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These comparisons showed that disc1 expression was around 1.8- and 2.4-fold higher respectively in the ptch1; ptch2 mutant and sibling pools compared with the smo b641 sibling pool (Fig. 3C,D), confirming that disc1 expression is responsive to increased levels of Shh signalling.

Mutant and sibling pools representing up to 24 different mutations were tested in parallel.

cDNA was generated from RNA of 72 hpf homozygous ste mutant and sibling embryos using oligo dT) primer and SuperScript III reverse transcriptase (Invitrogen).

The presence of tether cells was also confirmed by myo7aa expression; no difference was seen between eis mutant and sibling embryos (Fig. 1G,H).

Pka-C1BG02142 (P element insertion in the 5′ UTR) and Pka-C1EY08221 (P element insertion in the promoter) mutations were outcrossed into the iso31 background for 5 7 generations to generate mutant and sibling control progenies.

PCR was initially performed on mutant and sibling pools for genome scanning, and subsequently on the individuals that had been used for the pooling in order to confirm potential linkages to specific markers.

We analyzed the lifespan of multiple cohorts of female and male C32 mutants and sibling controls, finding that C32 flies in the w1118 background of either sex lived up to 100% longer than control w1118 flies (Figures 2A-2E).

The TC identifies differentially expressed transcripts between mutant embryos and sibling controls using a 3' end based RNAseq protocol.

Sequencing of genomic DNA (gDNA) from individual wild-type (n=2), mutant (n=2) and sibling (n=6: one homozygous wild type, five heterozygous) embryos from a cross between eis heterozygous parents confirmed genetic linkage of the SNP identified by HMFseq with the eis locus (Fig. 2E).

To biochemically confirm p53 activation, we probed protein extracts from controls (AB strain), esco2 mutant and esco2 sibling embryos for p53.

There were, however, no global elevations in apoptosis at 24 hpf between ethanol-treated mutants and sibling counterparts compared with their untreated mutants and siblings (supplementary material Fig. S5), suggesting that the pdgfra-ethanol interaction leads to an increase in neural-crest specific cell death.