Late emigrating neural crest cells migrate specifically to the exit points of cranial branchiomotor nerves

Morphological segmentation of the avian hindbrain into rhombomeres is also reflected by the emergent organisation of branchiomotor nerves. In each case, the motor neurons of these nerves lie in two adjacent rhombomeres (e.g. of the Vth nerve in r2 and r3, VIIth in r4 and r5 etc.), and their outgrowing axons emerge into the periphery through defined exit points in rhombomeres r2, r4 and r6, respectively. Sensory axons of the cranial ganglia also enter the neuroepithelium at the same points. Motor axon outgrowth through experimentally rotated rhombomeres has suggested that a chemoattractive mechanism, involving the exit points, may form a component of their guidance. Yet so far, nothing is known about the establishment of the exit points or the identity of the cells that form them. In this study, we describe a group of late emigrating cranial neural crest cells which populate specifically the prospective exit points. Using chimaeras in which premigratory chick neural crest had been replaced orthotopically by quail cells, a population of neural crest was found to leave the cranial neural tube from about stage 10+ onwards and to migrate directly to the prospective exit points. These cells define the exit points by stage 12+, long before either motor or sensory axons have grown through them. The entire neural crest population of exit point cells expresses the recently described cell adhesion molecule c-cad7. Further, heterotopic grafting experiments show that midbrain and spinal cord crest, grafted at late stages in place of r4 crest, share the same migratory behaviour to the facial nerve exit points and express the same markers as cells contributed by the native r4 crest. It was not possible to generate new exit points in odd numbered rhombomeres simply by experimentally increasing their (normally insignificant) amount of crest production. Initiation of the exit point region probably lies, therefore, in the neuroepithelium.