Fig. 2

Oogenic miRNAs localize in four spatial patterns in the germline. Dissected germlines, oriented left to right, with oocytes on the right. Asterisk marks the distal tip cell. Bright-field (left) images of germlines. Dark staining is indicative of positive probe expression. DNA (white, stained with DAPI, right panel) staining labels distinct germ cell nuclear stages. The schematic in each panel depicts miRNA localization, gray labeling highlights positive probe expression. a U6 small nucleolar RNA, expresses in all germ cell nuclei (arrowhead marks sperm) and the somatic gonad nuclei (arrow marks somatic sheath cells). b Pattern 1 miRNA, miR-39-3p, localizes to the cytoplasm of late pachytene cells, diplotene oocytes, diakinetic oocytes, and maturing oocytes, but not in sperm cells or somatic sheath cells. c Pattern 2 miRNA, miR-54-3p, localizes to cytoplasm of diplotene oocytes, diakinetic oocytes and maturing oocytes. d Pattern 3 miRNA, miR-229-5p localizes to cytoplasm of the maturing oocyte. Arrowhead marks sperm, which are negative for the miRNA. e Pattern 4 miRNA, miR-81-3p localizes to the cytoplasm of an oocyte undergoing fertilization in the spermatheca. None of the miRNAs positively localized to the somatic gonadal cells or sperm cells. Each experiment was performed three times, 75–100 gonads were dissected for each replicate and ~20–30 germlines were imaged for each replicate. Scale bar: 20 μM