Fgf8 and Gbx2 induction concomitant with Otx2 repression is correlated with midbrain-hindbrain fate of caudal prosencephalon

Chick/quail transplantation experiments were performed to analyse possible factors involved in the regionalisation of the midbrain-hindbrain domain. T he caudal prosomeres, expressing Otx2, were transplanted at stage HH10 into rostrocaudal levels of the midbrain-hindbrain domain, either straddling th e intra-metencephalic constriction (type 1 grafts), or at rostral and media l levels of pro-rhombomere A1 (type 2 and 3 grafts, respectively); thus, in all situations, one border of the graft was in contact with the host Gbx2- and Fgf8-expressing domains. The area containing the graft, recognised by Q CPN immunohistochemistry, was first analysed 48 hours after transplantation for (Otx2, Gbx2, En2 and Fgf8, Although in all three situations, a large p art of the graft maintained Otx2 expression, another part became Otx2 negat ive and was induced to express Gbx2 and Fgf8, These inductive events occurr ed exclusively at the interface between the Otx2-positive transplanted doma in and the ipsilateral host Gbx2-positive rhombomere 1, creating a new Otx2 -Gbx2 boundary within the grafted territory, In type 1 and 2 grafts, the in duced Fgf8 domain is in continuity with the host Fgf8 isthmic domain, where as for type 3 grafts, these two domains are separate. High levels of En2 ex pression were also induced in the area expressing Gbx2 and Fgf8, and Wnt1 a nd Pax2 expressions, analysed in type 3 grafts, were induced at the intragr aft Otx2-Gbx2 new boundary. Moreover at later embryonic stages, the graft d eveloped meso-isthmo-cerebellar structures. Thus, gene expressions induced in the grafted prosencephalon not only mimicked the pattern observed in the normal midbrain-hindbrain domain, but is followed by midbrain-hindbrain cy todifferentiation, indicating that not only Fgf8 but also confrontation of Otx2 and Gbx2 may play an essential role during midbrian-hindbrain regional isation.