THE CAUDAL LIMIT OF OTX2 GENE-EXPRESSION AS A MARKER OF THE MIDBRAIN HINDBRAIN BOUNDARY - A STUDY USING IN-SITU HYBRIDIZATION AND CHICK/QUAIL HOMOTOPIC GRAFTS/

Segmentation of the neural tube has been clearly shown in the forebrai n and caudal hindbrain but has never been demonstrated within the midb rain/hindbrain domain. Since the homeobox-containing gene Otx2 has a c audal limit of expression in this region, we examined, mainly in chick embryos, the possibility that this limit could represent an interneur omeric boundary separating either two cerebellar domains or the mesenc ephalic and cerebellar primordia. In situ hybridisation with chick or mouse Otx2 probes showed the existence of a transient Otx2-negative ar ea in the caudal mesencephalic vesicle, between stages HH10 and HH17/1 8 in chick, and at embryonic day 9.5 in mice. The first postmitotic ne urons of the mesencephalon sensu stricto, as labelled with an anti-bet a-tubulin antibody, overlay the Otx2-positive neuroepithelium with a p erfect match of the caudal limits of these two markers at all embryoni c stages analysed (until stage HH20). Chick/quail homotopic grafts of various portions of the midbrain/hindbrain domain have shown that the progeny of the cells located in the caudal mesencephalic vesicle at st age HH10 are found within the rhombomere 1 as early as stage HH14. Fur thermore, our results indicate that the cells forming the HH20 constri ction (coinciding with the caudal Otx2 limit) are the progeny of those located at the caudal Otx2 limit at stage HH10 (within the mesencepha lic vesicle), As a result, the Otx2-positive portion of the HH10 mesen cephalic vesicle gives rise to the HH20 mesencephalon, while the Otx2- negative portion gives rise to the HH20 rostral rhombomere 1. Long-sur vival analysis allowing the recognition of the various grisea of the c himeric brains strongly supports the view that, as early as stage HH10 , the caudal limit of Otx2 expression separates mesencephalic from ist hmo/cerebellar territories. Finally, this study revealed unexpected ro strocaudal morphogenetic movements taking place between stages HH10 an d HH16 in the mediodorsal part of the caudal Otx2-positive domain.