Comparing Differentiated Rod And Cone Transcriptomes Reveals Rax As A Controller Of A Defined Cone Gene Regulatory Program

de Prisco, Nicola (2017). Comparing Differentiated Rod And Cone Transcriptomes Reveals Rax As A Controller Of A Defined Cone Gene Regulatory Program. PhD thesis The Open University.

DOI: https://doi.org/10.21954/ou.ro.0000c6f8

Abstract

Purpose
Rods and cones convert light stimuli into signaling information for brain- mediated visual perception. Specific gene mutations primarily affecting both photoreceptor (PRs) types lead to retinal dystrophies, including rod-cone (retinitis pigmentosa) and cone-rod degeneration. Rods and cones are generated from common precursor cells and their differentiation program is strongly regulated by transcription factors (TFs) that control gene networks during development transitions and in the adult to maintain their identity and function.

The aim of this thesis was to investigate post-mitotic transcriptional regulation of rod- and cone-specific gene regulatory networks (GRNs). Specifically, retrieving transcription factor sets has biological relevance and may be instrumental for designing somatic gene transfer methodologies for both gene-based and regenerative therapeutic strategies.

Methods
We developed an Adeno-associated viral (AAV) vector-based transfer method to isolate rods and cones by fluorescence-based sorting. To isolate “pure” rod and cone populations from the same retinal samples we designed a double expression cassette (“double fluo”) encoding mCherry under the transcriptional control of the human G-protein coupled receptor Kinase 1 (GRK1) gene rods and cones-specific promoter elements of, and eGFP under the transcriptional control of rod-specific human Guanine Nucleotide Binding Protein Alpha Transducing (GNAT1) promoter elements. Porcine (Sus scrofa) retinae were injected with AAV 2/8 GRK1-mCherry_hGNAT1-eGFP, at the dose of 1x10¹² genome copies (GC) per eye (n=3). The animals were sacrificed 15 days after injection to evaluate fluorescence reporter based rod and cone photoreceptor identities by histological and FACS analysis. We evaluated, on sorted populations, rods and cone specific transcriptomes by RNA sequencing (RNA- seq) using Illumina Hiseq 1500 (n=3). Moreover, after overexpression of human Retina And Anterior Neural Fold Homeobox (RAX), using subretinal injection of AAV2/8 hGNAT1-hRAX2aeGFP at a dose of 5 x 10¹¹gc/eye we studied the GRN associated with RAX, by a second round of RNA-seq. Electroretinography (ERG) was used to correlate RAX-specific GRN with retina function.

Results
Histological analysis showed unambiguous expression of both mCherry and eGFP in rods, whereas cones showed exclusive expression of mCherry. Furthermore, cone arrestin (CAR) showed exclusive co-localization with the mCherry signal (cones), which was absent in red-green cells (rods). Photoreceptors specifically labeled were positive for both mCherry and eGFP (rods) or mCherry-only (cones) expressing Rhodopsin (RHO), Neural retina leucin zipper (NRL), Guanine Nucleotide Binding Protein Alpha Transducine 1 (GNAT1) (rod markers) and Short wavelength cone opsin (S-opsin), Medium wavelength cone opsin (M-opsin), Guanine Nucleotide Binding Protein Alpha Transducing 2 (GNAT2) (cone markers), respectively.

RNA-seq differential expression analysis (DEA) showed 198 differential expressed genes (DEGs), of which 68 genes were up-regulated and 128 genes down-regulated in rod versus cone analysis. Furthermore, 8 TFs were found differentially expressed in rods and 8 in cones. In the cone-specific TFs set, out of 3 known TFs (Rax, Sall3 and Six6), Rax was selected for further analysis. Rax was ectopically expressed in rods (Rax-rod) to reconstruct its GRN. Differential expression analysis identified 386 DEGs, of which 224 genes (58%) were up- and 162 (42%) down regulated. Clustering for biological processes gene ontology (BP-GO), showed enrichment in action potential genes. Furthermore, ERG showed a different light response of the hRax-treated retina. In particular, a decrease of the mesopic (dark adapted) and an increase of the photopic (light adapted) responses were found.

Conclusion
We showed that the “double fluo” provided a robust method to isolate and cross-compare rod and cone photoreceptors in the adult porcine retina upon photoreceptor somatic gene transfer via AAV vectors. Furthermore, RNA-seq data set analysis showed that the cone and rod specific transcriptome differs quantitatively by about 200 transcripts and that 8 TFs are photoreceptor specific In addition, the data show that post-mitotic rods are able to express cone genes upon Rax ectopic expression. Furthermore the data support a role of Rax in controlling cone-specific action potential gene set.