Abstract
Purpose :
Zebrafish possess a retinal zone with specialized neuronal circuits that enable high acuity vision similar to the human macula. The processes that govern the development of the high acuity zone (AZ) are currently unknown. Here, we tested whether retinoic acid signaling plays a role.
Methods :
Transgenic Tg[opn1sw1:GFP] zebrafish at 72 hours post fertilization (hpf) were exposed to the retinoic acid receptor (RAR) α agonist, AM580 or its vehicle, DMSO, until 120 hpf. Subsequently, confocal images were acquired of the temporal (includes the AZ), dorsal and nasal eye immunofluorescently labeled for UV cone opsin, which was used to measure the outer segment (OS) length. Eyes were also collected for RNA isolation to assess expression levels of known RAR targets (cyp26b1, nr2f5, srfp1a) by qRT PCR. Moreover, hybridization chain reactions (HCR) were performed for cyp26a1 and cyp26c1 on Tg[TP1:venus] or Tg[opn1sw1:GFP] zebrafish at 48, 72 or 120 hpf.
Results :
GFP-positive UV cone photoreceptors were present at 120 hpf in the AZ, dorsal and nasal regions of DMSO and AM580-treated Tg[opn1sw1:GFP] zebrafish eyes. However, the UV cone OS length was largely reduced in the AZ and to a lesser extend in the nasal region of AM580-treated eyes relative to DMSO controls, while the OS length did not differ in the dorsal retina. RARα activation by AM580 significantly upregulated cyp26b1, nr2f5 and srfp1a expression levels; however, their role in OS development is unclear. To assess how retinoic acid levels might be spatially regulated we determined the expression patterns of cyp26a1 and cyp26c1, genes encoding RA degrading enzymes. Expression of cyp26a1 was first observed in a subset TP1:venus-positive Müller glia in the temporal region at 60 hpf. At 72 and 120 hpf, a subset of Müller glia throughout the entire retina were cyp26a1-positive; however, labeling was more biased towards the temporal and nasal regions. In contrast, cyp26c1 was restricted to a small area in the nasal and temporal regions from 48 hpf. Co-labeling of cyp26c1 with TP1:venus from 72 hpf indicates expression in Müller glia. Both, cyp26a1 and cyp26c1 were not observed in Tg[opn1sw1:GFP]-positive UV cones.
Conclusions :
Reduced UV cone OS length following RARα activation suggests that low/absent RARα signaling is necessary for proper OS development predominantly in the AZ. RA availability in the AZ is potentially regulated by Müller glial cyp26a1 expression.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.