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.     

Establishment of dorsal-ventral polarity in the Drosophila embryo: the induction of polarity by the Toll gene product

Drosophila females that lack Toll gene activity produce dorsalized embryos, in which all embryonic cells behave like the dorsal cells of the wild-type embryo. Injection of wild-type cytoplasm into young Toll- embryos restores their ability to produce a normal dorsal-ventral pattern in a position-dependent manner. No matter where the cytoplasm is injected relative to the dorsal-ventral axis of the egg shell, the position of the injected cytoplasm defines the ventralmost part of the rescued pattern. Although injection of wild-type cytoplasm into mutants at six other dorsal-group loci also restores the ability to produce lateral and ventral structures, only Toll- embryos lack any residual dorsal-ventral polarity. Experiments suggest that the activity of the Toll product is normally regulated by other dorsal-group genes and that the function of the Toll product is to provide the source for a morphogen gradient in the dorsal-ventral axis of the wild-type embryo.     

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.     

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.     

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.     

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.     

Gastrulation defective, a complement factor C2/B-like protease, interprets a ventral prepattern in Drosophila

gastrulation defective (gd) encodes a serine protease required for specification of dorsal-ventral cell fates during Drosophila embryogenesis. Using RNA microinjection, I show that wild-type gd RNA can restore ventrolateral pattern elements with correct polarity with respect to egg shape in embryos lacking gd function. While low RNA concentrations restore ventrolateral pattern elements, higher concentrations ventralize the embryo. Gastrulation defective concentration has a rate-limiting effect on the domain of high Dorsal concentration but little effect upon the slope of the gradient. In embryos from pipe-null females, much higher RNA concentrations generate an ectopic axis oriented with respect to the site of injection. The data suggest that the Dorsal gradient is not directly determined by asymmetric cues in the eggshell but arises de novo within the perivitelline space as a consequence of self-regulatory properties of the protease cascade. A homology to the mammalian complement factors C2 and B is also described.     

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.     

Plasma membrane localization of the Toll protein in the syncytial Drosophila embryo: importance of transmembrane signaling for dorsal-ventral pattern formation

Formation of the Drosophila embryo's dorsal-ventral pattern requires the maternal product of the Toll gene. DNA sequence and genetic analyses together suggested that the Toll gene product is a transmembrane protein which communicates information from an extracytoplasmic compartment to the cytoplasm. Using antibodies as probes, we show that the Toll protein is a 135 x 10(3) Mr glycoprotein which is tightly associated with embryonic membranes. During the syncytial stage when dorsal-ventral polarity is established, the maternal Toll protein is associated with the plasma membrane around the entire embryo. During later embryonic stages, the Toll protein is expressed zygotically on many cell surfaces, possibly to promote cell adhesion. The plasma membrane localization of the Toll protein in the syncytial embryo suggests that transmembrane signaling from the extracellular perivitelline space to the cytoplasm is required for establishment of the embryonic dorsal-ventral pattern.     

Relocalization of the dorsal protein from the cytoplasm to the nucleus correlates with its function

dorsal is one of the maternally active dorsal-ventral polarity genes of Drosophila and is homologous to the vertebrate proto-oncogene c-rel. In wild-type embryos, the dorsal protein is found in the cytoplasm during cleavage. After the nuclei migrate to the periphery of the embryo, a ventral-to-dorsal gradient of nuclear dorsal protein is established. The formation of the nuclear gradient is disrupted in mutant embryos from other maternally active dorsal-ventral polarity genes: in dorsalized embryos only cytoplasmic protein is observed, while in ventralized embryos the nuclear gradient is shifted dorsally. My findings suggest that nuclear localization is critical for dorsal to function as a morphogen and that the distribution of the dorsal protein determines cell fate along the dorsal-ventral axis.     

Ventralization of the Drosophila embryo by deletion of extracellular leucine-rich repeats in the Toll protein.

Dorsoventral polarity of the Drosophila embryo is established by a signal transduction pathway in which the maternal transmembrane protein Toll appears to function as the receptor for a ventrally localized extracellular ligand. Certain dominant Toll alleles encode proteins that behave as partially ligand-independent receptors, causing embryos containing these proteins to become ventralized. In extracts of embryos derived from mothers carrying these dominant alleles, we detected a polypeptide of approximately 35 kDa in addition to full-length Toll polypeptides with antibodies to Toll. Our biochemical analyses suggest that the smaller polypeptide is a truncated form of Toll lacking extracellular domain sequences. To assay the biological activity of such a shortened form of Toll, we synthesized RNA encoding a mutant polypeptide lacking the leucine-rich repeats that comprise most of Toll's extracellular domain and injected this RNA into embryos. The truncated Toll protein elicited the most ventral cell fate independently of the wild-type Toll protein and its ligand. These results support the view that Toll is a receptor whose extracellular domain regulates the intrinsic signaling activity of its cytoplasmic domain.     

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.     

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.     

Spätzle regulates the shape of the Dorsal gradient in the Drosophila embryo

Dorsal-ventral polarity of the Drosophila embryo is established by a nuclear gradient of Dorsal protein, generated by successive gurken-Egfr and sp?tzle-Toll signaling. Overexpression of extracellular Sp?tzle dramatically reshapes the Dorsal gradient: the normal single peak is broadened and then refined to two distinct peaks of nuclear Dorsal, to produce two ventral furrows. This partial axis duplication, which mimics the ventralized phenotype caused by reduced gurken-Egfr signaling, arises from events in the perivitelline fluid of the embryo and occurs at the level of Sp?tzle processing or Toll activation. The production of two Dorsal peaks is addressed by a model that invokes action of a diffusible inhibitor, which is proposed to normally regulate the slope of the Dorsal gradient.     

Expression and regulation of Spätzle-processing enzyme in Drosophila

The Drosophila melanogaster Toll receptor controls embryonic dorsal-ventral axis formation and is crucial for the innate immune response. In both cases, Toll is activated by the enzymatically cleaved form of its ligand Sp?tzle (Spz). During axis formation, Spz is cleaved by the maternally provided serine protease Easter while the Sp?tzle-processing enzyme (SPE) activates Spz after infection. We confirm the role of SPE in immunity and show that it is a zygotic gene specifically expressed in immune tissues implying that the dual activation of Spz is achieved by differential spatiotemporal expression of two similar but distinct serine proteases.     

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.     

Pattern Formation: The link between ovary and embryo

The Drosophila gene nudel may encode a spatially restricted serine protease involved in producing the ligand for the receptor Toll and linking dorsal–ventral polarity in the egg chamber to the developing embryonic axis.     

Genetic Characterization of Tube and Pelle, Genes Required for Signaling between Toll and Dorsal in the Specification of the Dorsal-Ventral Pattern of the Drosophila Embryo

tube and pelle are two of the maternally transcribed genes required for dorsal-ventral patterning of the Drosophila embryo. Females homozygous for strong alleles of tube or pelle produce embryos that lack all ventral and lateral embryonic pattern elements. By analyzing the phenotypes caused by 24 pelle and 9 tube alleles, we have defined characteristic features of the two genes, including the extremely variable phenotypes of a number of tube alleles and the antimorphic character of a number of pelle alleles. Double mutant females carrying dominant ventralizing alleles of Toll and dorsalizing alleles of tube or pelle produce dorsalized embryos, suggesting that tube and pelle act downstream of the membrane protein Toll in the signaling pathway that defines the embryonic dorsal-ventral pattern. Both tube and pelle are also important zygotically for survival: at least 30% of the zygotes lacking either tube or pelle die before adult stages, while 90-95% of tube(-) pelle(-) double mutant zygotes die. We discuss the phenotypes of tube-pelle double mutants in the context of whether the two proteins interact directly.