| Summary: | The neural crest is a unique population of pluripotent cells that are crucial in vertebrate embryogenesis. In the trunk, NCCs migrate along a ventromedial pathway give rise to the dorsal root ganglia or the sympathetic ganglia (SG), or follow a dorsolateral pathway and give rise to melanocytes. Intriguingly, NCCs following the ventromedial pathway migrate in a metameric pattern through the rostral half somite and avoid the caudal somite and this is thought to dictate the metameric pattern of the SG. Static analyses have characterized the development of these structures, but timelapse imaging of NCCs in their normal environment could potentially reveal unidentified cellular and molecular interactions integral to SG development. However, because NCCs migrate deep within the embryo, it is challenging to track NCCs little is known about the cellular mechanisms mediating their migration, aggregation and differentiation. Here, we follow fluorescently labeled trunk NCCs using a novel sagittal explant culture system and timelapse confocal microscopy. We show trunk NCCs migrate in chain-like formations, and restriction to the rostral somite is not maintained once these cells arrive at the dorsal aorta. Instead, discrete SG only arise after an intermixing of cells along the ventral border of the somite followed by segregation into ganglia. The diverse cell migratory behaviors and active reorganization at the target sites suggest that cell-cell and cell-environment interactions are coordinated with dynamic molecular processes. In a screen for molecules expressed on NCCs during SG formation, we identified the cell adhesion molecule N-cadherin expressed once NCCs arrive adjacent to the dorsal aorta and form ganglia. Additionally, altering cadherin function drastically alters ganglia size. Additionally, we found EphrinB1 expression was absent adjacent to the dorsal aorta as NCCs had dispersed in this corridor, but strikingly up-regulated in the inter-ganglionic regions after discrete ganglia formation. The ephrinB1 receptor, EphB2, is also expressed on NCCs distributed adjacent to the dorsal aorta when discrete ganglia form. Altering ephrinB1/EphB2 signaling interferes with formation of the primary SG chain and blocks the formation of discrete ganglia. Taken together, these results indicate the importance of adhesive and inhibitory mechanisms in the formation of SG.
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