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.     

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.     

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.     

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.     

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.     

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.     

A novel pattern of follicular epithelium morphogenesis in higher dipterans

In fly ovaries, the follicular epithelium surrounding germline cells diversifies into several morphologically distinct cell subpopulations. This complex process is crucial for the formation of a regionally complex eggshell and establishment of polarity of the future embryo. Morphogenetic changes accompanying patterning of the follicular epithelium have been best characterized in the model fly, Drosophila melanogaster. Here, we analyze follicular epithelium diversification in the ovaries of Tachypeza nubila, a brachyceran fly closely related to the group Cyclorrhapha, which also includes Drosophila. We provide morphological evidence that in Tachypeza, the diversification process differs from that described in the Drosophila model system in several important respects: (i) follicle cells differentiate into five subpopulations (versus eight in Drosophila); (ii) only one of these subpopulations (i.e. border cells) is migratory (versus four in Drosophila); (iii) the main body follicle cells form a uniform epithelium with no distinct border between follicle cells covering the nurse cell compartment and the oocyte; (iv) chorionic material is deposited not only on the surface of the oocyte but also on the nurse cells; (v) there is no centripetal migration of the follicle cells; (vi) the resulting eggshell is morphologically simple with no regional specializations except for the micropylar apparatus at the anterior pole of the oocyte. Our findings provide novel insights into the evolution of the follicle cell patterning and functioning in dipterans. A critical analysis of these processes in different dipteran groups strongly indicates that in Tachypeza, follicular epithelium diversification follows a distinct pattern, novel for higher dipterans.     

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.     

Genetic variation for dorsal-ventral patterning of the Drosophila melanogaster eggshell

Patterning of the insect eggshell is an excellent system for exploring the molecular basis of phenotypic variation. In Drosophila melanogaster, two dorsal-anterior respiratory appendages are produced in response to signaling through the Epidermal growth factor receptor (Egfr). Previous work implicates Egfr pathway function in both intraspecific variation for dorsal appendage spacing (DAS) on the eggshell, as well as interspecific differences in dorsal appendage number and location. To test the hypothesis that genetic variation in Egfr contributes to variation in eggshell patterning, we have made use of naturally occurring intraspecific variation for DAS as a model quantitative trait. We found that there is substantial segregating genetic variation for DAS in D. melanogaster, and have tested for associations with 289 common polymorphisms in the Egfr locus. A marginal association was seen with two polymorphic sites in Egfr; however, we failed to replicate these findings in a second population, or in a modified quantitative complementation test designed to specifically test the effects of the putative polymorphisms. Therefore, we conclude that the polymorphisms we have identified in Egfr do not contribute to variation in DAS, and further work is required to understand the genetic architecture of this trait.