glaikit Is Essential for the Formation of Epithelial Polarity ... - Cell Press

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Nov 23, 2004 - John Dunlop,1,* Xavier Morin,1,3. Montserrat Corominas ..... Bloomington stock center, and the Hybridoma Bank for providing ily with extensive ...
Current Biology, Vol. 14, 2039–2045, November 23, 2004, 2004 Elsevier Ltd. All rights reserved.

DOI 10.1016/j .c ub . 20 04 . 10 .0 4 8

glaikit Is Essential for the Formation of Epithelial Polarity and Neuronal Development John Dunlop,1,* Xavier Morin,1,3 Montserrat Corominas,2 Florenci Serras,2 and Guy Tear1 1 Medical Research Council Centre for Developmental Neurobiology 4th floor, New Hunts House Guys Campus King’s College London London SE1 1UL United Kingdom 2 Departament de Genetica Facultat de Biologia Universitat de Barcelona Diagonal 645 02028 Barcelona Spain

Summary Epithelial cells have a distinctive polarity based on the restricted distribution of proteins and junctional complexes along an apical-basal axis. Studying the formation of the polarized ectoderm of the Drosophila embryo has identified a number of the molecules that establish this polarity. The Crumbs (Crb) complex is one of three separate complexes that cooperate to control epithelial polarity and the formation of zonula adherens [1, 2, 3]. Here we show that glaikit (gkt), a member of the phospholipase D superfamily, is essential for the formation of epithelial polarity and for neuronal development during Drosophila embryogenesis. In epithelial cells, gkt acts to localize the Crb complex of proteins to the apical lateral membrane. Loss of gkt during neuronal development leads to a severe CNS architecture disruption that is not dependent on the Crb pathway but probably results from the disrupted localization of other membrane proteins. A mutation in the human homolog of gkt causes the neurodegenerative disease spinocerebellar ataxia with neuropathy (SCAN1) [4], making it possible that a failure of membrane protein localization is a cause of this disease. Results and Discussion The gkt gene is initially expressed ubiquitously in the early embryonic ectoderm, after which its expression is restricted to neuroblasts and a subset of ganglion mother cells in stage 11 Drosophila embryos [5]. To investigate the role of gkt in the early embryo, we had to remove maternally derived gkt transcript because significant levels of transcript were detected in embryos homozygous for lethal mutant alleles of gkt (data not shown). Two lethal alleles of gkt were used for this analy*Correspondence: [email protected] 3 Current address: Developmental Biology Institute of Marseilles (IBDM), Case 907, Campus de Luminy, 13288 Marseille Cedex 9, France.

sis: the first, gktA12, was generated through the imprecise excision of the P element in the line l(2)k00223 [6] and consists of the deletion of a 58 bp section, which was partially replaced by a 35 bp random sequence upstream of the transcriptional start site of gkt (Figure 1A). The second allele, gktG85, was generated by EMS mutagenesis (Figure 1A). Animals homozygous for either gktA12 or gktG85 die at late embryonic/early larval stages. Neither gktA12 nor gktG85 was lethal in combination with alleles of the neighboring gene, mad. These results are in contrast to those for l(2)k00223, which was lethal when combined with mad alleles as well as with the gktA12 and gktG85 alleles (Figure 1B). This indicates that l(2)k00223 affects two complementation groups, mad and gkt, and no further analysis was performed with this allele. The gktG85 allele results in the loss of Gkt protein, which was confirmed in mosaic clones in the wing imaginal disc (Figures 1C–1E). Gkt transcript was removed from embryos via two methods: the germline clone (GLC) technique [7], the effectiveness of which was evident from the loss of transcript in an in situ hybridization (Figures 1F and 1G), and RNAi [8]. In the absence of maternal gkt, the same phenotype was observed by either technique: a loss of epidermal cuticle (Figures 2A and 2B) and a major disruption of the epidermis and central nervous system (CNS) (Figures 2C and 2D). The continuity of the embryonic epidermis was lost, probably due to cell death, thus exposing the underlying CNS. Overall, this disruption of the epidermis was very reminiscent of that caused by the loss of genes required for epithelial polarization [2,3]. To investigate the epidermal polarity in these embryos, we examined the localization of Ecadherin (Ecad), Zonula adherens (ZA) component [9] that is normally localized to the apical lateral membrane (ALM) (Figures 2E and 2F). Ecad levels were found to be reduced and no longer located circumferentially around the ALM of the epidermal cells, indicating that there indeed was a disruption to the polarization of the epidermal cells. The disruption in epidermal cell morphology was most evident after stage 10, at which point the majority of the epidermal cells lost their columnar appearance. We investigated the extent of the disruption to epidermal cell morphology by examining the localization of proteins that act as markers for epithelial polarization and are known to be necessary for the generation and maintenance of epithelial polarity. Within the embryonic ectoderm, there are three protein complexes that direct polarity formation. These complexes are represented by Crb or Bazooka (Baz), which both localize to the subapical region (SAR), and Discs large (Dlg), which is enriched in the ALM just basal to the ZA and SAR [9, 10, 11, 12]. In the gktGLC/RNAi embryos the wild-type Crb localization was severely disrupted (Figures 3A–3C). This loss of Crb was mirrored by a loss from the SAR of Stardust (Sdt) (data not shown), which has been shown to bind to Crb and act with Crb in the formation and maintenance of apical polarity in epithelial cells [13, 14]. Baz functions in a separate complex to that of Crb

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Figure 1. gkt Alleles (A) A schematic of the gkt gene locus (not to scale) shows the relative positions of the alleles used for the complementation analysis and of the genes mad, gkt, and CG3347, respectively. A small deletion (23 bp) and base pair substitution (35 bp; highlighted in red) are present in gktA12 upstream of the transcription start site of gkt. (B) Results of the complementation analysis using gktG85, gktA12, l(2)k00223, mad2, madB1, and KG05281. KG05281 is the closest lethal mutation proximal to gkt. (C) A merged image of a mosaic clone in the wing imaginal disc was generated with the gktG85 allele, with the clone marked by the loss of ␤-gal in the red channel and Gkt staining in the green channel. The clone is outlined with a dashed white line, and Gkt is absent from within the clone. (D) Single red channel from (C). (E) Single green channel from (C). (F) gkt in situ hybridization pattern in a wild-type precellularization embryo, which shows the presence of gkt transcript as a blue stain. (G) gkt in situ hybridization pattern in a gktGLC embryo, which was processed at the same time as that in (F) and has major reduction in gkt transcript.

and Sdt, even though it also localizes to the SAR [2, 3]. In stage 10 gktGLC/RNAi embryos, the localization of Baz was not severely affected, and Baz was still present

in an apical position in the lateral membrane (Figures 3D–3F). In older embryos the localization of Baz was no longer fully circumferential (data not shown), which was presumably an indirect consequence of the earlier loss of the Crb complex and epithelial polarity. Dlg is present in a complex with Scribble and Lethal giant larvae basal to the SAR, and in gktGLC/RNAi embryos it did not show a significant disruption in its pattern of localization; it remained enriched in the lateral region (Figures 3G–3I). In addition, the basal lateral membrane marker, Neurotactin (Nrt), was not affected in gktGLC embryos (data not shown). This analysis of polarized membrane markers suggests that appropriate apical localization of the Crb complex requires gkt, and thus the disruption in epithelial polarity in the absence of gkt is explainable in terms of Crb loss. The lack of any obvious cellularisation or early gastrulation defect, as well as the correct early localization of Armadillo, in the absence of gkt argues for a direct effect on the Crb complex. This does not eliminate the possibility that the effect on the Crb complex by gkt was via an intermediate mechanism involving a disruption in Ecad localization. One additional feature of gktGLC/RNAi embryos was a significant loss of secondary epithelial tissue, in contrast to results for crb11A22 embryos (data not shown). This may be due to a defect in the invagination of progenitor tissue or to an inability to rescue polarity after the initial disruption. Embryos that are homozygous for the gktG85 allele do not show any disruption in epithelial structure at stages 10-11, probably because of maternal rescue, whereas embryos homozygous for the null allele crb11A22 show a disruption in ectodermal cell polarity from stage 8, which typically includes a loss of the columnar morphology and a disruption in the distribution of junctional proteins [9, 15, 2, 3]. When one copy of crb was removed in gktG85/gktG85;crb11A22/⫹ embryos, these embryos exhibited a severe disruption in epithelial polarity (Figures 3J–3L). There was a similar enhancement of loss of epithelial polarity by gktG85 when one copy of sdt was removed from gktG85 embryos with a sdt allele (data not shown). There was no significant interaction between gkt alleles and either baz or dlg loss-of-function alleles. We extended the analysis of gkt’s role by examining the localization of more membrane proteins. gkt dsRNA was injected into lines that had been identified in a protein trap screen in which GFP is fused to particular membrane proteins [16], and the effect on their localization in epithelial cells was examined. Two membrane proteins whose localization was affected were Neuroglian (Nrg) and Lachesin (Lac), both members of the immunoglobulin superfamily [17, 18]. In wild-type embryos Nrg is localized to the lateral membrane of epidermal cells, whereas in gktRNAi embryos Nrg was present at reduced levels in the epidermal cells of gkt RNAi embryos (Figures 3M and 3N). This alteration in Nrg was not due to an indirect mechanism requiring the crb pathway because Nrg was still observed in the plasma membrane of crb11A22 homozygous embryos (Figure 3O). The loss from the lateral membrane of Nrg probably does not contribute to the loss of epithelial polarity because embryos null for Nrg have not been reported to exhibit polarity defects [19]. Lac behaved the same way as Nrg in that the loss of membrane localization was specific to the removal of gkt and not an indirect effect of the

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Figure 2. Epidermal Disruption (A and B) Cuticle preps of (A) wild-type and (B) gktRNAi embryos. (C and D) Nrt (red), marking the CNS, and Baz (green), marking the epidermis, are localized in (C) wild-type and (D) gktGLC st12-13 embryos. The arrowheads point to regions where the underlying CNS is revealed through loss of epidermal tissue. (E and F) En face view of Ecad localization in (E) wild-type and (F) gktGLC st11 embryos.

loss of polarity (data not shown). This indicates that the removal of gkt affects the localization of other proteins in addition to that of the Crb complex. The subcellular localization of Gkt was assayed with a rabbit polyclonal antibody raised against the C-terminal region of Gkt. Double staining with Crb indicated that Gkt did not colocalize with Crb at the SAR (Figures 4A–4F). Gkt was localized in the cytoplasm and did not appear in a discrete intracellular compartment such as the Golgi (Figures 4G–4I), nor was its distribution suggestive of the endoplasmic reticulum. This pattern of Gkt localization was also observed in newly delaminated neuroblasts, where Gkt was cytoplasmic and did not colocalize with the cortical marker Miranda (Figures 4J– 4L). The loss of Gkt can affect the localization of Crb, Sdt, and Nrg, which are present in the plasma membrane, whereas the localization of Gkt is cytoplasmic, sug-

gesting an indirect or transient molecular interaction between Gkt and these membrane proteins. In addition to the loss of epithelial polarity, removal of maternal gkt also had a severe effect on neuronal development, where there was an almost complete failure of normal axonal outgrowth (Figure 5B). This phenotype was apparent from early stages of axon outgrowth and was in contrast to that of crb11A22 mutant embryos, which have a relatively normal CNS at an equivalent stage (Figures 5C–5E). It was possible that these early defects were a result of a disruption in asymmetric cell divisions of neural progenitor cells, neuroblasts, which relies on the correct polarized localization of a number of proteins, including Baz, Inscuteable, and Pins [20]. The localization of these proteins was tested, and the proteins were found to be correctly distributed in dividing gktGLC/RNAi neuroblasts (data not shown), which

Figure 3. Loss of Epithelial Polarity (A–I) Lateral views of stage 9 stained epithelial layers that are wild-type (A, D, and G), from gkt RNAi injections (B, E, and H) and gkt germ line clones (C, F, and I). (A), (B), and (C) were stained for Crb, in red, and DNA, in blue. (D), (E), and (F) were stained with Baz, in green, and DNA, in blue. (G), (H), and (I) were stained with Dlg, in red, and DNA, in blue. (J–L) Lateral view of stage 11 epithelial layers stained with Dlg, in red, Baz, in green, and DNA, in blue, with the genotypes (J) wild-type, (K) crb11A22 homozygous, and (L) gktG85/ gktG85;crb11A22/⫹ . (M–O) Lateral view of stage 11 epithelial layers stained for GFP, in green, and DNA, in blue, in embryos expressing Nrg-GFP. (M) Homozygous Nrg-GFP, (N) homozygous NrgGFP after gktRNAi injections, and (O) nrgGFP/nrg-GFP;crb11A22/crb11A22.

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Figure 4. Localization of Gkt (A) En face view of stage 11 epithelial cells, with Gkt staining in green and Crb in red, and single channel views of Gkt (B) and Crb (C). (D) Lateral view of stage 11 epithelial cells, with Gkt staining in green and Crb in red, and single-channel views of Gkt (E) and Crb (F). (G) Lateral view of stage 11 epithelial cells, with Gkt staining in green and a golgi marker in red, and single-channel views of Gkt (H) and the golgi marker (I). (J) Lateral view of a stage 10 embryo stained with Gkt, in green, and Miranda, in red, and single-channel views of Gkt (K) and Miranda (L). The outline of the neuroblast in (J), (K), and (L) is marked by white dots.

suggests that gkt is not required for asymmetric positioning of determinants in neuroblasts. Crb is not expressed in the embryonic CNS, but Nrg and Lac are, which allowed their distribution to be assayed in this tissue after removal of gkt. Nrg and Lac both showed a reduction within the CNS, which in the case of Lac resulted in the loss of protein from the subset of neurons in which it is expressed (Figures 5F and 5G). Evidence for a specific role for gkt in neuronal development emerged from observations on axonal pathway formation in gktG85 homozygous embryos, in which maternal gkt transcript was available to complete early epithelial development. These embryos showed a defasciculation of axon bundles, both within the commissures and the longitudinals (Figures 5H and 5I). Staining with the neuronal specification markers, Even-skipped (Eve), Hunchback, and Engrailed revealed no alteration to their patterns of expression between wild-type embryos and homozygous gktG85 embryos (data not shown). This suggests that the axon defasciculation defects found in gktG85 homozygous embryos are not a consequence of incorrect neuronal specification. An alternative explanation is that the defects are more specific to the process of axon guidance; for example, one defect might be a failure to appropriately target membrane protein necessary for fasciculation to the axon cell surface. Support for this comes from the loss of Nrg and Lac within the CNS, and at least for Nrg there is a known role in axon guidance [19]. gkt is a member of the phospholipase D superfamily,

which catalyzes the cleavage of a phosphodiesterase bond by using an enzyme active site based around a duplicated HKD motif [21, 22]. It is likely that, if Gkt has an enzymatic activity, then the presence of ectopic Gkt will alter the signaling pathway in which it participates. Embryos in which either a Da-gal4 or a maternal gal4 driver caused overexpression of gkt in epithelial cells were stained with a variety of epithelial markers so that the consequence of increased Gkt activity in epithelial cells could be assessed. No effect on epithelial polarity was observed as a consequence of this ectopic gkt expression (data not shown). The effect of overexpressed Gkt on neuronal development was also tested with the neuronal-specific elav-Gal4 driver. A severe disruption in neuronal development was evident as a loss of organized axonal structure (Figures 5J and 5K). Abnormalities in the pioneer neurons could be seen from very early stages, but this was not reflected in a change in the pattern of expression of the specification marker Eve in stage 12 embryos (data not shown). This result indicates that overexpressed Gkt is capable of interfering with the normal differentiation of a neuron and would be consistent with Gkt acting as a rate-limiting step of a signaling pathway. It is clear that gkt has an important function during early epithelial and neuronal development; loss of gkt activity has serious deleterious consequences in both these tissues. During polarization of epidermal cells, gkt appears to be acting specifically on the crb pathway, but not on either the baz pathway or the dlg pathway.

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of the phospholipase D family of proteins are involved in the transport of membrane proteins [23, 24], which is consistent with the results presented here. Similarly, gkt may function in the embryonic CNS to transport a subset of proteins, including Nrg and Lac. It remains to be tested whether the CNS phenotypes generated after loss of gkt can be explained solely in terms of Nrg and Lac or whether there are additional affected membrane proteins that may also contribute. Interestingly, the human homolog of gkt, hTdp1, is affected in the neurodegenerative disease SCAN1 [4], which is caused by a point mutation in the enzyme active site of hTdp1. It is tempting to speculate that this disease may be caused by a disruption in membrane protein localization and that this disruption may lead to altered neuronal signaling and cell death in adults. An alternative hypothesis for Gkt function has come from study of the yeast homolog, Tdp1, which has been implicated in the rescue of inactive topoisomerase I covalently bound to DNA [25, 26]. The results reported here are not consistent with a role for gkt in DNA replication; there was no effect on neuroblast division in gktGLC/RNAi embryos, and the localization of Gkt is predominantly cytoplasmic. Furthermore, SCAN1 mainly affects postmitotic neurons, which would not be expected to be affected by a disruption in DNA replication [4]. For this reason, we refer to gkt as being a member of the phospholipase D superfamily and leave open the question of the substrate of gkt. The data presented here indicate a role for gkt in the localization of a subset of membrane proteins, and we favor a model where Gkt would function cytoplasmically to regulate protein transport or expression. Experimental Procedures

Figure 5. Disruption of CNS Development (A and B) Stage 17 embryos stained with anti-FasII, which labels three fascicle tracts either side of the ventral midline, in (A) wildtype and (B) gktGLC embryos. (C, D, and E) Stage 13 embryos stained for FasII, in (C) wild-type, (D) gktGLC, and (E) crb11A22 homozygous embryos. (F and G) Live stage 14 Lac-GFP-expressing embryos that were either not injected (F) or injected (G) with gkt dsRNA. (H and I) Stage 17 Sema2b-tau-myc embryos were stained for Myc to visualize cell bodies and axons in (H) wild-type and (I) gktG85 homozygous backgrounds. The arrowhead in (H) points to a normal commissure in which midline-crossing axons are tightly bundled, whereas in (I) the arrowheads mark commissures in which axons are no longer bundled together. (J and K) Overexpression of gkt in the CNS. Anti-FasII staining of stage 13 embryos that are (J) wild-type and (K) overexpressing gkt under the control of elav-gal4.

A possible link between gkt and the crb pathway is that gkt could be necessary for the transport of crb pathway components to the SAR. A role in membrane protein transport would be consistent with the loss of Nrg and Lac from the lateral epithelial membrane. The function of gkt in localizing membrane proteins such as the Crb complex probably involves the conserved HKD motif, but the potential substrate is not clear. Other members

Immunocytochemistry and Confocal Microscopy Primary antibodies used were as follows: mouse monoclonal antiCrb (Cq4, 1:10; Developmental Studies Hybridoma Bank), mouse monoclonal anti-Ecad (DCAD2, 1:10; Developmental Studies Hybridoma Bank), mouse monoclonal anti-Dlg (4F3, 1:10; Developmental Studies Hybridoma Bank), rabbit polyclonal anti-Baz (1:1000; a gift from E. Knust), mouse monoclonal anti-Nrt (BP106, 1:10; Developmental Studies Hybridoma Bank), rabbit polyclonal anti-GFP (1:1000; Molecular Probes), mouse monoclonal Drosophila Golgi marker (1:500; Calbiochem), mouse monoclonal anti-FasII (1D4, 1:10; Developmental Studies Hybridoma Bank), mouse monoclonal anti-C-Myc (9E10, 1:1000; Developmental Studies Hybridoma Bank), rabbit polyclonal anti-Gkt (1:1000). Secondary antibodies were conjugated to Cy3, Cy5, and fluorescein isothiocyanate (FITC) (Jackson ImmunoResearch Laboratories, West Grove, PA) and used at a dilution of 1:300. Embryos were fixed in 4% formaldehyde and 1⫻ PBS for 20 min. Embryos were mounted either in glycerol (after DAB staining) or Vectashield (after fluorescence staining; from Vector Labs). Laser scanning confocal microscopy was performed with either an Olympus Fluoview or a Zeiss LSM510.

Glaikit Antibody A polyclonal antibody against Gkt was raised in rabbits under standard conditions (Harlan Sera-lab, Loughborough, UK), with the purified C-terminal 20 kDa region of Gkt being fused to a 6xHis tag. The ability of the polyclonal Gkt antibody to recognize a specific protein of correct predicted size was confirmed by Western analysis of whole embryo extracts. Immunostaining with the anti-Gkt polyclonal antibody was performed at 1:1000 dilution with embryos that were fixed for 20 min in Bouin’s fixing fluid and then processed under standard conditions.

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Generation of Mutant Alleles of gkt Lethal alleles in gkt were generated through the imprecise excision of the P-lacW element present in the line l(2)k00223 [6], which is inserted 16 bp downstream of the transcriptional start site of the mad gene locus and 300 bp upstream of the transcriptional start site of the gkt gene locus. The imprecise excision line gktA12 was tested for complementation with previously characterized mad alleles. Molecular analysis was carried out on homozygous mutant embryos that had been selected for the lack of the fluorescent balancer chromosome Cyo (Kr-GFP), and the genomic sequence of the gkt region was ascertained by the direct sequencing of amplified PCR products. The allele gktG85 was generated by EMS mutagenesis and selected by lethality over the gktA12 allele.

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10. Drosophila Stocks Mutant strains already characterized and used here were crb11A22, sdtEH, dlgG3, and baz4. The lines Lac-GFP and Nrg-GFP were generated in a protein trap screen [16] and contain the GFP exon inserted into the genes lac and nrg, respectively. The ectopic expression of gkt was performed with the UAS/Gal4 system [27]. Germ line clones (GLC) were generated with the allele gktA12 once it had been recombined with a FRT40A chromosome. The induction of GLCs was performed by the FLP-DFS technique [6]. The line Sema2b-taumyc expresses the Tau-Myc fusion protein in the Sem2b-expressing neurons and was obtained from B. Dickson [28]. Homozygous embryos were identified with balancer chromosomes containing hblacZ or Kr-GFP. RNAi Templates for dsRNA synthesis were PCR generated to introduce T7 promoters at each end. Double-stranded RNA was generated with the MEGAscript kit (Ambion Inc., Austin, TX), and injection of embryos and immunohistochemistry were as described [8].

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Acknowledgments We would like to thank Susanna Romani and Bill Chia for their critical reading of the manuscript and Eli Knust, Barry Dickson, the Bloomington stock center, and the Hybridoma Bank for providing antibodies and fly stocks. This work was supported by the Biotechnology and Biological Sciences Research Council, a European Molecular Biology Organization long-term fellowship (J.D.), and the Marie Curie Fellowship (X.M.). Received: February 27, 2004 Revised: September 23, 2004 Accepted: September 23, 2004 Published: November 23, 2004 References 1. Bilder, D., Li, M., and Perrimon, N. (2000). Cooperative regulation of cell polarity and growth by Drosophila tumour suppressors. Science 289, 113–116. 2. Bilder, D., Schober, M., and Perrimon, N. (2003). Integrated activity of PDZ protein complexes regulates epithelial polarity. Nat. Cell Biol. 5, 53–58. 3. Tanentzapf, G., and Tepass, U. (2003). Interactions between the crumbs, lethal giant larvae and bazooka pathways in epithelial polarisation. Nat. Cell Biol. 5, 46–52. 4. Takashima, H., Boerkoel, C.F., John, J., Saifi, G., Salih, M.A.M., Armstrong, D., Mao, Y., Quiocho, F.A., Roa, B.B., Nakagawa, M., et al. (2002). Mutation of TDP1, encoding a topoisomerase I-dependent DNA damage repair enzyme, in spinocerebeller ataxia with neuronal neuropathy. Nat. Genetics 32, 267–272. 5. Dunlop, J., Corominas, M., and Serras, F. (2000). The novel gene glaikit, is expressed during neurogenesis in the Drosophila melanogaster embryo. Mech. Dev. 96, 133–136. 6. Roch, F., Serras, F., Cifuentes, F.J., Corominas, M., Alsina, B., Amoros, M., Lopez-Varea, A., Hernandez, R., Guerra, D., Cavicchi, S., et al. (1998). Screening of larval/pupal P-element induced

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