First published online 26 March 2008
doi: 10.1242/dev.018028
Development 135, 1669-1679 (2008)
Published by The Company of Biologists 2008
The YPWM motif links Antennapedia to the basal transcriptional machinery
Frédéric Prince1,
Tomonori Katsuyama2,
Yoshiteru Oshima2,
Serge Plaza3,
Diana Resendez-Perez4,
Meera Berry5,
Shoichiro Kurata2 and
Walter J. Gehring1,*
1 Biozentrum, University of Basel, Klingelberstrasse 70, CH-4056 Basel,
Switzerland.
2 ETH Zurich, Department of Biosystems, CH-4058 Basel, Switzerland.
3 CNS-Centre de Biologie du Developpement, 118 route de NARBONNE, Bat 4R3, 31062
Toulouse, France.
4 Facultad de Ciencas Biologicas UANL, Cuidad Universitaria, C.P. 66450,
Mexico.
5 Micromet AG, Am Klopferspitz 19, 82152 Martinsried/Munich, Germany.
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Fig. 1. The eye-to-wing transformation is specific for Antp and
dependent on the YPWM motif. (A) Ectopic wings (W) and legs (L)
induced on the head of ey-Gal4; UAS-Nact;
UAS-Antp flies. (B) Higher magnification of the ectopic wing
(W) in A, showing the marginal bristles of the triple row. (C) Higher
magnification of the antenna-to-leg transformation in A. The apical bristles
on the tibia indicate second leg identity (2nd L). (D) Head
of an ey-Gal4; UAS-Nact;
UAS-AntpAAAA fly showing no wing structures. (E)
Higher magnification of the antenna-to-leg transformation, showing the tibial
apical bristles (L). (F) Head of an ey-Gal4;
UAS-Nact; UAS-AntpQ50K fly. No ectopic
wings are formed. (G) Higher magnification of the transformed arista
with a claw (CL). (H) The head of an ey-Gal4;
UAS-Nact; UAS-Ubx fly shows no ectopic wing.
(I) Higher magnification of the ectopic leg with a claw (CL).
(J) Higher magnification of ectopic bristles induced in the eye part of
the head. (K,M,O,Q) Bright-field micrograph of
third instar discs and (L,N,P,R) the VG protein
distribution visualized by immunostaining of the corresponding disc to its
left. Ant, antennal discs; Eye, eye discs. (K-N) VG is expressed in the wing
pouch in wild-type wing (K,L) discs but not in the eye-antennal disc (M,N).
(O,P) VG is ectopically induced in the eye disc of ey-Gal4;
UAS-Nact; UAS-Antp larvae. (Q,R) There is no
detectable VG protein induced in eye-antennal disc of ey-Gal4;
UAS-Nact; UAS-AntpAAAA larvae.
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Fig. 2. Drosophila BIP2 interacts directly with ANTP via the YPWM
motif. (A) Schematic of the Antp constructs used fused to
the LexA DNA-binding domain. LexA-YPWM-HD consists of the YPWM motif, the
linker region and the HD with  -helices 1-3 (H1-H3). LexA-YPWM-N-term
consists of the YPWM motif, the linker and the N-terminal arm of the HD.
LexA-AAAA-N-term is the same construct as LexA-YPWM-N-term with the YPWM motif
substituted by four alanines. LexA-YPWM-consists of the YPWM motif and the
linker region. LexA-AAAA- is the same construct as LexA-YPWM- with the YPWM
motif substituted by four alanines. LexA- is an empty vector. (B) X-gal
filter-lift experiment. (C) Relative β-gal activity. The
β-gal experiment was repeated four times independently using three
samples of each interaction tested. (D) Glutathione-S-transferase (GST)
pull-down experiments. The ANTP-YPWM-HD-GST fusion protein (amino acid
279-356) was produced in E. coli and purified with glutathione
sepharose beads. The BIP2-235 protein (amino acids 853-1088) was produced in a
rabbit reticulocyte lysate and labelled with [35S]-methionine. The
BIP2 protein domain found in the yeast two-hybrid screen (BIP2-235) is also
able to interact with ANTP-HD fused to GST in vitro. The synthesis of the
BIP2-235 protein gives three bands, likely to be due to different methionine
start codons used for protein synthesis by the reticulocyte lysate. (E)
Co-immunoprecipitation of ANTP and BIP2. The BIP2 protein was tagged with the
hemaglutinin (HA) epitope and immunoprecipitated with an anti-HA antibody.
Co-immunoprecipitated ANTP protein was detected by using a mouse monoclonal
anti-ANTP antibody. Upon mutating the YPWM motif of ANTP, ANTP is not
co-immunoprecipitated with BIP2-HA, unlike the wild-type ANTP protein. The
larvae used were hs>bip2-HA, Antp (YHA), hs>bip2-HA,
AntpAAAA (AHA), hs>bip2, Antp (Y), hs>bip2,
AntpAAAA (A), and wild type (WT), as a control.
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Fig. 3. AntpCtx show similar adult phenotypes to ectopic
Antp in combination with Nact in eye imaginal
discs. (A,B) Head of AntpCtx an adult
showing an ectopic dorsal thoracic structure (Th in A) and wing tissue (W in
B) on the dorsal side. (C,D)
ey-Gal4>UAS-Nact; UAS-Antp head
showing some ectopic thoracic tissue (Th in C) and a wing (W in D) growing out
of the dorsal side. (E,F) Heads of
OK-107-Gal4>UAS-Antp flies showing the eye reduction phenotype.
(G) The addition of bip2 increases the frequency of formation
of ectopic wings (W) formed on the head of OK-107>UAS-bip2;
UAS-Antp flies. Antp is able to induce ectopic wings without
Nact.
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Fig. 4. The AntpCtx eye-antennal disc is transformed into
various parts of the wing disc. (A) Su(H)-lacZ staining on
an AntpCtx eye-antennal disc shows ectopic N
signaling (arrow). (B) Su(H)-lacZ staining on wild-type
eye-antennal discs showing active Notch signaling. (C)
AntpCtx eye-antennal disc showing ectopic VG protein (red)
and ectopic ANTP protein (green). (D,F,H,J)
AntpCtx eye-antennal disc.
(E,G,I,K) Wild-type eye-antennal disc. (D,E)
Ectopic VG (green) represses EYA (red) cell non-autonomously in the
AntpCtx eye-antennal disc (D). (F,G) Ectopic VG (green)
and ectopic EYG (eyg-lacZ; β-Gal in red) in the
AntpCtx eye-antennal disc (F) indicate a transformation
toward notum identity. (H,I) Ectopic VG (green) is co-expressed with ectopic
WG (wg-lacZ; β-Gal in red) in the AntpCtx
eye-antennal disc (H). The localization of the ectopic VG protein corresponds
to the dorsal eye region where the ectopic wings are formed. (J,K) Ectopic WG
(red) is co-expressed with ectopic DPP (dpp-lacZ; β-Gal in red)
in the AntpCtx eye-antennal disc, indicating
transformation to wing disc cells (J).
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Fig. 5. The bip24 mutant genetically interacts with
AntpCtx. (A) Head of an
AntpCtx/+; ciD/+ fly with an ectopic
wing (W) on the dorsal head. The wing shows the characteristic wing margin
bristles. (B) Head of an AntpCtx/+;
bip24/+ fly with the head capsule transformed into dorsal
thorax (Th). One eye is missing and one eye is strongly reduced. Flies showing
an ectopic wing on the dorsal head regularly show a normal or mildly reduced
eye and no head capsule-to-thorax transformation. Flies with a head
capsule-to-thorax transformation mostly show severely reduced or lost eyes.
The eye reduction was therefore used as a measure of the two phenotypes shown
above. (C) Analysis of eye-to-wing transformation and the strength of
eye reduction in the progeny of AntpCtx females crossed to
yw; bip24/ciD males. The number of F1
flies counted were: AntpCtx;+; ciD/+,
233; AntpCtx/+; bip24/+, 234. Only 4%
of the flies with a single bip2 gene copy
(AntpCtx;+; ciD/+) show ectopic wings
on their dorsal head, compared with 14% with two wild-type bip2 gene
copies (AntpCtx/+;
bip2+/bip2+; see
Table 2). Normal (slightly
reduced) sized eyes, reduced (strongly reduced) and missing eyes were counted.
Flies with a single bip2 gene copy show a stronger eye reduction
phenotype than do flies harbouring two gene copies.
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