Fig. S1. Pharmacological treatments controls. Drug treatment was initiated at 24 hpf and embryos were fixed at 48 hpf (A–D) or started at 30 hpf fixing embryos at 60 hpf (E–G). Cyclopamine effectively blocks the Shh pathway, as shown by the decreased expression of ptc1 in the midbrain area in comparison to its vehicle (EtOH). In contrast, purmorphamine treatment showed increased expression of ptc1 in comparison to the vehicle (DMSO). (E and G) Controls were developed at different times in order to match to their corresponding treatment. Anterior is towards left, lateral views are showed. Arrowhead indicates MHB. Scale bar: 50 μm. cyc, cyclopamine.

Fig. S2. Developing OT cells are sensitive to alterations of the Hh pathway. Sagittal confocal optical section analysis after pharmacological treatments at time frames as indicated. (A, D) Cyclopamine treatment results in diminished H3-P-positive cell number in the OT. (C, D) The opposite effect is achieved by using purmorphamine where the proliferative cell number increases significantly. Means significantly different in (C) P < 0.05. (E) Dorsal confocal sections show the location of the active proliferative zone within the OT at 60 hpf. The level in the z-axis for each image is represented by a blue line. (F) The ventral region, although showing strong POMC expression in the pituitary anlage, only reveals a few proliferating cells. Anterior is towards the left. Lateral views are shown from (A) to (C), and dorsal views in (E) and (F). Gray and white dashed lines represent the head limit and the eye position, respectively. e, eye; OT, optic tectum. Scale bar: 50 μm.

Fig. S3. Patterning of midbrain and cell death in smu embryos. (A, B) The expression of the MHB marker, fgf8, is normal in smu embryos, and (C, D) wnt1 demarcates the dorsal limit of the OT in a semi-circle similarly in WT and smu embryos at 36 hpf. (E, F) ptc1 expression in smu embryos is absent. At 72 hpf the expression of pax7 is preserved in smu (L, M). The proneural marker, neuroD, is reduced in the OT of smu mutants compared with WT (black arrowhead, in N vs O). (G–J) Whereas dead cells can be identified in the eye of smu mutant embryos (in both 36 and 48 hpf, white arrows), there is no increased cell death in the dorsal midbrain in smu embryos, as revealed by acridine orange staining (white arrowhead in H). (K) 48 hpf embryos treated with CuSO4 were shown as a positive control (Hernandez et al., 2007), indicating cell death of neuromasts located in the anterior lateral line (small white arrowhead). Anterior is towards left, the entire panel show lateral views. Scale bar: 100 μm.

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