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 Nudels 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, ndlLP-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).