Walker JC, Harland RM.

GM 42341 NIGMS NIH HHS , R01 GM042341 NIGMS NIH HHS

	Figure 1. miR-24a is expressed in the developing neural retina. In situ hybridization shows expression of pri-miR-24a in the embryonic eye anlagen and posterior mesenchyme (stage 19 [A] and stage 23 [B]. Expression continues in the neural retina as the optic cup develops and invaginates (stage 28 [C] and stage 31 [D]). (C, inset) Sense probe shows no expression. (E) RT–PCR for the primary miRNA from stages 7 to 31 shows that miR-24a expression begins at stage 19 and is highly expressed throughout maturation.
	Figure 2. miR-24a knockdown results in a reduction in eye size. (A–F′) Hematoxylin and eosin staining of 12-μm sections in embryos injected with 20 ng of 24aMO in one cell at the two-cell stage, injected half on the right. The knockdown of miR-24a causes a reduction in eye size that does not occur until after stage 26 (A), first becoming prevalent at stage 28 (B). The reduction in eye size becomes obvious in stage 31 (C), stage 34 (D), stage 40 (E), and stage 45 (F,F′) embryos (see also Supplemental Fig. 1). (G) The size of the eye was measured at stage 40 and the ratio of the injected eye to the uninjected eye in each embryo was used to classify the severity of phenotypes. miR-24a knockdown caused many embryos to have a smaller eye, an effect that was rescuable by coinjection of miR-24a duplex RNA, but not of miR-133b duplex RNA. A mismatch morpholino also had no effect on eye size ratio (n = 121). (H) 24aMO functionally represses miR-24a. A GFP construct with two miR-24a recognition elements (24aMRE) when injected alone strongly fluoresces, but shows significantly lower levels of fluorescence when coinjected with duplex miR-24a RNA (n = 9; error bars are the standard error of the mean [SEM]). This effect is dependent on the interaction between miR-24a and the miR-24a recognition elements, because mutating either abrogates the effect. 24aMO is able to rescue fluorescence of 24aMRE by blocking the function of miR-24a when coinjected. Similar results were obtained with multiple experiments. RFP without miR-24a recognition elements was always coinjected, so that a measure of GFP repression was expressed as the relative fluorescence of GFP/RFP.
	Figure 3. miR-24a knockdown has no effect on proliferation, but leads to an increase in apoptosis. (A) Immunohistochemistry for phosphorylated histone3 (PH3, in red) shows that there is no significant difference in proliferation between control eyes and 24aMO-injected eyes (n = 6 embryos per stage; 18 sections per embryo; sections are 12 μm; error bars are SEM). (B) Knockdown of miR-24a results in a significant increase in apoptosis at all stages assayed, measured by the number of TUNEL-positive nuclei per 20 (n = 6 embryos per stage; 11 sections per embryo; error bars are SEM).
	Figure 4. Knockdown of miR-24a causes up-regulation of Caspase9 protein and is rescuable by its inhibition. (A) RT–PCR of caspase9 on stage 28 heads shows no change in mRNA levels between uninjected and miR-24a knockdown embryos. (B) Western blot for Caspase9 on stage 28 heads shows a significant increase in protein levels when miR-24a function is blocked (n = 3 experiments; error bars are SEM). (C) Coinjection of Caspase inhibitors to rescue the miR-24a knockdown phenotype. Only a pan-Caspase inhibitor (All) or an inhibitor specific to Caspase9 rescued the reduction in eye size caused by injection of 24aMO (n ≥ 47 for each treatment; error bars are SEM).
	Figure 5. miR-24a targets the 3′ UTR of caspase9 and apaf1. (A) The 3′UTRs of X. laevis caspase9 and apaf1 have two putative miR-24a-binding sites, as measured by RNAhybrid (Kruger and Rehmsmeier 2006). (B) The addition of miR-24a causes GFP reporters with miR-24a recognition elements in their 3′UTRs to be repressed. GFP reporters with the 3′UTR of caspase9 and apaf1 show repression by miR-24a. Mutating the putative miR-24a-binding sites in these constructs (mutCasp9 and mutApaf1) abolishes the repression (n = 4 experiments; error bars are SEM). (C) Embryos injected with 2 ng of miR-24a or miR-24amm or nothing (control) were cultured in 15 mM hydroxyurea to induce apoptosis at the onset of gastrulation. Only miR-24a was able to significantly repress or delay the onset of apoptosis (n = 3 experiments; error bars are SEM).

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