DEV021444 Supplementary Material

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• Supplemental Figure S1 (Adobe PDF) -

  Fig. S1. mRNA targets of Wisp during late oogenesis. PAT assays of 30 mRNAs showing that Wisp has many targets. The poly(A) tail lengths of the 30 mRNAs were reduced in wisp^KG5287 stage 14 oocytes as compared with wild type. The top of each poly(A) tail is indicated. His4 has two poly(A) sites; both mRNAs are regulated by Wisp. sop mRNA was used as a control. Primer sequences are available upon request.

• Supplemental Figure S2 (Adobe PDF) -

  Fig. S2. PAP and Orb expression overlap during early oogenesis. Immunostaining of germarium and stage 3 egg chamber with (A) anti-PAP (1:500) and (B) anti-Orb (1:5000, acite produced from 6H4). (C) Merge. In addition to PAP nuclear expression, substantial amounts of PAP are present in the cytoplasm of germline cells. This cytoplasmic expression overlaps with Orb expression. Single confocal sections are shown.

• Supplemental Figure S3 (Adobe PDF) -

  Fig. S3. Lack of Bcd accumulation and premature mRNA destabilization in wisp mutant embryos. (A) Immunostaining of 0- to 1-hour embryos with anti-Bcd, showing that Bcd protein does not accumulate in wisp^12-3147/Df(1)RA47 embryos. Bcd protein gradient has previously been shown to be unaffected in wisp¹⁸¹ mutant embryos (Tadros et al., 2003). We could not reproduce this result using two different wisp mutant combinations, wisp^KG5287/Df(1)RA47 (Figure 7C) and wisp^12-3147/Df(1)RA47. In these two wisp mutant combinations, one of which corresponds to the null, Bcd protein does not accumulate in embryos (in a small number of embryos, faint amounts of unlocalized Bcd protein can be seen). (B) Quantification of nos mRNA levels in 0- to 1-hour and 2- to 3-hour wild-type and wisp^KG5287 embryos by qRT-PCR (real-time RT-PCR). nos levels were normalized with rp49 (RpL32). The ratio of nos mRNA/rp49 mRNA was set to 100 in 0- to 1-hour wild-type embryos. In the wild type, nos mRNA is destabilized in 2- to 3-hour embryos. In wisp mutant embryos, nos mRNA levels are low both in 0- to 1-hour and 2- to 3-hour embryos, showing that destabilization occurs prematurely in 0- to 1-hour embryos. Two experiments with independent RNA preparations are shown. qRT-PCRs were performed using the Lightcycler System (Roche Molecular Biochemical). Oligos were: nos, 5′-CGGAGCTTCCAATTCCAGTAAC and 5′-AGTTATCTCGCACTGAGTGGCT; rp49, 5′-CCAAGCACTTCATCCGCCACCAGTC and 5′-TCCGACCACGTTACAAGAACTCTCA. (C) RT-PCR of Hsp83 mRNA in 0- to 1-hour, 1- to 2-hour and 2- to 3-hour wild-type and wisp^KG5287/Df(1)RA47 embryos, showing that Hsp83 mRNA is destabilized earlier in wisp mutant embryos than in wild type. sop mRNA was used as a loading control. Maternal mRNA destabilization was previously reported to be prevented in wisp mutant embryos, using Hsp83 as a test mRNA (Tadros et al., 2003). Therefore, we analysed Hsp83 mRNA stability in wisp^KG5287/Df(1)RA47 embryos. We could not reproduce the lack of Hsp83 mRNA destabilization. By contrast, Hsp83 mRNA destabilization was premature in wisp^KG5287/Df(1)RA47 embryos, as was the case for destabilization of nos and osk mRNAs. Hsp83 oligos were 5′-GGAGAAGTACACTGAGGATG and 5′-CAGGCAGCTCCAGACCCTCC.

  Tadros, W., Houston, S. A., Bashirullah, A., Cooperstock, R. L., Semotok, J. L., Reed, B. H. and Lipshitz, H. D. (2003). Regulation of maternal transcript destabilization during egg activation in Drosophila. Genetics 164, 989-1001.