Purpose: To create the Tg(ccnb1:EGFP)(nt18) zebrafish line that spatially and temporally labels retinal progenitor cells with enhanced green fluorescent protein (EGFP) during zebrafish retinal development and regeneration.
Methods: We cloned the 1.5 kb promoter region of the zebrafish cyclin B1 (ccnb1) gene upstream of the EGFP gene in the Tol2 vector, which was used to generate the stable Tg(ccnb1:EGFP)(nt18) transgenic zebrafish line. Immunohistochemistry and in situ hybridization techniques verified that the ccnb1:EGFP transgene was expressed in retinal progenitor cells during retinal development, in the undamaged adult retina, and in the regenerating adult retina.
Results: At 36 h post-fertilization, both the enhanced green fluorescent protein (EGFP) and proliferating cell nuclear antigen (PCNA) expressions were observed throughout the developing transgenic retina, but they became restricted to the circumferential marginal zone by five days post-fertilization. In situ hybridization confirmed that this EGFP expression matched the cyclin B1 mRNA expression pattern. In comparison to the Tg(1016a1tubulin:EGFP) transgenic line that expresses EGFP in neuronal progenitor cells, the Tg(ccnb1:EGFP)(nt18) line more faithfully follows the rise and fall of PCNA expression through the developing retina and brain. In the adult retina, there are three cell types that continue to proliferate, the Müller glia in the inner nuclear layer, the rod precursor cells in the outer nuclear layer, and the stem cells in the circumferential marginal zone. In the Tg(ccnb1:EGFP)(nt18) retina, EGFP coexpressed with PCNA in all three of these proliferating cell types. Exposing the adult retina to constant intense light destroys the rod and cone photoreceptors and induces an increase in the number of proliferating Müller glia, which produces actively dividing neuronal progenitor cells that migrate to the outer nuclear layer (ONL) and replenish the lost photoreceptors. Following constant light damage, Tg(ccnb1:EGFP)(nt18) zebrafish expressed EGFP in both the proliferating Müller glia and the migrating neuronal progenitor cells.
Conclusions: The spatial and temporal patterning of EGFP expression in the Tg(ccnb1:EGFP)(nt18) line directly reflects the known locations of proliferating cells in the zebrafish retina, making it a useful marker to study the transient nature of neuronal progenitor cells during the development and regeneration of the zebrafish retina.