Comprehensive Gene Expression Profiling Protocol

Candidate genes (Supplementary Table 6) and gene markers used to characterise the drug phenotypes were amplified by two rounds of nested PCR using gene-specific primers and a T7 universal primer on cDNA from mixed developmental stages as initial template. Riboprobes were synthesized with the T7 enzyme (Ambion’s MEGAscript kit, #AM1334) and stored in hybridisation buffer at a concentration of 50 ng/μl at −20 °C. Colorimetric and fluorescent whole mount in situ hybridisation were performed following an established protocol^{3 (link),30 (link)}. After probe washes, samples for fluorescent in situ hybridisation were incubated with anti-DIG-POD Fab fragments (ROCHE, #11633716001) and a mouse anti-acetylated α-tubulin antibody (clone 6-11B-1, Millipore-446 Sigma, #MABT868, 1:500) to co-stain cell boundaries. After signal development with a Tyramide Signal Amplification kit (Akoya Biosciences, #NEL742001KT), these samples were washed several times in PTx and treated for secondary antibody incubation as described above. To characterise in silico gene expression dynamics during development, we used an available stage-specific RNA-seq dataset³³ , representing the average expression of the two replicates with the package pheatmap v.1.0.12 available in R, were colour intensity shows the z-score value for each gene.