Fig. 5. Mutagenesis of the Dorsocross locus and phenotypic rescue experiments. (A) Genomic map of the Dorsocross region showing known and predicted genes, deficiencies and P-insertions. Numbers indicate base pair coordinates of genomic sequences on chromosome 3L (BGPD release 3). Slashes indicate the omission of sequences owing to space limitations. Genes above the genomic line are transcribed from left to right and those below from right to left (introns are not displayed). EP(3)3556 was used for generating Df(3L)DocA and Df(3L)DocB, and EP(3)584 (male-sterile insertion in bol, located within the omitted sequence) for Df(3L)EP584MR2. Relevant breakpoints of previously known deficiencies were mapped by PCR from homozygous embryos. Dashes indicate uncertainty ranges of breakpoints. In situ hybridization confirmed the presence of mRNAs of all three Doc genes in Df(3L)Scf-R6, absence of Doc3 mRNA in Df(3L)Scf-R11, absence of Doc1 mRNA in Df(3L)29A6, and absence of all three Doc mRNAs in Df(3L)DocA and Df(3L)DocB homozygotes. Df(3L)DocA and Df(3L)DocB complement female sterility associated with smg¹, whereas Df(3L)Scf-R6 and Df(3L)Scf-R11 do not. Therefore upstream smg sequences (hatched), including alternative smg start sites, are dispensable. The sequences at the breakpoints of Df(3L)DocA, Df(3L)DocB and Df(3L)EP584MR2 will be made accessible in FlyBase. (B-D) Mid stage 12 embryos stained with anti-Kr antibody. (B) Embryo homozygous for Df(3L)DocA (DocA mutant; composite of two focal planes), which lacks Kr in the amnioserosa (arrowheads). Kr in the nervous system serves as an internal staining control (white asterisks). (C) Doc1+2+3 RNAi embryo, which is a wild-type embryo injected with a mix of Doc1, Doc2 and Doc3 dsRNA (3'-fragments downstream of T-box) and shows a phenotype as with DocA mutants. (D) Control wild-type embryo injected with buffer only. (E) Stage 14 DocA mutant embryo showing absence of Kr staining in the amnioserosa region (arrowhead) and incomplete germ band retraction. Somatic muscle staining of Kr in E-G is denoted by black asterisks. (F) Stage 14 DocA mutant embryo with forced expression of Doc2 in the early amnioserosa (via c381-GAL4). Kr expression in the amnioserosa is rescued to a significant degree (arrow), as well as extended temporally. Retraction defects are fully rescued in this and the majority of other embryos. (G) Stage 14 DocA mutant embryo with forced expression of Doc3 as in F. There is some rescue of Kr expression in the amnioserosa (arrow) but little rescue of the germ band retraction defects. (H-J) Cuticle preparations of unhatched first instar larvae visualized by dark-field optics. (H) Df(3L)DocA mutants have a u-shaped phenotype owing to the failure of germ band retraction. A similar cuticle phenotype is observed in Doc1+2+3 RNAi embryos (I), but not in the wild-type control (J). as, amnioserosa; fk, filzkörper.

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