Figure 6

Nanos2 knockdown quantification over time and phenotypic characterization of Nanos2 knockdown larvae. (A–C) RT-qPCR results showing comparable expression levels of Nanos2 mRNA in both controls, but a significant decrease (p value ≤ 0.01) of Nanos2 mRNA expression levels when comparing Nanos2 shRNA mixture-electroporated samples to the scrambled shRNA control at 1 dpf (A), 3 dpf (B), and 5 dpf (C). Bright field images of non-electroporated animals are shown to display the animals’ morphology at each timepoint of RNA extraction. In all cases, levels of Nanos2 expression in scrambled shRNA and Nanos2 shRNA mixture (mix) samples were quantified relative to the NE control. Bar heights represent mean values of three independent experiments and error bars show standard deviations. ns non-significant; ***p value ≤ 0.01. (D) Representative images of 3 dpf larvae showing mature nematocytes (white) on the larval surface for each of the three different conditions presented. Cell nuclei are stained in blue. Images were projected from confocal stacks of ~ 10 μm. (E) Box plot showing the number of mature nematocytes for each of the three conditions. Center lines show the medians; box limits indicate the 25th and 75th percentiles (first and third quartiles); whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles; outliers are represented by circles. For NE, n = 49; for scrambled shRNA, n = 53; for Nanos2 shRNA mixture (mix), n = 65. (F) Representative images of 3 dpf larvae showing the GLWamide⁺ neurons (yellow) on the larval aboral region for each of the three different conditions. Images were projected from confocal stacks of ~ 45 μm. (G) Box plot showing the number of GLWamide⁺ neurons for each of the three conditions. Box plot characteristics as in (B). For NE, n = 72; for scrambled shRNA, n = 68; for Nanos2 shRNA mixture, n = 75. Altogether, Nanos2 knockdown yields a significant reduction (p value ≤ 0.01) of mature nematocytes and a significant increase (p value ≤ 0.01) of GLWamide⁺ neurons in the 3 dpf larvae. Ns non-significant; ***p value ≤ 0.01. Scale bars 100 μm in (D) and 25 μm in (F).