First published online 26 November 2003
doi: 10.1242/dev.00920
Development 131, 25-36 (2004)
Published by The Company of Biologists 2004
rigor mortis encodes a novel nuclear receptor interacting protein required for ecdysone signaling during Drosophila larval development
Julie Gates1,*,
Geanette Lam1,
José A. Ortiz2,
Régine Losson3 and
Carl S. Thummel1,
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
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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.
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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).
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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.
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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.
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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.
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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).
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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).
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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).
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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.
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© The Company of Biologists Ltd 2004
