a valuable tool for dissecting the mechanisms underlying eye formation. Conversely, homozygous gal4 insertion results in a fully penetrant eyeless phenotype, allowing for the continued tracking of rx3-expressing cells within the developing brain. This transgenic line permits intersectional labelling with other UAS-driven transgenes, enabling further exploration of gene function. Remarkably, unlike previously published rx3 reporters that use medaka gene regulatory sequences, our line lacks ectopic expression and provides a more faithful recapitulation of endogenous rx3 expression. The tg(gSAIzGFFD2459B) line represents a versatile platform for both studying the developmental gene regulation of eye-fated cells and enhancing our understanding of genetic determinants in vertebrate organogenesis. By reporting rx3 transcriptional activity in both wild-type and mutant states, this transgenic tool supports the critical role of rx3 in eye morphogenesis and offers new avenues for investigating congenital disorders of the eye and brain.