Measuring the effects and rates of microsatellite instability in the morphogen concentration-sensitive enhancers of Drosophila. Clinton Rice, Albert J. Erives. Department of Biology, University of Iowa, Iowa City, IA.

   Developmental enhancers are important sites of functional evolution in animal genomes. The neurogenic ectoderm enhancers (NEEs) located at vnd, brk, vn, and rho drive expression of key regulators of embryonic development in response to maternal Dorsal, which acts as a morphogen to pattern the dorsal/ventral axis. Different NEEs are able to activate their target genes over different ranges of the Dorsal nuclear gradient. We have found that these enhancers are canonical for the Drosophila genus, and that the NEE at vnd is also present in mosquitos. Among the functional transcription factor binding sites that comprise NEEs are motif-invariant Dorsal and Twist binding sites, separated by an 8-18 bp spacer. Changes in the length of this spacer alter the concentration-response threshold, leading to wider or narrower expression stripe widths for reporter genes or correct expression stripe widths in embryos of different sizes. Spacer evolution apparently occurs via expansion and contraction of CA-microsatellite tracts within and around Twist binding sites. The four canonical NEEs are enriched in similar microsatellites that are presumed to be relic Twist binding sites and spacers. Here, we are using these microsatellite sites to measure both the deleterious effects and relative rates of mutations within the NEEs. Using multiplexed genotyping, we are assaying for length variants and sequencing to determine whether microsatellite expansion or contraction has occurred. To determine the level of selection acting at these loci, we are comparing the rates of microsatellite length variation between adults (i.e. post-selection) and failed embryos, as well as between microsatellite repeats inside and outside of the functional spacer region. We are also using DNA repair mutant spellchecker1 (spel1) to exacerbate microsatellite instability and improve our ability to measure relative rates of change.