Muscles in the Drosophila second thoracic segment are patterned ...

Research Paper Homeotic gene function in Drosophila thoracic muscle development Roy et al. 227
segment, or if the effectiveness with which the HT3 ectoderm
is transformed towards the T2 phenotype were
increased, thereby ‘overpowering’ Antp function in the
HT3 mesoderm. The second possibility may explain the
presence of IFM-like muscles, albeit at a very low frequency,
in the HT3 of animals that are abx, bx 3 , pbx in trans
with a deficiency for the entire BX-C [22].
Materials and methods
Fly stocks
Canton-S strain was used as the wild-type stock in all control experiments
unless otherwise mentioned. The UAS-Antp transgenic strain was generously
provided by Sean Carroll (University of Wisconsin, Madison). The
SG29.1 GAL4 line was generated in our laboratory by mobilization of a
pGAL4 element onto the scalloped locus. The UAS-lacZ and 24B-GAL4
lines were obtained from Andrea Brand and Norbert Perrimon (Harvard
Medical School, Boston, USA). The Antp w10 mutant stock was obtained
from the Drosophila Stock Centre, Bloomington, USA. Four-winged flies
that are homozygous for abx, bx 3 , pbx mutations in the Ubx gene were
generated as described previously [7]. The e22c-GAL4 strain was
obtained from the Drosophila Stock Centre, Bloomington, USA, and it
expresses GAL4 specifically in the ectoderm during embryogenesis [11].
We examined its ability to drive Antp expression using the UAS-Antp
transgene and Antp-specific antibodies and found that it could promote
uniform levels of Antp expression all over the embryonic ectoderm.
Mesodermal mosaic for Antp
In order to remove Antp specifically from the mesoderm and analyze its
effect on T2 muscle development, we collected embryos from a cross
of male flies of the genotype e22c-GAL4/ + ; Antp w10/+ with females of
the genotype UAS-Antp; Antp w10/+ . We were able to identify unambiguously
embryos that were homozygous for the Antp w10 mutation on
the basis of midgut morphology (Fig. 2e,f) and these embryos were
analyzed for rescue of T2 muscle pattern by staining with a rabbit antifly
muscle myosin antibody (n = 25). Approximately half of these
embryos (n = 11/25) showed rescue of muscle pattern in both
hemisegments of T2 because they had one copy of the e22c-GAL4
and one copy of the UAS-Antp transgenes.
Histochemical staining for -galactosidase activity
-galactosidase activity from the UAS-lacZ transgene was assayed by
standard procedures [15].
Immunohistochemistry and microscopy
Embryos, larval imaginal discs and pupal tissue were prepared for
immunohistochemistry as described previously [5,7]. The anti-Antp
monoclonal antibody was a gift from Danny Brower (University of
Arizona, Tucson) and was used at a dilution of 1:50. The rabbit anti-fly
muscle myosin antibody, for the detection of embryonic muscle patterns,
was used at a dilution of 1:500 and was obtained from Dan
Kiehart (Duke University, Durham). Fluorophore-conjugated appropriate
secondary antibodies were purchased from Jackson Immuno Research
Laboratory Inc. The Vectastain Elite Kit (Vector Laboratories) was used
for indirect immunoperoxidase assays. After immunolabelling, the
preparations were analyzed by laser scanning confocal or differential
interference contrast microscopy.
Analysis of IFM morphology
Adult hemithoraces were dehydrated through alcohol grades, cleared in
methyl salicylate, mounted in Canada Balsam and the morphology of the
IFMs was visualized using polarized light optics.
Acknowledgements
We thank A.H. Brand, N. Perrimon, S.B. Carroll and T. Kaufman for fly stocks;
D. Brower, D. Kiehart and R. White for antibodies; K. White for use of confocal
microscope and V. Rodrigues and anonymous reviewers for comments on
the manuscript. This work was supported by grants from the National Centre
for Biological Sciences and from the Department of Science and Technology,
Govt. of India.
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