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Fig. 8
Precise genetic boundaries define three major anteroposterior (AP) partitions.
(A-B′′) Triple fluorescent in situ hybridization combining Fezf-Pax4/6-Gbx in lateral (A) and dorsal views (A′) reveals clear-cut boundaries between the hypothalamo–prethalamic primordium (HyPTh) Fezf+, the Di-Mesencephalic primordium (DiMes) Pax4/6+, and the Rhombencephalo-Spinal primordium (RhSp) Gbx+ regions; details are summarized in a 3-D reconstruction (B,B′). A magnified view of Pax4/6 expression combined with DAPI showed that the DiMes domain consists of two rows of cells along the AP axis (asterisks)(A′′,B′′). (C-C′′) Triple fluorescent in situ hybridization combining Fezf-Six3/6-Gbx probes in lateral (C) and dorsal views (C′) confirms a rostral Fezf+ domain (HyPTh), a caudal Gbx+ domain (RhSp), and a double-negative domain in between characterized by Six3/6 expression (DiMes). A magnified view (C′′) helps to visualize a rostral domain with Six3/6 and Fezf coexpression (yellow staining, arrow) that we identified as the Rostral-HyPTh domain. (D-D′′) Double fluorescent in situ hybridization combining Rx and IrxB probes in lateral (D) and dorsal views (D´) show that Rx is expressed in the entire HyPTh territory, stopping caudally at the HyPTh/DiMes boundary; details can be observed in the magnified view (D′′). (E-E′′) Double fluorescent in situ hybridization combining FoxD and Pax4/6 probes in lateral (E), dorsal (E′), and magnified dorsal (E′′) views shows that the small territory expressing FoxD corresponds to the basal plate of the entire HyPTh primordium, stopping caudally at the DiMes border. (F-F′′) Double fluorescent in situ hybridization combining Nk2.2 and IrxB probes in lateral (F), dorsal (F′), and magnified dorsal (F′′) views determines that Nk2.2 is expressed only in the alar and basal plate of the Rostral-HyPTh and Intermediate-HyPTh domains, leaving a negative gap corresponding to the Caudal-HyPTh domain. (G-G′) Double fluorescent in situ hybridization combining Pax3/7 and Gbx probes identifies patches of Pax3/7 expression in the Caudal-HyPTh domain. (H,H′) Double fluorescent in situ hybridization combining Nkx2.1 and IrxB probes shows that Nk2.1 expression in the floor plate extends rostrally beyond the HyPTh/DiMes boundary. Scale bar in A-H′: 50 μm except A′′ and B′′, scale bar: 5 μm.