Effect of miR-10 knockdown and overexpression of endogenous Hox target transcripts A) Whole mount in situ hybridization with probes for hoxB1a, B2a, B3a, B4a and B5a on 24hpf embryos injected with morpholino reagent 1 or 2 (MO1 or MO2), miR-10 siRNA or non injected controls (NIC). To allow quantitative detection, embryos hybridized with the same probe were stained equally long and staining was continuously monitored and stopped before reaching signal saturation. HoxB1a and HoxB3a respond to both gain and loss of function (arrows) of miR-10 with a decrease and increase in expression levels respectively. HoxB2a, HoxB4a and HoxB5a are unresponsive to miR-10 overexpression or knockdown. B) Double whole mount in situ hybridization on 24 hr embryos using probes for hoxB1a and hoxB4a showing the different responses of the genes. Embryos were stained equally long till adequate staining was obtained for the hoxB4a probe. C) In situ hybridization with HoxA1a and HoxA3a on 24 hpf embryos injected with MO1, MO2 or miR-10 siRNA. HoxA1a responds to both overexpression and knockdown. The transcript level of HoxA3a does not respond to either overexpression or knockdown. D) In situ hybridization with HoxB1b on 24 hpf embryos morphant and overexpression embryos. There is strong upregulation of HoxB1b expression in the morphants but no downregulation is observed in the miR-10 siRNA injected embryos.
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