The product of the Drosophila easter gene, a member of the trypsin family of serine proteases, must be more active ventrally than dorsally 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 is active both dorsally and ventrally. The dominant 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 (Chasan, 1992).
The embryonic phenotypes produced by many of the eaD mutations have been described at the level of cuticle patterns and gastrulation behavior (Chasan, 1989; Jin, 1990). Focus was placed on five alleles that are classified as being ventralized (ea125.3, ea83l and ea5022) or lateralized (ea20n and ea5.13). These alleles are completely penetrant and show a spectrum of phenotypes, in contrast to the dorsalization caused by a loss of maternal easter function. Each eaD mutation is caused by a single amino acid substitution in a conserved region of the Easter catalytic domain (Jin, 1990; Chang, 2002).
Among the ventralizing alleles, embryos produced by ea125.3/+ females exhibit the weakest effects, with only slight expansion of ventral denticle bands (Chasan, 1989). Embryos laid by ea83l/+ and ea5022/+ females showed stronger phenotypes, characterized by the expansion of ventral denticle bands, reduction or absence of dorsolaterally derived structures, such as the filzkörper, and near absence of dorsal hairs (Chasan, 1989). Embryos laid by ea5022/+ females exhibit more disorganized denticles and more severe head deformities than embryos laid by ea83l/+ females. At gastrulation, embryos produced by females carrying all three alleles invaginated an apparently normal ventral furrow, but initiation of the lateral cephalic furrow is shifted to a more dorsal position and fewer dorsal folds are formed (Chang, 2002).
Embryos produced by females carrying the lateralizing alleles ea20n and ea5.13 show a marked reduction in dorsoventral asymmetry. Unlike the other eaD alleles, ea20n/+ females lay a mixture of ventralized and lateralized embryos, as determined by analyzing both differentiated cuticles and gastrulation movements. The ventralized embryos show a stronger phenotype than do embryos laid by ea83l/+ and ea5022/+ females. By comparison, all of the embryos laid by ea5.13/+ females show a reduction in both dorsal pattern elements and ventrally derived mesoderm, developing a cuticle with rings of ventral and lateral denticle bands (Chasan, 1989). At gastrulation, embryos from ea5.13/+ females fail to invaginate a ventral furrow and exhibit a widened head fold (Chang, 2002).
The phenotypes of embryos produced by eaD females were examined in the absence of wild-type maternal easter activity, in order to study the genetic dominance exerted by these eaD mutations. For the ventralizing alleles, embryos produced by eaD/+ and eaD/ea- females are virtually indistinguishable. By contrast, the embryos produced by ea20n/ea- and ea5.13/ea- females are markedly more elongated and have fewer ventral denticle bands than embryos laid by ea20n/+ and ea5.13/+ females. Notably, all the embryos laid by ea20n/ea- females develop a lateralized head fold during gastrulation, when compared with the mixed population laid by ea20n/+ females. In summary, the presence of a wild-type dose of easter is able to confer detectable dorsoventral asymmetry to embryos produced by the lateralizing eaD alleles (Chang, 2002).
Taken together, the analysis of gastrulation behavior and differentiated cuticles suggests that the five eaD mutations can be ordered in the following allelic series, with the strongest allele exhibiting the greatest loss of dorsoventral polarity: wild type>ea125.3>ea83l>ea5022>ea20n>ea5.13 (Chang, 2002).
The primary interest in the eaD mutations is to understand their effects on the Dorsal gradient. Although embryos could be stained directly for the expression of Dorsal protein, it was felt that changes in the shape of the gradient would be too subtle to discern and difficult to quantitate. Therefore, the expression domains were characterized of the zygotic genes zen, sog, rho and twist, each corresponding to a specific concentration range of nuclear Dorsal. In order to simplify comparisons between embryos, domain size was expressed as a percentage of the embryo circumference. The major points from these data are summarized below (Chang, 2002).
The zen gene is transcribed in the dorsal 38% of the wild-type embryo circumference, in those cells that lack nuclear Dorsal. In dorsalized embryos laid by ea- females, zen expression expands across the entire dorsoventral axis. By contrast, the zen domain is completely absent in embryos produced by eaD/+ and eaD/ea- females for both the ventralizing and lateralizing eaD alleles. Thus, even in embryos produced by the weakest ventralizing allele ea125.3, Dorsal protein is present in dorsal nuclei. In summary, analysis of zen expression shows that the dorsal domain, defined by the absence of nuclear Dorsal, is lost in all of the mutant embryos (Chang, 2002).
The sog gene is transcribed in two ventrolateral stripes that total 31% of the wild-type embryo circumference, with each stripe abutting the ventral domain defined by twist expression. In embryos produced by ea125.3 females, each domain is expanded, such that the total sog domain occupies 50% of the embryo circumference. In embryos produced by ea83l and ea5022 females, the sog domain is expanded across the dorsal midline, although in some of these embryos, the staining becomes weaker on the dorsal side; this region of lower expression might correspond to a concentration of nuclear Dorsal protein normally found in one or two nuclei at the dorsal sog boundary. Because the dorsal boundary is sometimes difficult to determine, only the ventral unstained domain was quantitated in these embryos (Chang, 2002).
In embryos laid by females carrying the lateralizing alleles, sog staining is significantly expanded. Consistent with the observations described above, a mixed population was observed among the embryos laid by ea20n/+ females; some embryos showed uniform sog staining, while others maintained a ventral unstained domain. By comparison, all embryos laid by ea20n/ea- females show uniform sog expression. Similarly, embryos produced by ea5.13/+ and ea5.13/ea- females showed sog staining across the dorsoventral axis (Chang, 2002).
The quantitation of the sog domain shows that the slope of the Dorsal gradient is flattened in all of these mutant embryos. In addition, a small but significant change in the size of the presumptive mesoderm is detected by quantitating the size of the ventral unstained domain. Embryos laid by ea83l/ea- and ea5022/ea- females show a decrease in the size of the ventral domain, when compared with the wild-type size seen in embryos laid by ea83l/+ and ea5022/+ females. In summary, the domain of low nuclear Dorsal, reflected by the activation of sog transcription, is expanded dorsally in all mutant embryos (Chang, 2002).
This decrease in the slope of the Dorsal gradient was further characterized by the analysis of rho expression, a marker corresponding to intermediate levels of nuclear Dorsal. The rho gene is transcribed in two ventrolateral stripes that total 18.5% of the wild-type embryo circumference. In contrast to the weak sog expression observed across the dorsal side in some mutant embryos, dorsal boundaries for the rho domain are well defined. The size of each rho domain is modestly expanded in embryos produced by females carrying the ventralizing alleles ea125.3, ea83l and ea5022. The size of each rho domain is further increased in embryos laid by ea20n/+ females, while the two stripes are fused into a single domain in embryos produced by ea20n/ea- females. Embryos produced by ea5.13/+ and ea5.13/ea- females express rho in all cells along the dorsoventral axis. As noted above for the quantitation of sog expression, the expansion of rho expression observed in embryos produced by ea83l/ea- and ea5022/ea- females are each accompanied by a decrease in the ventral unstained domain (Chang, 2002).
The twist gene is transcribed in cells that give rise to mesoderm. The Twist protein, visualized by anti-Twist antibodies, is expressed in the ventral 21.5% of the wild-type embryo circumference. A reduction in the size of the ventral domain of high nuclear Dorsal, inferred from Twist staining and the absence of sog and rho transcription, was observed in all mutant embryos, except for the weakest ventralizing allele ea125.3. In moderately ventralized embryos, this reduction was small but significant; in lateralized embryos, the ventral domain was completely absent. The size of the Twist domain is nearly the same as in wild type in embryos produced by females carrying the ventralizing alleles ea125.3, ea83l and ea5022. The Twist domain is slightly reduced in embryos laid by ea20n/+ females, while no Twist expression is detected in embryos laid by ea20n/ea- females. A very faint, reduced Twist domain was observed among some embryos laid by ea5.13/+ females, while no Twist expression was detected in embryos laid by ea5.13/ea- females. In the wild-type embryo, the size of the Twist domain is in good agreement with the ventral domain also defined by the absence of sog and rho expression. In the eaD mutant embryos, the Twist domain appears slightly larger than the ventral domain when determined by examining sog and rho transcription; some cells could be expressing low levels of both rho and Twist, as a consequence of a reduction in the slope of the Dorsal gradient. In no case was the ventral domain expanded in any of the embryos produced by the eaD alleles (Chang, 2002).
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date revised: 20 December 2006
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