A unified developmental timeline of pigmented ocular tissues in the mouse eye.

Pigmented tissues of the vertebrate eye are essential for optical performance, metabolic support, and tissue homeostasis, yet their coordinated development has been difficult to study because melanin interferes with histological and immunohistochemical analyses. Here, we apply a gentle hydrogen peroxide based bleaching method compatible with IHC for Rpe65, Rhodopsin, ZO-1, Cralbp/Rlbp1, S100b, and Cd34, and examined Dct/Tyrp2 expression, to generate a unified developmental atlas of all pigmented tissues in the mouse eye. We also re-analyzed published single-cell RNA-seq datasets from developing and adult mouse eyes to examine gene expression patterns across pigmented ocular tissues. Using daily samples from embryonic day 14.5 (E14.5) through postnatal day 14 (P14), we tracked the emergence and maturation of pigmented cell populations in the retinal pigment epithelium (RPE), choroid, ciliary body, and iris. The RPE, the earliest tissue to synthesize melanin, does not acquire continuous Rpe65 expression, apical-basal polarity, or full morphological maturity until eyelid opening. Neural crest-derived melanocytes in the choroid, ciliary body and iris undergo distinct maturation phases marked by early Dct expression, subsequent melanin synthesis, and late induction of S100b. In the innermost layer of the future iris epithelium, early Dct expression precedes postnatal pigmentation. Notably, we observe a coordinated burst of melanogenesis across multiple ocular and periocular tissues immediately after birth, suggesting regulation by systemic developmental transitions rather than light exposure. Together, these findings establish a comprehensive timeline for the maturation of pigmented ocular tissues, providing a clearer framework for studying eye development and disease.