In situ Hybridization of Enhancer Regions

Embryos were collected and subjected to in situ hybridization as described previously [12 (link)]. Enhancer regions were cloned in vector pSP72 (Promega), then digested with appropriate enzymes to make labeled run-off transcripts using T7 or SP6 RNA transcriptase, with the DIG RNA labeling kit (Roche Applied Science). The two probes produced by T7 or SP6 recognize the same enhancer region, but different strands. The genomic regions used for probes are listed in S10 Fig. In order to reduce staining variability when lines were to be compared, in situ hybridization was performed in parallel at the same time. Representative embryos from the stained populations are shown.