The developing neurovascular anatomy of the embryo: A technique of simultaneous evaluation using fluorescent labeling, confocal microscopy, and three-dimensional reconstruction
Gi. Taylor et al., The developing neurovascular anatomy of the embryo: A technique of simultaneous evaluation using fluorescent labeling, confocal microscopy, and three-dimensional reconstruction, PLAS R SURG, 108(3), 2001, pp. 597-604
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.