Functional analysis of the nuage, a unique germline organelle, in drosophila melanogaster 3

59 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 th

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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 1 (ORF1), possess protein-protein interaction properties (Gubitz et al., 2002; Hoogeveen and Oostra, 1997; Martin et al., 2004)

0.5kb

PBac{WH}CG15707 f06583

TUD 613-670aa

ZnF 512-539aa

Coiled-coil

17-37aa

a

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MNLEDISMIMKLFDSNMHKLQGNLRSYQTEMHQIHKELTEKLSHADLLYRSLIPLHDHLVASLSEVNAHVM KLNVQLHINRQSVRLGDYEYYEKSIDNPYSSIRSGLQAIEKPGCAEAICQSSKPAFVECLPSSTSEEVPVV AVQEASSTNQLDAISVVNENLSEERDATPQPLAVSKNMEETMPSNPFHEQLEGSLEEIPVGSKIVVETEKA NNPVRSEASAPATSDNQSLLAAKQGTQTIGTGICNKISKSTINMPNNWQLENPTEVTAASIEKVNKLPKSP RNRFLLPPKGGTETTRRDIYNQILKDMAAFPENTIVTAVLASVDVTDNCAYVAKWDESSDRIKKVLQRQLP LQELDQLPDYGDIFAVLDSINNIITRITINSSSAGGGYDAYLIDFGEHIHFDGNETIFKLPDDIKRLPAQA IRCDLINCDIANMHCFVNTYIKIRVHENNNSTLVAEPVIDRLSRPTKTNTTKYPAGITEDDMAMLNEIDES

TSDPLKAVLGFRPKDEQRICRHYDPKLNGCFKGNNCRFAHEPFAPNGATKDVELARALPETIFDTTVHFEI

GSIVGILITFINGPTEVYGQFLDGSPPLVWDKKDVPENKRTFKSKPRLLDIVLALYSDGCFYRAQIIDEFP SEYMIFYVDYGNTEFVPLSCLAPCENVDSFKPHRVFSFHIEGIVRSKNLTHQKTIECIEYLKSKLLNTEMN VHLVQRLPDGFLIRFLDDWKYIPEQLLQRNYAQVSQ

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)

b

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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

co-localisation Bar is 4 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

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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

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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 krimp f06583 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 krimp f06583 is a

loss-of-function allele Moreover, immunostaining of krimp f06583 ovary with anti-KRIMP indicated the loss of perinuclear foci (Figure 3.1.2b)

Figure 3.1.4 krimp f06583 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

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To confirm that krimp f06583 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 krimp f06583/Df(2R)Exel6063 exhibited female sterility and a similar extent of loss in KRIMP perinuclear staining to

that of homozygous krimp f06583 (Appendix IV) Hence, krimp f06583 was employed as a

loss-of-function allele to characterise krimp phenotypes in this thesis

In krimpmutant ovary, progression of oogenesis was compromised and degeneration of the egg chambers was observed from stage 8 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 3 and C(3)G dissociates to become extra-chromosomal (Page and Hawley, 2001)

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Figure 3.1.5krimp mutant exhibits meiotic progression defects Immunostaining with

a synaptonemal complex marker C(3)G (green), shows that the krimpoocyte nucleus fails

to compact into a karyosome (blue) Bar is 5 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 in100% (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 8 onwards mutant egg chambers (Figure 3.1.6; Neuman-Silberberg and Schupbach, 1996)

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Figure 3.1.6 krimp mutant exhibits oocyte polarity defects Ovary staining with

anti-GRK The level of GRK expression is downregulated (green arrowheads) and its

dorsal-anterior localisation is disrupted in stage 8 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 9 (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

newly-67

described nuage component KRIMP shares similarities in at least two or more

spindle-class 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 4 (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-of-function phenotypes, even in the presence of gain-of-functional KRIMP

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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

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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 KRIMP-Venus protein localises to the perinuclear regions of the germline cells, which co-localises 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 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 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 krimp f06583 mutant ovary are as a result of the loss

of CG15707 gene functions