PERSISTENCE OF RHOMBOMERIC ORGANIZATION IN THE POSTSEGMENTAL HINDBRAIN

Rhombomeres are morphological varicosities of the neural tube that are present between embryonic day (E) 1.5 and E5 and are characterised by compartment organisation, segmentally neuronal organisation and spati ally restricted patterns of gene expression. After E5, the segmented o rigins of the hindbrain become indistinct, while the adult hindbrain h as an longitudinal columnar nuclear organisation. In order to assess t he impact of the early transverse pattern on later longitudinal organi sation, we have used orthotopic quail grafts and in situ hybridisation to investigate the long-term fate of rhombomeres in the embryonic chi ck hindbrain. The uniformity of mixing between quail and chick cells w as first verified using short-term aggregation cultures. The dispersal of the progeny of individual rhombomeres (r) was then assessed by the unilateral, isochronic and orthotopic transplantation of either r2, r 3, r4, r5 or r6 from quail to chick at embryonic day E2. In addition, orthotopic, partial rhombomere grafts, encompassing an inter-rhombomer e boundary and adjacent rhombomere bodies were used to assess cell mix ing within rhombomeres. Operated embryos were incubated to either E7 o r Fill when chimaeric brains were removed. Quail cells were identified in whole mounts or serial sections using the quail-specific antibody QCPN. Subsequently, radial glia morphology was assessed either by immu nohistochemistry or DiI labelling. A series of fixed hindbrains betwee n E6 and E9 were probed for transcripts of Hoxa-2 and Hoxb-1. Fate-map ping reveals that the progeny of individual rhombomeres form stripes o f cells running dorsoventrally through the hindbrain. This pattern of dispersal precisely parallels the array of radial glia. Although the p ostmitotic progeny of adjacent rhombomeres spread to some extent into each others' territory in intermediate and marginal zones, there is li ttle or no mixing between rhombomeres in the ventricular zone, which t hus remains compartmentalised long after the rhombomeric morphology di sappears. Segmental gene expression within this layer is also maintain ed after E5. A more detailed analysis of mixing between proliferating cells, using partial rhombomere grafts, reveals that both mixing and g rowth are nonuniform within the ventricular layer, suggesting, in part icular, that longitudinal expansion within this layer is restricted. T ogether, these observations suggest that rhombomeres do not disappear at E5, as has previously been supposed, rather they persist in the ven tricular zone to at least E9, ensuring a continuity in the presumed se gmental cues that specify neuroepithelial cells in the hindbrain.