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.     

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.     

Ecdysone response genes govern egg chamber development during mid-oogenesis in Drosophila.

The steroid hormone ecdysone regulates larval development and metamorphosis in Drosophila melanogaster through a complex genetic hierarchy that begins with a small set of early response genes. Here, we present data indicating that the ecdysone response hierarchy also mediates egg chamber maturation during mid-oogenesis. E75, E74 and BR-C are expressed in a stage-specific manner while EcR expression is ubiquitous throughout oogenesis. Decreasing or increasing the ovarian ecdysone titer using a temperature-sensitive mutation or exogenous ecdysone results in corresponding changes in early gene expression. The stage 10 follicle cell expression of E75 in wild-type, K10 and EGF receptor (Egfr) mutant egg chambers reveals regulation of E75 by both the Egfr and ecdysone signaling pathways. Genetic analysis indicates a germline requirement for ecdysone-responsive gene expression. Germline clones of E75 mutations arrest and degenerate during mid-oogenesis and EcR germline clones exhibit a similar phenotype, demonstrating a functional requirement for ecdysone responsiveness during the vitellogenic phase of oogenesis. Finally, the expression of Drosophila Adrenodoxin Reductase increases during mid-oogenesis and clonal analysis confirms that this steroidogenic enzyme is required in the germline for egg chamber development. Together these data suggest that the temporal expression profile of E75, E74 and BR-C may be a functional reflection of ecdysone levels and that ecdysone provides temporal signals regulating the progression of oogenesis and proper specification of dorsal follicle cell fates.     

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.     

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.     

D-cbl, a negative regulator of the Egfr pathway, is required for dorsoventral patterning in Drosophila oogenesis

During Drosophila oogenesis, asymmetrically localized Gurken activates the EGF receptor (Egfr) and determines dorsal follicle cell fates. Using a mosaic follicle cell system we have identified a mutation in the D-cbl gene which causes hyperactivation of the Egfr pathway. Cbl proteins are known to downregulate activated receptors. We find that the abnormal Egfr activation is ligand dependent. Our results show that the precise regulation of Egfr activity necessary to establish different follicle cell fates requires two levels of control. The localized ligand Gurken activates Egfr to different levels in different follicle cells. In addition, Egfr activity has to be repressed through the activity of D-cbl to ensure the absence of signaling in the ventral most follicle cells.     

Mechanisms of Gurken-dependent pipe regulation and the robustness of dorsoventral patterning in Drosophila

The restriction of Pipe, a potential glycosaminoglycan-modifying enzyme, to ventral follicle cells of the egg chamber is essential for dorsoventral axis formation in the Drosophila embryo. pipe repression depends on the TGFalpha-like ligand Gurken, which activates the Drosophila EGF receptor in dorsal follicle cells. An analysis of Raf mutant clones shows that EGF signalling is required cell-autonomously in all dorsal follicle cells along the anteroposterior axis of the egg chamber to repress pipe. However, the autoactivation of EGF signalling important for dorsal follicle cell patterning has no influence on pipe expression. Clonal analysis shows that also the mirror-fringe cassette suggested to establish a secondary signalling centre in the follicular epithelium is not involved in pipe regulation. These findings support the view that the pipe domain is directly delimited by a long-range Gurken gradient. Pipe induces ventral cell fates in the embryo via activation of the Sp?tzle/Toll pathway. However, large dorsal patches of ectopic pipe expression induced by Raf clones rarely affect embryonic patterning if they are separated from the endogenous pipe domain. This indicates that potent inhibitory processes prevent pipe dependent Toll activation at the dorsal side of the egg.     

Temporal comparison of Broad-Complex expression during eggshell-appendage patterning and morphogenesis in two Drosophila species with different eggshell-appendage numbers

A central question in biology is how developmental mechanisms are altered to bring about morphological evolution. Drosophilids boast a remarkable diversity in eggshell-appendage number-from as few as one to as many as nine, depending on the species. Appendage patterning in Drosophila melanogaster is well characterized, inviting candidate-gene-based approaches that identify the developmental mechanisms underlying Drosophilid eggshell diversity. Previous studies show that a combination of Epidermal growth factor receptor (EGFR) and TGFbeta/BMP2,4 Decapentaplegic (DPP) signaling determines appendage fate in D. melanogaster. Broad-Complex expression integrates EGFR and DPP signaling and predicts future appendage position. Here we present our confocal analyses of BR-C immunofluorescence and appendage morphogenesis in Drosophila melanogaster (two appendages) and Drosophila virilis (four appendages). Our comparison suggests that differences in BR-C patterns among Drosophilids may be strongly influenced by anterior-posterior information.     

Notch signaling links interactions between the C/EBP homolog slow border cells and the GILZ homolog bunched during cell migration

In the follicle cell (FC) epithelium that surrounds the Drosophila egg, a complex set of cell signals specifies two cell fates that pattern the eggshell: the anterior centripetal FC that produce the operculum and the posterior columnar FC that produce the main body eggshell structure. We have previously shown that the long-range morphogen DPP represses the expression of the bunched (bun) gene in the anterior-most centripetal FC. bun, which encodes a homolog of vertebrate TSC-22/GILZ, in turn represses anterior gene expression and antagonizes Notch signaling to restrict centripetal FC fates in posterior cells. From a screen for novel targets of bun repression we have identified the C/EBP homolog slow border cells (slbo). At stage 10A, slbo expression overlaps bun in anterior FC; by stage 10B they repress each other's expression to establish a sharp slbo/bun expression boundary. The precise position of the slbo/bun expression boundary is sensitive to Notch signaling, which is required for both slbo activation and bun repression. As centripetal migration proceeds from stages 10B-14, slbo represses its own expression and both slbo loss-of-function mutations and overexpression approaches reveal that slbo is required to coordinate centripetal migration with nurse cell dumping. We propose that in anterior FC exposed to a Dpp morphogen gradient, high and low levels of slbo and bun, respectively, are established by modulation of Notch signaling to direct threshold cell fates. Interactions among Notch, slbo and bun resemble a conserved signaling cassette that regulates mammalian adipocyte differentiation.     

Role of Notch pathway in terminal follicle cell differentiation during Drosophila oogenesis

 During Drosophila oogenesis the body axes are determined by signaling between the oocyte and the somatic follicle cells that surround the egg chamber. A key event in the establishment of oocyte anterior-posterior polarity is the differential patterning of the follicle cell epithelium along the anterior-posterior axis. Both the Notch and epithelial growth factor (EGF) receptor pathways are required for this patterning. To understand how these pathways act in the process we have analyzed markers for anterior and posterior follicle cells accompanying constitutive activation of the EGF receptor, loss of Notch function, and ectopic expression of Delta. We find that a constitutively active EGF receptor can induce posterior fate in anterior but not in lateral follicle cells, showing that the EGF receptor pathway can act only on predetermined terminal cells. Furthermore, Notch function is required at both termini for appropriate expression of anterior and posterior markers, while loss of both the EGF receptor and Notch pathways mimic the Notch loss-of-function phenotype. Ectopic expression of the Notch ligand, Delta, disturbs EGF receptor dependent posterior follicle cell differentiation and anterior-posterior polarity of the oocyte. Our data are consistent with a model in which the Notch pathway is required for early follicle cell differentiation at both termini, but is then repressed at the posterior for proper determination of the posterior follicle cells by the EGF receptor pathway. Received: 5 November 1998 / Accepted: 14 December 1998     

Local Gurken signaling and dynamic MAPK activation during Drosophila oogenesis.

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

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.