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First published online 26 November 2003
doi: 10.1242/dev.00920

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rigor mortis encodes a novel nuclear receptor interacting protein required for ecdysone signaling during Drosophila larval development

Julie Gates^1,*, Geanette Lam¹, José A. Ortiz², Régine Losson³ and Carl S. Thummel^1,

¹ Howard Hughes Medical Institute, Department of Human Genetics, University of Utah School of Medicine, Salt Lake City, UT 84112-5331 USA
² Instituto de Neurociencias, Universidad Miguel Hernández-CSIC, Apdo.18, Ctra. Valencia Km 87, 03550-San Juan (Alicante), Spain
³ IGBMC, Dépt de Génétique Physiologique, BP 10142, 67404 Illkirch Cedex, CU Strasbourg, France

View larger version (53K): [in a new window]   Fig. 1. Lethal phenotypes of rig mutant larvae. (A) The majority of rig mutant larvae (l(2)k07839/Df(2R)exu1) die during the third instar and become stiff and extended in an apparent attempt to pupariate. (B) rig mutants that die as prolonged second instar larvae reach the size of a normal late third instar larva. The anterior spiracles of these prolonged second instar larvae end with a single club-like structure (arrow) rather than the multiple branches associated with a wild type third instar anterior spiracle (F, arrow). (C) Two cuticles are often seen when rig mutants die while molting. This larva died trapped inside the second instar cuticle, which had separated almost completely from the third instar cuticle (arrowhead). (D) This larva managed to shed its second instar cuticle from the anterior portion of its body, but died while trying to shed this cuticle further. A thin piece of cuticle extends from its mouthhooks to the point on the body where the second instar cuticle remains (arrowhead). (E,F) A y w early second instar larva (E) and y w late third instar larva (F) are shown as controls. All panels are lateral views with anterior to the right, except for E which is a dorsal view. All images were taken at the same magnification.

View larger version (137K): [in a new window]   Fig. 2. rig mutant larvae have defective mouthhooks. (A) The mouthhooks of control (+/Df(2R)exu1) second instar larvae have three to four large teeth, while control third instar larvae (B) have larger mouthhooks with numerous smaller teeth. (C) rig mutants (l(2)k07839/Df(2R)exu1) that die as prolonged second instar larvae have normal second instar mouthhooks. (D) The mouthhooks of rig mutant that die as third instar larvae are severely malformed. (E) rig mutants that die while trying to molt have two sets of mouthhooks, a normal second instar set (red arrows) and a malformed third instar set (black arrows).

View larger version (87K): [in a new window]   Fig. 3. rig exerts gene-specific effects on ecdysone-regulated gene expression. RNA was extracted from staged control (+/Df(2R)exu1) and mutant (l(2)k07839/Df(2R)exu1) second instar (0, 6, 12 or 18 hours after the first-to-second instar molt) larvae. Animals were resynchronized at the molt to the third instar and RNA was collected either at the molt (24 hours after the first-to-second instar molt) or 6 hours later (30 hours). Equal amounts of total RNA were fractionated by formaldehyde agarose gel electrophoresis and analyzed by northern blot hybridization. Radiolabeled probes were used to detect rig, E75A, ßFTZ-F1, E74A, E74B, EcR, and usp transcription. Hybridization to detect rp49 mRNA was used as a control for loading and transfer.

View larger version (74K): [in a new window]   Fig. 4. rig encodes a protein with multiple WD-40 repeats and an LXXLL motif. The nine WD-40 repeats identified by at least two of the three protein motif search engines used to analyze the Rig sequence are highlighted in yellow. The LXXLL motif that is associated with known nuclear receptor cofactors is boxed.

View larger version (43K): [in a new window]   Fig. 5. (A) Rig protein binds to DHR3, EcR, ßFTZ-F1, USP and SVP in vitro. In vitro synthesized 35S-labelled Rig was incubated in a batch assay with control GST alone (lane 2) or GST fusions to the indicated receptors in either the presence (+) or absence (–) of ligand (1 µM muristerone A, a potent activator of EcR). Bound Rig was resolved by SDS-PAGE and visualized by autoradiography. Lane 1 depicts 1/5 the amount of input Rig protein, marked by an arrow. (B) Rig binds in vitro to the FTZ-F1 ligand binding domain (LBD) in an AF-2-independent manner. In vitro synthesized 35S-labelled Rig was incubated in a batch assay with control GST alone (lane 2) or GST fusions to full-length FTZ-F1 (lane 3), full-length ßFTZ-F1 (lane 4), the ßFTZ-F1 LBD (E region, lane 5) or a mutated ßFTZ-F1 LBD in which the core of the AF-2 activation domain was deleted (AF-2, lane 6). Bound Rig was resolved by SDS-PAGE and visualized by autoradiography. Lane 1 depicts 1/5 the amount of input Rig protein, marked by an arrow.

View larger version (37K): [in a new window]   Fig. 6. Rig protein is expressed in the brains and salivary glands of second and early third instar larvae. Organs were dissected from rig mutant (l(2)k07839/Df(2R)exu1) (A-C) or control (l(2)k07839/CyO or Df(2R)exu1/CyO) (D-I) larvae staged at either 0-4 hours (A,D,G) or 16-24 hours (B,E,H) after the first-to-second instar larval molt, or 0-4 hours after the second-to-third instar larval molt (C,F,I). Brains (A-F) or salivary glands (G-I) were fixed and stained to detect Rig protein. The specificity of the antibodies was demonstrated by the absence of staining in rig mutant larvae (compare A-C with D-F). Background staining can occasionally be seen in the ring gland (B).

View larger version (69K): [in a new window]   Fig. 7. Rig protein is present in the cytoplasm of brain and imaginal disc cells. Brain complexes (A-C) and wing imaginal discs (D-F) dissected from late third instar larvae were fixed and stained to detect Rig protein (red). A mouse antihistone antibody was used to counterstain the nuclei (green).

View larger version (84K): [in a new window]   Fig. 8. Rig protein shuttles between the nucleus and cytoplasm in larval salivary gland cells. Larval salivary glands dissected from third instar larvae staged 18-24 hours (A-C), 24-30 hours (D-F), or 36-42 hours (G-I) after the molt, or newly formed prepupae (J-L), were fixed and stained to detect Rig protein (red). A mouse anti-histone antibody was used to counterstain nuclei (green).

View larger version (115K): [in a new window]   Fig. 9. Rig protein translocates from the cytoplasm to nucleus in larval midgut cells. Larval midguts dissected from third instar larvae staged 12-18 hours (A-C), 18-24 hours (D-F), or 24-30 hours (G-I) after the molt were fixed and stained to detect Rig protein (red). A mouse anti-histone antibody was used to counterstain nuclei (green). Arrows point to clusters of imaginal gut cells.