<i>Mei-P26</i> regulates PGC development.

<p>(A,B) Immunofluorescence images of embryos at stage 16, stained with anti-Vasa (red), anti-Tj (green) and anti-Fas3 (blue) antibodies. (A) Wild-type embryo. (B) Embryo injected with <i>mei-P26</i> dsRNA has a reduced number of PGCs in the gonads. Ventral view is shown with anterior to the left. Scale bars represent 50 µm. (C–E) Immunostaining of embryos at stage 11 stained with anti-Vasa (red) and anti-CycB antibodies (green). Lateral view is shown; scale bars represent 50 µm in C,E and 5 µm in D,F. (C) Wild-type control embryos. (D) Enlargement of the boxed region in (C). In the wild-type embryos, germ cells do not express CycB. (E) Embryo injected with <i>mei-P26</i> dsRNA. (F) Enlargement of the boxed region in (E). In embryos injected with <i>mei-P26</i> dsRNA, germ cells express CycB prematurely. (G–I) Immunofluorescence images of ovaries of third-stage larvae stained with anti-vasa (red), anti-Hts (green) and anti-Tj (blue) antibodies. Scale bars represent 20 µm. (G) Wild-type ovary. (H,I) Larval ovaries with reduced <i>mei-P26</i> function have rudimentary ovaries and contain few or no germ cells.(H) Ovaries of larvae treated with <i>mei-P26</i> dsRNA. (I) Ovaries of <i>nos-Gal4-VP16/UAS-mei-P26-shRNA</i> larvae. (J-K) Immunostaining of ovaries at third larval stage with anti-Vasa (red) and anti-pSmad antibodies (green). DAPI labels the nuclei (blue). (J) Wild-type ovary. (K) Larval ovary treated with <i>mei-P26</i> dsRNA. PGCs with reduced <i>mei-P26</i> function express the Dpp downstream transducer pMad (arrow). Scale bars represent 10 µm.</p

<i>Pbl</i> affects germ-cell development.

<p>(A,B) Immunostaining of a wild type (A) and a homozygous <i>pbl³</i> mutant embryo (B). <i>Pbl</i> mutants have fewer germ cells, misguided germ cells and abnormally compacted gonads. Vasa staining labels germ cells (red), Tj staining labels the somatic gonad precursor cells (green). Week Tj expression is also detectable in the cenrtral nervous system. The outline of the embryos is marked by Fas3 staining (blue). Scale bar represents 50 µm. (C–F) Immunofluorescence images of adult gonads. Wild type ovariole (C) and testis (E) contain high number of developing germ cells. <i>Nos-Gal4-VP16/UAS-pbl-shRNA</i> (TRiP.GL01092) ovariole (D) and testis (F) lack germ cells. Vasa staining labels germ cells (red), DAPI labels the nuclei (blue). Anterior is to the left. Scale bar represents 20 µm.</p

RNAi screen reveals genes required for embryonic germ cell development.

<p>(A–D) Frames from movie sequences show germ-cell development of wild type and dsRNA-injected embryos with abnormal germ cell development. Embryos express EGFP in the germ cells. All embryos are shown in dorsal view with anterior to the left. The scale bar represents 50 µm. (A) Control embryo injected with buffer. (B–D) Examples for various germ-line defects. (B) Embryo injected with <i>CG8116</i> dsRNA. Arrows indicate germ cells stuck in the midgut. (C) Embryo injected with <i>pbl</i> dsRNA. PGCs are scattered in the body cavity (arrowheads), their number is reduced and no embryonic gonads were formed. (D) Embryo injected with <i>neur</i> dsRNA shows gonad compaction defects. (E) Heat map representation of the RNAi phenotypes following hierarchical clustering. Color code represents penetrance of the phenotypic categories.</p

Mei-P26 is expressed in the embryonic and larval PGCs.

<p>(A–E) Immunofluorescence staining of embryos (A–C), of a third stage larval testis (D), and of a third stage larval ovary (E). Vasa staining (red) marks the germ cells, Mei-P26 staining (green) indicates the localization of Mei-P26 and Hts staining (blue) labels the fusomes in (D) or the spectrosomes in (E). (A–C) Immunofluorescence images of wild-type embryos. Throughout embryogenesis, Mei-P26 is detectable after formation of the pole cells in the germ line. (A) Lateral view of an embryo at stage 4. (B) Dorsal view of an embryo at stage 11. (C) Lateral view of an embryo at stage 13. Scale bars represent 50 µm. (D) Apical tip of a testis from a third-stage larva. Asterisk indicates the hub cells. Mei-P26 is weekly expressed in the GSCs (arrow) and accumulates in the in the nuclei of differentiating spermatocytes (arrowheads). Scale bar represents 20 µm. (E) Ovary of a wild-type third-stage larva. Mei-P26 accumulates in the germ cells. Anterior is at the top, scale bar represents 20 µm.</p

<i>Feo</i> is required for mitosis of larval germ cells.

<p>(A–D) Immunofluorescence images of third stage larval ovaries. Ovaries of larvae injected with <i>feo</i> dsRNA are rudimentary and contain fewer but larger germ cells than the wild type suggesting that the PGCs were unable to undergo mitotic divisions. All ovaries are shown with anterior to the top. Scale bar represents 20 µm. Vasa staining labels the germ cells (red), Tj staining labels the somatic intermingled cells (blue), Fas3 staining labels the anterior somatic cells in (A,B), Hts staining labels the germ-cell specific spherical spectrosomes in (C,D). (E,F) Immunofluorescence images of larval ovaries. (E) Wild-type ovary. (B) Expression of <i>feo</i>-shRNA in the germ line driven by the <i>nos-Gal4-VP16</i> driver induces PGCs with multiple centrosomes. Vasa staining labels the germ cells (red), γ-Tubulin staining labels the centrosomes (green), DAPI marks the nuclei (blue). Arrows indicate the centrosomes. Scale bar represents 20 µm. (G–I) Immunofluorescence images of first-stage larval testes. (G) Wild-type control testis. (H,I) Testes of a larva treated with <i>feo</i> dsRNA (G) and a <i>feo^EA86/Y</i> mutant (I) contain few, abnormally enlarged germ cells. Vasa staining labels the germ cells (red), Tj staining labels the somatic intermingled cells (blue) and Fas3 labels the hub cells (green). Scale bar represents 10 µm.</p

Feo is expressed in the larval germ cells.

<p>Localization of Feo in larval gonads. First-stage larval testes (A,B) and ovaries (C) were stained with anti-Feo (red), anti-α-Tubulin (green) and anti-Vasa (blue) antibodies. Arrows indicate the localization of Feo at the spindle midbody in dividing germ cells. (B) Enlargement of the boxed area in (A). Scale bar represents 10 µm in H,J and 5 µm in I.</p

Systematic simultaneous silencing reveals genetic interaction between <i>trio</i> and <i>Gap1</i>.

<p>Graph showing the quantification of the genetic interaction between <i>trio</i> and <i>Gap1</i>. Co-silencing of <i>trio</i> and <i>Gap1</i> increased the penetrance of the gonad formation defects induced by silencing of the genes separately (T-test, p<0.05). Bars represent the averaged penetrance of eight individual experiments. Error bars show standard deviation.</p

SALS regulates sarcomere growth via formin interactions.

(A-F) F-actin staining of myofibrils dissected from FL-SALS and ΔProR expressing IFMs in a mef2-Gal4/UAS-Dcr2 background (B, C) and upon DAAM knockdown (E, F) (24h AE). The controls used are UAS-Control1/+; mef2-Gal4/UAS-Dcr2 in A, and UAS-Control1/+; mef2-Gal4/UAS-DAAMRNAi in D. (G-L) F-actin staining of myofibrils dissected from FL-SALS and ΔProR expressing IFMs in a mef2-Gal4/UAS-Dcr2 background (H, I) and upon fhos knockdown (K, L) (96h APF). The controls used are UAS-Control1/+; mef2-Gal4/UAS-Dcr2 in G, and UAS-Control1/UAS-fhosRNAi; mef2-Gal4/UAS-Dcr2 in J. As UAS-Control1 we used UAS-Vang, a transgenic line with no significant effect on IFM development (for further details see M&M). (M-P) Quantification of sarcomere length (M, N) and sarcomere width (O, P) in mutant flies with the indicated genotype. The M and O panels refer to adult IFMs (24h AE), whereas the N and P panels apply to pupal IFMs (96h APF). P-values were calculated using two-tailed unpaired Student’s t-test with Welch’s correction or Mann-Whitney U test according to the normality (n.s., not significant P>0.05; **P≤0.01; ****P≤0.0001). n indicates the number of sarcomeres measured. Scale bars: 2 μm.</p

Myofibrillar distribution of the truncated SALS proteins.

(A-A’) A SALS antibody staining (cyan) in a salsnull, mef2-Gal4/+ (control) adult IFM myofibril reveals a strong accumulation in the H-zone and a weaker signal at the Z-disc. (B-E’) Anti-FLAG staining of myofibrils with the indicated genotype. Note that these myofibrils are derived from rescued flies in which the FLAG-tagged SALS protein forms are expressed in a null mutant background. As expected, the full length (FL) form exhibits a highly similar pattern (B’) as observed with the SALS antibody, and pattern of the ΔProR is also very similar to this (C’). In the cases of ProR-WH2-C-term and WH2-C-term a strong H-zone enrichment is detected, but the signal at the Z-disc signal is either absent or appears weaker (D’, E’) when compared to the FL pattern. Actin (in magenta) was used to highlight the sarcomeres in all samples. Scale bars: 2 μm. (TIF)</p

A Summary table of the SALS constructs with their different phenotypic effects in rescue and overexpression conditions.

A Summary table of the SALS constructs with their different phenotypic effects in rescue and overexpression conditions.</p