Analysis of RNA Interference Lines Identifies New Functions of Maternally-Expressed Genes Involved in Embryonic Patterning in Drosophila melanogaster

Abstract

Embryonic patterning in Drosophila melanogaster is initially established through the activity of a number of maternally expressed genes that are expressed during oogenesis. mRNAs from some of these genes accumulate in the posterior pole plasm of the oocyte and early embryo and localize further into RNA islands, which are transient ring-like structures that form around the nuclei of future primordial germ cells (pole cells) at stage 3 of embryogenesis. As mRNAs from several genes with known functions in anterior-posterior patterning and/or germ cell specification accumulate in RNA islands, we hypothesized that some other mRNAs that localize in this manner might also function in these developmental processes. To test this, we investigated the developmental functions of 51 genes whose mRNAs accumulate in RNA islands by abrogating their activity in the female germline using RNA interference. This analysis revealed requirements for ttk, pbl, Hip14, eIF5, eIF4G, and CG9977 for progression through early oogenesis. We observed dorsal appendage defects in a proportion of eggs produced by females expressing double-stranded RNA targeting Mkrn1 or jvl, implicating these two genes in dorsal-ventral patterning. In addition, posterior patterning defects and a reduction in pole cell number were seen in the progeny of Mkrn1 females. Because the mammalian ortholog of Mkrn1 acts as an E3 ubiquitin ligase, these results suggest an additional link between protein ubiquitination and pole plasm activity.

Keywords: embryonic patterning; germ plasm; localized mRNAs; oogenesis; pole cells.

Figure 1

Dark-field photographs of cuticle preparations of RNAi knockdown embryos. Three embryos are illustrated from each knockdown line to capture the range of phenotypic severity that was observed. Embryos are oriented with anterior to the left. Control wild-type (wt) embryos are shown in the top row. The phenotypes observed for each line are discussed in Results.

Figure 2

Embryos derived from RNAi knockdown mothers were stained for Vas protein (green) to visualize pole cells. Two embryos are shown for each knockdown line. For those that develop sufficiently, the embryo in the left panel is at the blastoderm stage, whereas the embryo in the right panel is at stage 10, which is the stage at which pole cells are in mid-migration or later. In many cases development does not progress normally beyond the blastoderm stage, and in these instances the embryo in the right panel represents what appears to be the latest stage of development achieved. In some cases, development ceases before cellularization, and then two representative embryos are shown. Wild-type embryos (wt), for comparison, are shown in the first row. The phenotypes observed for each line are discussed in Results.

Figure 3

Embryos derived from RNAi knockdown mothers were stained for Vas protein (green) to visualize pole cell migration defects. Wild-type embryos, for comparison, are shown in the first picture. The phenotypes observed for each line are discussed in Results.

Figure 4

Dark-field photographs of dorsal appendage defects of RNAi knockdown embryos. Two embryos are illustrated from each knockdown line to capture the range of phenotypic severity that was observed. Wild-type embryos (wt), for comparison, are shown in the first row. The phenotypes observed for each line are discussed in Results.

Figure 5

Ovaries derived from RNAi knockdown mothers that did not lay eggs were visualized by DAPI staining (blue). Wild-type ovaries, for comparison, are shown in the first picture, and the oocyte (oo), 15 nurse cells (nc), and follicle cells (fc) are labeled. The phenotypes observed for each line are discussed in Results. In the bottom right panel, a single wild-type ovariole and two entire ovaries from the Hip14 shRNA expressing line are photographed together to illustrate the difference in size and extent of development.

Figure 6

Analysis of the efficacy of knockdown of each gene by RT-PCR analysis. The name of the targeted gene and four lanes of gel are shown in each small picture. In each picture, cDNA prepared from wild-type embryos were added in lanes 1 and 2 and cDNA prepared from RNAi knockdown embryos were added in lanes 3 and 4. Primers amplifying the indicated gene were used in lanes 1 and 3 to compare cDNA level in wild-type and knockdown lines. Primers amplifying a control gene knockdown and primers of rp49 were used in lanes 2 and 4. Knockdown is most efficient when the band in lane 3 is absent or very much weaker than the band in lane 1, whereas the bands in lanes 2 and 4 are equally intense. The percentage value in each panel reports the following ratio of band intensities: (lane 3/lane 1) / (lane 4/lane 2). This value measures the efficiency of the knockdown when controlled for potential differences in the amount of RNA used for the PCR reaction in the control and knockdown lanes. 0% represents a total knockdown and 100% represents a completely ineffective knockdown. Band intensities were quantitated using ImageJ software.

Figure 7

del RNAi affects early pole plasm formation. Ovaries derived from RNAi knockdown mothers who lay eggs, but where pole cells do not form, were immunostained for Vas protein (green) and Osk protein (red) to visualize pole plasm formation. Nuclei were visualized by DAPI staining (blue). Wild-type ovaries, for comparison, are shown in the first row. del RNAi: Accumulation of VAS at the oocyte posterior and nuage in nurse cells are reduced while cytoplasmic nurse cell VAS levels are normal. Posterior OSK levels are also reduced.