How it is possible to maintain a segmented body plan after loss of a key developmental gene? Dr. Alys Jarvela, a biochemist, geneticist, and postdoctoral scholar in the Pick Lab at University of Maryland, presented her research to address this precise question.
When comparing segmentation mechanisms between the fruit fly, Drosophila melanogaster, and the mosquito, Anopheles stephensi, Dr. Jarvela strikingly found out that mosquitoes don’t have paired gene in their genomes. While insects typically have three pax3/7 genes, only two appeared to be in the many mosquito genomes she searched. These two genes have been identified as gooseberry (gsb) and gooseberry-neuro (gsb-n), as determined by phylogenetic analysis; neither of the pax3/7 genes found in mosquito genomes group with the paired genes (figure 1). Furthermore, gsb and gsb-n are syntenic – they occur adjacent to each other on the same chromosome – in the Anopheles genome, just as other gsb and gsb-n genes are in other insect genomes. Of course, Anopheles embryos and larvae retain a segmented body plan and segment polarity expression of engrailed despite the loss of prd. So, how do mosquitoes maintain their segmented body plan?
To address this question, Dr. Jarvela sought to understand the roles of some of the other segmentation gene orthologs in A. stephensi. Might there be differences in their expression patterns that could explain mosquitoes’ loss of prd? Dr. Jarvela used a technique known as in situ hybridization staining, which allows one to visualize which cells contain mRNA of interest. Imaging revealed the expression patterns of most of the segmentation genes she examined were nearly identical to the expression patterns of these genes in Drosophila.
She then wondered which transcription factor(s) might be taking the place of prd in the activation of engrailed, a segment polarity gene. Before she could try to identify the CREs dependent on Prd’s replacement in the mosquito, she first needed to map the Prd-dependent CREs in Drosophila. To do this, she conducted a classic enhancer-reporter screen. In this approach, a candidate CRE is placed upstream of some reporter gene, and this construct is then integrated into the Drosophila genome (Yeh et al. 1995). The ability of the candidate CRE to activate transcription of the reporter is then assayed by analyzing expression of the reporter (such as by in situ hybridization, described above). Using this technique, Dr. Jarvela was able to identify Prd-dependent engrailed regulatory regions. She then tested candidate Aste-engrailed CREs in Drosophila embryos, and was able to drive expression of a reporter in an engrailed-like pattern, demonstrating that these DNA sequences are very likely Aste-engrailed regulatory elements. Future work will look at which transcription factors might be responsible for binding these CREs in the absence of prd in mosquitoes.
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