TEMPORAL DYNAMICS OF PROGRAMMED GENOME REARRANGEMENT IN SEA LAMPREY EMBRYOGENESIS

Programmed genome rearrangement (PGR) in the sea lamprey (Petromyzon marinus) leads to the elimination of ~0.5Gb (20%) of the germline genome during early embryogenesis, yet its timing relative to the maternal-to-zygotic transition (MZT) remains unclear. In this study, we imaged embryos cleared at 3-hour intervals (up to 72 hours post fertilization (hpf)) using high-resolution lightsheet fluorescence microscopy and a custom deep-learning image-analysis pipeline (~80% accuracy) to quantify nuclei and micronuclei counts. Micronuclei, which are the hallmarks of PGR first appear between 24 to 27hpf (vs ~36hpf previously reported) indicating PGR starts earlier than previously reported in the sea lamprey. Micronuclei count per cell exhibited cyclical peaks and throughs at 9-hour intervals, suggesting synchronized waves of targeted chromosome elimination coordinated with mitosis, moving from the animal pole towards the vegetal pole, implying potential presence of directional signaling cues. Small molecule inhibition (MK5108) and CRISPR-Cas9 knockout of Aurora kinase A revealed that embryos arrested development at ~27hpf with reduced micronuclei, indicating that zygotic transcription initiates concurrently with PGR. Integration with RNA-seq revealed minor transcription peak at 12hpf and a major zygotic-activation peak at 24hpf, placing MZT within a 12–33hpf window that overlaps PGR onset. Finally, we established a novel live-imaging of intact embryos via H2B-GFP mRNA injection and custom agarose mounting, enabling 10-minute interval recordings for up to 48h with minimal phototoxicity. Collectively, this work refines the temporal map of lamprey PGR, defines its functional overlap with MZT, and provides the first live-imaging platform for dynamic genome-restructuring events in vertebrate embryos.