Figure 6
Figure 6. gata2 is not the only miR-451 target implicated in regulating erythrocyte maturation (A) Diagram of target blocking (TB) MO designed to protect the 2 gata2-3′UTR miR-451 binding sites a and b, and a control MO corresponding to sequences between sites a and b. (B,C) Results of a modification of the reporter assay outlined in Figure 4, for the purpose of validating the efficacy and specificity of the target blocking MOs. For each panel, the sensor RNA was as shown in green. Labels to left of each panel indicate rhodamine-traced miRNA (red labels) and the target blocking MO (MO-TB) delivered to the corresponding rows of embryos. Relative fluorescence intensity between the 2 rows of embryos in each panel assays for miRNA-mediated GFP-sensor-RNA repression. (B) Left panel: a combination of MO-TBs to sites a + b (bottom row of embryos) protect the GFP- gata2-3′UTR sensor RNA from miR-451-mediated repression (top row of embryos). Right panel: MO-TB control does not protect against miR-451-mediated repression. (C,D) Panels illustrating other experimental outcomes for different GFP sensor RNA, miRNA and MO-TB combinations as summarized in the Table (D), using a comparative scale for GFP fluorescence (+ to ++++). The color-coding of the gata2-3′UTR variants in the sensor mRNA column refers to panel A. (E,F) gata2 is not the only miR-451 target responsible for the erythrocyte immaturity of mnr. gata2 target-blocking MOs interfere with the gata2-3′UTR/miR-451 interaction in vivo, verified by persistence of gata2 transcripts in the anterior intermediate cell mass (ICM) of 24-hpf embryos (▾ compared with control ▿). Note that there are 3 controls for comparing the level of gata2 expression: (i) the parallel-processed embryos that received TB-MO-control; (ii,iii) 2 internal controls within each embryo provided by (ii) the comparison of expression in the anterior ICM versus the posterior ICM, and (iii) the comparison between ICM gata2 expression (which in the anterior ICM is exposed to endogenous miR-451 regulation), and the neuronal gata2 expression (which is not exposed to endogenous miR-451 expression). (F) Tabulates the anterior ICM level of gata2 expression by categories (+++, anterior ICM expression as strong as posterior ICM expression; ++, anterior ICM expression less than posterior ICM expression; +, anterior ICM expression absent or less than 30% of posterior ICM expression). n = number of embryos. Despite the effect on gata2 expression, gata2 target-blocking MOs did not affect erythrocyte maturation as assessed either morphologically or by the N:C area ratio. N:C ratio data are mean plus or minus SE for n independent experiments. Figure S12 further demonstrates the reproducibility of these data by presenting them as scatterplots and providing additional fields of representative cells.

gata2 is not the only miR-451 target implicated in regulating erythrocyte maturation (A) Diagram of target blocking (TB) MO designed to protect the 2 gata2-3′UTR miR-451 binding sites a and b, and a control MO corresponding to sequences between sites a and b. (B,C) Results of a modification of the reporter assay outlined in Figure 4, for the purpose of validating the efficacy and specificity of the target blocking MOs. For each panel, the sensor RNA was as shown in green. Labels to left of each panel indicate rhodamine-traced miRNA (red labels) and the target blocking MO (MO-TB) delivered to the corresponding rows of embryos. Relative fluorescence intensity between the 2 rows of embryos in each panel assays for miRNA-mediated GFP-sensor-RNA repression. (B) Left panel: a combination of MO-TBs to sites a + b (bottom row of embryos) protect the GFP- gata2-3′UTR sensor RNA from miR-451-mediated repression (top row of embryos). Right panel: MO-TB control does not protect against miR-451-mediated repression. (C,D) Panels illustrating other experimental outcomes for different GFP sensor RNA, miRNA and MO-TB combinations as summarized in the Table (D), using a comparative scale for GFP fluorescence (+ to ++++). The color-coding of the gata2-3′UTR variants in the sensor mRNA column refers to panel A. (E,F) gata2 is not the only miR-451 target responsible for the erythrocyte immaturity of mnr. gata2 target-blocking MOs interfere with the gata2-3′UTR/miR-451 interaction in vivo, verified by persistence of gata2 transcripts in the anterior intermediate cell mass (ICM) of 24-hpf embryos (▾ compared with control ▿). Note that there are 3 controls for comparing the level of gata2 expression: (i) the parallel-processed embryos that received TB-MO-control; (ii,iii) 2 internal controls within each embryo provided by (ii) the comparison of expression in the anterior ICM versus the posterior ICM, and (iii) the comparison between ICM gata2 expression (which in the anterior ICM is exposed to endogenous miR-451 regulation), and the neuronal gata2 expression (which is not exposed to endogenous miR-451 expression). (F) Tabulates the anterior ICM level of gata2 expression by categories (+++, anterior ICM expression as strong as posterior ICM expression; ++, anterior ICM expression less than posterior ICM expression; +, anterior ICM expression absent or less than 30% of posterior ICM expression). n = number of embryos. Despite the effect on gata2 expression, gata2 target-blocking MOs did not affect erythrocyte maturation as assessed either morphologically or by the N:C area ratio. N:C ratio data are mean plus or minus SE for n independent experiments. Figure S12 further demonstrates the reproducibility of these data by presenting them as scatterplots and providing additional fields of representative cells.

Close Modal
This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal