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Fig. S5 Immunostaining of Zn5, Islet1/2 and GFAP, and quantification of Zn5+ cell patterning defects. A. At 24 hpf, Islet1/2 staining is increased in srn mutants compared to WT embryos, in primary motor neurons and Rohon-Beard neurons, identified based on their morphology and location in the spinal cord. Dashed lines indicate segment boundaries. B. At 48 hpf, in WT embryos, Zn5+ cells are also Islet1/2+. In srn mutants, Islet1/2 expression is reduced and majority of Zn5+ cells are not Islet1/2+. C. Zn5 and GFAP immunostaining in WT and srn mutants at 48 hpf, showing the spatial relationship between these markers. D. GFAP staining in HuC:GFP embryos at 48 hpf, showing the spatial relationship between neuron cell bodies and GFAP+ processes in the spinal cord. Scale bar = 40 μm. E–H. Quantification of Zn5+ cell patterning defects. There are 3–5 Zn5+ cell at every 20 μm interval in WT and des mutants, 1–9 in srn, dla mutants and medium dose DAPT treated embryos, and 0–3 in mib and high dose DAPT treated embryos, consistent with clumping and gaps in the spinal cord. Data from a representative embryos is shown in E and G. The distribution of all embryos is shown in F and H (4–9 embryos for each; Kolmogorov-Smirnov test, p<0.05).