gurken
The antenno-maxilary complex (AMC) forms the chemosensory system of the Drosophila larva and is involved in gustatory and olfactory perception. It has been shown that a mutant allele of the homeodomain transcription factor Prospero (prosVoila1, V1), presents several developmental defects including abnormal growth and altered taste responses. In addition, many neural tracts connecting the AMC to the central nervous system (CNS) were affected. These earlier reports on larval AMC did not argue in favour of a role of pros in cell fate decision, but strongly suggested that pros could be involved in the control of other aspect of neuronal development. In order to identify these functions, microarray analysis was performed of larval AMC and CNS tissue isolated from the wild type, and three other previously characterised prospero alleles, including the V1 mutant, considered as a null allele for the AMC. A total of 17 samples were first analysed with hierarchical clustering. To determine those genes affected by loss of pros function, a discriminating score was calculated reflecting the differential expression between V1 mutant and other pros alleles. A total of 64 genes were identified in the AMC. Additional manual annotation using all the computed information on the attributed role of these genes in the Drosophila larvae nervous system, enabled identification of one functional category of potential Prospero target genes known to be involved in neurite outgrowth, synaptic transmission and more specifically in neuronal connectivity remodelling. The second category of genes found to be differentially expressed between the null mutant AMC and the other alleles concerned the development of the sensory organs and more particularly the larval olfactory system. Surprisingly, a third category emerged from this analyses and suggests an association of pros with the genes that regulate autophagy, growth and insulin pathways. Interestingly, EGFR and Notch pathways were represented in all of these three functional categories. It is propose that Pros could perform all of these different functions through the modulation of these two antagonistic and synergic pathways. The current data contribute to the clarification of the prospero function in the larval AMC and show that pros regulates different function in larvae as compared to those controlled by this gene in embryos. In the future, the possible mechanism by which Pros could achieve its function in the AMC will be explored in detail (Guenin, 2010).
In the AMC, Prospero is expressed in a cluster of cells (composed of neuronal and support cells, but not glial cells) that emerge during embryonic life and are maintained until the end of the larval stages. In embryos, Pros was reported to be involved in cell fate decision and in cell-cycle control. By contrast, earlier data from the larval AMC rather suggested that pros could assume more restricted functions, such as the control of neuron-specific functions. The present study confirms this hypothesis and shows that in the chemosensory organs dedicated to larval olfactory and gustatory sensing, prospero could regulate genes involved in neurite outgrowth and synaptic transmissionz (Guenin, 2010).
Since pros was clearly shown to control axonal and dendritic outgrowth, the possibility cannot be excluded that the connection of pros with several genes that drive synaptic activity could be the indirect consequence of its involvement in neurite outgrowth control. In this respect, it is interesting to mention that a recent study showed that axon targeting of the R7 Drosophila photoreceptor cells to their synaptic partner requires R7-specific transcription factor Prospero. That study proposed that Pros could promote cell-type-specific expression of sensory receptors and cell-surface proteins regulating synaptic target specificity (Guenin, 2010).
As previously mentioned, some of the genes identified in this functional class are also involved in neural connectivity remodelling. How can this be achieved if the AMC is completely histolysed? In fact, in Drosophila, not all sensory neurons degenerate; Some larval neurons persist and remodel to take on a new role in the adult system. During the metamorphosis larval arbors of these neurons are pruned back and new adult-specific arbors are generated through a subsequent period of outgrowth. It seems that the neurites of these persistent larval neurons are used to partly guide axons of adult sensory neurons towards and within the CNS. Therefore, histolysis and remodelling are two processes that are achieved during metamorphosis and could concern distinct neurons (Guenin, 2010).
Does Pros play any role in AMC neuronal remodelling? The question cannot actually be answered. However, it has been previously reported that the insulin and epidermal growth factor signalling pathways, as well as ubiquitin-specific proteases are all required for the regulation of Drosophila neuronal remodelling. Interestingly, all of these components emerge clearly from the current analysis (Guenin, 2010).
Actually, no work was done on the Drosophila larvae anterior sense organ in order to check whether some of the sensory neurons (which have also an embryonic origin) persist and remodel to take place in the adult olfactory or gustatory system. Therefore, the question is left open. At least the answers will provide important insights into the mechanisms that govern developmental plasticity in insect nervous systems (Guenin, 2010).
In summary, these data collected from larval AMC and the previous genome wide expression profiling done on embryos confirms that pros is associated with the regulation of neuronal specific genes. In this respect, it is essential to note that except for a few genes (126), most of the Pros target genes identified (~1000) were not represented the current microarrays. For this reason, and because the current experiments were performed on isolated individual larval tissues, it is not possible to determine whether the genes identified by these authors are specifically expressed in embryos and/or in tissues other than AMC (Guenin, 2010).
In Drosophila, the insulin/TOR signalling pathway is divided into two branches. The insulin and its downstream effectors P13 and FOXO (forkhead box) represent one branch of this pathway, while the other branch acts through the TOR family of Serine-Threonin kinases. It has been shown that the insulin/TOR signalling pathway inhibits autophagy and controls growth by regulating ribosome biogenesis and protein biosynthetic capacity. It has been demonstrated that the TOR pathway is a nutritional checkpoint that participates in the systemic control of larval growth emanating from the Fat body (Guenin, 2010).
Microarray analysis has revealed a group of highly correlated pros candidate genes (correlation index: 0.9) that are either controlled by the insulin/TOR signalling pathway or are directly involved in the signalling cascade. This is the case for Ash2 which was found to be regulated by TOR signalling. Similarly, FK506-bp1 affects autophagy through the modulation of FOXO and Lk6 was reported to be a direct FOXO Target. Therefore it seems that in the larval AMC, Pros could be associated with growth, autophagy and nutrient sensing through the regulation of genes that are directly or indirectly linked to the insulin/TOR pathway. Interestingly, TOR was found to be differentially expressed in the V1 pros mutant in the CNS (Guenin, 2010).
As described previously, loss of pros function in the AMC induced several alterations including axon pathfinding defects and abnormal growth and taste responses. This is consistent with microarray results showing that in the larval AMC, Pros expression is associated with the regulation of genes involved in the control of neurite outgrowth, mediation of growth and autophagy and in the organisation and function of the olfactory system. The mechanism by which all of these functions are achieved by pros in the AMC is presently not known but EGFR and/or Notch pathways could play a central role. Several lines of evidence are in favour of this hypothesis (Guenin, 2010).
Four ligands are known to bind EGFR receptor: Keren, Gurken, Spitz, and Vein. Two of these were identified as potential targets of Prospero: Keren in both larval AMC and CNS and Gurken (Grk) in the larval CNS only. Moreover, Notch and EGFR were identified as putative Pros target genes in both embryos and larval AMC, indicating that they could play a central role (Guenin, 2010).
It has been reported that EGFR signalling is required for the development of some of the neurons and cuticular structures present in the AMC. In this respect, it is interesting to point out that EGFR involvement has been reported during the development of mouse gustatory epithelia in the palate and tongue (Guenin, 2010).
The expression of Notch, EGFR and Pros have been shown to be tightly linked. It has been demonstrated that normal levels of Pros expression in photoreceptor R7 cells in the Drosophila eye require EGFR signalling as well as Notch activation. In addition, a recent analysis has shown that in R7 cells, Notch and EGFR cooperate in a complex way to promote pros transcription (Guenin, 2010).
Although these data suggest that Notch and EGFR could play a central role in the mechanism by which Prospero carries out its function in the larval AMC, this hypothesis has still to be validated. In the future, it will be of great interest to explore in detail the mechanism by which all of these functions are accomplished by the homeodomain transcription factor Prospero (Guenin, 2010).
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 (DERAF 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 18oC, 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).
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).
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, mbGrkDelta19AAmyc, 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).
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 (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 sec5E13 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 sec5E13 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 sec5E13 oocytes. Much Gurken remains in the
vicinity of the nucleus, but very little is present in the membrane. The cytoplasmic Gurken in sec5E13 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 sec5E13 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 sec5E13 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 sec5E13 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, sec5E13 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
sec5E13 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
sec5E13 oocytes enhances the phenotype of the
sec5E13 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
sec5E13 phenotype by further slowing the delivery of
membrane to the cell surface (Murthy, 2004).
Examining sec5E13 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%) sec5E13 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 sec5E13 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 sec5E13 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).
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 rab6D23D 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 BicDmom background. Interestingly, in such BicDmom 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 Khc7.288 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).
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).
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 dPds5cohiba mutants is consistent with a role of Pds5 in chromosome cohesion during Drosophila meiosis. Like Spo76, the dPds5cohiba 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 dPdscohiba 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, dPdscohiba 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),
Glycosphingolipids (GSLs) are present in all eukaryotic membranes and are implicated in neuropathologies and tumor progression in humans. Nevertheless, their in vivo functions remain poorly understood in vertebrates, partly owing to redundancy in the enzymes elongating their sugar chains. In Drosophila, a single GSL biosynthetic pathway is present that relies on the activity of the Egghead and Brainiac glycosyltransferases. Mutations in these two enzymes abolish GSL elongation and yield oogenesis defects, providing a unique model system in which to study GSL roles in signaling in vivo. This study used egghead and brainiac mutants to show that GSLs are necessary for full activation of the EGFR pathway during oogenesis in a time-dependent manner. In contrast to results from in vitro studies, it was found that GSLs are required in cells producing the TGFα-like ligand Gurken, but not in EGFR-expressing cells. Strikingly, it was found that GSLs are not essential for Gurken trafficking and secretion. However, this study characterized the extracellular Gurken gradient and showed that GSLs affect its formation by controlling Gurken planar transport in the extracellular space. This work presents the first in vivo evidence that GSLs act in trans to regulate the EGFR pathway and shows that extracellular EGFR ligand distribution is tightly controlled by GSLs. This study assigns a novel role for GSLs in morphogen diffusion, possibly through regulation of their conformation (Pizette, 2009).
Glycosphingolipids (GSLs) are ubiquitous components of eukaryotic cell membranes. They consist of a variable oligosaccharide chain attached to a ceramide lipid backbone (Cer) that tethers them to the lumenal leaflet of membranes. GSLs are mainly synthesized from Ceramide in the Golgi apparatus by a stepwise process in which unique glycosyltransferases add monosaccharides to a growing lipid-linked oligosaccharide chain. They are subsequently exported towards the plasma membrane, their principal location, where they are enriched together with cholesterol in membrane microdomains. The expression of a particular GSL is differentially regulated according to the developmental stage, the cell type and its differentiation state. The role of vertebrate GSLs has mostly been addressed in vitro. These studies indicate that cell-surface GSLs participate in adhesion through the binding in trans of lectins or other GSLs. GSLs can also bind in cis to directly modulate the activity of receptor tyrosine kinases (RTKs) at the plasma membrane. GSLs are also thought to be involved in vesicular transport along the exocytic and endocytic pathways, sorting proteins into different compartments. Lastly, the presence of GSLs in membrane microdomains, which are considered as signaling platforms, may underlie many of their functions (Pizette, 2009).
There is, however, little in vivo evidence to support any of these presumed functions. In S. cerevisiae, mutants abolishing all GSL synthesis fail to show defects in intracellular trafficking. In C. elegans, GSLs appear to be dispensable throughout life. In mammals, the vast majority of GSLs are built on glucosylceramide (GlcCer), and synthesis branches at the level of the third glycosyl residue to yield three classes (lacto-, globo- and ganglioseries). Knockout of the mouse GlcCer synthase gene (Ugcg) leads to early embryonic lethality, for unclear reasons; assessing the effects of knocking out downstream glycosyltransferases is complicated by redundancy in these genes and between different GSLs. Nonetheless, disrupting the ganglioseries pathway produces mice that display neurological abnormalities after birth. Interestingly, in humans, mutations affecting GSL synthesis and degradation trigger severe neuropathologies. Therefore, GSLs are at least required for proper function of the adult nervous system, but no firm link has yet been established between this requirement and their proposed cellular roles (Pizette, 2009).
Drosophila GSLs are simpler in structure than their vertebrate counterparts, with a single biosynthetic pathway described to date, giving rise to a family of differentially elongated molecules. Egghead and Brainiac are glycosyltransferases responsible for GSL biosynthesis in the fly, catalyzing the addition of the second and third glycosyl residues of the GSL oligosaccharide chain ). There is no redundancy in these enzyme functions and no alternate biosynthetic pathway. Hence, egh and brn mutants are devoid of elongated GSLs and provide a useful model system for studies of GSL functions in vivo. Importantly, mutations in each gene are lethal and cause identical phenotypes during oogenesis and embryogenesis that are reminiscent of loss-of-function in the Notch receptor and EGF RTK (EGFR) pathways. Since the expression of a GSL-dedicated human galactosyltransferase in Drosophila egh mutants rescues their viability and fertility in a brn-dependent fashion, these data indicate that Drosophila GSLs are essential for development, perhaps by modulating signaling (Pizette, 2009).
During Drosophila oogenesis, activation of the EGFR pathway primarily depends on Gurken (Grk), an EGFR ligand similar to vertebrate TGFα, that is secreted by the oocyte. The EGFR-Grk couple acts twice to polarize the follicular epithelium as well as the future embryo along both anteroposterior (AP) and dorsoventral (DV) axes. Despite ubiquitous expression of EGFR in follicle cells, its activation is spatially restricted by asymmetric Grk localization. In early oogenesis, grk mRNA and protein are enriched at the posterior pole of the oocyte, and Grk activates EGFR in neighboring follicle cells, inducing them to adopt a posterior fate. At mid-oogenesis, these cells signal back to the oocyte, resulting in a reorganization of its cytoskeleton, a redistribution of oocyte maternal determinants along the AP axis, and the movement of the nucleus towards the anterior oocyte cortex. Since grk RNA remains associated with the oocyte nucleus, a new restricted source of Grk is created to limit the highest activation of EGFR to the adjacent follicle cells, instructing them to assume a dorsal identity. Respiratory appendages are eggshell structures derived from dorsolateral follicular cells and their examination is an excellent means to monitor EGFR signaling. Indeed, mild Grk or EGFR loss-of-function causes a fusion of the respiratory appendages owing to the absence of the dorsal-most cells (weak ventralization). By contrast, a more severe reduction in EGFR signaling abrogates the formation of these structures (complete ventralization) (Pizette, 2009 and references therein).
This study addresses the role of GSLs in EGFR signaling during oogenesis using egh and brn mutants. First, it was shown that GSLs exert a temporal control on the level of activation of EGFR. No evidence was found of a role for GSLs in the direct modulation of EGFR activity, but instead GSLs act at the level of the EGFR ligand Grk. Despite reports of GSL function in trafficking, the results indicate that GSLs are dispensable for Grk export to the plasma membrane and for its secretion. However, by observing the gradient of secreted Grk, this study shows that GSLs control Grk diffusion in the extracellular space (Pizette, 2009).
Once Grk is secreted, GSLs are necessary for its efficient diffusion in the extracellular space. The possibility cannot be dismissed that GSLs might have a
slight influence on Grk secretion, a decrease in Grk secretion cannot explain
the observed changes in the extracellular Grk gradient shape in the
brn mutant or the higher levels of secreted Grk that accumulate above
the source, as compared with the wild type. GSLs are thus crucial for the
formation of a Grk gradient that is able to achieve maximal activation of
EGFR (Pizette, 2009).
Surprisingly, GSLs were found to be involved only in the final step of Grk
signaling during the establishment of the DV axis. Prior to DV patterning, Grk
activates EGFR to set the AP axis of the egg. It was
observed, however, that AP polarity is not compromised in egh and brn alleles. The analysis of the distribution of the intermediate product mactosylceramide (MacCer) during oogenesis also supports the idea that the GSL biosynthetic pathway is not active at the time AP patterning is established. Grk signaling therefore seems to be more sensitive to GSL function for the determination of DV fates (Pizette, 2009).
Even during this process, there appear to be differential requirements for
GSL activity. DV patterning proceeds in two distinct temporal phases of EGFR
signaling, but only the second is under the control of GSLs. In a first phase
(between stages 8 and 10a), the EGFR pathway establishes embryonic DV polarity
and dorsal follicle cell fates. This phase culminates in the induction of
rho1 transcription in DA follicle cells and depends on paracrine
signaling mediated by Grk. This initial phase is not overtly affected in
brn mutants as evidenced by the observation that their embryos have a normal DV axis and it was found that rho1 expression was still induced. By contrast, the second phase of EGFR signaling is triggered by rho1 expression and
corresponds to an amplification of EGFR activity needed to split the RA.
This phase is disrupted in the brn mutant because the expression of
rho1 and aos is not upregulated, as exemplified by the
fusion of the RA (Pizette, 2009).
According to Wasserman and Freeman, the amplification phase is independent
of Grk and relies on autocrine EGFR signaling. However, this study shows that GSLs, unlike the other molecules implicated in this process, act in the germ line to regulate the distribution of extracellular Grk. This indicates that this phase is not solely autocrine and that there is still a need for Grk-mediated paracrine signaling (Pizette, 2009).
At stage 10a, the vitelline membrane is already being deposited as
vitelline bodies in the extracellular space between the oocyte and the
follicular epithelium. Morphological data show that these bodies have not yet
fused, leaving space for a number of interdigitating microvilli emanating from
the oocyte membrane and the apical side of the follicle cells. Since the
brn mutation specifically affects Grk signaling at this stage, it is
proposed that GSLs play a role in Grk accessibility to follicle cells and that
this is likely to be mediated through the microvilli. In support of this, at
stages at which elongated GSLs are not essential (AP patterning and the onset
of DV patterning), the oocyte plasma membrane is closely apposed to the apical
side of the follicle cells. This, therefore, supports the hypothesis that GSLs
are only required when Grk is not easily accessible to its receptor (Pizette, 2009).
This study provides the first experimental description of the wild-type
extracellular Grk gradient at stage 10a and uncovers an unexpected feature: at the dorsal
midline, past the source, high and steady levels of Grk are maintained over
about half of the AP axis length. This result is at odds with a mathematical
modeling of the Grk gradient that predicted a shallow decrease from anterior
to posterior. However, from a strong and constant level of Grk over half
the dorsal midline, it is expected that the expression domains of the Grk primary target
genes have an identical width along most of the AP axis. This is precisely
what is observed for kekkon-1 and pipe, which are clearly
EGFR primary target genes. It is thus suggested that a stripe-shaped source of
extracellular Grk along the dorsal midline, rather than a point-like source of
Grk above the oocyte nucleus, is more efficient in accommodating patterning
across the entire epithelium (Pizette, 2009).
Besides contributing to the shape of the Grk gradient, high Grk levels
along the dorsal midline might serve to upregulate rho1 expression,
leading to higher EGFR activity, aos expression and splitting of the
RA primordium. Indeed, in the absence of elongated GSLs,
weak rho1 expression is retained but it is not upregulated or
refined. Grk signaling is necessary for this step. Since,
in the brn mutant, there is a reduction in the high levels of
extracellular Grk along the dorsal midline, it is proposed that a low Grk
threshold is sufficient to initiate and maintain rho1 transcription
(as well as the spatial regulation of EGFR), whereas a
higher Grk threshold increases rho1 expression levels (Pizette, 2009).
What could be the basis for the discrepancy between these results and the
mathematical modeling of the Grk gradient? In the latter, EGFR expression was assumed to be uniform throughout the follicular epithelium. However, this study showed that at stage 10a, EGFR levels are lower along part of the dorsal midline in a
region coincident with that of high Grk levels. Furthermore, it was found that
decreasing Grk binding to EGFR increased Grk spreading. Therefore,
at the dorsal midline, the reduction in EGFR levels might saturate receptor
occupancy. This could allow a large quantity of Grk to remain unbound,
facilitating its movement toward the posterior pole (Pizette, 2009).
The most striking result of this study is that GSLs shape the extracellular Grk gradient and play a role in Grk diffusion without apparently interfering with the
regulation of Grk diffusion by EGFR. But
what could that role be? Grk movement in the extracellular space between the
oocyte and the follicular epithelium is complicated by the formation of the
vitelline membrane. Grk could either be released into the
extracellular space or it could remain associated with the oocyte plasma
membrane and localize to its microvilli. These alternatives could not be distinguished, since immunofluorescent staining is of insufficient
resolution and the extracellular space was not well preserved in the
immunoelectron microscopy experiments. Others have nevertheless reported the
presence of Grk on microvilli. GSLs could therefore be important for Grk targeting to microvilli versus flat portions of the membrane. This, however, is unlikely
because Grk still activates EGFR in the brn mutant, indicating that it can
encounter its receptor. By contrast, what Grk fails to do in the mutant
context is to concentrate along the dorsal midline at a distance from its
point of secretion. This suggests that GSLs function in the planar transport
of Grk along the AP axis, from one oocyte microvillus to the next, a
hypothesis supported by the fact that the oocyte microvilli were found to be
oriented parallel to the AP axis (Pizette, 2009).
An intriguing property of secreted Grk in the brn mutant context
is that it is detected by extracellular staining and not conventional
immunostaining. In an effort to understand the basis for this, it was found that
secreted Grk is sensitive to the presence of detergent and to temperature,
suggesting that its conformation relies on the presence of GSLs once it
reaches the cell surface. Interestingly, GSLs induce a conformational change in the amyloid β-protein upon its release from the plasma membrane. It is thus
possible that under the experimental conditions, Grk conformation is not fully
restored, modifying its ability to diffuse (Pizette, 2009).
In this case, how could the two processes be linked? There is increasing
evidence that the spreading of secreted molecules depends on elaborate events
involving their multimerization and/or incorporation into higher-order
structures such as lipoprotein particles. It is therefore tempting to speculate that a change in secreted Grk conformation that depends on plasma membrane GSLs reflects its packaging into special structures that are required for its efficient transport along microvilli. Since mammalian GSLs can be shed from the plasma membrane and are found
circulating with secreted lipoprotein particles,
GSLs could enhance Grk spreading by delivering it to these particles (Pizette, 2009).
Another, non-mutually exclusive means by which GSLs could affect Grk
diffusion is linked to their enrichment in plasma membrane microdomains. Since
GSLs can interact with proteins through their oligosaccharide chain, GSLs
could bind Grk, or a Grk co-factor, sorting Grk into such domains. It has been
reported that Grk is potentially palmitoylated, and
palmitoylation is one of the signals that target proteins to membrane
microdomains. Because Grk recruitment to these domains has not been
addressed and because the detection of extracellular Grk by biochemical means
in ovaries has so been so far elusive, understanding how GSLs regulate Grk diffusion will have to await the generation of better tools (Pizette, 2009).
Vesicle trafficking plays a crucial role in the establishment of cell polarity in various cellular contexts, including axis-pattern formation in the developing egg chamber of Drosophila. The EGFR ligand, Gurken (Grk), is first localized at the posterior of young oocytes for anterior-posterior axis formation and later in the dorsal anterior region for induction of the dorsal-ventral (DV) axis, but regulation of Grk localization by membrane trafficking in the oocyte remains poorly understood. This study reports that Syntaxin 1A (Syx1A) is required for efficient trafficking of Grk protein for DV patterning. Syx1A is associated with the Golgi membrane and is required for the transportation of Grk-containing vesicles along the microtubules to their dorsal anterior destination in the oocyte. These studies reveal that the Syx1A dependent trafficking of Grk protein is required for efficient EGFR signaling during DV patterning (Tian, 2013).
The localization of grk mRNA and its protein product in the oocyte is crucial for the establishment of both the AP and dorsal-ventral axes. grk mRNA and protein are
localized at the posterior of the oocyte during early oogenesis to activate EGFR signaling
in the posterior follicle cells, which in turn send a mysterious signal back to initiate AP
axis formation in the oocyte. On the basis of this new AP axis, grk mRNA and protein
are subsequently localized at the anterior-dorsal corner of the oocyte to induce dorsalventral pattern formation. The localization of grk transcripts depends on the
microtubules in the oocyte. These transcripts are transported to the dorsal-anterior
destination along the microtubules by Dynein in nonmembranous transport particles and
are statically anchored by Dynein within cytoplasmic structures called sponge bodies. Grk protein is believed to be translated from its localized mRNA
and is translocated into endoplasmic reticulum; afterward it travels through the Golgi
complex and reaches the plasma membrane to be secreted. This study reports the demonstration that localization of Grk protein to
the dorsal-anterior region of the oocyte depends on membrane trafficking, differing from
the nonmembranous particle transport of its mRNA during this stage. This finding is based on the study of a newly identified hypomorphic allele of
Syx1A whose germ-line clones have defective dorsal follicle-cell specification and
abnormal Grk protein localization after stage 7. Interestingly, grk mRNA localization is normal until stage 9 in these germ-line clones. The mislocalization of Grk protein is therefore not a result of its mRNA mislocalization, at least between stages 7 and 9, indicating that the role of Syx1A in Grk protein trafficking is specific (Tian, 2013).
Using kek-lacZ as a reporter, it was found that Syx1ASH0113 germ-line clones strongly
disrupted EGFR signaling in the dorsal anterior follicle cells-many egg chambers
showed complete loss of dorsal expression of Kek-lacZ-but the majority of mature
eggs developed from these clones showed only shortened dorsal appendages, a phenotype
indicating disruption of dorsal EGFR signaling but less severe than that of egg chambers
with no expression of kek-lacZ in follicle cells adjacent to the oocyte nucleus. This
phenotypic discrepancy probably arises because only a small fraction of syx1A germ-line clones develop into mature eggs, and those eggs represent the least marked phenotype.
Indeed, many germ-line clones were observed with oocytes smaller than those of wild-type
egg chambers at the same developmental stage, perhaps indicating a general role of
Syx1A in membrane growth that is essential for oocyte development (Tian, 2013).
Although no defect was detected in Syx1A clones in Grk posterior localization and
signaling to activate EGFR in the posterior follicle cells, it cannot be ruled out that that Syx1A has no role in the posterior localization of Grk protein. In the
germ-line clone of a null allele of syx1A, syx1Aδ229, in contrast, oogenesis is arrested at around stages 5-6, preventing examination of the effect of
Grk-EGFR signaling in the posterior follicle cells. The new syx1A mutant allele
is most likely a hypomorphic allele, sequencing analysis of the open reading frame (ORF)
region of the syx1A gene in homozygous syx1Aδ229 mutants did not find any changes in the ORF region, suggesting the mutation is probably at the regulatory region. Nonetheless, the findings suggest that localization of Grk to the dorsal anterior region depends more heavily on Syx1A-dependent membrane trafficking.
Rab6 is known in mammals to promote trafficking at the level of the Golgi
apparatus and is colocalized with the Golgi and trans-Golgi markers. Previously, a Rab6-mediated exocytic pathway has been shown to be involved in Grk trafficking in germ-line cells during oogenesis. The current studies suggest that Rab6 has a role similar to that of
Syx1A for Grk localization at the dorsal-anterior corner of the oocyte after stage 7. Also,
Rab6 appears not to be needed for Grk localization at the posterior before stage 7, a
pattern suggesting the functional correlation between Rab6 and Syx1A in Grk trafficking
in the oocyte. Consistently, Syx1A and Rab6 can form puncta in the nurse cells and
oocytes and are colocalized in some of these puncta, and colocalization of these two
proteins with Golgi marker GalT was observed. Furthermore, the mislocalization of
Syx1A and Grk in rab6 germ-line clones or of Rab6 and Grk in syx1A germ-line clones indicates that Syx1A and Rab6 can act together for Grk trafficking. Attempts were made to perform a coimmunoprecipitation study to determine whether these two proteins are
physically associated in the oocyte, but no obvious physical interaction between them
was detected, suggesting that they may not interact directly in the Grk
trafficking (Tian, 2013).
This study demonstrated the important role vesicle tracking plays in a specific
localization of Grk in the oocyte. This process requires the both Syx1A and Rab6, which
traffic along the microtubules in the oocyte. About 11 Syntaxin proteins occur in
Drosophila, and they are involved in many different cellular
processes. For example, Drosophila Syx5 is required for Golgi reassembly after cell
division and for translocation of proteins to the apical membrane, and
Syx 16 is ubiquitously expressed, appears to be localized to the Golgi apparatus, and may
selectively regulate Golgi dynamics. Syx 1A is a critical component of
the SNARE complex and is essential for synaptic vesicle fusion. The finding that the mutant for Syx1A can cause such a strong phenotype in
the Grk trafficking is exciting. Future studies will determine whether other Syntaxin
molecules have similar roles in the oocyte (Tian, 2013).
Home page: The Interactive Fly © 1997 Thomas B. Brody, Ph.D.
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| Effects of Mutation
| References
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