EGFR signaling modulates synaptic connectivity via Gurken

Synaptic target selection is critical for establishing functional neuronal circuits. The mechanisms regulating target selection remain incompletely understood. This study describes a role for the EGF receptor and its ligand Gurken in target selection of octopaminergic Type II neurons in the Drosophila neuromuscular system. Mutants in happyhour, a regulator of EGFR signaling, form ectopic Type II neuromuscular junctions. These ectopic innervations are due to inappropriate target selection. It was demonstrated that EGFR signaling is necessary and sufficient to inhibit synaptic target selection by these octopaminergic Type II neurons, and that the EGFR ligand Gurken is the post-synaptic, muscle-derived repulsive cue. These results identify a new pathway mediating cell-type and branch-specific synaptic repulsion, a novel role for EGFR signaling in synaptic target selection, and an unexpected role for Gurken as a muscle-secreted repulsive ligand (Naylor, 2011).

Synaptic target selection is a critical step in establishing functional neural circuits. The molecular mechanisms governing this selection have not yet been fully explored. The observation that hppy mutants have an increased frequency of ectopic octopaminergic Type II NMJs has resulted in identification of a pathway critical for synaptic target recognition in these neurons. EGFR signaling pathway was found to be required to prevent the development of inappropriate synaptic contacts. This inhibitory signal is mediated by muscle-derived EGFR ligand, Gurken, working through the EGF receptor in type II motoneurons. This mechanism sculpts the neuronal wiring pattern in a cell-type and branch-specific manner (Naylor, 2011).

There are many signaling pathways that influence the innervation pattern of Drosophila motor neurons. These findings identify a novel role for EGFR signaling in mediating a repulsive guidance cue to Type II neurons. EGF has previously been demonstrated to regulate axon growth and guidance. For example, EGF positively regulates Sema-3a levels in the cornea and interacts with NCAM-180 to promote neuritogenic activity. However, these data are the first to demonstrate that an EGF receptor and ligand provide a synaptic targeting signal (Naylor, 2011).

Ectopic Type II NMJs were found in hppy mutants, as well as when a dominant negative EGFR was expressed in Type II neurons or its ligand Gurken was knocked down in the muscle targets. This presents an apparent contradiction. hppy is described as a negative regulator of EGFR (Corl, 2009), and its phenotype in this system is suppressed by a hypomorphic mutation in rolled (ERK), consistent with hppy functioning as a negative regulator of EGFR signaling. Why then does loss of hppy have the same phenotype as inhibition of the receptor or ligand? It was posited that the well-described strong negative feedback induced by EGFR signaling may be the explanation. A model is proposed in which activation of the EGFR pathway mediates a signal that inhibits the formation or stabilization of Type II NMJs. In hppy mutants, however, loss of negative regulation would allow for excess activation of the EGFR that would induce a quick, strong and long-lasting negative feedback activity early in development, essentially turning off EGFR signaling in cells expressing happyhour. The result would be loss of the synaptic inhibitory signal mediated by EGFR at the stage when these Type II neurons are extending to their targets and the promotion of ectopic synapse formation. It is appreciated that this model is speculative, and that an alternative is that hppy and rolled are regulating a pathway that is distinct from the EGFR/gurken pathway (Naylor, 2011).

By what mechanism does EGFR signaling affect synapse formation in Type II neurons? Presumably, there is a molecular program downstream of EGFR activation that modifies the Type II neuron such that it does not form and/or maintain an NMJ with an inappropriate target. These changes could occur at the level of the cell body or locally within individual branches. It is unlikely that cell body changes are central to the phenotype because such neuron-wide mechanisms could not easily be translated into branch-specific behavior. In contrast, local effects of EGFR signaling within neurites could explain such specificity. The cell biological mechanism mediating the branch-specific inhibition is not known. Possibilities include alterations in the local translation or membrane insertion of synaptogenic molecules, local modulation of cytoskeletal dynamics, or failure to properly prune Type II connections (Naylor, 2011).

Not only is the EGFR mediating a branch-specific effect, but it is also cell-type specific. The Type II motoneuron MNSNb/d-II and the Type Is motoneuron MNSNb/d-Is travel together and presumably encounter the same cues across the hemisegment, however they generate different innervation patterns. This implies that these two types of neurons have developed cell-type specific repertoires of receptors or signaling pathways that shape their target choices (Naylor, 2011).

While the data indicate a role for EGFR signaling in Type II synaptic target selection it is also likely that Type II target selection has multiple components. The phenotypes occur at a relatively low penetrance, so it is likely that complementary and combinatorial guidance cues function with the EGFR pathway to shape target selection of Type II neurons (Naylor, 2011).

Gurken has been studied exclusively in the developing oocyte and has no known roles in other tissues. Hence, it is surprising that Gurken conveys the repulsive signal from muscle to the octopaminergic Type II neurons. In support of a function in muscle, the modEncode RNA-seq project has found that Gurken transcript is enriched 3.5 fold in larval body wall muscles. While Gurken may be secreted from all muscles, a model is preferred in which localized expression in a muscle subset shapes the branching pattern of the innervating motoneuron. In this model, Gurken released from muscles 6 and 7, as well as other targets that should not be innervated, would locally inhibit synaptogenesis, blocking the formation of ectopic connections while allowing for the normal innervation at muscles 12 and 13. This model is consistent with findings that knockdown of Gurken in muscle results in ectopic NMJs while localized overexpression in the normal target cell inhibits formation of appropriate NMJs. While these functional data are strong, the model must remain speculative because it has not been possible to determine the localization of Gurken using currently available reagents. Future studies will investigate how this Gurken/EGFR pathway is integrated with the recently defined semaphorin- and activity-dependent mechanisms that also play an important role in shaping synaptic target selection in these neurons (Naylor, 2011).

Evolutionary changes in TGFalpha distribution underlie morphological diversity in eggshells from Drosophila species

Drosophila eggshells display remarkable morphological diversity among species; however, the molecular origin of this structural diversification is mostly unknown. This study analyzed the dorsal ridge (DR), a lumen-like structure along the dorsal side of eggshells, from numerous Drosophila species. This structure varies in length and width across species, and is absent from D. melanogaster eggshells. DR formation with distinct spatiotemporal changes in epidermal growth factor receptor (EGFR) activation, which acts as a key receptor in eggshell patterning. Changes in the distribution of the TGFalpha-like ligand Gurken (GRK), a crucial ligand for axis formation, underlies EGFR activation and DR formation in D. willistoni. Furthermore, this study demonstrates that GRK from D. willistoni rescues a grk-null D. melanogaster fly and, remarkably, it is also sufficient to generate a DR-like structure on its eggshell (Niepielko, 2014).


In early stages the GRK transcript is found in a crescent-shaped spread lining the posterior end of the oocyte. By stage 8, when the oocyte nucleus moves to its central position at the future dorsal-anterior region of the oocyte, the GRK transcript has a more diffuse pattern and also accumulates along the anterior margin of the oocyte. During stage 9, when the oocyte nucleus has reached its cortical anterior position, GRK transcript shows a perinuclear accumulation lining the cortex of the oocyte surrounding the dorsal and anterior side of the ooctye nucleus (Neuman-Silberberg, 1994).

In mid-oogenesis the gurken transcript becomes spatially localized to the future dorsal-anterior cortex of the oocyte. To analyze the distribution pattern of Gurken protein, antibodies were prepared against Gurken. The distribution pattern of the Gurken protein is decribed in wild-type ovaries and in ovaries from a number of dorsal-ventral patterning mutants. The protein is exclusively found at the future dorsal cortex where it colocalizes with the memjbrane associated F-actin. The protein is more broadly localized than is the mRNA. In fs(1)k10, squid, and cappuccino mutants, Gurken protein is mislocalized and resides at the anterior margin of the oocyte, around the entire circumference of the egg chamber. One major form of the Gurken protein is detected, which likely corresponds to the unprocessed protein (Neuman-Silberberg, 1996).

During Drosophila oogenesis, Gurken, a protein associated with the oocyte nucleus, activates the Drosophila EGF receptor in the follicular epithelium. Gurken first specifies posterior follicle cells, which in turn signal back to the oocyte to induce the migration of the oocyte nucleus from a posterior to an anterior-dorsal position. From this location Gurken signals again to specify dorsal follicle cells, which give rise to dorsal chorion structures, including the dorsal appendages. If Gurken signaling is delayed and starts after stage 6 of oogenesis, the nucleus remains at the posterior pole of the oocyte. Eggs develop with a posterior ring of dorsal appendage material that is produced by main-body follicle cells expressing the gene Broad-Complex. They encircle terminal follicle cells expressing variable amounts of the TGFbeta homolog, decapentaplegic. By ectopically expressing decapentaplegic and using clonal analysis with Mothers against dpp, it has been shown that Decapentaplegic signaling is required for Broad-Complex expression. Thus, the specification and positioning of dorsal appendages along the anterior-posterior axis depends on the intersection of both Gurken and Decapentaplegic signaling. This intersection also induces rhomboid expression and thereby initiates the positive feedback loop of EGF receptor activation, which positions the dorsal appendages along the dorsal-ventral egg axis (Peri, 2000).

Nuclear movement is essentially a self-induced event. Grk localized with the nucleus activates Egfr in posterior-terminal cells. Consequently, these cells acquire the ability to signal back to the oocyte, reorganize the oocyte cytoskeleton and thus initiate nuclear movement to the dorsal-anterior pole where Grk signals again. The timing of posterior fate induction and back signaling is still elusive since the molecular nature of these processes has not been analysed. Grk signaling as late as midstage 6 leads to normal anterior llocaliztion of the nucleus. This means that posterior-fate induction followed by back signaling can occur close to the time when nuclear movement normally takes place. Starting from midstage 6, Grk signaling becomes insufficient to promote nuclear movement, although up to midstage 7 an influence on nuclear movement can be detected. This transition is probably caused by changes both in the follicular epithelium and in the oocyte. During this period the posterior terminal follicle cells appear to lose their competence to respond to Grk signaling, since they start to show mixed anterior/posterior terminal fates, which might result in a lack of back signaling. cni codes for a small hydrophobic protein that is required for normal Grk signaling by controlling the transport of Grk protein to the plasma membrane of the oocyte. Egg chambers with partial posteriorization, mutant for hypomorphic grk or cni alleles show normal nuclear behaviour; this indicates that back signaling has caused correct cytoskeletal rearrangements. It is therefore concluded that in the case of late Grk signaling the response of the oocyte to back signaling is also impaired so that cytoskeletal rearrangements necessary for nuclear movement are incomplete (Peri, 2000).

If Grk activation occurs too late to induce nuclear movement, or if the movement is incomplete so that the nucleus resides in an intermediate position, Grk remains localized with the nucleus and Grk signals from there to the nearby follicular epithelium. The resulting patterns of kekkon, Broad-Complex and pipe expression are dictated by the nuclear position and suggest that no intrinsic DV polarity in egg chambers exists. This is especially obvious when completely symmetric rings of dorsal appendage (DA) material are ectopically induced from a posteriorly localized nucleus. In this situation, a set of marker genes corresponding to the entire DV axis of the follicular epithelium is expressed along the AP axis, indicating that there is no principal bias to the way these genes are activated or repressed in main-body follicle cells. Similar conclusions have been drawn from the study of mago nashi mutants, although in this case only the expression of the primary Egfr target kek, which is not a dedicated DV patterning gene, has been analysed. Together these observations demonstrate that the movement of the nucleus is the sole determinant of the orthogonal orientation of the body axes and that it stochastically determines the position of the dorsal side of the egg (Peri, 2000).

In these experiments the spatial and temporal expression pattern of the endogenous Grk signal was manipulated to probe the competence of the follicular epithelium. If activation of Grk signaling occurs after midstage 7, the oocyte nucleus remains in the posterior: Grk then signals to a region that includes both the terminal follicle cells and an abutting ring of main-body follicle cells. Simultaneous Grk signaling to both cell populations clearly shows their different developmental responses: the terminal cells form either anterior or posterior structures, while the encircling ring of cells forms dorsal cell types characterized at the molecular level by repression of pipe and by the activation of BR-C. This definitely excludes the possibility that posterior and dorsal cell-fate specification is controlled by timing so that late Grk signaling always has a dorsalising effect, irrespective of the cell group receiving the signal. The observation confirms earlier studies, which showed that terminal and main body follicle cells have different default states in the absence of signaling and respond differently upon ectopic activation of Ras or Egfr. It also demonstrates that the different responses of terminal and main-body follicle cells to Grk signaling are not strictly separated in time, such that the former have entirely lost their ability to react when the latter are competent. Posteriorization is seen occurring simultaneously with dorsal fate induction (Peri, 2000).

However, whenever dorsal fates (i.e. dorsal appendages) are found together with posterior chorion structures, a closer inspection of these egg chambers reveals that the posteriorization is incomplete and dpp, normally only found anteriorly, is still present at the posterior pole. The possibility of mixed populations consisting of anterior and posterior follicle cells, a situation also caused by certain hypomorphic cni and grk alleles, is probably linked to the fact that the terminal cells are not a homogenous cell group. They seem to be divided into three subgroups by Grk-independent patterning mechanisms. These subgroups might have different sensitivities toward Grk signaling (Peri, 2000).

After nuclear movement, Grk signaling has at least two effects on follicle cell patterning that are essential for later embryonic development and egg morphology. (1) It leads to pipe repression and thereby defines the region of the egg from which the embryonic DV axis emerges; (2) it induces the formation of such anterior-dorsal chorion specialization, like the DAs. While the former action of Grk occurs along the entire AP axis, the latter is confined to approximately the anterior third of the mainbody follicle cells. How is this difference in range of Grk action achieved? According to a first model, timing might play a crucial role. pipe repression starts earlier than BR-C activation; in the intervening time the egg chamber grows and the follicle cells continue to migrate over the oocyte nucleus. Thus, different egg chamber geometries at the time of pattern induction might explain the differences in range of the signal. According to another model, an anterior-posterior prepattern is established within the follicular epithelium, which allows Grk’s induction of dorsal appendage fates only in anterior main-body follicle cells. This model was first proposed in a study in which an activated version of Egfr was expressed uniformly in all main-body follicle cells. Despite uniform activation of primary Egfr targets like kek, other target genes were only activated in proximity to anterior terminal follicle cells. This leads to the suggestion of a signal emanating from anterior terminal follicle cells, which modulates the response towards Egfr activation in the main-body follicle cells. The results presented here clearly favor the second model and identify Dpp as the actual signaling molecule that prepatterns the main body follicle cells. Residual dpp expression in posterior terminal cells explains the difference between the hs-cni and mago phenotypes, and most importantly, ectopic posterior dpp expression in egg chambers with posterior Grk signaling is sufficient to induce DA formation. Currently, the relative contribution of Dpp signaling to the specification of the DAs and operculum, respectively cannot be assessed. Previous observations suggest that high levels of Dpp repress dorsal appendage and promote operculum formation. Analysis of follicle cell clones lacking Mad function, however, demonstrates that Dpp signaling is required for BR-C expression and suggests that lower levels of Dpp, insufficient for operculum formation, are likely to specify DAs (Peri, 2000).

The finding that rho expression in the follicular epithelium cannot be induced by Grk alone, but also requires Dpp, shows that both cell-fate specification and cell-fate patterning are controlled by the intersection of the two pathways. Loss-of-function clones have been used to demonstrate that rho and spi are not required for dorsal appendage formation per se but that they are necessary to separate the two appendages and to position them dorsolaterally. Since this patterning mechanism involves the self-amplification of Egfr activation and includes the diffusible ligand Spi, the process must be under tight spatial control to prevent runaway activation in the follicular epithelium. While the localization of Grk limits the process along the DV axis, it is proposed that a Dpp gradient emanating from anterior-terminal cells prevents spreading of the Grk signal along the AP axis of the main-body follicle cells (Peri, 2000).

Graded maternal Short gastrulation protein contributes to embryonic dorsal-ventral patterning by delayed induction: Expression of tld and tok is ventrally restricted by the TGF-α ligand Gurken

Establishment of the dorsal–ventral (DV) axis of the Drosophila embryo depends on ventral activation of the maternal Toll pathway, which creates a gradient of the NFkappaB/c-rel-related transcription factor Dorsal. Signaling through the maternal BMP pathway also alters the dorsal gradient, probably by regulating degradation of the IkB homologue Cactus. The BMP4 homologue decapentaplegic (dpp) and the BMP antagonist short gastrulation (sog) are expressed by follicle cells during mid-oogenesis, but it is unknown how they affect embryonic patterning following fertilization. This study provides evidence that maternal Sog and Dpp proteins are secreted into the perivitelline space where they remain until early embryogenesis to modulate Cactus degradation, enabling their dual function in patterning the eggshell and embryo. Metalloproteases encoded by tolloid (tld) and tolkin (tok), which cleave Sog, are expressed by follicle cells and are required to generate DV asymmetry in the Dpp signal. Expression of tld and tok is ventrally restricted by the TGF-α ligand encoded by gurken, suggesting that signaling via the EGF receptor pathway may regulate embryonic patterning through two independent mechanisms: by restricting the expression of pipe and thereby activation of Toll signaling and by spatially regulating BMP activity (Carneiro, 2006).

This study has shown that sog, dpp, and tld act during oogenesis to promote the formation of dorsal anterior structures of the eggshell and to establish the embryonic DV axis. According to a proposed model, Sog is produced in follicle cells and is processed into different forms depending on DV location and stored in the perivitelline space. These forms of Sog then persist until early stages of embryogenesis at which time they act by a delayed induction mechanism to alter signaling mediated by maternally derived Dpp. It is proposed that an asymmetric distribution of Sog peptides is produced through the action of the ventrally localized Tld and Tok metalloproteases. Different forms of Sog act locally to inhibit Dpp signaling ventrally (e.g., N-Sog) or diffuse over considerable distances to concentrate Dpp dorsally (e.g., full-length Sog or C-Sog). According to this model, a dorsal-to-ventral gradient of Dpp activity is formed in the perivitelline space that counteracts and sharpens the inverse gradient of nuclear dorsal (Carneiro, 2006).

An important finding in this study is that Sog protein produced by follicle cells is secreted into the perivitelline space where it persists until the end of oogenesis and early embryogenesis, prior to initiation of zygotic sog expression. One way maternal Sog fragments might influence DV patterning in the embryo is to modify zygotic Dpp signaling. However, maternal Dpp signaling is involved in establishing the relative positions of the ventral mesoderm versus the lateral neuroectodermal territories, while zygotic Dpp activity determines the relative positions of dorsal and lateral domains. These distinct phenotypes suggest that maternal Sog acts by modulating the maternal rather than the zygotic component of Dpp signaling (Carneiro, 2006).

This analysis also suggests that the Dpp synthesized by follicle cells is secreted into the perivitelline space and stored there until advanced stages of oogenesis. These maternally synthesized Sog and Dpp proteins may act on the embryo following fertilization when signaling through the Toll pathway is initiated. Several lines of evidence support this hypothesis. (1) Through epistatic analysis, it was shown that maternal Dpp does not act upstream of the Toll receptor. Therefore, genes expressed in the follicle cell epithelium that regulate DV patterning exclusively via the Toll pathway should not be targets of maternal Dpp signaling. Alternatively, undescribed non-Toll mediators of DV patterning could potentially be targets of maternal Dpp in the follicular epithelium. (2) Blocking Tkv receptor function or reducing maternal Dpp activity (by 8xhssog, in follicle cells has no effect on the pattern of pip expression. It has been shown that maternal dpp does not alter grk expression. Thus, no evidence was found that the embryonic effects here described in this study are due to alterations in patterning of the follicular epithelium. (3) Maternal dpp signaling increases the levels of Cactus protein in the embryo by a mechanism that is independent of Toll. Finally, inhibition of Tkv with tkvDN expressed with an early maternal driver alters the embryonic expression domains of ventral and lateral genes such as vnd and snail, which are targets of dorsal activation but not of zygotic BMP signaling. tkvDN expression also alters expression of DV genes in lateralized embryos, which lack dorsal ectoderm and early zygotic dpp expression. In aggregate, these various data support the view that maternal dpp and sog act by delayed induction on the embryo itself. The possibility cannot be ruled out, however, that the embryonic DV phenotypes described in this study result from the combined effects of direct and indirect maternal dpp signaling with the predominant effect being direct (Carneiro, 2006).

Delayed inductive activities have been proposed for a variety of proteins synthesized during oogenesis. For example, activation of the terminal system relies on delayed inductive activity of the secreted product of the torsolike gene (tsl), which is expressed by follicle cells at the two poles of the oocyte and associates with the vitelline membrane. ndl has a dual action on chorion integrity and embryonic patterning. The embryonic patterning function of ndl is thought to be mediated by Nudel protein that is secreted into the perivitelline space where it associates with the embryonic plasma membrane and initiates a proteolytic cascade. It is proposed that Sog and Dpp secreted by follicle cells also serve two roles. First, they contribute to patterning the follicle cell epithelium and chorion, and secondly, they are transferred to and stored in the perivitelline space where it is proposed that they function after fertilization to modify Toll patterning in the embryo (Carneiro, 2006).

During embryogenesis, Sog protein diffuses dorsally from the neuroectoderm and may carry Dpp dorsally in a complex with Tld, Tsg, and Scw, resulting in the generation of peak Dpp activity in the dorsal midline. The spatial distribution of maternal Sog, Dpp, Tld and Tok during oogenesis could also create asymmetric BMP activity. Since tld and tok are expressed only in ventral follicle cells, a ventral-to-dorsal gradient of Sog fragments is likely to be produced. Because cleavage of Sog by Drosophila Tld and Tok is dependent on the amount of Dpp, cleavage of Sog by Tld and Tok should be increased near the source of Dpp, generating an oblique gradient of Sog fragments in the egg chamber. The existence of such a gradient is supported by the greater staining seen in anterior ventral cells with the anti-Sog 8A antiserum during stage 10B. However, greater asymmetry may exist as a result of differential distribution of an array of Sog fragments throughout the egg chamber. Unfortunately, visualization of such asymmetry would be hard to achieve due to limitations in the ability to recognize several fragments by existing Sog antisera (Carneiro, 2006).

The analysis of marked sog− and tld− follicle cell clones suggests that the mobility of Sog fragments in the extracellular compartment may contribute to creating a maternal Dpp activity gradient. Such clones resulted in different Sog staining patterns in the perivitelline space adjacent to the clones depending on where they were located along the DV axis. The staining pattern observed with the 8A antibody suggests that ventrally generated N-Sog cleavage products may be less diffusible than intact Sog or than C-Sog and remain restricted to their site of production. In contrast, full-length Sog and C-Sog fragments appear to diffuse more readily (Carneiro, 2006).

Diffusion of Dpp may also contribute to patterning the eggshell. The expression of dpp in anterior follicle cells is consistent with its role in the formation of dorsal anterior chorionic structures. An anterior-to-posterior gradient of Dpp activity in dorsal regions of the egg chamber is suggested by the Dpp-dependent activation of the A359 enhancer trap and graded repression of bunched along the AP axis. In addition, BR-C expression is lost in mad− clones away from the source of Dpp. sog is likely to contribute to establishing this BMP gradient since ventral sog−clones act non-cell-autonomously to decrease the size of the operculum. Since ventral tld− clones also alter the extent and angle of the operculum, Tld may process Sog to generate a fragment that diffuses and carries Dpp to a dorsal anterior location, concentrating and thus enhancing Dpp activity. Further evidence that a fragment with such activity exists derives from the observation that overexpression of a C-terminal Sog fragment generates chorionic phenotypes that strongly resemble dpp overexpression (Carneiro, 2006).

A dorsally produced form of Sog also appears to participate in patterning the eggshell since sog− clones located dorsally result in fusion of dorsal appendages along the dorsal midline. DV positioning of the dorsal appendages depends on several factors, most critically on EGFR signaling. In contrast, mild overexpression of dpp generates fusion of the dorsal appendages. Considering the well-established role of Sog in modulating Dpp activity, the fused appendage phenotype generated by dorsal sog− clones most likely reflects the loss of Dpp antagonism exerted by Sog (Carneiro, 2006).

In addition to the activities described above, N-Sog fragments which remain ventrally restricted could exert Supersog-like activity, antagonizing BMPs while acquiring resistance to further cleavage and degradation by Tld. This ventrally restricted activity most likely patterns the embryo but does not affect dorsal positioning of eggshell structures, which depends on the combined activity of Dpp/BMPR signaling and dorsally generated Grk/EGFR signals (Carneiro, 2006).

The assortment of Sog fragments in egg chambers is very similar to that in the embryo. Full-length and processed forms of Sog generated by Tld during oogenesis might remain asymmetrically distributed during embryogenesis and exert distinct activities. This hypothesis is in agreement with the effect of tld− and sog− follicle cell clones on the embryo. In the majority of cases, tld− follicle cell clones result in ventralized cuticles, indicating that Tld generates some activity that synergizes with Dpp. Reciprocally, the great majority of sog− follicle cell clones result in dorsalized cuticles and embryos, indicating that Sog primarily acts by antagonizing Dpp. Since only ventral sog− clones generate cuticle defects, ventrally produced Sog presumably generates a ventralizing activity that blocks Dpp locally. In contrast, since in a minority of cases ventral shifts are observed in embryonic gene expression domains resulting from sog− clones, as well as a minority of dorsalized cuticles from tld− clones, there may also be a form of Sog that can enhance Dpp signaling. This positive BMP promoting activity could be generated ventrally, as suggested above in the case of chorion patterning (Carneiro, 2006).

A model depicting the proposed effects of different Sog forms on formation of the chorion and embryonic patterning is presented. According to this model, ventrally restricted Tld cleaves Sog near the Dpp source in ventral anterior follicle cells generating N-Sog and C-Sog. It is suggested that N-Sog fragments remain restricted near ventral anterior cells to antagonize Dpp, while C-Sog fragments diffuse dorsally concentrating Dpp in dorsal anterior cells that direct formation of the operculum. This asymmetric production of Sog molecules would generate a dorsal-to-ventral gradient of Dpp, with the highest levels dorsally near the anterior Dpp source. Although direct visualization of the predicted resulting Dpp gradient in the embryo is hard to achieve with the tools available, it is proposed that such a similarly oriented gradient persists until early embryogenesis based on the asymmetric pattern of Dpp-GFP distribution during late oogenesis and the observed alterations in embryonic gene expression domains resulting from modifications in maternal Dpp signaling (Carneiro, 2006).

The slope of the Dl nuclear gradient ultimately defines the extent of the mesoderm (Mes), neuroectoderm (NE), and dorsal ectoderm (DE). A uniform increase or decrease in nuclear Dl along the DV axis can only alter the extent of the Mes and DE and positioning of the NE, while a change in the slope of the gradient will modify the extent of NE territories such as the vnd expression domain. Under all conditions that Dpp signaling was altered, modifications were observed in the width of the vnd domain. This suggests that graded maternal Dpp signaling helps determine the slope of the dorsal gradient. Earlier studies suggested that Dpp inhibits Cactus degradation and as a consequence decreases Dl translocation into the nucleus. Increased Dpp signaling should result in more Dl retained in the cytoplasm, with consequent narrowing of the mesoderm and ventral shift in lateral and dorsal expression domains. Conversely, inhibition of Dpp signaling would result in increased levels of Dl becoming available for nuclear translocation. Considering the proposal that maternal Dpp is highest dorsally, and that Cactus may also act to prevent Dl diffusion along the DV axis, decreasing Dpp should lower Cactus levels in dorsal–lateral regions of the embryo and result in the redistribution of free Dl from ventral to lateral regions. As a consequence of this redistribution of Dl, there would be a slight decrease in Dl levels ventrally and an increase laterally that would have the net effect of flattening the gradient. Such a mechanism would require a certain degree of mobility of dorsal dimers in the syncytial blastoderm. In future studies, it will be interesting to determine the relative mobilities of Dl/Cactus complexes in the cytoplasm (Carneiro, 2006).

Maternal BMP signaling may also increase the robustness of dorsal patterning. The prevailing view of DV patterning is that signaling through the Toll pathway is sufficient to generate threshold-dependent activation of several dorsal target genes along the entire DV axis. Activation of Toll triggered by the ON/OFF pip expression pattern must be transformed into a ventrally centered gradient of Toll signaling. Several mechanisms may contribute to generate this gradient, based on autoregulatory feedback mechanisms. Although the Toll system may be internally robust, regulatory inputs from other signaling pathways could also contribute further to its stability, such as suggested for the wntD pathway and for maternal Dpp. While a significant body of evidence supports the standard view that establishment of the dorsal gradient through the Toll pathway is central to DV axis specification, the maternal Dpp pathway may constitute an important secondary mechanism that sharpens and ensures robustness and stability of the dorsal gradient in response to a rapidly changing embryonic environment (Carneiro, 2006).

The initiating event in maternal DV patterning is localized activation of the Grk/EGFR pathway in dorsal cells. Grk functions by restricting the expression of both pip and tld/tok, providing two potentially independent means for spatially regulating the activity of Toll and Dpp. This dual action of the Grk/EGFR pathway is consistent with analysis in which it was found that embryonic cuticles from gd−; grk−; Tl[3] mothers displayed a phenotype distinct from those collected from gd−; Tl[3] mothers. While cuticles from both genotypes had denticle belts surrounding the entire circumference of the embryo, cuticles from gd−; grk−; Tl[3] mothers were more elongated than those from gd−; Tl[3] mothers and exhibited a more ventral character. This suggests that grk provides an additional signal for asymmetry downstream or in parallel to gd. It is suggested that the hypothetical system proposed acts downstream of grk/EGFR and in parallel to Toll may be the Dpp pathway (Carneiro, 2006).

Quantifying the Gurken morphogen gradient in Drosophila oogenesis

Quantitative information about the distribution of morphogens is crucial for understanding their effects on cell-fate determination, yet it is difficult to obtain through direct measurements. A parameter estimation approach was developed for quantifying the spatial distribution of Gurken, a TGFα-like EGFR ligand that acts as a morphogen in Drosophila oogenesis. Modeling of Gurken/EGFR system shows that the shape of the Gurken gradient is controlled by a single dimensionless parameter, the Thiele modulus, which reflects the relative importance of ligand diffusion and degradation. By combining the model with genetic alterations of EGFR levels, the value of the Thiele modulus was estimated in the wild-type egg chamber. This provides a direct characterization of the shape of the Gurken gradient and demonstrates how parameter estimation techniques can be used to quantify morphogen gradients in development (Goentoro, 2006).

From the standpoint of the analysis of Drosophila oogenesis, quantitative characterization of the Gurken gradient enables more detailed models of pattern formation initiated by this morphogen. For example, it is now possible to quantify the distinct thresholds in the Gurken signal which define boundaries of the expression of a large number of Gurken targets in the follicular epithelium. When normalized to the maximal concentration at the dorsal side, the Gurken gradient drops to 63% at one-third dorsal, which coincides roughly with the boundary of the dorsal genes (such as kekkon and sprouty), 22% at the boundary of pipe expression, and 10% at the ventral midline. This description of the Gurken gradient provides the first step toward the quantitative model of pattern formation in the follicular epithelium and will allow an assessment of the threshold of gene expression for target genes of the Egf receptor (Goentoro, 2006).

The role of Dpp and its inhibitors during eggshell patterning in Drosophila: Dpp acts together with Grk in a concentration-dependent manner to specify the identity and position of at least two distinct follicle cell types

The Drosophila eggshell is patterned by the combined action of the epidermal growth factor [EGF; Gurken (Grk)] and transforming growth factor ß [TGF-ß; Decapentaplegic (Dpp)] signaling cascades. Although Grk signaling alone can induce asymmetric gene expression within the follicular epithelium, the ability of Grk to induce dorsoventral polarity within the eggshell strictly depends on Dpp. Dpp, however, specifies at least one anterior region of the eggshell in the absence of Grk. Dpp forms an anteriorposterior morphogen gradient within the follicular epithelium and synergizes with the dorsoventral gradient of Grk signaling. High levels of Grk and Dpp signaling induce the operculum, whereas lower levels of both pathways induce the dorsal appendages (DAs). Evidence is presented that the crosstalk between both pathways occurs at least at two levels. First, Dpp appears to directly enhance the levels of EGF pathway activity within the follicular epithelium. Second, Dpp and EGF signaling collaborate in controlling the expression of Dpp inhibitors. One of these inhibitors is Drosophila sno (dSno), a homolog of the Ski/Sno family of vertebrate proto-oncogenes, which synergizes with daughters against dpp and brinker to set the posterior and lateral limits of the region, giving rise to dorsal follicle cells (Shravage, 2007).

The results show that Dpp has Grk-independent and Grk-dependent functions in the follicular epithelium. Even in the absence of Grk, Dpp is required to specify a group of anterior follicle cells that surround the micropyle. All dorsal follicle cells that contribute to a morphologically visible polarization of the eggshell require the combined action of Grk and Dpp. Within the region giving rise to dorsal follicle cells, Dpp acts together with Grk in a concentration-dependent manner to specify the identity and position of at least two distinct follicle cell types (Shravage, 2007).

In the absence of Dpp, Grk can still activate kekkon and repress pipe. Thus, Dpp is not required for Grk signaling per se. It is suggested that Dpp signaling rather activates transcription factors or causes chromatin modifications that allow Grk to induce dorsal target genes involved in follicle cell specification (Shravage, 2007).

Mirror might be such a transcription factor that is activated by Dpp and confers the ability to adopt dorsal fates to a ring of anterior follicle cells. mirror acts downstream of Grk and probably also downstream of Dpp in specifying dorsal follicle cells. However, mirror expression alone leads only to the formation of DA material. Thus, it is likely that mirror only provides the general potential to produce dorsal follicle cells. Additional inputs from Dpp and EGF signaling are needed to produce the full set of dorsal follicle cell fates. This scenario suggests two phases of Dpp signaling. An early phase demarcates the region in which Grk induces dorsal follicle cell fates. This might require only one (low level) threshold of Dpp signaling and is likely to be mediated through activation of mirror. A later phase establishes distinct dorsal follicle cell fates. Here, Dpp acts as a morphogen in combination with EGF signaling (Shravage, 2007).

The results presented in this study suggest that high levels of EGF and Dpp signaling correspond to regions II and III of the operculum, whereas lower levels of both pathways correspond to the DAs. With regard to region III of the operculum that separates the two DAs, the assumption appears to contradict a model based on results that showed that Grk signaling induces the expression of rhomboid (rho), which in turn activates Spitz, a second TGF-α-like molecule. This leads to an amplification of EGF signaling. Highest signaling levels centered at the dorsal midline lead to the induction of the inhibitor argos (aos), which antagonizes Spitz. This in turn lowers the levels of EGF signaling along the dorsal mildline. According to this model, high levels of EGF signaling promote DA, lower levels operculum region III formation. However, the expression patterns of kek, which result from Grk or Dpp overexpression, appear to contradict this model. Indeed, it is believed that the regulatory loop of rho and aos is not required to establish the operculum or DA fates per se. The pattern of BR-C expression is not significantly altered in rho or aos mutant follicle cell clones. However, rho and aos might contribute to patterning processes that are required for the morphogenesis and, as a result of this, for splitting of the DAs. DA extension (tube formation) has been shown to require the collaboration of rho-expressing floor cells and BR-C-expressing roof cells. The rho-expressing floor cells are part of the Fas3 expression domain that separates the BR-C domains. These rho-expressing cells have to form a separate stripe on each side of the dorsal midline to allow the splitting of the DAs. It is suggested that the rho/aos regulatory loop is required to generate two distinct stripes of late-rho expression within the dorsal Fas3 domain. The result is a splitting of the DAs accompanied by the establishment of a region of Fas3 cells that do not express rho, and thus give rise to region III of the operculum (Shravage, 2007).

The establishment of the region giving rise to dorsal follicle cells and its subdivision into operculum and DA-producing cells is an intriguing problem of two-dimensional patterning. The pattern of cell fates depends on the concentration-dependent read-out of two orthogonal signaling gradients (EGF and Dpp). This read-out is complex because the signaling pathways themselves appear to influence each other. (1) There is evidence for a direct influence of Dpp on EGF signaling; (2) the Dpp inhibitors brk and dSno are targets of both pathways, and (3) rho is also a target of both pathways (Shravage, 2007).

Evidence for a direct crosstalk between both pathways is provided by the analysis of kek expression. kek appears to be a primary target gene of EGF signaling, since basal levels of its expression are independent of Dpp. However, an enhancement of kek expression was observed upon dpp overexpression in stage 10A prior to the activation of rho and aos. Moreover, the stage 10B expression patterns of rho and aos do not correlate with the observed changes in kek expression. Thus, these changes cannot be caused by the secondary modulation of the EGF signaling profile. Therefore, a direct crosstalk between both pathways is suggested. This could be because of a Dpp receptor-dependent activation of the ras/MAPK cascade. A TGF-ß receptor-dependent activation of the MAPK cascade has been observed in several vertebrate cell types. One could imagine that the triangular-shaped domain of Fas3 expression, which defines the anterior and dorsal borders of the BR-C domain, is specified by high levels of EGF signaling brought about by a Dpp-dependent enhancement of MAPK signaling. A confirmation of this model would necessitate direct monitoring of MAPK activity upon altered Dpp signaling (Shravage, 2007).

The border between operculum and DAs is also crucially dependent on brk. In brk mutant follicle cell clones, Fas3 expression expands at the expense of the BR-C domains. However, brk expression is upregulated within a broad domain at the dorsal side that also includes the Fas3-expressing region separating the BR-C domains. Although brk represses Fas3 expression in lateral regions allowing BR-C expression, brk is unable to repress Fas3 at the dorsal midline. This suggests that Fas3 expression, which is predominantly dependent on high levels of EGF signaling, cannot be repressed by brk, whereas Fas3 expression in more lateral regions predominantly dependent on Dpp signaling is repressed by brk (Shravage, 2007).

The hemi-circular boundary of the total region giving rise to dorsal chorion fates appears to be defined by a constant value reflecting the sum or the product of EGF and Dpp signaling. The cis-regulatory elements of dSno represent a sensitive sensor for this dual input. At the dorsal midline, lower amounts of Dpp signaling are required to activate dSno than in lateral regions, and the opposite holds true for EGF signaling. During brain development in flies and in several contexts in vertebrates Sno is involved in the control of cell proliferation that has been shown to be crucially dependent on the relative levels of TGF-ß and EGF signaling. It is conceivable that for spatial patterning of the follicular epithelium dSno uses regulatory elements that are derived from a more basic function in the control of cell proliferation in other tissues. The follicle cell expression of dSno might provide a convenient experimental setting to dissect such regulatory elements (Shravage, 2007).

The fact that loss of dSno causes only mild defects is because of redundancy. A combination of three Dpp inhibitors appears to be involved in establishing the border between dorsal follicle cells and the remainder of the mainbody follicular epithelium. brk clones alone have no effect on the position of this border because they cause only a replacement of the DAs by operculum. dad mutant clones seem to lack patterning defects altogether. However, already removing one copy of these inhibitors in a homozygous dSno mutant background leads to an enlargement of operculum and a posterior shift of the DAs. Weak phenotypic effects of dSno have recently been reported for wing vein formation. Wing vein formation, too, represents a developmental context in which several Dpp inhibitors collaborate (Shravage, 2007).

The dSno mutation that was generated deletes a highly conserved protein domain that is responsible for the interaction with Smad proteins in vertebrates and with Medea in flies. The knockout mutations in mice are based on the deletion of this domain. Thus, this dSno mutation should represent a null allele. However, an unusual complexity of the dSno locus has been reported and a deletion is described that suggests that dSno is lethal, in variance to other findings. However, a truncation allele has been described lacking an important part of the conserved Smad interaction domain that, like the currently described allele, is viable. Because the possibility exists that the previously described deletion affects other genes in the chromosomal region of dSno, the question of lethality of dSno requires further analysis (Shravage, 2007).

Loss of dSno in the follicular epithelium does not result in changes in dpp expression or pMAD distribution. Whereas a feedback on dpp expression was not expected, possible changes in pMAD distribution might be below the level of detection of staining protocol. However, there are two other possible explanations. First, in brain development dSno has been shown to be a mediator of Baboon (Activin), rather than Dpp signaling. To investigate whether this also holds true for the follicular epithelium large baboon (Activin type I receptor) mutant follicle cell clones were generated. These clones did not show patterning defects, suggesting that dSno does not act via Baboon signaling with regard to follicle cell patterning. Second, the failure to detect changes in pMAD distribution might follow from the molecular mechanism of Sno action. A core feature of the inhibitory function of Sno proteins results from their ability to bind to the common Smad (Smad4). This binding prevents (or modulates) the interaction with phosphorylated R-Smads required for the transcriptional control of target genes. If this mechanism applies to DSno, the loss of dSno would not change the phosphorylation state of MAD and, if the interaction between DSno and Medea occurred predominantly in the nucleus, there would also be no significant change in the nuclear accumulation of pMAD (Shravage, 2007).

The salvador-warts-hippo pathway is required for epithelial proliferation and axis specification in Drosophila

In Drosophila, the body axes are specified during oogenesis through interactions between the germline and the overlying somatic follicle cells. A Gurken/TGF-alpha signal from the oocyte to the adjacent follicle cells assigns them a posterior identity. These posterior cells then signal back to the oocyte, thereby inducing the repolarization of the microtubule cytoskeleton, the migration of the oocyte nucleus, and the localization of the axis specifying mRNAs. However, little is known about the signaling pathways within or from the follicle cells responsible for these patterning events. It study shows that the Salvador Warts Hippo (SWH) tumor-suppressor pathway is required in the follicle cells in order to induce their Gurken- and Notch-dependent differentiation and to limit their proliferation. The SWH pathway is also required in the follicle cells to induce axis specification in the oocyte, by inducing the migration of the oocyte nucleus, the reorganization of the cytoskeleton, and the localization of the mRNAs that specify the anterior-posterior and dorsal-ventral axes of the embryo. This work highlights a novel connection between cell proliferation, cell growth, and axis specification in egg chambers (Meignin, 2007).

Multicellular organisms develop through an orchestrated temporal and spatial pattern of cell behavior, which is controlled by cell-to-cell signaling. In Drosophila melanogaster, the establishment of the embryonic axes occurs in the oocyte and depends on a sequence of signals between the germline and the somatic cells. First, Gurken (Grk) signals from the oocyte to the adjacent follicle cells (FCs), in which Torpedo (Top, EGFR) is activated, and this signal instructs them to adopt a posterior identity. The posterior FCs (PFCs) then send an unidentified signal back to the oocyte, leading to the movement of the nucleus from the posterior to the dorsoanterior (DA) corner and the repolarization of the microtubule (MT) cytoskeleton, with the minus ends at the anterior and lateral cortex and the plus ends at the posterior. This repolarization results in the localization of the mRNAs that encode key patterning factors. grk mRNA is next to the nucleus at the DA corner of the oocyte. At this corner, Grk instructs the overlying FCs to adopt dorsal fates. In contrast, oskar (osk) and bicoid (bcd) mRNAs are localized at the posterior and anterior pole, respectively, thus defining the anterior posterior (AP) embryonic axis and the germ cells. Although several genes are required in the FCs to control these events, little is known about the signaling pathways within and from the FCs (Meignin, 2007).

One of the genes required for axis formation during oogenesis is the tumor suppressor merlin (mer). However, it is not known whether Mer influences axis specification directly or what signaling pathways lie downstream of Mer. In other tissues, Mer is known to activate the Salvador Warts Hippo (SWH) pathway, which is a tumor-suppressor pathway. Inhibition of the SWH pathway leads to a characteristic overgrowth phenotype in adult organs because of an overproliferation of cells, increased cell growth, and defects in apoptosis. To test whether the SWH pathway is required in the function of Mer in axis formation, the localization of grk, bcd, and osk mRNA was examined in egg chambers with warts (wts) and hippo (hpo) mutant FCs. wts and hpo encode two serine/threonine kinases that are core components of this pathway. In both cases, grk mRNA is mislocalized at the posterior, osk mRNA is mislocalized at the center, and bcd mRNA is mislocalized at the posterior and anterior poles. The mislocalization of these mRNAs could be due to failure of the MTs to repolarize, as has been previously shown in grk/EGFR and mer mutants. In wild-type oocytes, the MTs are organized in an AP gradient. In contrast, in egg chambers with hpo mutant FCs, the MTs are distributed diffusely all over the oocyte cytoplasm. Considering these results, together with previous characterizations of similar phenotypes, it is concluded that the oocyte cytoskeleton in mutant egg chambers for the SWH pathway is disorganized with the MT plus ends at the center and the minus ends at the anterior and posterior poles. These defects resemble those described in oocytes lacking the Grk signal. In wts mutants, however, Grk protein is detected at the posterior pole, where grk mRNA is mislocalized. This demonstrates that the axis-specification defects in wts mutant egg chambers are not a consequence of the absence of Grk protein (Meignin, 2007).

It was shown that mer is required in the FCs for the repolarizing signal back to the germline and consequently for the migration of the oocyte nucleus from the posterior to the DA corner. Similarly, when mutant FC clones were generated for wts, hpo, and expanded (ex), an activator of the SWH pathway, the oocyte nucleus fails to migrate to the anterior. Another protein that is upstream of the SWH pathway is the giant atypical cadherin fat (ft). However, egg chambers with ft mutant FCs show no defects in oocyte polarity, and both the nucleus and Staufen (Stau) [a marker for osk mRNA] are always properly localized. In other epithelia, hpo and wts are required to repress the activity of Yorkie (Yki) and overexpression of yki phenocopies loss-of-function mutations of hpo and wts. Similarly, it was found that overexpression of yki in the FCs also causes the mislocalization of Stau and the oocyte nucleus. These results indicate that the SWH pathway, with the exception of Ft, might be required for the repolarizing signal back from the FCs to the oocyte (Meignin, 2007).

Because this signal is sent by the PFCs, whether the SWH pathway is required only in these cells was analyzed. In egg chambers with wild-type PFCs within an otherwise hpo or wts mutant epithelium, as well as in hpo, wts, and ex germline clones, the oocyte polarity is unaffected. However, in egg chambers with hpo mutant PFCs in an otherwise wild-type epithelium, the oocyte nucleus is mislocalized. It was also observed that when only a few cells at the posterior are mutant, Stau localizes in the region of the oocyte that faces the posterior wild-type cells. The SWH pathway is not required in the polar cells for axis determination because egg chambers with hpo or wts mutant PFCs and wild-type polar cells show oocyte polarity defects. It is concluded that the SWH pathway is required only in the PFCs to induce axis specification in the oocyte (Meignin, 2007).

In contrast to the monolayered wild-type epithelium, anterior and posterior, but not lateral, hpo and wts mutant cells form a bilayered, and occasionally a multilayered, epithelium. Given that the SWH pathway is required to control proliferation in epithelia of imaginal discs, whether the bilayered epithelium is a result of overproliferation was analyzed. At stage 6 of oogenesis, wild-type FCs undergo a Notch-dependent switch from a mitotic cell cycle to an endocycle. For this reason, phosphohistone 3 (PH3), a marker for mitotic cells, is detected only until that stage and never later. In contrast, hpo mutant anterior and posterior FCs are often positive for PH3 at stage 7-10B, indicating that these cells are still dividing. Similar results are obtained in yki overexpressing FCs. Taken together, these findings show that the SWH pathway is required for the control of proliferation at the anterior and posterior FCs (Meignin, 2007).

The formation of a multilayered epithelium was also observed in stage 3-5 mutant FCs, although the number of dividing cells is similar to that of the wild-type. It has been recently shown that the aberrant orientation of the mitotic spindle in the FCs results in the formation of a multilayered epithelium. Therefore the orientation of the mitotic spindle was analyzed in wild-type and hpo mutant cells. It was observed that, contrary to wild-type cells, the mitotic spindle in mutant FCs is often at an angle or perpendicular to the membrane. This aberrant orientation disrupts the remaining daughter cells within the same plane, thereby resulting in a bilayered epithelium (Meignin, 2007).

Often, tumor suppressors are important for the polarity of the epithelia. To determine whether this is the case for the SWH pathway, the atypical (novel) Protein Kinase C (nPKC), an apical marker, and Disc large (Dlg), a lateral marker, were examined in the FCs. In wild-type cells, as well as in hpo mutant FCs that maintain a monolayer epithelium, nPKC and Dlg localize at the apical and lateral membrane, respectively. However, when the mutant epithelium forms several layers of cells, nPKC and Dlg are often mislocalized, with a reduction of the nPKC staining and an expansion of the Dlg-positive membrane. Nevertheless, a certain degree of the polarity in these cells is maintained because nPKC is always apical in the cells that are in contact with the oocyte (Meignin, 2007).

Because SWH pathway mutant cells do not exit mitosis and keep dividing, it is possible that their differentiation is impaired. To address this question, the expression of Fasciclin III (FasIII) and eyes absent (eya) were analyzed in wild-type and wts and hpo mutant FCs. FasIII and Eya are downregulated in a Notch-dependent manner in the main-body FCs after stage 6 of oogenesis. However, the levels of FasIII in hpo mutant PFCs and Eya in wts mutant PFCs remain high after stage 6. To further assess the effect of the SWH pathway on the Notch-dependent maturation of the FCs, the expression of Hindsight (Hnt), a transcription factor that is upregulated by Notch signaling in all FCs was examined.. In hpo posterior FC clones, this Hnt upregulation is blocked. Contrary to notch clones, however, hpo lateral and anterior clones do not show defects in FasIII, Eya, or Hnt expression. Furthermore, border, centripetal, and stretched cells that are mutant for hpo migrate normally. Considering all these results together, it is concluded that the SWH pathway is essential for the PFCs to fully differentiate (Meignin, 2007).

The findings described above, together with the proliferation defects in hpo and wts mutant cells, suggest that the SWH pathway is required for Notch signaling. To test whether this is the case, the expression of universal Notch transcriptional reporters was analyzed in wild-type and hpo mutant FCs. In wild-type egg chambers, the Notch reporter E(spl)mß7-lacZ is expressed in all FCs upon Notch activation at stage 6 of oogenesis. In contrast, it was found that in hpo mutant cells, the levels of E(spl)mß7-lacZ are weakly reduced in 53% of the clones and normally expressed in the rest. It has been shown that in wing imaginal discs, mer and ex are required to control Notch localization in the cell and consequently its activity. Similarly, the subcellular distribution of Notch is affected in hpo mutant FCs. Contrary to the wild-type, in which Notch accumulates in the apical membrane, Notch expands to other membranes and is often detected in clusters in hpo clones. The results point out that hpo is essential in the PFCs for the Notch-dependent expression of several differentiation markers, such as FasIII, Eya, and Hnt, and for Notch subcellular localization. These observations and the weak defects on the Notch reporters support a function of the SWH pathway in modulating Notch signaling (Meignin, 2007).

Because the SWH pathway is required for the polarization of the oocyte, as well as for the differentiation of the PFCs, whether the mutant cells are competent to respond to Grk and indeed adopt a posterior fate was analyzed. Dystroglycan (DG) is expressed in all FCs at early stages of oogenesis, but upon Grk signaling, DG forms an AP gradient with lower levels at the PFCs. The fact that this Grk-dependent gradient of DG is also observed in the hpo mutant epithelia suggests that the mutant cells are responsive to the Grk signaling. Similarly, when hpo clones affect only a portion of the PFCs, the posterior fate marker pointed is expressed as in the wild-type in 40% of the cases. However, in 60% of the egg chambers with partial hpo posterior clones, and in all cases when all the PFCs are mutant, the expression of pointed is abolished. These results illustrate that hpo is required to fully process the Grk/EGFR signal in the PFCs. Conversely, in grk mutant egg chambers, the Hpo-dependent expression of Hnt is not affected, suggesting that the EGFR pathway is not required for the activation of the SWH pathway in the PFCs (Meignin, 2007).

Considering all these results together, it is concluded that the SWH pathway is involved in the Notch- and Gurken-dependent maturation of the PFCs. Whether the SWH pathway modulates this maturation directly or indirectly, for example by affecting membrane properties, needs to be further investigated (Meignin, 2007).

To study whether the oocyte polarity defects in egg chambers with FCs mutants for the SWH pathway are a consequence of the FCs proliferation and differentiation defects, egg chambers with ex and ft mutant PFCs were analyzed. Egg chambers with ft PFCs occasionally form a bilayer, although they never have defects in oocyte polarity, suggesting that the morphological disruption of the epithelia in itself does not block the repolarizing signal. Egg chambers with ex PFCs show weak defects in the epithelium, with a bilayer rarely formed and restricted to only a few mutant cells, but Stau is never properly localized. However, Hnt is not properly expressed in stage 7 ex mutant FCs, suggesting that the mislocalization of Stau is a consequence of the ex mutant cells being undifferentiated at the stage when the repolarizing signal is sent to the oocyte. These results suggest that the defects in oocyte polarity are probably due to a lack of proper differentiation of FCs in SWH mutant egg chambers (Meignin, 2007).

This study has analyzed the requirement of the SWH pathway during oogenesis. Several of the components of this pathway, but not ft, are required in the PFCs to induce the axis specification in the germline. The defects in oocyte polarity, however, are probably due to a lack of proper differentiation of the PFCs in SWH mutant egg chambers. In addition, the pathway is required in the terminal cells to control their proliferation. It has already been shown that terminal follicle cells are different from lateral follicle cells. The distinct spatial requirement of the SWH pathway for differentiation and proliferation is another feature that distinguishes the terminal from the lateral FCs, and the posterior from the anterior FCs. These results point out that this dual function of the SWH pathway might be achieved by modulation of the Notch and EGFR signals. In conclusion, the SWH pathway lies at the intersection of two signaling pathways and is permissive for the signal that is sent from the follicle cells to repolarize the oocyte.

Salvador-warts-hippo signaling promotes Drosophila posterior follicle cell maturation downstream of notch

The Salvador Warts Hippo (SWH) network limits tissue size in Drosophila and vertebrates. Decreased SWH pathway activity gives rise to excess proliferation and reduced apoptosis. The core of the SWH network is composed of two serine/threonine kinases Hippo (Hpo) and Warts (Wts), the scaffold proteins Salvador (Sav) and Mats, and the transcriptional coactivator Yorkie (Yki). Two band 4.1 related proteins, Merlin (Mer) and Expanded (Ex), have been proposed to act upstream of Hpo, which in turn activates Wts. Wts phosphorylates and inhibits Yki, repressing the expression of Yki target genes. Recently, several planar cell polarity (PCP) genes have been implicated in the SWH network in growth control. This study shows that, during oogenesis, the core components of the SWH network are required in posterior follicle cells (PFCs) competent to receive the Gurken (Grk)/TGFβ signal emitted by the oocyte to control body axis formation. These results suggest that the SWH network controls the expression of Hindsight, the downstream effector of Notch, required for follicle cell mitotic cycle-endocycle switch. The PCP members of the SWH network are not involved in this process, indicating that signaling upstream of Hpo varies according to developmental context (Polesello, 2007).

Body axis formation is a critical stage of development in most multicellular organisms. In Drosophila melanogaster, the anteroposterior (AP) body axis is determined by the polarization of the developing oocyte. The egg chamber is composed of 16 germ cells (15 nurse cells plus the oocyte) and the follicular epithelium. Specification of the AP axis requires active transport of several mRNAs along the microtubule network, thereby resulting in asymmetric mRNA and protein localization inside the oocyte. For example, bicoid (bcd) and oskar (osk) mRNAs localize to and control the formation of the anterior and posterior poles, respectively. This process is initiated through bidirectional signaling between the oocyte and the adjacent follicle cells. In midoogenesis egg chambers, grk mRNA is localized between the oocyte nucleus and the plasma membrane at the presumptive posterior pole and targets the Grk signal to the posterior follicle cells (PFCs) only. Grk is believed to be the ligand for the Torpedo/DER (EGFR) signaling pathway, which controls PFC identity. Once they are specified, the PFCs send an unknown signal back to the oocyte; this signal is required to establish oocyte posterior polarity (Polesello, 2007).

Mer, which has recently been proposed to be part of the SWH network in tissue-size control, has been suggested to play a role in signal back. Therefore whether other members of this network could play a role in body axis formation was addressed. It was tested whether hpo, like mer, is required in PFCs to control oocyte polarity by generating FLP/FRT mitotic clones of mutant cells in the egg chamber was tested with either a kinase-dead (hpoJM1) or a truncating (hpoBF33) allele of hpo. These two alleles behave similarly in all subsequent experiments (Polesello, 2007).

In wild-type egg chambers, the RNA-binding proteins Staufen (Stau) and Osk are localized in a crescent at the posterior pole of the oocyte. When the PFCs were mutant for hpo (visualized by the lack of GFP), both Osk and Stau are mislocalized. If all PFCs were mutant, both Stau and Osk were found in the middle of the oocyte or were absent in some cases for Osk. When hpo clones affected only a portion of the PFCs, Stau was mislocalized almost exclusively in the mutant part, showing the importance of the crosstalk between PFCs and the oocyte (Polesello, 2007).

In hpo germline clones, Stau localization is unaffected if the PFCs are wild-type, suggesting that Hpo is not required for secretion of the Grk signal by the oocyte. Similarly, hpo activity in polar cells is not sufficient to rescue hpo PFC phenotypes because chambers with mutant PFCs and wild-type (GFP-positive) polar cells show disrupted Stau localization. Together, these data suggest that hpo is required in the PFCs to control oocyte polarity (Polesello, 2007).

By using Stau localization as a readout, it was found that like mer and hpo, ex, sav, mats, wts, and yki are playing a role in PFCs to control oocyte polarity, suggesting that 'canonical' Hpo signaling is responsible for the observed phenotype. In contrast, fat (ft) and discs overgrown (dco) are not required in PFCs to control oocyte polarity. This suggests that the core components of the SWH network but not the SWH-associated PCP genes are required for anteroposterior axis formation (Polesello, 2007).

The microtubule cytoskeleton plays an active role in the correct localization of posterior determinants such as Osk mRNA and Stau. Therefore, whether the microtubules are normally organized when the PFCs were mutant for hpo was tested. The oocyte nucleus is initially positioned at the posterior pole (up to stage 6) and migrates to an anterodorsal localization in a microtubule-dependent manner after the signal back from the PFCs (stages 7-14). The oocyte nucleus fails to migrate to an anterodorsal position in 50% of egg chambers with PFC hpo clones. The expression of a tubulin-GFP fusion protein was drived in the germline to visualize the microtubule network. In control oocytes, tubulin-GFP forms a regular network of filaments with a stronger accumulation at the anterior pole corresponding to the nucleation site. Egg chambers with hpo mutant PFCs present ectopic Tubulin-GFP accumulation at the posterior pole of the oocyte. Apart from this defect, the general aspect of the microtubule network is normal in egg chambers with hpo PFC clones, even when the oocyte nucleus has failed to migrate to the anterior end. Finally, microtubule polarity was examined by using both Nod-βGalactosidase (Nod-βGal, minus end marker-anterior) and Kinesin-βGalactosidase (Kin-βGal, plus end marker-posterior) fusion proteins. When the PFCs were mutants for hpo, Nod-βGal was present at both poles or only at the posterior of the oocyte when the nucleus failed to migrate. When all PFCs were hpo mutant, Kin-βGal localization was in a diffuse cloud in the middle of the ooplasm. As for Stau, only half of the Kin-βGal was normally localized when only part of the PFCs were hpo mutant. Together these data support the idea that core components of the SWH pathway are required in the PFCs to build oocyte polarity, controlling microtubule-network orientation (Polesello, 2007).

Because the SWH network is known to control cell number, a phosphorylated Histone 3 (PH3) antibody was used to follow cell division in the follicle cells. During egg-chamber development, follicle cells undergo normal mitotic divisions up to stage 6, thereby giving rise to ~650 follicle cells surrounding the germ cells. Follicle cells then switch from mitotic cycles to three rounds of endoreplication cycles (endocycles) during stages 7-10A. Thus, follicle cells normally stop proliferating after stage 6, as assayed by the absence of PH3-positive cells. hpo PFC clones still contained PH3-positive cells until stage 10B. This excess proliferation observed in hpo mutant cells gives rise to both a reduction of the size of follicle cell nuclei (reduced endocycling) and formation of double layers of cells at the posterior of the egg chamber. Formation of extra layers in the follicular epithelium has been reported to result from misorientation of the mitotic spindle. Normally, the mitotic spindle is parallel to the surface of the germline cells but appears randomly oriented in hpo mutant PFCs because both parallel and perpendicularly oriented spindles were observed. This defect in the mitotic-spindle orientation is probably responsible for the double-layer formation. The proliferation defect specifically affects PFCs because reduced nuclei, ectopic PH3 foci or double layers were not obvious elsewhere. Finally, it was found that loss of the core components of the SWH network, but not of ex for which the proliferation defect is weaker, produced a double cell layer (Polesello, 2007).

In imaginal discs, loss of SWH pathway genes leads to increased expression of Yki target genes. Whether this is also the case in PFCs was tested. As expected, disruption of SWH activity in PFCs gave rise to an increase in ex expression, although no changes were detected in DIAP1 or cycE expression. ex upregulation was restricted to the PFCs in both wts mutant cells and yki gain-of-function experiments. These results suggest that core components of the SWH network specifically control proliferation of a particular subset of follicle cells required for body axis establishment (Polesello, 2007).

Because hpo mutant PFCs were still dividing after stage 6, whether hpo loss of function could affect PFC polarity was assessed. Armadillo (Arm) and Discs large (Dlg) normally label the adherens junctions and the lateral region of the cell, respectively. In hpo mutant PFCs, these were found all around the cells. In addition, the level of Arm, atypical Protein Kinase C (aPKC), and phosphorylated Moesin (P-Moe) were increased. Nevertheless, some aspects of the polarity in these cells were preserved because aPKC was still localized in the apical domain facing the oocyte (Polesello, 2007).

Grk signals via the EGF receptor Torpedo (Top) and activates the Ras signaling pathway, specifying the PFC identity. The PFC fate can be followed by the expression of the Ras target pointed (pnt-LacZ). In the absence of hpo, pnt-LacZ expression was disrupted in most but not all PFC clones. Nevertheless, hpo mutant PFCs were still able to activate the Jak/STAT pathway in response to a signal emerging from the polar cells, (monitored with a STAT reporter suggesting that the polarity defect observed in hpo mutant PFCs does not affect their ability to receive secreted signals in general. wts mutant PFCs were negative for the dpp-LacZ reporter, a specific marker of the anterior follicle cell fate (stretch and centripetal cells), suggesting that when the SWH pathway is compromised, the PFCs are not merely transformed into anterior cells. In addition, it was found that hpo mutant PFCs present characteristics of immature cells such as maintenance of Fasciclin III (FASIII) and eyes absent (eya) expression. Normally, the level of these two genes is downregulated when the follicle cells switch from mitotic cycles to endocycles. It is noted that, when hpo mutant PFCs were FASIII positive, they did not express pnt-LacZ and vice versa. In addition, it was found that pnt-LacZ-positive hpo mutant PFCs have normal Stau localization. This suggests that the primary defect in hpo mutant cells is the failure to mature. In the rare cases where hpo mutant PFCs mature properly, they are competent to transduce the Grk signal, and oocyte polarity is normal (Polesello, 2007).

Notch (N) is required in the follicle cells for the mitotic-endocycle switch that occurs at stage 6 and for controlling follicle cell identity. N mutant follicle cells, like hpo mutant PFCs, keep proliferating because they are stuck in an immature state and continue to express undifferentiated markers such as FASIII. Recently, members of the SWH network were reported to modulate N activity by affecting its subcellular localization. N protein, which localizes to the apical part of the follicle cells, is downregulated at midoogenesis. This downregulation is delayed in wts and hpo mutant PFCs, possibly causing a defect in N signaling. Hindsight (Hnt), a target of N, which starts to be expressed in all follicle cells at stage 7 after N activation, was examined. Expression of Hnt in hpo mutant PFCs is compromised. In addition, it was found that the expression of Cut, which is normally inhibited by Hnt at stage 7, was maintained in hpo and wts clones up to stage 10. Finally, whether the modulation of N activity by the SWH network was direct was tested by looking at the expression of direct N reporters. no obvious reduction of the m7-LacZ reporter was found in hpo PFC clones. However, because of the perdurance of the β-galactosidase protein, this type of reporter is more suitable to follow increases rather than decreases in signaling. It therefore cannot be entirely rule out that the SWH network might directly affect Notch activity. Nevertheless, together these data show that inactivation of the SWH network compromises the regulation of downstream targets of Notch such as Hnt and Cut. As is the case for FASIII, misregulation of these genes is restricted to the PFCs in a SWH mutant background (Polesello, 2007).

Because of this spatial restriction of SWH activity to PFCs, whether the SWH network could be part of the Torpedo/Ras pathway acting downstream of the Grk signal was tested. ras, wts double loss-of-function clones were examined. ras, wts clones present characteristics of both ras and wts single-mutant clones, namely upregulation of Dystroglycan (DG), as observed in ras clones, and maintenance of FASIII protein, as observed in wts clones. In addition, grk mutant egg chambers present only DG upregulation but no FASIII modification and no substantial change in ex expression. It is therefore concludes that the SWH network and EGFR/Ras signaling are likely to act in parallel to control respectively PFC maturation and identity and that Grk is not the ligand that controls the SWH network activation (Polesello, 2007).

A last concern was to test whether the SWH network is involved in the PFC signal back that controls oocyte polarity. To tackle this point, attempts were made to uncouple the possible signal back to the oocyte from the PFC maturation phenotypes. ex loss of function, which affects Stau localization but presents a very reduced proliferation rate and double-layer formation compared to other SWH members, was examined. Unfortunately, ex loss of function still affected Arm, FASIII, and Cut protein levels in the PFCs, in particular at midoogenesis, when both the N and Grk signals act. Therefore mer, cut double mutants were generated. In theory, this should force the cells to differentiate (lack of cut) and still affect SWH activity (lack of mer). As expected, whereas mer loss of function alone elicited both Cut upregulation and Stau mislocalization, mer/cut PFC clones were able to induce normal oocyte polarity, manifested by correct Stau localization. It is concluded that the activity of the SWH network is required to control PFC maturation, but this pathway is probably not involved in the signal-back process (Polesello, 2007).

In conclusion, this study has shown that the core components of the SWH network are required specifically to allow the maturation of the PFCs receiving the Grk signal, thus controlling AP body axis formation. The PFC defect is due to a lack of Hnt expression in response to Notch signaling. Because the function of the SWH network is restricted to the PFCs, one interesting speculation is that it is an added layer of Notch regulation specific to PFCs, which, given their crucial role in initiating body axis formation, need robust control of signaling. Placing this regulatory element in complement and in parallel to the signal that initiates PFC specification (Grk) would ensure, in cooperation with the Unpaired signal (Jak/STAT pathway) from the polar cells, a tight and robust boundary between the PFCs and the rest of the follicle cells (Polesello, 2007).

Finally the results make a clear distinction between the core components of the SWH network (hpo, sav, wts, mats, and yki) and mer, ex on one hand and the PCP genes (ft and dco) on the other. It is speculated that the core components are used in a variety of contexts during development, whereas the PCP genes are restricted to organ-size specification (Polesello, 2007).

A combinatorial code for pattern formation in Drosophila oogenesis

Two-dimensional patterning of the follicular epithelium in Drosophila oogenesis is required for the formation of three-dimensional eggshell structures. Analysis of a large number of published gene expression patterns in the follicle cells suggests that they follow a simple combinatorial code based on six spatial building blocks and the operations of union, difference, intersection, and addition. The building blocks are related to the distribution of inductive signals, provided by the highly conserved epidermal growth factor receptor and bone morphogenetic protein signaling pathways. The validity of the code is demonstrated by testing it against a set of patterns obtained in a large-scale transcriptional profiling experiment. Using the proposed code, 36 distinct patterns were distinguished for 81 genes expressed in the follicular epithelium, and their joint dynamics were characterize over four stages of oogenesis. The proposed combinatorial framework allows systematic analysis of the diversity and dynamics of two-dimensional transcriptional patterns and guides future studies of gene regulation (Yakoby, 2008b).

Drosophila eggshell is a highly patterned three-dimensional structure that is derived from the follicular epithelium in the developing egg chamber. The dorsal-anterior structures of the eggshell, including the dorsal appendages and operculum, are formed by the region of the follicular epithelium, which is patterned by the highly conserved epidermal growth factor receptor (EGFR) and bone morphogenetic protein (BMP) signaling pathways. The EGFR pathway is activated by Gurken (GRK), a transforming growth factor α-like ligand secreted by the oocyte. The BMP pathway is activated by Decapentaplegic (DPP), a BMP2/4-type ligand secreted by the follicle cells stretched over the nurse cells (Yakoby, 2008b).

Acting through their uniformly expressed receptors, these ligands establish the dorsoventral and anteroposterior gradients of EGFR and DPP signaling and control the expression of multiple genes in the follicular epithelium. Under their action, the expression of a Zn finger transcription factor, Broad (BR), evolves into a pattern with two patches on either side of the dorsal midline. The BR-expressing cells form the roof (upper part) of the dorsal appendages. Adjacent to the BR-expressing cells are two stripes of cells that express rhomboid (rho), a gene that is directly repressed by BR and encodes ligand-processing protease in the EGFR pathway. These cells form the floor (lower part) of the appendages (Yakoby, 2008b).

The patterns of genes expressed during the stages of egg development that correspond to appendage morphogenesis are very diverse. At the same time, inspection of a large number of published patterns suggests that they can be 'constructed' from a small number of building blocks. For instance, the T-shaped pattern of CG3074 is similar to the domain 'missing' in the early pattern of br, while the two patches in the late pattern of br appear to correspond to the two 'holes' in the expression of 18w. Based on a number of similar observations, it was hypothesized that all of the published patterns could be constructed from just six basic shapes, or primitives, which reflect the anatomy of the egg chamber and the spatial structure of the patterning signals (Yakoby, 2008b).

In computer graphics, representation of geometrical objects in terms of a small number of building blocks is known under the name of constructive solid geometry, which provides a way to describe complex shapes in terms of just a few parameters -- the types of the building blocks, such as cylinders, spheres, and cubes, their sizes, and operations, such as difference, union, and intersection. Thus, information about a large number of structures can be stored in a compact form of statements that contain information about the types of the building blocks and the operations from which these structures were assembled. This study describes a similar approach for two-dimensional patterns and demonstrate how it enables the synthesis, comparison, and analysis of gene expression at the tissue scale (Yakoby, 2008b).

The six building blocks used in the annotation system can be related to the structure of the egg chamber and the spatial distribution of the EGFR and DPP signals. The first primitive, M (for 'midline'), is related to the EGFR signal. It reflects high levels of EGFR activation and has a concave boundary, which can be related to the spatial pattern of GRK secretion from the oocyte. The second primitive, denoted by D (for 'dorsal'), reflects the intermediate levels of EGFR signaling during the early phase of EGFR activation by GRK, and is defined as a region of the follicular epithelium that is bounded by a level set (line of constant value) of the dorsoventral (DV) profile of EGFR activation. The boundary of this shape is convex and can be extracted from the experimentally validated computational model of the GRK gradient. The third primitive, denoted by A (for 'anterior'), is an anterior stripe which is obtained from a level set of the early pattern of DPP signaling in the follicular epithelium. This pattern is uniform along the DV axis, as visualized by the spatial pattern of phosphorylated MAD (P-MAD). Thus, the D, M, and A primitives represent the spatial distribution of the inductive signals at the stage of eggshell patterning when the EGFR and DPP pathways act as independent AP and DV gradients (Yakoby, 2008b).

Each of the next two primitives, denoted by R (for 'roof') and F (for 'floor'), is composed of two identical regions, shaped as the respective expression domains of br and rho, and reflect spatial and temporal integration of the EGFR and DPP pathways in later stages of eggshell patterning. The mechanisms responsible for the emergence of the F and R domains are not fully understood. It has been shown that the R domain is established as a result of sequential action of the feedforward and feedback loops within the EGFR and DPP pathways. The formation of the F domain requires the activating EGFR signal and repressive BR signal, expressed in the R domain. Thus, at the current level of understanding, the R and F domains should be viewed as just two of the shapes that are commonly seen in the two-dimensional expression patterns in the follicular epithelium. The sixth primitive, U (for 'uniform'), is spatially uniform and will be used in combination with other primitives to generate more complex patterns (Yakoby, 2008b).

While a number of patterns, such as those of jar and Dad, can be described with just a single primitive, more complex patterns are constructed combinatorially, using the operations of intersection (∩), difference ( ), and union (∪) For example, the dorsal anterior stripe of argos expression is obtained as an intersection of the A and D primitives (A∩D). The ventral pattern of pip is obtained as a difference of the U and D primitives (U D). The pattern of 18w is constructed from the A, D, and R primitives, joined by the operations of union and difference (A∪D R). For a small number of published patterns, the annotations reflect the experimentally demonstrated regulatory connections. For example, the U D annotation for pip reflects that actual repression of pip by the dorsal gradient of EGFR activation. For a majority of genes, the annotations should be viewed as a way to schematically represent a two-dimensional pattern and as a hypothetical description of regulation (Yakoby, 2008b).

The geometric operations of intersection, difference, and union can be implemented by the Boolean operations performed at the regulatory regions of individual genes. Boolean operations evaluate expression at each point and assign a value of 0 (off) or 1 (on). As an example, consider a regulatory module, hypothesized for argos, that performs a logical AND operation on two inputs: the output of the module is 1 only when both inputs are present. When both of the inputs are spatially distributed, the output is nonzero only in those regions of space where both inputs are present, leading to an output that corresponds to the intersection of the two inputs. Similarly, a spatial difference of the two inputs can be realized by a regulatory module that performs the ANDN (ANDNOT) operation. This is the case for pip, repressed by the DV gradient of GRK signaling and activated by a still unknown uniform signal. Finally, a regulatory module that performs an OR operation is nonzero when at least one of the inputs is nonzero. When the inputs are spatially distributed, the output is their spatial union (Yakoby, 2008b).

Boolean operations on primitives lead to patterns with just two levels of expression (the gene is either expressed or not). In addition to Boolean logic, developmental cis-regulatory modules and systems for posttranscriptional control of gene expression can perform analog operations, leading to multiple nonzero levels of output. Consider a module that adds the two binary inputs, shaped as the primitives. The output is nonzero in the domain shaped as the union of the two primitives, but is characterized by two nonzero levels of expression. This type of annotation is reserved only for those cases where the application of Boolean operations would lead to a loss of the spatial structure of the pattern (such as the A + U expression pattern of mia at stage 11 of oogenesis. For example, the union of the A and U primitives is a U primitive, whereas the sum of these primitives is an anterior band superimposed on top of a spatially uniform background (Yakoby, 2008b).

Signaling pathways guide organogenesis through the spatial and temporal control of gene expression. While the identities of genes controlled by any given signal can be identified using a combination of genetic and transcriptional profiling techniques, systematic analysis of the diversity of induced patterns requires a formal approach for pattern quantification, categorization, and comparison. Multiplex detection of gene expression, which has a potential to convert images of the spatial distribution of transcripts into a vector format preferred by a majority of statistical methods, is currently feasible only for a small number of genes and systems with simple anatomies. This paper presents an alternative approach based on the combinatorial construction of patterns from simple building blocks (Yakoby, 2008b).

In general, the building blocks can be identified as shapes that are overrepresented in a large set of experimentally collected gene expression patterns. This approach can be potentially pursued in systems where mechanisms of pattern formation are yet to be explored. At the same time, in well-studied systems, the building blocks can be linked to identified patterning mechanisms. This study chose six primitives based on the features that are commonly observed in real patterns and related to the structure of the tissue as well as the spatial distribution of the inductive signals. A similar approach will be useful whenever a two-dimensional cellular layer is patterned by a small number of signals, when cells can convert smoothly varying signals into spatial patterns with sharp boundaries, and when the regulatory regions of target genes have the ability to combinatorially process the inductive signals. One system in which this approach could be feasible is the wing imaginal disc, which is patterned by the spatially orthogonal wingless and DPP morphogens (Yakoby, 2008b).

The six primitives are sufficient to describe the experimentally observed patterns during stages 10-12 of oogenesis. A natural question is whether it is possible to accomplish this with a smaller number of primitives. Two of the primitives, R and F, could be potentially constructed from the D, M, and A primitives, which are related to the patterns EGFR and DPP activation during the earlier stages of eggshell patterning. Specifically, recent studies of br regulation suggest that the R domain is formed as a difference of the D, A, and M patterns (Yakoby, 2008a). Furthermore, the formation of the F domain requires repressive action in the adjacent R domain. With the R and F domains related to the other four primitives, the size of the spatial alphabet will be reduced even further (from six to four), but at the expense of increasing the complexity of the expressions used to describe various spatial patterns (Yakoby, 2008b).

Previously, the question of the diversity of the spatial patterns has been addressed only in one-dimensional systems. For example, transcriptional responses to the Dorsal morphogen gradient in the early Drosophila embryo give rise to three types of patterns in the form of the dorsal, lateral, and ventral bands. This work provides an attempt to characterize the diversity and dynamics of two-dimensional patterns. Thirty-six qualitatively different patterns were constructed, and it is proposed that each of them can be constructed using a compact combinatorial code. The sizes of the data sets from the literature and from transcriptional profiling experiments are approximately the same (117 and 96 patterns, respectively. Based on this observation, it is expected that discovered patterns will be readily described using this annotation system (Yakoby, 2008b).

A gene expressed in more than one stage of oogenesis is more likely to appear in different patterns, and it was found that groups of genes sharing the same pattern at one time point are more likely to scatter in the future than to stay together. More detailed understanding of the dynamics of the spatial patterns of the EGFR and DPP pathway activation is crucial for explaining these trends and the two observed scenarios for the emergence of complex patterns. A gene that makes its first appearance as a complex pattern, such as the A∩D pattern of argos at stage 10B, can be a direct target of the EGFR and DPP signal integration. In contrast, a gene such as Cct1, which changes from the A to the R pattern, can be a dedicated target of DPP signaling alone, and changes as a consequence of change in the spatial pattern of DPP signaling. Future tests of such hypotheses require analysis of cis-regulatory modules responsible for gene regulation in the follicular epithelium. While only a few enhancers have been identified at this time, this categorization of patterns should accelerate the identification of enhancers for a large number of genes (Yakoby, 2008b).

Proposed for the spatial patterns of transcripts, these annotations can also describe patterns of protein expression, modification, and subcellular localization. For example, the stage 10A patterns of MAD phosphorylation and Capicua nuclear localization can be accurately described using the A and U D annotations, respectively. The ultimate challenge is to use the information about the patterning of the follicular epithelium to explore how it is transformed into the three-dimensional eggshell. A number of genes in the assembled database encode cytoskeleton and cell adhesion molecules, suggesting that they provide a link between patterning and morphogenesis. It is hypothesized that the highly correlated expression patterns of these genes give rise to the spatial patterns of force generation and mechanical properties of cells that eventually transform the follicular epithelium into a three-dimensional eggshell (Yakoby, 2008b).

Effects of Mutation or Deletion

Changes in the dorsalizing germ-line signal affect the embryonic dorsoventral pattern. A reduction in strength of the germ-line signal as produced by mutations in gurken or torpedo does not change the slope of the embryonic dorsoventral morphogen gradient, but causes a splitting of the gradient ventrally. This leads to embryos with two partial dorsoventral axes. A change in distribution of the germ-line signal as caused by fs(1)K10, squid and orb mutations leads to a shift in the orientation of the embryonic dorsoventral axis relative to the anterior-posterior axis. In extreme cases, this results in embryos with a dorsoventral axis almost parallel to the anterior-posterior axis. These results imply that gurken, unlike other localized cytoplasmic determinants, is not directly responsible for the establishment of cell fates along a body axis, but that it restricts and orients an active axis-forming process which occurs later in the follicular epithelium or in the early embryo (Roth, 1994).

Mutations in gurken and torpedo cause a ventralization in the follicle cell epithelium during Drosophila oogenesis and in the pattern of the embryo that develops in the resultant egg. Both genes lie midway in an epistatic series between fs(1)K10 and dorsal; the mutations block the dorsalization normally observed in K10 eggs but have no effect on the phenotype of embryos derived from dorsal mothers. Analysis of germ-line mosaics demonstrates that both ovarian and embryonic phenotypes will be produced when either the gurken+ gene is removed from the germ line or torpedo+ is removed from the follicular soma. This shows that the dorsoventral pattern of the Drosophila egg chamber depends on the transfer of spatial information from the germ line to the somatic follicle cells, and from somatic cells to the oocyte (Schupbach, 1987).

The Drosophila eggshell, which has a pair of chorionic appendages (dorsal appendages) located asymmetrically along both the anterior/posterior and dorsal/ventral axes, provides a good model to study signal instructed morphogenesis. Broad-Complex, a gene encoding zinc-finger transcription factors, is essential for the morphogenesis of dorsal appendages and is expressed in a bilaterally symmetrical pattern in the lateral-dorsal-anterior follicle cells during late oogenesis. This pattern of expression is induced and specified along the dorsoventral axis by an epidermal growth factor receptor signaling pathway, which in the oocyte includes Gurken, a localized transforming growth factor alpha-like molecule. In the surrounding somatic follicle cells, Torpedo, the Drosophila EGF receptor homolog that functions as the target of Gurken specifies BR-C expression. Mutants that result in the mislocalization of Gurken, such as fs(1)K10, induce a BR-C late expression pattern that is expanded to the ventral follicle cells surrounding the oocyte. This expanded BR-C expression results in expansion of the dorsal appendages to the ventral region. Four extra copies of grk gene increase the gap between the two groups of BR-C expressing cells to about 8 cells wide, in comparison with the 4-cell-wide gap in wild type, resulting in a widened dorsal gap between the two dorsal appendages. A decrease in Grk-Egfr signaling in a topQY1 mutation in Egfr results in BR-C expression in the dorsal most follicle cells, leading to a fusion of the dorsal appendages in the dorsal-most region. The Egfr target gene pointed regulates the number of BR-C expressing cells. Ectopic expression of pnt decreases the number of BR-C expressing cells, suggesting that Pnt regulates BR-C expression. The precisely localized expression of BR-C along the AP axis requires a separate signaling pathway, initiated by a transforming growth factor-beta homolog, Decapentaplegic, in nearby follicle cells. These two signaling pathways (Gurken functioning from the oocyte and Dpp functioning from the follicle cells) co-ordinately specify patches of follicle cells to express the Broad-Complex in a unique position with respect to both DV and AP axes respectively, and which, in turn direct the differentiation of the dorsal appendages in the correct position on the eggshell (Deng, 1997).

Brainiac functions in modulating the Gurken-EGF-R interaction during genesis of the follicular epithelium. brainiac is expressed in germ cells at the time follicle cells first surround the nurse cell-oocyte complex. grk and brainiac exhibit dosage-sensitive interactions. Double heterozygotes of weak mutants lay eggs with partially fused dorsal appendages, while single mutants are completely wildtype. Animals heterozygous for strong alleles show slight defects in patterning of follicular epithelium, while double mutants lay completely ventralized eggs completely lacking dorsal appendages. Ovarioles from females homozygous for weak alleles of grk or brn resemble wild type in terms of follicular cell development. In contrast, egg chambers from animals homozygous for strong alleles consistently display both fused egg chambers and gaps in the follicular epithelium, frequently uncovering over half of the egg chamber. It is concluded that grk acts in concert with brn to achieve the migration of prefollicular cells to surround each nurse cell-oocyte complex and to form a continuous epithelium (Goode, 1996).

A set of dorsal follicle cells is patterned by the oocyte in a cell-cell signaling event occurring at stages 8 and 9 when the germinal vesicle (nucleus) migrates to the dorsal anterior of the oocyte. The anterodorsally positioned oocyte nucleus produces Gurken mRNA, a proposed ligand for the Epidermal growth cell receptor gene present on the overlying follicle cells. Activating Egfr transmits a signal through a Raf-dependent signaling pathway to generate anterior dorsal follicle cell fates, resulting in the respective specializations of the eggshell, including the dorsal appendages. A ventral follicle cell subpopulation that does not experience induction by Gurken produces molecular cues for a different inductive event, directing embryonic dorsal-ventral embryonic axis formation (Dobens, 1997 and references).

A Drosophila sequence homologous to the mammalian growth factor-stimulated TSC-22 gene was isolated in an enhancer trap screen for genes expressed in anterodorsal follicle cells during oogenesis. In situ hybridization reveals that bunched transcripts localize to the anterior dorsal follicle cells at stages 10-12 of oogenesis. Additional staining is evident in the border cells at the nurse cell/oocyte border and in a group of posterior polar follicle cells. The centripetally migrating follicle cells, just anterior to the stained columnar cells of the anterodorsal patch do not stain. Changes in bun enhancer trap expression in genetic backgrounds that disrupt the grk/Egfr signaling pathway suggest that bun is regulated by growth factor patterning of dorsal anterior follicle cell fates. In fs(1)K10 mutant egg chambers, dorsal follicle cell fates expand at the expense of ventral follicle cell fates, presumably due to mislocalization of GRK mRNA from the anterodorsal portion of the oocyte to more ventral positions. In fs(1)K10 females, expression of bunched expands ventrally, with two maxima in the anterodorsal anteroventral follicle cells, diminishing laterally. In stage 10 follicles from Egfr mutants expression of bun is lost from the dorsal anterior; reduced bun expression is shifted to more posterior follicle cells. Egg chambers from a gurken mutant completely lack dorsal appendages. No bunched expression is seen in the dorsal anterior follicle cells from stage 10 gurken mutant egg chambers. Clonal analysis shows that bun is required for the proper elaboration of dorsal cell fates leading to the formation of the dorsal appendages. Eggs from bunched mutants are shortened and their dorsal appendages are short and often wide, with branched and split ends (Dobens, 1997).

Preliminary evidence indicates the bunched is sensitive to decapentaplegic levels in the follicle cells. It is therefore thought that normal bunched expression in the dorsal anterior follicle cells is the result of combined action of the Egfr receptor for Grk and serine/threonine kinase receptors (see Thick veins and Punt) for Decapentaplegic (Dobens, 1997).

Directed cell migration is important for many aspects of normal animal development, but little is known about how cell migrations are guided or the mechanisms by which guidance cues are translated into directed cell movement. Evidence is presented that signaling mediated by the epidermal growth factor receptor (Egfr) guides dorsal migration of border cells during Drosophila oogenesis. The transforming growth factor-alpha (TGF-alpha)-like ligand Gurken appears to serve as the guidance cue. To mediate this guidance function, Egfr signals via a pathway that is independent of Raf-MAP kinase and is specific for the Egfr receptor (Duchek, 2001).

Border cells constitute a cluster of 6 to 10 specialized somatic follicle cells that perform a stereotypic migration during Drosophila oogenesis. At the beginning of stage 9, border cells delaminate from the anterior follicular epithelium and initiate their migration between the germline derived nurse cells, toward the oocyte. About 6 hours later, at stage 10, the border cells reach the oocyte and then migrate dorsally toward the germinal vesicle (GV). The migration of border cells is essential for female fertility; however, it is not known what guides this migration. Spatial information may be provided by the surrounding tissue in the form of cell-associated or secreted guidance cues, for example, as attractive gradients. The posterior and dorsal migration phases might be guided by separate cues, or by a single cue and a fixed migration path (Duchek, 2001).

To identify guidance cues, the following was taken into consideration: The gradient of spatial information would be perturbed if a key attractant or repellant were uniformly overexpressed. This would be expected to cause the cells to migrate inefficiently as there would be no difference between signaling in the front and the back of the cell. To identify genes capable of perturbing border-cell migration when expressed uniformly, a modular misexpression screen was performed with the P element EPg. Expression was induced in the germline (nanosGAL4:VP16 ) and in the border cells themselves (slboGAL4). Of 8500 independent insertion lines, three showed defects in border-cell migration but no detectable morphological abnormalities in the egg chamber. In one of these, EPg35521, the single EPg element is inserted in such a way that it drives expression of the gene encoding the neuregulin-like EGFR ligand Vein. Border-cell migration is affected both when Vein is expressed in the germline tissue and when it is expressed in the border cells themselves, as might be expected of a secreted molecule (Duchek, 2001).

To determine whether the effect on migration is specific to Vein or common to Egfr ligands, secreted forms of the TGF-alpha-like ligands Gurken and Spitz were expressed in border cells. Both affect border-cell migration, with the potent ligand secreted Spitz having the strongest effect. Border-cell expression of an activated, ligand-independent, form of Egfr [lambda-top] also severely affects migration. Thus, constitutive stimulation of Egfr signaling in border cells effectively inhibits their migration (Duchek, 2001).

To determine whether Egfr signaling is required for normal border-cell migration, a dominant negative form of the receptor (DN-DER) or the transmembrane Egfr inhibitor Kekkon-1 was expressed in border cells. Both specific Egfr inhibitors severely inhibit dorsal migration of border cells, with only minor effects on the initial posterior migration. Most eggs from these females do not hatch and appear unfertilized. This phenotype mimics loss of border-cell function, suggesting that the dorsal aspect of migration may be essential. The requirement for Egfr in border cells was confirmed by looking at clones of Egfr mutant cells. When all outer border cells were mutant for Egfr, the cluster remained in the center of the egg chamber at stage 10, whereas 90% of wild-type clusters were found dorsally. When mixed clusters with both wild-type and mutant cells move dorsally, the wild-type cells are in the front. Thus, Egfr signaling is required specifically for dorsal border-cell migration (Duchek, 2001).

When border cells migrate dorsally, activating ligands for Egfr are produced by the oocyte (Gurken) and, in response to Gurken, by dorsal follicle cells (Vein and Spitz). Dorsal migration still occurs when dorsal follicle cells are mutant for vein, spitz , or rhomboid, which is required for Spitz activation. Thus, although ectopic expression of Vein or activated Spitz proteins can affect border-cell guidance, neither is required for the process. Removing Egfr from patches of dorsal follicle cells, which renders them unable to activate secondary signals, also has no effect. In contrast, dorsal migration is perturbed in gurken mutants. Ovaries from grkDC/grk2b6 mutant females show a range of defects. In mildly affected egg chambers where the GV has moved anterior and dorsal, border cells complete posterior migration but fail to migrate dorsally. In stage-10 oocytes, Gurken protein is detected in a membrane-associated gradient with the highest level at the dorsal anterior over the GV. These results are most consistent with Gurken serving as the dorsal guidance cue, although contributions from other Egfr ligands cannot be excluded (Duchek, 2001).

Next, an examination was performed to see which intracellular signaling pathways downstream of Egfr might mediate the effect on border-cell migration. Egfr signaling has been shown to regulate growth and differentiation during Drosophila development via activation of the Raf-MAP kinase (MAPK) pathway. Moderate activation of this pathway is observed in migrating border cells at both phases of migration, particularly in the leading cells. Mammalian tissue culture studies have indicated, however, that mitogenic and migration-inducing activities of Egfr and other receptor tyrosine kinases (RTKs) may occur via different pathways, prompting further investigation (Duchek, 2001).

To investigate the role of the Raf-MAPK pathway, clonal analysis was performed with a raf null mutant (phl11). When all outer border cells are mutant, migration is normal during stage 9. Mutant clusters are very rarely recovered at stage 10, but dorsal migration can occur. Expression of an activated form of Raf (RafGOF) in border cells results in robust activation of MAPK but has no effect on border-cell migration. Finally, expression of an activated form of the Drosophila fibroblast growth factor (FGF) receptor Heartless strongly activates MAPK in border cells but has no effect on migration. This contrasts with the effect of Egfr. Thus, the effects of Egfr signaling on border-cell migration appear to be specific (not elicited by all RTKs) and independent of Raf-MAPK (Duchek, 2001).

The small guanosine triphosphatase Ras can link RTKs to MAPK pathway or other pathways. Dominant negative Ras (RasN17) and activated Ras (RasV12) moderately affect posterior and dorsal border-cell migration, indicating that Ras has a role in both migrations. Phosphatidylinositol 3-kinase (PI3K) has been implicated directly as regulator of chemotaxis in different systems. However, expression of dominant negative or activated forms of the Drosophila PI3K catalytic subunit (p110DN and p110CAAX) does not affect border-cell migration. Phospholipase C-gamma (PLC-gamma), which can bind directly to RTKs via its SH2 domain, may mediate effects on movement of tissue culture cells. In the Drosophila genome, there appears to be only one PLC-gamma, encoded by the small wing (sl) locus. Null mutants in sl do not affect border-cell migration. Thus, neither PI3K nor PLC-gamma appear to be key mediators downstream of Egfr in this context (Duchek, 2001).

Border cells are sensitive to Egfr signaling from the onset of migration, which suggests that the posterior migration may be guided by a similar RTK signal. Activated Heartless has no effect on migration. breathless mutant border cells migrate normally, and overexpression of the ligand Branchless has no effect. In addition, border cells mutant for dof, which is required for signal transduction by both FGF receptors, migrate normally. Thus, neither of the two Drosophila FGF receptors, Breathless and Heartless, perform this role (Duchek, 2001).

The RTKs of the EGF receptor family are required for growth, survival, differentiation, and migration of various cell types during animal development. EGF signaling also stimulates growth and metastatic potential of human tumors, as well as proliferation and motility of tissue culture cells. These results demonstrate that Egfr signaling can direct cell migration in vivo. Egfr acts as a guidance receptor for border cells during oogenesis and is specifically required for the second phase of their migration. Another RTK with similar signaling properties may serve this function for the first phase of migration. Evidence has been presented that guidance effects of Egfr are mediated by a noncanonical signaling pathway. The challenge is now to determine which pathways and molecules downstream of Egfr translate guidance information into directed cell movement in vivo (Duchek, 2001).

Dorsal-ventral polarity of the Drosophila embryo is established by a nuclear gradient of Dorsal protein, generated by successive gurken-Egfr and spätzle-Toll signaling. Overexpression of extracellular Spätzle dramatically reshapes the Dorsal gradient: the normal single peak is broadened and then refined to two distinct peaks of nuclear Dorsal, to produce two ventral furrows. This partial axis duplication, which mimics the ventralized phenotype caused by reduced gurken-Egfr signaling, arises from events in the perivitelline fluid of the embryo and occurs at the level of Spätzle processing or Toll activation. The production of two Dorsal peaks is addressed by a model that invokes the action of a diffusible inhibitor, which is proposed to normally regulate the slope of the Dorsal gradient (Morisato, 2001).

The shape of the Dorsal gradient is dramatically changed in embryos laid by females carrying mutations in the gurken-Egfr signaling pathway. Not only do these embryos expand Twist expression, as a consequence of a reduction in the dorsalizing signal that establishes egg chamber asymmetry, but they exhibit two distinct peaks within the Twist domain that give rise to two ventral furrows. In the experiments described here, this partial axis duplication is not evident during oogenesis, because pipe RNA was found to be expressed in a single broad domain in follicle cells. The production of two Dorsal peaks could be mimicked by injecting high levels of spz RNA into the pre-cellular embryo cytoplasm, suggesting that pattern refinement occurs during embryogenesis. It is suggested that while the size of the ventral domain is expanded in grk and Egfr ovarian egg chambers, the partial axis duplication observed in mutant embryos is caused by reactions occurring later in the embryo (Morisato, 2001).

It may have been easier to imagine how the selection of one or two gradient peaks would involve signaling within the follicular epithelium, because spatial information could then be stably maintained and transmitted by cells. The elaboration of the two dorsal appendages in the Drosophila eggshell results from a series of such intercellular signaling events. Activation of Egfr by Gurken stimulates transcriptional induction of Argos, a secreted Egfr inhibitor, which then downregulates Egfr activity in the initial central domain, leaving two lateral domains of signaling (Morisato, 2001).

In fact, the findings described in this paper argue that events involving the diffusion of an extracellular morphogen not only regulate the gradient slope, but perhaps unexpectedly, determine the position and number of maxima within the axis in response to the broad cues generated during oogenesis. Reaction-diffusion models have been applied to analyze the respective contributions of the gurken-Egfr and spätzle-Toll pathways in generating embryonic pattern. The current studies provide experimental support for this theoretical work, and present opportunities for understanding the underlying mechanisms (Morisato, 2001).

Formation and maintenance of the Dorsal gradient appear dynamic. The shape of the Dorsal gradient in the wild-type embryo does not change markedly after nuclear translocation is first detected. In embryos laid by grk females or embryos expressing high levels of Spätzle, however, the shape of the Dorsal gradient is subtly modified. In particular, the minimum lying between the two Dorsal peaks becomes deeper in older embryos. This observation suggests that signaling takes place over a period of time, and explains how an initial asymmetry, in the form of the broad stripe of pipe, might be gradually refined into a gradient of positional information (Morisato, 2001).

In embryos produced by grk females, it is inferred that Spätzle processing is occurring at wild-type levels, but the reaction is distributed over a broader domain. The ventral region becomes sufficiently expanded such that the difference between the diffusion rates of processed Spätzle and the inhibitor can reshape the ventral domain itself. In particular, rapid diffusion of the inhibitor results in a lower concentration at each border, compared with the center of the domain. This change in the ratio of processed Spätzle to inhibitor eventually produces a peak at each border of the expanded domain. By this reasoning, an expanded ventral domain never generates more than two peaks because there are never more than two borders (Morisato, 2001).

Embryos synthesizing high levels of precursor Spätzle increase the amount of processed Spätzle, thereby expanding the domain of high nuclear Dorsal. In contrast to embryos produced by grk females, where a wild-type level of processed Spätzle is distributed over a broader area, an increased level of processed Spätzle appears to generate a broader domain in these injected embryos. Pattern refinement is observed only at the highest levels of Spätzle production, perhaps because only in this situation can the minimum domain size be created (Morisato, 2001).

The complexity of the patterning process is underscored by the observation that partial axis duplication can be induced by both an increase and decrease in spz dosage, depending on the extent of pipe expression dictated by gurken-Egfr signaling. A deeper understanding of this dynamic behavior will probably require the application of mathematical approaches (Morisato, 2001).

Evidence is presented for the following model, which accounts for many of the observations described above. The initial shape of the gradient (at t0) is established by the proteolytic activation of Spätzle in a relatively broad domain, reflecting the ventral region of the egg chamber that expresses pipe RNA. It is proposed that the Spätzle processing reaction generates an inhibitor that negatively regulates the production of the ventral signal, possibly at the level of Easter protease activity or the interaction between processed Spätzle and Toll. Whereas processed C-terminal Spätzle is believed to bind to Toll quickly and show limited movement after cleavage, it is postulated that the hypothetical inhibitor undergoes broader diffusion. In the wild-type embryo, inhibitor action is responsible for establishing the region of high nuclear Dorsal, corresponding to the Twist domain, to be narrower than the ventral region of the egg chamber expressing pipe RNA. The final shape of the Dorsal gradient (at t1) is generated over time by the opposing effects of processed Spätzle and the inhibitor (Morisato, 2001).

A feedback loop between Wolbachia and the Drosophila gurken mRNP complex influences Wolbachia titer

Although much is known about interactions between bacterial endosymbionts and their hosts, little is known concerning the host factors that influence endosymbiont titer. Wolbachia bacterial endosymbionts are globally dispersed throughout most insect species and are the causative agent in filarial nematode-mediated disease. gurken (grk), a host gene encoding a crucial axis determinant, has a cumulative, dosage-sensitive impact on Wolbachia growth and proliferation during Drosophila oogenesis. This effect appears to be mediated by grk mRNA and its protein-binding partners Squid and Hrp48/Hrb27C, implicating the grk mRNA-protein (mRNP) complex as a rate-limiting host factor controlling Wolbachia titer. Furthermore, highly infected flies exhibit defects that match those occurring with disruption of grk mRNPs, such as nurse cell chromatin disruptions and malformation of chorionic appendages. These findings suggest a feedback loop in which Wolbachia interaction with the grk mRNP affects both Wolbachia titer and grk mRNP function (Serbus, 2011).

The major findings of this study are that host grk has a cumulative, dosage-sensitive impact on Wolbachia titer. This impact does not appear to be related to the Grk protein, invoking a role for the grk mRNP. Accordingly, Wolbachia exhibit association with a grk mRNP protein, and disrupting known protein constituents of the grk mRNP affects Wolbachia titer analogously to grk disruptions. Highly infected flies also have defects analogous to grk mRNP disruptions, including defects in nurse cell chromatin structure and dorsal appendage formation. These findings suggest that Wolbachia interaction with the grk mRNP has a significant impact on both Wolbachia titer and grk mRNP function (Serbus, 2011).

One of the surprising outcomes from this study is the microtubule-independent impact of grk on Wolbachia titer. Disruptions of microtubules and cytoplasmic dynein have been shown to disrupt Wolbachia distribution and density in oogenesis. One interpretation of this study is that Wolbachia are transported along microtubules into the oocyte where Wolbachia replicate preferentially at the oocyte anterior end. A role for grk in regulating Wolbachia titer initially appeared consistent with that scenario. Grk signaling is crucial for proper microtubule orientation in oogenesis, and grk mRNPs are known to be transported by the microtubule-based motor, cytoplasmic dynein. Thus grk could be argued to directly or indirectly affect Wolbachia transport toward a replication-promoting area of the oocyte. However, the results of this study indicate that the impact of grk on Wolbachia titer is independent of these models. grk has a comparable repressive effect on Wolbachia titer in both nurse cells and the oocyte throughout oogenesis, although grk is primarily known to affect oocyte microtubules. The effects of grk on Wolbachia titer are detected before any known influence of grk on microtubules in oogenesis. Furthermore, colcemid tests indicate that the impact of grk on Wolbachia titer is largely independent of microtubules in both nurse cells and the oocyte. This indicates that grk affects Wolbachia titer primarily through a different mechanism (Serbus, 2011).

A microtubule-independent role for grk could be explained by a previously unrecognized function for grk mRNA or Grk protein. An initial genetic tests did not differentiate between these possibilities because the grk mutants used disrupt both mRNA and protein, and the grk overexpression tests should elevate both mRNA and protein loads. However, this issue is addressed by using well-established mutations in translational repressors sqd and hrb27C, which encode components of the grk mRNP complex that repress grk translation. The reduction in Wolbachia titer seen in sqd and hrb27C mutants ultimately suggest that Wolbachia density does not correlate with Grk protein availability in the cytoplasm. An alternative possibility is that the grk mRNP complex has a function in regulating Wolbachia titer. The appearance of Wolbachia associated with GFP-Sqd in fixed samples and live imaging are also consistent with a possible interaction between Wolbachia and mRNP components (Serbus, 2011).

One of the issues raised by this study is specificity of the grk effect because Sqd and Hrb27C are hnRNP proteins that are not exclusive to the grk mRNP complex. Thus, it is also possible that distinct mRNPs with a protein composition similar to grk mRNPs also contribute to Wolbachia titer control. However osk mRNP complexes are thought to share many components with grk mRNPs, including Sqd and Hrb27C, yet genetic disruptions of osk that reduce both mRNA and protein load did not induce a striking reduction in Wolbachia titer in a preliminary screen. Although this study does not rule out a role for other mRNPs, it suggests that the grk-related effects on Wolbachia titer are not necessarily a general property of host mRNPs (Serbus, 2011).

One of the remaining questions is how a grk mRNP exerts an influence on Wolbachia titer. Electron microscopy evidence shows no indication of a mortality-based effect. The significant increase in Wolbachia doublets detected in grk mutant nurse cells might be informative, however. Perhaps grk mutants prevent final abscission of the Wolbachia membrane during binary fission. Another interpretation is that upon completion of binary fission, the Wolbachia daughter cells remain trapped within the single original host vacuole, subjecting the bacteria to competition for limited nutrient resources. Either scenario would be consistent with a role for host grk in promoting Wolbachia growth and proliferation. Future studies are needed to address how directly this defect might be attributable to the grk mRNP complex (Serbus, 2011).

An association of Wolbachia with GFP-Sqd raises the possibility that grk mRNPs affect Wolbachia biochemistry or trafficking though a direct mechanism. It is also possible that the impact of grk mRNPs on Wolbachia is facilitated by intermediate factors. For example, Sqd has been shown to coimmunoprecipitate with the retinoblastoma Rb protein from Drosophila cell culture and ovarian extract. Rb is known to bind and repress E2F family transcription factors. Thus, grk mRNPs might affect Dp/E2F-based host transcriptional activation patterns that support Wolbachia trafficking and/or replication. Furthermore, Sqd binds Cup, a translational repressor that is required for localization of grk mRNA. Cup has been shown to interact with Nup154, a member of a protein family that supports nuclear pore assembly and nuclear import processes. This scenario provides another route by which grk mRNPs might affect availability of host products relevant to Wolbachia titer regulation (Serbus, 2011).

One of the additional questions raised by this study is its applicability to neglected tropical diseases. It is known that Wolbachia endosymbionts of Onchocerca volvulus, Wuchereria bancrofti and Brugia malayi contribute significantly to African river blindness and lymphatic filariasis. Because elimination of Wolbachia from these nematodes disrupts both the filarial host and manifestations of disease, any insight into Wolbachia titer control is potentially useful. The recently sequenced Brugia genome does not appear to encode a grk gene, but it has possible homologs for hrb27C and sqd, as well as for the grk mRNA-binding proteins Bruno/Aret, Imp and Orb. Thus, a speculative possibility is that Brugia cells might also harbor grk-like mRNPs that exert an influence on Wolbachia titer (Serbus, 2011).

This study raises the further question of whether intracellular pathogens could interact similarly with host mRNP components. Viruses such as hepatitis C have been shown to bind host mRNP proteins and use them to facilitate viral protein synthesis and viral replication. Although preliminary, there are some hints that pathogenic bacteria interact with host mRNPs as well. A number of genome-wide RNAi screens have been done in Drosophila tissue culture to assess the effect of host factors on Listeria, Mycobacterium, Chlamydia and Francisella infection. This work indicated that disruption of certain splicing or translation initiation factors correlated with reductions in intracellular Listeria, Chlamydia and Francisella infection levels. The datasets also indicate that disruption of the grk mRNP component hrb27C, or Brain Tumor, a suppressor of Hunchback translation, reduced Francisella and Listeria infection loads. It will be of great interest to see whether future studies find Wolbachia interactions with grk mRNP components to be representative of a generalized titerinfluencing mechanism shared by other intracellular symbionts and pathogens (Serbus, 2011).

During oogenesis, Wolbachia are not only positioned where key developmental events occur, but also rely on the same transport mechanisms as many of the morphogens that control these events. For example, early in oogenesis, anterior localization of Wolbachia occurs at the same time and position as that of the patterning events that establish the anterior- posterior axis. Later in oogenesis, both Wolbachia and host germline determinants rely on the motor protein kinesin-1 to concentrate at the posterior pole. In both of these situations, despite relying on the same transport mechanisms and occupying the same position as the host patterning molecules, Wolbachia do not interfere with these essential developmental events. This suggests that Wolbachia achieve a balance in which titer is maximized without disrupting oocyte development. Support for this comes from the finding that Wolbachia with abnormally high titer produce defects in dorsal appendage formation. Because this event relies on the Grk signaling pathway, one possibility is that this occurs as a consequence of a disruptive association of Wolbachia with grk mRNP components. This would create a selective pressure to establish more moderate Wolbachia levels within the host. Thus, the functional interaction between Wolbachia and grk provides a molecular example for how the interests of host and Wolbachia success can be achieved (Serbus, 2011).

Other symbiotic organisms have been shown to direct morphogenetic processes in the host. Vibrio fischerei induce formation of the light-producing organ in squid and Rhizobium induce root nodule formation in leguminous plants for nitrogen fixation. Perhaps the interaction between Wolbachia and grk represents a step toward the evolution of symbiosis in which Wolbachia also become integral to regulation of host morphogenesis (Serbus, 2011).


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gurken: Biological Overview | Regulation | Factors affecting Gurken RNA localization and translation | Developmental Biology | Effects of Mutation

date revised: 21 November 2016 

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