Evidence is presented showing that the Gd protein is processed. An EcoRV restriction fragment from the gd cDNA (amino acids 347-548) was cloned in-frame into pBS SK and used to express protein. Bacterially derived protein was harvested from inclusion bodies and used as an immunogen in rabbits. Western blots of extracts from ovaries and early embryos revealed three cross reacting bands (34, 30, and 27.5 kDa) that are smaller than the 60-kDa full length protein observed in in vitro translation experiments. All three peptides are completely absent in extracts from gd7 females whereas extracts of gd5 and gd6 lack the two larger bands and gd4 possess all three. Extracts of carefully staged embryos and ovaries show that (1) the crossreacting polypeptides are most abundant in the ovaries; (2) that the level of protein decreases from the moment of egg laying, and (3) that it is essentially gone by 4 h (Konrad, 1998).
To prove that Gd is an active serine protease, it is necessary to demonstrate its enzymatic activity in vitro. For this purpose, a recombinant form of Gd was made using cultured Drosophila S2 cells. The culture medium of these cells after transfection with a plasmid encoding Gd contains a major polypeptide of 66 kDa, which is detectable with polyclonal antibodies raised against a Gd bacterial fusion protein. This polypeptide, which was not detected in culture medium of mock-transfected cells, is similar in size to the polypeptide made by in vitro translation and the baculovirus system from a gd cDNA. These data indicate that the 66-kDa polypeptide secreted by transfected S2 cells is the Gd protein (Han, 2000).
To test whether Gd is proteolytically activated, the recombinant Gd protein was treated with trypsin, which cleaves after (that is, C terminal to) basic residues. Trypsin was chosen because of the possibility that Gd is activated in vivo by the Nudel protease, which is predicted to be specific for basic residues and is the only known protease in the Toll signaling pathway that could function before Gd. The culture medium containing recombinant Gd was incubated with increasing amounts of trypsin bound to Sepharose beads. After the immobilized trypsin was removed by centrifugation, the supernatant was analyzed by Western blot. This treatment generated 4-5 smaller polypeptides, including a prominent one of about 29 kDa (Han, 2000).
It was necessary to determine whether full-length Gd or any of the smaller polypeptides generated by trypsin digestion is an active serine protease. Since the amount of protein is apparently not sufficient for enzyme assays using synthetic substrates, a more sensitive affinity labeling method was used for detecting serine protease activity. Specifically, biotinylated peptidyl chloromethylketones, synthetic inhibitors of trypsin-like serine proteases that react covalently with active proteases but not inactive zymogens, were used. After the supernatants from trypsin digestion were incubated with these reagents, biotinylated proteins in the supernatants were visualized by avidin blot. The predominant biotinylated polypeptide appears to be the 29-kDa fragment of Gd, suggesting that this polypeptide represents an active serine protease. The polypeptide corresponding to full-length Gd was not affinity labeled. The same experiment was performed with a recombinant form of the Gd S-A protein, in which the catalytic serine was mutated to alanine. As expected, the 29-kDa polypeptide generated by trypsin digestion of the mutant protein did not react with the affinity reagents. These results strongly suggest that the 29-kDa polypeptide generated by trypsin digestion of the wild-type Gd protein is an active serine protease (Han, 2000).
To test whether Gd, Snake, and Easter function in a proteolytic cascade, as suggested by genetic studies, their biochemical activities were examined by coexpression in Drosophila S2 cells. In many experiments, inactive forms of Easter and Snake were used in which the catalytic serine had been mutated to an alanine residue. This strategy was adopted because the active forms seemed to be unstable after zymogen cleavage and thus difficult to detect. To test first whether Gd activates Snake, the processing of catalytically inactive Snake (SNKS-A) was examined by Western blotting after it was expressed with Gd. Because previous studies had suggested that Gd activity depends on proteolytic processing, the activities of two truncated Gd forms were initially examined. GdDeltaN1 lacks amino acids 1 through 211 after signal sequence cleavage, where Lys-211 is the nearest basic residue N-terminal to the conserved catalytic domain and thus might serve as a cleavage site for Gd activation. GdDeltaN2 lacks amino acids 1 through 253, corresponding to a potential cleavage site suggested by Konrad (1998) at the beginning of the conserved catalytic-domain sequence. Expression of SNKS-A with either of these Gd forms resulted in the appearance of a 40-kDa C-terminal Snake polypeptide, approximately the size predicted for the Snake catalytic domain of 29 kDa plus a Myc-based C-terminal epitope tag of 9.5 kDa. In contrast, it was found that Snake cleavage does not occur when the Snake substrate contains a mutation in the zymogen-activation site. These results suggest that Gd cleaves Snake at its zymogen-activation site. This result suggests that Snake is cleaved by Easter within the prodomain, which could either enhance or inhibit further Snake activation by Gd as part of a positive or negative feedback loop (LeMosy, 2001).
Surprisingly, the Gd zymogen is also able to induce the cleavage of SNKS-A to generate the 40-kDa Snake catalytic domain, although at lower levels than those seen with the truncated Gd forms. This observation led to a test of whether coexpressing the zymogen forms of all three proteases could result in the activation of Easter. It was found that a catalytically inactive Easter substrate (EAS-A) is cleaved when expressed with both Gd and Snake but not when expressed with either Gd or Snake alone. EAS-A is cleaved to a 35-kDa form representing a C-terminal fragment, as judged by its reactivity with an antibody against a C-terminal FLAG-epitope tag. This C-terminal fragment migrates similarly to EADeltaN. In addition, mutation of the zymogen-activation site in EAS-A eliminates production of the 35-kDa polypeptide when the mutant protein is expressed with Gd and Snake. These results suggest that, when expressed with Gd and Snake, Easter can be cleaved at its zymogen-activation site and therefore activated (LeMosy, 2001).
To determine whether Snake is responsible for the cleavage of EAS-A in this experiment, EAS-A was expressed with either preactivated Snake (SNKDeltaN), or preactivated Gd (GDDeltaN1). SNKDeltaN promotes efficient conversion of EAS-A to the 35-kDa C-terminal form, whereas GdDeltaN1 is unable to promote cleavage of this substrate, supporting the conclusion that Snake is the protease that activates Easter. In these experiments, it was not possible to directly demonstrate that Easter becomes an active protease because the catalytically inactive EAS-A substrate was used. However, similar experiments using wild-type Easter provided indirect evidence that a proteolytically active Easter is generated. When the wild-type Easter zymogen was expressed with both Gd and Snake, but not either alone, a fragment of 32 kDa lacking the C-terminal FLAG tag was the most abundantly cleaved form of Easter detected, although the 35-kDa form could still be detected, albeit weakly. These results suggest that the active Easter protease is unstable and may undergo further processing in a reaction that requires its own proteolytic activity. In further support of the conclusion that both Snake and Easter become activated when expressed with the Gd zymogen, it was found that both the 40-kDa Snake catalytic-domain fragment generated by Gd and the 50-kDa Snake product generated by Easter could be detected in cells expressing all three wild-type zymogens (LeMosy, 2001).
These results suggest that the Gd zymogen has some activity against Snake, but that the processing of Gd gives rise to more active forms. To determine whether the Gd zymogen undergoes proteolytic processing when it promotes the activation of Snake and Easter, the processing of Gd was examined by Western blotting. When expressed alone, Gd exists predominantly as the full-length zymogen, although minor processed forms can be detected. However, when expressed with Snake or both Snake and Easter, two cleavage products at 46 kDa and 50 kDa are seen prominently, whereas little cleavage is seen if Gd is expressed with Easter. Both processed forms react with an antibody to a C-terminal HA tag present in the protein, indicating that they represent C-terminal fragments of Gd, and the 46-kDa cleavage product is similar in size to GdDeltaN1 (LeMosy, 2001).
The cleavage of Gd in these reactions depends on Gd's own catalytic activity as well as that of Snake. This result suggests that Gd can promote its own activation, perhaps through self-cleavage or positive feedback regulation involving the downstream proteases. To test whether Gd is cleaved by itself, Snake, or Easter, the catalytically inactive Gd substrate (GDS-A) was coexpressed with either preactivated GdDeltaN2, SNKDeltaN, or EADeltaN. Whether Gd is cleaved by Nudel's protease domain, NDL-PD was examined. All of the proteases appear capable of cleaving Gd. However, SNKDeltaN generates both 46- and 50-kDa forms of cleaved Gd, previously seen when the Gd, Snake, and Easter zymogens are coexpressed, whereas NDL-PD and GdDeltaN2 primarily produces the 46-kDa form and EADeltaN primarily produces the 50-kDa form (LeMosy, 2001).
Earlier studies suggested that Gd activity is restricted to the ventral side of the embryo by the action of pipe as well as nudel. To test whether proteolytic activation of Gd is regulated by these genes, processing of Gd was examined in wild-type and mutant backgrounds. Western blot analysis of Gd present in wild-type ovary extracts identified a polypeptide of 64 kDa, the size predicted for the Gd zymogen. This polypeptide is absent in ovary extracts derived from gd9 mutant females and is replaced by a smaller polypeptide, whereas in several other gd mutants, no specific Gd polypeptide could be detected. In no case were 27- through 34-kDa Gd species detected in ovary extracts, as has been described by Konrad (1998). Together, these findings suggest that Gd is present principally as a full-length zymogen during oogenesis (LeMosy, 2001).
To avoid strong background bands in the 46-kDa region of gels that complicated detection of endogenous Gd, a transgenic fly strain expressing the HA-tagged Gd was used in in vitro experiments; this construct rescues wild-type gd function in gd mutant embryos. As in the case with endogenous Gd, a single 64-kDa band was detected corresponding to the GdHA zymogen. In contrast to the undetectable level of Gd processing observed during oogenesis, a dramatically different picture is seen in embryogenesis. In extracts of laid eggs containing GdHA, a major species of 46 kDa was detected, similar in size to the smaller of the two cleaved Gd forms seen in vitro, in addition to the Gd zymogen. The 46-kDa form is strongly detected in the first hour of embryogenesis and persists for the first 3 h of embryonic development (LeMosy, 2001).
The processing of GdHA was examined in nudel and pipe mutant backgrounds. The 46-kDa form could not be detected in ndl mutant eggs; this finding is consistent with genetic data suggesting that the Nudel protease is required for the activity of Gd and the other proteases. In contrast, the 46-kDa cleaved Gd product is detected in pipe mutant eggs. Surprisingly, this result suggests that Gd processing in vivo occurs independently of the spatial cue generated by the action of pipe (LeMosy, 2001).
The localization of Gd during oogenesis was examined to test the possibility that prelocalization of Gd to a ventral site is important for determining the spatial distribution of the ventralizing signal. After its secretion in late oogenesis, Gd is present uniformly within the perivitelline space surrounding the oocyte. The distribution of Gd is indistinguishable from that of Nudel, suggesting that Gd is localized at the oocyte surface. In biochemical fractionation experiments, Gd was found in a 16,000 × g pellet fraction from which it could be released only by strong denaturing agents or high pH. These findings suggest that the Gd zymogen, apparently like the Snake and Easter zymogens, is uniformly distributed in the perivitelline space and, like Nudel, is not freely diffusible within this compartment (LeMosy, 2001).
Injection experiments involving the use of dominant active Easter (Chasan, 1992) and Snake (Smith, 1994), as well as injection of perivitelline (PV) fluid from dorsal mutant embryos into gd mutant embryos, have lead to production of ventral elements at the site of injection, rather than in the normal ventral region (Stein, 1992). These data suggest that D/V polarity is established by asymmetric presentation of the Toll ligand to the oocyte. PV fluid from dorsal mutant embryos (thought to be depleted of Spätzle ligand because of the presence of the Toll receptor) can rescue D/V polarity in snake and easter mutant embryos. This same PV fluid cannot restore normal ventral structures to either gd or nudel embryos. In contrast, injection of PV fluid from Toll mutant embryos (thought to contain active Spätzle ligand) into gd embryos produces ventral structures at the site of injection (Stein, 1992). The same fluid injected into snake or easter embryos produces embryos with normal polarity, independent of the site of injection (Stein, 1992). Because the temperature-sensitive period for both Nudel and Gd action includes a period before fertilization when the initial D/V asymmetry is known to be established (Konrad, 1988), it is possible that these two gene products cooperate to form a localized anchor for a Spätzle activating complex (Konrad, 1998 and references).
Embryos lacking the maternal activity of gd (hereafter referred to as gd mutant embryos) fail to undergo ventral and lateral development, therefore developing the dorsalized phenotype evident during gastrulation and in the cuticle pattern. To test whether ventral and lateral development in gd mutant embryos can be rescued, these embryos were injected with synthetic RNA encoding the Gd protein. Injection of a low concentration of gd RNA results in a high percentage of complete rescue, with embryos showing the wild-type pattern of gastrulation movements and cuticular structures, including some that hatched out of the eggshell. Complete rescue with normal dorsoventral polarity is observed whether the RNA is injected into the prospective ventral or dorsal side of the embryo as defined by the egg shape. The level of complete rescue was comparable to what has been observed in experiments involving the injection of snake RNA into snake mutant embryos and easter RNA into easter mutant embryos. Rescue of ventral and lateral development in gd mutant embryos by injection of gd RNA suggests that the Gd protein is required after fertilization to establish the embryonic dorsoventral axis (Han, 2000).
An examination was carried out to see whether a high level of gd RNA could elicit ventral and lateral development in the absence of other genes that, like gd, act at the beginning of the Toll signaling pathway. Injection of a high concentration of gd RNA results in ventral and lateral development in embryos from strongly dorsalizing mutants of nudel and pipe; however, these embryos develop a lateralized phenotype lacking the dorsoventral asymmetry of ventralized embryos. The response to injection is stronger in pipe mutant embryos than in nudel mutant embryos; most of the latter remain dorsalized. With a moderately dorsalizing windbeutel mutant, injection of gd RNA results in most embryos becoming ventralized and a smaller number of embryos becoming lateralized. Because gd can induce ventral and lateral development in the absence of normal nudel, pipe, or windbeutel activity, gd must act downstream of these genes. Injection of a high level of gd RNA does not elicit ventral and lateral development in snake mutant embryos, consistent with the previous finding that gd acts upstream of snake (Han, 2000).
To determine whether wild-type (wt) gd transcripts can rescue embryos from gd mothers, gd RNA at different concentrations was microinjected into embryos lacking gd function. Injection of wt gd RNA restores ventrolateral pattern elements such as filzkorper or ventral denticles, in a concentration-dependent manner. While no ventrolateral pattern elements were observed in uninjected embryos, when injected with 1 µg/ml RNA, 19% of embryos exhibited filzkorper. At 100 µg/ml RNA, 2% of embryos exhibited filzkorper and 44% exhibited ventral denticles. At 1 mg/ml, 31% of the embryos exhibited ventral denticles and the number of embryos with no cuticle increased to 68%. The increase in the no-cuticle phenotype is consistent with high concentrations of gd RNA specifying mesodermal cell fate, which does not make cuticle (DeLotto, 2001).
The phenotype of gd rescued embryos differs from snk RNA and ea RNA rescued embryos. Microinjection of snk and ea RNA can rescue completely the phenotype of embryos from snk and ea mothers and normalize D/V cell fates, producing hatching larvae. However, in no case has ventralization been observed for high concentrations of wt snake or ea RNA. While gd RNA restores ventrolateral pattern elements in a concentration-dependent manner the pattern was never normalized. Furthermore, while snk and ea RNA rescue shows no position dependence, the rescue observed with gd RNA is largely localized to the injection site. Embryos injected with moderate RNA concentrations produce ventrally open cuticles with split ventral denticles near the site of injection (DeLotto, 2001).
To determine whether microinjection of gd RNA transcripts could alter D/V cell fates in wt embryos, gd RNA was injected into OregonR embryos and the pattern was analyzed. After injection both the gastrulation pattern and larval cuticles were locally ventralized and all injected embryos failed to hatch. To determine how gd RNA microinjection alters the fate map, embryos were injected with gd RNA, and then either probed with antibodies against Twist, a mesodermal cell fate marker, or hybridized in situ for rhomboid, a ventrolateral cell fate marker. In wt embryos, Twist is expressed as ventral stripe, 16-18 cells wide along the anterior-posterior length of the blastoderm in the future mesoderm. The first domain of rhomboid expression in wild-type embryos is two ventrolateral stripes between 6 and 8 cells wide, within the neuroectodermal primordium. Since early lateral rhomboid expression is limited to a defined intermediate concentration of Dorsal, it has been used as a sensitive indicator of the slope and position of the Dorsal gradient (DeLotto, 2001).
To determine how gd RNA concentration affects the Dorsal gradient, RNA was injected at 50 µg/ml at the posterior of embryos from gd9/gd9 or wt females. Embryos were allowed to develop and in situ hybridized with rhomboid. Injected wt embryos exhibit two ventrolateral stripes, however they are shifted dorsally near the site of injection. The width of the rho stripe is identical to that of wt embryos. When examined for Twist expression, expression of Twist is expanded dorsally whereas at the anterior it is closer to normal. Thus, increasing Gd dosage in wt embryos locally expands the mesodermal anlagen without altering the slope of the Dorsal gradient (DeLotto, 2001).
gd RNA transcripts were injected into an embryo from a gd9/gd9 mother. Uninjected gd9/gd9 embryos show no lateral rhomboid expression at cellular blastoderm. Over the dorsal and ventrolateral regions of the gd injected embryo, rho is expressed as a uniform stripe approximately six cells wide, shifted dorsally near the site of injection. However, away from the injection site on the ventral side, the stripe broadens to cover a large part of the ventral region. The primary consequence of increasing gd concentration is to shift the position of the boundary between mesoderm and ventral neurogenic ectoderm dorsally, while the width of the ventral neurogenic ectodermal primordium remains constant. However, the ventral part of the egg responds more to an equivalent gd RNA concentration than dorsal or ventrolateral parts of the embryo as rho expands. In all injected embyros, the transition from stripe to broad expression is sharp and at the same D/V position. This increased response on the ventral side defines a previously unidentified stripe-like ventral zone (DeLotto, 2001).
Pipe is a candidate for defining an asymmetric cue directing D/V pattern during embryogenesis. To test whether gd RNA ventralization requires pipe, gd RNA was injected into embryos from pip664/pip664 females. At low gd RNA concentrations, no effect was seen. However, at 100 µg/ml, small ectopic patches of ventral denticles were observed. Between 100-500 µg/ml localized expression of Twist appeared close to the injection site. When hybridized in situ for rho, either one or two radial stripes spanning the D/V axis were generated. In wt embryos a third rho stripe appears in the future amnioserosa very shortly after the two lateral stripes appear. When two rho stripes were observed in embyros from pipe-null mothers, the distance between them was the same as the distance between the lateral rho stripes and the amnioserosal rho stripe. All injected embryos stained for Twist show only one patch of Twist expression near the injection site. When it appears, the second rho stripe correlates with amnioserosal rho expression. It is concluded that high gd RNA concentrations in a pipe-null background induces an ectopic axis with the ventral pole defined by the point of injection. Embryos from snk229-, ea1- and spz197-null females were microinjected with 100 µg/ml gd RNA and remained dorsalized, indicating that this effect requires their activities (DeLotto, 2001).
The data presented here suggest that the shape of the Dorsal gradient is not directly determined by asymmetric cues in the eggshell but rather arises within the perivitelline space as a consequence of self-regulatory properties of the protease cascade triggered by Gd. Localized phenotypic rescue is consistent with the idea that Gd is membrane bound. Since Gd can produce an ectopic axis in a pipe-null background, pipe activity is not required for binding. Binding of Gd to a surface within the PVS is therefore independent of the spatial control of activation that is normally ventrally restricted and requires Pipe (DeLotto, 2001).
The spatial activation of Gd must be regulated subsequent to and independent of binding. This could be explained via interaction of Gd with a Pipe-modified proteoglycan. Since pipe is a heparan sulfate 2-O-sulfotransferase and its expression is restricted to the ventral third of the somatic follicle cell, it presumably modifies a somatically expressed proteoglycan. Ventrally restricted modification by Pipe of this proteoglycan could control Gd activation. It is known that complement and coagulation pathways can be both positively and negatively regulated by interaction with heparan sulfate proteoglycans (DeLotto, 2001).
Since direct quantitative measurement of the Dorsal gradient is difficult, one of the most sensitive markers for the position and slope of the Dorsal gradient, rho expression, was used. The data suggest that a self-regulating patterning mechanism exists, which is independent of Gd concentration and can determine the slope of the gradient. The injection phenotype is not exactly like that of embryos from females carrying mutations in gurken and torpedo (Egfr). In these mutations, while rho stripes are shifted dorsally, the mesoderm splits into two ventral furrows. This splitting has not been observed in any gd RNA injections (DeLotto, 2001).
An intriguing observation from these injections is that of a discontinuity along the width of the embryo. A ventral region appears to be more sensitive than lateral and dorsal parts of the embryo are to Gd activity. While over most of the ventrolateral and dorsal surfaces a uniform width rho stripe is generated, away from the point of injection, rho is expressed in a broad ventral patch. The transition between stripe and broad zone is sharp, bilaterally symmetrical and always at the same relative position in injected embryos. These results suggest the existence of a ventral prepattern in the form of a broad stripe-like zone with distinct boundaries (DeLotto, 2001).
Han (2000), while also reporting ventralization by gd microinjection, has indicated that microinjection of gd RNA into embryos from gd7/gd7 mothers can also normalize and rescue some embryos to hatching. While this differs from the observations in gd4 and gd9 embryos, it is possible that the ability to rescue gd7 is an allele-specific effect. gd7, while an amorphic allele, results from a single amino acid change of Gly44 to Asp in contrast to gd4 and gd9, which are deletions and truncations, respectively. Allele-specific effects are observed at gd (DeLotto, 2001 and references therein).
The RNA concentration required to produce an ectopic ventralizing signal in pipe-null embryos is significantly greater than that necessary to 'rescue' an embryo from a gd-null mother. In the pipe injections, it is proposed that high local concentrations of Gd become proteolytically active at the ectopic site by 'titrating out' regulatory serpins that normally control the cascade. Serpins specifically regulating this pathway have not yet been identified, but there is evidence for a serpin that binds Easter and evidence for a serpin regulating Spätzle processing in innate immunity. While the Dorsal gradient formed in injected pipe nulls is somewhat broader than in wt, it is still formed independently of Pipe. Therefore, the part of the pathway responsible for shaping the Dorsal gradient must have self-regulating properties that are independent of the ventral prepattern (DeLotto, 2001).
The data suggest a model in which the Dorsal gradient is shaped dynamically via a proteolytic activation cascade during embryogenesis starting with a ventral stripe prepattern. The prepattern can be visualized as initially consisting of: (1) a broad ventral zone or stripe in which Gd activity is potentiated, and (2) the remainder of the PVS in which Gd or the other proteases are inhibited. An interesting possibility is that the ventral stripe zone results from the modification of a uniformly distributed inhibitory substance through pipe post-translational modification. The sharp boundaries of the zone identified in these experiments suggests the ventral prepattern is not already graded. The fine-tuning of the slope of the Dorsal gradient may involve complex interactions within the proteolytic cascade downstream of Gd or via feedback to Gd. The results presented in this study are consistent with Gd being a protease that has a pivotal role in interpreting the D/V prepattern as well as initiating and shaping the Dorsal gradient during embryogenesis (DeLotto, 2001).
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. In this study, 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 (LeMosy, 2006).
The results establish that spatial control by pipe occurs at or above the level of Easter zymogen activation, rather than through direct promotion of Easter's catalytic activity against Spätzle. While Easter zymogen activation could be the first spatially controlled reaction, a complex interaction between GD and Snake has been shown to present a particularly attractive target for regulatory control. For example, an engineered auto-activating allele of Snake still requires function of the GD pro-domain for signaling, and a ventral region of the perivitelline space appears to be more sensitive to levels of GD introduced by microinjection than is the dorsal side. GD and Snake appear to interact in a way that stimulates GD catalytic activity in vitro, so it is possible that a ventral cofactor enriching the local concentrations of these components could greatly enhance the cascade. Further biochemical studies of GD and Snake processing in vivo should aid in defining the mechanism of spatial control in this developmentally important protease cascade (LeMosy, 2006).
Easter and Snake both share significant structural homology with extracellular trypsin-like serine proteases. Experiments using dominant active forms of Snake and Easter show that Snake activates Easter, which in turn activates the Spätzle ligand. In combination with the somatically expressed genes, windbeutel, pipe, and nudel, Gd activates Snake in a location-dependent manner that marks the future ventral cells. The recent cloning of nudel indicates that Ndl is a large (350 kDa) extracellular glyco-protein with motifs suggesting that it might bind to extracellular matrix as well as to other proteins. Nudel also contains a serine protease catalytic domain. The occurrence of several protease-like proteins both upstream (Nudel) and downstream (Snake, Easter) of Gd may be responsible for the multiply processed Gd peptides observed on Western blots. The physical location of the Nudel protein (including whether it is incorporated as a component of the vitelline membrane) is not yet known, although the fragility of the ndl embryos suggests that Ndl may be required for stability of the vitelline membrane. Gd mRNA is expressed in follicle cells beginning at about stage 10 and may be graded in a ventral to dorsal manner, although it is expressed uniformly in the nurse cell/oocyte complex. The location of Gd remains to be determined. Four of the five currently identified genes needed for asymmetric activation of the Spz ligand encode secreted members of the serine protease superfamily. It is proposed that Gd functions as part of an anchored complex that triggers a proteolytic activation hierarchy involving Nudel, Snake, and Easter, resulting in localized activation of Spz ligand and asymmetry along the D/V axis (Konrad, 1998 and references).
Three serine protease zymogens -- Gastrulation defective (Gd), Snake (Snk) and Easter (Ea) -- and a nerve growth factor-like growth factor ligand precursor, Spätzle, are required for specification of dorsal-ventral cell fate during Drosophila embryogenesis. The proteases have been proposed to function in a sequential activation cascade within the extracellular compartment called the perivitelline space. Biochemical interactions between these four proteins have been examined using a heterologous co-expression system. The results indicate that the three proteases do function in a sequential activation cascade, that Gd becomes active and initiates the cascade and that interaction between Gd and Snk is sufficient for Gd to cleave itself autoproteolytically. The proteolytically active form of Ea cleaves Gd at a different position, revealing biochemical feedback in the pathway. Both Gd and Snk bind to heparin-Sepharose, providing a link between the pipe-defined ventral prepattern and the protease cascade. These results suggest a model of the cascade in which initiation is by relief from inhibition, and spatial regulation of activity is due to interaction with sulfated proteoglycans (Dissing, 2001).
nudel, a somatically required dorsal group gene, encodes a 320 kDa mosaic protein with a centrally located serine protease catalytic domain. It has been suggested that nudel might activate a dorsal-ventral proteolytic cascade. This possibility was investigated using the assay system by co-expressing either the full-length form of Nudel, or a constitutively active Nudel serine protease catalytic chain (ndlDeltan) in different combinations with the four proteins described here. In all experiments conducted thus far, no reproducible effect of Nudel or nudDeltan upon any of the other proteins nor an effect of any of the other proteins upon Nudel-specific polypeptides was observed. Consequently there is no evidence for direct biochemical interaction of Nudel with Gd, Snk, Ea or Spz and therefore no direct role for Nudel within the protease cascade can be ascribed. Nudel apparently does not directly activate the cascade but rather is required earlier for proper establishment or maintenance of the ventral prepattern (Dissing, 2001).
Gd appears to play a critical role in the proteolytic cascade since it can initiate the cascade yet does not appear to require classical zymogen activation in order to do so. The data suggest that exposure to the zymogen form of Snk is sufficient for Gd to become active and activate Snk, triggering the cascade, and for Gd to generate lower molecular weight polypeptides. With respect to a mechanism of activation, it is interesting to note that Gd bears some similarity to mammalian complement factors C2 and B. These proteases have novel activation mechanisms requiring complex formation and a conformational change as a prerequisite to activation. An alternative explanation for how Gd functions is that it has some intrinsic activity as a zymogen. Upon binding to and activating Snk, it then proteolytically processes itself to generate lower molecular weight inactive forms (Dissing, 2001).
Activated Ea can proteolytically process Gd, suggesting that a second form of feedback occurs within the cascade. Ea cleaves Gd at a novel position to generate a Gd polypeptide that is slightly larger than the predominant band generated by Gd itself. The significance of processing by Ea is not altogether clear from the data. However, it is reasonable to assume that cleavage by Ea is a way to modify Gd's biochemical properties. It is proposed that Ea may feed back negatively on the precursor form and/or the active form of Gd. This would provide a means of down-regulating the protease cascade to prevent amplification from 'running away', resulting in overproduction of the ventralizing signal (Dissing, 2001).
Since Gd can autoactivate the cascade, a requirement for an upstream protease to activate the cascade need not be postulated. Rather, the data might argue that a mechanism exists to prevent Gd from becoming activated too early in embryogenesis before the Toll receptor is completely expressed on the plasma membrane. This idea has been incorporated into a biochemical model of the cascade. The existence of an inhibitory factor that prevents Gd from activating Snk is proposed. The inhibitory factor must itself be inactivated in a spatially or temporally regulated way for proper activation of the cascade. However, the net result must be to permit the activation of Gd within the ventral perivitelline space at the proper time for accurate elaboration of the ventralizing signal. This region of the perivitelline space may correspond to the ventral stripe prepattern described from gd mRNA injection experiments (Dissing, 2001).
The ability of both Gd and Snk to bind to heparin-Sepharose suggests that their activities may be regulated in vivo by sulfated proteoglycans. Since pipe expression in somatic follicle cells is ventrally restricted and the gene encodes a heparan sulfate 2-O-sulfotransferase, sulfate modification of an as yet unknown proteoglycan may provide the ventral cue in the egg. Gd and Snk may interact directly with this sulfated proteoglycan and this interaction may provide the ventral restriction to activation of the cascade (Dissing, 2001).
It has been suggested that the protease cascade may enable an initial asymmetry in the form of a ventral stripe prepattern to be converted into a graded distribution of processed Spz ligand. The potential for both (1) amplification with subsequent steps and (2) feedback after activation could enable the cascade to self-regulate the shape of the Dorsal protein gradient. This property could provide plasticity in the patterning process and a means of compensating for minor variation in the size and shape of individual embryos. Such a mechanism would also be sufficiently adaptable that it could be conserved evolutionarily. The data suggest some remarkable similarities between the dorsal-ventral protease cascade and the classical complement and blood coagulation pathways (Dissing, 2001).
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 Spatzle (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. This study shows 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 (see Figure 4: Model for ventral processing of Ea by 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 (Cho, 2012).
The results demonstrate that GD provides two essential functions in the dorsoventral (DV) pathway: activation of the Snk protease and ventrally localized Ea cleavage. These findings support a model in which processing of GD generates a fragment with a C-terminal protease domain and N-terminal sequences that interact with the sulfated ventral cue to localize GD to the ventral region of the PVS. GD may bind directly to carbohydrates associated with vitelline membrane protein that have been sulfated as a result of Pipe enzymatic action in ventral cells of the follicle layer. Consistent with this possibility, GD protein has been shown to bind to heparin and to anionic components of a highly purified Drosophila eggshell matrix preparation (Cho, 2012).
It is proposed that ventrally localized GD binds to both Ea and Snk and plays a direct role in promoting an interaction between them. It is possible that a single GD molecule binds either to Ea or to Snk and that ventral localization of GD acts to concentrate GD-bound Ea and Snk and bring them into proximity. Alternatively, GD bound only to Snk or Ea may undergo a conformational change when it interacts with the Pipe-sulfated ventral cue that results in an enhancement of Snk proteolytic activity or an increased susceptibility of Ea to cleavage by Snk. Lastly, GD bound simultaneously to both Ea and Snk may respond to the ventral cue by undergoing a conformational change that brings Snk and Ea into productive juxtaposition and results in Ea cleavage. A mechanism in which GD interacts with Pipe and the sulfated ventral cue to promote productive interaction between Ea and Snk can also explain the results of RNA injection studies. Injection of very high levels of in vitro synthesized RNA encoding the GD zymogen could lateralize or reorient the polarity of embryos produced by pipe mutant females. Those results were interpreted to indicate that Pipe is normally required for activation of GD on the ventral side of the embryo, but that at high concentrations, GD could become enzymatically active by an alternative Pipe-independent mechanism. The current results suggest instead that high concentrations of GD can promote interactions between Easter and Snake, or conformational changes in those proteins that lead to Snake-mediated cleavage of Easter, even in the absence of Pipe and the ventral cue. In conclusion, by demonstrating that the GD serine protease is localized within the ventral PVS in a Pipe-dependent manner and that the interaction of GD with the Pipe-sulfated ventral cue enables it to bring about the ventrally restricted processing of Easter, the work reported in this study explains how ventrally localized sulfotransferase activity in the follicle cell layer leads to spatially localized activation of the Toll receptor and to the formation of the Drosophila embryonic DV axis (Cho, 2012).
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