PROJECT SUMMARY Our goal is to discover how closely related species adopted unrelated axis determinants for specifying the anterior embryo. Many aspects of animal development are conserved between species as different as humans and flies, but some key regulators change very rapidly over the course of evolution for unknown reasons. Explaining such unexpected plasticity in developmental gene networks will help us understand the basic science of developmental robustness and congenital disease in all animals, including humans. Comparing multiple closely related species is a powerful approach for understanding causes of plasticity in gene networks. This approach is extremely difficult to implement with vertebrate model organisms but can be readily accomplished in insects. Flies (Diptera) are particularly suitable because they include Drosophila melanogaster, one of the leading model organisms in developmental biology, and because other dipteran species that can be cultured in a laboratory setting are amenable to functional studies. Our research in a variety of dipteran model organisms during the previous funding period established that these closely related species use a broad range of anterior determinants (ADs) for establishing embryo polarity and anterior-specific gene expression, most likely through the formation of long-range transcription factor gradients with morphogen-like activity. This discovery enables us to examine why key developmental regulators can be highly unstable in evolution. While Drosophila’s AD (Bicoid) is a classic morphogen model, it is not known whether its mechanism of action can be generalized nor how the evolutionary transition to Bicoid-dependent pattern formation was achieved. The first of these two questions will be addressed by identifying and characterizing chromatin and gene targets of the AD in the moth fly Clogmia albipunctata, Cal-Opamat (Aim 1) and of the AD in the common midge Chironomus riparius, Panish (Aim 2). This will be done by testing for AD-dependent chromatin accessibility in stage-matched embryos with or without reduced AD activity, using ATAC-seq and RNAi. These experiments will be complemented as needed by ChIP-seq, and by characterizing the expression and function of predicted target genes in vivo. The second question, concerning the transition to Bicoid- dependent pattern formation, will be addressed by determining the mechanism of anterior specification in the soldier fly Hermetia illucens (Aim 3). This organism was chosen because it is the closest tractable outgroup to the clade of species with bicoid. A pilot study revealed several AD candidates, including Hil-Stau, the ortholog of Staufen. This RNA-binding protein binds bicoid mRNA in early Drosophila embryos. To identify Hermetia’s AD, anterior-localized and Hil-Stau-binding mRNAs will be determined by expression studies, co- immunoprecipitation, sequencing, and their function will be examined by RNAi. The determination of the anterior- speci...