Functional analysis of the Drosophila embryonic germ cell transcriptome by RNA interference

In Drosophila melanogaster, primordial germ cells are specified at the posterior pole of the very early embryo. This process is regulated by the posterior localized germ plasm that contains a large number of RNAs of maternal origin. Transcription in the primordial germ cells is actively down-regulated until germ cell fate is established. Bulk expression of the zygotic genes commences concomitantly with the degradation of the maternal transcripts. Thus, during embryogenesis, maternally provided and zygotically transcribed mRNAs determine germ cell development collectively. In an effort to identify novel genes involved in the regulation of germ cell behavior, we carried out a large-scale RNAi screen targeting both maternal and zygotic components of the embryonic germ line transcriptome. We identified 48 genes necessary for distinct stages in germ cell development. We found pebble and fascetto to be essential for germ cell migration and germ cell division, respectively. Our data uncover a previously unanticipated role of mei-P26 in maintenance of embryonic germ cell fate. We also performed systematic co-RNAi experiments, through which we found a low rate of functional redundancy among homologous gene pairs. As our data indicate a high degree of evolutionary conservation in genetic regulation of germ cell development, they are likely to provide valuable insights into the biology of the germ line in general

A Functional Genomic Screen Combined with Time-Lapse Microscopy Uncovers a Novel Set of Genes Involved in Dorsal Closure of Drosophila Embryos

Morphogenesis, the establishment of the animal body, requires the coordinated rearrangement of cells and tissues regulated by a very strictly-determined genetic program. Dorsal closure of the epithelium in the Drosophila melanogaster embryo is one of the best models for such a complex morphogenetic event. To explore the genetic regulation of dorsal closure, we carried out a large-scale RNA interference-based screen in combination with in vivo time-lapse microscopy and identified several genes essential for the closure or affecting its dynamics. One of the novel dorsal closure genes, the small GTPase activator pebble (pbl), was selected for detailed analysis. We show that pbl regulates actin accumulation and protrusion dynamics in the leading edge of the migrating epithelial cells. In addition, pbl affects dorsal closure dynamics by regulating head involution, a morphogenetic process mechanically coupled with dorsal closure. Finally, we provide evidence that pbl is involved in closure of the adult thorax, suggesting its general requirement in epithelial closure processes

A Drosophila mir-282 mikroRNS gén szerkezeti és funkcionális jellemzése

A sejtek dinamikusan változó génkifejeződési mintázatainak kialakításában a szabályozó fehérjék szerepe már régóta ismert. Az utóbbi bő egy évtizedben azonban RNS alapú regulátor komplexeket azonosítottak, melyek szintén részt vesznek a génaktivitás szabályozásában. A mikroRNS-ek (miRNS) endogén, egyszálú szabályzó RNS-ek, melyek hajtű alakú átmeneti transzkriptumból keletkeznek. Az érett, körülbelül 22 nukleotid hosszú miRNS-ek néhány fehérjével komplexet alkotva poszt-transzkripcionális szinten fejtik ki negatív szabályozó hatásukat. PhD dolgozatom a bioinformatikai módszerekkel előrejelzett mir-282 nevű Drosophila melanogaster miRNS gén molekuláris és fenotípusos jellemzésével foglalkozik. A Drosophila melanogaster mir-282 génjének bizonyítottuk transzkripciós aktivitását, kifejeződésének és elsődleges szerkezetének leírásával alátámasztottuk a korábbi gén előrejelzést.Újonnan indukált mir-282-re specifikus deléció vizsgálatával bizonyítottuk, hogy a mir-282 lókusz funkcionális transzkriptumot kódol, mely befolyásolja az élethosszt, az életképességet és a petehozamot.Megvizsgáltuk a mir-282 potenciális célgénjeit is, és a mir-282 negatív génszabályozó funkcióját a rutabaga gén esetében kísérletesen bizonyítottuk

<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

<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

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

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>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