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First published online 5 May 2004
doi: 10.1242/dev.01143

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Cell-autonomous roles of the ecdysoneless gene in Drosophila development and oogenesis

¹ Department of Molecular Biology, University of South Bohemia, and Institute of Entomology ASCR, Ceske Budejovice 37005, Czech Republic
² Department of Biology, University of North Carolina Greensboro, Greensboro, NC 27402, USA

View larger version (21K): [in a new window]   Fig. 1. Map of the ecd locus. The interval of ecd is delimited by the right breakpoints of deletions Df(3L)Aprt201 and Df(3L)R+R2 within the cytological region 62D5-D7 (top). Ten predicted genes (BDGP) located in this interval are shown. The filled boxes represent coding regions and the lines between them denote introns, arrows indicate the orientation of each gene. Five genomic fragments used for the preparation of rescue constructs are indicated by the black lines. CG5714 (underlined), present in the rescuing fragments E5 and S4, is the ecd gene.

View larger version (125K): [in a new window]   Fig. 2. Ecdysoneless is conserved throughout eukaryotes. Alignment of the Drosophila (Dm) Ecd protein (Swiss-Prot Q9W032) with human (Hs) SGT1 (O95905), and its closest relatives from Arabidopsis thaliana (At; Q9LSM5) and Schizosaccharomyces pombe (Sp; Q9US49), using Clustal W. Black shading indicates amino acid identity, and gray shading indicates similarity if present in two or more of the aligned sequences. The putative ATP/GTP-binding site (P-loop) is marked by the horizontal arrow. Asterisks indicate the positions of the premature termination codons in alleles ecd2, ecdg24 and ecdl(3)23. The arrowhead indicates the conserved proline 656, which is mutated to serine in ecd1.

View larger version (46K): [in a new window]   Fig. 3. Structure and expression of mutant Ecd forms. (A) Schematic representation of mutant versions of the Ecd protein in the four studied ecd alleles. Numbers are amino acid positions; asterisks denote stop codons. A 4-bp deletion in the -ray induced mutant ecdg24 changes the reading frame, resulting in the indicated amino acid substitutions and a stop codon. (B) Western blot of the wild-type and mutant Ecd forms. A low amount of maternal Ecd is still visible in the first instar, but not in the second instar homozygous ecd2 larvae. The truncated Ecd version is detected by the anti-Ecd antibody in second instar ecdl(3)23 larvae.

View larger version (135K): [in a new window]   Fig. 4. Molting defects of ecd mutants. (A) Presence of the second instar (smaller arrows) and third instar (larger arrows) mouth hooks in an ecd1/ecd2 heterozygous larva kept at 29°C. (B) Two pairs of mouth hooks in an ecdg24/Df(3L)R+R2 hemizygote that died during the first molt. The first instar mouth hooks are indicated by small arrows and the second instar ones are indicated by larger arrows. (C) First instar cuticle attached through the first instar mouth hooks (arrow) to the new cuticle in an ecdg24 hemizygote.

View larger version (94K): [in a new window]   Fig. 5. Developmental expression of ecd. (A) Northern blot analysis showing expression of an ecd mRNA in embryos (E), in first (L1), second (L2) and third (L3) instar larvae, in wandering larvae (W), in white puparia (WP), in pupae (P) and in egg-laying females (add female symbol). Bottom panel shows a control re-hybridization of the blot with the constitutively expressed gene mbf1. (B) Western blot with the anti-Ecd antibody detects Ecd in unfertilized eggs from virgin mothers (Unf) and embryos 0-2 hours and 12-24 hours after egg laying.

View larger version (113K): [in a new window]   Fig. 6. ecd is expressed in specific steroidogenic, non-steroidogenic and reproductive organs. (A-F) The anti-Ecd antibody strongly stains the lateral lobes of a third instar ring gland (A); a confocal image (B) shows that Ecd is in the cytoplasm. Low signal is seen in the ring gland during the second instar (C, arrows). Activity of an ecd-lacZ reporter in the ring gland of second instar (D) and late third instar (E) larvae is visualized with X-gal staining. Arrowhead in D marks the medial corpora allata region. (F) Anti-Ecd antibody staining shows Ecd expression targeted to the lateral ring gland of a Feb36-Gal4; ecd2, P[UAS-ecd] second instar larva. (G-I) A third instar CNS shows moderate Ecd levels (G) compared with the lateral ring gland (A). Ecd is abundant in the eye-antennal (H) and wing (I) imaginal discs. (J-N) The anti-Ecd antibody shows strong expression in both the ovary (J) and the testis (K) of third instar larvae. High levels of Ecd occur in the nurse cells of stage 10 egg chambers (L, arrow). The same stage nurse cells also accumulate an ecd transcript, as shown by in situ hybridization (M). Ecd is present primarily on the apical end of the adult testis (N). Except for F, staining was performed on white1118 animals.

View larger version (71K): [in a new window]   Fig. 7. Ecd-deficient cells do not survive in proliferating imaginal discs. ecd2/ecd2 (top row) and mbf1/mbf1 (bottom row) null mutant clones were induced during the first larval instar. Only the sister ecd+/+ clones, marked by the intensive expression of P[w+, ub-GFP], are found in the adult eye (A), eye-antennal discs (B) and wing discs (C), whereas ecd– cells are absent. Clones lacking mbf1, P[w+, ub-GFP] are maintained in all these imaginal tissues (D-F, arrows).

View larger version (69K): [in a new window]   Fig. 8. Egg chambers carrying clones of ecd2/ecd2 follicle cells fuse together. The normal number of 15 nurse cells in a control vitellogenic egg chamber (A) frequently doubles (B) when ecd– clones are induced in adult females. Presence of two (C,D) or more (F) oocytes confirms egg chamber fusions. Fasciclin III expression appears at the ends of a double egg chamber, but not at the fusion line (E). Multiple FasIII signals may result from a fusion of several cysts (G). (A,A') Images show the same wild-type egg chamber. (D,D',E,E') Images show Orb, FasIII and Ecd proteins in the same egg chambers. Large clones of ecd– cells are clearly visible (D') and smaller clones are emphasized with yellow lines (E'). Except for DNA staining with DAPI (A,B), all images are confocal sections. Arrows indicate Orb-positive oocytes; arrowheads indicate FasIII-positive polar follicle cells. Anterior is to the left.

View larger version (105K): [in a new window]   Fig. 9. Loss of ecd in the germline causes previtellogenic arrest of egg chambers. (A,B) ecd2/ecd2 germline clones (arrowheads) can be distinguished from an ecd+/+ clone (arrow) by the lack of staining with the anti-Ecd antibody. All of these clones arrest because of the loss of Ecd, or because of the ovoD1 mutation. (C,D) Mosaic egg chambers containing both Ecd– and Ecd+ nurse cells were identified with the anti-Ecd antibody three days after ecd2 clones had been induced in adult females. Only such mosaic egg chambers formed vitellogenic oocytes (asterisk). (C,C') Confocal sections through the same egg chamber.

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