BIOLOGICAL OVERVIEW

Dorsoventral polarity of the Drosophila embryo requires maternal spätzle-Toll signaling to establish a nuclear gradient of Dorsal protein. The easter gene encodes a serine protease that generates processed Spätzle, which in turn acts as the Toll ligand. The shape of the Dorsal gradient is altered in embryos produced by females carrying dominant alleles of easter (ea^D) that do not require cleavage of the zymogen for activity. By examining the expression domains of the zygotic genes zen, sog, rho and twist, which are all targets of nuclear Dorsal, it has been shown that the slope of the Dorsal gradient is progressively flattened in stronger ea^D alleles. In the wild-type embryo, activated Easter is found in a high molecular weight complex called Ea-X, which is hypothesized to contain a protease inhibitor. In ea^D embryo extracts, an Easter form corresponding to the free catalytic domain is detected; this form is never observed in wild type. Mutant ea^D proteins retain protease activity, as determined by the production of processed Spätzle both in the embryo and in cultured Drosophila cells. These experiments suggest that the ea^D mutations interfere with inactivation of catalytic Easter, and imply that this negative regulation is essential for generating the wild-type shape of the Dorsal gradient (Chang, 2002).

Dorsoventral patterning of the Drosophila embryo begins in the ovarian egg chamber, where the developing oocyte is surrounded by an epithelium of somatically derived follicle cells. During mid-oogenesis, activation of the EGF Receptor (Egfr) in the follicle cells is spatially restricted by the dorsally localized distribution of its ligand encoded by the gurken (grk) gene. As a consequence, transcription of pipe, which apparently encodes a glycosaminoglycan modifying enzyme required for establishing embryonic polarity, is restricted to a broad stripe on the ventral side of the egg chamber (Chang, 2002 and references therein).

Spatial information originating in the follicular epithelium is later transmitted to the embryo through the perivitelline space, which lies between the vitelline layer of the eggshell and the embryo plasma membrane. The establishment of embryonic polarity is dependent on the ventrally restricted activation of the uniformly distributed receptor Toll. The ligand for Toll is apparently encoded by the spätzle gene, which produces a protein containing a C-terminal cystine knot motif found in many vertebrate growth factors. The Spätzle protein is secreted into the perivitelline space as an inactive precursor, and is cleaved into the active ligand through the activity of a serine protease cascade that includes the products of the genes nudel, gastrulation defective, snake and easter (ea) (Chang, 2002 and references therein).

Activation of Toll initiates an intracellular signaling pathway that results in the nuclear translocation of the transcription factor encoded by dorsal, a member of the NF-kappaB/rel family. Dorsal is initially present throughout the embryonic cytoplasm, where it is retained by the inhibitory IkappaB protein encoded by cactus. Signaling on the ventral side leads to the proteolysis of Cactus, thereby releasing Dorsal. Along the dorsoventral axis, high levels of Dorsal protein are present in ventral nuclei, progressively lower levels in lateral nuclei and no detectable protein in dorsal nuclei. The shape of the Dorsal gradient is characterized by the size of the ventral domain (measured by the number of nuclei expressing peak Dorsal) and a distinct slope (assessed by the number of nuclei that lie between highest and lowest nuclear Dorsal). Changing the shape of the Dorsal gradient causes patterning defects that lead to embryonic lethality (Chang, 2002 and references therein).

The Dorsal gradient subdivides the axis into distinct domains by setting the expression limits of key zygotic regulatory genes, which are responsible for initiating the differentiation of various tissues. High levels of nuclear Dorsal lead to the transcription of twist in mesodermal precursor cells. The Twist protein is itself expressed in a graded fashion in the most ventral 16-18 cells, and this domain can be subdivided into smaller threshold responses. Intermediate levels of nuclear Dorsal activate the transcription of short gastrulation (sog) in two lateral stripes flanking the ventral Twist domain, each about 14-16 cells wide. The rhomboid (rho) gene is transcribed in a ventral subset of 8-10 cells in each sog domain. The zerknüllt (zen) gene is transcribed in the dorsal ~40% of the embryo circumference, in the region where Dorsal is absent from nuclei. Changes in the Dorsal gradient can be characterized by examining the expression domains of these zygotic genes (Chang, 2002).

How is the shape of the Dorsal gradient regulated? Two classes of mutations produce particularly interesting effects on the wild-type shape. In the first class, embryos produced by grk^- and Egfr^- females show two peaks of nuclear Dorsal separated by a shallow ventral minimum. These ventralized embryos proceed to gastrulate with two ventral furrows instead of the single wild-type ventral furrow. This phenotype can be mimicked by overexpression of Spätzle, suggesting that partial axis duplication arises from events in the perivitelline fluid of the embryo. Despite the dramatic reshaping of the ventral domain in these mutant embryos, the slope of the Dorsal gradient remains wild type (Chang, 2002 and references therein).

In the second class, dominant alleles of easter (ea^D) cause a more symmetric distribution of nuclear Dorsal (Steward, 1989). As a consequence, females carrying ea^D mutations produce ventralized embryos, in which ventrolateral structures are expanded at the expense of dorsal structures, or lateralized embryos, in which dorsoventral polarity is largely lost (Chasan, 1989; Jin, 1990; Chang, 2002).

The easter gene encodes the final member of the protease cascade required to activate Spätzle. Easter is initially synthesized as an inactive zymogen containing an N-terminal pro-domain and a C-terminal catalytic domain. Proteolytic cleavage at the activation site between these two domains by Snake presumably generates active Easter in vivo (Chasan, 1992; Dissing, 2001; LeMosy, 2001). Yet, in wild-type embryo extracts, active Easter is found in a high molecular weight complex called Ea-X, which is hypothesized to contain a protease inhibitor of the serpin family (Misra, 1998). Easter is proposed to be active only on the ventral side of the embryo. The ea^D mutations, which map to conserved regions within the catalytic domain (Jin, 1990), somehow cause a loss of this spatial regulation (Chang, 2002 and references therein).

Changes in the shape of the Dorsal gradient caused by a group of representative ea^D alleles mutations have been characterized by examining the expression domains of the zygotic genes zen, sog, rho and twist. Within the allelic series, dorsoventral asymmetry is progressively lost and the slope of the Dorsal gradient flattens. When production of activated Easter was examined in ea^D embryo extracts, an Easter form corresponding to the free catalytic domain is detected; this free catalytic domain is never observed in wild type. The Ea^D catalytic domain exhibits protease activity, as measured by its ability to generate processed Spätzle in the embryo. In the case of the strongest lateralizing ea^D allele, protease activity is detected several hours after the blastoderm stage in perivitelline fluid transfer experiments. Finally, mutant Ea^D proteins expressed in cultured Drosophila S2 cells cleave precursor Spätzle. These data suggest that the ea^D mutations interfere with Easter inactivation by the inhibitor X, and support a model in which regulation by X is required for shaping the Dorsal gradient (Chang, 2002).

Dominant ventralizing and lateralizing easter mutations cause profound changes in the shape of the Dorsal gradient, as visualized by the expression of zygotic marker genes. The increasing severity of ea^D phenotypes, initially determined by examining cuticle patterns and gastrulation movements, are correlated with a decrease in the slope of the Dorsal gradient. In both ventralized and lateralized embryos, the dorsal zen domain is absent, replaced by an expanded lateral sog domain. In lateralized embryos, the ventral twist domain is also absent, with the lateral sog domain expanded along the entire dorsoventral axis (Chang, 2002).

Ventralized embryos produced by grk^- and Egfr^- females exhibit an expansion of the ventral Twist domain at the expense of the dorsal domain, while maintaining a wild-type slope of the Dorsal gradient, as assessed by the size of the rho and sog domains. By contrast, in the ventralized embryos produced by ea^83l/ea^- and ea⁵⁰²²/ea^- females, the slope of the Dorsal gradient is flattened, leading to broader domains of rho and sog expression. This change is accompanied by a decrease, rather than an increase, in the size of the ventral Twist domain. The ea^D ventralized phenotype thus appears to arise from a redistribution of the ventral signal. This change in the shape of the Dorsal gradient is even more dramatic in lateralized embryos, leading to a loss of detectable dorsoventral polarity (Chang, 2002).

Monitoring the expression of target genes enabled the analysis of the Dorsal gradient, but also placed a limit on resolution. For example, the phenotypes of embryos laid by ea²⁰ⁿ/ea^- and ea^5.13/ea^- females initially appeared identical when assessed by sog RNA expression. A more refined image of the shape of the Dorsal gradient emerged after monitoring rho RNA expression, which responds to a narrower concentration range of nuclear Dorsal. Although embryos laid by ea^5.13/ea^- females appear symmetric, it remains formally possible that residual polarity could be detected if a marker corresponding to an even narrower range of nuclear Dorsal were available (Chang, 2002).

Embryos produced by ea^125.3/ea^-, ea^83l/ea^-, ea⁵⁰²²/ea^- and ea²⁰ⁿ/ea^- females show varying degrees of dorsal-ventral asymmetry. The presence of a wild-type dose of easter causes a slight expansion of the Twist domain and a slight reduction in the rho domains, thereby producing a shift towards the normal shape of a Dorsal gradient. The Dorsal gradient in embryos laid by ea^D/+ females reflects a partial contribution from each easter allele, rather than a simple superimposition of the gradient shapes observed in embryos laid by ea^D/ea^- and +/ea^- females. This behavior could be explained if only a set amount of Easter zymogen (wild type and mutant combined) could be cleaved by a limiting amount of activated Snake (Chang, 2002).

Although embryos laid by ea^5.13/+ and ea^5.13/ea^- females can be distinguished by their cuticles, the expression of marker genes in these embryos is quite similar. In particular, formation of the Twist domain is largely inhibited in embryos laid by ea^5.13/+ females. It remains to be determined whether the stronger dominance exhibited by ea^5.13 can be explained by the simple dose argument presented above, or whether Ea^5.13 is interfering with the proper formation of the Dorsal gradient by a more active mechanism (Chang, 2002).

The experiments described above suggest how the spectrum of phenotypes observed in ventralized and lateralized embryos can be explained by separately considering two distinct properties of the Easter protein: (1) inactivation by inhibitor X, and (2) Easter protease activity (Chang, 2002).

In both ventralized and lateralized embryos, the shape of the Dorsal gradient is altered by the absence of the dorsal zen domain, which is replaced by an expanded sog domain. The studies suggest that this phenotype arises from the failure of activated Easter to be properly regulated after zymogen cleavage: the protease domain fails to form a complex with X and remains active. This interpretation is consistent with earlier studies (Jin, 1990) that characterized the effects of changing ea^D dose. Injection of ea^83l and ea^125.3 RNA into ea^- embryos produces a ventralized phenotype, while lower levels of the same RNA rescues to hatching, suggesting that the Easter produced by these ventralizing alleles are defective in negative regulation (Chang, 2002).

The stability of the Ea-X complex suggests that X might be a serpin, reacting with the active site serine of Easter (Misra, 1998). Most mutations that map in the Easter catalytic domain would be expected to affect both protease activity and the interaction with inhibitor X. It is suggested that the ventralizing ea^D alleles (as exemplified by ea^83l) form a special class of mutations that retain catalytic activity, but affect inhibition by X. These studies imply that regulation of Easter following zymogen activation is required for maintaining polarity during formation of the Dorsal gradient. If activated Easter were capable of diffusion, X may play a primarily kinetic role to maintain the initial asymmetry of zymogen activation, by inhibiting activated Easter before its diffusion to the dorsal side (Chang, 2002).

In lateralized embryos, the Dorsal gradient appears even less polar. Both the dorsal zen and the ventral twist domains are absent, with the lateral sog domain expanded along the entire dorsoventral axis. The experiments described above suggest that in addition to their failure to be inactivated by X, the lateralizing alleles also partially reduce Easter protease activity. Since the ea^D mutations map to conserved regions within the protease domain, it is not surprising that some of these alleles show effects on catalytic activity. This point is most clearly observed for the case of the Ea^5.13DeltaN protein, which exhibits less Spätzle processing activity than wild-type EaDeltaN upon expression in cultured S2 cells. In the embryo, this weaker Ea^5.13 protease is apparently unable to generate the level of processed Spätzle required for nuclear translocation of Dorsal that leads to twist transcription (Chang, 2002).

The final amount of processed Spätzle generated in the embryo depends on both Easter specific activity and the length of time the enzyme remains active. In the case of embryos produced by ea^5.13 females, perivitelline transfer experiments detect stable Ea^5.13 activity many hours after cellularization. Despite the lower specific activity of Ea^5.13 suggested by the experiments in cultured cells, the prolonged time of Easter action results in a higher level of processed Spätzle in embryos. Similarly, the ea²⁰ⁿ mutation appears to cause a significant decrease in specific activity, as suggested by a reduction in the combined size of the domains expressing rho RNA and Twist in embryos produced by ea²⁰ⁿ/ea^- females compared with embryos laid by other ea^D/ea^- females. Yet, nearly wild-type amounts of processed Spätzle are observed in ea²⁰ⁿ embryo extracts, probably because Ea²⁰ⁿ fails to be quickly inactivated by inhibitor X (Chang, 2002).

At a broader level, the analysis of ea^D mutations underscores the important relationship between timing of signal production and generation of spatial pattern. Previous studies demonstrated that cleavage of Spätzle is required by Easter for Toll activation. The experiments described here refine this model by showing that the wild-type shape of the Dorsal gradient requires high levels of Easter protease activity during a brief period of time. Failure to properly inactivate Easter leads to a loss of the dorsal domain of the axis, thereby leading to a ventralized phenotype. When this defect is coupled with a reduction in Easter protease function, wild-type levels of processed Spätzle are eventually produced in the embryo, but the shape of the Dorsal gradient becomes more symmetric (Chang, 2002).

GENE STRUCTURE

cDNA clone length - 1429

Bases in 5' UTR - 203

Exons - 3

Bases in 3' UTR - 47

PROTEIN STRUCTURE

Amino Acids - 392

Structural Domains

easter gene codes for an extracytoplasmic serine protease (Chasan, 1989). For information about protease structure and mechanism of action see InterPro entry IPR001314.

date revised: 25 November 2002

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