Pathfinding Pattern Of Branchiomotor Axons Running On Neural Tube During Early Chick Development

Segmentation is a widely employed strategy in development. In the head, the segmentation of the neuroepithelium of the hindbrain coordinates the development of the cranial motor nerves. Particularly in hindbrain, the branchiomotor nerves reflect this rhombomeric organization. The branchiomotor axons grow dorsally away from the floor plate, project laterally within the neuroepithelium to exit to the periphery. During extending in the neural tube, the projecting branchiomotor axons are guided by diffusible cues including repulsive, attractive and adhesive properties, and aided by guideposts, such as nerve exit points, glial cells etc. However, after exiting to the exit point, nothing is known what determine the pathfinding pattern of branchiomotor axons. The accessory nerve, XI cranial nerve, is a pure motor nerve. It displays a unique organization in that its somata located in an extended region of the neural tube, forming a large rostro-caudal extending nucleus, and its axons turn immediately after exiting the neural epithelium cranially, and ascendant along the developing neural tube before they extend ventrally into the periphery. Due to the unique pathfinding pattern, the cranial accessory axons were used as a model to explore the pathfinding pattern determination of the branchiomotor axons. In our research, the roles of elongation of rhomobomere 8, neural crest cells, neuroepithelium were revealed in guiding pathfinding pattern of branchiomotor axons. Firstly, we investigated whether the extended nucleus domain arises by an elongation of rhomobomere 8 or whether this was due to a migration of neurons along the neural tube which had originated from rhombomere 8. Secondly we determined whether accessory nerve root patterning was influenced by environmental cues derived from the mesoderm or whether it represented an intrinsic property of the neural tube. After the homotopic transplantation of a rhombomere 8 segment, we did not observe an elongation of the grafted tissue. Furthermore migration of cells within the neural tube was not detected. After the heterotopic tranplantation of a rhombomere 8 segment from occipital to cervical and thoracic level, we observed the typical accessory nerve root pattern. In contrast a cervical neural tube graft was not able to give rise to the typical accessory nerve root pattern when transplanted to hindbrain level. The absence of somites did not alter the accessory nerve roots in the operated region. Our results reveal that neurons within rhombomere 8 maintain their axial position within the neural tube in the occipital region. The formation of the accessory nerve root pattern is an intrinsic property of the neural tube. Based on these results, we detect whether the motor axon pattern was determined by neuroepithelium and (or) neural crest. Since Sox 10 signal is essential for the migration of nascent neural crest cells, the mutant mouse SoxlOlacZ/lacZ experiment suggests that the accessory pattern extends at the same pattern as wild-type. Then, the mutant mouse WntlCre-/- was used expectantly to delete all the neural crest cells. We found the same result displayed as mutant mouse SoxlOlacZ/lacZ. Using neural crest cells marker HNK-1, we found some neural crest left in the rhombomere 8 (r8). The data suggest the SoxlOlacZ/lacZ and WntlCre-/- mutant mouse can not inhibit the migration of all the neural crest cells. May be the remanent crest cells is sufficient to carry out the hypothetic axon guidance role of neural crest. Elimination of neural crest source by surgical or Noggin Beads implantation results in accessory axons pathfinding vertically extend dorsally and ventrally. Furthermore, the neural crest transplantation experiment between trunk and r8 was used to validate the result. We found that the accessory and spinal axon patterns were not affected by graft. Further, the heterotopic graft of dorsal neural tube between trunk and r8, which concludes neural crest and neuroepithelium was used to reveal whether the neuroepithelium owns a role of axon guidance. The data tell us that in the trunk level, the grafted r8 neuroepithelium repelled the spinal axons extend directly to periphery. Coincidentally, the grafted trunk neuroepithelium allows accessory axons vertically extending. The neuroepithelium therefore contains guidance cues for axon extending. Together, we conclude that the neuraoepithelium may drive neural crest cells to instruct accessory axons running on the neural tube.