Abstract
Purpose :
The molecular and cellular mechanisms governing the formation of a high acuity area (HAA), also known as fovea, during retinal development remain mostly unknown. It has previously been described that retinoic acid (RA) is modulated during development of the chick HAA by exclusion of RA in the central retina (presumptive HAA), a critical process for complete absence of rods and concomitant peak cellular density in the ganglion cell layer in this area. While the highest density of retinal ganglion cells (RGCs) is a well- established attribute of mature HAAs, little is known about progression and acquisition of this RGCs peak density during development.
Methods :
To identify any HAA-specific attributes regarding RGCs during retinal development, chick retinas across multiple time points were used (embryonic day E4.5, E6, E12 and E20). As a first step, all-trans RA was injected vitreously in ovo at E4.5 (predicted “foveogenesis” period) followed by bulk RNAseq analysis; this pharmacological-genetic approach could reveal RGCs specific gene expression responses after RA treatment. Following this, longitudinal immunofluorescence analyses were conducted by the use of unreported RGCs antibodies and evaluated by advanced imaging of flat-mounts reconstructions, culminating in the acquisition of RGCs isodensity maps and subsequent examinations.
Results :
Bulk RNASeq experiments comparing all-trans RA- and DMSO-injected retinas were carried out by dissection of central retinas 16 hours after in ovo injections, revealing differentially expressed genes (fold change>1.5) mostly related to RGCs differentiation on both directions, either up- or downregulated. Longitudinal immunofluorescence studies on control flat-mount retinal preparations revealed a limited number of antibodies, such as Islet 1/2 and Satb2, labeling the RGCs at early time points (E6), while a more robust and general expression with multiple markers is present at both mid- (E12) and late-stage retinas (E20). In addition, through evaluation of isodensity maps, we detected an enriched cellular area in centro-ventronasal region using different RGCs markers starting at E12, which moves towards a more centronasal region (presumptive HAA) in late stage retinas.
Conclusions :
By longitudinal evaluation of RGC population on flat-mounts of developing chick retinas using various markers, our study reveals the progression and acquisition of RGCs peak density at the HAA.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.