Misexpression of argos, an inhibitor of EGFR signaling in oogenesis, leads to the production of bicephalic, ventralized, and lateralized Drosophila melanogaster eggs

Epidermal growth factor receptor (EGFR) signaling pathways are frequently involved in generating cell fate diversity in a number of organisms. During anterior-posterior and dorso-ventral polarity in the Drosophila egg chamber and eggshell, EGFR signaling leads to a number of determinative events in the follicle cell layer. A high level of Gurken signal leads to the expression of argos in dorsal midline cells. Lateral follicle cells, receiving a lower level of Gurken signal, can continue to express the Broad-Complex (BR-C) and differentiate into cells which produce chorionic appendages. Misexpression of argos in mid-oogenesis causes the midline cells to retain expression of BR-C, resulting in a single fused large appendage. Evidence that argos can directly repress Gurken-induced EGFR signaling is seen when premature expression of argos is induced earlier in oogenesis. It represses the Gurken signal at stage 5-6 of oogenesis which determines posterior follicle cells and occasionally leads to eggs with anteriors at both ends. We propose that the Gurken signal at stage 9 of oogenesis induces follicle cells to take on two fates, dorsal midline and lateral, each producing different parts of the eggshell and that argos is one of the key downstream genes required to select between these two fates.     

The role of brinker in eggshell patterning

Drosophila oogenesis provides a useful system to study signal transduction pathways and their interactions. Through clonal analysis, we found that brinker (brk), a repressor of Dpp signaling, plays an important role in the Drosophila ovary, where its function is essential for dorsal appendage formation. In the absence of brk, operculum fates are specified at the expense of dorsal appendage fates. Brk is expressed by most of the oocyte associated follicle cells, starting from stage 8 of oogenesis. Transforming Growth Factor beta (TGFbeta) signaling represses brk expression in both the early stage egg chambers and in the anterior follicle cells. In brk mutant follicle cell clones at the dorsal anterior region, Broad Complex (BR-C) expression is down-regulated in a larger domain than in wild type. We show that BR-C is required for dorsal appendage development. In large anterior BR-C mutant clones, dorsal appendages are absent, and instead, the eggshell has an enlarged operculum like region at the anterior. In addition, we show that the Epidermal Growth Factor (EGF) receptor signaling represses the TGFbeta signaling in oogenesis by up-regulating brk expression. From our results and previously published data, it appears that anterior follicle cells integrate the levels of EGF receptor activation and TGFbeta receptor activation. Operculum fate results when the sum of the level of activation of both pathways reaches a threshold level, and reduction of activity of one pathway can be compensated to some extent by increase in the other pathway.     

The role of Dpp and its inhibitors during eggshell patterning in Drosophila

The Drosophila eggshell is patterned by the combined action of the epidermal growth factor [EGF; Gurken (Grk)] and transforming growth factor beta [TGF-beta; Decapentaplegic (Dpp)] signaling cascades. Although Grk signaling alone can induce asymmetric gene expression within the follicular epithelium, here we show that 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. We provide evidence 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.     

The Drosophila JNK pathway controls the morphogenesis of the egg dorsal appendages and micropyle.

During Drosophila oogenesis, the formation of the egg respiratory appendages and the micropyle require the shaping of anterior and dorsal follicle cells. Prior to their morphogenesis, cells of the presumptive appendages are determined by integrating dorsal-ventral and anterior-posterior positional information provided by the epidermal growth factor receptor (EGFR) and Decapentaplegic (Dpp) pathways, respectively. We show here that another signaling pathway, the Drosophila Jun-N-terminal kinase (JNK) cascade, is essential for the correct morphogenesis of the dorsal appendages and the micropyle during oogenesis. Mutant follicle cell clones of members of the JNK pathway, including DJNKK/hemipterous (hep), DJNK/basket (bsk), and Djun, block dorsal appendage formation and affect the micropyle shape and size, suggesting a late requirement for the JNK pathway in anterior chorion morphogenesis. In support of this view, hep does not affect early follicle cell patterning as indicated by the normal expression of kekkon (kek) and Broad-Complex (BR-C), two of the targets of the EGFR pathway in dorsal follicle cells. Furthermore, the expression of the TGF-beta homolog dpp, which is under the control of hep in embryos, is not coupled to JNK activity during oogenesis. We show that hep controls the expression of puckered (puc) in the follicular epithelium in a cell-autonomous manner. Since puc overexpression in the egg follicular epithelium mimics JNK appendages and micropyle phenotypes, it indicates a negative role of puc in their morphogenesis. The role of the JNK pathway in the morphogenesis of follicle cells and other epithelia during development is discussed.     

EGFR-dependent network interactions that pattern Drosophila eggshell appendages

Similar to other organisms, Drosophila uses its Epidermal Growth Factor Receptor (EGFR) multiple times throughout development. One crucial EGFR-dependent event is patterning of the follicular epithelium during oogenesis. In addition to providing inductive cues necessary for body axes specification, patterning of the follicle cells initiates the formation of two respiratory eggshell appendages. Each appendage is derived from a primordium comprising a patch of cells expressing broad (br) and an adjacent stripe of cells expressing rhomboid (rho). Several mechanisms of eggshell patterning have been proposed in the past, but none of them can explain the highly coordinated expression of br and rho. To address some of the outstanding issues in this system, we synthesized the existing information into a revised mathematical model of follicle cell patterning. Based on the computational model analysis, we propose that dorsal appendage primordia are established by sequential action of feed-forward loops and juxtacrine signals activated by the gradient of EGFR signaling. The model describes pattern formation in a large number of mutants and points to several unanswered questions related to the dynamic interaction of the EGFR and Notch pathways.     

mtv shapes the activity gradient of the Dpp morphogen through regulation of thickveins

Drosophila wings are patterned by a morphogen, Decapentaplegic (Dpp), a member of the TGFbeta superfamily, which is expressed along the anterior and posterior compartment boundary. The distribution and activity of Dpp signaling is controlled in part by the level of expression of its major type I receptor, thickveins (tkv). The level of tkv is dynamically regulated by En and Hh. We have identified a novel gene, master of thickveins (mtv), which downregulates expression of tkv in response to Hh and En. mtv expression is controlled by En and Hh, and is complementary to tkv expression. In this report, we demonstrate that mtv integrates the activities of En and Hh that shape tkv expression pattern. Thus, mtv plays a key part of regulatory mechanism that makes the activity gradient of the Dpp morphogen.     

Differential requirement of EGFR signaling for the expression of defective proventriculus gene in the Drosophila endoderm and ectoderm

A homeobox gene, defective proventriculus (dve), is expressed in various tissues including the ventral ectoderm and midgut. Here, we show the expression pattern of dve in the ventral ectoderm, in which dve expression is induced by Spitz, a ligand for Drosophila epidermal growth factor receptor (EGFR). In spitz mutants, dve expression is only lost in the ventral ectoderm and overexpression of Spitz induces ectopic dve activation in the ventral ectoderm. Dve expression in the middle midgut depends on Decapentaplegic (Dpp) signaling, while expression of a dominant-negative form of Drosophila EGFR (DER(DN)) also causes a marked decrease in dve expression in the middle midgut. Furthermore, heterozygous mutation of thick veins (tkv), a Dpp receptor, strongly enhances the effect of DER(DN). These results indicate that EGFR signaling is crucial for dve expression in the ventral ectoderm and is required in the middle midgut where it cooperates with Dpp signaling.     

Versatility in signalling: multiple responses to EGF receptor activation during Drosophila oogenesis

The Drosophila epidermal growth factor receptor (EGFR) is active in different tissues and is involved in diverse processes such as patterning of the embryonic ectoderm, growth and differentiation of imaginal discs and cell survival. During oogenesis, the EGFR is expressed in the somatic follicle cells that surround individual oocyte-nurse cell complexes. In response to germline signals, the follicle cells differentiate in a complex pattern, which in turn leads to the establishment of the egg axes. Two recent reports have shown that the strategies used to pattern posterior follicle cells are different from those used to pattern dorsal follicle cells. In posterior follicle cells, EGFR activity is translated into an on-off response, whereas, in dorsal follicle cells, patterning mechanisms are initiated and refined by feedback that modulates receptor activity over time.     

Combined activities of Gurken and decapentaplegic specify dorsal chorion structures of the Drosophila egg

During Drosophila oogenesis Gurken, 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. Here, 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 homologue, decapentaplegic. By ectopically expressing decapentaplegic and clonal analysis with Mothers against dpp we show 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.     

Species-specific activation of EGF receptor signaling underlies evolutionary diversity in the dorsal appendage number of the genus Drosophila eggshells

In Drosophila melanogaster, the patterning of dorsal appendages on the eggshell is strictly controlled by EGFR signaling. However, the number of dorsal appendages is remarkably diverse among Drosophila species. For example, D. melanogaster and D. virilis have two and four dorsal appendages, respectively. Here we show that during oogenesis the expression patterns of rhomboid (rho) and argos (aos), positive and negative regulators of EGFR signaling, respectively, were substantially different between D. melanogaster and D. virilis. Importantly, the number and position of both the rho expression and MAPK activation were consistent with those of the dorsal appendages in each species. Despite the differences in the spatial expression, these results suggest that the function of EGFR signaling in dorsal appendage formation is largely conserved between these two species. Thus, our results link the species-specific activation of EGFR signaling and the evolution of eggshell morphology in Drosophila.     

Evolution of BMP signaling in Drosophila oogenesis: a receptor-based mechanism

The bone morphogenetic protein (BMP) signaling pathway is a conserved regulator of cellular and developmental processes in animals. The mechanisms underlying BMP signaling activation differ among tissues and mostly reflect changes in the expression of pathway components. BMP signaling is one of the major pathways responsible for the patterning of the Drosophila eggshell, a complex structure derived from a layer of follicle cells (FCs) surrounding the developing oocyte. Activation of BMP signaling in the FCs is dynamic. Initially, signaling is along the anterior-posterior (A/P) axis; later, signaling acquires dorsal-ventral (D/V) polarity. These dynamics are regulated by changes in the expression pattern of the type I BMP receptor thickveins (tkv). We recently found that signaling dynamics and TKV patterning are highly correlated in the FCs of multiple Drosophila species. In addition, we showed that signaling patterns are spatially different among species. Here, we use a mathematical model to simulate the dynamics and differences of BMP signaling in numerous species. This model predicts that qualitative and quantitative changes in receptor expression can lead to differences in the spatial pattern of BMP signaling. We tested these predications experimentally in three different Drosophila species and through genetic perturbations of BMP signaling in D. melanogaster. On the basis of our results, we concluded that changes in tkv patterning can account for the experimentally observed differences in the patterns of BMP signaling in multiple Drosophila species.     

Sprouty is a general inhibitor of receptor tyrosine kinase signaling.

Sprouty was originally identified as an inhibitor of Drosophila FGF receptor signaling during tracheal development. By following the capacity of ectopic Sprouty to abolish the pattern of activated MAP kinase in embryos, we show that Sprouty can inhibit other receptor tyrosine kinase (RTK) signaling pathways, namely the Heartless FGF receptor and the EGF receptor. Similarly, in wing imaginal discs, ectopic Sprouty abolishes activated MAP kinase induced by the EGF receptor pathway. Sprouty expression is induced by the EGFR pathway in some, but not all, tissues in which EGFR is activated, most notably in follicle cells of the ovary, the wing imaginal disc and the eye disc. In the ovary, induction of sprouty expression follows the pattern of EGFR activation in the follicle cells. Generation of homozygous sprouty mutant follicle-cell clones demonstrates an essential role for Sprouty in restricting EGFR activation throughout oogenesis. At the stage when dorso-ventral polarity of the follicle cells is established, Sprouty limits the ventral expansion of the activating Gurken signal. Later, when dorsal appendage fates are determined, reduction of signaling by Sprouty facilitates the induction of inter-appendage cell fates. The capacity of Sprouty to reduce or eliminate accumulation of activated MAP kinase indicates that in vivo it intersects with the pathway upstream to MAP kinase. The ability of ectopic Sprouty to rescue lethality caused by activated Raf suggests that it may impinge upon the pathway by interacting with Raf or downstream to it.