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.     

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.     

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.     

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.     

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.     

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.     

A gradient of nuclear localization of the dorsal protein determines dorsoventral pattern in the Drosophila embryo

The dorsoventral axis of the Drosophila embryo is determined by a morphogen gradient established by the action of 12 maternal-effect genes: the dorsal group genes and cactus. One of the dorsal group genes, dorsal (dl), encodes the putative morphogen. Although no overall asymmetry in the distribution of dorsal protein is observed, a gradient of nuclear concentration of dl protein is established during cleavage stages, with a maximum at the ventral side of the egg. At the dorsal side of the egg, the protein remains in the cytoplasm. Nuclear localization of the dl protein, and hence gradient formation, is blocked in dorsalizing alleles of all of the other dorsal group genes, while in ventralizing mutants nuclear localization extends to the dorsal side of the egg. A correlation between dl protein distribution and embryonic pattern in mutant embryos indicates that the nuclear concentration of the dl protein determines pattern along the dorsoventral axis.     

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.     

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.     

Genes that control dorsoventral polarity affect gene expression along the anteroposterior axis of the Drosophila embryo

At least 13 genes control the establishment of dorsoventral polarity in the Drosophila embryo and more than 30 genes control the anteroposterior pattern of body segments. Each group of genes is thought to control pattern formation along one body axis, independently of the other group. We have used the expression of the fushi tarazu (ftz) segmentation gene as a positional marker to investigate the relationship between the dorsoventral and anteroposterior axes. The ftz gene is normally expressed in seven transverse stripes. Changes in the striped pattern in embryos mutant for other genes (or progeny of females homozygous for maternal-effect mutations) can reveal alterations of cell fate resulting from such mutations. We show that in the absence of any of ten maternal-effect dorsoventral polarity gene functions, the characteristic stripes of ftz protein are altered. Normally there is a difference between ftz stripe spacing on the dorsal and ventral sides of the embryo; in dorsalized mutant embryos the ftz stripes appear to be altered so that dorsal-type spacing occurs on all sides of the embryo. These results indicate that cells respond to dorsoventral positional information in establishing early patterns of gene expression along the anteroposterior axis and that there may be more significant interactions between the different axes of positional information than previously determined.     

An autoregulatory feedback loop directs the localized expression of the Drosophila CPEB protein Orb in the developing oocyte

The RRM-type RNA binding protein Orb plays a central role in the establishment of polarity in the Drosophila egg and embryo. In addition to its role in the formation and initial differentiation of the egg chamber, orb is required later in oogenesis for the determination of the dorsoventral (DV) and anteroposterior (AP) axes. In DV axis formation, Orb protein is required to localize and translate gurken mRNA at the dorsoanterior part of the oocyte. In AP axis formation, Orb is required for the translation of oskar mRNA. In each case, Orb protein is already localized at the appropriate sites within the oocyte before the arrival of the mRNAs encoding axis determinants. We present evidence that an autoregulatory mechanism is responsible for directing the on site accumulation of Orb protein in the Drosophila oocyte. This orb autoregulatory activity ensures the accumulation of high levels of Orb protein at sites in the oocyte that contain localized orb message.     

The Toll/IL-1 receptor binding protein MyD88 is required for Xenopus axis formation

The Toll/Dorsal pathway regulates dorsoventral axis formation in the Drosophila embryo. We had previously obtained evidence that a homologous pathway exists in Xenopus, however, its role during normal frog development had not been established. Here we report the cloning of Xenopus MyD88 (XMyD88), whose mammalian homologs are adaptor proteins linking Toll/IL-1 receptors and IRAK kinases. We show that in the frog embryo overexpression of a dominant-negative form of XMyD88 blocked Toll receptor activity, specifically inhibited axis formation and reduced expression of pivotal organizer genes. The observed stage-dependency of interference suggests a function for maternal XMyD88 soon after fertilization. We conclude that XMyD88 activity is required for normal Spemann organizer formation, implying an essential role for maternal Toll/IL-1 receptors in Xenopus axis formation.     

DmMyD88 controls dorsoventral patterning of the Drosophila embryo

MyD88 is an adapter protein in the signal transduction pathway mediated by interleukin-1 (IL-1) and Toll-like receptors. A Drosophila homologue of MyD88 (DmMyD88) was recently shown to be required for the Toll-mediated immune response. In Drosophila, the Toll pathway was originally characterized for its role in the dorsoventral patterning of the embryo. We found that, like Toll, DmMyD88 messenger RNA is maternally supplied to the embryo. Here we report the identification of a new mutant allele of DmMyD88, which generates a protein lacking the carboxy-terminal extension, normally located downstream of the Toll/IL-1 receptor domain. Homozygous mutant female flies lay dorsalized embryos that are rescued by expression of a transgenic DmMyD88 complementary DNA. The DmMyD88 mutation blocks the ventralizing activity of a gain-of-function Toll mutation. These results show that DmMyD88 encodes an essential component of the Toll pathway in dorsoventral pattern formation.     

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.     

Drosophila pipe protein activity in the ovary and the embryonic salivary gland does not require heparan sulfate glycosaminoglycans

The Drosophila pipe gene encodes ten related proteins that exhibit amino acid sequence similarity to vertebrate heparan sulfate 2-O-sulfotransferase. One of the Pipe isoforms, which is expressed in the ventral follicular epithelium, is a key determinant of embryonic dorsoventral polarity, suggesting that Pipe-mediated sulfation of a heparan sulfate proteoglycan provides a spatial cue for dorsoventral axis formation. We used several approaches to investigate this possibility in the work described here. We determined the nucleotide alterations in 11 different pipe alleles. Ten of the mutations specifically affect the pipe isoform that is expressed in the ovary. Among these ten mutations, two alter an amino acid in the putative binding site for 3'-phosphoadenosine 5'-phosphosulfate, the universal sulfate donor. Using Alcian Blue, a histochemical stain that detects sulfated glycans, we observed a novel, pipe-dependent macromolecule in the embryonic salivary glands. Genes known to participate in the formation of heparan sulfate in Drosophila are not required for the production of this material. To investigate whether a heparan sulfate proteoglycan is involved in pipe function in dorsoventral patterning, we generated females carrying follicle cell clones mutant for heparan sulfate synthesis-related genes. Embryos from follicles with mutant clones did not exhibit a dorsalized phenotype. Taken together, our data provide evidence that Pipe acts as a sulfotransferase, but argue against the hypothesis that the target of Pipe is a heparan sulfate glycosaminoglycan.