The developing neurovascular anatomy of the embryo: A technique of simultaneous evaluation using fluorescent labeling, confocal microscopy, and three-dimensional reconstruction

The close spatial relationship between peripheral nerves and blood vessels in the adult is well known. However, evidence supporting the congruent deve lopment of these structures in embryos remains anecdotal. Neurovascular rel ationships also have been shown to be conserved in other vertebrates. This homology suggests that either peripheral nerves or blood vessels, or both, might have fundamental morphogenetic roles during embryologic development. Both peripheral nerves and blood vessels have been independently implicated as etiologic agents in the pathogenesis of congenital disabilities, and se veral congenital anomalies fit their distribution patterns. This article pr esents a technique for the simultaneous visualization of peripheral nerves and blood vessels at different stages in the developing embryo. The forelim bs of 310 quail embryos were dissected over a 1-year period. Peripheral ner ves were labeled with the neural crest and axon antibody, HNK-1, followed b y fluorescein-conjugated secondary antibodies. Blood vessels were labeled b y a perfusion technique using the fluorescent dye, dioctadecyl-tetramethyli ndocarbocyanine. Specimens were processed and imaged in whole-mount with co nfocal microscopy, and images were reconstructed using three-dimensional mo deling software. Both nerves and blood vessels seem to undergo a highly ste reotypic sequence of development in the embryonic quail forelimb. Furthermo re, the existence of a close spatial relationship between nerves and blood vessels suggests either a high degree of developmental interdependence or s hared patterning mechanisms. This technique permits further evaluation of t he possible role peripheral nerves and blood vessels might play in the path ogenesis of congenital disabilities and provides a starting point for furth er studies aimed at elucidating the means by which peripheral nerves and bl ood vessels are patterned in the forelimb of the avian embryo.