Molecular timekeeping and temporal cell fate specification in C. elegans - ABSTRACT/SUMMARY Development is an inherently dynamic process that operates on diverse timescales. Coordinating gene expression, cell fate determination, and tissue growth is crucial in development, but our understanding is limited. Through genetic analyses of C. elegans temporal patterning, we have discovered a molecular clock, composed of circadian clock orthologs, that coordinates cell fate specification to developmental pace. It accomplishes this task by generating oscillatory patterns of transcription that exhibit highly reproducible onset phases, durations, and amplitudes. This clock regulates the transcriptional dosage of key microRNA (miRNA) genes that program temporal cell fate transitions during development. The experiments outlined in this proposal aim to understand the molecular underpinnings of this clock by determining the structures of three separate complexes (protein::protein and protein::DNA) that assemble and disassemble in a specific order to tailor dynamic miRNA transcriptional patterns. We will then use this knowledge to engineer clock component mutations that alter features of these dynamic interactions. We will validate how these novel mutants affect complex assembly in vitro and how they disturb development in vivo. Specifically, we hypothesize that these tailored mutations, both loss-of- and gain-of-function, will alter miRNA transcriptional output by disrupting specific features of oscillatory transcription (phase, amplitude, or duration). We will quantify changes in miRNA expression in these mutants by directly monitoring miRNA transcriptional dynamics in mutant animals using a new state-of-the-art imaging platform. This imaging system will also enable us to directly correlate changes in transcriptional dynamics to alterations in temporal stem cell division patterns during development. In addition, we aim to use biochemical strategies to identify the ligands that coordinate clock transcription factors. Identifying these ligands will reveal how miRNA expression patterns sync across tissues to organize temporal patterning. Notably, many of the physical interactions between the clock components we aim to study in the nematode system are conserved between the mammalian orthologs. Therefore, these studies will not only shed light on how the timing of gene expression contributes to developmental robustness but also may illuminate how transcription is coordinated with metabolic changes during development.
