Rhombomere transplantation repatterns the segmental organization of cranial nerves and reveals cell-autonomous expression of a homeodomain protein

The developing vertebrate hindbrain consists of segmental units known as rhombomeres. Hindbrain neuroectoderm expresses 3′ Hox 1 and 2 cluster genes in characteristic patterns whose anterior limit of expression coincides with rhombomere boundaries. One particular Hox gene, referred to as Ghox 2.9, is initially expressed throughout the hindbrain up to the anterior border of rhombomere 4 (r4). Later, Ghox 2.9 is strongly upregulated in r4 and Ghox 2.9 protein is found in all neuroectodermal cells of r4 and in the hyoid crest cell population derived from this rhombomere. Using a polyclonal antibody, Ghox 2.9 was immunolocalized after transplanting r4 within the hindbrain. Wherever r4 was transplanted, Ghox 2.9 expression was cell-autonomous, both in the neuroectoderm of the graft and in the hyoid crest cell population originating from the graft. In all vertebrates, rhombomeres and cranial nerves (nerves V, VII+VIII, IX, X) exhibit a stereotypic relationship: nerve V arises at the level of r2, nerve VII+VIII at r4 and nerves IX-X extend caudal to r6. To examine how rhombomere transplantation affects this pattern, operated embryos were stained with monoclonal antibodies E/C8 (for visualization of the PNS and of even-numbered rhombomeres) and HNK-1 (to detect crest cells and odd-numbered rhombomeres). Upon transplantation, rhombomeres did not change E/C8 or HNK-1 expression or their ability to produce crest cells. For example, transplanted r4 generated a lateral stream of crest cells irrespective of the site into which it was grafted. Moreover, later in development, ectopic r4 formed an additional cranial nerve root. In contrast, transplantation of r3 (lacks crest cells) into the region of r7 led to inhibition of nerve root formation in the host. These findings emphasize that in contrast to spinal nerve segmentation, which entirely depends on the pattern of somites, cranial nerve patterning is brought about by factors intrinsic to rhombomeres and to the attached neural crest cell populations. The patterns of the neuroectoderm and of the PNS are specified early in hindbrain development and cannot be influenced by tissue transplantation. The observed cell-autonomous expression of Ghox 2.9 (and possibly also of other Hox genes) provides further evidence for the view that Hox gene expression underlies, at least in part, the segmental specification within the hindbrain neuroectoderm.