Genetic analysis of the developmental potential of photoreceptor progenitors in zebrafish

Abstract

Purpose : In zebrafish, mutations of tbx2a or tbx2b result in a cell fate switch of UV-sensitive cone progenitors into rods. Loss of thrb2 results in the absence of red-sensitive cones and an increased number of UV-sensitive cones. This project addresses the developmental potential of photoreceptor progenitors by gene targeting of multiple transcription factors.

Methods : One-cell stage wildtype and tbx2b^p25bbtl (lor) embryos were injected with gRNA/Cas9 complex targeting exon 1 and 2 of tbx2a or exon 1 of thrb2. UV cones were visualized using the Tg(-5.5sws1:EGFP) reporter line. Other cell types were visualized by immunolabeling. Images were captured using a Nikon confocal microscope. Photoreceptors numbers were quantified in a 3500 µm² area near the optic nerve, and data analyzed using an ANOVA with type II sum of squares on a linear model.

Results : Germline mutations of tbx2a were recovered in exons 1 or 2 and are predicted to cause premature stop codons upstream of the DNA binding domain; large deletions spanning from exon 1 to exon 2 were recovered and are predicted to disrupt the DNA binding domain. Compound heterozygous and homozygous tbx2a^-/- mutant larva (n=5) had intermediate numbers of UV cones (59.7 ± 33.9, p=0.0003) and rods (28.6 ± 13.2, p=0.4) when compared to wildtype (n=6, UV= 107.2 ± 13.9; rod=14.3 ± 17.7) and tbx2b^p25bbtl (n=7, UV= 2.8 ± 2.0, p<0.0001; rod=125.2 ± 20.1, p<0.0001). The presence of a single tbx2b^p25bbtl allele on a tbx2a^-/- background resulted in a further reduction in UV cones. Larvae homozygous for tbx2a^-/- and tbx2b^p25bbtl showed a complete absence of UV cones and a significant interaction between tbx2a and tbx2b affecting rod number (91.5 ± 9.0, n=6, p=0.002). The larvae also displayed an unexpected otolith phenotype. No changes in immunolabeling for other cell types were observed. Gene targeting of thrb2 on either the tbx2b^p25bbtl or tbx2a^-/-/ tbx2b^p25bbtl background resulted in loss of red cones and increased numbers of UV cones and rods.

Conclusions : The lack of UV cones and an otolith phenotype suggest functional redundancy of tbx2a/tbx2b in photoreceptor and auditory development. Targeting thrb2 suggests that tbx2 is dispensable for UV cone specification, and that the red cone progenitors have the potential to differentiate as UV cones and rods. Ongoing work tests the mechanisms of tbx2a and tbx2b interaction in photoreceptor development.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.