Fig 1. Screen for miRNAs that enhance the hypoxic response.

(A) Left: Schematic representation of the HRE-LacZ reporter. The enhancer derived from the murine lactate dehydrogenase-A (ldh-A), which contains two HIF-responsive elements (HREs) and one cyclic AMP-responsive element (CRE), controls the expression of β-galactosidase in a HIF/Sima-dependent manner. Right: The reporter was not expressed in embryos with the btl-Gal4 driver alone in normoxia or mild hypoxia (11% O₂), and was induced in these embryos exposed to strong hypoxia (5% O₂, arrow); hypoxic induction of the reporter was suppressed by the expression of sima RNAi. In embryos homozygous for the fga⁹ mutant allele, the HRE-LacZ reporter was strongly induced even in normoxia, but this induction was largely reduced by simultaneous expression of sima RNAi. Expression of a fatiga RNAi, which has a modest effect, was not sufficient to induce reporter expression in normoxia, but provoked induction at 11% O₂ and enhancement of the response at 5% O₂. Reporter expression in these conditions was also suppressed by coexpression of sima RNAi. The staining observed at the anterior tip of the embryo (arrowhead) is hypoxia-independent. (B) Design of the screen. A collection of 93 miRNAs was overexpressed with btl-Gal4 in stage 14–17 embryos that also bear the HRE-LacZ reporter. The negative control expressed a btl-Gal4 driver along with the HRE-LacZ reporter, but not a miRNA. Embryos were exposed to 11% O₂ for 4 h, after which X-gal staining was performed.