Expression patterns of CYP26A1, FGF8, CDKN1A , and NPVF in the developing rhesus monkey retina

**Background:** The fovea centralis is a specialized region of the primate retina responsible for high-acuity vision, characterized by a high density of cone photoreceptors and no rods. The mechanisms directing foveal development are poorly understood, partly due to the lack of a fovea in common animal models. Recent transcriptomic studies have identified genes enriched in the developing primate macula, including CYP26A1, FGF8, CDKN1A, and NPVF. In chickens, the retinoic acid pathway regulates FGF8 expression to direct development of a rod-free area. However, the cellular expression patterns and dynamics of these genes in the developing primate retina were unknown. This study systematically characterizes the expression of these genes in the rhesus monkey retina from the first to third trimester.

**Methods:** Fetal rhesus monkey retinas were collected at gestational ages spanning the first (40, 50 days), second (60, 75, 80, 110 days), and third (140, 145 days) trimesters. Samples were fixed, paraffin-embedded, and sectioned. RNAscope in situ hybridization was used to detect CYP26A1, FGF8, CDKN1A, and NPVF mRNA. Immunohistochemistry was performed with antibodies against cell-type markers (e.g., BRN3 for RGCs, CRX for photoreceptors, RLBP1 and glutamine synthetase for Müller glia, PCNA for proliferating cells). Hematoxylin and eosin staining assessed retinal morphology. EdU labeling identified proliferating cells. Quantifications of retinal length and ganglion cell layer thickness were performed using Student's t-test.

**Key Results:** - The temporal retina (foveal region) was larger than the nasal retina at 40 days (1.93-fold, p=0.001) and had a thicker ganglion cell layer (p=0.011). - Neurogenesis followed a fovea-to-periphery gradient: RGC genesis completed by 50 days in the fovea, while bipolar cells and Müller glia appeared in the foveal center by 50 days. ATOH7 was downregulated in the fovea by 50 days. - CYP26A1 was highly expressed in foveal progenitors (PCNA+ and EdU+) at 40 and 50 days, with lower expression in the nasal retina and around the optic nerve head. At later stages (110-140 days), CYP26A1 expression was restricted to RLBP1+ glutamine synthetase+ Müller glia in the macula (fovea, parafovea, perifovea), but not in peripheral Müller glia. - FGF8 was expressed in a gradient from the optic nerve head to the periphery at early stages, not enriched in the fovea. At later stages, low levels were detected in the ONL and INL. - CDKN1A expression began at 50 days in the foveal center and optic nerve head, colocalizing with RLBP1. By 75 days, it extended throughout the temporal retina, and by 140 days, it was expressed in Müller glia across the entire retina. - NPVF expression began at 50 days in a few cells at the macula center, colocalizing with RLBP1 and GS. Expression extended laterally over time, remaining predominantly in the temporal retina by 140 days.

**Clinical Implications:** This study provides critical insights into the molecular and cellular dynamics of foveal development in a nonhuman primate model. The finding that CYP26A1 is expressed in foveal progenitors and later in macular Müller glia suggests a role for retinoic acid metabolism in foveal specialization. The lack of fovea-specific enrichment for FGF8, CDKN1A, and NPVF indicates that these genes are not exclusive foveal markers but are associated with Müller glia maturation. These results highlight the unique molecular identity of macular Müller glia, which may have implications for understanding retinal diseases affecting the macula, such as age-related macular degeneration. The data also underscore the developmental advancement of the fovea and provide a foundation for future studies on the mechanisms of foveal patterning and potential therapeutic strategies for retinal repair.