Whole-mount double fluorescent in situ hybridisation was performed in wild-type embryos at 24 hpf for her2 and her12 (A), her4 and her12 (B) and hes5 and her12 (C). Brackets in lateral views and dotted lines in transverse views denote the extent of the spinal cord. Note that her2 and her4 expression in notochord cells is indicated by asterisks. n = 6 embryos per staining. Scale bars: 20 μm.

Whole-mount double fluorescent in situ hybridisation was performed in wild-type embryos at 24 hpf for her2 and her12 (A), her4 and her12 (B) and hes5 and her12 (C). Brackets in lateral views and dotted lines in transverse views denote the extent of the spinal cord. Note that her2 and her4 expression in notochord cells is indicated by asterisks. n = 6 embryos per staining. Scale bars: 20 μm.

Wild-type embryos were treated with LY-411575 or cyclopamine for 0, 1, 2, 4, 6, 8, or 10 hr, and fixed at 24 hpf. Whole mount in situ hybridisation was then performed for her12 in LY-411575 treated embryos and ptc2 in cyclopamine treated embryos. Brackets denote the extent of the spinal cord. n = 15 embryos per staining. Scale bar: 20 μm.

ptc2:Kaede and her12:Kaede embryos were photoconverted at 24 hpf (A), 48 hpf (B) and 72 hpf (C) and imaged 6 hr post-conversion. Each line (grey) represents Kaede^green fluorescent intensity along the mediolateral or dorsoventral axis (see Figure 2A) of one embryo. The red line denotes the average profile of all embryos (n = 4). The max intensity axes are 0–50%, while the DV/ML axes display the full extent of the transverse section. The position of the spinal canal in the dorsoventral profiles is denoted by a dotted line. (A) The signalling profiles during the ‘signalling activation’ phase. (B) The signalling profiles during the ‘signalling consolidation’ phase. (C) The signalling profiles during the ‘signalling termination’ phase. (D) The average profiles of all three phases plotted together. The relevant spinal canal position in the dorsoventral profile is marked by the matching coloured dotted line.

Continuation of the time course described in Figure 2, where her12:Kaede and ptc2:Kaede embryos were photoconverted at 84 hpf or 90 hpf, and imaged 6 hr post-conversion. Lateral views of confocal projections and transverse views of single slices are shown. Kaede^g panels show de novo synthesised Kaede^green after the time of photoconversion, while the merge panels show both previous Kaede^red expression and new Kaede^green expression. Brackets in lateral views and dotted lines in transverse views denote the extent of the spinal cord. n = 4 embryos per timepoint. Scale bars: 20 μm.

Whole-mount double fluorescent in situ hybridisation was performed in wild-type embryos for her12 and ptc2 (A), sox2 and her12 (B), and sox2 and ptc2 (C) at either 24, 48, or 72 hpf. Dotted lines denote the extent of the spinal cord. n = 6 embryos per staining. Scale bars: 20 μm.

(A) Whole-mount in situ hybridisation was performed in mindbomb mutants or their sibling controls for ptc2 expression at 30 hpf. n = 15 embryos per staining. (B) Embryos were injected with the morpholino targeting both rbpja and rbpjb (rbpja/b^MO) at the one-cell stage, and whole mount in situ hybridisation was performed in rbpja/b^MO-injected and uninjected control embryos for ptc2 expression at 24 hpf. n = 35 embryos per staining. Brackets in the lateral views and dotted lines in the transverse views denote the extent of the spinal cord. Arrows indicate ptc2 expression in somites. Scale bars: 20 μm.

(A) Whole-mount in situ hybridisation was performed in mindbomb mutants or their sibling controls for ptc2 expression at 30 hpf. n = 15 embryos per staining. (B) Embryos were injected with the morpholino targeting both rbpja and rbpjb (rbpja/b^MO) at the one-cell stage, and whole mount in situ hybridisation was performed in rbpja/b^MO-injected and uninjected control embryos for ptc2 expression at 24 hpf. n = 35 embryos per staining. Brackets in the lateral views and dotted lines in the transverse views denote the extent of the spinal cord. Arrows indicate ptc2 expression in somites. Scale bars: 20 μm.

hsp:rSmoM2-tRFP embryos and wild-type controls (A), or hsp:Gal4; UAS:NICD embryos and wild-type controls (B) were heat shocked at 11 hpf and stained for the expression of ptc2 and her12 at 24 hpf. Brackets in lateral views and dotted lines in transverse views denote the extent of the spinal cord. Arrows indicate ptc2 expression in somites. Note that expression of hsp:rSmoM2-tRFP resulted in an expansion of her12 expression in the vasculature compared to control embryos (arrowheads in A). The n number for each staining is shown. Scale bars: 20 μm.

(A) Wild-type embryos were incubated in LY-411575 from 20 hpf for 1, 2 or 3 hr and DMSO for the entire duration. Embryos were stained for the expression of ptc2 or sox2. Brackets in lateral views and dotted lines in transverse views denote the extent of the spinal cord. Arrows indicate ptc2 expression in the somites. The n number for each staining is shown. Scale bars: 20 μm. (B) Transverse sections of embryos were taken from the experiment in A and the extent of the expression domain of ptc2 and sox2 was measured and quantified as a percentage of the spinal cord. To directly compare changes in ptc2 and sox2 expression domains, the mean of the DMSO treated group was used as the ‘control maximum’ and all values were normalized as a percentage of their relevant control maximum. Each data point represents the average expression domain percentage of one embryo. n = 8 embryos per condition. Data are plotted with mean ± SD. Statistics: Mann-Whitney U test. Asterisks representation: p-value<0.001 (***).

(A) Schematic representation of the manipulation to the Hh pathway caused by ectopic expression of rSmoM2-tRFP. The point of manipulation is highlighted in green with an asterisk. (B) Experimental design in C. (C) hsp:rSmoM2-tRFP or wild-type control embryos were heat shocked at 20 hpf, and then incubated in either DMSO or LY-411575 until fixation at 30 hpf. Whole mount in situ hybridisation was performed for ptc2 and olig2. Brackets in lateral views and dotted lines in transverse views denote the extent of the spinal cord. Arrows indicate ptc2 expression in somites. The n number for each staining is shown. Scale bars: 20 μm.

(A) Schematic representation of the manipulation to the Hh pathway caused by the loss of primary cilia in iguana mutants. The point of manipulation is highlighted in green with an asterisk. (B) Experimental design in C. (C) iguana mutant and sibling control embryos were incubated in either DMSO or LY-411575 at 20 hpf until fixation at 30 hpf. Whole mount in situ hybridisation was performed for ptc2 and olig2. Brackets in lateral views and dotted lines in transverse views denote the extent of the spinal cord. Arrows indicate ptc2 expression in somites. The n number for each staining is shown. Scale bars: 20 μm.

Wild-type embryos were treated with DMSO, cyclopamine, or LY-411575 from 20 to 30 hpf, and stained with gli2a, gli2b or gli3 probes. Brackets in lateral views and dotted lines in transverse views denote the extent of the spinal cord. Arrows indicate gli2a expression in somites. Note that in LY-411575 treated embryos, gli3 expression is absent in most of the ventral spinal cord but is maintained in the very dorsal region. n = 15 embryos per staining. Scale bars: 20 μm.

Wild-type embryos were treated with DMSO, cyclopamine, or LY-411575 from 20 to 30 hpf, and stained with gli2a, gli2b or gli3 probes. Brackets in lateral views and dotted lines in transverse views denote the extent of the spinal cord. Arrows indicate gli2a expression in somites. Note that in LY-411575 treated embryos, gli3 expression is absent in most of the ventral spinal cord but is maintained in the very dorsal region. n = 15 embryos per staining. Scale bars: 20 μm.