Role of Neural Crest Cells in Establishing Corneal Transparency During Embryonic Development in Mice

Corneal transparency emerges during embryogenesis through the coordinated organization of neural crest-derived cells (NCCs) and extracellular matrix (ECM), yet the temporal and structural basis of this process remains incompletely defined. Here, we provide a multimodal, spatiotemporal analysis of mouse corneal development from embryonic Day 10 (E10) to birth (P0), combining whole-mount and sectioned immunofluorescence, tissue clearing with three-dimensional (3D) imaging, second harmonic generation (SHG) microscopy, full-field optical coherence microscopy (FFOCM), and transmission electron microscopy (TEM). We show that early periocular mesenchyme is characterized by broad expression of neural crest-associated markers (Sox9, HNK1), followed by progressive spatial restriction and downregulation as cells populate the corneal stroma. Sox10-positive cells remain primarily associated with developing nerves and are largely excluded from the stromal compartment. Concomitantly, stromal cells undergo marked morphological transitions, from rounded to progressively flattened and elongated phenotypes, accompanying stromal expansion. Collagen deposition is first detected in the subepithelial region around E12 and increases thereafter, with SHG and TEM analyses revealing progressive organization and compaction of fibrillar networks. Quantitative ultrastructural analysis indicates dynamic changes in interfibrillar spacing during development, consistent with ongoing ECM remodeling. The emergence of a posterior-to-anterior gradient in cell morphology and matrix organization suggests a spatially coordinated maturation process across the stroma. Together, these findings provide an integrated structural framework linking NCCs dynamics, stromal cell differentiation, and ECM organization during murine corneal development. This work establishes a quantitative and multiscale atlas of corneal morphogenesis that informs how tissue architecture compatible with transparency is progressively established in vivo.