Holes in the Plasma Membrane Mimic Torso-Like Perforin in Torso Tyrosine Kinase Receptor Activation in the Drosophila Embryo.

Receptor tyrosine kinase (RTK) pathways play central roles in development, and, when abnormally activated, they can lead to pathological conditions, including oncogenesis. Thus, RTK activation, mediated by ligand binding, is under tight control, a critical step being the conversion of an inactive precursor into the active form of the ligand. A variety of mechanisms have been shown to be involved in this conversion; however, little attention has been paid to how mechanical phenomena may impinge on this process. Here we address this issue by studying Torso, an RTK activated at both poles of the Drosophila embryo at the blastoderm stage. Torso activation is induced by a cleaved form of Trunk, a growth factor-like protein, but it also requires the accumulation of the Torso-like (Tsl) protein at both ends of the blastoderm. Tsl is the only known protein in Drosophila bearing a membrane attack complex/perforin (MACPF) domain-a motif present in proteins involved in pore formation at cell membranes. However, while different hypotheses have been put forward to account for the function of Tsl in Torso receptor activation, little is known about its molecular role and whether it indeed contributes to membrane pore formation. Here, we show that mechanically induced holes in the Drosophila embryo can substitute for Tsl function. These results suggest that Tsl is required for an exchange between the interior of the Drosophila embryo and its surrounding milieu and that mechanically induced cell injuries may contribute to abnormal RTK activation.

The trigger (and the restriction) of Torso RTK activation.

The Torso pathway is an ideal model of receptor tyrosine kinase systems, in particular when addressing questions such as how receptor activity is turned on and, equally important, how it is restricted, how different outcomes can be generated from a single signal, and the extent to which gene regulation by signalling pathways relies on the relief of transcriptional repression. In this regard, we considered it pertinent to single out the fundamental notions learned from the Torso pathway beyond the specificities of this system (Furriols and Casanova 2003 EMBO J. 22, 1947-1952. ( doi:10.1093/emboj/cdg224 )). Since then, the Torso system has gained relevance and its implications beyond its original involvement in morphogenesis and into many disciplines such as oncogenesis, hormone control and neurobiology are now acknowledged. Thus, we believe that it is timely to highlight additional notions supported by new findings and to draw attention to future prospects. Given the late development of research in the field, we wish to devote this review to the events leading to the activation of the Torso receptor, the main focus of our most recent work.

Transfer of Dorsoventral and Terminal Information from the Ovary to the Embryo by a Common Group of Eggshell Proteins in Drosophila.

The Drosophila eggshell is an extracellular matrix that confers protection to the egg and also plays a role in transferring positional information from the ovary to pattern the embryo. Among the constituents of the Drosophila eggshell, Nasrat, Polehole, and Closca form a group of proteins related by sequence, secreted by the oocyte, and mutually required for their incorporation into the eggshell. Besides their role in eggshell integrity, Nasrat, Polehole, and Closca are also required for embryonic terminal patterning by anchoring or stabilizing Torso-like at the eggshell. Here, we show that they are also required for dorsoventral patterning, thereby unveiling that the dorsoventral and terminal systems, hitherto considered independent, share a common extracellular step. Furthermore, we show that Nasrat, Polehole, and Closca are required for proper Nudel activity, a protease acting both in embryonic dorsoventral patterning and eggshell integrity, thus providing a means to account for the role of Nasrat, Polehole, and Closca. We propose that a Nasrat/Polehole/Closca complex acts as a multifunctional hub to anchor various proteins synthesized at oogenesis, ensuring their spatial and temporal restricted function.

Accumulation of the Drosophila Torso-like protein at the blastoderm plasma membrane suggests that it translocates from the eggshell.

The eggshell serves as a depository for proteins that play an important role in early embryonic development. In particular, the Drosophila eggshell is responsible for transferring asymmetries from the egg chamber to specify the regions at both ends of the embryo through the uneven activation of the Torso (Tor) receptor in its membrane. This process relies on the restricted expression of the gene torso-like (tsl) in subpopulations of follicle cells during oogenesis and its protein accumulation at both poles of the eggshell, but it is not known how this signal is transmitted to the embryo. Here, we show that Tsl accumulates at the embryonic plasma membrane, even in the absence of the Tor receptor. However, during oogenesis, we detected Tsl accumulation only at the eggshell. These results suggest that there is a two-step mechanism to transfer the asymmetric positional cues from the egg chamber into the early embryo: initial anchoring of Tsl at the eggshell as it is secreted, followed by its later translocation to the egg plasma membrane, where it enables Tor receptor activation. Translocation of anchored determinants from the eggshell might then regulate the spatial and temporal control of early embryonic developmental processes.

Germline and somatic vitelline proteins colocalize in aggregates in the follicular epithelium of Drosophila ovaries.

Nasrat and Polehole, two Drosophila proteins related functionally and by sequence, are secreted from the oocyte and incorporated into the vitelline membrane, where they play a role in the integrity of the same and in the activation of embryonic Torso RTK. In addition, they also accumulate in a punctate pattern in the follicular epithelium. Here we show that their accumulation at the follicle cells depends on their gene expression in the germline, indicating that these proteins move from the oocyte to the follicle cells in a process that does not require endocytosis. Finally we used cell markers to examine the distribution of these proteins at the follicle cells and show they accumulated in aggregates with vitelline membrane proteins in close association with the plasmatic membrane. We propose that these aggregates represent spatially restricted sinks for vitelline membrane proteins that fail to be incorporated into vitelline bodies and later on into the vitelline membrane.

Deadpan contributes to the robustness of the notch response.

Notch signaling regulates many fundamental events including lateral inhibition and boundary formation to generate very reproducible patterns in developing tissues. Its targets include genes of the bHLH hairy and Enhancer of split [E(spl)] family, which contribute to many of these developmental decisions. One member of this family in Drosophila, deadpan (dpn), was originally found to have functions independent of Notch in promoting neural development. Employing genome-wide chromatin-immunoprecipitation we have identified several Notch responsive enhancers in dpn, demonstrating its direct regulation by Notch in a range of contexts including the Drosophila wing and eye. dpn expression largely overlaps that of several E(spl) genes and the combined knock-down leads to more severe phenotypes than either alone. In addition, Dpn contributes to the establishment of Cut expression at the wing dorsal-ventral (D/V) boundary; in its absence Cut expression is delayed. Furthermore, over-expression of Dpn inhibits expression from E(spl) gene enhancers, but not vice versa, suggesting that dpn contributes to a feed-back mechanism that limits E(spl) gene expression following Notch activation. Thus the combined actions of dpn and E(spl) appear to provide a mechanism that confers an initial rapid output from Notch activity which becomes self-limited via feedback between the targets.

Conserved and divergent elements in Torso RTK activation in Drosophila development.

The repeated use of signalling pathways is a common phenomenon but little is known about how they become co-opted in different contexts. Here we examined this issue by analysing the activation of Drosophila Torso receptor in embryogenesis and in pupariation. While its putative ligand differs in each case, we show that Torso-like, but not other proteins required for Torso activation in embryogenesis, is also required for Torso activation in pupariation. In addition, we demonstrate that distinct enhancers control torso-like expression in both scenarios. We conclude that repeated Torso activation is linked to a duplication and differential expression of a ligand-encoding gene, the acquisition of distinct enhancers in the torso-like promoter and the recruitment of proteins independently required for embryogenesis. A combination of these mechanisms is likely to allow the repeated activation of a single receptor in different contexts.

Control of germline torso expression by the BTB/POZ domain protein pipsqueak is required for embryonic terminal patterning in Drosophila.

Early embryogenesis in Drosophila melanogaster is controlled by maternal gene products, which are deposited in the egg during oogenesis. It is not well understood how maternal gene expression is controlled during germline development. pipsqueak (psq) is a complex locus that encodes several nuclear protein variants containing a PSQ DNA-binding domain and a BTB/POZ domain. Psq proteins are thought to regulate germline gene expression through epigenetic silencing. While psq was originally identified as a posterior-group gene, we show here a novel role of psq in embryonic terminal patterning. We characterized a new psq loss-of-function allele, psq(rum), which specifically affects signaling by the Torso (Tor) receptor tyrosine kinase (RTK). Using genetic epistasis, gene expression analyses, and rescue experiments, we demonstrate that the sole function impaired by the psq(rum) mutation in the terminal system is an essential requirement for controlling transcription of the tor gene in the germline. In contrast, the expression of several other maternal genes, including those encoding Tor pathway components, is not affected by the mutation. Rescue of the psq(rum) terminal phenotype does not require the BTB/POZ domain, suggesting that the PSQ DNA-binding domain can function independently of the BTB/POZ domain. Our finding that tor expression is subject to dedicated transcriptional regulation suggests that different maternal genes may be regulated by multiple distinct mechanisms, rather than by a general program controlling nurse-cell transcription.

closca, a new gene required for both Torso RTK activation and vitelline membrane integrity. Germline proteins contribute to Drosophila eggshell composition.

The Drosophila eggshell is a specialised extracellular matrix (ECM) that surrounds and protects the oocyte and the embryo until its eclosion. In addition, the vitelline membrane, the innermost layer of the eggshell, holds the local determinant required to activate the Torso RTK pathway, which establishes the embryonic terminal regions. Here we report the identification and characterisation of closca, a gene encoding a new member of a group of proteins that act non-redundantly in vitelline membrane biogenesis and in Torso signalling. We also show that the Nasrat protein, another member of this group, is incorporated into the vitelline membrane, thereby indicating that the eggshell is a shared ECM that receives contributions from both follicle cells and the germline. This observation also provides a new scenario that accounts for the long known contribution of germline products to vitelline membrane biogenesis and to the follicle cell-dependent activation of the Torso receptor.

Two distinct but convergent groups of cells trigger Torso receptor tyrosine kinase activation by independently expressing torso-like.

Cell fate determination is often the outcome of specific interactions between adjacent cells. However, cells frequently change positions during development, and thus signaling molecules might be synthesized far from their final site of action. Here, we analyze the regulation of the torso-like gene, which is required to trigger Torso receptor tyrosine kinase activation in the Drosophila embryo. Whereas torso is present in the oocyte, torso-like is expressed in the egg chamber, at the posterior follicle cells and in two separated groups of anterior cells, the border cells and the centripetal cells. We find that JAK/STAT signaling regulates torso-like expression in the posterior follicle cells and border cells but not in the centripetal cells, where torso-like is regulated by a different enhancer. The border and centripetal cells, which are originally apart, converge at the anterior end of the oocyte, and we find that both groups contribute to trigger Torso activation. Our results illustrate how independently acquired expression of a signaling molecule can constitute a mechanism by which distinct groups of cells act together in the activation of a signaling pathway.