BIOLOGICAL OVERVIEW

Two signals initiate the events that will engender dorso-ventral patterning in the egg and in the embryo (Schumpbach, 1994). The first prepares the scene for the second. It is a signal from the oocyte to follicle cells (maternal cells surrounding the egg) to enter a dorsal differentiation program, and it requires the ligand Gurken and the receptor Torpedo. This first signal negatively delimits the ventral zone within the follicle cell epithelium, restricting the formation of the ligand of the second signal to a spatially restricted area in the ventral part of the egg. Having its boundaries prepared, the second signal awaits activation upon fertilization. It is this second signal that involves the ligand Spätzle and the receptor Toll.

Spätzle is one of 11 dorsal group genes identified by their completely dorsalized phenotype, lacking ventral, lateral and dorsolateral pattern elements. The dorsal group genes activate the Dorsal transcription factor, expressed in the ventral part of the embryo and responsible for ventralization of the embryo.

The area around the egg is filled with a perivitelline fluid capable of restoring dorsal-ventral polarity to mutant easter, snake and Spätzle embryos. This has been demonstrated by injecting perivitelline fluid into the perivitelline space of mutant eggs (Stein, 1992).

One component of the perivitelline fluid is zymogen serine protease, a member of the trypsin family coded for by the easter gene. Easter is one of three proteins in the perivitelline fluid required for Dorsal protein activation. Spätzle is the only dorsal group gene upstream of the Toll receptor that is required for Easter to exert its effect on the dorsal-ventral pattern (Chasen, 1992). Easter is responsible for activating Spätzle, so that Spätzle can then interact with Toll. The Easter protein activates Spätzle by proteolysis, producing a ligand that can bind the Toll receptor. Toll in turn activates Dorsal in a process involving a change in location of Dorsal protein from the cytoplasm to the nucleus.

The cloning of the spätzle gene necessitated employment of a reversion to the wild type of a dominant mutant of spätzle. Why a dominate mutant? Whatever the cause of a dominant mutation, inactivation of such a mutation might involve destruction of the gene itself. The dominant mutant allele was mutated by a viral insertion element (P) integrated in the midst of the spätzle gene, thereby inactivating it. Use of a P insertion element allows for the recovery of the chromosome element in which it inserts (by fishing for the P element).

A genomic library was constructed from the P revertant type, and a P positive clone was identified that hybridized with the same genome region to which spätzle maps (Marisato, 1994). This is proof that the P vector contains DNA homologous to spätzle. In a different lab, spätzle activity was purified using an assay for ventralization by perivitelline fluid (Schneider, 1994).

The antifungal defense of Drosophila is controlled by the spätzle/Toll/cactus gene cassette. A loss-of-function mutation in the gene encoding a blood serine protease inhibitor, Spn43Ac, has been shown to lead to constitutive expression of the antifungal peptide Drosomycin, and this effect is mediated by the spätzle and Toll gene products. Spätzle is cleaved by proteolytic enzymes to its active ligand form shortly after immune challenge; cleaved Spätzle is constitutively present in Spn43Ac-deficient flies. Hence, Spn43Ac negatively regulates the Toll signaling pathway, and Toll does not function as a pattern recognition receptor in the Drosophila host defense (Levashina, 1999).

Flies carrying ethylmethane sulfonate-induced mutations in the necrotic (nec) locus were used. The locus, which maps at position 43A, generates three transcripts encoding putative serine protease inhibitors of the serpin family. The nec mutants exhibit brown spots along the body and the leg joints, corresponding to necrotic areas in the epidermis. This mutant phenotype is rescued by a single transgenic copy of one of the serpin genes, Spn43Ac. Because the absence of a functional Spn43Ac serpin may affect proteolytic cascades involved in the host defense of Drosophila, the level of expression of the antimicrobial peptide genes were examined in nec mutants. All genes were induced 6 hours after challenge in wild-type (WT) flies; however, in nec mutants the gene encoding drosomycin is strongly expressed in the absence of immune challenge. The expression is further enhanced by immune challenge. The gene encoding the peptide metchnikowin, which has both antibacterial and antifungal activities, also exhibits constitutive expression in nec mutants, although the response is less marked than for drosomycin. In contrast, no constitutive expression is observed by genes encoding diptericin and cecropin A1, whose expression is either independent of the Toll signaling pathway or requires a signal from an additional pathway, depending on the immune deficiency (imd) gene (Levashina, 1999).

Overexpression of the Spn43Ac gene in nec flies abolishes the constitutive expression of drosomycin, whereas overexpression of a different serpin gene from the same cluster, Spn43Aa, has no effect on this phenotype. In a Tl or spz loss-of-function background, the nec-mediated constitutive expression of drosomycin is abolished, indicating that Spn43Ac acts upstream of spz and Tl. However, when the nec mutation is combined with gastrulation defective (gd) or snake (snk) loss-of-function mutations, constitutive expression of drosomycin is still observed, confirming that these proteases are not necessary for the Toll-controlled antifungal response. Furthermore, the constitutive expression of drosomycin is not affected when the nec mutation is in an imd mutant background, suggesting that the imd-mediated expression of the antibacterial peptide genes is independent of the proteolytic cascade controlled by Spn43Ac (Levashina, 1999).

The expression of the Tl gene and that of the downstream genes in the signaling cascade is up-regulated by immune challenge. The transcription of the Spn43Ac gene is up-regulated by immune challenge. This up-regulation is not observed in a Tl loss-of-function background. Conversely, Tl gain-of-function mutants exhibit a constitutive expression of Spn43Ac. In imd mutants, the up-regulation of Spn43Ac by immune challenge is similar to that in wild-type flies. Thus, Spn43Ac is an immune-responsive gene, and its expression is under the positive control of the Toll pathway. This could represent a negative feedback mechanism to shut down the activation of Toll by inhibiting the upstream proteolytic cascade (Levashina, 1999).

To function as a negative regulator of the Toll pathway upstream of Spätzle and Toll, Spn43Ac should be present in the hemolymph of adult flies. Indeed, immunoblotting with an antiserum directed against recombinant Spn43Ac reveals a band of ~60 kD in the blood of WT flies. This band is absent from the hemolymph of flies deficient for the Spn43Ac gene. The size of the mature Spn43Ac protein predicted from the cDNA sequence is smaller (52kD) than the size of the immunoreactive protein, possibly reflecting posttranslational modifications (because serpins are generally glycoproteins). After immune challenge, a band of ~50 kD is observed, which may correspond to the Spn43Ac serpin that has undergone cleavage by activated protease or proteases (Levashina, 1999).

During dorsoventral patterning of the embryo, the 382-residue Spätzle protein is cleaved to a 106-residue COOH-terminal active ligand form. Experiments on the putative proteolytic cleavage of Spätzle in the host defense have not been reported so far, and protein extracts from naive and immune-challenged flies were examined by protein immunoblotting, using two polyclonal antisera directed against recombinant COOH-terminal Spätzle. These antisera recognize the full-length Spätzle protein and a smaller COOH-terminal fragment of 16- to 18-kD. In experiments with unchallenged flies, a band corresponding to a protein of 40 to 45 kD is detected in denatured extracts. It is also present in extracts of hemolymph. One hour after immune challenge, the 40- to 45-kD band has disappeared, whereas an immune-reactive doublet of ~16- to 18-kD is apparent, which is assumed to correspond to the processed form of Spätzle protein. The Spätzle protein has glycosylation sites, which may account for slightly larger molecular sizes than predicted from the cDNA sequences. The 16- to 18-kD doublet is detected in unchallenged nec flies, together with the 40- to 45-kD protein corresponding to uncleaved Spätzle. This result is in agreement with the working hypothesis that in nec mutants the absence of the functional serpin leads to the constitutive cleavage of Spätzle. Finally, the strong signal of the 40- to 45-kD form of Spätzle together with that of the 16- to 18-kD form in nec mutants confirms at the protein level that the expression of the spz gene is regulated by a positive-feedback loop (Levashina, 1999).

These data indicate that in the absence of a functional product of the Spn43Ac serpin gene in the blood of adult flies, the Spätzle protein is spontaneously cleaved, leading to constitutive activation of the Toll signaling pathway. This phenotype can be rescued, either by a functional Spn43Ac transgene or by a spz- or Tl-deficient background. It is not known whether the protease, which cleaves Spätzle, is a direct target of the serpin (Levashina, 1999).

Conceptually, the activation of Spätzle by blood protease zymogens is similar to the coagulation cascade in the horseshoe crab, which can be activated by binding of LPS to an upstream multidomain recognition protein. Several serpins, which fall into the same class as Spn43Ac, can specifically inhibit the proteases of the coagulation cascade (Levashina, 1999 and references therein).

These results, and the parallels with the horseshoe crab coagulation cascade, imply that non-self recognition is an upstream event. Toll does not qualify as a bona fide pattern recognition receptor in Drosophila, in contrast to what has been proposed for Toll-like receptors in mammals. The actual pattern recognition receptor, which initiates the cascade leading to the cleavage of Spätzle and activation of Toll, remains to be identified. Genetic aberrations and deficiencies of mammalian serpin genes have been correlated with clinical syndromes, such as pulmonary emphysema, angioedema, and coagulopathies, as a result of inappropriate inhibition of their respective target proteases. The demonstration that a serpin functions in the regulation of the Drosophila immune response highlights the similarities between innate immunity in insects and mammals and reinforces the idea of a common ancestry of this system (Levashina, 1999 and references therein).

GENE STRUCTURE

Toll, the receptor for Spätzle and the spätzle gene are tightly linked, both found within 0.4cm of one another (Morisato, 1994).

Bases in 5' UTR - 864

Bases in 3' UTR - 298

PROTEIN STRUCTURE

Two transcripts are found, one of 2.1 kb and a second of 2.3 kb. The 2.3 kb transcript codes for an extra 73 amino acids inserted into the N-terminal region of the protein (Morisato, 1994).

Amino Acids - 253

Structural Domains

The spätzle gene encodes a novel secreted protein that appears to require activation by a proteolytic processing reaction, controlled by genes acting upstream of spätzle in the genetic pathway. A deletion mutant that retains only the C-terminal 106 amino acids of spz can activate Toll in the absence of the genes normally required for SPZ activity. This domain includes 7 of the 9 cysteine residues of SPZ. The activity of the C-terminal domain is inhibited in the full-length precursor. Because the deletion that retains the C-terminal 168 amino acids rescues the spz mutant phenotype with normal polarity, and requires easter for activity, it is inferred that this truncated protein retains the inhibitory activity (Morisato, 1994).

Biochemical interactions underlying the generation of the ventralising signal during Drosophila embryogenesis were investigated by the expression of recombinant Easter and Spatzle proteins. An active form of Easter protease cleaves the Spatzle protein, generating a carboxyterminal polypeptide fragment which, when microinjected into the perivitelline space of a spatzle deficient embryo, directs production of ventrolateral pattern elements. This Spatzle carboxyterminal fragment is a disulfide-linked dimer. Modelling suggests that the core disulfide bonds and dimer arrangement of this fragment are highly similar to vertebrate nerve growth factor. Each polypeptide contains three intrachain disulfide bridges in a 1-3, 2-6 and 3-7 arrangement. Cysteine number five is assigned to a single interchain disulfide bridge resulting in the 24-kD dimer. The disulfide involved in dimerization contributes significantly but is not absolutely required for the activity of this protein. It is concluded that the 24-kD Spatzle carboxyterminal dimer must adopt a parallel (head-to-head) structure that is similar to NGF. Thus Spatzle may be considered a member of a new family of neurotrophin-like signaling molecules in invertebrate development (DeLotto, 1998).

EVOLUTIONARY HOMOLOGS

The ligand for the Toll receptor is thought to be Spätzle, a secreted protein that is activated by proteolytic cleavage. trunk, a gene required for activity of the Torso receptor, encodes a protein that resembles SPZ in several respects. In particular, the sequence suggests that TRK is a secreted protein and that it contains an internal site for proteolytic cleavage. Furthermore, the carboxy-terminal domain of TRK has a similar arrangement of cysteines to that of SPZ. It has been proposed that trk encodes an extracellular ligand involved in specifying terminal body pattern and suggested by analogy with SPZ that a cleaved form of TRK constitutes the ligand for the Torso receptor (Casanova, 1995).

The Spatzle/Toll signaling pathway controls ventral axis formation in Drosophila by generating a gradient of nuclear Dorsal protein. Dorsal controls the downstream regulators dpp and sog, whose patterning functions are conserved between insects and vertebrates. Although there is no experimental evidence that upstream events are conserved as well, the following question was posed: can a vertebrate embryo respond to maternal components of the fly Dorsal pathway? A dorsalizing activity is demonstrated for the heterologous Easter, Spatzle and Toll proteins in UV-ventralized Xenopus embryos; dorsalization is inhibited by a co-injected dominant Cactus variant. Thus the epistatic relationships between upstream and downstream components of the Drosophila dorsoventral (d/v) pathway are maintained in the frog, as is evident from the inhibtion of Spz and Easter activity by the dominant Cactus mutation. It is concluded that the Dorsal signaling pathway is a component of the conserved d/v patterning system in bilateria (Armstrong, 1998).

Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.

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