DEV02729 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. Body patterning and pole-cell-forming activities of osk and osk^imm transgenes in D. melanogaster. (A) Exon/intron organization of osk and osk^imm genes. The coding regions are darkly shaded, and the 3′UTRs are in a lighter shade. Both Posk⁺ and Posk^imm contain their own coding region and 3′UTR, while the Posk^imm3′mel transgene contains the osk^imm coding region and osk 3′UTR. The exon junctions of osk^imm were confirmed by sequencing osk^imm cDNA from D. immigrans embryos. (B,C) Quantitation of the body patterning (B) and pole cell forming (C) activities of osk and osk^imm transgenes.

• Supplemental Figure 2 -

  Fig. S2. Embryonic localization of osk and osk^imm mRNAs. (A-G) Embryos probed with an osk RNA probe (left panels) or an osk^imm RNA probe (right panels). (A) osk⁵⁴/Df embryo, showing absence of osk mRNA localization. (B) w¹¹¹⁸ embryo; the absence of signal from the localized osk mRNA demonstrates the absence of cross hybridization between osk and osk^imm mRNAs. (C,D,F) Detection of endogenous osk mRNA in Posk⁺ (C), Posk^imm3′mel (D), or Posk^imm (F) embryos. (E,G) Detection of transgenic osk^imm mRNA in Posk^imm3′mel (E) or Posk^imm (G) embryos. (H) RNase protection assay used to detect the level of osk^immexpression in each representative transgenic line. The osk^immprobe is specific for osk^imm as there is no band detected in lanes 1 and 2. The rp49 mRNA was detected as a loading control.

• Supplemental Figure 3 -

  Fig. S3. Oskar distribution during pole cell development and migration. (A-H′). Detection of D. melanogaster Osk in embryos from egg-lay (A) to arrival of the pole cells at the gonad (H). Paired panels (e.g. A and A′) show Osk in A-H and double labeling of Osk (red) and GFP-Aub (green) in A′-H′. GFP-Aub is strictly cytoplasmic, and its absence demarcates nuclei after pole cells have formed. In early embryos (A, egg-lay; B, pole bud formation; C, pole cell formation), Osk is cytoplasmic in small particles, and also appears in nuclei starting at syncytial blastoderm stage (C). A and A′ show the entire posterior end of the embryo, while all other panels focus on a portion of the extreme posterior covering several pole cells. During cellular blastoderm (D) and gastrulation (E) stages, Osk appears in the periphery of spheres in the cytoplasm. By early migration of the pole cells (F), Osk begins to be harder to detect. In pole cells during later migration (G) and arrival at the gonad (H), Osk is difficult to detect and only appears in small foci. Note that in F′-H′, cytoplasmic polar granules are still detectable by GFP-Aub and have fragmented into smaller particles. (I-I′′). Nuclear bodies of pole cells at gastrulation, stained for Osk (I), GFP-Aub (I’) and merge (I’’). Arrowheads point to the nuclear bodies, which do not contain GFP-Aub. (J-J′′). Polar granules and nuclear bodies are also detectable using Vasa-GFP (J′), which colocalizes with Osk (J); merge (J′′). Scale bars: 5 μm.

• Supplemental Figure 4 -

  Fig. S4. Conservation of Osk amino acid sequences. (A) Graphic representation of the identity between Osk^imm and six other Drosophila Osk proteins, as well as Osk from A. gambiae. The graph is divided into four sections, according to regions of high or low homology. Asterisks indicate the position of missense mutations: osk³⁰¹, osk²⁵⁵, osk^6B10, osk⁸⁸ and osk¹⁶⁶, respectively (Kim-Ha et al., 1991; Breitwieser et al., 1996). Above the graph, long and short Osk are depicted as they relate to the regions in the graph. Below the graph, specific ranges of amino acid sequence identity are shown. The percentages in parentheses refer to the identity of A. gambiae as compared to the other Osk sequences. (B) Direct comparison of the sequences of Osk^imm and Osk. Identical residues are highlighted.