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Genes to Cells (2009) 14, 1067-1077. doi:10.1111/j.1365-2443.2009.01335.x
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Essential requirement for RING finger E3 ubiquitin ligase Hakai in early embryonic development of Drosophila

 Masako Kaido1,a, Housei Wada1, Masayo Shindo1,b and Shigeo Hayashi1,2,*

1 Riken Center for Developmental Biology, 2-2-3 Minatojima-minamimachi, Chuo-ku, Kobe 650-0047, Japan
2 Department of Biology, Graduate School of Science, Kobe University, Kobe 657-8501, Japan


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Experimental procedures
 References
 
Hakai is a RING finger type E3 ubiquitin ligase that is highly conserved in metazoans. Mammalian Hakai was shown to bind and ubiquitinate the intracellular domain of E-cadherin, and this activity is implicated in down-regulation of E-cadherin during v-Src-induced cellular transformation. To evaluate this model in vivo, we studied the function of the Drosophila homologue of Hakai. In cultured S2 cells, Drosophila Hakai and E-cadherin (Shotgun) formed a complex in a way distinct from the interaction described for mammalian counterparts. Hakai null mutants died during larval stages but this lethality could be offset by a HA-tagged Hakai construct. While zygotic Hakai function was dispensable for cell proliferation and differentiation in the wing disc epithelium, maternal Hakai mutants showed a variety of defects in epithelial integrity, including stochastic loss of E-cadherin expression and reduction of aPKC; defects in cell specification and cell migration were also observed. No increase of E-cadherin, however, was observed. Regulation of multiple target proteins under control of Hakai is, therefore, essential for early embryonic morphogenesis in Drosophila.


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Experimental procedures
 References
 
Dynamic exchanges of cell adhesion molecules in cell–cell junctions are essential for concerted movement of cells during morphogenesis. E-cadherin is a key organizer of the adherens junction of epithelial tissues and much attention has been focused on molecules regulating localization and stability of E-cadherin (Takeichi 1991; Miyoshi & Takai 2008). In epithelial Madin-Darby canine kidney (MDCK) cells, activation of v-Src downregulates E-cadherin activity and dissociates cell–cell adhesion (Behrens et al. 1993; Takeda et al. 1995), mimicking epithelial-to-mesenchymal transformation in cancer. Hakai is a RING finger type E3 ubiquitin ligase with affinity to the intracellular domain of E-cadherin (Fujita et al. 2002). A model was proposed that Hakai is involved in negative regulation of E-cadherin by v-Src in MDCK cells (Fujita et al. 2002); according to this model, Hakai binds and ubiquitinates tyrosine-phosphorylated E-cadherin, and induces its endocytosis. Although additional works with cultured cells reported results consistent with the model (Shen et al. 2008), gene disruption studies have yet to be reported for mice or other vertebrates. Thus, it is not clear to what extent this mechanism contributes to the regulation of cell adhesion in living organisms. To address this issue, we isolated a Drosophila counterpart of Hakai and studied its function using genetic and biochemical approaches.


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Experimental procedures
 References
 
Hakai forms a complex with E-cadherin in Drosophila cells

Blast searches identified CG10263 as the only Drosophila gene with significant homology to mammalian Hakai in their RING finger domain (68% homology in a 110-amino acid region). CG10263 encodes three splice variants that share the common N-terminal region including the RING finger domain (Fig. 1a). To test if these proteins interact with Drosophila E-cadherin, we expressed a genomic fragment of CG10263 in Drosophila S2 cells. Antibodies against CG10263 or N-terminally tagged HA epitope detected two bands, likely corresponding to CG10263-PD and CG10263-PA/PC (Fig. 1b, data not shown). Immunoprecipitation with anti-CG10263 antibody recovered coexpressed E-cadherin–GFP fusion protein, and anti-GFP recovered CG10263 proteins, suggesting that E-cadherin and Hakai associate with each other (Fig. 1b). Similar recovery of the two bands of CG10263 proteins also suggests that the protein interaction involves a region shared by CG10263-PD and CG10263-PA/PC, including the RING finger domain. To determine if CG10263 is involved in protein ubiquitination, ubiquitination status of proteins associated with CG10263 was examined. Immunoprecipitates of E-cadherin included a number of ubiquitinated proteins, and their amount increased with coexpression of CG10263 (Fig. 1c). Immunoprecipitates of CG10263 also copurified ubiquitinated proteins (Fig. 1c). Based on the structural homology, E-cadherin binding and its association with ubiquitination activity, we concluded that CG10263 is the Drosophila counterpart of mammalian Hakai and, hereafter, was renamed Hakai.


Figure 1
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  		Figure 1  Characterization of the Drosophila hakai gene product. (a) Genomic organization of CG10236. Exon–intron structures of three isoforms are indicated. Boxes filled black, red, blue, yellow represent coding region and grey boxes represent noncoding sequences. Red box corresponds to the RING finger domain shared by all isoforms. Yellow and blue boxes correspond to the sequence unique to the long (CG10236-RD) and short (CG10236-RA and -RB) isoforms respectively. Thin lines correspond to introns and intergenic regions. The genomic region included in the HA-tagged rescue construct, and the deleted regions in the hakai1 mutation are indicated in the bottom two lines. (b) Association of Hakai and E-cadherin in Drosophila S2 cells. Drosophila E-cadherin-GFP (DEFL, functional C-terminal GFP fusion) and Hakai were coexpressed in S2 cells and immunoprecipitation assays were performed. The anti-E-cadherin antibody detected a 150-kDa band of the extracellular domain. Both of the long and short isoforms of Hakai were coprecipitated by the antibody against the GFP tag of DEFL. (c) Ubiquitination assay. DEFL and Hakai were coexpressed with HA-tagged ubiquitin, and cell lysates were prepared for immunoprecipitation assays. Immunoprecipitation with anti-GFP detected major ubiquitinated bands below the 100 kDa marker. It is likely that those bands include the intracellular fragment of DEFL (see right hand panel) and Armadillo. In addition, numerous bands with slower mobility were detected. Bands migrating below the 50 kDa marker are heavy chains of IgG.

 
To map the domain in E-cadherin required for interaction with Hakai, we made a series of deletions in the cytoplasmic domain of E-cadherin (Fig. 2a). These deletion proteins were stably expressed (Fig. 2b), and localized to the plasma membrane (data not shown). Deletion of position 1369–1499 (del 1, including the β-catenin-binding domain) or the entire cytoplasmic domain (TM–GFP) impaired the activity to stabilize Armadillo, the Drosophilaβ-catenin, as previously reported (Fig. 2b; Oda & Tsukita 1999b). Unexpectedly, we found that del1 and TM-GFP proteins were recovered in anti-Hakai immunoprecipitates with efficiencies comparable to the full-length E-cadherin. Replacement of the GFP tag with a Myc tag in full-length or cytoplasmic domain-deleted versions of E-cadherin did not alter their ability to interact with Hakai (Fig. 2d). As a control for antibody specificity, unfused GFP protein was coexpressed with Hakai, but it was not recovered by anti-Hakai antibody (Fig. 2e). These results suggest that the cytoplasmic domain of Drosophila E-cadherin is entirely dispensable for association with Drosophila Hakai proteins. As the structure of Drosophila Hakai proteins predicts cytoplasmic localization, its interaction with Drosophila E-cadherin is likely indirect, involving at least one additional component that bridges the cytoplasmic Hakai protein and the extracellular or transmembrane domain of E-cadherin. This result was surprising as the cytoplasmic fragment of mouse E-cadherin was shown to associate with the mammalian Hakai protein (Fujita et al. 2002).


Figure 2
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  		Figure 2  Mapping of the E-cadherin region required for association with Hakai. (a) Structure of deletion derivatives of E-cadherin-GFP construct DEFL. This protein is cleaved at position 1010 (cleavage site) in the extracellular domain to form a heterodimer consisting of noncovalently associated extracellular and C-terminal fragments (Oda & Tsukita 1999b). (b) Expression of deletion mutants. In the upper panel, anti-E-cadherin detected the 150 kDa extracellular fragment. Minor bands of slower mobility are uncleaved proteins. Lower panel shows β-galactosidase coexpressed as an internal control for transfection efficiency, and endogenous Armadillo protein that was stabilized by association with the intracellular domain of E-cadherin. The blotted filter was sequentially probed by two antibodies. (c) Association of Hakai and deletion derivatives of DEFL. Immunoprecipitation of Hakai recovered coexpressed DEFL and its deletion derivatives. Arm was also recovered by DEFL and del4. (d) Myc-tagged E-cadherin (Full) and its deletion derivative lacking the entire intracellular domain (TM) both precipitated with Hakai. (e) Control for antibody specificity. Immunoprecipitation with anti-Hakai did not recover unfused GFP.

 
Intracellular localization of Hakai

Expression of hakai in embryos was examined by in situ hybridization. High level of maternal transcript was detected in blastderm stage embryos (Fig. 3b), and the transcript persisted up to stage 14 (Fig. 3d, f, h), consistent with previous report (Tomancak et al. 2002; BDGP gene expression database http://www.fruitfly.org). We also noted high hakai expression in migrating endoderm (anterior and posterior midgut, asterisks in Fig. 3h). To visualize the intracellular distribution of Hakai, we stained S2 cells transfected with the HA-Hakai construct (Fig. 3i,i'). Both anti-Hakai and anti-HA staining revealed identical signals distributed broadly throughout membranous structures in the cytoplasm, perinuclear region, and the plasma membrane, and this localization was identical to untagged Hakai protein (Fig. 3i,j, and data not shown). When coexpressed with E-cadherin, HA-Hakai was found enriched at the cell–cell contact interface sites (Fig. 3j,j', arrow).


Figure 3
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  		Figure 3  Expression of hakai in Drosophila embryos and S2 cells. HA-Hakai was detected with anti-HA antibody. (a–h) Embryos hybridized with sense (S) or antisense (AS) probe of hakai. (a, b) Stage 5; (c, d) stage 7; (e, f) stage 8; (g, h) stage 14. Asterisk indicates midgut primordia (endoderm). (i) Expression of Hakai was detected with anti-Hakai antibody. (i') DIC image. (j) S2 cells coexpressing HA-Hakai (magenta) and DEFL (green, j'). HA antibody was used to detect of Hakai. DEFL induced tight cell adhesion (arrow) where high accumulation of Hakai was detected. (k) Lateral ectoderm of stage 15 embryo expressing HA-Hakai by the daughterless-Gal4 driver. (l) High magnification image of HA-Hakai stained embryos, co-stained with DAPI (green). (L', L'') Single channel images. Bar: 2 µm. (m–t) Apical (m, q) and basal (n–p, r–t) views of ectodermal cells stained for E-cadherin, HA-Hakai and Rab11. Single and double label views are shown. Arrows indicate vesicular structures coexpressing E-cadherin and HA-Hakai.

 
We then examined embryonic expression of Hakai. Our anti-Hakai antibody failed to detect histochemically the endogenous Hakai epitope, so we stained embryos expressing HA-Hakai under the control of the ubiquitous daughterless-Gal4 driver. As shown below, HA-Hakai was able to offset the zygotic lethality of hakai null mutation. At low magnification, HA-Hakai appeared to be enriched around the nucleus (Fig. 3k); higher magnification views revealed punctuate structures in the cytoplasm and the perinuclear region (Fig. 3l,l'), and the signal distribution was distinct from the chromosome stained with DAPI (Fig. 3l''). To determine the identity of the punctuate cytoplasmic HA-Hakai patterns, the embryos were co-stained with anti-E-cadherin and the recycling endosome marker Rab11 (Fig. 3m–t). In addition to its localization in the adherens junction (Fig. 3m), E-cadherin existed as intercellular vesicles with a fraction of them colocalizing with Rab11 (33%, n = 90, Fig. 3o), as previously reported (Langevin et al. 2005; Lock & Stow 2005). Furthermore, a significant fraction of E-cadherin-positive intracellular vesicles were found to overlap with Hakai (25%, n = 92, Fig. 3n). On the other hand, we detected no overlap of Hakai and Rab11 (0%, n = 90, Fig. 3p). Co-staining with early endosome marker Rab5 and late endosome marker Rab 7, and GFP markers for Golgi apparatus and lysosome revealed no significant overlap with HA-Hakai (data not shown). These results indicate that Hakai coexists with E-cadherin in an intracellular vesicle compartment; the identity of those vesicles remains to be determined.

Isolation of Hakai null mutation

We isolated a deletion allele (hakai1) of hakai by P-element excision from the semi-viable insertion strain CG10263KG01389. hakai1 lacks the coding region covering the N-terminus and the RING finger domain, and was considered to be a null allele (Fig. 1a). Most homozygous hakai1 mutant embryos completed embryogenesis and become motile larvae, but died at some point during their larval stage, and no viable adults were recovered. This zygotic lethality was partially offset by HA-Hakai expressed by the ubiquitous daughterless promoter (41% of expected viability). The rescued flies exhibited minor morphological abnormalities including small wings, kinked legs and short life-span. Nevertheless, many of the rescued males and females were fertile and produced normal progenies when crossed with wild-type flies, suggesting that the genomic HA-hakai construct encodes functional hakai products. Zygotic hakai function is, therefore, essential for larval viability in Drosophila.

To characterize the potential roles of hakai in cell adhesion, we stained homozygous hakai1 embryos with a set of markers including adherens junction protein E-cadherin and Arm, septate junction protein Dlg and Fasciculin 3, and subapical domain protein Crb and aPKC. We also performed a time-lapse analysis of the tracheal system labelled with the adherens junction marker alpha-catenin-GFP (Oda & Tsukita 1999a). There were no cases where we were able to detect obvious abnormalities in protein distributions and tissue morphology (data not shown). We next assessed postembryonic functions of hakai by mosaic analysis. The cross was designed so that both hakai1 homozygous and +/+ sister clones were simultaneously marked with different markers (Fig. 4d). In third instar wing discs, the cells in hakai1 clones proliferated at a rate comparable to their wild-type twin clones (Fig. 4a,b). In those clones, expression pattern of E-cadherin and Dlg did not change (Fig. 4c and data not shown). Adult flies carrying hakai1 clones developed wings with normal size and shape; the only defect we noted was occasional duplications of mechanosensory bristles. We thus concluded that most of the major morphogenetic events of the wing disc epithelia could precede without zygotic function of hakai, although its role in larval viability and sensory morphogenesis remains to be elucidated.


Figure 4
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  		Figure 4  Somatic hakai-mutant cells in the wing imaginal disc. Wing imaginal disc with expression of Kusabira Orange (red) and E-cadherin (grey) (a), of Kusabira Orange and Myc (green) (b), and E-cadherin alone (c) is shown. Homozygous hakai1-mutant cells were induced by the MARCM mitotic recombination technique (d). In a, mutant cells were positively marked by the expression of Kusabira Orange. In b, mutant cells were additionally labelled by 2x copy Myc epitope, and +/+ cells in sister clones were marked by the lack of the Myc epitope. Mutant cells occupied a large fraction of the wing pouch, suggesting that the mutation did not compromise cell proliferation. Expression of E-cadherin and epithelial architecture was not altered in mutant territories.

 
Maternal Hakai function is essential for epithelial integrity

To address the role of Hakai in early embryogenesis, we performed germline mosaic analyses. Hakai-mutant germlines completed normal oogenesis, but most of fertilized embryos died before hatching. Cuticle preparations of these embryos revealed phenotypes ranging from poorly formed denticles to a near complete absence of exoskeletons. An example shown in Fig. 5b revealed failure in head involution and dorsal closure, suggesting epithelial formation and cell movement required for those morphogenetic processes are impaired. Discontinuity or complete loss of denticle belts suggests segmentation was also affected.


Figure 5
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  		Figure 5  Ectodermal phenotypes of embryos lacking maternal Hakai activity. (a, b) Cuticle preparation of control and severe class of hakai1 embryos. Mutant embryos showed defects in dorsal cuticle and head structure. Ventral denticle belts were discontinuous. (c) zygotic hakai1/+ embryo derived from hakai1 egg. Expression of wg-lacZ showed defective segmentation and ectodermal organization. (d, e) Structure of dorsal ectoderm revealed by E-cadherin staining. hakai1 embryos showed disorganized dorsal epidermis. (f, g) Ectodermal cells undergoing tracheal invagination (magenta, E-cadherin; green DAPI). Control embryo at stage 11 showed tightly organized epithelia. In hakai1 embryos, a number of cells that lost E-cadherin dissociated from the ectoderm (arrows and a cell cluster in the tracheal pit). Segmental groove was also abnormal. (h, i) Stage 16 embryos stained with E-cadherin and aPKC. Mild class hakai1 embryos lost aPKC expression.

 
We next examined mutant embryos with a set of cell surface markers to examine the status of cell adhesion. Paternal hakai+ chromosome was marked with wg-lacZ to distinguish its zygotic genotype. Both of the embryos with or without the paternal copy of hakai showed morphological defects. The phenotypes ranged widely, from severe classes showing segmentation defects (Fig. 5c) and loss of epithelial integrity (Fig. 5g), to mild classes showing reduction of a subset of cell surface markers (Fig. 5i). We noted that, in rare cases, paternally rescued escapers developed to adulthood, suggesting a significant contribution of paternal (zygotic) hakai expression in development.

In severe-class mutants, even maternal hakai1 mutants with zygotic hakai+ chromosome showed segmentation defects as revealed by the expression of the wingless-LacZ marker (Fig. 5c). The dorsal ectoderm of the severe class of maternal and zygotic hakai mutants (m–z–) consisted of epithelial cells of varying size and morphology clearly distinct from the uniformly stretched epithelial cell morphology seen in control embryos (Fig. 5d,e). This phenotype was also previously observed in embryos defective in dorsal closure (reviewed in Harden 2002), and likely accounts for the failure in dorsal closure. In addition, severe-class embryos showed deteriorated epithelial integrity (Fig. 5c,g); cells frequently lost expression of E-cadherin and detached from the body wall epithelia (Fig. 5g, arrowheads). Those abnormal cells were most prominent in epithelial regions undergoing major morphological changes, such as tracheal invagination and segmental furrow formation (Fig. 5g). In mild-class embryos that reached stage 16, the epithelial integrity appeared relatively normal as revealed by apically localized expression of E-cadherin (Fig. 5i). Expression of Crb, Dlg and Fas3 was also normal (data not shown). However, those embryos showed significantly reduced expression of the subapical marker aPKC (Fig. 5i').

Hakai function in midgut morphogenesis

Based on the expression of hakai RNA in midgut primordia (Fig. 3h), we next asked if Hakai functions in midgut formation. Midgut of control embryos consists of yolk covered by endodermal epithelia with round nuclei. The central cells of midgut epithelia have further grown to contain especially large polyploid nuclei (tentatively called middle midgut endoderm, mme). Ventral side of midgut is associated with longitudinal belts of visceral mesoderm consisting of thin elongated cells (Fig. 6a,c). In hakai m–z– mutants, endoderm cell layer and associated visceral mesoderm remain separated in anterior and posterior parts, whereas mme appeared in the anterior edge of stalled posterior midgut (Fig. 6b,d). Those observations suggest that endoderm and visceral mesoderm failed to reach proper positions in hakai m–z– mutants, leading to defect in midgut formation.


Figure 6
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  		Figure 6  Defective midgut morphogenesis in hakai mutants. Control (a, c) and hakai1 m–z– (b, d) embryos stained with Dlg (grey scale or magenta) and DAPI (green). (c, d) Twofold magnified views of the midgut part of a and b respectively. mme: middle midgut endoderm, end: endoderm, vm: visceral mesoderm.

 
Time-lapse analyses revealed requirement of Hakai for F-actin organization and cell migration

To further characterize the phenotype of the mild-class hakai mutants, we performed time-lapse analysis of tracheal cells marked with the F-actin marker GFP-moesin to monitor the dynamics of F-actin—rich filopodia (Chihara et al. 2003). Second chromosome insertions of btl-Gal4 and UAS-GFP-moesin were recombined with hakai1, and males carrying the recombinant chromosome were crossed with females with germline clones of hakai1. Thus, only m–z–hakai mutants were labelled with GFP expression. Embryos that reached the stage of dorsal branch elongation (stage 15) were selected for imaging of migrating tracheal cells. In hakai mutants, the dorsal branch terminus was misshapen and misrouted (Fig. 7b), and some cells were only loosely associated with rest of the branch (Fig. 7b, arrow). Duplication of terminal cells was also frequently observed (Fig. 7b, asterisk, 28% of dorsal branch, n = 32 from four time-lapse recorded embryos. Control: 0%, n = 60, six embryos). Abnormally long filopodia were found extending in the wrong direction, and disintegrated or accumulated large amounts of F-actin (Fig. 7b, arrowhead). Taken together, these time-lapse imaging results suggest that hakai is required for F-actin organization in migrating tracheal cells.


Figure 7
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  		Figure 7  Time-lapse analysis of tracheal branch migration. Images of embryos expressing the F-actin marker GFP-moesin under the control of btl promoter were captured by confocal time-lapse microscopy (Movies S1 and S2 in Supporting Information). Dorsal views of embryos at stage 15 are shown (anterior, left). Different time points of segments outlined by the dashed-square are shown below. Dorsal branches from each hemi-segment migrated toward dorsal midline and go through tube fusion (60' of a). DT indicates dorsal trunk. In hakai1 embryos (b), duplicated terminal branch (asterisk), loosened cell–cell interface (arrow), misdirected and disintegrating filopodia (arrowhead) are shown.

 

   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Experimental procedures
 References
 
In this work, we characterized the Drosophila counterpart of mammalian Hakai protein, which has E-cadherin-binding and associated ubiquitination activities. We demonstrated that maternal Hakai products are essential for maintaining integrity of ectodermal epithelia, and F-actin based cell motility and cell shape change in tracheal cells. The reduction of E-cadherin levels in the cells detaching from the body wall, and the down-regulation of aPKC suggests that Hakai is required for proper function of cell surface proteins. In addition, defects in midgut morphogenesis suggest that Hakai function is also required for endoderm and mesoderm morphogenesis.

Our data allowed us to identify the stage when Hakai function is required. Most of the maternal hakai mutants, with and without the paternal hakai+ chromosome, are embryonic lethal. About one-half of them showed the severe-class phenotype exhibiting abnormalities in epithelial architecture, and the rest reached late embryonic stages with an apparently normal distribution of E-cadherin localization. Only a small fraction of mutants with paternal Hakai function escaped embryonic lethality and reached adulthood. Removal of hakai function during the larval stage did not affect the epithelial architecture of wing imaginal discs. We thus conclude that Hakai function is most vital in the early stages of embryogenesis and its contribution declines in later stages.

Observations of Hakai mutant embryos allowed us to assess the proposed model of Hakai function in the down-regulation of E-cadherin (Fujita et al. 2002). Despite the defects in epithelial morphogenesis, overall level and distribution of E-cadherin within cells that retained epithelial integrity appeared within normal range. We also performed cell surface biotinylation experiments on S2 cells overexpressing Hakai, but did not detect significant reduction of E-cadherin at the cell surface (MK, unpublished observation). In addition, the apparent absence of a direct interaction between Hakai and the intracellular domain of Drosophila E-cadherin suggests a different mode of association between these proteins. We thus concluded that Drosophila Hakai does not play a major and direct role in down-regulation of E-cadherin levels.

The graded expressivity of the hakai-mutant phenotype may be understood in the context of temporal regulation of cell adhesive structures. Assembly of cell–cell junctional complexes in Drosophila proceeds in a stepwise manner. The initial asymmetry is laid out during cellularization and matures progressively into final forms by assembling the adherens junction and septate junction (reviewed in Tepass et al. 2001). HA-Hakai was absent from cell–cell interfaces, but colocalized with E-cadherin in cytoplasmic vesicles that are different from known endosomal compartments labelled with Rab5, Rab7 or Rab11. One possibility is that Hakai associates with E-cadherin in a synthetic pathway and regulates a process that is important in the early stages of junctional complex assembly.

A large-scale protein interaction screen has identified a number of proteins that interact with Drosophila Hakai in yeast two-hybrid assay (Giot et al. 2003; BioGrid database, http://www.thebiogrid.org/index.php). One of the potential Hakai interacters listed in the database is aPKC. The phenotype observed where aPKC localization is absent from the apical domain may reflect a mechanism involving direct protein interaction. Another Hakai-interacting protein included in the list was TNF-like protein IMD (immune deficiency, Georgel et al. 2001), and interaction between IMD and Hakai has recently been demonstrated (A. E. Brown and P. Ligoxygakis, in preparation). Furthermore, we found that endoderm migration was defective in hakai mutants. It is known that endoderm migration depends on proper association with visceral mesoderm (Martin-Bermudo et al. 1999). The high level of hakai mRNA expression in endoderm suggests that Hakai is required in endodermal cells for their migration over the substrate of visceral mesoderm. The defective distribution of visceral mesoderm may reflect an independent requirement for Hakai in visceral mesoderm migration. As endoderm epithelia and visceral mesoderm do not express E-cadherin, additional Hakai target protein must be playing a key role in cell adhesion and migration in endoderm and visceral mesoderm development. Interactions of multiple target proteins may be involved in the Hakai-dependent regulation of epithelial development and the isolation of the hakai mutation reported here is anticipated to accelerate research in this direction.


   Experimental procedures
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
References
 
Fly stocks

Standard procedures for Drosophila handling and staining were used (Sullivan et al. 2000). hakai1 allele was produced by mobilization of P-element insertion CG10263KG01389 (Bellen et al. 2004), and PCR screening for detecting a deletion. A recombinant chromosome carrying P{piM}36F hakai1 FRT40A was used for mosaic analysis. Stock used for mosaic analyses were, y w ubx-flp; tubP-GAL80 FRT40A /CyO; da-Gal4 UAS-KO [MARCM clone (Lee & Luo 2001), KO: Kusabira Orange, (Karasawa et al. 2004), ubx-flp was kind gift from Jurgen Knoblich, Institute of Molecular Biotechnology, Vienna], and w hs-flp; P{piM}36F hakai1FRT40A/CyO-wg-lacZ and 2 x ovoD1 2L FRT40A/CyO [germ line clone; (Chou et al. 1993)]. P-element transformation was performed by standard procedure (Rubin & Spradling 1982). UAS-KO was constructed by inserting a Kusabira Orange fragment (MBL, Nagoya, Japan) into pUAST (Brand & Perrimon 1993). HA-hakai was constructed by PCR-amplifying a 1862-base pair fragment of the hakai genomic region spanning from the first methionine codon to 98 base pairs downstream of the hakai transcript, and inserting it downstream of the HA tag. The sequence was confirmed by sequencing. GFP markers for Golgi apparatus (P{UAS-Grasp65-GFP}2; Bloomington Stock Center) and lysosome [UAS-GFP-LAMP; (Pulipparacharuvil et al. 2005)] were coexpressed with HA-Hakai by da-Gal4 for subcellular localization analyses.

Antibodies, in situ hybridization and imaging

Rabbit anti-Hakai antibody was raised against a peptide GCHHTLKKGTPHQSES of Hakai, and purified using a peptide affinity column (Peptide Institute Inc., Osaka, Japan). Anti-E-cadherin (DCAD2), Arm (N2 7A1), Crb (Cq4) and Dlg (4F3) were obtained from Developmental Studies Hybridoma Bank (DSHB, University of Illinois). Other antibodies used were: anti-aPKC and anti-Drosophila E-cadherin extracellular domain (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-HA (Roche Diagnostics, Basel, Switzerland), anti-GFP (MBL), and anti-beta galactosidase (Promega Corporation, Madison, WI, USA). Fluorescent images were captured by confocal microscope (Olympus FV1000; Olympus Corporation, Tokyo Japan). Time-lapse movies were taken with Olympus FV1000. Twenty z-sections were taken every 3 min and processed as described (Kato & Hayashi 2008). Whole mount in situ hybridization was performed by standard protocol with digoxigenin-labelled RNA probes (Sullivan et al. 2000).

Cell culture and immunoprecipitation assays

Drosophila S2 cells were maintained in Schneider’s Insect Medium (Invitrogen, USA) supplemented with 10% FCS. Transfection was performed with Effecten Transfection Reagent (Qiagen, Tokyo, Japan). Protein expression vectors based on pUAST (Brand & Perrimon 1993) were coexpressed with actin-Gal4 plasmid pWAGal4 (Gift from Yasushi Hiromi, National Institute of Genetics, Mishima Japan). For the ubiquitination assay, HS-HA-ubiquitin was cotransfected and cells were heat shock-treated for 30 min at 37 °C, 2 h before harvest (Sakata et al. 2004). Cell lysates were prepared in RIPA buffer, and immunoprecipitation was performed with antibodies and affinity beads conjugated with anti-GFP (MBL), protein A (GE Healthcare), and anti-HA (Roche).


   Acknowledgements
 
We thank Yasuyuki Fujita, Hideki Nakagoshi and members of the Hayashi lab for discussions, Nao Niwa for help in in situ hybridization, Yash Hiromi for pWAGal4 plasmid, Jurgen Knoblich, Bloomington Stock Center, and Kyoto Stock Center for fly stocks, DSHB (University of Iowa) for antibodies. This work was supported in part by a Research Grant for Genome Research from MEXT Japan (SH).


   Footnotes
 
Communicated by: Isao Katsura Back

aPresent address: Kanazawa University, Frontier Science Organization 13-1 Takaramachi, Kanazawa Ishikawa 920-8641, Japan. Back

bPresent address: Servus, LLC., Edogawa, Tokyo, Japan. Back

* shayashi{at}cdb.riken.jp


   References
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
 References
 
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Received: 28 April 2009
Accepted: 11 June 2009





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