Melanopsin mRNAs isoforms in the rabbit retina

Abstract

Purpose : Rabbit pups (kits) are a natural model for synchronization of rhythms by food and its posterior shift to photoentrainment. Because the activity of the kits is not synchronized with the light-dark cycle, but by the breastfeeding from the mother, is interesting whether any photoreceptor involved in the photoentrainment can be expressed at the early stages of development. We previously provided evidence on the topic by showing that OPN4 gene expression reaches high levels before Thy1, the marker gene for ganglion cells. This could mean that the cells expressing melanopsin are primarily due to as photoreceptors rather than being projecting neurons. If this would be the case, melanopsin might be constantly expressed at all early stages and probably by some cell types aside ganglion cells within the rabbit retina. To elucidate this question we wanted to design a tool for studying the OPN4 mRNA location at the cell level, as a first result, we obtained evidence for the occurrence of different OPN4 mRNA isoforms in this tissue

Methods : We obtained 8 eyes from two months old rabbits, which were used in physiology experiments not related with this project. RNA was isolated from each retina and RT-PCR was assayed. The primers used were designed from exons 3 to 10 as well as exons 4 to 10 in the deduced rabbit OPN4 gene sequence according to GenBank, primers for Thy1 PCR amplification spanned exon 3, GADPH gene was used as a PCR control. All OPN4 PCR products were excised from gel, cloned in pGEMTeasy™ and sequenced. In silico analysis was performed with BioEdit and MEGA software.

Results : Sequence analysis showed that Thy1 and GAPDH genes were amplified from all samples. RT-PCR for OPN4 gene resulted in two products using either primer mix, 3-10 or 4-10; a longer one consisting of 1180 bp and 1037 bp, as well as a shorter of 1067 pb and 924 bp, respectively. All of them were confirmed as OPN4 gene by sequence alignment compared to that from GenBank. In silico analysis showed that the shorter PCR products lack exon 8 sequence, corresponding to a 37 aminoacids deletion at the C-terminus.

Conclusions : In the rabbit retina there are two mRNAs encoding OPN4, this raises the possibility of the occurrence of melanopsin isoforms as has been found for rodent retinae. The question arises regarding whether the different OPN4 mRNAs are expressed by several cell types.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.