Animal development requires the activity of tightly coordinated programs of gene expression, known as "gene regulatory networks." Changes in these networks, which can occur at the level of DNA sequences that regulate genes, or of regulatory proteins that bind to these sequences, are important mediators of evolution. This project investigates in detail a poorly-understood set of evolutionary mechanisms in which a gene regulatory network's activity is shifted to a new tissue, and a different network appears to take over its role in the old tissue. The research focuses on nervous system development in the embryos of mosquitoes and fruit flies, taking advantage of two highly tractable research organisms with a mature selection of experimental tools, along with novel computational methods the investigators have developed for identifying gene regulatory sequences. The results will provide insight into how regulatory networks evolve and will build a foundation for in-depth functional analysis of the consequences of such evolution, over a greater evolutionary distance than has been possible in most previous studies. As an added impact, the work will lead to increased knowledge of the developmental biology of the Zika, dengue, and yellow fever vector mosquito Aedes aegypti and generate new tools and resources for the study and experimental manipulation of this important organism. The project will also support training of scientists at the graduate, undergraduate, and high school levels. Aspects of the project will form the basis for a new research program for local (city) high school students, centered around genetic model organisms.

Gene regulatory networks (GRNs) are composed of transcription factors (TFs) and cis-regulatory modules (CRMs, 'enhancers'). This project will interrogate a novel mode of GRN evolution using the mosquito Aedes aegypti. In this "repeal, replace, and redeploy" mode of evolution, a GRN functioning in the embryonic nervous system midline in the fly Drosophila and other insects (the "Sim GRN") has been "redeployed" to lateral regions in A. aegypti while "repealed" from and potentially "replaced" in the midline. The altered gene expression observed in A. aegypti appears to be the result of trans-dependent redeployment of the GRN, stemming from cis-mediated changes in the expression of key regulatory TFs. This includes in particular the "master" TF Sim, which in the nervous systems of all other studied insects is confined to the midline. The current project investigates the molecular details of how GRN evolution has occurred. Specific aims are to: (1) Characterize the development of the late A. aegypti embryonic central nervous system with respect to cell identities and molecular markers in both medial and lateral regions. (2) Perform reciprocal transgenic analysis in D. melanogaster and A. aegypti of CRMs from both species which regulate genes of the Sim GRN. Orthologous pairs of CRMs will be identified using the PI's SCRMshaw algorithm. (3) Identify the specific mechanisms leading to the evolved patterns of gene expression using a combination of genetics, ectopic gene expression, and mutagenesis of regulatory sequences to define specific cis- and trans- regulatory changes between the two species.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

National Science Foundation (NSF)
Division of Integrative Organismal Systems (IOS)
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Anthea Letsou
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Suny at Buffalo
United States
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