nudel

DEVELOPMENTAL BIOLOGY

Oogenesis in adult females

Nudel messenger RNA is expressed almost exclusively in follicle cells. Expression is first detectable at a low level in stage 7 of oogenesis and reaches a peak level during stages 9 and 10, when follicle cells are known to express genes for vitelline envelope proteins. In the majority of egg chambers, Nudel mRNA is asymmetically distributed, with higher levels found in ventral (as opposed to dorsal) follicle cells. In about a third of egg chambers, the distribution of Nudel mRNA is not noticeably asymmetric (Hong, 1995).

Effects of Mutation or Deletion

Females lacking normal nudel activity produce embryos that become dorsalized. The cuticle produced by nudel mutants is twisted and lacks both ventral and lateral structures. In addition to becoming dorsalized, eggs produced by nudel mutants are unusually fragile and therefore very sensitive to manipulation. This additional phenotype has not been described for mutations in the two other somatically required genes: pipe and windbeutel. Thus the nudel gene product could be a componenet of the vitelline envelope required for structural integrity of the egg (Hong, 1995).

A temperature sensitive allele of nudel was used to define the time period of Nudel functional involvement in establishment of polarity. The temperature-sensitive period for nudel function begins roughly at stage 7 of ooogenesis, just before the beginning of yolk deposition and the expression of genes encoding vitelline envelope proteins by the follicle cells. The period for nudel function ends roughly 2 hours after fertilization. Thus nudel function is required not just during oogensis but also during early stages of embryogenesis (Hong, 1995).

Mutations in the nudel gene cause one of two distinct maternal-effect defects. Class I mutations cause early arrest of embryonic development. Here, most embryos cease development prior to the syncytial blastoderm stage. Eggs produced by class I mutants are very fragile and sensitive to manipulation. These eggs exhibit an "oozy eggshell" phenotype in which contents appear to slowly leak out upon chorion removal. A structural defect in the vitelline envelope may therefore be responsible for the fragility of eggs laid by class I mutants. Class II nudel mutations result in a dorsalization of the embryo. In heterozygotes of class I and II alleles, the early developmental arrest phenotype of class I alleles is never observed. Whether the class I and II alleles complement each other depends on which allelic combinations are tested. A striking biphasic distribution of phenotypes is observed in some class I/class II heterozygotes in which no intermediately dorsalized embryos are present. Three of the four class II alleles have point mutations in the central protease domain suggesting that these mutants fail to activate the protease cascade. The fragile mutants are thought to be defective in assembling of the vitelline envelope (Hong, 1996).

The establishment of embryonic dorsoventral polarity in Drosophila depends on a signaling mechanism in which the signal for ventral development is locally produced. This mechanism requires the activity of the nudel gene in ovarian follicle cells, which provide dorsoventral positional information to the embryo. The nudel gene product, a large mosaic protein with a central serine protease domain, has been proposed to act as a local trigger for a protease cascade that produces the ventral signal. The serine protease activity of the Nudel protein is shown to be essential for embryonic dorsoventral polarity; the active Nudel protease is generated by autoproteolytic cleavage of a zymogen form. To examine the function of the central serine protease domain of Nudel, two point mutations having predictable effects on protease function were introduced separately into a genomic nudel transgene. One mutation (S1332A) alters the essential catalytic serine residue, which eliminates enzymatic activity of serine proteases, while the second mutation (R1144L) alters the P1 residue in the predicted zymogen cleavage site, which is predicted to alter the cleavage specificity required for zymogen activation without adversely affecting catalytic activity of the protease. These transgenic nudel constructs were assayed for their ability to rescue embryos derived from female flies lacking expression from the endogenous nudel gene. Such embryos arrest early in development, apparently due to an additional requirement for the Nudel protein in the structural integrity of the embryo. A single copy of the wild-type nudel transgene fully rescues nudel activity to give hatching larvae. In contrast, both site-directed mutants are able to rescue the early arrest phenotype but are inactive in the establishment of dorsoventral polarity, yielding completely dorsalized embryos. These results strongly suggest that, despite its unusual structure, Nudel is an authentic serine protease regulated by zymogen cleavage and that NudelÂ’s protease activity is essential for the establishment of embryonic dorsoventral polarity (LeMosy, 1998).

While the Nudel protease domain is detectable only in the 250 kDa polypeptide in mature ovarian egg chambers, four additional Nudel protease forms are found in extracts of laid fertilized eggs. The smallest and most abundant of these is a 33 kDa form similar in size to the Nudel serine protease domain generated by in vitro translation, suggesting that it might contain the protease domain with very little surrounding sequence. New bands at 38 kDa and 75-80 kDa are also seen, while the 250 kDa polypeptide appears to be reduced in abundance compared to ovaries. These smaller Nudel polypeptides are transiently present during embryogenesis, disappearing by 2 hours of development at 22°C. Based upon the previously defined temperature-sensitive period for nudel function, extending 1-2 hours into embryogenesis, one or more of these embryonic Nudel protease forms is likely to be the catalytically active Nudel protease required for the establishment of dorsoventral polarity. Partial processing of Nudel occurs during oogenesis. Activation of the Nudel protease is independent of the other known proteases involved in dorsoventral polarity establishment and appears to occur symmetrically on the surface of the embryo. Western blot examination of Nudel in extracts of fertilized eggs derived from females mutant for the pipe, windbeutel, gastrulation defective, snake, easter or Toll genes shows normal processing of Nudel protease, as well as C-terminal and N-terminal Nudel polypeptides. Thus, Nudel protease activation appears to proceed independent of known components of the Toll signaling pathway that act outside the embryo (LeMosy, 1998).

Secreted Nudel lies in a layer distinct from the nascent vitelline membrane, visualized by double-labeling with Nudel protease domain antibody and antibody to the Sv23 vitelline membrane protein. Counterstaining with rhodamine-phalloidin to label the cortical actin cytoskeleton of the oocyte shows that secreted Nudel is always closely apposed to the oocyte surface. Nudel is likely to be fixed at this surface, since Nudel polypeptides are largely insoluble in ovary extracts without the addition of denaturing agents. A change in Nudel protease localization that depends on Nudel protease activation was detected in the earliest laid embryos. The activated patterns of Nudel polypeptides and immunolocalization are also detected in laid unfertilized eggs, suggesting that ovulation may somehow lead to Nudel protease activation. Ovulation normally immediately precedes fertilization and results in many physiologic changes in the egg, such as resumption of meiosis, so linking Nudel protease activation to ovulation could ensure that Nudel protease is active in early embryogenesis (LeMosy, 1998).

In the absence of direct evidence for the cleavage of Gastrulation defective protein by Nudel protease, the data suggest an alternative role for Nudel protease: that the biologically relevant substrate for Nudel protease is Nudel itself. Processing of both N-terminal and C-terminal portions of Nudel are blocked in the absence of Nudel protease but occurs normally in the absence of the downstream dorsal-group proteases, suggesting that Nudel protease may itself proteolyze other regions of the Nudel protein. Cleavage of extracellular matrix proteins by matrix metalloproteases has been shown to reveal functions of these proteins that are not present in the intact molecules. Similarly, cleavage of Nudel by Nudel protease could activate a distinct portion of Nudel, such as the LDL-receptor ligand-binding motifs, which subsequently interact with other proteases in the Toll signaling pathway. These findings suggest that Nudel protease activation initiates the protease cascade that produces the ventral signal, but that spatial regulation occurring downstream of Nudel protease activation localizes the cascade to the ventral side of the embryo (LeMosy, 1998).

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. Certain mutant nudel alleles (designated Class I alleles) demonstrate the structurally defective eggs and early embryonic arrest seen for a null allele of nudel, while others (Class II alleles) exhibit only the dorsalized phenotype characteristic of the genes involved in the Toll signaling pathway. Complementation is observed between certain Class I and Class II alleles, suggesting that Nudel's structural and patterning functions involve distinct regions of the structurally modular Nudel protein. Because the Class II (dorsalizing) alleles are characterized by defects in the function of the Nudel protease, the complementation analysis argues that Nudel protease activity is involved only in dorsoventral patterning and is dispensable for the structural integrity of the egg. Evidence is provided 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 (LeMosy, 2000a).

The expression of Nudel containing a protease active site mutation predicted to eliminate all protease activity results in neutral red-permeable eggs containing dorsalized embryos, rather than the severely fragile eggs containing developmentally arrested embryos typical of Class I nudel alleles. This finding suggests that there is a significant, additional structural requirement for the nonprotease regions of Nudel. However, biochemical analysis of vitelline membrane biogenesis does not detect any additional defect in the ndl¹⁴ mutant generally deficient for Nudel protein when compared to the ndl¹¹¹ mutant lacking only Nudel protease function. One possibility is that Nudel is structurally required only for vitelline membrane crosslinking, with the Class I phenotype representing a more complete and catastrophic loss of crosslinking. Alternatively, there may be an earlier structural requirement for a nonprotease region of Nudel during oogenesis that was not detected by biochemical assays. Consistent with the latter possibility, modest structural abnormalitieshave been observed in Class I nudel mutant egg chambers, such as the presence of F-actin-containing inclusion bodies within the oocyte and separation of the oocyte plasma membrane from the vitelline membrane, that could reflect abnormalities of the extracellular matrix or of oocyte adhesion (LeMosy, 2000a).

Crosslinking of the vitelline membrane at the onset of embryogenesis is thought to be performed by a peroxidase-type enzyme, based upon the presence of cross-linked dityrosine and trityrosine residues in hydrolysates of vitelline membranes prepared from laid eggs, but not ovaries. Supporting this mechanism of crosslinking in Drosophila, there is strong evidence that the outer chorion layer of the eggshell is crosslinked by an endogenous peroxidase in late oogenesis. Crosslinking reactions are tightly regulated by controlling the availability or activity of the crosslinking enzyme or by controlling the availability of the substrate. For example, the chorion peroxidase is incorporated into the forming eggshell but does not act until the follicle cells secrete H2O2, a hydrogen acceptor required for the crosslinking reaction. In contrast, the sea urchin fertilization envelope is rapidly generated and crosslinked by an ovoperoxidase that is secreted together with other structural components of the envelope and H2O2. While the vitelline membrane is preformed prior to cross-linking, like the chorion, no peroxidase appears to be associated with this structure during oogenesis. The vitelline membrane might be cross-linked by a mechanism involving incorporation of an active peroxidase into a preexisting scaffold at the onset of embryogenesis (LeMosy, 2000a and references therein).

A likely role for the Nudel protease in crosslinking is the proteolytic activation of a crosslinking enzyme. Such proteolytic activation has been documented for several types of cross-linking enzymes but remains more speculative for the peroxidases. Proteolytic cleavage upon secretion has been demonstrated for the sea urchin ovoperoxidase. While the significance of this cleavage is unknown, conservation of the cleavage site among three sea urchin ovoperoxidases and a Drosophila peroxidase, peroxidasin, suggests that cleavage might be important for peroxidase function. Alternatively, the Nudel protease could be involved in another aspect of a crosslinking reaction, such as the release of H2O2 from the oocyte or cleavage of a vitelline membrane protein to generate a form capable of being cross-linked. In any case, the identification of nudel as a gene required for crosslinking of the vitelline membrane and the description of a mutant phenotype associated with a defect in this specific step of vitelline membrane biogenesis should facilitate future biochemical and genetic studies of this process (LeMosy, 2000a and references therein).

A compelling question arises from this work: what is the relationship between Nudel's functions in eggshell crosslinking and embryonic patterning? Previous work established that catalytic activity of the Nudel protease domain is essential for embryonic dorsoventral patterning, while it has now been shown that Nudel protease activity is also required for vitelline membrane crosslinking. One possibility is that the Nudel protease cleaves distinct substrates that act independently in vitelline membrane crosslinking and in dorsoventral patterning, e.g., a peroxidase and a protease zymogen in the dorsoventral protease cascade. In this two-pathway model, the involvement of the Nudel protease in eggshell biogenesis is irrelevant to its role in dorsoventral patterning. A potentially more interesting possibility is that Nudel protease acts in only one pathway with dorsoventral patterning dependent upon Nudel's activity in a crosslinking reaction; in this model, the Nudel protease would not directly cleave a protease zymogen in the dorsoventral protease cascade. Several lines of evidence suggest that if crosslinking is required for dorsoventral patterning, this requirement is likely to be specific rather than due to general leakiness of the vitelline membrane. Studies of the downstream components of the dorsoventral protease cascade have shown that preactivated forms of the Snake and Easter proteases can function in the perivitelline space of nudel mutant embryos: these studies suggest that the endogenous components are not lost by leakage through the defective vitelline membrane of these embryos. This argument is further supported by the finding that certain mutant alleles of the terminal-group gene, fs(1)Nasrat, produce eggs with leaky vitelline membranes within which embryos develop with normal dorsoventral polarity (LeMosy, 2000a and references therein).

Crosslinking of the vitelline membrane could directly lead to the creation of a specific matrix structure that is necessary for the function of one or more of the dorsoventral proteases. An analogy for this is found in the fibrinolytic protease cascade in mammals, in which crosslinked fibrinogen and fibrin act as catalysts to dramatically increase the conversion of a serine protease zymogen, plasminogen, to its active form, plasmin. Crosslinked fibrinogen and fibrin have this property, while monomeric fibrinogen does not, because polymerization of fibrinogen exposes binding sites for the serine proteases tissue plasminogen activator (tPA) and plasminogen that appear to orient the active site of tPA with the zymogen activation site of plasminogen. Similarly, vitelline membrane crosslinking involving Nudel protease could expose binding sites on the inner surface of the vitelline membrane that are involved in the formation of a zymogen activation complex for the dorsoventral protease cascade. A variant of the one-pathway model is that the Nudel protease activates crosslinking of not only the vitelline membrane but also a matrix structure at the plasma membrane where Nudel resides and that dorsoventral patterning is dependent on the latter crosslinking event. Distinguishing among these possibilities will be important to the long-term goal of understanding how the Toll signaling pathway is initiated and ventrally restricted within the embryonic perivitelline space (LeMosy, 2000a).

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. Biochemical and immunolocalization studies are described 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 (class II) result in a failure to activate the protease domain of Nudel. These 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 the analyses also reveal an important role for a region adjacent to the protease domain in Nudel protease function. Mutations causing egg fragility and early embryonic arrest (class I) 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, potential mechanisms for the complementary and antagonistic interactions between the nudel alleles are suggested (LeMosy, 2000b).

Class I mutations result in relative loss of Nudel from the extracellular space. Although the class I mutations cause a variety of defects in Nudel protein structure and processing, they share the common feature that there is a substantial decrease in the total amount of Nudel present within the extracellular space where it is presumably required. This quantitative defect is likely to be the root cause of the class I phenotypes of egg fragility and early embryonic arrest. Similar phenotypes of egg flaccidity and early developmental arrest can arise from such varied defects as decreased yolk uptake and loss of structural proteins of the eggshell. The localization of Nudel at the oocyte surface is consistent with involvement in such functions as vitelline membrane biogenesis or oocyte adhesion to an extracellular matrix (LeMosy, 2000b).

The C-terminal 402 amino acids of Nudel appear not to be essential for association with the oocyte surface (ndl¹⁸ protein), while protein sequences or post-translational modifications in the N-terminal half of Nudel may be required for this association (ndl¹⁶ protein). The oocyte surface localization of mutant Nudel proteins (ndl¹⁵, ndl¹⁷, ndl¹⁸, ndl^LP-2) that appear not to have undergone proteolytic processing or extensive carbohydrate addition suggests that some post-translational modifications are not essential for secretion or surface binding. However, these defective proteins may be unstable in the extracellular space (ndl¹⁵ protein), perhaps due to the absence of carbohydrates that may protect glycoproteins from proteolysis or due to failure to assemble into an extracellular matrix (LeMosy, 2000b).

Perhaps the biggest surprise is that, despite these gross defects in Nudel expression and biogenesis, most of the class I alleles are able to complement the class II ndl⁰⁴⁶ allele. With the exception of ndl¹¹, where no Nudel could be detected, each of the class I alleles that is able to complement the class II ndl⁰⁴⁶ allele exhibits either the presence of a polypeptide containing the Nudel protease domain or the presence of a Nudel polypeptide large enough to contain the protease domain. This latter result is in the case of the very weakly expressing ndl¹⁶ and ndl¹⁷ alleles (where the weak protease domain antibody failed to detect a specific polypeptide). Among these complementing alleles, the degree of complementation correlates with the secretion level of the class I protein. Together, these findings are consistent with the idea that the complementing class I alleles are able to deliver Nudel protease to the extracellular space. It appears that a very small amount of functional Nudel protease, present within a variety of mutant Nudel precursors, is sufficient to complement the defective Nudel protease made by the ndl⁰⁴⁶ allele (LeMosy, 2000b).

Class II mutations compromise Nudel protease function. Consistent with previous studies suggesting that catalytic activity of the Nudel protease is essential for the establishment of dorsoventral polarity and that the Nudel protease is auto-activated, all of the class II mutations have been found to have defects in Nudel protease activation, and all but one could be ascribed to mutations within the serine protease catalytic domain itself. The exception, ndl⁹, which affects a cysteine N-terminal to the protease domain, is particularly intriguing. This mutation is predicted to disrupt a disulfide bond linking the protease catalytic domain to a potential N-terminal regulatory domain and might also affect the structure of this prodomain. The steep temperature dependence of the function of the ndl⁹ protein would be consistent with impaired thermal stability of interactions between the protease domain and an N-terminal domain that is not covalently linked in the ndl⁹ protein. This N-terminal domain could be required for Nudel protease binding to cofactors or substrates, similar to the N-terminal regulatory domain of enterokinase that is essential for interactions of this serine protease with its macromolecular substrate, trypsinogen (LeMosy, 2000b).

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date revised: 24 July 2001 

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