Abstract
Purpose :
Cell specification is a fundamental process that underlies the formation of tissues and organs. In the retina, photoreceptor specification must coordinate with development of other retinal cells and eyecup formation. Photoreceptors have defined subtypes which differ in spectral sensitivity, morphology, wiring, and gene expression. Transcription factors (TFs) modulate genetic programs to specify each photoreceptor subtype. Seminal work in mice identified key TFs required for the determination of rods (Mears, 2001, Cheng, 2006) and L cones (Ng, 2001, Roberts, 2005) but work in other species has highlighted that photoreceptor fate is complex and our understanding is still incomplete (Oel et al., 2020; Ochi et al., 2004, Alvarez-Delfin, 2009; Duval et al., 2018; Ogawa at al., 2019). The purpose of our research is to identify mechanisms that control photoreceptor fate during development.
Methods :
We performed deep transcriptomic profiling (RNAseq) of photoreceptors—by manually collecting pools of the five photoreceptor subtypes of the adult zebrafish retina—to determine which transcription factors are differentially expressed. Then we developed a platform to perform CRISPR-based reverse-genetic screens to identify which transcription factors control photoreceptor fate determination.
Results :
We identified Tbx2 as a critical TF for photoreceptor fate determination. In zebrafish, tbx2b is implicated in UV-cone specification (Alvarez-Delfin, 2009): mutations in this gene lead to loss of UV cone progenitors, which are routed to become rods. tbx2 was duplicated in teleosts, and RNAseq revealed that tbx2b is expressed by UV and S cones, and tbx2a is expressed by UV and L cones. TBX2 is highly conserved across vertebrates and is highly expressed by S cones in primate (Peng, 2019) and by UV cones in chicken (Yamagata, 2021). Using FØ-CRISPR screening techniques (Hojishima, 2019), we generated tbx2b mutant zebrafish larvae that recapitulate the known phenotype. Surprisingly, tbx2a mutants display the same phenotype, suggesting that tbx2b and tbx2a act independently and are both required for UV-cone specification. Furthermore, we found that tbx2a acts in L cones to represses M-cone fate (Sandkam et al., 2020) and that tbx2b acts in S cones to also repress M-cone fate.
Conclusions :
This early success substantiate these methods as an efficient and flexible platform to study mechanisms of fate determination in photoreceptors.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.