γCOP Is Required for Apical Protein Secretion and Epithelial Morphogenesis in Drosophila melanogaster

Background There is increasing evidence that tissue-specific modifications of basic cellular functions play an important role in development and disease. To identify the functions of COPI coatomer-mediated membrane trafficking in Drosophila development, we were aiming to create loss-of-function mutations in the γCOP gene, which encodes a subunit of the COPI coatomer complex. Principal Findings We found that γCOP is essential for the viability of the Drosophila embryo. In the absence of zygotic γCOP activity, embryos die late in embryogenesis and display pronounced defects in morphogenesis of the embryonic epidermis and of tracheal tubes. The coordinated cell rearrangements and cell shape changes during tracheal tube morphogenesis critically depend on apical secretion of certain proteins. Investigation of tracheal morphogenesis in γCOP loss-of-function mutants revealed that several key proteins required for tracheal morphogenesis are not properly secreted into the apical lumen. As a consequence, γCOP mutants show defects in cell rearrangements during branch elongation, in tube dilation, as well as in tube fusion. We present genetic evidence that a specific subset of the tracheal defects in γCOP mutants is due to the reduced secretion of the Zona Pellucida protein Piopio. Thus, we identified a critical target protein of COPI-dependent secretion in epithelial tube morphogenesis. Conclusions/Significance These studies highlight the role of COPI coatomer-mediated vesicle trafficking in both general and tissue-specific secretion in a multicellular organism. Although COPI coatomer is generally required for protein secretion, we show that the phenotypic effect of γCOP mutations is surprisingly specific. Importantly, we attribute a distinct aspect of the γCOP phenotype to the effect on a specific key target protein.


Supporting Information Text S1. Identification of mutations and removal of background mutations.
We generated three isogenic γCOP P{lArB}A383.2M3 lines and found that these three lines were all homozygous viable (although in average only 31% of the expected homozygous flies were eclosing) and weakly fertile (egg-laying activity was lower than observed for wild type; data not shown). In addition, the flies were smaller and lacked the posterior cross-vein (data not shown).
In our P-element remobilization experiment we started with all three different isogenic P{lArB}A383.2M3 lines and set up an initial 844 crosses, with single males, harboring both the P{lArB}A383.2M3 insertion and the P-element transposase ( Figure 1A). Finally, the P-element remobilization experiment yielded 1108 stably balanced excision chromosomes, which had lost the ry + marker and therefore likely the P{lArB}. Of these lines, 277 were lethal, the others were homozygous viable and crossveinless. Since γCOP had been found to be an essential gene in other organisms [30], we expected a Drosophila γCOP null mutant to be embryonic lethal; thus, novel γCOP alleles should be found among the lethal lines. By screening through a large number of these embryonic lethal lines using a PCR assay, we identified a few γCOP mutants harboring deletions (deletions 6, 8 and 10; Figure 1B-C, E; Materials and Methods). In order to identify more and larger deletions, we were looking for suitable lines to be used in complementation assays: We used a jump-out deletion of the distal neighboring gene pygo [34], Df(3R)pygo 11-3 , which also deletes parts of the γCOP gene ( Figure 1E), a pygo allele, pygo 130 , which specifically affects the pygo locus [34][35] and an independent γCOP allele, which had become available in the meantime, called kg06383 (Flybase). By testing the remaining 144 embryonic lethal excision lines in complementation assays with pygo 130 , Df(3R)pygo 11-3 and γCOP kg06383 and our PCR-based assay, we identified additional γCOP and γCOP pygo double mutants (lines 5, 12, 577 and 677  Figure 1E).
We were also analyzing the homozygous viable lines, to find evidence that the P-element had precisely excised in these lines. Indeed by the single fly PCR method using primer pairs flanking the original P-element insertion (e.g. cop14 and cop2rev (Materials and Methods)), we revealed that in 51 of 93 (51/93) homozygous viable lines, the P-element had precisely excised.
In only one line, which unfortunately was lost, a tiny deletion was detected. In another 10 lines, part of the P-element seemed to have remained in place as the PCR signal was bigger than in wild type; sequencing a few of these lines confirmed that only internal P-element deletions were present (data not shown). The remaining 31 lines were not further analyzed. This initial PCR analysis confirmed that in most homozygous viable lines the P-element had excised precisely or only small parts of it had stayed in place (and only the ry + marker was lost). Sequencing two of these excision alleles confirmed that indeed the entire P-element was precisely removed (data not shown). However, these lines were still crossveinless (cv) and to our big surprise were female sterile (fs). Crossing these excision lines to the lethal lines showed that most isolated lines were female sterile and crossveinless. This suggested to us that the crossveinless phenotype was due to a second mutation present in the original stock and that a third mutation was induced through the jump-out procedure. We considered this likely due to a second transposable element present in the background, which was not seen by our in situ hybridization experiment to polytene salivary gland chromosomes, using a ry probe [33]. In summary, not only γCOP deletion alleles but also γCOP-unassociated female sterile or potentially also lethal hits on chromosome 3 could be present in the lethal excision lines.
In complementation assays with the newly available independent γCOP allele kg06383 (Flybase), we found that only 81 of the 144 of the lethal lines, as well as our PCR-identified γCOP deletions, did not complement kg06383 and thus represent γCOP alleles. The others seemed to be lethal due to second hits present on chromosome 3, which were not associated with the γCOP locus. There is also no complementation observed between the γCOP P{lArB}A383.2M3 allele and kg06383 or the other newly identified γCOP deletions, indicating that the γCOP P{lArB}A383.2M3 allele is indeed a hypomorphic γCOP allele (data not shown).
The presence of background mutations could severely disturb a functional analysis of γCOP. Therefore, the background mutations (fs and cv) were removed. We mapped them roughly by analyzing meiotic recombination events between the multiply marked rucuca chromosome (Materials and Methods, data not shown) and the P{lArB}A383.2M3 and found the cv phenotype proximal to e and distal to ry (where two known cv mutations map (cv-c and cv-d (Flybase)) and the fs mutation on 3L. Then, we removed the background mutations by replacing all segments of 3L and all sequences proximal to e s with an isogenic rucuca chromosome. This resulted in lines like e.g. ru 1 h 1 th 1 st 1 cu 1 sr 1 e s γCOP 10 . Furthermore, we removed most of the rucuca makers again by recombination with either an isogenic FRT82B or another isogenic chromosome 3. In this way, lines like e.g. FRT82B sr 1 e s γCOP 10 or FRT82B e s γCOP 10 were obtained. Subsequently, these lines were tested in complementation assays to verify the absence of both the background fs and the cv mutation and used in our further analysis. Although we have replace almost the entire third chromosome, we can a priori not know whether potential lethal mutations have also been present or and if so, been removed, for they could principally be located distal to ebony. Therefore, rescue experiments were performed to prove that indeed no other lethal mutation than the one in γCOP was present on these chromosomes ( Figure 2).