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Two distinct domains of Bicoid mediate its transcriptional downregulation by the Torso pathway

Florence Janody^1,*, Rachel Sturny¹, Valérie Schaeffer^2,, Yannick Azou¹ and Nathalie Dostatni^1,,¶

¹ Developmental Biology Institute of Marseille, Laboratoire de Génétique et Physiologie du Développement, Université de la Méditerrannée, Luminy, Case 907, 13288 Marseille Cedex 09, France
² Department of Biology, New York University, 100 Washington Square East, New York, NY 10003, USA
^* Present address: Skirball Institute of Biomolecular Medicine, 540 First Avenue, New York, NY 10016, USA
Present address: EMBL, Meyerhofstrasse 1, 69117 Heidelberg, Germany
Present address: UMR218, Curie Institute-Section of Research, Pavillon Pasteur, 26 rue d’Ulm, 75 248 Paris Cedex 05, France

View larger version (62K): [in a new window]   Fig. 1. Bcd-GCN4 is insensitive to inhibition by the Torso pathway. (A) The Bcd protein and its chimeric derivatives were wild-type Bcd protein (1) composed of the histidine/proline-rich repeat (H/P, amino acids 11-42), the homeodomain (HD, amino acids 91-151), the serine/threonine-rich domain (ST, amino acids 152-252), the glutamine-rich domain (Q, amino acids 252-300), the alanine-rich domain (A, amino acids 300-349) and the acidic C-terminal domain (C, amino acids 340-489); the Bcd-GCN4 and Bcd-GCN4C proteins in which three copies of the GCN4 activation domain (amino acids 54-141) have been fused to Bcd, respectively, downstream of the ST domain (2) or at its C terminus (3). (B-D) Cuticle preparation of embryos. Anterior is at the top. (E-J) In situ hybridisations on whole-mount embryos at the beginning of cellularisation with hb (E,G,I) and otd (F,H,J) probes. Embryos were from wild-type females (B,E,F), from females carrying one copy of wild-type bcd and one copy of the bcd-GCN4 transgene (C,G,H) and from females carrying two wild type copies of bcd and the bcd-GCN4C transgene (D, I and J).

View larger version (59K): [in a new window]   Fig. 2. Heterologous activation domains are not affected by the Torso pathway. (A) Structure of the fusion proteins between the Gal4 DNA-binding domain and one (1) or three (2) copies the GCN4 activation domain (amino acids 54-141), the VP16 activation domain (amino acids 413-490) (3) and the B42 activation domain (amino acids 1-79) (4). (B) transfections were performed with 1 µg of the UAS-CAT reporter and with 1 µg of pPAC (-) or its producer derivatives for the Gal4-GCN4 (1), Gal4-3GCN4 (2), Gal4-VP16 (3) and Gal4-B42 (4) proteins. (C-F) In situ hybridisations were performed on blastoderm embryos with a lacZ probe. Embryos were from female carrying the Gal4-GCN4 (C), Gal4-3GCN4 (D), Gal4-VP16 (E) and Gal4-B42 (F) transgenes. Females were crossed with males carrying the UASp-lacZ transgene. Anterior is to the left in this and subsequent figures showing in situ hybridized whole mounts.

View larger version (21K): [in a new window]   Fig. 3. The serine-threonine-rich domain of Bcd is an activation domain. (A) Structure of the Bcd deletion variants. (B) Transfections were performed with 1 µg of the Bcd3-CAT reporter and with 5 µg of pPAC (1) or its producer derivatives for the Bcd (2), QAC (3), HP/HD (4) and HD/ST (5) proteins.

View larger version (31K): [in a new window]   Fig. 4. trans-acting potential of Bcd domains in S2 cells. (A) Structure of the fusion proteins between the Gal4 DNA-binding domain and simple or multiple copies of the Bcd domains. (B) Transfections were performed with 1 µg of the UAS-CAT reporter and with 1 µg of pPAC (-) or its producer derivatives for the Gal4-Q (1), Gal4-3Q (2), Gal4-QA (3), Gal4-Q3A (4), Gal4-3Q3A (5), Gal4-3A (6), Gal4-C (7), Gal4-3C (8), Gal4-3A3C (9), Gal4-3Q3C (10) and Gal4-3ST (11) proteins.

View larger version (76K): [in a new window]   Fig. 5. trans-acting potential of the Bcd domains in the embryo. In situ hybridisations were performed on blastoderm embryos with a lacZ probe. Embryos were from females carrying the Bcd-Gal4 (Bellaïche et al., 1996) (A), Gal4-3ST (B,E), Gal4-3C (C,F), Gal4-2Q (D), Gal4-3A (G), Gal4-3A3C (H), Gal4-3Q3A (I) and Gal4-3Q3C (J) transgenes. Females were crossed with males carrying the UASp-lacZ transgene. Females were torPM (E,F).

View larger version (131K): [in a new window]   Fig. 6. AC is insensitive to inhibition by Torso. In situ hybridisations were performed on blastoderm embryos with a lacZ probe. Embryos were from bcdE1 females carrying two copies of the AC (A), QAC (B), A (C) and C (D) transgenes (Schaeffer et al., 1999), from wild-type females (E), from bcdE1 females carrying one copy of AC (F), from tor4021 females (G), and from tor4021 bcdE1 females carrying one copy of AC (H). Females were crossed with males carrying the UASp-lacZ transgene.