No requirement for localized Nudel protein expression in Drosophila embryonic axis determination

Drosophila embryonic dorsal-ventral polarity is defined by a maternally encoded signal transduction pathway. Gastrulation Defective, Snake, and Easter comprise a serine protease cascade that operates in the perivitelline space to generate active ligand for the Toll receptor, which resides in the embryonic membrane. Toll is activated only on the ventral side of the embryo. Spatial regulation of this pathway is initiated by the ventrally restricted expression of the sulfotransferase Pipe in the follicular epithelium that surrounds the developing oocyte. Pipe is thought to modify a target molecule that is secreted and localized within the ventral region of the egg and future embryo, where it influences the activity of the pathway such that active Toll ligand is produced only ventrally. A potential substrate for Pipe is encoded by nudel, which is expressed throughout the follicle cell layer and encodes a large, multi-functional secreted protein that contains a serine protease domain as well as other structural features characteristic of extracellular matrix proteins. A previous mosaic analysis suggested that the protease domain of Nudel is not a target for Pipe activity as its expression is not required in pipe-expressing cells, but failed to rule out such a role for other functional domains of the protein. To investigate this possibility, we carried out a mosaic analysis of additional nudel alleles, including some that affect the entire protein. Our analysis demonstrated that proteolytically processed segments of Nudel are secreted into the perivitelline space and stably localized, as would be expected for the target of Pipe, However, we found no requirement for nudel to be expressed in ventral, pipe-expressing follicle cells, thereby eliminating Nudel as an essential substrate of Pipe sulfotransferase activity.     

The nudel protease of Drosophila is required for eggshell biogenesis in addition to embryonic patterning

The dorsoventral axis of the Drosophila embryo is defined by a ventral signal that arises within the perivitelline space, an extracellular compartment between the embryo plasma membrane and the vitelline membrane layer of the eggshell. Production of the ventral signal requires four members of the serine protease family, including a large modular protein with a protease domain encoded by the nudel gene. Here we provide evidence that the Nudel protease has an integral role in eggshell biogenesis. Mutations in nudel that disrupt Nudel protease function produce eggs having vitelline membranes that are abnormally permeable to the dye neutral red. Permeability varies among mutant nudel alleles but correlates with levels of Nudel protease catalytic activity and function in embryonic dorsoventral patterning. These mutations also block cross-linking of vitelline membrane proteins that normally occurs upon egg activation, just prior to fertilization. In addition, Nudel protease autoactivation temporally coincides with vitelline membrane cross-linking and can be triggered in mature eggs in vitro by conditions that lead to egg activation. We discuss how the Nudel protease might be involved in both eggshell biogenesis and embryonic patterning.     

Localized serine protease activity and the establishment of Drosophila embryonic dorsoventral polarity

Drosophila embryo dorsoventral polarity is established by a maternally encoded signal transduction pathway in which three sequentially acting serine proteases, Gastrulation Defective, Snake and Easter, generate the ligand that activates the Toll receptor on the ventral side of the embryo. The spatial regulation of this pathway depends upon ventrally restricted expression of the Pipe sulfotransferase in the ovarian follicle during egg formation. Several recent observations have advanced our understanding of the mechanism regulating the spatially restricted activation of Toll. First, several protein components of the vitelline membrane layer of the eggshell have been determined to be targets of Pipe-mediated sulfation. Second, the processing of Easter by Snake has been identified as the first Pipe-dependent, ventrally-restricted processing event in the pathway. Finally, Gastrulation Defective has been shown to undergo Pipe-dependent, ventral localization within the perivitelline space and to facilitate Snake-mediated processing of Easter. Together, these observations suggest that Gastrulation Defective, localized on the interior ventral surface of the eggshell in association with Pipe-sulfated eggshell proteins, recruits and mediates an interaction between Snake and Easter. This event leads to ventrally-restricted processing and activation of Easter and consequently, localized formation of the Toll ligand, and Toll activation.     

A ventrally localized protease in the Drosophila egg controls embryo dorsoventral polarity

Drosophila embryo dorsoventral (DV) polarity is defined by serine protease activity in the perivitelline space (PVS) between the embryonic membrane and the inner layer of the eggshell. Gastrulation Defective (GD) cleaves and activates Snake (Snk). Activated Snk cleaves and activates Easter (Ea), exclusively on the ventral side of the embryo. Activated Ea then processes Sp?tzle (Spz) into the activating ligand for Toll, a transmembrane receptor that is distributed throughout the embryonic plasma membrane. Ventral activation of Toll depends upon the activity of the Pipe sulfotransferase in the ventral region of the follicular epithelium that surrounds the developing oocyte. Pipe transfers sulfate residues to several protein components of the inner vitelline membrane layer of the eggshell. Here we show that GD protein becomes localized in the ventral PVS in a Pipe-dependent process. Moreover, ventrally concentrated GD acts to promote the cleavage of Ea by Snk through an extracatalytic mechanism that is distinct from GD's proteolytic activation of Snk. Together, these observations illuminate the mechanism through which spatially restricted sulfotransferase activity in the developing egg chamber leads to localization of serine protease activity and ultimately to spatially specific activation of the Toll receptor in the Drosophila embryo.     

Biochemical defects of mutant nudel alleles causing early developmental arrest or dorsalization of the Drosophila embryo

The nudel gene of Drosophila is maternally required both for structural integrity of the egg and for dorsoventral patterning of the embryo. It encodes a structurally modular protein that is secreted by ovarian follicle cells. Genetic and molecular studies have suggested that the Nudel protein is also functionally modular, with a serine protease domain that is specifically required for ventral development. Here we describe biochemical and immunolocalization studies that provide insight into the molecular basis for the distinct phenotypes produced by nudel mutations and for the interactions between these alleles. Mutations causing loss of embryonic dorsoventral polarity result in a failure to activate the protease domain of Nudel. Our analyses support previous findings that catalytic activity of the protease domain is required for dorsoventral patterning and that the Nudel protease is auto-activated and reveal an important role for a region adjacent to the protease domain in Nudel protease function. Mutations causing egg fragility and early embryonic arrest result in a significant decrease in extracellular Nudel protein, due to defects in post-translational processing, stability, or secretion. On the basis of these and other studies of serine proteases, we suggest potential mechanisms for the complementary and antagonistic interactions between the nudel alleles.     

Activation of Snake in a serine protease cascade that defines the dorsoventral axis is atypical and pipe-independent in Drosophila embryos

During Drosophila embryogenesis, establishment of ventral and lateral cell fates requires spatial regulation of an extracellular serine protease cascade composed of Nudel, Gastrulation Defective (GD), Snake, and Easter. Pipe, a sulfotransferase expressed ventrally during oogenesis, sulfates secreted targets that somehow confer positive spatial input to this cascade. Nudel and GD activation are pipe-independent, while Easter activation requires pipe. The effect of pipe on Snake activation has been unknown. Here we show that Snake activation is cascade-dependent but pipe-independent. These findings support a conclusion that Snake’s activation of Easter is the first spatially regulated step in the dorsoventral protease cascade.     

Synthesis of the sulfate donor PAPS in either the Drosophila germline or somatic follicle cells can support embryonic dorsal-ventral axis formation

The establishment of dorsal-ventral (DV) polarity in the Drosophila embryo depends upon a localized signal that is generated in the perivitelline space of the egg through the action of a serine proteolytic cascade. Spatial regulation of this pathway is determined by the expression of the pipe gene in a subpopulation of ventral follicle cells in the developing egg chamber. The Pipe protein exhibits homology to vertebrate glycosaminoglycan sulfotransferases. In a previous study, we demonstrated that embryonic DV polarity depends upon the sulfotransferase activity of Pipe. Surprisingly, however, our results also indicated that formation of the embryonic DV axis does not require the synthesis of the high-energy sulfate donor, 3'-phosphoadenosine 5'-phosphosulfate (PAPS) in the follicle cells in which Pipe is presumed to function. Here, we resolve this apparent paradox by demonstrating that dorsalized embryos are only produced by egg chambers in which both germline and follicle cells lack PAPS synthetase activity. Thus, PAPS produced either in the germline or in the follicular epithelium can support the requirement for Pipe sulfotransferase activity in embryonic DV patterning. This finding indicates the existence of a conduit for the movement of PAPS between the germline and the follicle cells, which highlights a previously unappreciated mechanism of soma/germline cooperation affecting pattern formation.     

The polarity of the dorsoventral axis in the Drosophila embryo is defined by an extracellular signal

Twelve maternal effect loci are required for the production of Drosophila embryos with a correct dorsoventral axis. Analysis of mosaic females indicates that the expression of the genes nudel, pipe, and windbeutel is required in the somatic tissue, presumably in the follicle cells that surround the oocyte. Thus, information coming from outside the egg cell influences dorsoventral pattern formation during embryogenesis. In transplantation experiments, the perivitelline fluid from the compartment surrounding the embryo can restore dorsoventral pattern to embryos from females mutant for nudel, pipe, or windbeutel. The positioning of the transplanted pervitelline fluid also determines the polarity of the restored dorsoventral axis. We propose that the polarizing activity, normally present at the ventral side of the egg, is a ligand for the Toll receptor. Presumably, local activation of the Toll protein by the ligand initiates the formation of the nuclear concentration gradient of the dorsal protein, thereby determining dorsoventral pattern.     

Multiple extracellular activities in Drosophila egg perivitelline fluid are required for establishment of embryonic dorsal-ventral polarity.

Twelve maternal effect genes (the dorsal group and cactus) are required for the establishment of the embryonic dorsal-ventral axis in the Drosophila embryo. Embryonic dorsal-ventral polarity is defined within the perivitelline compartment surrounding the embryo by the ventral formation of a ligand for the Toll receptor. Here, by transplantation of perivitelline fluid we demonstrate the presence of three separate activities present in the perivitelline fluid that can restore dorsal-ventral polarity to mutant easter, snake, and sp?tzle embryos, respectively. These activities are not capable of defining the polarity of the dorsal-ventral axis; instead they restore structures according to the intrinsic dorsal-ventral polarity of the mutant embryos. They appear to be involved in the ventral formation of a ligand for the Toll protein. This process requires serine proteolytic activity; the injection of serine protease inhibitors into the perivitelline space of wild-type embryos results in the formation of dorsalized embryos.     

The Maternal Nudel Protein of Drosophila Has Two Distinct Roles Important for Embryogenesis

The nudel gene is maternally required to define dorsoventral polarity of the Drosophila embryo. It encodes an unusual mosaic protein with a protease domain that may trigger the protease cascade required for ventral development. We describe phenotypic and molecular analyses of nudel mutations that provide further insight into nudel protein function. Surprisingly, nudel mutations primarily cause either dorsalized embryos in which dorsal cell fates are expanded over ventral and lateral cell fates or fragile eggs that fail to develop beyond early embryonic stages. The nudel protein is therefore required not only for embryonic dorsoventral polarity but also for structural integrity of the egg. Complementation and antagonistic interactions between nudel alleles suggest that the nudel protein is functionally modular and that protein-protein interactions are important for nudel protein function. Three nudel mutations that produce dorsalized embryos map to the protease domain of nudel, suggesting that this domain is specifically required for defining embryonic dorsoventral polarity. Finally, certain combinations of nudel alleles simultaneously produce completely dorsalized and normal embryos yet very few embryos of intermediate mutant phenotypes. The unusual biphasic distribution of phenotypes may indicate that nudel activity above a threshold is required to generate embryonic dorsoventral polarity.     

Spatial regulation of developmental signaling by a serpin

An extracellular serine protease cascade generates the ligand that activates the Toll signaling pathway to establish dorsoventral polarity in the Drosophila embryo. We show here that this cascade is regulated by a serpin-type serine protease inhibitor, which plays an essential role in confining Toll signaling to the ventral side of the embryo. This role is strikingly analogous to the function of the mammalian serpin antithrombin in localizing the blood-clotting cascade, suggesting that serpin inhibition of protease activity may be a general mechanism for achieving spatial control in diverse biological processes.     

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

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

Spatially dependent activation of the patterning protease, Easter

The dorsoventral axis of the Drosophila embryo is established by the activating cleavage of a signaling ligand by a serine protease, Easter, only on the ventral side of the embryo. Easter is the final protease in a serine protease cascade in which initial reaction steps appear not to be ventrally restricted, but where Easter activity is promoted ventrally through the action of a spatial cue at an unknown step in the pathway. Here, biochemical studies demonstrate that this spatial control occurs at or above the level of Easter zymogen activation, rather than through direct promotion of Easter's catalytic activity against the signaling ligand.     

Windbeutel is required for function and correct subcellular localization of the Drosophila patterning protein Pipe

Drosophila embryonic dorsal-ventral polarity originates in the ovarian follicle through the restriction of pipe gene expression to a ventral subpopulation of follicle cells. Pipe, a homolog of vertebrate glycosaminoglycan-modifying enzymes, directs the ventral activation of an extracellular serine proteolytic cascade which defines the ventral side of the embryo. When pipe is expressed uniformly in the follicle cell layer, a strong ventralization of the resulting embryos is observed. Here, we show that this ventralization is dependent on the other members of the dorsal group of genes controlling dorsal-ventral polarity, but not on the state of the Epidermal Growth Factor Receptor signal transduction pathway which defines egg chamber polarity. Pipe protein expressed in vertebrate tissue culture cells localizes to the endoplasmic reticulum. Strikingly, coexpression of the dorsal group gene windbeutel in those cells directs Pipe to the Golgi. Similarly, Pipe protein exhibits an altered subcellular localization in the follicle cells of females mutant for windbeutel. Thus, Windbeutel protein enables the correct subcellular distribution of Pipe to facilitate its pattern-forming activity.     

The polarisation of the anterior-posterior and dorsal-ventral axes during Drosophila oogenesis.

Recent work on Drosophila oogenesis has begun to reveal how the first asymmetries in development arise and how these relate to the later events that localise the positional cues which define the embryonic axes. The Cadherin-dependent positioning of the oocyte creates an anterior-posterior polarity that is transmitted to the embryo through the localisation and localised translation of bicoid, oskar, and nanos mRNA. In contrast, dorsal-ventral polarity arises from the random migration of the nucleus to the anterior of the oocyte, where it determines where gurken mRNA is translated and localised. Gurken signalling then defines the embryonic dorsal-ventral axis by restricting pipe expression to the ventral follicle cells, where Pipe regulates the production of an unidentified cue that activates the Toll signalling pathway.