3 Results 3.1 Characterisation of a novel nuage component Krimper (KRIMP) 3.1.1 KRIMP is a nuage component By comparing the expression profiles of isolated tumour GSCs, induced by the loss of bam expression or the overexpression of decapentaplegic, to that of the somatic cells, CG15707 (hereby known as krimper) was identified as one of the potential candidate genes that is highly expressed in the GSCs (Kai et al., 2005). krimper (krimp) encodes a protein predicted to contain a coiled-coil domain, a CCCH-type zinc finger motif and a tudor domain (Figure 3.1.1). Tudor domain-containing proteins, such as the Drosophila TUD, SPN-E, mouse Ring finger protein 17 (RNF17), and Mouse Tudor Repeat-1 (MTR-1), are reported to play essential roles in both female and male germlines (Boswell and Mahowald, 1985; Chuma et al., 2006; Gillespie and Berg, 1995; Pan et al., 2005). Proteins harbouring CCCH-type zinc fingers are known to bind mRNAs (Lai and Blackshear, 2001; Lai et al., 2000; Lai et al., 2002). Coiled-coil domain-containing proteins, which include Fragile Mental X Retardation Protein (FMRP), Gemin5, and Open Reading Frame (ORF1), possess protein-protein interaction properties (Gubitz et al., 2002; Hoogeveen and Oostra, 1997; Martin et al., 2004). a AUG STOP Coiled-coil ZnF TUD 17-37aa 512-539aa 613-670aa 0.5kb PBac{WH}CG15707f06583 59 MNLEDISMIMKLFDSNMHKLQGNLRSYQTEMHQIHKELTEKLSHADLLYRSLIPLHDHLVASLSEVNAHVM KLNVQLHINRQSVRLGDYEYYEKSIDNPYSSIRSGLQAIEKPGCAEAICQSSKPAFVECLPSSTSEEVPVV AVQEASSTNQLDAISVVNENLSEERDATPQPLAVSKNMEETMPSNPFHEQLEGSLEEIPVGSKIVVETEKA NNPVRSEASAPATSDNQSLLAAKQGTQTIGTGICNKISKSTINMPNNWQLENPTEVTAASIEKVNKLPKSP RNRFLLPPKGGTETTRRDIYNQILKDMAAFPENTIVTAVLASVDVTDNCAYVAKWDESSDRIKKVLQRQLP LQELDQLPDYGDIFAVLDSINNIITRITINSSSAGGGYDAYLIDFGEHIHFDGNETIFKLPDDIKRLPAQA IRCDLINCDIANMHCFVNTYIKIRVHENNNSTLVAEPVIDRLSRPTKTNTTKYPAGITEDDMAMLNEIDES TSDPLKAVLGFRPKDEQRICRHYDPKLNGCFKGNNCRFAHEPFAPNGATKDVELARALPETIFDTTVHFEI GSIVGILITFINGPTEVYGQFLDGSPPLVWDKKDVPENKRTFKSKPRLLDIVLALYSDGCFYRAQIIDEFP SEYMIFYVDYGNTEFVPLSCLAPCENVDSFKPHRVFSFHIEGIVRSKNLTHQKTIECIEYLKSKLLNTEMN VHLVQRLPDGFLIRFLDDWKYIPEQLLQRNYAQVSQ b Figure 3.1.1 KRIMP is a nuage component. (a) Gene structure of krimp. krimp contains two exons and is predicted to contain a coiled-coil domain, a CCCH-type zinc finger motif, and a tudor domain. A piggyBac insertion (f06583), inserted at 35 bp upstream of krimp ORF, results in female sterility. (b) Schematic representation of the modular structure of KRIMP. The coiled-coil domain, CCCH-type (underlined) zinc finger motif and tudor domain are highlighted in yellow, magenta, and cyan, respectively. Although krimp was identified as a highly expressed mRNA in the GSCs from the microarray analysis, immunostaining of KRIMP indicates a wide expression in germline cells, including the differentiating germ cells in the germarium and egg chambers (Figure 3.1.2a). KRIMP appears to localise to perinuclear foci reminiscent of the nuage (Figure 3.1.2b). In fact, co-staining of KRIMP with a well-known nuage component VAS shows an overlap of virtually all KRIMP and VAS foci in the nuage (Figure 3.1.2b). Unlike VAS, which is both a nuage and pole plasm component (Hay et al., 1988; Lasko and Ashburner, 1990), KRIMP is detected only in perinuclear foci and not in the pole plasm (Figure 3.1.2a). 60 a b Figure 3.1.2 Subcellular localisation of KRIMP in D. melanogaster germline cells. (a) KRIMP localises to the perinuclear regions of the germline cells in the Drosophila ovary. Ovaries were immunostained with anti-KRIMP (green) and anti-VAS (red). Bar is 20 µm. KRIMP perinuclear foci co-localise with VAS foci. All ovarioles are orientated with the anterior to the left. (b) Closer view of a nurse cell nucleus confirms the colocalisation. Bar is µm. Homologues of KRIMP can be identified in the Drosophilidae family, including the melanogaster group and others such as D. virilis and D. grimshawi (Figure 3.1.3). Although no close orthologs of KRIMP are found in the higher vertebrates, several mouse tudor-domain proteins RNF17, TDRD1, TDRD3, and TDRD6 are reported to localise to the chromotoid body (Chuma et al., 2003; Goulet et al., 2008; Pan et al., 2005; Vasileva et al., 2009). Among those, one of them may potentially be functional homologues of KRIMP that have evolved diversely. 61 Figure 3.1.3 Homologues of KRIMP are identified in the Drosophilidae family. A ClustalW alignment of D. melanogaster KRIMP and its homologues shows 42% identity and 60% similarity to D. ananassae; and 36% identity and 55% similarity to D. virilis. Identities and gaps are indicated by asterisks and dashes, respectively. 62 3.1.2 krimp mutant exhibits spindle-class phenotype A piggyBac transposable element inserted at 35 bp upstream of the krimp ORF was identified as a possible krimp mutant allele, where females homozygous for krimpf06583 were sterile. Northern blotting analysis revealed the absence of the 2.5 kb krimp transcript in the mutant ovary (Figure 3.1.4), indicating that krimpf06583 is a loss-offunction allele. Moreover, immunostaining of krimpf06583 ovary with anti-KRIMP indicated the loss of perinuclear foci (Figure 3.1.2b). Figure 3.1.4 krimpf06583 is a loss-of-function allele. Northern analysis indicates the expected transcript size of ~2.5 kb in the control ovary. No detectable transcript is observed in krimp mutant ovary. 63 To confirm that krimpf06583 is a suitable mutant allele for the characterisation of the krimp phenotypes, this allele was placed over an available deletion that uncovers krimp genomic region, Df(2R)Exel6063. Transheterozygotes krimpf06583/Df(2R)Exel6063 exhibited female sterility and a similar extent of loss in KRIMP perinuclear staining to that of homozygous krimpf06583 (Appendix IV). Hence, krimpf06583 was employed as a loss-of-function allele to characterise krimp phenotypes in this thesis. In krimp mutant ovary, progression of oogenesis was compromised and degeneration of the egg chambers was observed from stage onwards (data not shown). A closer examination of krimp mutant ovary revealed meiotic progression and oocyte polarity defects that are commonly seen in the nuage component mutants spn-E, vas, and mael (Findley et al., 2003; Page and Hawley, 2001; Styhler et al., 1998). krimp mutant oocyte nucleus failed to form a compact karyosome and the synaptonemal marker C(3)G, remained chromosomal (Figure 3.1.5). This is in contrast to the wild-type oocyte nucleus, which compacts into a karyosome by stage and C(3)G dissociates to become extrachromosomal (Page and Hawley, 2001). 64 Figure 3.1.5 krimp mutant exhibits meiotic progression defects. Immunostaining with a synaptonemal complex marker C(3)G (green), shows that the krimp oocyte nucleus fails to compact into a karyosome (blue). Bar is µm. An examination of the D/V marker GRK, indicated a loss of D/V polarity in krimp mutant oocytes. The level of GRK expression was markedly reduced in 100% (n = 30) of the mutant ovarioles and its localisation to the anterior-dorsal corner of the oocyte was affected in 93% (n = 61) of stage onwards mutant egg chambers (Figure 3.1.6; Neuman-Silberberg and Schupbach, 1996). 65 Figure 3.1.6 krimp mutant exhibits oocyte polarity defects. Ovary staining with antiGRK. The level of GRK expression is downregulated (green arrowheads) and its dorsalanterior localisation is disrupted in stage egg chamber. Bar is 20 µm. Lastly, precocious translation of osk mRNA was also observed in 80% (n = 55) of krimp mutant ovarioles (Figure 3.1.7). In the wild-type, osk mRNA is transcribed at the onset of oogenesis but translation is only initiated at stage (Figure 3.1.7; St Johnston, 1993). This is consistent with the osk silencing defects reported previously for armi, mael, aub, and spn-E mutants (Figure 3.1.7, (Cook et al., 2004). Taken together, the newly66 described nuage component KRIMP shares similarities in at least two or more spindleclass phenotypes with the other nuage component mutants armi, spn-E, vas, aub, and mael. To ensure that krimp phenotypes are the result of disrupting primary gene functions, I expressed a Venus(YFP)-tagged version of KRIMP protein under the control of an Upstream Activating Sequence (UASp) promoter in krimp mutant. By crossing the flies harbouring the UASp-krimp-venus transgene to flies that express the nosgal4VP16 transgene, Galactosidase (GAL4) binds the UASp promoter and drives the expression of KRIMP-Venus in a germline-specific manner (Appendix V; Fischer et al., 1988; Phelps and Brand, 1998). Using this UAS/GAL4 system, KRIMP-Venus protein was visualised as perinuclear foci that co-localised with endogenous VAS perinuclear foci in the wild-type ovary, therefore paralleling the localisation of endogenous KRIMP protein (Figure 3.1.8). However, when compared to endogenous KRIMP expression, more diffuse cytoplasmic KRIMP-Venus was observed (Figure 3.1.8), suggesting that the Venus-tag affected KRIMP localisation to the perinuclear nuage slightly. Although nosgal4VP16 overexpresses UASp-krimp-venus transgene, we did not see any gain-offunction phenotypes, even in the presence of functional KRIMP. 67 Figure 3.1.7 Nuage component mutants exhibit precocious osk translation. Ovary staining with anti-OSK. In wild-type, osk is translated at stages 7-9. Precocious translation of osk mRNA is observed in the nuage component mutants, as indicated by green arrowheads. Bar is 20 µm. 68 Figure 3.1.8 KRIMP-Venus(YFP) localises to the perinuclear nuage in the ovary. Immunostaining with anti-GFP (in green) and anti-VAS (in red) indicates that KRIMPVenus protein localises to the perinuclear regions of the germline cells, which colocalises with VAS perinuclear foci. Bar is 10 µm. UASp-krimp-venus transgene that was driven by nosgal4VP16 could fully rescue the female sterility defect in krimp mutant, with the compaction of the oocyte nucleus into a karyosome by stage in 100% (n = 44) of the egg chambers (Figure 3.1.9a), an accurate repression of osk translation in 100% (n = 24) of the ovarioles (Figure 3.1.9c), a normal GRK expression in 100% (n = 23) of the ovarioles, and the localisation of GRK to the anterior-dorsal corner of the oocyte in 97% (n = 31) of stage egg chambers (Figure 3.1.9d). This indicates that the fusion protein is fully functional and KRIMP localisation to the perinuclear nuage is essential for proper meiosis and oocyte polarity specification. Hence, all the observed phenotypes in krimpf06583 mutant ovary are as a result of the loss of CG15707 gene functions. 69 [...]... krimp mutant, with the compaction of the oocyte nucleus into a karyosome by stage 6 in 100% (n = 44) of the egg chambers (Figure 3. 1. 9a) , an accurate repression of osk translation in 100% (n = 24) of the ovarioles (Figure 3. 1.9c), a normal GRK expression in 100% (n = 23) of the ovarioles, and the localisation of GRK to the anterior-dorsal corner of the oocyte in 97% (n = 31 ) of stage 8 egg chambers (Figure...Figure 3. 1.8 KRIMP-Venus(YFP) localises to the perinuclear nuage in the ovary Immunostaining with anti-GFP (in green) and anti-VAS (in red) indicates that KRIMPVenus protein localises to the perinuclear regions of the germline cells, which colocalises with VAS perinuclear foci Bar is 10 µm UASp-krimp-venus transgene that was driven by nosgal4VP16 could fully rescue the female sterility defect in krimp... corner of the oocyte in 97% (n = 31 ) of stage 8 egg chambers (Figure 3. 1.9d) This indicates that the fusion protein is fully functional and KRIMP localisation to the perinuclear nuage is essential for proper meiosis and oocyte polarity specification Hence, all the observed phenotypes in krimpf065 83 mutant ovary are as a result of the loss of CG15707 gene functions 69 . localises to the perinuclear nuage in the ovary. Immunostaining with anti-GFP (in green) and anti-VAS (in red) indicates that KRIMP- Venus protein localises to the perinuclear regions of the germline. immunostaining of KRIMP indicates a wide expression in germline cells, including the differentiating germ cells in the germarium and egg chambers (Figure 3. 1. 2a) . KRIMP appears to localise to perinuclear. the mutant ovary (Figure 3. 1.4), indicating that krimp f065 83 is a loss -of- function allele. Moreover, immunostaining of krimp f065 83 ovary with anti-KRIMP indicated the loss of perinuclear