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Fig. 1 Two Nkx6 proteins are dynamically expressed in PMNs. (A) Schematic of Islet and Nkx6 protein dynamics during PMN subtype specification at the 8- to 12-somite level. At 11 hpf, MiP and CaP both express Islet1 (blue) and Nkx6 (green asterisks). At 14 hpf, CaP and MiP maintain Nkx6 and Islet1 expression and CaP has initiated expression of Islet2 (red). At 16 hpf, Islet1 is downregulated in MiP (gray); CaP has downregulated expression of Nkx6 and Islet1, but continues to express Islet2. By 18 hpf, MiP has reinitiated expression of Islet1. At 24 hpf, neither MiP nor CaP expresses Nkx6; MiP still expresses Islet1, and CaP still expresses Islet2. (B,C) Dorsal views of 10-hpf embryos. (B) nkx6.1 mRNA is expressed in many medial neurectodermal cells in the pMN domain. (C) nkx6.2 mRNA is expressed in the same domain as nkx6.1. (D,E) Lateral views of 18-hpf embryos showing that (D) nkx6.1 and (E) nkx6.2 mRNA is expressed in the ventral spinal cord; however, some of the ventral-most cells are nkx6-negative (asterisks). (F-I) Lateral views of Islet and Nkx6 co-expression. MiP and CaP were identified by soma position relative to somite boundaries (dotted lines). (F) Single optical slice showing that all ventrally located PMNs express Islet and Nkx6 proteins at 14 hpf, whereas dorsally located Rohon-Beard neurons express only Islet. (G-I) Projected z-stacks showing localization of Nkx6 and Islet Abs in the ventral neural tube. (G) By 16 hpf, CaPs no longer express Nkx6. (H) At 17 hpf, MiPs (arrows) express Nkx6, whereas CaPs are Nkx6-negative. Inset is a 3D rendering of the boxed area; cells co-expressing Nkx6 and Islet (arrows in H) were identified by the Velocity software program (see Materials and methods) and are pseudo-colored blue. The slight differences between the boxed area in H and the inset are due to the differences in angle of visualization: H is a direct lateral view, whereas the inset is rotated (see axes in inset). (I) By 19 hpf, MiPs (arrows) have also downregulated Nkx6. Scale bars: 50 µm in B-E; 20 µm in F-I.