Protein Interactions

To explore factors involved in the enrichment of the Nudel transcript in ventral follicle cells, nudel expression was examined in egg chambers from females mutant for genes that act genetically upstream of nudel. Weakly ventralizing Egf-r (torpedo) mutants do not alter the ventral enrichment of nudel transcript. In contrast, nudel RNA is uniformly distributed in most of the egg chambers produced by strongly dorsalizing fs(1)K10 mutations. A similar result was obtained with egg chambers from females homozygous for a pipe mutation, resulting in strongly dorsalized embryos (Hong, 1995). '

Analysis of mosaic females indicates that the expression of the genes nudel, pipe, and windbeutel is required in the somatic tissue, presumably in the follicle cells that surround the oocyte. Thus, information coming from outside the egg cell influences dorsoventral pattern formation during embryogenesis. In transplantation experiments, the perivitelline fluid from the compartment surrounding the embryo can restore dorsoventral pattern to embryos from females mutant for nudel, pipe, or windbeutel. The positioning of the transplanted pervitelline fluid also determines the polarity of the restored dorsoventral axis. It is proposed that the polarizing activity, normally present at the ventral side of the egg, is a ligand for the Toll receptor. Presumably, local activation of the Toll protein by the ligand initiates the formation of the nuclear concentration gradient of the Dorsal protein, thereby determining dorsoventral pattern (Stein, 1991).

The product of the Drosophila easter gene, a member of the trypsin family of serine proteases, needs to be more active ventrally than dorsally in order to promote normal embryonic polarity. The majority of the Easter protein in the embryo is present in the unprocessed zymogen form and appears to be evenly distributed in the extracellular space, indicating that the asymmetric activity of wild-type Easter must arise post-translationally. A dominant mutant form of easter that does not require cleavage of the zymogen for activity (ea delta N) is active both dorsally and ventrally. The ea delta N mutant bypasses the requirement for five other maternal effect genes, indicating that these five genes exert their effects on dorsal-ventral patterning solely by controlling the activation of the Easter zymogen. It is proposed that dorsal-ventral asymmetry is initiated by a ventrally-localized molecule in the vitelline membrane that nucleates an Easter zymogen activation complex, leading to the production of ventrally active Easter enzyme (Chasen, 1992).

At least three of the dorsal group genes (snake, easter and gastrulation defective) encode secreted serine proteinases which probably function during early development in the perivitelline compartment of the embryo. The Easter proteinase is homologous in its light chain sequence to the hemocyte proclotting enzyme (PCE) of the Japanese horseshoe crab Tachypleus tridentatus. PCE is the terminal member of a proteolytic cascade activated in response to microbial polysaccharides and acts to cleave coagulogen, an invertebrate equivalent of fibrinogen. On the basis of this homology the overall primary structure of the easter proteinase, its mode of activation and its substrate specificity can be predicted. The result also suggests that Easter functions zygotically in hemocytes in a Drosophila defensive response analogous to that found in Tachypleus. The Toll receptor protein is absent in early cleavage embryos but accumulates rapidly at the syncytial blastoderm stage, the developmental stage at which its function is required. This finding suggests that translation of Toll mRNA is regulated in response to fertilization and egg deposition. These two observations are consistent with a model of dorso-ventral pattern formation in which a proteolytic cascade is activated uniformly in the perivitelline compartment of the embryo and causes the release of ventrally localised ligands of the Toll receptor. A possible alternative model in which a proteolytic cascade is activated in response to a ventrally restricted signal is also discussed (Gay, 1992).

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 (as demonstrated by the transplantation of perivitelline fluid) are found three separate activities present in the perivitelline fluid that can restore dorsal-ventral polarity to mutant easter, snake, and spatzle 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 (Stein, 1992).

An examination has been made of the effect of a preactivated form of the Snake protease on the generation of dorsal-ventral polarity. SP6 RNA microinjection experiments reveal that different cell fates acquired at cellular blastoderm can be specified by the amount and spatial distribution of activated Snake protein. These results support a protease cascade model in which localized activation of uniformly distributed protease proenzymes leads to the spatially restricted production of ligand in the perivitelline space on the ventral side of the embryo (Smith, 1994a).

Mutant alleles of snake were cloned and sequenced, revealing two types of lesions: point mutations that alter the amino acid sequence (snk073 and snkrm4) and point mutations that alter the splicing (snk229 or snk233) of intron 1 of the mRNA from the normal 3' end of the intron to a cryptic site. snake mutant embryos derived from homozygous mothers can be fully rescued by injection of RNA transcripts of the wild-type snake cDNA. RNA phenotypic rescue and site-directed mutagenesis experiments indicate that Snake requires the serine, histidine and aspartic acid of the catalytic triad for normal activity. Deletion experiments show that an acidic proenzyme domain is required for Snake rescue activity to be uniformly distributed throughout the embryo. A second proenzyme domain, called the disulfide knot, appears to be essential for normal regulation of Snake. Transcripts encoding only the proenzyme polypeptides of either Snake or Easter can dorsalize wild type embryos. A model is proposed in which the proenzyme determinants of both the Snake and Easter enzymes mediate interaction between the serine proteases and other components of the dorsal-ventral patterning system (Smith, 1994b).

The easter gene, required for the development of all lateral and ventral pattern elements in the Drosophila embryo, appears to encode an extracellular serine protease. Dominant easter alleles increase the number of cells that give rise to lateral and ventral structures. Nine dominant and four recessive mutations are caused by single amino acid substitutions at conserved sites in the putative serine protease catalytic domain. The activity of dominant products was assayed by injecting in vitro synthesized transcripts from the dominant alleles into young embryos. The results suggest that the dominant easter products cleave the normal substrate, but fail to respond to a spatially asymmetric regulator (Jin, 1990).

Establishment of dorsoventral polarity within the Drosophila embryo requires extraembryonic positional information generated during oogenesis. The spatial restriction of Toll activation requires earlier signaling events that occur during oogenesis to determine the dorsoventral pattern of the follicular epithelium. The genes windbeutel, pipe, and nudel are required within the somatic follicle cells of the ovary for production of this spatial cue. Using a novel follicle cell marker system, the effect of mutant follicle cell clones has been evaluated on the embryonic dorsoventral pattern. No spatially localized requirement for nudel activity is found. These results imply that wild-type ndl activity in subpopulations of follicle cells, regardless of their position, must be sufficient to mediate its dorsoventral patterning function. In contrast, windbeutel and pipe are required only within a restricted ventral region of the follicular epithelium. This ventral region can determine lateral embryonic cell fates nonautonomously, indicating that spatial information originating ventrally is subsequently refined, perhaps via diffusion, to yield the gradient of positional information that determines the embryonic dorsoventral pattern (Nilson, 1998).

A determination was made of the minimum clone width along the dorsoventral axis that leads to local elimination of the entire twist stripe. This allowed the definition of a region along the ventral side of the follicular epithelium where wind activity in the follicle cells is required in order to induce embryonic twist expression. Loss of wind acivity in a 1-2 cell wide region does not eliminate the entire twist stripe in the corresponding region of the embryo. However, when wind activity is lost from a region approximately 4-6 cells wide overlying the ventral midline region, local loss of the entire twist expression stripe results. This analysis leads to an initial estimate that wind activity is required in a ventral region approxiately 4-6 follicle cells wide along the dorsoventral axis to induce a normal pattern of twist expression in the embryo. Although these results demonstrate a localized requirement for wind and pipe, they do not imply that either the expression of these genes or their activity is spatially restricted. Rather, these results identify a functionally distinct region within the ventral follicular epithelium that is required to establish the embryonic Dorsal gradient. A likely interpretation is that this region defines a spatially localized process that ultimately generates active Toll ligand. Interestingly, this area corresponds well to the width and postion of the twist expression domain (Nilson, 1998).

The dorsal-ventral asymmetry of the Drosophila embryo is established by a signal that is transmitted through the uniformly distributed Toll receptor, resulting in a graded relocation of Dorsal into the nuclei of the blastoderm embryo. gastrulation defective is one of the earliest acting of the maternally required genes that is crucial to this process. The gastrulation defective gene has been cloned and characterized and found to encode a protein structurally related to the serine protease superfamily, which also includes the Snake, Easter, and Nudel proteins. These data provide additional support for the involvement of a protease cascade in generating an asymmetric signal (i.e., asymmetric Spatzle activity) during the establishment of dorsal-ventral polarity in the Drosophila embryo (Konrad, 1998).

Injection experiments involving the use of dominant active Easter and Snake, as well as injection of perivitelline (PV) fluid from dorsal mutant embryos into gd mutant embryos, leads to production of ventral elements at the site of injection, rather than in the normal ventral region. 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. The same fluid injected into snake or easter embryos produces embryos with normal polarity, independent of the site of injection. This result strongly suggests that Gd is a key component required for establishing the localized activation of Spätzle and thus the asymmetric activation of Toll. 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 , it is possible that these two gene products cooperate to form a localized anchor for a Spätzle activating complex (Konrad, 1998 and references therein).

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 therein).

Three serine protease zymogens -- Gastrulation defective (GD), Snake (Snk) and Easter (Ea) -- and a nerve growth factor-like growth factor ligand precursor, Spaetzle, 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).

nudel: Biological Overview | Developmental Biology | Effects of Mutation | References

date revised: 24 October 98

Home page: The Interactive Fly © 1997 Thomas B. Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.