View larger version (88K):
[in a new window]
|
Fig. 2. Murine Tsh genes rescue the cuticular phenotype of the
tsh8 null mutant. Both the homeotic transformation and the
segment polarity phenotype are restored. (A) Wild-type larval cuticle showing
the three thoracic (T1, T2, T3) and the eight abdominal segments. (B) Close-up
of the T1-T2 region. Arrows indicate the beard (b) in the T1 segment. (C)
Homozygous tsh8 loss-of-function cuticular phenotype; the
T1-specific denticles are missing (homeotic transformation of the T1 segment
into the labial segment) (Röder et
al., 1992 ) and naked and denticled cuticular regions are reduced
in the trunk. (D-G) Rescue of tsh8 by Tsh genes. Close-up
of T1-T3 region is on the right. (D) At 22°C in tsh8
homozygotes, 69B>Tsh1 expression results in partial rescue of
tsh8 phenotype. The T1 segment is weakly restored
(denticles form only on one side in this photograph) and the size of naked
cuticle is comparable with wild type (compare with A-C). At 22°C,
Tsh3 display similar levels of rescue (not shown), whereas
Tsh2 has very little effect (see text). (E-G) At 29°C, the three
Tsh genes perfectly restore the naked cuticle regions in the trunk although
the T1, on the basis of the morphology of the beard and T1 denticle belt, is
not completely rescued (compare with B). This defective differentiation was
used to distinguish tsh8 homozygotes. Homeotic
transformation of the labial segment into T1 segment is highlighted by ectopic
denticles in the head (additional T1 and arrows). (H,I) Increased dose of
mouse Tsh reveals a segment polarity phenotype similar to tsh. (H)
69B>tsh larvae raised at 25°C exhibit ectopic naked cuticle in
the trunk (in contrast to 69B>tsh at 18°C in
Fig. 1D), particularly along
the ventral midline. (I) Combination of two independent insertions of
UAS-Tsh1 (69B>2xTsh1) also causes ectopic naked cuticle
(similar results were obtained with Tsh2 and Tsh3, data not
shown). Most of the denticles of the thoracic and ectopic T1 segments are
absent. (J) Combination of UAS-Tsh1 with UAS-Tsh2
(69B>Tsh1;Tsh2) gives similar results (as for UAS-Tsh1
with UAS-Tsh3 and for UAS-Tsh2 with UAS-Tsh3, not
shown).
|
25% of 69B>Tsh2
(n=170). (D-G) Regulation of mod expression by Tsh genes.
(D) mod expression revealed by in situ staining in a stage 11
wild-type embryo. mod mRNA are detected in the labium (arrow). (E)
Reduced mod expression in the epidermis of the labial segment upon
ectopic expression of tsh (69B>tsh) or Tsh genes as
illustrated in F by Tsh1. Reduction of mod expression occurs
in roughly 75% of 69B>Tsh1 (n=291) and
69B>Tsh3 (n=170) and 50% of 69B>Tsh2
(n=230) embryos.
PLDLS embryos (top, compare with
Fig. 3E). (B) pABgal-mouse
Tsh1, 2, and 3 (0.025 and 0.1 µg) alone repress pGL2-5xUAS-Luc reporter
activity (0.25 µg). (C) GST-pull down assay showing the interaction between
in vitro translated mouse Ctbp1 and GST-mouse Tsh fusion proteins.
Non-specific interaction is shown with GST alone. (D) In an independent
experiment from B, co-expression of 0.1 µg mouse Ctbp1 with 0.1 µg
Gal4-mouse Tsh fusions leads to a synergistic inhibition. Deletion of the
putative CtBP interaction motif in pABgal-mouse Tsh1 reduces the activity of
the reporter from 5 to 2.5-fold and abolishes the synergistic repression in
the presence of mouse Ctbp1. Mouse Ctbp1 alone does not affect the reporter
activity (not shown). Data are presented as fold repression relative to
luciferase activity, normalized to the ß-galactosidase activity, of the
reporter in presence of empty pABgal and pcDNA3.myc.