3956267
Drosophila eggshell is patterned by sequential action of feedforward and feedback loops
During Drosophila oogenesis, patterning activities of the EGFR and Dpp pathways specify several subpopulations of the follicle cells that give rise to dorsal eggshell structures. The roof of dorsal eggshell appendages is formed by the follicle cells that express Broad (Br), a zinc-finger transcription factor regulated by both pathways. EGFR induces Br in the dorsal follicle cells. This inductive signal is overridden in the dorsal midline cells, which are exposed to high levels of EGFR activation, and in the anterior cells, by Dpp signaling. We show that the resulting changes in the Br pattern affect the expression of Dpp receptor thickveins (tkv), which is essential for Dpp signaling. By controlling tkv, Br controls Dpp signaling in late stages of oogenesis and, as a result, regulates its own repression in a negative-feedback loop. We synthesize these observations into a model, whereby the dynamics of Br expression are driven by the sequential action of feedforward and feedback loops. The feedforward loop controls the spatial pattern of Br expression, while the feedback loop modulates this pattern in time. This mechanism demonstrates how complex patterns of gene expression can emerge from simple inputs, through the interaction of regulatory network motifs.
1092306
Patterning and morphogenesis of the follicle cell epithelium during Drosophila oogenesis.
CONTENTS Introduction . .... Morphology of Drosophila oogenesis ........................................ Egg chamber formation and early tollicle cell differentiation .. ... Polarity determination and germ line-soma interactions during oogenesis .................. Origin of polarity AP axis formation DVaxis formation Follicle cell migration and eggshell morphogenesis ...................................................... Border cell migration Migration over the oocyte Centripetal migration Dorsal-anterior follicle cell migration and dorsal appendage formation Summary ................................................................................... ................. 541 541 542 544 .................. ...............................
40231108
The Drosophila shell game: patterning genes and morphological change.
What are the mechanisms that convert cell-fate information into shape changes and movements, thus creating the biological forms that comprise tissues and organs? Tubulogenesis of the Drosophila dorsal eggshell structures provides an excellent system for studying the link between patterning and morphogenesis. Elegant genetic and molecular analyses from over a decade provide a strong foundation for understanding the combinatorial signaling events that specify dorsal anterior cell fates within the follicular epithelium overlying the oocyte. Recent studies reveal the morphogenetic events that alter that flat epithelial sheet into two tubes; these tubes form the mold for synthesizing the dorsal appendages--eggshell structures that facilitate respiration in the developing embryo. This review summarizes the mutant analyses that give insight into these patterning and morphogenetic processes.
39757637
Germ line and soma cooperate during oogenesis to establish the dorsoventral pattern of egg shell and embryo in Drosophila melanogaster
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 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.
40063504
The TGF-beta signaling pathway is essential for Drosophila oogenesis.
We examine roles of signaling by secreted ligands of the TGF-beta family during Drosophila oogenesis. One family member, the DPP ligand encoded by the decapentaplegic (dpp) gene, is required for patterning of anterior eggshell structures. This requirement presumably reflects the expression pattern of dpp in an anterior subset of somatic follicle cells: the centripetally migrating and the nurse cell-associated follicle cells. Similar requirements are also revealed by mutations in the saxophone (sax)-encoded receptor, consistent with the idea that DPP signaling is, at least in part, mediated by the SAX receptor. A loss of germline sax function results in a block in oogenesis associated with egg chamber degeneration and a failure of the transfer of nurse cell contents to the oocyte, indicating that TGF-beta signaling is required for these events. Some phenotypes of sax mutations during oogenesis suggest that SAX responds to at least one other TGF-beta ligand as well in the posterior follicle cells.
13026511
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.
36029574
Two signalling pathways specify localised expression of the Broad-Complex in Drosophila eggshell patterning and morphogenesis.
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. We show that the 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 is induced and specified along the dorsoventral axis by an epidermal growth factor receptor signalling pathway, which includes a localised transforming growth factor-alpha like molecule, Gurken, in the oocyte and the Drosophila EGF receptor homologue, Torpedo, in the surrounding somatic follicle cells. Furthermore, the precisely localised expression of BR-C along the AP axis requires a separate signalling pathway, initiated by a transforming growth factor-beta homologue, Decapentaplegic, in nearby follicle cells. These two signalling pathways, one from the oocyte and the other from the follicle cells, co-ordinately specify patches of follicle cells to express the Broad-Complex in a unique position in respect to both major axes, which in turn directs the differentiation of the dorsal appendages in the correct position on the eggshell.
18691381
The function of the broad-complex during Drosophila melanogaster oogenesis.
The Broad-Complex (BR-C) is an early ecdysone response gene that functions during metamorphosis and encodes a family of zinc-finger transcription factors. It is expressed in a dynamic pattern during oogenesis. Its late expression in the lateral-dorsal-anterior follicle cells is related to the morphogenesis of the chorionic appendages. All four zinc-finger isoforms are expressed in oogenesis, which is consistent with the abnormal appendage phenotypes resulting from their ectopic expression. We investigated the mechanism by which the BR-C affects chorion deposition by using BrdU to follow the effects of BR-C misexpression on DNA replication and in situ hybridization to ovarian mRNA to evaluate chorion gene expression. Ectopic BR-C expression leads to prolonged endoreplication and to additional amplification of genes, besides the chorion genes, at other sites in the genome. The pattern of chorion gene expression is not affected along the anterior-posterior axis, but the follicle cells at the anterior of the oocyte fail to migrate correctly in an anterior direction when BR-C is misexpressed. We conclude that the target genes of the BR-C in oogenesis include a protein essential for endoreplication and chorion gene amplification. This may provide a link between steroid hormones and the control of DNA replication during oogenesis.
9026083
Drosophila Rhomboid-1 Defines a Family of Putative Intramembrane Serine Proteases
The polytopic membrane protein Rhomboid-1 promotes the cleavage of the membrane-anchored TGFalpha-like growth factor Spitz, allowing it to activate the Drosophila EGF receptor. Until now, the mechanism of this key signaling regulator has been obscure, but our analysis suggests that Rhomboid-1 is a novel intramembrane serine protease that directly cleaves Spitz. In accordance with the putative Rhomboid active site being in the membrane bilayer, Spitz is cleaved within its transmembrane domain, and thus is, to our knowledge, the first example of a growth factor activated by regulated intramembrane proteolysis. Rhomboid-1 is conserved throughout evolution from archaea to humans, and our results show that a human Rhomboid promotes Spitz cleavage by a similar mechanism. This growth factor activation mechanism may therefore be widespread.
15914672
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.
11142401
Capicua regulates follicle cell fate in the Drosophila ovary through repression of mirror
The dorsoventral axis of the Drosophila egg is established by dorsally localized activation of the epidermal growth factor receptor (Egfr) in the ovarian follicular epithelium. Subsequent positive- and negative-feedback regulation generates two dorsolateral follicle cell primordia that will produce the eggshell appendages. A dorsal midline domain of low Egfr activity between the appendage primordia defines their dorsal boundary, but little is known about the mechanisms that establish their ventral limit. We demonstrate that the transcriptional repressor Capicua is required cell autonomously in ventral and lateral follicle cells to repress dorsal fates, and functions in this process through the repression of mirror. Interestingly, ectopic expression of mirror in the absence of capicua is observed only in the anterior half of the epithelium. We propose that Capicua regulates the pattern of follicle cell fates along the dorsoventral axis by blocking the induction of appendage determinants, such as mirror, by anterior positional cues.
14668129
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.
1306949
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 factorβ [TGF-β; 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.
27594062
Relief of gene repression by torso RTK signaling: role of capicua in Drosophila terminal and dorsoventral patterning.
Differentiation of the embryonic termini in Drosophila depends on signaling by the Tor RTK, which induces terminal gene expression by inactivating at the embryonic poles a uniformly distributed repressor activity that involves the Gro corepressor. Here, we identify a new gene, cic, that acts as a repressor of terminal genes regulated by the Tor pathway. cic also mediates repression along the dorsoventral axis, a process that requires the Dorsal morphogen and Gro, and which is also inhibited by Tor signaling at the termini. cic encodes an HMG-box transcription factor that interacts with Gro in vitro. We present evidence that Tor signaling regulates terminal patterning by inactivating Cic at the embryo poles. cic has been evolutionarily conserved, suggesting that Cic-like proteins may act as repressors regulated by RTK signaling in other organisms.
2426344
The drosophila dorsoventral patterning gene gurken produces a dorsally localized RNA and encodes a TGFα-like protein
Cell-cell interactions in the Drosophila ovary play a crucial role in the establishment of dorsoventral polarity of both the egg shell and the future embryo. Torpedo/DER (top/DER), a homolog of the vertebrate epidermal growth factor receptor, is required for this signaling process in the somatic cells of the ovary. In contrast, gurken (grk), which also functions in this pathway, is required in the germline. We cloned the grk gene and found that it encodes a TGF alpha-like protein. Grk is, therefore, likely to be a ligand of top/DER, activating the receptor during oogenesis. During oogenesis, the grk transcript becomes asymmetrically localized to the dorsal corner of the oocyte. We propose that the dorsal localization of grk RNA results in a spatially restricted ligand that asymmetrically activates the receptor.
14106786
Ectopic activation of torpedo/Egfr, a Drosophila receptor tyrosine kinase, dorsalizes both the eggshell and the embryo.
The Drosophila gene torpedo/Egfr (top/Egfr) encodes a homolog of the vertebrate Epidermal Growth Factor receptor. This receptor is required several times during the life cycle of the fly for the transmisson of developmental cues. During oogenesis, Top/Egfr activation is required for the establishment of the dorsal/ventral axis of the egg and the embryo. To examine how ectopic Top/Egfr activation affects cell fate determination, we constructed an activated version of the protein. Expression of this activated form (lambda top) in the follicle cells of the ovary induces dorsal cell fates in both the follicular epithelium and the embryo. Different levels of expression resulted in different dorsal follicle cell fates. These dorsal cell fates were expanded in the anterior, but not the posterior, of the egg, even in cases where all the follicle cells covering the oocyte expressed lambda top. The expression of genes known to respond to top/Egfr activation, argos (aos), kekkon1 (kek 1) and rhomboid (rho), was also expanded in the presence of the lambda top construct. When lambda top was expressed in all the follicle cells covering the oocyte, kek 1 and argos expression was induced in follicle cells all along the anterior/posterior axis of the egg chamber. In contrast, rho RNA expression was only activated in the anterior of the egg chamber. These data indicate that the response to Top/Egfr signaling is regulated by an anterior/posterior prepattern in the follicle cells. Expression of lambda top in the entire follicular epithelium resulted in an embryo dorsalized along the entire anterior/posterior axis. Expression of lambda top in anterior or posterior subpopulations of follicle cells resulted in regionally autonomous dorsalization of the embryos. This result indicates that subpopulations of follicle cells along the anterior/posterior axis can respond to Top/Egfr activation independently of one another.
7466642
Quantitative analysis of the GAL4/UAS system in Drosophila oogenesis
The GAL4/UAS system is extensively used for targeted gene expression in Drosophila, but the strength of the GAL4 drivers and their effects on target genes are rarely quantified. Quantitative information about the strength of the perturbations introduced by the GAL4/UAS system would further expand the usefulness of the GAL4/UAS system in studying gene functions and developmental processes. We have developed an assay to determine the relative level of expression for target genes tagged with green fluorescent protein (GFP). Our assay enables the relative quantitation of fluorescent proteins within specific cell types and developmental time windows in living eggs/embryos, and permits the analysis of samples from a broad expression range. We illustrate the assay using a panel of four GAL4 drivers and three UAS responder lines in Drosophila oogenesis, discuss the issues associated with the interpretation of the quantitative data, and correlate our results with the analysis of the GAL4/UAS system at the transcript level. The imaging‐based strategy described here can be used to quantify other GAL4 drivers in Drosophila and other organisms. genesis 44:66–74, 2006. Published 2006 Wiley‐Liss, Inc.
5644643
Analysis of genetic mosaics in developing and adult Drosophila tissues.
We have constructed a series of strains to facilitate the generation and analysis of clones of genetically distinct cells in developing and adult tissues of Drosophila. Each of these strains carries an FRT element, the target for the yeast FLP recombinase, near the base of a major chromosome arm, as well as a gratuitous cell-autonomous marker. Novel markers that carry epitope tags and that are localized to either the cell nucleus or cell membrane have been generated. As a demonstration of how these strains can be used to study a particular gene, we have analyzed the developmental role of the Drosophila EGF receptor homolog. Moreover, we have shown that these strains can be utilized to identify new mutations in mosaic animals in an efficient and unbiased way, thereby providing an unprecedented opportunity to perform systematic genetic screens for mutations affecting many biological processes.
19979092
Identifying loci required for follicular patterning using directed mosaics.
We have developed a 'directed mosaic' system in Drosophila by using the GAL4 system to control the expression of the yeast recombinase, FLP, in a spatial and temporal fashion. By directing FLP expression, we show that it is possible to efficiently and specifically target loss-of-function studies for vital loci to the developmental pathway of interest. A simple F1 adult phenotypic screen demonstrated that most adult tissues can be analyzed with this approach. Using GAL4 lines expressed during oogenesis, we have refined the system to examine the roles of vital loci in the development of the follicular epithelium. We have identified essential genes involved in egg chamber organization, cell migration and cell shape. Further, we have used this technique to gain insights into the role of the Drosophila EGF receptor pathway in establishing the egg axes. Finally, using different UAS-FLP, GAL4 and existing FRT lines, we have built stocks that permit the analysis of approximately 95% of the genome in follicular mosaics.
1521076
The torso receptor tyrosine kinase can activate raf in a ras-independent pathway
Activation of the receptor tyrosine kinase (RTK) torso defines the spatial domains of expression of the transcription factors tailless and huckebein. Previous analyses have demonstrated that Ras1 (p21ras) operates upstream of the D-Raf (Raf1) serine/threonine kinase in this signaling pathway. By using a recently developed technique of germline mosaics, we find that D-Raf can be activated by torso in the complete absence of Ras1. This result is supported by analysis of D-Raf activation in the absence of either the exchange factor Son of sevenless (Sos) or the adaptor protein drk (Grb2), as well as by the phenotype of a D-Raf mutation that abolishes binding of Ras1 to D-Raf. Our study provides in vivo evidence that Raf can be activated by an RTK in a Ras-independent pathway.
25768626
Mosaic analyses reveal the function of Drosophila Ras in embryonic dorsoventral patterning and dorsal follicle cell morphogenesis.
In Drosophila melanogaster, the Ras signal transduction pathway is the primary effector of receptor tyrosine kinases, which govern diverse developmental programs. During oogenesis, epidermal growth factor receptor signaling through the Ras pathway patterns the somatic follicular epithelium, establishing the dorsoventral asymmetry of eggshell and embryo. Analysis of follicle cell clones homozygous for a null allele of Ras demonstrates that Ras is required cell-autonomously to repress pipe transcription, the critical first step in embryonic dorsoventral patterning. The effects of aberrant pipe expression in Ras mosaic egg chambers can be ameliorated, however, by post-pipe patterning events, which salvage normal dorsoventral polarity in most embryos derived from egg chambers with dorsal Ras clones. The patterned follicular epithelium also determines the final shape of the eggshell, including the dorsal respiratory appendages, which are formed by the migration of two dorsolateral follicle cell populations. Confocal analyses of mosaic egg chambers demonstrate that Ras is required both cell- and non cell-autonomously for morphogenetic behaviors characteristic of dorsal follicle cell migration, and reveal a novel, Ras-dependent pattern of basal E-cadherin localization in dorsal midline follicle cells.
43478668
Refining GAL4‐driven transgene expression in Drosophila with a GAL80 enhancer‐trap
We constructed an enhancer‐trap element, P{GAL80}, that encodes the yeast GAL80 repressor to refine expression of transgenes driven by the binary GAL4/UAS system. GAL80 blocks GAL4 activity by binding to its transcriptional activation domain. We screened GAL80 enhancer‐traps for repression of GAL4‐induced green fluorescent protein (GFP) in the intact larval nervous system. We selected one line that repressed GFP in a large set of cholinergic neurons. This line was used to refine GFP expression from a set of over 200 neurons to a subset of 20 neurons in a preselected GAL4 line. Expression of tetanus neurotoxin, a potent blocker of neurotransmitter release, in these 20 neurons reproduced an aberrant larval turning behavior previously assigned to the parental set of 200 neurons. Our results suggest that targeted GAL80 expression could become a useful means of spatially refining transgene expression in Drosophila. genesis 39:240–245, 2004. © 2004 Wiley‐Liss, Inc.
41583218
Analysis of cell migration using whole-genome expression profiling of migratory cells in the Drosophila ovary.
Cell migration contributes to normal development and homeostasis as well as to pathological processes such as inflammation and tumor metastasis. Previous genetic screens have revealed signaling pathways that govern follicle cell migrations in the Drosophila ovary, but few downstream targets of the critical transcriptional regulators have been identified. To characterize the gene expression profile of two migratory cell populations and identify Slbo targets, we purified border cells and centripetal cells expressing the mouse CD8 antigen and carried out whole-genome microarray analysis. Genes predicted to control actin dynamics and the endocytic and secretory pathways were overrepresented in the migratory cell transcriptome. Mutations in five genes, including ttk, failed to complement previously isolated mutations that cause cell migration defects in mosaic clones. Functional analysis revealed a role for the Notch-activating protease Kuzbanian in border cell migration and identified Tie as a guidance receptor for the border cells.
12232483
Regulatory mechanisms required for DE-cadherin function in cell migration and other types of adhesion
Cadherin-mediated adhesion can be regulated at many levels, as demonstrated by detailed analysis in cell lines. We have investigated the requirements for Drosophila melanogaster epithelial (DE) cadherin regulation in vivo. Investigating D. melanogaster oogenesis as a model system allowed the dissection of DE-cadherin function in several types of adhesion: cell sorting, cell positioning, epithelial integrity, and the cadherin-dependent process of border cell migration. We generated multiple fusions between DE-cadherin and α-catenin as well as point-mutated β-catenin and analyzed their ability to support these types of adhesion. We found that (1) although linking DE-cadherin to α-catenin is essential, regulation of the link is not required in any of these types of adhesion; (2) β-catenin is required only to link DE-cadherin to α-catenin; and (3) the cytoplasmic domain of DE-cadherin has an additional specific function for the invasive migration of border cells, which is conserved to other cadherins. The nature of this additional function is discussed.
2775838
Systems-level questions in Drosophila oogenesis.
This paper describes computational and experimental work on pattern formation in Drosophila egg development (oogenesis), an established experimental model for studying cell fate diversification in developing tissues. Epidermal growth factor receptor (EGFR) is a key regulator of pattern formation and morphogenesis in Drosophila oogenesis. EGFR signalling in oogenesis can be genetically manipulated and monitored at many levels, leading to large sets of heterogeneous data that enable the formulation of increasingly quantitative models of pattern formation in these systems.
18053449
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.
11663510
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. We have developed a parameter estimation approach for quantifying the spatial distribution of Gurken, a TGFalpha-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, we have estimated the value of the Thiele modulus 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.
14484273
Sequential activation of the EGF receptor pathway during Drosophila oogenesis establishes the dorsoventral axis.
Previous work has demonstrated a role for the Drosophila EGF receptor (Torpedo/DER) and its ligand, Gurken, in the determination of anterioposterior and dorsoventral axes of the follicle cells and oocyte. The roles of DER in establishing the polarity of the follicle cells were examined further, by following the expression of DER-target genes. One class of genes (e.g. kekon) is induced by the DER pathway at all stages. Broad expression of kekon at the stage in which the follicle cells migrate posteriorly over the oocyte, demonstrates the capacity of the pathway to pattern all follicle cells except the ventral-most rows. This may provide the spatial coordinates for the ventral-most follicle cell fates. A second group of target genes (e.g. rhomboid (rho)) is induced only at later stages of oogenesis, and may require additional inputs by signals emanating from the anterior, stretch follicle cells. The function of Rho was analyzed by ectopic expression in the stretch follicle cells, and shown to induce a non-autonomous dorsalizing activity that is independent of Gurken. Rho thus appears to be involved in processing a DER ligand in the follicle cells, to pattern the egg chamber and allow persistent activation of the DER pathway during formation of the dorsal appendages.
7089767
An Autoregulatory Cascade of EGF Receptor Signaling Patterns the Drosophila Egg
Intercellular signaling through the EGF receptor (EGFR) patterns the Drosophila egg. The TGF alpha-like ligand Gurken signals from the oocyte to the receptor in the overlying somatic follicle cells. We show that in the dorsal follicle cells this initial paracrine signaling event triggers an autocrine amplification by two other EGFR ligands, Spitz and Vein. Spitz only becomes an effective ligand in the presence of the multitransmembrane domain protein Rhomboid. Consequent high-level EGFR activation leads to localized expression of the diffusible inhibitor Argos, which alters the profile of signaling. This sequential activation, amplification, and local inhibition of the EGFR forms an autoregulatory cascade that leads to the splitting of an initial single peak of signaling into two, thereby patterning the egg.
28864722
Hrs mediates downregulation of multiple signalling receptors in Drosophila
Endocytosis and subsequent lysosomal degradation of activated signalling receptors can attenuate signalling. Endocytosis may also promote signalling by targeting receptors to specific compartments. A key step regulating the degradation of receptors is their ubiquitination. Hrs/Vps27p, an endosome‐associated, ubiquitin‐binding protein, affects sorting and degradation of receptors. Drosophila embryos mutant for hrs show elevated receptor tyrosine kinase (RTK) signalling. Hrs has also been proposed to act as a positive mediator of TGF‐β signalling. We find that Drosophila epithelial cells devoid of Hrs accumulate multiple signalling receptors in an endosomal compartment with high levels of ubiquitinated proteins: not only RTKs (EGFR and PVR) but also Notch and receptors for Hedgehog and Dpp (TGF‐β related). Hrs is not required for Dpp signalling. Instead, loss of Hrs increases Dpp signalling and the level of the type‐I receptor Thickveins (Tkv). Finally, most hrs‐dependent receptor turnover appears to be ligand independent. Thus, both active and inactive signalling receptors are targeted for degradation in vivo and Hrs is required for their removal.
5998789
Oogenic function of the myogenic factor D-MEF2: negative regulation of the decapentaplegic receptor gene thick veins.
The myogenic factor D-MEF2 is required for the proper differentiation of muscle cells during Drosophila embryogenesis and the correct patterning of indirect flight muscles assembled during later metamorphosis. In addition to these essential myogenic functions, mutant D-mef2 adult females are weakly fertile and produce defective eggs. D-MEF2 is expressed in nurse and follicle cells of the wild-type egg chamber. We have analyzed the D-mef2 oogenic phenotype and show that the gene is required for the normal patterning and differentiation of the centripetally migrating follicle cells that are crucial for development of the anterior chorionic structures. D-mef2 alleles exhibit a genetic interaction with a dominant-negative allele of thick veins (tkv), which encodes a type I receptor of the Decapentaplegic-signaling pathway. tkv RNA is overexpressed in D-mef2 mutant egg chambers, and, conversely, forced expression of D-mef2 represses tkv expression. These results indicate a role for D-MEF2 in the regulation of tkv gene expression and Decapentaplegic signal transduction that are essential for proper determination and/or differentiation of the anterior follicle cells. Additionally, they demonstrate a vital function for the D-MEF2 transcription factor in multiple genetic pathways during Drosophila development.
26535726
Pointed, an ETS domain transcription factor, negatively regulates the EGF receptor pathway in Drosophila oogenesis.
Spatially regulated activation of the Drosophila epidermal growth factor (EGF) receptor by its ligand, Gurken, is required for establishment of the dorsal/ventral axis of the oocyte and embryo. During mid-oogenesis, Gurken is concentrated at the dorsal-anterior of the oocyte and is thought to activate the EGF receptor pathway in adjacent follicle cells. In response to this signal, dorsal follicle cell fate is determined. These cells further differentiate into either appendage-producing or midline cells, resulting in patterning in the dorsal follicle cell layer. We show here that Pointed, an ETS transcription factor, is required in dorsal follicle cells for this patterning. Loss of pointed results in the loss of midline cells and an excess of appendage-forming cells, a phenotype associated with overactivation of the EGF receptor pathway in the dorsal region. Overexpression of pointed leads to a phenotype similar to that generated by loss of the EGF receptor pathway. This suggests that Pointed normally down-regulates EGF receptor signaling in the midline to generate patterning in the dorsal region. Interestingly, pointed expression is induced by the EGF receptor pathway. These data indicate a novel antagonistic function for Pointed in oogenesis; in response to activation of the EGF receptor, pointed is expressed and negatively regulates the EGF receptor pathway, possibly by integrating information from a second pathway.
3144048
Border of Notch activity establishes a boundary between the two dorsal appendage tube cell types.
Boundaries establish and maintain separate populations of cells critical for organ formation. We show that Notch signaling establishes the boundary between two types of post-mitotic epithelial cells, the Rhomboid- and the Broad-positive cells. These cells will undergo morphogenetic movements to generate the two sides of a simple organ, the dorsal appendage tube of the Drosophila egg chamber. The boundary forms due to a difference in Notch levels in adjacent cells. The Notch expression pattern mimics the boundary; Notch levels are high in Rhomboid cells and low in Broad cells. Notch(-) mutant clones generate an ectopic boundary: ectopic Rhomboid cells arise in Notch(+) cells adjacent to the Notch(-) mutant cells but not further away from the clonal border. Pangolin, a component of the Wingless pathway, is required for Broad expression and for rhomboid repression. We further show that Broad represses rhomboid cell autonomously. Our data provide a foundation for understanding how a single row of Rhomboid cells arises adjacent to the Broad cells in the dorsal appendage primordia. Generating a boundary by the Notch pathway might constitute an evolutionarily conserved first step during organ formation in many tissues.
17769723
Top–DER- and Dpp-dependent requirements for the Drosophila fos/kayak gene in follicular epithelium morphogenesis
The Drosophila fos (Dfos)/kayak gene has been previously identified as a key regulator of epithelial cell morphogenesis during dorsal closure of the embryo and fusion of the adult thorax. We show here that it is also required for two morphogenetic movements of the follicular epithelium during oogenesis. Firstly, it is necessary for the proper posteriorward migration of main body follicle cells during stage 9. Secondly, it controls, from stage 11 onwards, the morphogenetic reorganization of the follicle cells that are committed to secrete the respiratory appendages. We demonstrate that DER pathway activation and a critical level of Dpp/TGFbeta signalling are required to pattern a high level of transcription of Dfos at the anterior and dorsal edges of the two groups of cells that will give rise to the respiratory appendages. In addition, we provide evidence that, within the dorsal-anterior territory, the level of paracrine Dpp/TGFbeta signalling controls the commitment of follicle cells towards either an operculum or an appendage secretion fate. Finally, we show that Dfos is required in follicle cells for the dumping of the nurse cell cytoplasm into the oocyte and the subsequent apoptosis of nurse cells. This suggests that in somatic follicle cells, Dfos controls the expression of one or several factors that are necessary for these processes in underlying germinal nurse cells.
39753993
Hox Control of Organ Size by Regulation of Morphogen Production and Mobility
Selector genes modify developmental pathways to sculpt animal body parts. Although body parts differ in size, the ways in which selector genes create size differences are unknown. We have studied how the Drosophila Hox gene Ultrabithorax (Ubx) limits the size of the haltere, which, by the end of larval development, has ∼fivefold fewer cells than the wing. We find that Ubx controls haltere size by restricting both the transcription and the mobility of the morphogen Decapentaplegic (Dpp). Ubx restricts Dpp's distribution in the haltere by increasing the amounts of the Dpp receptor, thickveins. Because morphogens control tissue growth in many contexts, these findings provide a potentially general mechanism for how selector genes modify organ sizes.
4392409
A genome-wide transgenic RNAi library for conditional gene inactivation in Drosophila
Forward genetic screens in model organisms have provided important insights into numerous aspects of development, physiology and pathology. With the availability of complete genome sequences and the introduction of RNA-mediated gene interference (RNAi), systematic reverse genetic screens are now also possible. Until now, such genome-wide RNAi screens have mostly been restricted to cultured cells and ubiquitous gene inactivation in Caenorhabditis elegans. This powerful approach has not yet been applied in a tissue-specific manner. Here we report the generation and validation of a genome-wide library of Drosophila melanogaster RNAi transgenes, enabling the conditional inactivation of gene function in specific tissues of the intact organism. Our RNAi transgenes consist of short gene fragments cloned as inverted repeats and expressed using the binary GAL4/UAS system. We generated 22,270 transgenic lines, covering 88% of the predicted protein-coding genes in the Drosophila genome. Molecular and phenotypic assays indicate that the majority of these transgenes are functional. Our transgenic RNAi library thus opens up the prospect of systematically analysing gene functions in any tissue and at any stage of the Drosophila lifespan.
7169406
Network motifs: theory and experimental approaches
Transcription regulation networks control the expression of genes. The transcription networks of well-studied microorganisms appear to be made up of a small set of recurring regulation patterns, called network motifs. The same network motifs have recently been found in diverse organisms from bacteria to humans, suggesting that they serve as basic building blocks of transcription networks. Here I review network motifs and their functions, with an emphasis on experimental studies. Network motifs in other biological networks are also mentioned, including signalling and neuronal networks.
15323746
EDL/MAE regulates EGF-mediated induction by antagonizing Ets transcription factor Pointed
Inductive patterning mechanisms often use negative regulators to coordinate the effects and efficiency of induction. During Spitz EGF-mediated neuronal induction in the Drosophila compound eye and chordotonal organs, Spitz causes activation of Ras signaling in the induced cells, resulting in the activation of Ets transcription factor Pointed P2. We describe developmental roles of a novel negative regulator of Ras signaling, EDL/MAE, a protein with an Ets-specific Pointed domain but not an ETS DNA-binding domain. The loss of EDL/MAE function results in reduced number of photoreceptor neurons and chordotonal organs, suggesting a positive role in the induction by Spitz EGF. However, EDL/MAE functions as an antagonist of Pointed P2, by binding to its Pointed domain and abolishing its transcriptional activation function. Furthermore, edl/mae appears to be specifically expressed in cells with inducing ability. This suggests that inducing cells, which can respond to Spitz they themselves produce, must somehow prevent activation of Pointed P2. Indeed hyperactivation of Pointed P2 in inducing cells interferes with their inducing ability, resulting in the reduction in inducing ability. We propose that EDL/MAE blocks autocrine activation of Pointed P2 so that inducing cells remain induction-competent. Inhibition of inducing ability by Pointed probably represents a novel negative feedback system that can prevent uncontrolled spread of induction of similar cell fates.
14783869
The Transmembrane Molecule Kekkon 1 Acts in a Feedback Loop to Negatively Regulate the Activity of the Drosophila EGF Receptor during Oogenesis
We have identified the Drosophila transmembrane molecule kekkon 1 (kek1) as an inhibitor of the epidermal growth factor receptor (EGFR) and demonstrate that it acts in a negative feedback loop to modulate the activity of the EGFR tyrosine kinase. During oogenesis, kek1 is expressed in response to the Gurken/EGFR signaling pathway, and loss of kek1 activity is associated with an increase in EGFR signaling. Consistent with our loss-of-function studies, we demonstrate that ectopic overexpression of kek1 mimics a loss of EGFR activity. We show that the extracellular and transmembrane domains of Kek1 can inhibit and physically associate with the EGFR, suggesting potential models for this inhibitory mechanism.
2404299
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.
4413371
Argos inhibits epidermal growth factor receptor signalling by ligand sequestration
The epidermal growth factor receptor (EGFR) has critical functions in development and in many human cancers. During development, the spatial extent of EGFR signalling is regulated by feedback loops comprising both well-understood activators and less well-characterized inhibitors. In Drosophila melanogaster the secreted protein Argos functions as the only known extracellular inhibitor of EGFR, with clearly identified roles in multiple stages of development. Argos is only expressed when the Drosophila EGFR (DER) is activated at high levels, and downregulates further DER signalling. Although there is ample genetic evidence that Argos inhibits DER activation, the biochemical mechanism has not been established. Here we show that Argos inhibits DER signalling without interacting directly with the receptor, but instead by sequestering the DER-activating ligand Spitz. Argos binds tightly to the EGF motif of Spitz and forms a 1:1 (Spitz:Argos) complex that does not bind DER in vitro or at the cell surface. Our results provide an insight into the mechanism of Argos function, and suggest new strategies for EGFR inhibitor design.