Collaborative Research: Imaging the transition: Temporal control of zygotic genome activation by the nuclear to cytoplasmic ratio

Early embryonic development is characterized by a dramatic transition from cellular dependence on RNAs that were in the egg prior to fertilization to RNAs produced by the embryo itself. This process is known as zygotic genome activation (ZGA). The timing of ZGA is linked to changes in cell size, specifically the ratio of nuclear content (DNA) to cytoplasmic volume (also known as the nuclear-to-cytoplasmic or N/C ratio), which increases as cells replicate their DNA without growth. How cells measure this ratio and use it to control gene expression is a fundamental biological question that remains poorly understood. This project will address it using fruit fly embryos as a model system, combining cutting-edge live imaging with genetic and genomic approaches to reveal the molecular logic by which cells sense their size and activate transcription accordingly. Understanding how cells coordinate gene expression with cell size has broad implications for developmental biology and human health, as defects in this process can lead to developmental disorders and are implicated in diseases such as cancer. This collaborative project will also provide interdisciplinary training opportunities for graduate students at the interface of genomics, quantitative imaging, and computational biology, addressing workforce development in biotechnology and biomedicine. The project will also include educational outreach activities that engage K-12 students with key concepts in basic biology and genetics. This project will use Drosophila embryos to uncover the molecular mechanisms by which the N/C ratio controls the timing of zygotic genome activation. In Aim 1, the investigators will use existing RNA-seq data sets from embryos arrested at low N/C ratios to systematically identify genes that directly respond to the N/C ratio to initiate zygotic transcription. A subset of the resulting candidate genes will be selected for quantitative live imaging using the MS2/MCP transcription reporter system to determine specific transcriptional parameters (e.g., probability and timing of transcriptional activation) that respond to changes in the N/C ratio. Mathematical modeling will be used to extract quantitative transcriptional parameters from the data and determine what aspects of transcription respond to the changing N/C ratio. In Aim 2, the investigators will leverage the genome-wide set of N/C ratio-sensitive genes to identify cis-regulatory sequences and trans-acting factors that confer N/C ratio sensitivity. Bioinformatic analysis will be used to identify DNA motifs enriched in the regulatory regions of N/C ratio-sensitive genes, and systematic enhancer dissection will define the minimal sequences sufficient for N/C ratio-dependent transcription. Candidate transcription factors identified through motif enrichment and enhancer analysis will be experimentally manipulated to test whether altering their concentrations shifts the N/C ratio threshold for gene activation, both at individual loci and on a genome-wide scale. The outcomes will provide new mechanistic understanding of how cells measure their N/C ratio and coordinate transcriptional activation during a critical developmental stage. 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. NSF Award ID: 2534079 | Program: 01002627DB NSF RESEARCH & RELATED ACTIVIT | Principal Investigator: Amanda Amodeo | Institution: Dartmouth College, HANOVER, NH | Award Amount: $690,177 View on NSF Award Search: https://www.nsf.gov/awardsearch/show-award/?AWD_ID=2534079 View on Research.gov: https://www.research.gov/awardapi-service/v1/awards/2534079.html