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COP9 signalosome subunits 4 and 5 regulate multiple pleiotropic pathways in Drosophila melanogaster

Efrat Oron^1,*, Mattias Mannervik², Sigal Rencus^3,*, Orit Harari-Steinberg¹, Shira Neuman-Silberberg⁴, Daniel Segal^3, and Daniel A. Chamovitz^1,

¹ Department of Plant Sciences, Tel Aviv University, Tel Aviv 69978, Israel
² Department of Developmental Biology, Wenner-Gren Institute, Stockholm University, S-106 91 Stockholm, Sweden
³ Department of Molecular Microbiology and Biotechnology, Tel Aviv University, Tel Aviv 69978, Israel
⁴ Molecular Genetics of Development, Faculty of Health Sciences, Ben-Gurian University of the Neger, Beer-Sheva 84105, Israel
^* These authors contributed equally to this manuscript

View larger version (28K): [in a new window]   Fig. 1. Lethality of csn4null and csn5null mutants. Mutant homozygous individuals, identified as non-GFP, and non-mutant heterozygote siblings, identified as GFP positive, were scored for viability each day following laying. n=120 for each strain.

View larger version (13K): [in a new window]   Fig. 2. The CSN is maternally deposited and is present until late embryogenesis. (A) Unfertilized eggs from virgin wild-type females were homogenized in gel-filtration buffer and total soluble protein was fractionated over a Superose 6 gel-filtration column (Pharmacia). Fractions (0.5 ml) were examined for the presence of CSN5 or CSN7 as indicated by immunoblot analysis with anti-CSN5 or anti-CSN7 affinity-purified antibodies. Positions of size markers are shown. (B) Proteins from csn5null late-stage embryos (identified as non-kr-GFP) and heterozygote siblings (identified as kr-GFP positive) were analyzed for CSN5 and CSN7 as above.

View larger version (180K): [in a new window]   Fig. 3. CSN4 and CSN5 are essential for oogenesis. Ovaries (A-C), egg chambers (F-H), and the most developed stage oocytes (D,E,I-L) are shown for the following genotypes: wild type (A,D); w/w hs-FLP; FRT csn4+/FRT OVOD, heat-shocked (E); w/w hs-FLP; FRT csn4null/FRT OVOD, non heat-shocked (H,I); w/w hs-FLP; FRT csn4null/FRT OVOD, heat-shocked (B,F,J); w/w hs-FLP; FRT csn5null/FRT OVOD, heat-shocked (G,K); w/w hs-FLP; csn5P/FRT OVOD, heat-shocked (C,L). (A,D) Wild-type control; (E) negative control showing that heat shock does not effect oogenesis; (H,I) OVOD-derived oocytes. (B,C,F,G,J-L) Germline clones of csn4null (B,F,J) csn5null (G,K) and csn5p (C,L). csn5null ovaries are essentially indistinguishable from csn4null ovaries and are not shown. Staging of oocytes was according to position in ovariole.

View larger version (102K): [in a new window]   Fig. 4. Dorsoventral patterning defects in csn5p mutant embryos. Embryos were hybridized with digoxigenin-labeled antisense RNA probes and are oriented with dorsal upwards and anterior towards the left (A,B,G,H) except C-F, which are dorsal views. Csn5p mutant embryos were derived from csn5p germline clones. (A,B) Wild-type (wt) and mutant (csn5p) embryos undergoing cellularization after hybridization with a snail probe. In the mutant embryo, the snail expression domain forms on the ventral side as in wild type, but has a wavy border. (C,D) Cellularizing wild-type and mutant embryos after hybridization with a rhomboid probe. rhomboid is expressed in two lateral stripes in the presumptive neuroectoderm. In the mutant embryo, the two lateral stripes are wavy. (E,F) Wild-type and mutant embryo at the onset of gastrulation hybridized with the rhomboid probe. In addition to the two lateral stripes, rhomboid is expressed in the dorsal ectoderm in response to the dpp signaling molecule at this stage. In the csn5p mutant embryo, the dorsal ectoderm staining is expanded, suggesting that CSN5 normally restricts dpp signaling. (G,H) Cellularizing wild-type and mutant embryos hybridized with a tolloid probe. tolloid expression is normally excluded from the ventral side of the embryo by the Dorsal protein. In the mutant embryo, the pattern is severely distorted, with tolloid expression observed in parts of both the ventral and dorsal half of the embryo.

View larger version (65K): [in a new window]   Fig. 5. Anteroposterior patterning in embryos derived from csn5p germline clones. Embryos, oriented with dorsal upwards and anterior towards the left, were hybridized with digoxigenin-labeled antisense RNA probes. (A,B) giant expression in wild-type (A) and mutant (B) embryo undergoing cellularization. In the mutant embryo, giant expression is tilted towards the anterior. (C,D) Cellularizing wild-type and mutant embryo after hybridization with a knirps probe. knirps expression is tilted towards the anterior in the mutant embryo. (E-G) even-skipped expression in wild-type and mutant embryos undergoing cellularization. The seven-stripe even-skipped pattern is altered to a variable extent in csn5p mutant embryos. The embryo in F has an expanded stripe 2 and diminished stripe 5, whereas the embryo in G is more severely affected. (H-J) Embryos undergoing germband elongation hybridized with an engrailed probe. The mutant embryos in I,J have failed to undergo the cell movements of gastrulation. The normal engrailed pattern includes 14 stripes that extend along the length of the germband. In mutant embryos, a striped engrailed pattern can no longer be observed.

View larger version (116K): [in a new window]   Fig. 6. Axis patterning in csn4 and csn5 mutant oocytes. Oocytes were hybridized with digoxigenin-labeled antisense RNA probes. (A-D) oskar expression in wild-type (A), csn4null (B), csn5null (C) and csn5P (D) mutant oocytes. (E-H) bicoid expression in wild-type (E), csn4null (F), csn5null (G), and csn5P (H) mutant oocytes. (I-M) gurken expression in wild-type (I,J), csn4null (K), csn5null (L) and csn5P (M) mutant oocytes. Late (I) and early (J) gurken patterns are shown for the wild type. The arrow indicates the posterior.

View larger version (31K): [in a new window]   Fig. 7. csn4null and csn5null mutants are hypersensitive to MMS. First instar larvae of csn4null, csn5null, and their respective heterozygotic siblings were fed with yeast extract supplemented with methyl methane sulfonate (MMS). After treatment, the larvae were observed for viability. Viability=(xM/tM)/(xc/tc)x100, where xM is the number of living MMS-treated individuals, tM is the total number of MMS-treated individuals (including dead individuals), xc is the number of living non-treated individuals and tc is the total number of non-treated individuals for each strain. Starting numbers were 180 larvae for each strain.

View larger version (80K): [in a new window]   Fig. 8. Unique phenotypes of csn mutants. Dissected mouth hooks from a (A) third instar wild-type larva, similar to csn4+/null heterozygote larva, and from a (B) third instar csn4null homozygote larva. (C) Third instar csn4null homozygote larva attached to the second instar molt (arrow). (D,E) csn5null homozygote larvae showing melanotic capsules.

View larger version (19K): [in a new window]   Fig. 9. Gel filtration analysis of the COP9 signalosome and its subunits in the wild-type and mutant strains. Third instar larvae from wild-type and mutant strains were homogenized in gel-filtration buffer and total soluble protein was fractionated over a Superose 6 gel-filtration column (Pharmacia). Fractions (0.5 ml) were examined for the presence of CSN4, CSN5 or CSN7, as indicated, by immunoblot analysis with anti-CSN4, anti-CSN5 or anti-CSN7 affinity-purified antibodies. Positions of size markers are shown.