Determining the Migration Behavior of Retinal Progenitor Cells in the Embryonic Eye Field of Xenopus laevis

Abstract Given the critical role of progenitor cells staying within the eye field transcription factor (EFTF) signaling niche for normal eye development, we hypothesized that retinal progenitor cells (RPCs) differentiate within their initial region of inception during eye development. To investigate this, we utilized EosFP, a photoconvertible protein, as a lineage tracer in the model organism Xenopus laevis. By employing confocal laser microscopy for photoconversion, we labeled cells within elongated rectangular regions that encompassed both the eye field and the adjacent tissues. In a separate set of embryos, we identified which portions of these rectangular regions harbored cells destined to become part of the mature eye versus those that would form the surrounding tissues, tracing their development from stage 15 to stage 35. This allowed us to create a fate map of the stage 15 embryo using EosFP to accurately locate and label the eye field to address our hypothesis. With the eye field delineated using our lineage tracer, we further employed EosFP to label RPCs within individual quadrants of the developing eye. Tracking these RPCs from stage 15 to stage 35, we observed the retinal cells organizing into three principal layers of cell bodies, mirroring the layered neuroanatomy characteristic of the mature retina. We observed the red-labeled RPCs proliferated but remained predominantly within their quadrant of inception, with no dispersion into other, unlabeled quadrants of the eye by stage 35. These findings corroborate our hypothesis that RPCs undergo differentiation within their initial locations in the eye field. Our study illuminates the cellular dynamics of eye development in Xenopus laevis and introduces a novel method for lineage tracing of stem cell populations during embryonic development. Competing Interest Statement The authors have declared no competing interest. Footnotes Updated to reflect sole authorship. The previous co-author has been removed at the corresponding author's discretion. Minor methods/citation updates added: (1) a cross-reference to the related protocol paper (Grell, in preparation) at the start of the Methods "Injection of Photoconvertible Lineage Tracer" section; (2) a citation to the now-published companion preprint (Grell, 2026, doi: 10.64898/2026.05.03.722558) in the Discussion to support a developmental-normalcy observation, with corresponding reference list update. No data, figures, or interpretive content have been changed.