gurken

Targets of Activity

Dorsoventral polarity in the egg chamber involves the localization of maternal Gurken mRNA to the dorsal side of the oocyte. Gurken is secreted by the oocyte and diffuses to dorsal follicle cells. This localized signal from the oocyte to the follicle cells appears to initiate a cascade of events: dorsal follicle cell differentiation, and delimiting and orienting the future dorsoventral axis of the embryo (Schupbach, 1994).

The expression pattern of the gene rhomboid in the follicle cells is altered in ovaries of females containing extra copies of gurken. A change in gurken dosage in otherwise wild-type ovaries is sufficient to alter the number of somatic follicle cells directed to the dorsal fate. Therefore the gurken-torpedo signaling process plays an instructive role in oogenesis. It induces dorsal cell fates in the follicle cell epithelium and it controls the production of maternal components that will direct the embryonic dorsoventral pattern after fertilization (Neuman-Silberberg, 1994).

At stage 10 of oogenesis, mirror is expressed in anterior-dorsal follicle cells, and this is dependent upon the Gurken signal from the oocyte. The fringe gene is expressed in a complementary pattern in posterior-ventral follicle cells at the same stage. Ectopic expression of mirror represses fringe expression, thus linking the epidermal growth factor receptor (Egfr) signaling pathway to the Fringe signaling pathway via Mirror. The Egfr pathway also triggers the cascade that leads to dorsal-ventral axis determination in the embryo. twist was used as an embryonic marker for ventral cells. Ectopic expression of mirror in the follicle cells during oogenesis ultimately represses twist expression in the embryo, and leads to phenotypes similar to those that occur due to the ectopic expression of the activated form of Egfr. Thus, mirror also controls the Toll signaling pathway, leading to Dorsal nuclear transport. In summary, the Mirror homeodomain protein provides a link that coordinates the Gurken/Egfr signaling pathway (initiated in the oocyte) with the Fringe/Notch/Delta pathway (in follicle cells). This coordination is required for epithelial morphogenesis, and for producing the signal in ventral follicle cells that determines the dorsal/ventral axis of the embryo (Zhao, 2000).

mirror is expressed in a dynamic pattern during oogenesis. This includes expression in the germarium and in centrally-located follicle cells at stage 6 and the anterior-dorsal and centripetal follicle cells at stage 10. This paper concentrates on the regulation and function of mirr expression at stage 10 of oogenesis. At this time, mirr is expressed in the cells in the follicular epithelium that receive the Grk signal. This signal is known to be required in these cells to establish the dorsal-ventral axis of the egg and embryo. In grk-mutant ovaries mirr expression disappears or is occasionally reduced to a small group of follicle cells at the dorsal midline. In fs(1)k10 mutant ovaries, where grk transcripts diffuse from anterior-dorsal towards anterior-ventral within the oocyte at stage 9, all anterior follicle cells receive the Grk signal and an expanded expression of mirr is observed. Thus mirr expression is downstream of the Grk signal, and those cells that have activated Egfr signaling at this stage of oogenesis appear to respond by activating this homeodomain transcription factor. It is still not clear whether the early expression of mirr in the germarium requires the Grk/Egfr signaling. Grk/Egfr signaling activity does not always activate the mirr gene, since mirr is not expressed in posterior follicle cells at stage 7 of oogenesis, when grk signaling occurs at the posterior of the egg chamber. Other factors must, therefore, determine the way in which follicle cells, in different positions and with different developmental histories, respond to the grk signal (Zhao, 2000).

fringe (fng), which encodes a glycosyltransferase-like secreted protein, is involved in different developmental processes, such as the development of the wing and the eye and oogenesis. Its expression at stage 10 of oogenesis is restricted to the ventral and posterior follicle cells, and it is not observed in those cells that express mirr. So the expression patterns of mirr and fng are complementary, and both these patterns are defined by the position of Egfr activation in response to the Grk signal (Zhao, 2000).

To test whether the complementary expression of mirr and fng depend upon Egfr signaling at stage 10, their expression patterns were examined in flies expressing activated or dominant negative forms of Egfr. When the activated form of Egfr (DER^AF or lambdatop) is expressed in the anterior follicle cells, all those cells now express mirr, but not fng. When the dominant negative form of Egfr (DER ^DN) is expressed in those follicle cells surrounding the oocyte, but not the centripetal follicle cells, the anterior-dorsal follicle cells no longer express mirr. The expression domain of fng expands to include the anterior-dorsal cells in these mutants. Thus, the expression of both mirr and fng are either positively or negatively regulated by the activation of Egfr, and a complementary expression pattern is maintained in all these experiments. Experiments show that Grk/Egfr signaling is required to activate the expression of mirr in anterior-dorsal and centripetal follicle cells, which in turn represses the expression of fng in those cells. As a result, fng is only expressed in the posterior and ventral follicle cells, where it is required for the normal morphogenesis of the follicle cell layer (Zhao, 2000).

To investigate whether the ventrally restricted expression of the pipe gene results from regulation of transcriptional initiation, an examination was carried out of the ability of 5' sequences from the pipe gene to drive E. coli lacZ expression in a ventral pattern. Since fragment A includes the 5' end of the putative pipe cDNA, it is considered likely that this 5' end also contains promoter and enhancer/silencer elements required for the correct expression of pipe. Transformants carrying lacZ downstream of fragment A were generated and their ovaries dissected and stained for beta-galactosidase activity. Stage 10 egg chambers exhibit ventral beta-galactosidase staining that is virtually identical in distribution to that seen for the PIPEmRNA itself. When the same transgenic insert is introduced into the gurken mutant maternal background, uniform beta-galactosidase activity is detected. These observations confirm that the ventrally restricted expression of pipe arises through transcriptional regulation that is mediated via Grk/Egfr signaling (Sen, 1998).

To establish a regulatory link between the transcriptional regulation of kekkon-1 in the follicle cell epithelium and the Grk/Egfr signaling pathway, kek-lacZ expression was examined in mutants for grk, egfr, and the kinase Draf that is both necessary and sufficient for Egfr signaling in follicle cells. In each case, the dorsal-anterior gradient of kek-lacZ expression is abolished or severely diminished, revealing that kek-lacZ expression is transcriptionally regulated by the Grk/Egfr/Draf pathway. Expression of an activated form of Draf results in the ubiquitous expression of kek-lacZ within the follicular epithelium (Ghiglione, 1999).

To further analyze the role of the Grk/Egfr pathway in the transcriptional control of kek-lacZ, the subcellular localization of GRK mRNAs was mislocalized by disrupting the oocyte cytoskeletal network. Treatment of the oocyte with colchicine, an inhibitor of microtubule polymerization, leads to mislocalization of the oocyte nucleus and its associated GRK mRNAs, which correlates with ectopic kek-lacZ expression as well as KEK1 mRNA expression. Altogether, these results demonstrate that Grk is not only necessary but sufficient to regulate the spatial expression of kek1 (Ghiglione, 1999).

During Drosophila oogenesis, Gurken, a TGF-alpha like protein localized close to the oocyte nucleus, activates the MAPK cascade via the Drosophila EGF receptor (Egfr). Activation of this pathway induces different cell fates in the overlying follicular epithelium, specifying the two dorsolaterally positioned respiratory appendages and the dorsalmost cells separating them. Signal-associated internalization of Gurken protein into follicle cells demonstrates that the Gurken signal is spatially restricted and of constant intensity during mid-oogenesis. Gurken internalization can first be observed in all posterior follicle cells, abutting the oocyte from stage 4 to 6 of oogenesis. At the same time MAPK activation evolves in a spatially and temporally dynamic way and resolves into a complex pattern that presages the position of the appendages. Therefore, different dorsal follicle cell fates are not determined by a Gurken morphogen gradient. Instead they are specified by secondary signal amplification and refinement processes that integrate the Gurken signal with positive and negative feedback mechanisms generated by target genes of the Egfr pathway (Peri, 1999).

The Gurken homolog was cloned from D.virilis, separated from D. melanogaster by about 600 Myr. The two proteins possess a conserved domain structure. However, continuous stretches of high similarity at the sequence level are limited to a few short domains including the signal peptide and the transmembrane and intracellular domains. The overall identity of both proteins is 40% and their similarity including equivalent substitutes is 46%. Gurken mRNA is tightly localized to the dorsal anterior corner of the D. virilis oocytes at stages 8 to 10 of oogenesis. The only detectable difference is the retention of a significant amount of GRK mRNA at the posterior pole of D. virilis oocytes when the bulk of the RNA is already found at the dorsal anterior corner of the oocyte. This is actually also observable with D. melanogaster GRK mRNA and protein, albeit at a quantitatively much lower level (Peri, 1999).

In wildtype egg chambers rhomboid expression pattern can first be detected in a broad domain centered on the anterodorsal corner of the oocyte at the transition from stage 9 to stage 10a of oogenesis, at a time when MAPK activation cannot yet be detected in the follicular epithelium. Thereafter, RHO mRNA starts to be downregulated dorsally, beginning at the anterior margin of the domain. Dowregulation proceeds until the remnants of the first broad expression domain are reduced to a wide ring. rho expression is, at that time, retained only in a small patch in the dorsalmost cells and in two stripes extending laterally towards the nurse cell border. In these cells rho expression is then strongly upregulated and a refinement process begins that leads to a final pattern consisting of two L-shaped domains. Just as rho refinement starts in the dorsalmost part of the egg chamber, MAPK activation in the follicular epithelium first exceeds the detection threshold in exactly the same cells. During the following expansion of the MAPK domain, the strong activation at the leading edge seems to coincide with the refining stripes of rho expression. As the MAPK activation pattern reaches its final spectacle shape, again the dorsal and anterior portions of the MAPK staining pattern are associated with rho expression. In conclusion, rho expression precedes the detection of MAPK activation and both staining patterns are spatially and temporally well correlated. Rhomboid thus may be one of the factors responsible for generating the amplification and modulation of the initial Gurken signal (Peri, 1999).

Gurken signaling also induces the expression of a negative element in the Egfr pathway. Argos expression comes too late to explain the dynamic evolution of the MAPK and rhomboid patterns during stage 10 of oogenesis, but sprouty is expressed early enough to be part of the regulatory network controlling the Egfr pathway activation at stage 10. sprouty is induced in all posterior follicle cells abutting the oocyte at stage 4 to 6 of oogenesis. sty is absent from egg chambers lacking Gurken function, and thus sty may be one factor required to counteract the potential rho-dependent autoactivation of the Egfr pathway (Peri, 1999).

How does MAPK activation influence the morphogenesis of the developing egg shell? The gene Broad-complex is expressed and required in the anlagen of the dorsal respiratory appendages. It has been suggested, therefore, that Br-C acts as a marker specifying appendage fate. Gurken signaling leads to a repression of Br-C in the dorsalmost cells of the follicular epithelim. Br-C expression disappears in a sharply delineated dorsal-anterior patch coincident with the first observable MAPK activation. Later, when the MAPK activation refines to its distinct 'spectacle shape', Br-C expression is confined to two lateral domains showing weak MAPK activation surrounded by rings strongly staining for activated MAPK. Based on the striking complementarity of these patterns, it is proposed that at this stage Br-C is repressed by high levels and activated by low levels of MAPK activation. In addition, Br-C expression does not by itself specify dorsal appendage fate, as it is visible in duplicated anterior ringlike domains in completely ventralized ovaries that do not possess appendages. This adds a caveat to using Br-C expression as a fate marker for dorsoventral positions. It is proposed that Br-C instead is a general marker expressed in cells that undergo morphogenetic changes, consistent with its expression in the embryo and during earlier stages of oogenesis. It is proposed that Gurken initiates secondary processses in the follicular epithelium that modulate and amplify the initial activation of the Egfr pathway (Peri, 1999).

Axis formation in Drosophila depends on correct patterning of the follicular epithelium and on signaling between the germ line and soma during oogenesis. A method for identifying genes expressed in the follicle cells with potential roles in axis formation is described. Follicle cells are purified from whole ovaries by enzymatic digestion, filtration, and fluorescence-activated cell sorting (FACS). Two strategies are used to obtain complementary cell groups. In the first strategy, spatially restricted subpopulations are marked for FACS selection using a green fluorescent protein (GFP) reporter. In the second, cells are purified from animals mutant for the epidermal growth factor receptor ligand gurken (grk) and from their wild-type siblings. cDNA from these samples of spatially restricted or genetically mutant follicle cells is used in differential expression screens employing PCR-based differential display or hybridization to a cDNA microarray. Positives are confirmed by in situ hybridization to whole mounts. These methods are found to be capable of identifying both spatially restricted and grk-dependent transcripts. Results from pilot screens include (1) the identification of a homologue of the immunophilin FKBP-12 with dorsal anterior expression in egg chambers; (2) the discovery that the ecdysone-inducible nuclear hormone receptor gene E78 is regulated by grk during oogenesis and is required for proper dorsal appendage formation, and (3) the identification of a Drosophila homolog of the human SET-binding factor gene SBF1 with elevated transcription in grk mutant egg chambers (Bryant, 1999).

FKBP-12 has been shown to bind to TGF-beta type I receptors and has been proposed, on the basis of cell culture studies, to act as a regulated inhibitor of TGF-beta type I signaling. The Drosophila TGF-beta homolog dpp is expressed in anterior follicle cells and is required for the formation of anterior chorion structures. Follicle cell patterning may provide an instructive system in which to study the interactions between TGF-beta signaling, FKBP-12-like proteins, and the Egfr pathway, which is required for the induction of the FKBP-12 homolog and other genes in the dorsal anterior follicle cells. Consistent with a role for the FKBP-12 homolog in modulating dpp signaling, preliminary overexpression studies show defects in anterior chorion structures (Bryant, 1999).

Having determined that E78B is expressed under Egfr control in the follicle cells that form the dorsal appendages, it was asked whether E78B has a function in these cells. Eggs collected from viable E78 mutant flies and examined for defects in the chorion have been found to have shortened and/or broadened dorsal appendages. The eggshells of flies expressing E78B under control of a heat shock promoter were also examined, and they were found to have fused dorsal appendages. E78 has previously been shown to be inessential for viability and fertility. These observations of an incompletely penetrant defect in dorsal appendage morphogenesis suggests that E78 has a partially redundant role in follicle cell migration or patterning. Its role in larval chromosome puffing is similarly redundant: mutants reduce the size of late puffs but do not abolish them. The identification of a role for E78 illustrates an advantage of molecular expression screening over loss-of-function female sterile screens, which are unable to detect genes with redundant requirements (Bryant, 1999).

The cDNA clone GM04985 shows greater signal on microarray hybridization with a posterior-derived follicle cell probe (posterior-derived follicle cells are subject to Gurken signaling) than with a probe prepared from follicle cells not subject to Gurken signaling. It was therefore predicted that GM04985 would be preferentially expressed in the posterior follicle cells; this prediction was borne out by in situ hybridization. GM04985 hybridizes to the posterior follicle cells during stages 6-9. Sequence from the 5' end shows that the clone is a novel splice variant of the ecdysone-inducible nuclear hormone receptor superfamily gene E75, in which a unique region (base pairs 1-344 of the sequence) is spliced into the exon 2/3 junction of the E75A/B common region. The remainder of the clone was sequenced, and found to match the common exons 3, 4, and 5 of E75A/B, with a poly(A) region beginning at base pair 4574 of the E75A sequence. Thus, the variant appears to encode a receptor that lacks the N-terminal DNA-binding zinc fingers encoded by the unique regions and exon 2 of E75A/B. E75 transcripts have also been observed to have dorsal anterior expression in stage 10 egg chambers; in agreement with this observation, GM04985 hybridizes to the dorsal anterior of stage 10 chambers (Bryant, 1999). E75 has been shown to react positively to Gurken signaling (William Segraves, personal communication cited in Bryant, 1999).

A fragment of a second ecdysone-responsive nuclear hormone receptor superfamily gene, E78, was amplified in differential display reactions from sorted grk plus but not from grk minus cells. The fragment hybridizes to dorsal anterior follicle cells and is absent from the dorsal follicle cells of grk ovaries. The use of specific probes shows that only one of the two E78 transcripts, E78B, is expressed in these cells. Staining for E78B is detected from stage 10B onward. This gene was not hitherto known to be under the control of the Egfr pathway, and the results demonstrate that the methods used in this study can correctly identify follicle cell transcripts whose expression is regulated by grk. Eggs were collected from viable E78 mutant flies and examined for defects in the chorion. Eggs were found that had shortened and/or broadened dorsal appendages. The eggshells of flies expressing E78B under control of a heat shock promoter were also examined, and they were found to have fused dorsal appendages. E78 is known to be inessential for viability and fertility. The observation of an incompletely penetrant defect in dorsal appendage morphogenesis suggests that E78 has a partially redundant role in follicle cell migration or patterning. Its role in larval chromosome puffing is similarly redundant: mutants reduce the size of late puffs but do not abolish them. The identification of a role for E78 illustrates an advantage of molecular expression screening over loss-of-function female sterile screens, which are unable to detect genes with redundant requirements (Bryant, 1999).

Recent studies in vertebrates and Drosophila have revealed that Fringe-mediated activation of the Notch pathway has a role in patterning cell layers during organogenesis. In these processes, a homeobox-containing transcription factor is responsible for spatially regulating fringe (fng) expression and thus directing activation of the Notch pathway along the fng expression border. This may be a general mechanism for patterning epithelial cell layers. At three stages in Drosophila oogenesis, mirror (mirr) and fng have complementary expression patterns in the follicle-cell epithelial layer, and at all three stages loss of mirr enlarges, and ectopic expression of mirr restricts, fng expression, with consequences for follicle-cell patterning. These morphological changes are similar to those caused by Notch mutations. Ectopic expression of mirr in the posterior follicle cells induces a stripe of rhomboid (rho) expression and represses pipe (pip), a gene with a role in the establishment of the dorsal-ventral axis. Ectopic Notch activation has a similar long-range effect on pip. These results suggest that Mirror and Notch induce secretion of diffusible morphogens; a TGF-beta (encoded by dpp) has been identified as one such molecule in the germarium. mirr expression in dorsal follicle cells is induced by the EGF-receptor (EGFR) pathway and mirr then represses pipe expression in all but the ventral follicle cells, connecting Egfr activation in the dorsal follicle cells to repression of pipe in the dorsal and lateral follicle cells. These results suggest that the differentiation of ventral follicle cells is not a direct consequence of germline signaling, but depends on long-range signals from dorsal follicle cells, and provide a link between early and late events in Drosophila embryonic dorsal-ventral axis formation (Jordan, 2000).

Local activation of Notch in a number of developmental systems is achieved by spatially restricted expression of a homeodomain protein that either represses or induces fng expression, generating a border of fng expressing and non-expressing cells. It is less clear how the initial asymmetric expression of the homeobox protein is generated. Because the dorsal anterior expression of mirr is characteristic of a number of genes regulated by the Egfr pathway, mirr expression was analyzed in mutants that lack Gurken, one of the ligands for Egfr. In these egg chambers, the dorsal anterior pattern of mirr expression is reduced or lost, showing that activation of the Egfr pathway is necessary for mirr expression. However, the patterns of oogenesis in the germaria at stage 6 and in the centripetally migrating cells are unaltered, indicating that either another Egfr ligand or another pathway regulates mirr expression at these stages (Jordan, 2000).

Results from several developmental systems have led to the idea that the trio of a homeobox gene, FNG and Notch are fundamental to organogenesis. It is suggested that Mirr, Fng and Notch are part of a conserved mechanism for dividing epithelial cell layers into domains; it is thought that such a mechanism is not restricted to organogenesis. Furthermore, the data suggest that Mirr integrates the Egfr and Notch pathways in oogenesis: mirr transcription is induced by the Egfr pathway, and Mirr in turn spatially regulates fng expression leading to a Notch activation border. Finally, it is proposed that the link between Egfr pathway signaling in the dorsal follicle cells and the differentiation of the ventral follicle cells suggested by genetic studies is mediated by Mirr. The Egfr pathway induces mirr expression, which leads to creation of a Notch-Fng border in lateral follicle cells from which molecules are secreted that repress pipe expression. Pipe regulates the activity of a protease cascade that activates Toll and ultimately determines the dorsal-ventral pattern of the Drosophila embryo. These data show that expression of pip in the ventral follicle cells is not a direct consequence of a graded germline signal by Gurken, but depends on Mirr-dependent long-range signals from dorsal follicle cells. Mirr therefore connects the well-studied events in early and late Drosophila dorsal-ventral axis formation (Jordan, 2000).

The Drosophila fos (Dfos)/kayak gene is a key regulator of epithelial cell morphogenesis during dorsal closure of the embryo and fusion of the adult thorax. It is also required for two morphogenetic movements of the follicular epithelium during oogenesis: (1) it is necessary for the proper posteriorward migration of main body follicle cells during stage 9; (2) it controls, from stage 11 onwards, the morphogenetic reorganization of the follicle cells that are committed to secrete the respiratory appendages. Egfr pathway activation and a critical level of Dpp/TGFß signaling are required to pattern a high level of transcription of Dfos at the anterior and dorsal edges of the two groups of cells that will give rise to the respiratory appendages. In addition, evidence is provided that, within the dorsal-anterior territory, the level of paracrine Dpp/TGFß signaling controls the commitment of follicle cells towards either an operculum or an appendage secretion fate. Finally, it is shown that Dfos is required in follicle cells for the dumping of the nurse cell cytoplasm into the oocyte and the subsequent apoptosis of nurse cells. This suggests that in somatic follicle cells, Dfos controls the expression of one or several factors that are necessary for these processes in underlying germinal nurse cells (Dequier, 2001).

The data show that determination and localization of the Dfos columnar expression pattern requires both Egfr pathway activation and a precise level of paracrine Dpp signalling. The alteration of Dfos expression in mutants affecting different components of the Egfr pathway shows clearly that Grk-dependent Egfr activation and secondary Spitz- dependent Egfr amplification and refinement are necessary to determine the Dfos columnar expression pattern. Nonetheless, colchicine feeding experiments demonstrate that Grk-dependent Egfr activation is not sufficient to induce Dfos transcription in CFC, as is the case for the Egfr target gene kekkon. However, alteration of Dfos expression resulting from either a reduction of the Dpp level or its over-increase throughout the columnar epithelium, provides direct evidence that this signalling process is also required for proper patterning of Dfos expression (Dequier, 2001).

In C532-GAL4/UAS-dpp females grown at 18^oC, a slight increase in the level of Dpp accumulation in CFC induces multiple patches of cells showing a pattern of BR-C Z1 and DFos accumulation reminiscent of that of respiratory appendage secreting units in wild-type egg chambers. Strikingly, these patches were located at the lateral and posterior peripheries of the DAFC territory, which is consistent with the hypothesis that the central-most CMFC are the localized source of a Dpp gradient. In addition, the results indicate that ectopically provided Dpp in FLP-out clones represses BR-C Z1 and DFos accumulation in both dpp-expressing cells and those located within a radius of one to two cells, thus providing a direct evidence that Dpp acts in a paracrine manner to repress expression of the BR-C Z1and Dfos genes (Dequier, 2001).

The observation that the Dpp-dependent repression of BR-C Z1 is restricted to DAFC suggests that it is mediated by a component of the Dpp-signalling pathway, i.e. either a Dpp receptor or a Smad cofactor expressed differentially in DAFC. It has been shown that among the known Dpp receptors, Saxophone and Punt are ubiquitously expressed in CFC whereas Thick-vein is expressed in a row of anterior follicle cells. In a preliminary investigation of the pattern of expression of the Drosophila Smad genes in follicle cells, it was observed that medea is expressed from stage 11 onwards in two patches of CFC that may correspond to RASFC. However, whereas the medea gene is required for Dfos transcription in the main body follicle cells during stage 9, it appears to be fully dispensable for the Dfos columnar expression pattern. Work is currently in progress to investigate the pathway involved in the restriction of the Dpp-dependent repression of BR-C Z1 to DAFCs (Dequier, 2001).

Protein Interactions

Factors affecting Gurken mRNA splicing

Alternative splicing is used by metazoans to increase protein diversity and to alter gene expression during development. However, few factors that control splice site choice in vivo have been identified. Half pint (Hfp; FlyBase designation Poly-U-binding splicing factor) regulates RNA splicing in Drosophila. Females harboring hypomorphic mutations in hfp lay short eggs and show defects in germline mitosis, nuclear morphology, and RNA localization during oogenesis. hfp encodes the Drosophila ortholog of human PUF60 and functions in both constitutive and alternative splicing in vivo. In particular, hfp mutants display striking defects in the developmentally regulated splicing of ovarian tumor (otu). Furthermore, transgenic expression of the missing otu splice form can rescue the ovarian phenotypes of hfp. hfp is also required for efficient splicing of gurken mRNA and in the splicing of eukaryotic initiation factor 4E (eIF4E), which binds to the seven-methylguanosine cap at the 5' end of messenger RNAs and is a limiting factor in translation initiation (Van Buskirk, 2002).

The dorsal appendage phenotypes of hfp mutant eggs are suggestive of perturbations in DV patterning of the egg chamber. Since Hfp interacts with Enc, which is required for grk mRNA localization and Grk protein accumulation (Hawkins, 1997), it was asked whether grk expression is also affected in hfp mutants. In wild-type stage 9 egg chambers, grk mRNA is localized to a region overlying the oocyte nucleus, and Grk protein is thus restricted to the adjacent plasma membrane, where it induces dorsal follicle cell fates. In a small fraction of hfp mutant egg chambers, grk RNA appears to be poorly expressed or undetectable, which would account for the production of eggs with reduced dorsal egg shell structures. More striking, however, is that in 52% of the stage 9/10 hfp⁹ mutant egg chambers in which grk mRNA can be detected, the transcript is present in an anterior ring. Thus, hfp appears to be required for both the production of wild-type levels of grk transcript and, like enc, for proper grk mRNA localization. However, unlike enc, this mislocalized grk mRNA in hfp mutants gives rise to detectable Grk protein. This ring of Grk protein would be predicted to induce dorsal fates in all anterior follicle cells, accounting for the observed dorsalized eggs of hfp (Van Buskirk, 2002).

Since hfp encodes a predicted splicing factor, experiments were carried out to determine whether the grk RNA mislocalization in hfp is due to a splicing defect that leads to the production of a grk transcript lacking proper localization signals. The grk gene encodes a single identified transcript, and, in RT-PCR analysis of poly(A) RNA from wild-type ovaries, a single grk product is detected. In hfp mutants, a larger product is also detected, suggestive of inefficient removal of one of the grk introns. Using a series of primers, maintenance specifically of the third intron in a fraction of grk transcripts was detected in hfp mutants. This type of splicing defect is unlikely to account for the mislocalized grk RNA observed in hfp oocytes, since the unspliced transcript does not lack any sequences found in the wild-type transcript. Furthermore, previous studies have shown that insertion of a fragment of the lacZ gene into the grk cDNA at a position analogous to the third intron does not disrupt normal RNA localization. Thus, it appears that, while Hfp is required for efficient grk splicing, this is not the cause of the RNA localization defect (Van Buskirk, 2002).

Gurken protein processing: The role of Rhomboid and Star

The mechanism of activation of the Epidermal growth factor receptor (Egfr) by the transforming growth factor alpha-like molecule, Gurken (Grk) has been examined. Grk is expressed in the oocyte and activates the Egfr in the surrounding follicle cells during oogenesis. Expression of either a membrane bound form of Grk (mbGrk), or a secreted form of Grk (secGrk), in either the follicle cells or in the germline, activates the Egfr. In tissue culture cells, both forms can bind to the Egfr; however, only the soluble form can trigger Egfr signaling, which is consistent with the observed cleavage of Grk in vivo. The two transmembrane proteins Star (S) and Brho (rhomboid-2) potentiate the activity of mbGrk. These two proteins collaborate to promote an activating proteolytic cleavage and release of Grk. After cleavage, the extracellular domain of Grk is secreted from the oocyte to activate the Egfr in the follicular epithelium (Ghiglione, 2002).

Grk is cleaved in the germline. An important question is where exactly the cleavage of the Grk precursor occurs? Other studies have concluded that the cleavage of Spitz occurs in the TM and depends on the 15 amino acid stretch located between the EGF and TM domains. The Grk dibasic signal (R₂₄₀ and K₂₄₁) is not the cleavage site because its mutation does not abolish this event. However, mbGrkDelta19AA^myc, in which the 19 amino acid (Y₂₂₄ to V₂₄₂) located between the EGF and TM domains have been deleted, is no longer cleaved, suggesting that this sequence is directly or indirectly involved in the processing (Ghiglione, 2002).

The results do not rule out the hypothesis that Grk cleavage occurs in the TM domain as proposed for Spi. The high conservation between the Spi and Grk TM domains, in addition to aberrant Grk localization observed with different grk alleles affecting this TM domain, reveals its importance. Moreover, the cleaved product of Grk that is released in the medium, after co-expressing mbGrk+S+Rho-1/Brho in S2 cells, has a slightly higher mobility that the engineered secGrk. Thus, it is possible that mbGrk is cleaved within the TM domain and that proteolysis depends on the 19 amino acid interval (Ghiglione, 2002).

These results reflect the importance of the Grk TM domain for proper processing and routing through the secretory pathway. mbGrk processing is probably tightly regulated and leads to efficient Grk secretion, contrary to engineered secGrk, which is poorly secreted from the oocyte and which acts mainly intracellularly (Ghiglione, 2002).

The recent findings that Star and Brho, a Rho-related protein, are expressed in the oocyte led to an investigation of whether they are involved in Grk activation during oogenesis. Star and Rho proteins have been proposed to be involved in the processing and activation of Spi; however, because they have no obvious motifs that predict their biochemical functions, their roles in ligand maturation and/or secretion have remained obscure (Ghiglione, 2002).

The analysis of these proteins in the context of Grk signaling has provided numerous insights into the relationships between these transmembrane proteins. The in vivo data strongly suggest that the expression level of Star and Brho is very high in the oocyte, thus leading to an efficient cleavage and secretion of Grk. However, Star and Rho-1 are probably expressed at low level in the follicle cells. Indeed, the presence of Star in this epithelium using an anti-Star antibody has not been detected, whereas they clearly show a strong staining in the germline. The presence of both endogenous Star and Rho-1 in follicle cells explains why overexpression of mbGrk in this epithelium leads to a weak dorsalization of the eggs. Nevertheless removing one copy of Star is sufficient to completely suppress this phenotype. This confirms the observation that overexpression of mbGrk on its own is not able to activate the Egfr in vivo, as supported by the in vitro study. Overexpression experiments in follicle cells indicate a strong synergy between mbGrk, Star, and Brho, as previously observed for Spi. Further, co-expression of Star and Rho-1/Brho is sufficient for Grk cleavage and secretion in S2 cells, strongly suggesting that they are the only proteins required for this process. In addition, these tissue culture experiments reveal that Star and Rho-1/Brho are not obligate cofactors for this cleavage, because co-expression of mbGrk with Rho-1/Brho is sufficient to catalyze this proteolytic event. Star is not required for Rho-1/Brho-mediated proteolytic cleavage in S2 cells, but the soluble Grk extracellular domain is no longer detected in the medium from these cells, indicating that the function of Srar is necessary for trafficking/secretion of the ligand. However, Star is not able to cleave Grk in absence of Rho-1/Brho. Altogether, these results show that the functions of Rho-1/Brho and Star are distinct, which explains their co-dependence and synergism in vivo (Ghiglione, 2002).

Rho-1/Brho may facilitate Grk proteolysis either by activating or recruiting a yet unknown protease. By analogy to the processing of mammalian Egfr ligands, Grk cleavage may be catalyzed by an ADAM-like metalloprotease. Although these molecules are present in Drosophila, nothing is known yet about their functions. An alternative hypothesis, is that despite the absence of known protease domains in their sequences, Rho-1 and Brho themselves may have proteolytic activity. The subcellular localization of Brho, as observed for mature TACE (ADAM17), is predominantly in intracellular compartments. In addition, and directly relevant to this hypothesis, Presenilins, which define another subfamily of seven-pass transmembrane proteins, have been proposed to encode proteases. In Drosophila, Presenilin may be directly responsible for the proteolysis of the intra-transmembrane domain of Notch (Ghiglione, 2002 and references therein).

One of the striking feature of Rho-related proteins is that amino acid sequence conservation is most prominent in the predicted TM regions that contain some invariant charged residues. This suggests the presence of a hydrophilic pocket that might constitute an enzymatic active site or a channel, as observed in Presenilins. This model is further supported by the recent finding that the TM domain of Spi is important for its functional interaction with Rho-1 (Ghiglione, 2002 and references therein).

rho-related genes have been found in organisms from diverse kingdoms including C. elegans, rat, human, Arabidopsis, sugar cane, yeast and bacteria. The data suggest that Brho, like Rho-1, promotes Egfr signaling by activating TGFalpha-like ligands. Since RTKs have not been found in plants, yeast or bacteria, the rho-related genes in these organisms presumably serve other functions. It will be interesting to determine whether the activities of these Rho-related proteins are similar to those of Rho-1 and Brho, such as promoting the processing of proteins (Ghiglione, 2002).

Mosaic analysis of Star, both in the germline and in follicle cells, together with the Star antisense experiment, demonstrate that Star is required in follicle cells for Spi-dependent Egfr activation, and in the germline for Grk-dependent Egfr activation. Tissue culture experiments suggest that Star is not involved in Grk proteolysis, but instead in post-cleavage trafficking or secretion of the ligand. The intracellular localization of Star is also consistent with a role for Star at a step that follows the Brho-dependent cleavage, because it was found that Star is predominantly very close to, or at the plasma membrane, while Brho localizes to the Golgi. The role of Star, however, is not yet resolved because the results contrast with the ER localization of Star in the oocyte described by others. Interestingly, unlike Rho-1 and Brho, Star is probably involved in other processes as well. For example, Star has been identified as a suppressor of Delta, one of the Notch ligands. Delta encodes a transmembrane protein that is cleaved by the Kuzbanian metalloprotease, and the extracellular fragment antagonizes the function of the membrane-bound Delta protein as an activating Notch ligand. In the case of Notch signaling, a reduction of Star gene activity might lead to a reduced release of the extracellular Delta fragment, and thus enhance Delta signaling (Ghiglione, 2002 and references therein).

Finally, understanding the function of Star and Rho-1/Brho in Grk processing is relevant to studies of the mammalian ligands of the EGFR family as well, because TGFalpha may also be processed in vivo before receptor binding. Thus, although further work is needed to fully understand the biochemical function of Star, and Rho-1/Brho, these studies have provided a number of insights into the mechanism of action of these molecules (Ghiglione, 2002).

Drosophila has three membrane-tethered epidermal growth factor (EGF)-like proteins: Spitz, Gurken and Keren. Spitz and Gurken have been genetically confirmed to activate the EGF receptor, but Keren is uncharacterized. Spitz is activated by regulated intracellular translocation and cleavage by the transmembrane proteins Star and the protease Rhomboid-1, respectively. Rhomboid-1 is a member of a family of seven similar proteins in Drosophila. Four of the rhomboid family members have been examined: all are proteases that can cleave Spitz, Gurken and Keren, and all activate only EGF receptor signaling in vivo. Star acts as an endoplasmic reticulum (ER) export factor for all three. The importance of this translocation is highlighted by the fact that when Spitz is cleaved by Rhomboids in the ER it cannot be secreted. Keren activates the EGF receptor in vivo, providing strong evidence that it is a true ligand. These data demonstrate that all membrane-tethered EGF ligands in Drosophila are activated by the same strategy of cleavage by Rhomboids, which are ancient and widespread intramembrane proteases. This is distinct from the metalloprotease-induced activation of mammalian EGF-like ligands (Urban, 2002).

Gurken function is restricted to oogenesis where it is required to polarize both major axes of the egg. Recent evidence strongly suggests that Gurken undergoes proteolytic processing in vivo: Gurken is released from the oocyte and is internalized by follicle cells, exists exclusively in a cleaved form in oocytes, and an uncleavable mutant form is inactive. Gurken can be processed directly by Rhomboid proteases 1-4. In the tissue culture assay, Rhomboid protease activity is required for Gurken cleavage and secretion. Although Rhomboid-1 is not required in the female germline, the specific expression of Rhomboid-2 in the early oocyte suggests that a Rhomboid might have a role in Gurken processing. Star can translocate Gurken from the ER to the Golgi apparatus in cell culture and, in some cases, enhance Gurken secretion. Together, these results strongly suggest that Gurken activity, like that of Spitz, is at least partially regulated by Star-dependent ER to Golgi transport. The regulation of Gurken activity, however, also depends on the transmembrane protein Cornichon. Recent evidence in yeast and Drosophila suggests that Cornichon is an ER export factor, raising the question of the relative roles and significance of Star and Cornichon (Urban, 2002).

Cornichon and Gurken protein secretion

During Drosophila oogenesis, localization of the transforming growth factor alpha (TGF alpha)-like signaling molecule Gurken to the oocyte membrane is required for the establishment of polarity in the egg and embryo. To test Gurken domain functions, full-length and truncated forms of Gurken were expressed ectopically using the UAS/Gal4 expression system, or in the germline using the endogenous promoter. GrkdeltaC, containing a deletion of the cytoplasmic domain, localizes to the oocyte membrane and can produce functional signals. GrkdeltaTC, which lacks the transmembrane and cytoplasmic domains, retains signaling ability when ectopically expressed in somatic cells. However, in the germline, the GrkdeltaTC protein accumulates throughout the oocyte cytoplasm and cannot signal. Several strong gurken alleles contain point mutations in the transmembrane region. It has been concluded that secretion of Gurken requires its transmembrane region; a model is proposed in which the gene cornichon mediates this process (Queenan, 1999).

Cornichon is a small integral membrane protein that is required in the germline (Roth, 1995). Recent results demonstrate that ERV14, a cornichon homolog in yeast (Powers, 1998), is required for the specific transport of the transmembrane protein Axl2p to the plasma membrane. Erv14p is found in COPII-containing vesicles that bud from the ER. In erv14 mutants, Axl2p remains within the ER, while other secretory proteins are transported at wild-type rates, indicating that the role of Erv14p is to specifically promote the exit of Axl2p from the ER. Similarly, Cornichon may be required to allow Gurken to exit the ER and be routed to the oocyte surface. The class of gurken alleles in which Gurken protein is mislocalized contain molecular lesions that cluster in or near the transmembrane region. Since Cornichon is predicted to be a transmembrane protein, it is possible that Cornichon and Gurken directly interact via the Gurken transmembrane domain. Alternatively, the effects of Cornichon on Gurken secretion could be more indirect (Queenan, 1999 and references therein).

The exocyst component Sec5 is required for membrane traffic and Gurken trafficking in the Drosophila ovary

The exocyst (Sec6/8) complex is necessary for secretion in yeast and has been postulated to establish polarity by directing vesicle fusion to specific sites along the plasma membrane. The complex may also function in the nervous system, but its precise role is unknown. Exocyst function was investigated in Drosophila with mutations in one member of the complex, sec5. Null alleles die as growth-arrested larvae, whose neuromuscular junctions fail to expand. In culture, neurite outgrowth fails in sec5 mutants once maternal Sec5 is exhausted. Using a trafficking assay, impairments were found in the membrane addition of newly synthesized proteins. In contrast, synaptic vesicle fusion was not impaired. Thus, Sec5, although not involved in vesicle transport, nevertheless differentiates between two forms of vesicle trafficking at the membrane: trafficking for cell growth and membrane protein insertion depend on sec5, whereas transmitter secretion does not. In this regard, Sec5 differs from the homologs of other yeast exocytosis genes that are required for both neuronal trafficking pathways (Murthy, 2003). Sec5 is also required for membrane traffic and polarity in the Drosophila ovary. During oogenesis, Sec5 localization undergoes dynamic changes, correlating with the sites at which it is required for the traffic of membrane proteins. Germline clones of sec5 possess defects in membrane addition and the posterior positioning of the oocyte. Additionally, the impaired membrane trafficking of Gurken, the secreted ligand for the EGF receptor, and Yolkless, the vitellogenin receptor, results in defects in dorsal patterning and egg size. However, the cytoskeleton is correctly oriented. It is concluded that Sec5 is required for directed membrane traffic, and consequently for the establishment of polarity within the developing oocyte (Murthy, 2004).

The shift in Sec5 localization from the posterior of the oocyte to the anterior during stage 7 parallels a shift in the directed secretion of Gurken. Secreted at the posterior margin before stage 7, Gurken thereafter signals from an anterior corner of the oocyte to adjacent follicle cells. Those cells that receive the highest levels of Gurken repress the differentiation of the dorsal lateral follicle cells, thus creating a space between two lateral patches of cells that will form the appendages. Because females with sec5^E13 germlines lay eggs with fused dorsal appendages, a role for Sec5 in Gurken signaling was hypothesized (Murthy, 2004).

In early stages, both wild type and sec5^E13 germlines appropriately accumulated Gurken in the oocyte. After stage 7, however, Gurken is mislocalized in granules throughout the mutant oocytes. In stage 10 egg chambers, when Gurken is present at the dorsoanterior membrane of the oocyte in wild type, a substantial amount of Gurken is observed in granules scattered throughout the cytoplasm of sec5^E13 oocytes. Much Gurken remains in the vicinity of the nucleus, but very little is present in the membrane. The cytoplasmic Gurken in sec5^E13 oocytes is not coincident with a marker for the ER, Boca , indicating that the block in the directed trafficking of Gurken is at a later step of the pathway (Murthy, 2004).

Eggs derived from sec5^E13 homozygous germlines, are typically flaccid, small and, by Nomarski optics, devoid of yolk granules. Yolk proteins, however, are synthesized in fat bodies and follicle cells (which were not homozygous for the mutation) and are subsequently imported into the oocyte by endocytosis after binding to the vitellogenin receptor, Yolkless. A defect in the trafficking of Yolkless to the oocyte surface might therefore explain the decreased yolk content of the sec5 oocytes (Murthy, 2004).

In wild-type germlines, Yolkless is diffusely distributed until stage 8, whereupon, induced by an unknown signal, Yolkless translocates from the ooplasm to the cortex. At stage 7, Yolkless was detectable within both control and sec5^E13 oocytes. At stage 8 in the mutant, however, the majority of the receptor does not go to the surface, and remains cytoplasmic through stage 10. The mistrafficking of Yolkless, like the general disruption of membranes in the sec5 null allele, indicates that Sec5 is not only required for Gurken localization, but rather is of general significance for the membrane trafficking of many germline proteins (Murthy, 2004).

Although the Gurken and Yolkless mislocalizations are probably due to a defect in membrane trafficking, these phenotypes might be secondary to a defect in the concurrent reorganization of the oocyte, which includes the reorientation of the microtubule cytoskeleton, the movement of the oocyte nucleus to the anterior cortex of the oocyte, and the localization of Gurken mRNA and protein near the nucleus (Murthy, 2004).

To investigate this possibility, the localization was examined of several proteins restricted to the posterior pole of the oocyte: Oskar, Par-1 and a kinesin-ß-gal fusion. In both control and sec5^E13 germlines, all three proteins accumulate properly at the posterior pole in stage 8-10 oocytes. Dynein Heavy Chain (Dhc), also localizes to the posterior end of late stage oocytes. This marker also was normal in the mutants, accumulating first in the oocytes of early stage egg chambers and after stage 8 at the posterior end of the oocyte (Murthy, 2004).

To examine directly the polarity of the microtubules, sec5^E13 oocytes were imaged that expressed in the germline a marker for the minus ends of microtubules, a fusion of the head domain of Nod (no-distributive disjunction) to GFP. At stages 7 and 10, Nod-GFP is concentrated at the anterior end of the oocyte in both wild type and mutant. The correct positioning of the minus ends in sec5^E13 was also demonstrated with FITC-conjugated alpha-tubulin. Thus, the defective trafficking of Gurken and Yolkless cannot be secondary to microtubule defects (Murthy, 2004).

It was observed, however, that the overexpression of Nod-GFP in sec5^E13 oocytes enhances the phenotype of the sec5^E13 allele alone: the oocyte nucleus is often displaced from the cortex, membranes between cells are absent, the development of the follicle epithelium is disturbed and no eggs of this genotype are laid. These defects are never observed in Nod-GFP expressing lines that are not mutant for sec5. It is possible that the overexpression of the Nod motor domain impairs microtubule-based transport, thereby enhancing the sec5^E13 phenotype by further slowing the delivery of membrane to the cell surface (Murthy, 2004).

Examining sec5^E13 egg chambers, it was noted that the oocyte nucleus was sometimes mislocalized. The nucleus invariably moved to the anterior, as in wild type, but was not closely associated with the dorsoanterior plasma membrane. A three dimensional composite image was assembled from individual z sections of stage 10 egg chambers and rotated to reveal the relationship of the nucleus to the plasma membrane. This analysis confirmed that the nucleus was not always adjacent to the dorsal membrane: eight out of 39 (21%) sec5^E13 oocytes had a mispositioned nucleus, but none of 40 wild-type oocytes (Murthy, 2004).

Bicaudal-D (Bic-D) is a cytosolic protein that interacts with the dynein-dynactin complex, and participates in the cortical anchoring of the nucleus. Bic-D localized normally in sec5^E13 oocytes throughout oogenesis. In early stages, Bic-D is at the microtubule minus ends at the oocyte posterior, and by stage 6 relocalizes to the anterior rim, preceding the arrival of the nucleus. Subsequently, Bic-D concentrates above the nucleus. Even when the oocyte nucleus is displaced from the dorsal cortex, Bic-D remaines near the nucleus, indicating that its nuclear association is not sufficient to attach the nucleus to the dorsal cortex. Because no alteration of the microtubule cytoskeleton was in sec5^E13 germline clones, nor gross mislocalization of Bic-D, the lack of a tight association of the nucleus with the membrane must have other causes (Murthy, 2004).

The trafficking of the Gurken protein provides at present the best example of an identified membrane protein whose selective, Sec5-dependent localization is crucial to proper development. The final deposition of Gurken is likely to arise from a combination of mechanisms, including the transport of the oocyte nucleus to an anterior corner, the nearby localization of Gurken mRNA, the microtubule-dependent transport of Gurken protein to the cortex, and the insertion of both pre-existing and newly synthesized Gurken into the plasma membrane by vesicle fusion. The presence of displaced Gurken protein in the posterior regions of the mutant ooplasm may be an indirect result of blocked membrane fusion after which Gurken-containing vesicles may drift away from their normal target. Gurken trafficking, however, also indicates that Sec5 and the exocyst cannot be the only cues that direct vesicle fusion: Sec5 localizes along the entire anterior lateral rim of the oocyte, but Gurken is inserted only at that section adjacent to the nucleus. Furthermore, when the nucleus and Gurken transcripts are mislocalized by cytoskeletal changes, some Gurken signaling occurs ectopically, near the misplaced nucleus, and away from the major concentration of Sec5. Thus, the localization of Sec5 should be viewed as one of several layers of likely mechanisms for directing membrane proteins (Murthy, 2004).

In mediating the traffic of multiple membrane proteins, including both Gurken and Yolkless, Sec5 is clearly in a distinct category from Cornichon and Boca, proteins that act in the ER. These proteins are needed for the correct transport of individual proteins and appear to act at earlier trafficking steps. Gurken is retained inside the cell in cornichon mutants, although vitellogenesis proceeds normally. Boca, however, is required for the trafficking of Yolkless and other LDL receptor family proteins to the membrane, but does not influence Gurken traffic. These highly specific deficits, which are likely to occur upon exiting from the ER, are distinct from the more general disruption of traffic in sec5 mutants (Murthy, 2004 and references therein).

Many forms of membrane traffic to the cell surface now appear to depend on the exocyst. In multicellular organisms, these include vesicles derived from the trans-Golgi network (TGN) carrying newly synthesized proteins or mediating neurite outgrowth. However, not all forms of exocytosis depend on the exocyst. The fusion of synaptic vesicles at nerve terminals persists in sec5 mutants in which other trafficking events are blocked (Murthy, 2003) and apical protein delivery in MDCK cells is resistant to a block by antibodies to exocyst components. The essential differences between exocyst dependent and independent exocytotic events remain unclear (Murthy, 2004 and references therein).

Rab6 and BicD function together to ensure the correct delivery of secretory pathway components, such as the TGFα homolog Gurken, to the plasma membrane

The Drosophila oocyte is a highly polarized cell. Secretion occurs towards restricted neighboring cells and asymmetric transport controls the localization of several mRNAs to distinct cortical compartments. This study describes a role for the Drosophila ortholog of the Rab6 GTPase, Drab6, in establishing cell polarity during oogenesis. Drab6 localizes to Golgi and Golgi-derived membranes and interacts with BicD. Evidence is provided that Drab6 and BicD function together to ensure the correct delivery of secretory pathway components, such as the TGFα homolog Gurken, to the plasma membrane. Moreover, in the absence of Drab6, osk mRNA localization and the organization of microtubule plus-ends at the posterior of the oocyte were both severely affected. These results point to a possible connection between Rab protein-mediated secretion, organization of the cytoskeleton and mRNA transport (Januschke, 2007).

In vertebrate cells, Rab6 is associated with the Golgi and the trans-Golgi network (TGN) membranes. To investigate the subcellular localization of Drab6 in the Drosophila germ line, the expression pattern was monitored of transgenic lines expressing Drab6 fused to GFP and RFP. It was observed that during oogenesis, the global distribution of Drab6 evolved. Drab6 first accumulated transiently in a central position during stages 7/8, then is uniformly distributed at the beginning of stage 9 to end up juxtaposed to the entire oocyte cortex. It is noteworthy that promoters of different strengths give similar expression patterns. In addition, the genomic null allele rab6^D23D is fully rescued by the different lines expressing Drab6 (Januschke, 2007).

Drab6 does not colocalize extensively with ER membranes (labeled with PDI-GFP). Instead, it seems to be differentially associated with two types of Golgi units as revealed by its association with Lava Lamp (Lva and GalT). Lva, a cis-Golgi marker, colocalizes with Drab6, mainly at the cortex of the oocyte and in nurse cells. A GFP trap protein corresponding to a UDP-galactose:beta-N-acetylglucosamine beta-1,3-galactosyltransferase (GalT), enriches predominantly in Golgi membranes, exhibits a distribution similar to that of GFP-Drab6: it accumulates in the center of the oocyte at stage 8, where it colocalizes with Drab6, and is later confined to the cortex. Importantly, the distribution of Lva and GalT is similar in both matαtubGFP-Drab6, ubiRFP-Drab6 and control oocytes. Given that Lva and GalT markers are not present in the Golgi cisternae that are evenly distributed throughout the oocyte, as documented by electron microscopy (EM) analysis, they might be the hallmark of distinct functional Golgi units, with Drab6 being able to interact with both types of Golgi. Unlike Lva, the distribution of which is only mildly affected, GalT and acetylglucosamine-modified proteins [detected by the wheat germ agglutinin lectin (WGA)] expressed by Golgi structures are abnormally distributed in Drab6 mutants. Moreover, ultrastructural analysis by EM revealed that the ER is abnormally swollen in Drab6 mutant oocytes, and that the Golgi mini-stacks are markedly curved, with partially inflated cisternae (Januschke, 2007).

These morphological effects led to an investigation of the role of Drab6 in the secretory pathway. The polarized secretion of the TGFα-like growth factor Grk was monitored. Grk secretion is restricted to the anterodorsal corner through a rapid transit from the ER towards the Golgi apparatus. In GFP-Drab6-rescued egg chambers, Grk and Drab6 colocalize. In Drab6 mutant oocytes, grk mRNA localization is the same as in wild type. Grk protein, however, is slightly more abundant than in controls and an important fraction extends ventrally. Polarized secretion of Grk lead to the formation of two dorsal appendages on the egg shell. In the absence of Drab6, mislocalized Grk induces ventralization, instead of a dorsalization (multiple dorsal appendages on the egg shell) as observed when Grk is ectopically secreted. Hence, this argues for a specific failure of Grk delivery to the plasma membrane. This phenotype is specific to Drab6 because it could be fully rescued by the GFP-Drab6 transgene (Januschke, 2007).

Next, the intracellular localization of Grk was examined in the absence of Drab6. Grk accumulated frequently in large ring-like particles in the Drab6 mutant, but not in control oocytes. These Grk 'rings', similar to those of yolk granules, did not contain Lva, suggesting that Grk is not blocked in the Golgi. Grk actually accumulates in Drab6 mutants on vesicles stained by LysoTracker, which labels either lysosomes or late endosomes containing yolk granules. Hence, two independent approaches suggest that Grk is not blocked in the Golgi, but is mislocalized to post-Golgi compartments, probably endosomes (Januschke, 2007).

Interestingly, the secretory impairment was also confirmed by Lycopersicon esculentum tomato lectin (LE) detecting modified proteins in the Golgi. In the absence of Drab6, LE revealed abnormal vesicular structures in the oocyte and nurse cells that fail to reach the cortex. EM analysis also demonstrated rupture of the plasma membrane between neighboring nurse cells. Finally, it was observed that GFP-Drab6-rescued egg chambers exhibit an accumulative enrichment of Drab6 at the plasma membrane during oogenesis, which is particularly evident in nurse cells. This is consistent with the involvement of Drab6 in secretion towards the plasmalemma (Januschke, 2007).

The existence of three important and novel aspects of Drab6 function during oogenesis has been established as follows: (1) Consistent with its localization in vertebrate cells, Drab6 is predominantly localized to the Golgi complex in Drosophila, but overlaps with Golgi markers that have distinct localizations, suggesting that Drab6 might associate with distinct functional Golgi units. Drab6 might also play a role in membrane exchange between Golgi and ER and in Golgi organization, according to EM analysis, which is again consistent with known functions of mammalian Rab6. (2) By controlling the migration of Golgi units towards the cell cortex, Drab6 controls the delivery of membrane to the plasmalemma, as shown in Drab6 mutants in which glycosylated proteins labeled by WGA and LE lectins accumulate in large vesicular structures. This pattern is similar to the mislocalization profile of Grk in the absence of Drab6. (3) In the oocyte, Drab6 is required for the anterodorsal secretion of Grk, which leads to the differentiation of the follicle cells required for the morphogenesis of the dorsal appendages of the egg shell. In the absence of Drab6, it was observed that Grk is mislocalized to late endosomal or lysosomal compartments, demonstrating that Drab6 also affects post-Golgi traffic. In vertebrates, one of the Rab6 isoforms (Rab6A') is also involved in endosome-to-Golgi transport. Additionally, a role for Ypt6p (the only copy of Rab6 in the yeast S. cerevisiae) has also been documented as being involved in fusion of endosome-derived vesicles with the late Golgi (Siniossoglou, 2001). It remains to be established whether Drab6 functions directly in the secretory pathway or if the effects observed in Drab6 mutants on post-Golgi trafficking are a consequence of defects in endosome-to-Golgi trafficking (Januschke, 2007).

In order to identify potential Drab6-binding proteins, a yeast two-hybrid screen was performed using as bait Drab6Q71L, a GTPase-deficient mutant. Sixty-two distinct truncated clones of BicD, lacking parts of the amino-terminus, interacted with Drab6Q71L. The intersection of all identified fragments defined a minimal interacting domain, mapping to amino acids 699-772 in the coiled-coil motif H4 of BicD, shown for murine BicD to interact with the mammalian Rab6. In order to validate this interaction, glutathione S-transferase (GST) pull-down assays were performed, using lysates from wild-type ovaries. GST-Drab6 specifically retained BicD; GST alone and GST-Rab1 did not bind BicD. Furthermore, preloading GST-Drab6 with the non-hydrolyzable GTP analog, GTP-γ-S, yielded an improved interaction with BicD. It is concluded, therefore, that in vitro, BicD interacts through its carboxy-terminus preferentially with the active form of Drab6 (GTP-bound), as has been shown for mammalian Rab6 (Januschke, 2007 and references therein).

Time-lapse recording showed that in the oocyte and nurse cells, RFP-Drab6 and BicD-GFP colocalize to multiple large aggregates with low dynamics. Further GFP-Drab6 accumulation in the center depends on the presence of BicD during stage 8, as observed in a BicD^mom background. Interestingly, in such BicD^mom oocytes, Grk is found in ring-like structures remote from the nucleus, as observed in Drab6 mutant oocytes (Januschke, 2007).

Since BicD and Rab6 have been shown to be involved in MT-based transport, experiments were conducted to discover whether Drab6-positive structures require MTs for movement. Time-lapse microscopy revealed that large aggregates are less dynamic than the highly motile small particles. Colchicine MT depolymerization severely reduced the movement of Drab6 particles, which form large clusters, indicating that Drab6 is actively transported along MTs. The MT motors Kinesin I [Kinesin heavy chain (Khc)] and Dynein have been shown to be involved in polarizing the Drosophila oocyte. Inactivating the Dynein complex by the overexpression of Dynamitin prevents accumulation of Drab6 at the oocyte cortex, but does not significantly reduce Drab6 movements. By contrast, in Khc^7.288 germ line clones, Drab6 does not localize in the center of the oocyte during stage 7/8 but forms abnormal aggregates around the mispositioned nucleus. For reasons not currently fully understood, the speed of Drab6 particles is significantly reduced compared with controls or Dynamitin-overexpressing oocytes (Januschke, 2007).

Drab6 and BicD interact in a yeast two-hybrid screen and in GST pull-down assays and colocalize in vivo. Moreover, there are indications that Drab6 requires BicD for correct subcellular localization, which suggests that Drab6 interacts with BicD in Drosophila as it does in mammals. Strikingly, it was found that lack of each protein compromises Grk secretion in a very similar way. Overexpression of Dynamitin, to impair Dynein function, induces ectopic accumulation of Grk and ventralization of the egg shell (Januschke, 2002). Therefore, in Drosophila, BicD/Dynein and Drab6 are likely to be involved together in Grk secretion to the anterodorsal corner of the oocyte (Januschke, 2007).

It is important to mention that colocalization of the two proteins is limited. Moreover, lack of BicD or Drab6 yields different phenotypes. BicD mutation affects oocyte determination and the position of the oocyte nucleus, but has no impact on MT organization in mid-oogenesis, which is not the case in the Drab6 mutant. A genetic interaction between BicD's co-factor Egalitarian and Kinesin I has already been demonstrated, suggesting that Drab6 might interact with Dynein and Kinesin I via BicD (Januschke, 2007).

Interestingly, it was noticed that in the absence of Drab6, osk mRNA is not correctly localized in the oocyte. gurken and bicoid mRNAs are, however, unaffected, and osk mRNA localization to the oocyte center is frequent when the MT network is not correctly polarized. In Drab6 mutant oocytes, the defective posterior localization of the MT plusend marker Khc-ß-Gal indicates a defect in MT organization (Januschke, 2007).

Given that Drab6 is required for late Grk signaling at the anterodorsal corner of the oocyte, it might also be involved in early germ line to soma signaling mediated by Grk, which controls MT organization. This is thought unlikely. In the absence of this signaling, posterior follicle cells differentiate into anterior follicle cells and, as a consequence, the posterior structure of the egg shell, the aeropyle, is substituted with an anterior structure, the micropyle. An aeropyle is always observed at the posterior of eggs derived from Drab6 mutant oocytes. Additionally, removing Drab6 from the posterior follicle cells does not affect oocyte polarity. Hence, Drab6 is possibly involved in MT organization at the posterior pole. Interestingly, Rab6 family interactors such as Rab6IP2/ELKS are capable of interacting with CLASPs at the cortex of HeLa cells, suggesting a link between Rab6 protein and MT organization at the cortex (Januschke, 2007).

Efficient Gurken protein trafficking requires trailer hitch, a component of a ribonucleoprotein complex localized to the ER in Drosophila

Translational control of localized messenger mRNAs (mRNAs) is critical for cell polarity, synaptic plasticity, and embryonic patterning. While progress has been made in identifying localization factors and translational regulators, it is unclear how broad a role they play in regulating basic cellular processes. Drosophila trailer hitch (tral) has been identified as required for the proper secretion of the dorsal-ventral patterning factor Gurken, as well as the vitellogenin receptor Yolkless. Surprisingly, biochemical purification of Tral reveals that it is part of a large RNA-protein complex that includes the translation/localization factors Me31B and Cup as well as the mRNAs for endoplasmic reticulum (ER) exit site components, that regulate exit of proteins from the ER. This complex is localized to subdomains of the ER that border ER exit sites. Furthermore, tral is required for normal ER exit site formation. These findings raise exciting new possibilities for how the mRNA localization machinery could interface with the classical secretory pathway to promote efficient protein trafficking in the cell (Wilhelm, 2005).

One of the key events in dorsal-ventral patterning is the localization of grk mRNA to the dorsal-anterior region. The localization of grk mRNA in turn causes the trafficking of Grk protein to be confined to dorsal-anterior endoplasmic reticulum (ER)-Golgi units. It is this localized secretion of Grk that instructs the dorsal follicle cells to assume a dorsal cell fate. These dorsal follicle cells then secrete the proper eggshell components to generate a dorsal appendage. The dorsal-ventral patterning defect of the tral mutants suggests that tral might regulate some aspect of the localization or secretion of Grk. In wild-type egg chambers, Grk protein is expressed homogeneously throughout the oocyte during stages 6–7 and then is found only in small puncta near the plasma membrane in the dorsal-anterior region of the oocyte during stages 8-10. These small Grk puncta are known to coincide with sites of exit from the ER. In both tral¹ homozygotes and tral¹ hemizygotes, abnormally large Grk puncta were observed in 48% of homozygotes and 63% of hemizygotes during stages 6-8. This suggests that mutations in tral disrupt some aspect of Grk trafficking through the secretory pathway (Wilhelm, 2005).

Conceivably, tral mutants could affect Grk trafficking either by interfering with the proper localization/translational control of the grk message, by disrupting the microtubule cytoskeleton, or by blocking the normal trafficking of Grk protein through the secretory pathway. To test these possibilities, the localization of grk mRNA was assayed in tral mutant egg chambers by in situ hybridization. The localization of grk mRNA to the dorsal-anterior region of the oocyte during stages 8-10 is normal in tral mutant egg chambers. This result argues against defects in grk mRNA localization being responsible for the Grk trafficking defect observed in tral mutants (Wilhelm, 2005).

The fact that grk mRNA is correctly localized argues that the normal polarity of the microtubule cytoskeleton is intact in tral mutants; a polarized microtubule network is essential for grk mRNA localization. To confirm that the microtubule polarity is intact, whether the localization of Osk protein to the posterior is normal in tral mutant egg chambers was examined. Because the correct localization of Osk protein to the posterior requires both normal microtubule polarity and the proper localization of osk mRNA, this assay should reveal any functional defects in either the microtubule polarity or the transport of osk mRNA. Whereas large Grk puncta accumulate in the oocytes of tral mutants, Osk protein is present at the posterior of the oocyte. Consistent with this result, mutations in tral do not affect the normal anterior-posterior gradient of microtubule density in stage 9 egg chambers. Thus, mutations in tral do not affect either microtubule polarity or the transport of the grk and osk messages. This result may seem paradoxical, since grk signaling early in oogenesis is required to establish the microtubule polarity of the oocyte. However, the establishment of microtubule polarity is less sensitive to changes in the level of grk signaling than dorsal appendage formation. Because none of the tral alleles cause complete ventralization of the eggshell, it is not surprising that it has been possible to selectively affect dorsal appendage formation without altering the microtubule polarity of the oocyte (Wilhelm, 2005).

To rule out that large Grk puncta are due to a defect in Grk translational control, the distribution of large Grk puncta was examined during stages 8-10. If there were a defect in translational repression of grk mRNA, Grk protein should accumulate broadly throughout the oocyte. While some large Grk puncta are mislocalized to the side of the nucleus facing away from the oocyte cortex, both normal sized and large Grk puncta are restricted to the dorsal-anterior region of the oocyte. Because a defect in translational control would be expected to yield high levels of Grk protein throughout the oocyte, this result argues that the large Grk foci are not due to a loss of translational repression of the grk message. Because the polarity of the microtubule cytoskeleton and the localization/translation of grk mRNA appear normal in tral mutant egg chambers, the hypothesis was tested that the formation of large Grk puncta in tral mutants is due to a defect in the trafficking of Grk (Wilhelm, 2005).

In a variety of systems, ER exit sites are closely associated with Golgi units, presumably due to the role of ER trafficking in establishing and maintaining the Golgi. Because previous work established that small Grk puncta are coincident with ER exit sites, also known as the transitional ER, the effects of tral mutants on the distribution of Grk and its association with the Golgi were examined (Herpers, 2004). In wild-type egg chambers, the majority of Grk protein is present in small puncta that are closely associated with an individual Golgi complex that is positive for the Golgi marker Lava lamp. However, in tral mutants, the large Grk puncta have lost their intimate association with the Golgi. This suggested that the formation of large Grk foci might be due to a defect in ER exit (Wilhelm, 2005).

The COPII complex, which is required for ER-to-Golgi trafficking, is known to label discrete sites on the ER. Furthermore, a number of experiments have implicated these COPII sites and the regions surrounding them in exit from the ER. Using GFP-Sar1 as a marker for COPII complex formation, the distribution of ER exit sites was examined in wild-type and tral hemizygous egg chambers. GFP-Sar1 is distributed in small puncta throughout the nurse cells and oocyte in wild-type egg chambers. However, this organization is severely disrupted in tral¹/Df(3L)ED4483 egg chambers. In these egg chambers, the GFP-Sar1 is found in abnormally large puncta similar to those observed for Grk protein. Thus, tral is required for normal ER exit site distribution and morphology. The accumulation of Grk in large foci that are not correctly associated with the Golgi, together with the role of tral in organizing ER exit sites, argues that the disruption of ER exit sites in tral mutants leads to a functional defect in ER-Golgi trafficking. It is this disruption of ER-Golgi trafficking that likely underlies the failure in dorsal-ventral patterning observed in tral mutants (Wilhelm, 2005).

If tral plays a general role in ER exit site function, one would expect to observe defects in the trafficking of other secreted proteins. In order to test this, the effects of tral mutants on the trafficking of the vitellogenin receptor Yl were examined. Previous work on Yl has shown that in wild-type egg chambers, Yl protein is distributed homogeneously throughout the ER of the oocyte with an occasional small puncta until stage 8, when all of the Yl protein is transported to the plasma membrane (Schonbaum, 2000). Homozygous tral¹ oocytes showed no obvious disruption of Yl trafficking. However, 75% of hemizygous tral¹ oocytes showed Yl foci within the oocyte during stages 6-9. Therefore, tral is required for the trafficking of proteins besides Grk and likely plays a general role in promoting exit from the ER (Wilhelm, 2005).

It was next asked whether the Grk and Yl foci are distinct in tral¹ hemizygous oocytes. Immunostaining for both Grk and Yl revealed that the large foci for each protein are separate. This suggests that the two proteins use separate trafficking pathways that both require tral. The observation that the trafficking of Grk is more sensitive to decreases in tral function than the trafficking of Yl is consistent with this idea (Wilhelm, 2005).

A maternal screen for genes regulating Drosophila oocyte polarity uncovers new steps in meiotic progression

Meiotic checkpoints monitor chromosome status to ensure correct homologous recombination, genomic integrity, and chromosome segregation. In Drosophila, the persistent presence of double-strand DNA breaks (DSB) activates the ATR/Mei-41 checkpoint, delays progression through meiosis, and causes defects in DNA condensation of the oocyte nucleus, the karyosome. Checkpoint activation has also been linked to decreased levels of the TGFα-like molecule Gurken, which controls normal eggshell patterning. This easy-to-score eggshell phenotype was used in a germ-line mosaic screen in Drosophila to identify new genes affecting meiotic progression, DNA condensation, and Gurken signaling. One hundred eighteen new ventralizing mutants on the second chromosome fell into 17 complementation groups. This study describes the analysis of 8 complementation groups, including Kinesin heavy chain, the SR protein kinase cuaba (CG8174), the cohesin-related gene dPds5/cohiba, and the Tudor-domain gene montecristo. These findings challenge the hypothesis that checkpoint activation upon persistent DSBs is exclusively mediated by ATR/Mei-41 kinase and instead reveal a more complex network of interactions that link DSB formation, checkpoint activation, meiotic delay, DNA condensation, and Gurken protein synthesis (Barbosa, 2007),

In this study, a clonal screen was used to identify genes regulating meiotic progression in Drosophila. Instead of testing directly for defects in meiosis, an easy-to-score eggshell phenotype was used that is produced when the levels or activity of the morphogen Grk are affected. This allowed an efficient screen of a large number of mutant lines and identification of germ-line-specific genes as well as genes with essential functions. The number of new genes identified is likely less than the total number of 2R genes required for Grk synthesis and function since mutations were discarded that blocked oogenesis. Of the eight genes described in this study, five show meiotic phenotypes. dPds5, nds, and mtc delay meiotic restriction to the oocyte, although only dPds5 and nds genetically interact with mei-W68 and mei-41, respectively. trin and blv affect the morphology of the karyosome in spite of normal timing in meiotic restriction. This confirms the effectiveness of the screening method for meiotic genes. Genetic and developmental analysis of the newly identified genes provides evidence for new regulatory steps in a network that coordinates Drosophila meiosis and oocyte development (Barbosa, 2007),

One complementation group, cohiba, identifies the Drosophila homolog of Pds5p in Schizosaccharomyces pombe, Spo76 in Sordaria macrospore, and BimD in Aspergillus nidulans, which have been found associated with the cohesion complex of mitotic and meiotic chromosomes. Depletion of Pds5 affects not only cohesion but also condensation in meiotic prophase. The unique 'open chromatin' karyosome defect observed in dPds5^cohiba mutants is consistent with a role of Pds5 in chromosome cohesion during Drosophila meiosis. Like Spo76, the dPds5^cohiba phenotype is suppressed by Spo11 (mei-W68) mutations defective in DSB formation. This suggests that dPds5 is necessary to maintain the structure of the meiotic chromosomes after DSBs are induced. However, in contrast to known DSB repair genes, the meiotic delay and oocyte patterning defects of dPds^cohiba mutants are not due to activation of ATR/Mei-41-dependent checkpoint. One possibility is that the ATR downstream effector kinase dChk2 is activated via an alternative pathway, such as the Drosophila ataxia-telangiectasia mutated (ATM) homolog, which indeed activates dChk2 in the early embryo independently of ATR. Alternatively, dPds^cohiba mutants may activate a checkpoint that measures cohesion rather than DSB breaks. The only other cohesion protein characterized in Drosophila is the product of the orientation disruptor (ord). ORD plays a role in early prophase I by maintaining synaptic chromosomes and allowing interhomolog recombination. More importantly and perhaps similar to dPds5, ORD seems not to be required for DSB repair. However, in contrast to dPds5 mutants, karyosome morphology is normal in ord mutants, and an eggshell polarity phenotype has not been reported. Although required for chromatid cohesion, dPds5 and ORD might play complementary roles in SC dynamics: ORD may stabilize the SC in the oocyte, whereas dPds5 may be required for the disassembly of synapses as one of the pro-oocytes regresses from meiosis (Barbosa, 2007),

The screen identified mutations in montecristo (mtc) that affect the restriction of meiosis to the oocyte. It has been proposed that this delay reflects the activation of the ATR/Mei-41 checkpoint pathway. Similar to dPds5, Mtc may control the regression from pachytene in those cyst cells that will not adopt the oocyte fate. The delayed meiotic restriction observed in mtc mutants occurs, however, independently of DSB formation or Mei-41 checkpoint activation. Mtc contains a Tudor domain. In other Tudor-domain proteins, this domain has been shown to interact with methylated target proteins. Identification of specific Mtc targets may clarify its role in meiotic restriction and oocyte patterning (Barbosa, 2007),

A particularly intriguing and novel phenotype is uncovered by mutations in indios (nds). By delaying meiotic restriction and activating Mei-41 without affecting the karyosome morphology, nds mutants separate checkpoint activation leading to Grk decrease from checkpoint activation controlling karyosome compaction. The nds phenotype also occurs independently of DSBs, suggesting that the trigger that leads Nds to trigger checkpoint activation is not DNA breaks. The fact that nds mutants are extremely sensitive to Mei-41 dosage further suggests that Nds activity may specifically control a branch of the Mei-41 checkpoint regulating Grk activity. In contrast to nds, trin mutants do not delay meiotic restriction and show defects in the karyosome in spite of normal Grk levels. Like mutants in src64B and tec29, which show a similar phenotype, Trin may mediate chromatin remodeling in the oocyte by regulating the actin cytoskeleton. In this context, the DV phenotype of eggs from trin mutants may be an indirect effect due to defects in actin cytoskeleton function. The production of collapsed eggs by trin mutant germ-line clones is consistent with this idea (Barbosa, 2007),

Finally, blv mutants show striking similarity to vas mutants with respect to lack of sensitivity to DSB formation, no evident delays of meiotic restriction, or karyosome and Grk phenotypes. Blv may thus act downstream or independent of the Mei41/ATR checkpoint, and its further characterization may help to understand the effector side of the meiotic checkpoint pathway (Barbosa, 2007),

Previous knowledge pointed to Drosophila meiosis as a linear progression of events from homologous chromosome pairing and recombination to meiotic restriction, karyosome formation, and eggshell patterning, with DSB repair as the main checkpoint linking meiosis to Grk signaling. By uncoupling some of these events, this study suggests the existence of a more complex network that links the surveillance of meiotic progression to oocyte patterning (Barbosa, 2007),

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