Download Tom Buckman and John P Masly Department of Biology, University

Characterizing the development of the posterior lobe in Drosophila
Tom Buckman and John P Masly
Department of Biology, University of Oklahoma
Abstract
The posterior lobe is a reproductive structure that surrounds the male
genitalia in some Drosophila species. It has been shown that variation in the
shape and size of this structure affects reproductive success in mating
experiments, indicating that the posterior lobe may lead to reproductive
isolation and eventually speciation. Learning about the genetics controlling
the development of this structure is important for understanding how
evolution shapes morphology. We have identified a candidate gene, CG14567,
which has been shown by previous experiments to likely play an important
role in specifying posterior lobe size. Here, we looked for its expression in the
developing lobe using in situ hybridization. Furthermore, we used
immunohistochemistry on D. melanogaster to easily visualize the cell
membranes in these developing structures. Once these cells can be visualized
well enough for analysis, we will be able to determine the changes that occur
during development that give rise to such diverse phenotypes.
A recently-evolved, novel structure of the genitalia that
affects reproductive success
Methods
enhancing visualization of individual cell morphology using
immunohistochemistry on transgenic Drosophila lines
Visualizing the expression of a candidate gene, CG14567, using molecular
cloning and in situ hybridization
Fluorescent Antibody staining of cell membranes
by binding to a DE-cadherin-GFP fusion protein
Cloning of CG14567 into a plasmid construct
We insert a PCR-amplified copy of the CG14567 coding region into a commercially available plasmid
construct, which is then amplified by E. coli until there are hundreds of millions of copies of the
plasmid
Performing in vitro transcription to produce modified RNA
probes
In order to make the cell membranes of individual cells more brightly fluorescent, we
use immunohistochemistry to enhance the signal produced by these proteins during
microscopic analysis.
Results
The posterior lobe is located near the terminal genitalia in Drosophila
Significant variation in posterior lobe size and shape in
closely related species
We then use the promoter sites in the multiple cloning site of the plasmid vector to initiate
transcription of modified RNA strands that are complimentary to CG14567 transcripts.
There is wide variation in the shape and size of the posterior lobe in the D. melanogaster
species complex.
Hybridization of RNA probe to mRNA in developing Drosophila
Potential causes:
Changes in cell polarity
Varying cell size
Apoptosis
Changes in cell shape
Differing numbers of cells
As seen in the above figure, the
DE-cadherin-GFP fusion protein is
visible on the cell membranes of
developing posterior lobe cells.
The signal has yet to be amplified
by immunohistochemistry to date.
Future aims
What cellular-level changes occur during development cause
the diverse morphologies in these sister species?
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The coding region of CG14567 was
successfully cloned, but all
experiments have provided no
evidence of CG14567 expression in
developing pupal tissue.
Once the modified RNA probe is obtained, it is washed into the tissue of the Drosophila pupa.
Any sequence of messenger RNA present in the tissue that is complimentary to the probe will
become hybridized with it, and will fluoresce.
We plan to continue pursuing both experimental routes in order to come
to proper conclusions about both the expression of CG14567 and the
morphology of individual cells in the developing posterior lobe.
• Troubleshoot potential problems with in situ hybridization technique,
and implement a positive control to ensure protocol works.
• Continue immunohistochemical experiments, and perform
computational analysis of cell morphology.
• Conduct more immunohistochemical experiments for individuals
whose expression of candidate genes has been knocked down.