1 Wellcome Trust/Cancer Research UK Gurdon Institute and Department of Anatomy, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QN, UK
2 Institute of Genetics, University of Mainz, J.-J.-Becherweg 32, 55128 Mainz, Germany
Author for correspondence (e-mail: n.brown{at}welc.cam.ac.uk)
Accepted 1 November 2004
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Summary |
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Key words: Adhesion, Apical, Drosophila, Microtubule nucleation, ZP domain
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Introduction |
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Although the role of integrins in mediating cell adhesion to the ECM is well documented (Bökel and Brown, 2002), the wing epithelial cells make a second type of contact with the ECM that is much less well understood. The wing cells also adhere to an ECM formed on the outer surface of the wing, which ultimately forms the structure of the adult wing. This apical ECM (aECM) is also known as the cuticle and forms the invertebrate exoskeleton (Andersen, 1979
). Electron-microscopic (EM) analysis of pupal wing development (Tucker et al., 1986
; Mogensen and Tucker, 1987
; Mogensen et al., 1989
) has shown cell junctions at both basal and apical surfaces of the cell; the junctions on the basal surface presumably contain integrins, whereas the junctions on the apical surface contain receptors that have yet to be identified. Large microtubule bundles run from the apical junctions to the basal junctions. Because cells in the dorsal epithelium are attached via the basal junctions to cells in the ventral epithelium, the microtubule bundles appear to run continuously from one cell layer to the other, leading to their description as transalar arrays, even though they are interrupted by the basal junctions. There is evidence to suggest that the apical junctions contain a microtubule-organizing centre: at the time that these microtubules are formed, the cells lack a centrosome, the minus ends of the microtubules are positioned at the apical junctions and the microtubules have a more irregular diameter, consisting of 15-16 protofilaments rather than the usual 13, suggesting that they are nucleated in an different way than from a centrosome. Thus, the formation of apical junctions with the aECM might play a role in the assembly of the transalar arrays.
Cells with large bundles of microtubules spanning the apical-basal axis of the cell are also found in vertebrates, for example the neuroepithelial cells of the inner ear (Henderson et al., 1994; Henderson et al., 1995
). These cells are thought to provide an essential mechanical link between the oscillating extracellular membranes on the apical and basal sides that is necessary for their function in hearing. The apical tectorial membrane has molecular similarities with the insect aECM, because both contain proteins with a zona pellucida (ZP) domain, first identified in proteins of the mouse zona pellucida (Bork and Sander, 1992
). Thus, the tectorial membrane proteins
- and ß-tectorin in mammals (Killick et al., 1995
; Legan et al., 1997
), and the putative cuticle and transmembrane protein Dumpy (Dp) in Drosophila (Wilkin et al., 2000
) contain ZP domains. Mice carrying a targeted deletion of
-tectorin show severe defects in the tectorial membrane (Legan et al., 2000
). Flies carrying dumpy mutations display wing blisters (Prout et al., 1997
) and defects of the cuticle (Wilkin et al., 2000
) (this paper). Recent work has shown that ZP domain containing transmembrane domains can be cleaved to release the extracellular domain (Litscher et al., 1999
), which can then oligomerize into extracellular filaments (Jovine et al., 2002
). Although this suggests that ZP domain proteins are major components of the aECM, it does not rule out a possible alternative role as transmembrane receptors linking the aECM to the cytoskeleton. Recently, two additional ZP-domain proteins have been found to be part of the aECM of the pupal wing, encoded by the genes miniature and dusky (Roch et al., 2003
). The study of these proteins showed that the composition of the aECM has a profound effect on the control of cell shape in the pupal wing. Despite the identification of these three proteins, we still know relatively little about the composition of the Drosophila aECM and how it is linked to the apical surface and the underlying cytoskeleton.
In order to identify new proteins involved in morphogenesis of the wing, screens were performed for mutations that cause wing blisters (Prout et al., 1997; Walsh and Brown, 1998
). Four genes isolated from these screens [short stop (shot, also known as kakapo), rhea, steamer duck and blistery] encode cytoskeletal linkers that clearly play a role in the link between integrin junctions and the cytoskeleton (Brown et al., 2002
; Röper et al., 2002
; Clark et al., 2003
; Torgler et al., 2004
). One gene, dumpy, did not appear to be involved in integrin function but was instead suggested to be part of the aECM (Wilkin et al., 2000
). Here, we describe the characterization of two additional genes that appear to function with Dumpy in the link between the apical cell surface and the aECM.
We find that the two wing-blister genes, piopio (pio) and papillote (pot), encode proteins with a full and a partial ZP domain, respectively. We demonstrate that these two proteins, in addition to Dumpy, are essential for the integrity of the inner layer of the aECM and its link to the apical surface of the epidermis. We also find evidence that Pio is required for the microtubule organizing activity of the apical junctions. The identification of these genes therefore provides insight into the novel connection between aECM and the cytoskeleton.
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Materials and Methods |
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Microtubules were visualized using a pUASp::GFP65S- Tubulin84B insertion on the third chromosome (Grieder et al., 2000
). Expression of upstream activation sequence (UAS) constructs was directed to the developing wing using the 69B (Brand and Perrimon, 1993
) or CY2 (Queenan et al., 1997
) Gal4 driver lines; 69B was also used for the embryonic epidermis.
Wing clones were introduced by heat shocking larvae in food vials for 2.5 hours in a 37°C water bath at 12-36 hours after hatching. To mark clones morphologically on chromosome arm 2R, the shaVAI51 allele was introduced into FRT42D kakV168 and FRT42D pioV132 chromosomes. The MARCM (Lee and Luo, 1999) strategy was used to mark mutant clones positively on chromosome X and chromosome arm 2L.
Molecular characterization of piopio and papillote
The region containing piopio candidate region was narrowed to a 300 kb interval between the distal breakpoint of Df(2R)px2 and the proximal breakpoint of Df(2R)bsV100 (a deficiency recovered as a blistered allele) (Walsh and Brown, 1998) by PCR amplification of short test fragments from single embryos homozygous for either deficiency. PCR amplification of the predicted gene CG3541 (Adams et al., 2000
) from genomic DNA of pioV132/SM5 yielded a shorter, polymorphic fragment. Sequencing revealed that this was caused by a 1707 bp deletion removing the first two coding exons of the predicted gene (bases -512 to +1192 relative to the ATG). A 22 kb BamHI genomic fragment flanking CG3541 was cloned into a P-element vector and introduced into the genome to test for rescue. Sequencing of several cDNAs confirmed the predicted intron/exon structure and coding region of CG3541. However, the 3' untranslated region (UTR) of pio exceeded the predictions by about 500 bp and there are several alternative splice forms of the 5' untranslated region (UTR) (see submitted sequence, AY862157). The UAS-pioGFP construct was generated by PCR amplification of the pio open reading frame and 5' UTR from cDNA and cloned between a UAS promoter and the green fluorescent protein (GFP) coding sequence. A chimeric transmembrane protein was generated by replacing the cytoplasmic tail of the Torso receptor tyrosine kinase (a type-I transmembrane protein) with that of Pio, as done previously with the integrin-ß subunit cytoplasmic tail (Martin-Bermudo and Brown, 1999
). The short Pio cytoplasmic tail was amplified and cloned into EcoRI/SpeI sites in the vector pWR UAS::torsoD. Sequences at the fusion junction are:
Pio: RTFAIAIAIAGLILMLAVVAAVLCIMARRSTKTVSNSGSSI-YS;
Torso: RGVLLSEGNMVKLVLFIIVPICCILMLCSLTFCRRNRSE-VQAL;
fusion: RGVLLSEGNMVKLVLFIIVPICCILMLCRILCIMARRST-KTVS.
The region containing pot was mapped by inclusion in Df(1)RA47 but not Df(1)KA7 or Df(1)N105. Sequence-tagged sites (STSs) derived from cosmid ends (Kimmerly et al., 1996) were mapped relative to the breakpoints of these deficiencies by single-embryo PCR, limiting the pot candidate region to a 73 kb interval between the STSs Dm0452 and Dm1866. From our phenotypic analysis, we expected an apical protein, and this interval includes a predicted gene [CG2467 (Adams et al., 2000
)] encoding a protein with a signal peptide and a partial ZP domain. Sequencing of PCR products from heterozygous potP53/FM6 genomic DNA revealed a 13 bp deletion in the CG2467 gene, causing a frame shift after the last base encoding amino acid 686, which results in truncation of the mutant protein after an additional 15 unrelated residues. A 15kb SpeI/EclXI genomic fragment encompassing CG2467 was inserted into a P-element vector and introduced into the genome. To generate the Pot-CFP fusion construct, the cyan fluorescent protein (CFP) coding region (Clontech) was amplified and inserted into the pot genomic fragment using BamHI and EcoRI sites introduced beforehand by PCR. Sequencing of cDNAs confirmed the predicted exon-intron boundaries and the extent of the pot coding region, but the length of the 3' UTR again exceeded the prediction (see submitted sequence, AY862156). Domain analysis was performed with SMART (Schultz et al., 1998
) and PFAM (Bateman et al., 2002
).
Immunohistochemistry and microscopy
Confocal images were collected using a Biorad Radiance 2000/Nikon E800 microscope with 40x/1.30 oil and 60x/1.40 oil objectives. Fluorophores used were either GFP and CFP or Alexa 568 and Alexa 488 dyes. GFP/CFP images were obtained directly from live embryos or unfixed pupal wings dissected from the pupal case and mounted in Vectashield or Voltalef oil. To perform whole-mount antibody staining on pupal wings, the wings were dissected, heat fixed in boiling PBS for 10 seconds, cut several times across the wing blade using microsurgical scissors and postfixed in 4% formaldehyde in PBS for 2 hours. After washing extensively, standard antibody-staining procedures were applied. To image wing morphology, Nomarski-interference-contrast images were collected at the wavelength of the excitation laser and overlaid with GFP images collected in parallel. Antibodies were used at the following dilutions: guinea-pig anti-Kakapo (courtesy of T. Volk, Weizmann Institute, Rehovot, Israel) 1:250; mouse anti-acetylated--tubulin (Sigma), 1:500; fluorescent secondary antisera (Vector Labs), 1:500. Ultrastructural analyses were carried out as described previously (Prokop et al., 1998a
) on a Zeiss EM900 electron microscope. Figures were assembled in Photoshop (Adobe) and labelled with FreeHand (Macromedia).
Protein chromatography and detection
Wing lysates were generated by dissecting wings from transgenic and control pupae [either lacking any GFP or containing GFP alone expressed from the ubiquitin gene promoter (Luschnig et al., 2004)], shock freezing them on dry ice and grinding them in lysis buffer (PBS supplemented with 100 mM NaCl, 0.5% Triton X-100, 0.2% sodium dodecyl sulfate (SDS)] containing 1x concentrated Complete Protease Inhibitor Mix (Roche). Proteins from the supernatant were then separated on 10% or 12.5% polyacrylamide gels. Pio-GFP and Pot-CFP fusion proteins were detected using monoclonal anti-GFP antibody (Abcam; Vector Laboratories) and horseradish-peroxidase-coupled secondary antibodies (Jackson ImmunoResearch), followed by chemoluminescence detection (ECL; Pharmacia Biotech).
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Results |
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Molecular characterization of pio and pot
To discover the nature of the proteins encoded by pio and pot, we characterized them at the molecular level. Using deficiencies in the region, pio was mapped to a 300 kb region. As the mutant alleles were produced by X-rays, which often induce deletions, we scanned the genes in that interval for deletions by PCR. A deletion removing the first two coding exons of the predicted gene CG3541 (Adams et al., 2000) was found in the allele pioV132 (Fig. 1A), suggesting that this gene corresponds to pio. To confirm this, a rescue construct containing 22 kb of genomic DNA encompassing the CG3541 transcription unit was constructed, introduced into the genome and tested for the ability to complement pio mutations. The construct fully rescued the lethality of pio mutations and therefore we conclude that CG3541 corresponds to pio.
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Analysis of cDNAs identified in the expressed sequence tag (EST) project (Rubin et al., 2000) showed that different pio transcripts arise from alternative starts of transcription but that all transcripts produce the same protein. The sequence of the 462-amino-acid Pio protein shows that it is a transmembrane protein containing an extracellular ZP domain and a short cytoplasmic domain (Fig. 1A). ZP domains (Bork and Sander, 1992
) were first identified in the proteins that compose the mouse zona pellucida and have since been found in several proteins secreted from the apical side of cells. Other ZP-domain proteins that have a similar structure to Pio, with a signal peptide and transmembrane domain, have been shown to be cleaved by furin-type proteases, thus secreting the extracellular domain (Litscher et al., 1999
). The Pio protein contains a putative furin cleavage site (RPKR, residues 352-355) immediately following the ZP domain (amino acids 66-351), suggesting that its ZP domain might be secreted. The deletion in the pioV132 allele removes the start of translation as well as the signal peptide and therefore this allele is a null mutation.
The pot gene was similarly mapped to a 73 kb region using overlapping deficiencies. Within the interval of genomic DNA containing pot, we found that CG2467 (Adams et al., 2000) contained a short deletion in the potP53 allele, which causes a frame shift. As described for pio, we confirmed that we had identified the right candidate by constructing and testing a rescue construct encompassing this gene (Fig. 1B), and therefore conclude that CG2467 corresponds to pot. EST analysis showed a single pattern of splicing for pot cDNA, which encodes a 963-amino-acid protein with an N-terminal signal peptide and region with weak homology to ZP domains (Fig. 1B). However, the Pot sequence contains an inserted transmembrane domain disrupting the region with ZP-domain homology, and the degree of sequence conservation drops after this insertion. We therefore conclude that Pot contains a highly divergent, partial ZP domain containing a well-conserved first third of this domain, immediately followed by a transmembrane domain. Because there are no predicted furin cleavage sites between the putative extracellular part of the ZP domain and the transmembrane helix, Pot is probably an integral membrane protein. Thus, both Pio and Pot share with Dp a ZP domain, which is linked to function in the aECM.
Both pio and pot are conserved in insects, with clear orthologues identifiable in the mosquito, honeybee and silkmoth (data not shown). Sequence similarity is particularly pronounced within the ZP domains of both proteins and, in the case of Pio, the short cytoplasmic tail (Fig. 1A). Although other animals have ZP-domain-containing proteins, there do not appear to be orthologues of pio and pot, suggesting either that they fulfil an insect-specific function or that their functional equivalents in other species cannot be detected by their primary sequence.
Apical distribution of Papillote and Piopio in the pupal wing
To analyse the subcellular localization of Pot protein, we generated a CFP-tagged version, by inserting the CFP coding sequence between the last amino acid of pot and the stop codon in a genomic rescue construct containing the transcription unit and flanking DNA with putative regulatory elements. This construct and its untagged version were able fully to rescue the lethality of all pot alleles, demonstrating that the CFP fusion does not impair function. Confocal microscopy revealed that in the developing wing Pot-CFP was localized to the enlarged apical surface of these cells, where it was evenly distributed (Fig. 2A). We did not detect expression of Pot-CFP at an earlier stage of wing development in the larval imaginal disc (not shown).
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Because the pio gene is substantially larger than pot, we used a cDNA to construct a version of Pio that was C-terminally tagged with GFP and expressed it with a UAS promoter (UAS::pio-GFP); the CY2 Gal4 driver was used to drive expression of Pio-GFP in the pupal wing. Like Pot, Pio was found at the apical surface of the wing epithelial cells (Fig. 2B,D), but in punctate structures rather than being evenly distributed. The dots are enriched preferentially along the apicolateral circumference of the cells (Fig. 2B). The microtubule transalar arrays have a similar distribution when viewed from the apical surface (Fig. 2C, Fig. 3). Neither Pio nor Pot was found at the basal integrin-mediated junctions, where proteins such as GFP-tagged integrin-linked kinase are localized (Fig. 2E) (Zervas et al., 2001). Thus, both Pio and Pot are found on the apical surface, where Dp is also thought to function.
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The C-terminal CFP and GFP tags allowed us to assess the processing of Pio and Pot. As mentioned above, Pio, but not Pot, has a consensus recognition site for furin proteases, which are thought to cleave other ZP-domain-containing proteins. When PioGFP was examined by western blotting of pupal wing lysates, bands of a size consistent with both cleaved and uncleaved protein were detected at similar levels (Fig. 1C). In combination with the distribution of the GFP-tagged protein, this suggests that some Pio remains unprocessed and is associated with the punctate apical structures, and that some is cleaved. We cannot assess the distribution of the cleaved form of Pio-GFP, because it no longer contains the GFP moiety. By contrast, Pot-CFP does not appear to be cleaved at all (Fig. 1C), consistent with the lack of extracellular furin cleavage sites.
pio and pot mutant phenotypes in the wing
The apical localization of Piopio and Papillote, together with the fact that they contain ZP domains, suggested that they contribute to the aECM or its link to the epidermis. The wing blisters produced by clones pot or pio mutant wing cells might therefore be due to a separation of the aECM from the epithelial surface rather than a separation between the basal surfaces of the two epithelial sheets, as seen in integrin mutant clones (Brabant et al., 1996). To test this we used tubulin-GFP (Grieder et al., 2000
) to label the two epithelial layers. Transalar microtubule bundles were readily detected in the intervein cells of late pupal wings of wild-type flies (Fig. 3A). We marked pio mutant clones with a mutant in shavenoid that eliminates the hair produced by each hair cell (Taylor et al., 1998
) but does not by itself cause wing blisters (Fig. 3B). Although using the combination of tubulin-GFP and shavenoid failed to show the expected detachment between epithelia and aECM, it revealed a surprising phenotype for pio mutations.
We found that pio mutant cells completely lacked the transalar microtubule arrays (Fig. 3C,D). The loss of Pio and the microtubules resulted in an expansion of the mutant cell and a shortening of the wild-type cells in the opposing epithelial layer (Fig. 3D), suggesting that microtubules might be required for a balance in tension between the two layers. Despite the dramatic effect on the transalar arrays, the two epithelial sheets remained associated and the integrity of both epithelia was intact in the absence of Pio. This finding prompted us to examine microtubule organization in cells mutant for shot, a wing-blister gene that encodes a microtubule binding protein of the spectraplakin family (Röper et al., 2002). Shot is localized to apical and basal ends of the microtubule bundles in the embryonic tendon cells (Gregory and Brown, 1998
; Strumpf and Volk, 1998
) and we found that has a comparable distribution at the ends of the transalar microtubule bundles (Fig. 3F). In the absence of Shot in the embryo, muscle contraction ruptured the tendon cells and these cells had their microtubules detached from integrin junctions at the basal surface (Prokop et al., 1998b
). However, we did not detect any change to the transalar arrays in shavenoid marked clones of shot mutant cells in the pupal wing, nor did the mutant cells rupture or detach from the opposing wild-type cells during pupal stages (Fig. 3E).
We examined additional wing-blister mutants using the MARCM technique (Lee and Luo, 1999) to mark the mutant clones, so that only mutant cells express tubulin-GFP. In this technique mitotic recombination results in clones of cells that are homozygous for a given mutant that have also lost a transgene ubiquitously expressing the transcriptional repressor Gal80. This relieves the Gal80-mediated repression of Gal4-driven expression of a marker under the control of the UAS promoter (in this case UAS::tubulin-GFP) in the mutant cell clone. Cells mutant for pot had apparently normal transalar arrays (Fig. 3G,H), as did cells mutant for dp, the third wing-blister gene encoding a ZP-domain protein (Wilkin et al., 2000
) (data not shown). In clones of cells lacking the ßPS integrin subunit (myospheroid mutant) (Brower and Jaffe, 1989
), the separation between the cell layers was visible but the transalar microtubule bundles still extended along the apicobasal axis of the detached cells (Fig. 3I). Therefore, Pio appears to be uniquely required for the formation or maintenance of the transalar arrays. Because Pio has transmembrane and cytoplasmic domains, and some Pio remains uncleaved (Fig. 1), one hypothesis is that Pio recruits microtubule-nucleating machinery to apical junctions. This is consistent with the similarity between the punctate contacts of the microtubule bundles on the apical surface and the punctate distribution of Pio (Fig. 2B,C). We tested whether the short cytoplasmic domain of Pio could recruit microtubules on its own by fusing it to a heterologous transmembrane protein. However, expression of this chimeric protein in pupal wings did not perturb microtubule organization, nor was it able to rescue the loss of the transalar arrays in the pio mutant cells (data not shown).
As mentioned above, in the absence of integrins, the wing blister appears within the pupal wing (Brabant et al., 1996). By contrast, in the absence of Dp, Pio or Pot, the blister appears shortly after eclosion and we did not see any separation between the cell layers of the pupal wing. This demonstrates that the loss of these genes did not severely alter integrin function because, if it did, we would expect to see an early blister, as also found when the integrin-associated protein talin is removed (Brown et al., 2002
). Unfortunately, we also did not see evidence for the expected detachment between the apical surface of the clone and the aECM. We therefore examined the phenotype in mutant embryos to test the role of these proteins in the formation or attachment of the aECM.
Detachment of apical ECM from the epidermis in pio, pot and dp mutant embryos
Mutations in each of the wing-blister genes encoding ZP-domain proteins are embryonically lethal when homozygous. By expressing tubulin-GFP in the epidermis, a separation between the epidermis and the cuticle was visible in all three mutant backgrounds (Fig. 4C,D, for pio only). In contrast to the wing, we did not see any specific failure in the assembly of microtubules in the tendon cells of the pio mutant embryos (data not shown; see ultrastructural analysis below), suggesting that Pio might specifically be required when microtubules are nucleated from the apical junctions in the pupal wing in the absence of centrosomes (Tucker et al., 1986).
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At the ultrastructural level, embryos homozygous for pio, pot or dp mutations displayed identical defects (Fig. 5). In each case, a separation of the aECM (cuticle) from the apical surface of the epidermis was observed. Defects were observed in the innermost layer of the cuticle, rather than in the cellular adhesive structures.
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In wild-type embryos (Fig. 5C), the aECM, or cuticle, consists of a complex, multilayered, largely proteinaceous epicuticle that overlays the bilayered endocuticle (Fig. 5A,B). The endocuticle is composed of a thick, electron-light chitin layer [containing the chitin polysaccharide fibrils and associated proteins (Andersen, 1979)] and a thin, basal, darker layer called the deposition zone (Kaznowski et al., 1985
). Contact between the epidermis and the cuticular deposition zone mostly occurs in form of microvilli on the apical surface of the epidermal cells. In each of the mutant embryos, the chitin layer of the endocuticle (Fig. 5D-F, asterisk) has disintegrated, especially towards its basal side. An electrondense layer, presumably corresponding to a defective deposition zone, was faint and discontinuous compared with the wild-type deposition zone, and its typical tight contact to the chitin layer above was lost (Fig. 5D-F', black arrowheads). On the apical surface of the mutant epidermal cells, the typical microvilli can still be seen, often attached to the remaining fragments of the deposition zone. All elements of the external epicuticle appeared to be properly formed in the mutant embryos and remained in tight contact with the upper layers of the endocuticle. Thus, dp, pio and pot are required for the normal formation of the innermost layer of the aECM and its attachment to the epidermis.
Junctional structures within the epidermal cells appeared normal in dp, pio and pot mutant embryos. Because of the disruption of the transalar arrays in wing cells lacking Pio, we closely examined the apical connection of the microtubules in all three mutants, but they appeared normal. As in the wild type (Fig. 5C"), the apical and basal junctions of tendon cells in the mutant embryos were connected by large microtubule bundles (Fig. 5D"-F", white arrowheads) and, at the apical side, long tonofilaments extended away into the extracellular space (Fig. 5D"-F", white chevrons). Normally, tonofilaments anchor in the cuticle but, in all three mutant backgrounds, they appear to have been pulled out. Consistent with the proposed apical function of these proteins, the basal integrin-containing adhesive junctions of the tendon cells appeared normal (black curved arrows in Fig. 5C"-F"). In addition, the attachment of the basal surface of the rest of epidermal cells to the basement membrane [which is not dependent on integrins (Prokop et al., 1998a)], was also indistinguishable from the wild type (data not shown). In conclusion, the ultrastructural analysis has confirmed that the function of the ZP-domain-containing proteins Dumpy, Piopio and Papillote is restricted to the apical surface of the epidermal cells, where each of the proteins is required for the attachment to and/or assembly of the inner layer of the aECM.
Despite the similarities between the defects observed at the EM level, the embryonic mutant phenotypes of the three genes are not identical. The cuticle of homozygous pot embryos appeared faint after the internal tissues were dissolved with Hoyer's mountant (Walsh and Brown, 1998), suggesting that the aECM is not as resistant to this treatment, whereas the cuticle of pio or dp embryos appeared normal (data not shown). The normally straight main tracheal trunk appeared twisted and broken in places in pio (Fig. 4A,B) or dp mutant embryos, owing to problems in the intercalatory movements during tracheal morphogenesis (Jazwinska et al., 2003
), but this phenotype was not found in pot mutant embryos (data not shown). These findings, combined with the observation that Pio is required for the formation of the transalar arrays whereas the others are not, indicated that these three proteins do not work together as a simple functional unit but that each has distinct functions.
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Discussion |
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Molecular functions of Pio, Pot and Dp
An extracellular site of function for the Pio, Pot and Dp proteins is consistent with their structure. All three possess a signal peptide and a ZP domain that is thought to mediate the formation of extracellular protein fibres or meshworks (Jovine et al., 2002). In addition, Pio was also molecularly characterized by a group studying tracheal morphogenesis in the Drosophila embryo (Jazwinska et al., 2003
) and shown to be localized on the apical lumen of the trachea. Conceptual translation of the pio and dp open reading frames predicts proteins with a transmembrane domain and a short cytoplasmic tail, and thus these proteins might function as transmembrane receptors. However, transmembrane ZP-domain proteins can be post-translationally cleaved at recognition sites of furin-like endopeptidases, as shown for the mammalian ZP2 and ZP3 proteins (Litscher et al., 1999
) and for the fly ZP-domain protein NompA, which is required for the adhesion of peripheral mechanoreceptors (Chung et al., 2001
). Both Dp and Pio possess a putative cleavage site immediately following the ZP domain. We found that the Pio-GFP fusion protein was cleaved and that the size of the tagged C-terminal cleavage residue was consistent with cleavage of Pio at the furin site. Owing to the gigantic size of Dp, corresponding experiments are almost impossible, but it seems likely that similar proteolytic processing occurs. This would suggest that at least a proportion of Dp and Pio is cleaved and therefore released from the apical surface, where it could contribute to the aECM. The divergent ZP domain of Pot is interrupted by a predicted transmembrane domain and the putative furin cleavage site is not present in this protein, suggesting that Pot remains associated with the apical plasma membrane.
Ultrastructural analysis of the cuticle of embryos lacking Dp, Pio or Pot showed an identical defect, with the disruption of the cuticle limited to the chitinous endocuticle. The most-basal deposition zone appears faint and sometimes discontinuous, and layers within the endocuticle immediately above disintegrate, causing a gap to appear between the remainder of the cuticle and the underlying epidermis. Neither mutation affects assembly of the epicuticle or its attachment to the endocuticle. The unique faint cuticle phenotype of pot was not distinguishable at the EM level but fits with the even distribution of pot in the aECM of the wing. The EM phenotype of these three genes is clearly distinct from that seen in coracle mutations, in which the epicuticle splits away from the endocuticle (Lamb et al., 1998). Instead, the disruption of the endocuticle seen in pio, pot or dp is reminiscent of the defects in the tectorial membrane of mice carrying a targeted deletion in the ZP-domain-containing protein
-tectorin (Legan et al., 2000
). In such mice, the tectorial membrane detaches from the apical side of the underlying neuroepithelium. In addition, the finely striated matrix surrounding the collagen fibrils of the tectorial membrane is lost (Legan et al., 2000
), coinciding with an absence of immunoreactivity for the ECM proteins otogelin (Cohen-Salmon et al., 1997
) and ß-tectorin (Killick et al., 1995
), which is structurally similar to Pio. In the zona pellucida itself, which gave rise to the name of the protein family, three ZP-domain-containing proteins form a three-dimensional meshwork, and it has been suggested that ZP1 cross-links fibres containing the ZP2 and ZP3 proteins (Greve and Wassarman, 1985
; Jovine et al., 2002
). A similar cooperative interaction between the three proteins could explain why mutations in pio, dp or pot cause indistinguishable defects in the association of the apical surface of the embryonic epidermis with the aECM or cuticle.
In addition to their shared function, each protein appears to provide unique functions. Dumpy is a gigantic protein, with hundreds of epidermal-growth-factor-like (EGF) repeats combined with additional repeated domains. These are likely to provide interaction sites for binding to other aECM components. Pot contains an unusual half-ZP domain, followed by a transmembrane domain. This could help to keep the aECM closely apposed to the plasma membrane. A role in the assembly of a rigid aECM is also plausible, because Pot-CFP was readily detected in pupal wings but not in imaginal discs within the larva, which contain a more diffuse aECM (Brower et al., 1987). Alternatively, it is possible that the hydrophobic region could form a tight interaction domain rather than a transmembrane domain, which induces dimers of Pot for secretion. Pio has the unique role of contributing to the formation or stability of the microtubule transalar arrays in the wing. Ultrastructural studies have shown that the apical junctions are associated with the microtubule nucleating activity required to form the transalar arrays (Mogensen and Tucker, 1987
; Mogensen et al., 1989
). These microtubules have a greater circumference than those nucleated in these cells at an earlier stage when a centrosome is still present (Tucker et al., 1986
), suggesting that they are nucleated in a different way. Thus, Pio could play a role in the microtubule-organizing activity of the apical junctions.
The simplest way for Pio to contribute to microtubule organization is if a proportion of the protein remained an uncleaved transmembrane protein, and the cytoplasmic domain interacted with proteins that form the apical microtubule organising centre. Our efforts to test this by expressing a chimeric transmembrane protein containing the Pio cytoplasmic domain to see whether it perturbed microtubule organization were not successful. However, it should be noted that the conserved cytoplasmic domain is rich in residues that can be phosphorylated (Fig. 1A), so extracellular interactions might be essential to trigger modification of the cytoplasmic domain so that it can make the relevant intracellular interactions. Two findings support this model. First, C-terminally tagged Pio was readily detectable both by fluorescence and western blotting, whereas the cleaved C-terminal fragments from murine ZP2 and ZP3 were found to be rapidly cleared (Litscher et al., 1999; Qi et al., 2002
). Second, Pio was found in discrete points on the apical surface, similar to the points where microtubules were concentrated, in contrast to Pot, which was evenly distributed over the apical surface. However, we cannot at present rule out an alternative model in which the extracellular domain of Pio is a ligand for an unknown transmembrane receptor that regulates microtubule function.
In addition to pio, pot and dp, the Drosophila genome contains another 14 genes encoding ZP-domain-containing proteins (PFAM entry PF00100) (Bateman et al., 2002). Two of these genes, miniature and dusky, have recently been shown to play a role in the development of the wing that is substantially different to the one described here (Roch et al., 2003
). They are also likely to be components of the aECM but, in contrast to the wing-blister phenotype, the absence of these proteins leads to defects in the cell-shape changes that occur during pupal development. This phenotype highlights the role of the aECM in morphogenetic processes, as also seen with Pio and Dp during tracheal morphogenesis (Jazwinska et al., 2003
). Five of the ZP-domain-containing proteins in the genome are closely related to NompA, in that they also contain extracellular PAN modules (Tordai et al., 1999
). NompA is restricted in function to the nervous system, where it plays a role in linking the neuron to the aECM of the dendritic caps of the mechanoreceptors (Chung et al., 2001
). Thus, although ZP-domain-containing proteins in Drosophila have consistent roles at the apical surface of the cell, a range of distinct functions are mediated by these proteins.
Mutations in pio are the first to be identified that lead to defects in the formation of the transalar arrays in Drosophila. We were surprised to find that loss of Shot, which contains a microtubule-binding domain, did not perturb the organization of the bundled microtubules. Therefore, further analysis of Pio function might lead to an understanding of how microtubules can be nucleated at plasma-membrane junctions independent of centrosomes, a process that is likely to be conserved across evolution.
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Acknowledgments |
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Footnotes |
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Present address: The Wellcome Trust Centre for Cell-Matrix Research, The University of Manchester, Oxford Road, Manchester, M13 9PT, UK
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References |
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