The Drosophila JNK pathway controls the morphogenesis of imaginal discs during metamorphosis.

In Drosophila, the Jun-N-terminal Kinase-(JNK) signaling pathway is required for epithelial cell shape changes during dorsal closure of the embryo. In the absence of JNK pathway activity, as in the DJNKK/hemipterous (hep) mutant, the dorsolateral ectodermal cells fail both to elongate and move toward the dorsal midline, leading to dorsally open embryos. We show here that hep and the JNK pathway are required later in development, for correct morphogenesis of other epithelia, the imaginal discs. During metamorphosis, the imaginal discs undergo profound morphological changes, giving rise to the adult head and thoracic structures, including the cuticle and appendages. hep mutant pupae and pharate adults show severe defects in discs morphogenesis, especially in the fusion of the two lateral wing discs. We show that these defects are accompanied by a loss of expression of puckered (puc), a JNK phosphatase-encoding gene, in a subset of peripodial cells that ultimately delineates the margins of fusing discs. In further support of a role of puc in discs morphogenesis, pupal and adult hep phenotypes are suppressed by reducing puc function, indicative of a negative role of puc in disc morphogenesis. Furthermore, we show that the small GTPase Dcdc42, but not Drac1, is an activator of puc expression in a hep-dependent manner in imaginal discs. Altogether, these results demonstrate a new role for the JNK pathway in epithelial morphogenesis, and provide genetic evidence for a role of the peripodial membrane in disc morphogenesis. We discuss a general model whereby the JNK pathway regulates morphogenesis of epithelia with differentiated edges.     

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.     

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.     

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.     

JNK signaling pathway required for wound healing in regenerating Drosophila wing imaginal discs

We have examined wound healing during regeneration of Drosophila wing imaginal discs fragments by confocal microscopy and assessed the role of components of the JNK pathway in this process. After cutting, columnar and peripodial epithelia cells at the wound edge start to close the wound through formation and contraction of an actin cable. This is followed by a zipping process through filopodial protrusions from both epithelia knitting the wound edges from proximal to distal areas of the disc. Activation of the JNK pathway is involved in such process. puckered (puc) expression is induced in several rows of cells at the edge of the wound, whereas absence of JNK pathway activity brought about by hemipterous, basket, and Dfos mutants impair wound healing. These defects are accompanied by lowered or loss of expression of puc. In support of a role of puc in wound healing, hep mutant phenotypes are rescued by reducing puc function, whereas overexpression of puc inhibits wound healing. Altogether, these results demonstrate a role for the JNK pathway in imaginal disc wound healing, similar to that reported for other healing processes such as embryonic dorsal closure, thoracic closure, and adult epithelial wound healing in Drosophila. Differences with such processes are also highlighted.     

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.     

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.     

Requirements of Lgl in cell differentiation and motility during Drosophila ovarian follicular epithelium morphogenesis

The epithelial follicle cell layer over the egg chamber in Drosophila ovary undergoes patterning and morphogenesis at oogenesis. These developmental processes are essential for constructing the eggshell and establishing the body axes of the egg and resultant embryo, thereby being crucial for the egg development. We have previously shown that lethal(2)giant larvae (lgl), a Drosophila neoplastic tumor suppressor gene (nTSG) is required for the posterior follicle cell (PFC) fate induction during antero-posterior pattern formation of the follicular epithelium. In this report, we further characterize lgl in this epithelium patterning and the morphogenetic changes of specified border cells. Genetic interactions of lgl with discs large (dlg) and scribble (scrib), another two nTSGs in specifying the PFC fate reveal a cooperative role of this group of genes. Meanwhile, we find that loss of lgl function causes failure of follicle cells at the anterior to differentiate properly. The clonal analysis further indicates that lgl is necessary not only for the border cell differentiation, but also for control of the collective border cell migration via presumably modulating the apico-basal polarity and cell adhesion. Overall, we identify Lgl as an essential factor in regulating differentiation and morphogenetic movement of the ovarian epithelial follicle cells.     

Requirements of Lgl in cell differentiation and motility during Drosophila ovarian follicular epithelium morphogenesis

The epithelial follicle cell layer over the egg chamber in Drosophila ovary undergoes patterning and morphogenesis at oogenesis. These developmental processes are essential for constructing the eggshell and establishing the body axes of the egg and resultant embryo, thereby being crucial for the egg development. We have previously shown that lethal(2)giant larvae (lgl), a Drosophila neoplastic tumor suppressor gene (nTSG) is required for the posterior follicle cell (PFC) fate induction during antero-posterior pattern formation of the follicular epithelium. In this report, we further characterize lgl in this epithelium patterning and the morphogenetic changes of specified border cells. Genetic interactions of lgl with discs large (dlg) and scribble (scrib), another two nTSGs in specifying the PFC fate reveal a cooperative role of this group of genes. Meanwhile, we find that loss of lgl function causes failure of follicle cells at the anterior to differentiate properly. The clonal analysis further indicates that lgl is necessary not only for the border cell differentiation, but also for control of the collective border cell migration via presumably modulating the apico-basal polarity and cell adhesion. Overall, we identify Lgl as an essential factor in regulating differentiation and morphogenetic movement of the ovarian epithelial follicle cells.     

M6 membrane protein plays an essential role in Drosophila oogenesis

We had previously shown that the transmembrane glycoprotein M6a, a member of the proteolipid protein (PLP) family, regulates neurite/filopodium outgrowth, hence, M6a might be involved in neuronal remodeling and differentiation. In this work we focused on M6, the only PLP family member present in Drosophila, and ortholog to M6a. Unexpectedly, we found that decreased expression of M6 leads to female sterility. M6 is expressed in the membrane of the follicular epithelium in ovarioles throughout oogenesis. Phenotypes triggered by M6 downregulation in hypomorphic mutants included egg collapse and egg permeability, thus suggesting M6 involvement in eggshell biosynthesis. In addition, RNAi-mediated M6 knockdown targeted specifically to follicle cells induced an arrest of egg chamber development, revealing that M6 is essential in oogenesis. Interestingly, M6-associated phenotypes evidenced abnormal changes of the follicle cell shape and disrupted follicular epithelium in mid- and late-stage egg chambers. Therefore, we propose that M6 plays a role in follicular epithelium maintenance involving membrane cell remodeling during oogenesis in Drosophila.     

The neurogenic locus brainiac cooperates with the Drosophila EGF receptor to establish the ovarian follicle and to determine its dorsal-ventral polarity.

We have characterized the function of a new neurogenic locus, brainiac (brn), during oogenesis. Homozygous brn females lay eggs with fused dorsal appendages, a phenotype associated with torpedo (top) alleles of the Drosophila EGF receptor (DER) locus. By constructing double mutant females for both brn and top, we have found that brn is required for determining the dorsal-ventral polarity of the ovarian follicle. However, embryos from mature brn eggs develop a neurogenic phenotype which can be zygotically rescued if a wild-type sperm fertilizes the egg. This is the first instance of a Drosophila gene required for determination of dorsal-ventral follicle cell fates that is not required for determination of embryonic dorsal-ventral cell fates. The temperature-sensitive period for brn dorsal-ventral patterning begins at the inception of vitellogenesis. The interaction between brn and DER is also required for at least two earlier follicle cell activities which are necessary to establish the ovarian follicle. Prefollicular cells fail to migrate between each oocyte/nurse cell complex, resulting in follicles with multiple sets of oocytes and nurse cells. brn and DER function is also required for establishing and/or maintaining a continuous follicular epithelium around each oocyte/nurse cell complex. These brn functions as well as the brn requirement for determination of dorsal-ventral polarity appear to be genetically separable functions of the brn locus. Genetic mosaic experiments show that brn is required in the germline during these processes whereas the DER is required in the follicle cells. We propose that brn may be part of a germline signaling pathway differentially regulating successive DER-dependent follicle cell activities of migration, division and/or adhesion and determination during oogenesis. These experiments indicate that brn is required in both tyrosine kinase and neurogenic intercellular signaling pathways. Moreover, the functions of brn in oogenesis are distinct from those of Notch and Delta, two other neurogenic loci that are known to be required for follicular development.