Fig. 5. Shh induces 5-HT neurons via Gata2. (A-I) Whole-mount immunofluorescence, thin section immunofluorescence or whole-mount in situ hybridization on chick hindbrain 3-4 days after electroporation of Smo-M2.IRES.GFP. (A,B) Whole-mount immunofluorescence for 5-HT (A,B; red), merged with GFP (B, green); only the electroporated side is shown in B. (C,D) Immunofluorescence on thin (10-12 µm) sections through r1 for 5-HT (C,D; red), merged with GFP (D, green). Arrow (A,B) shows corresponding point in both panels. (E,F) Whole-mount in situ hybridization for Gata2 (E) and Lmx1b (F). Endogenous 5-HT neurons (en.) versus ectopic 5-HT neurons (ect.) are bracketed (A,E). (G-I) Immunofluorescence on thin (25 µm) sections through r1, with antibodies against Gata2 (red); higher magnification through the width of the neural tube shows co-localization of Gata2 with GFP (H, green) and 5HT (I, blue). (J-M) Immunofluorescence 3 days after electroporation of dominant-negative Gata2 (DN-G2), either with Smo-M2 (J,K) or alone (L-M). (J,K) Whole-mount immunofluorescence for 5-HT (J,K; red), merged with GFP (J, green). Only the electroporated side of the hindbrain is shown in J and an arrow indicates the absence of ectopic neurons. (L,M) Immunofluorescence on thin (10-12 µm) sections through the ventral neural tube at r1 of the hindbrain for 5-HT (L,M; red), merged with 3C2 (M, green). Arrows indicate the population of 5-HT neurons on the control (left) side and their absence on the electroporated (right) side. An arrowhead indicates the absence of 5-HT (L, red) in cells expressing DN-G2 (L, green) on the control side. The midbrain (MB), mid-hindbrain boundary (MHB, arrow) are indicated on wholemounts. fp, floor plate; rp, roof plate.