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doi: 10.1242/10.1242/dev.00161

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Regulation of Easter activity is required for shaping the Dorsal gradient in the Drosophila embryo

Andy J. Chang^* and Donald Morisato^,

Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, USA
^* Present address: Department of Anatomy, University of California, San Francisco, CA 94143, USA
Present address: The Evergreen State College, Lab I Room 3009, Olympia, WA 98505, USA

View larger version (71K): [in a new window]   Fig. 1. Cuticles of embryos laid by mutant easter females. Cuticles of a wild-type first instar larva (A) and dorsalized embryo laid by an ea- (ea4/ea5022rxl) female (B) are shown in dark-field illumination for comparison with embryos produced by eaD females (with indicated maternal genotypes). Cuticles are arranged in order of decreasing dorsoventral polarity from weakly ventralized (C,D), moderately ventralized (E-H) to lateralized phenotypes (I-L). For the lateralizing eaD alleles, there is a notable reduction in dorsoventral polarity in embryos laid by eaD/ea- females (J,L) when compared with those produced by eaD/+ females (I,K). When evident, cuticles are oriented anterior towards the left, dorsal side upwards.

View larger version (62K): [in a new window]   Fig. 2. Expression of sog RNA is expanded in embryos laid by eaD females. Blastoderm embryos laid by females with indicated genotypes were visualized for expression of sog mRNA by in situ hybridization. Cross-sections at 50% egg length are shown. The sog domain is expanded in embryos produced by eaD females (C-L), compared with embryos laid by wild-type (A) or +/ea- (B) females. Dorsal expansion across the dorsal midline is observed in all embryos (E-L), except for those produced by females carrying the weakly ventralizing allele ea125.3 (C,D). Ventral expansion is most notable in embryos laid by ea20n/ea-, ea5.13/+ and ea5.13/ea- females (J-L). With the exception of the ea5.13 allele, ventral expansion of sog expression is increased in embryos laid by eaD/ea- females compared with those produced by eaD/+ females (C-J), and is particularly dramatic for the ea20n allele (I,J). When evident, sections are oriented dorsal side upwards.

View larger version (62K): [in a new window]   Fig. 3. Expression of rho RNA is expanded in embryos laid by eaD females. Blastoderm embryos laid by females with indicated genotypes were visualized for expression of rho mRNA by in situ hybridization. Cross-sections at 50% egg length are shown. The rho domain is expanded in embryos produced by eaD females (C-L), compared with embryos laid by wild-type (A) or +/ea- (B) females. Dorsal expansion is evident in all embryos, while ventral expansion is most pronounced in embryos laid by ea20n/ea-, ea5.13/+ and ea5.13/ea- females (J-L). In ea83l, ea5022 and ea20n embryos, ventral expansion of rho expression is increased in embryos laid by eaD/ea- females compared with those produced by eaD/+ females (E-J). This change is most significant in ea20n embryos, where the two ventrolateral rho domains are fused into one ventral domain when a wild-type copy of easter is absent (I,J). When evident, sections are oriented dorsal side upwards.

View larger version (24K): [in a new window]   Fig. 4. eaD mutations cause a decrease in dorsoventral asymmetry. These composite diagrams represent embryo cross-sections (with the dorsal side upwards) that depict the expression domains of zygotic markers summarized in Table 1. Color intensity corresponds to the concentration of nuclear Dorsal, with darker colors denoting zygotic markers activated by higher concentrations of Dorsal. An expansion of rho and sog expression results from a decrease in the slope of the Dorsal gradient.

View larger version (67K): [in a new window]   Fig. 5. Detection of Easter catalytic domain in eaD embryo extracts. Extracts were prepared from 0-4 hour embryos laid by wild-type, eaN/+, ea8/ea5022rx1, ea83l/ea5022rx1 and ea5.13/ea5022rx1 females. Protein samples were separated on a 10% SDS polyacrylamide gel. The immunoblot was probed with anti-Easter antibodies. The Easter zymogen, the Easter catalytic domain (Ea CD), and a higher Mr form (Ea-X) are indicated by arrows. In this particular blot, the Easter zymogen co-migrates with a prominent cross-reacting 45 kDa band. In the eaD alleles, an increased level of the Easter catalytic domain and a corresponding decrease in Ea-X level is observed.

View larger version (48K): [in a new window]   Fig. 6. eaD protease activity assessed by level of embryonic processed Spätzle. Extracts were prepared from 0-4 hour embryos laid by spz-, wild-type, and +/ea- (+/ea5022rx1) females on the left and by eaD/+ and eaD/ea- females on the right. Protein samples were separated on a 12.5% SDS polyacrylamide gel. The immunoblot was probed with antibodies specific to the C-terminal domain of the Spätzle protein. Full-length Spätzle proteins are indicated by the bracket and the C-terminal processed Spätzle form is denoted by the arrow. Some variation in the distribution of full-length isoforms was observed in different extract preparations. The asterisk marks a crossreacting band that serves as a loading control. The amount of processed Spätzle in embryos laid by ea83l, ea5022 and ea20n females appears comparable or slightly lower than in wild-type, while the level appears to be slightly higher in embryos produced by ea5.13 females.

View larger version (24K): [in a new window]   Fig. 7. eaD protease activity measured in cultured S2 cells. Co-expression of precursor Spätzle and EaN in cultured S2 cells generates processed Spätzle, with the level of cleavage dependent on the amount of EaN expressed. (A) S2 cells were transfected with the following amounts of DNA: (1) 2 µg eaN; (2) 2 µg spz; (3) 2 µg spz + 2 µg eaN; (4) 2 µg spz + 0.4 µg eaN; (5) 2 µg spz + 0.1 µg eaN; (6) 2 µg spz + 2 µg ea83lN; (7) 2 µg spz + 0.4 µg ea83lN; (8) 2 µg spz + 0.1 µg ea83lN; (9) 2 µg spz + 2 µg ea5.13N; (10) 2 µg spz + 0.4 µg ea5.13N; (11) 2 µg spz + 0.1 µg ea5.13N. Conditioned medium was collected after overnight induction of the metallothionein promoter. Protein samples were separated on a 15% SDS polyacrylamide gel and the immunoblot probed with antibodies against full-length Spätzle protein. (B) Protein samples from the transfected cells described above were separated on a 10% SDS polyacrylamide gel and the immunoblot probed with antibodies against Easter protein. The level of Spätzle processing carried out by Ea83lN is indistinguishable from wild-type EaN. By comparison, Ea5.13N shows weaker protease activity, although it appears to be expressed at higher levels.