Spatiotemporal arising of molecular and functional blood-brain barrier properties in the developing Gallus gallus optic tectum

The cerebrovascular endothelium possesses unique properties that strictly regulate the neural microenvironment. While mammalian Blood Brain Barrier (BBB) development is well-characterized, the avian embryo ( Gallus gallus ) offers a unique, highly accessible model for real-time developmental imaging and manipulation. However, the precise molecular and functional timeline of BBB maturation in the avian brain remains poorly explored. Here, we provide the first comprehensive spatiotemporal characterization of BBB development in the Gallus gallus optic tectum (OT). We performed a multi-modal analysis between developmental stages HH32 and HH44, combining transcriptional analysis of BBB-associated genes, confocal and lightsheet microscopy, and quantitative functional assays for permeability and efflux activity. We identified a critical developmental window between stages HH36 and HH40 that marks the transition to a specialized barrier. This period is characterized by the significant upregulation of tight junction proteins ( CLDN5 , OCLN ) and efflux transporters ( ABCB1 ), accompanied by the downregulation of fenestration markers ( PLVAP ). Also, tight junction assembly protein ZO-1 undergoes a progressive redistribution from a discontinuous pattern to continuous linear expression along the vessel walls during this period. Functionally, we demonstrate that paracellular restriction to 10 kDa dextran develops in a region-dependent manner, while quantitative assays reveal a sharp reduction in Evans Blue permeability starting at HH38. Lastly, we detected the onset of P-glycoprotein efflux activity from HH36 onwards. Altogether, our findings redefine the chicken embryo as a tractable model for barrier genesis research, pinpointing the HH36-40 developmental window as the discrete period where the molecular and functional BBB properties are acquired. This establishes a robust baseline for future mechanistic investigations into neurovascular development and pathology.