Knock down analysis reveals critical phases for specific oskar noncoding RNA functions during Drosophila oogenesis

Abstract

The oskar transcript, acting as a noncoding RNA, contributes to a diverse set of pathways in the Drosophila ovary, including karyosome formation, positioning of the microtubule organizing center (MTOC), integrity of certain ribonucleoprotein particles, control of nurse cell divisions, restriction of several proteins to the germline, and progression through oogenesis. How oskar mRNA acts to perform these functions remains unclear. Here, we use a knock down approach to identify the critical phases when oskar is required for three of these functions. The existing transgenic shRNA for removal of oskar mRNA in the germline targets a sequence overlapping a regulatory site bound by Bruno1 protein to confer translational repression, and was ineffective during oogenesis. Novel transgenic shRNAs targeting other sites were effective at strongly reducing oskar mRNA levels and reproducing phenotypes associated with the absence of the mRNA. Using GAL4 drivers active at different developmental stages of oogenesis, we found that early loss of oskar mRNA reproduced defects in karyosome formation and positioning of the MTOC, but not arrest of oogenesis. Loss of oskar mRNA at later stages was required to prevent progression through oogenesis. The noncoding function of oskar mRNA is thus required for more than a single event.

Keywords: oskar; MTOC; karyosome; noncoding RNA; oogenesis; shRNA.

Figure 1

Knockdown of osk mRNA in oogenesis. (A) Diagram of the osk gene showing the positions of osk-shRNA target sites. RNA sequences directly below show the overlap between a Bru1 binding site (BRE) and the TRiP project osk-shRNA (GL01101) target. RNA sequences at bottom show the sequences mutated (red) in the osk^syn transgene at the site of the osk-shRNA#3 target, with nucleotides arranged by codon. (B) Levels of osk mRNA, assayed by qPCR, from ovaries of females in which the MAT GAL4 driver was present in combination with the osk-shRNA indicated. Error bars indicate standard deviations. (C) Rates of egg laying by females in which the MAT GAL4 driver was present in combination with the osk-shRNA indicated, except for the osk^N/osk^N genotype. For the sample tested in the presence of the osk^syn transgene the osk background was osk^N/osk⁺ to compensate for the transgenic copy of osk. Values above 3 eggs/female/hour are truncated (jagged bar end): the w¹¹¹⁸ stock used as a wild type control often has a slightly lower rate of egg laying than various experimental strains. (D) Ovarioles showing the timing of oogenesis arrest from osk mutant and KD. Scale bar is 100 µm.

Figure 2

Comparison of GAL4 drivers for activity during oogenesis. (A) Rates of egg laying by females in which the indicated GAL4 driver was present in combination with transgenes for the KD of osk or bru1. (B) Expression patterns from the combination of UAS-GFP and the indicated GAL4 driver. Each panel shows a single ovariole from germarium (left) to stage 8 (right). Panel (B’) is the same set of panels with gain increased in the RGB green channel. Scale bar is 100 µm.

Figure 3

osk mRNA can be removed by KD early in oogenesis. (A) MAT and NGT driver activity in early oogenesis. Shown are the most anterior portions of ovarioles, with the germarium to the left and individual egg chambers to the right. Scale bar is 10 µm. Only the RGB green channel is shown in the panels at right. (B) In situ detection of osk mRNA at early stages of oogenesis. For both stages the osk mRNA signal from the left column is shown by itself in the right column. For stage 4 the levels of osk mRNA in the KD with the NGT driver were variable and two examples indicative of this variation are shown. Scale bars are 10 µm. (C) Quantitation of osk mRNA levels at early stages of oogenesis from in situ hybridization images (representative examples in panel B). For the stage 4 analysis imaging conditions were chosen to best reveal differences in low levels of osk mRNA in the osk mutant and KDs; consequently, there was signal saturation (pixel intensity of 255) for the wild-type sample. P-values were derived from the Wilcoxon rank-sum test. For all pairwise comparisons P < 0.01, with two exceptions: in the germarium/Stage 1 samples P < 0.1 for w¹¹¹⁸ vs MAT≫osk-shRNA#3 and for osk⁰ vs NGT≫osk-shRNA#3.

Figure 4

Disruption of MTOC from KD of osk in early oogenesis. (A) Representative stage 3-4 egg chambers from females with the genotypes indicated. Each egg chamber is oriented with posterior, the position of the oocyte, to the right. Left column: Complete egg chambers stained for gamma-tubulin (green) and DNA (red). Scale bar is 10 µm. Middle column: Images from the left column with only the gamma-tubulin signal (white). Right column: enlargements of the regions outlined in yellow in the middle column, containing the oocyte. (B) Measurements of the areas bounded by foci of gamma-tubulin in the oocyte (see Material and methods). At least 19 oocytes were analyzed for each genotype. The P-values were derived from the Wilcoxon rank-sum test: *** P < 0.01. (C) Representative stage 5–6 egg chambers from females with the genotypes indicated. Each egg chamber is oriented with posterior to the right. Left: complete egg chambers stained for gamma-tubulin (green) and DNA (red). Scale bar is 10 µm. The posterior portion with the oocyte is shown to the right with only the gamma-tubulin signal (white).

Figure 5

Disruption of karyosome formation from KD of osk in early oogenesis. (A) Representative stage 3–4 egg chambers from females with the genotypes indicated. Each egg chamber is oriented with posterior to the right. Left: complete egg chambers stained for DNA (red) and lamin (green) to outline the nuclei. Scale bar is 10 µm. Right: enlargements of the oocyte nuclei from the left images. (B) Representative stage 5–6 egg chambers from females with the genotypes indicated. Presented as in panel A, except that the scale bar is 20 µm. (C) Proportion of stage 3–4 oocytes of the indicated genotypes with normal karyosomes, defined as a single cluster of DNA in the oocyte nucleus. The number (n) of oocytes analyzed for each genotype is indicated. The P-values were derived from the Wilcoxon rank-sum test: *** P < 0.01. (D) Proportion of stage 5–6 oocytes of the indicated genotypes with normal karyosomes, defined as a single cluster of DNA in the oocyte nucleus. The P-values were derived from the Wilcoxon rank-sum test: *** P < 0.01.