UC Santa Cruz

All living organisms have biological clocks that drive and coordinate repetitive developmental processes. These intrinsic timing devices are essential for animal development and survival but, in many cases, the underlying mechanisms are poorly understood. C. elegans larval development is an ideal system in which to study this problem. C. elegans employ two distinct but interrelated biological timing mechanisms to regulate progression through larval development. The heterochronic pathway ensures that stage-specific cellular events happen at the correct time and in the correct order, while a molt timer ensures the proper timing of each molt in relation to developmental progression. Chapter one of this work describes our investigations into the mechanisms employed by C. elegans to regulate and coordinate developmental progression and molting. lin-42, the C. elegans homolog to the circadian clock protein PERIOD2 (PER2), is unique among heterochronic genes for its dual role in regulating both stage-specific cellular events, and the precise timing and execution of molting, and is thought to coordinate molt cycles with developmental progression. To investigate the mechanisms through which LIN-42 regulates and coordinates developmental timing in C. elegans, we looked to the well-characterized mechanisms employed by its mammalian homolog PER2. The circadian period in mammals is determined by the stability of PER2 and the stability is largely determined through its interaction with Casein Kinase I  (CK1). Our work shows that CK1δ can bind and phosphorylate a LIN-42 substrate in vitro with similar kinetics compared to a PER2 substrate and that these interactions depend on conserved kinase binding domains. Further, we show that deletion of the SYQ and LT domains, two highly conserved alpha helices within the conserved kinase binding domain, results in dysregulation of LIN-42::GFP expression in vivo. However, severe developmental abnormalities and molting defects are only observed once those deletions are extended to encompass the whole kinase binding domain. Together, these data suggest that PER2/LIN-42 phosphorylation by CK1δ kinases is evolutionarily conserved and that this mechanism may be employed to coordinate developmental timing in C. elegans.When investigating essential systems and critical gene products, we often cannot employ classical genetic approaches to study gene function. Critical regulators of developmental progression and timing often function with distinct co-regulators in various developmental contexts and may have reiterative functions throughout development. In knockout or null mutants, if disruption of the first developmental event results in embryonic or larval lethality, later functions will be missed. Chapter 2 of this work describes a set of genetic tools that we produced to complement existing reagent and augment the use of the auxin inducible degron (AID) system in C. elegans. The AID system is a powerful tool to conditionally deplete proteins. We generated a set of single-copy, tissue-specific and pan-somatic TIR1-expressing C. elegans strains carrying a co-expressed blue fluorescent reporter to enable use of both red and green channels in experiments. We also generated a set of plasmids for constructing repair templates to generate fluorescent protein::AID fusions through CRISPR/Cas9-mediated genome editing. Together, these reagents will complement existing TIR1 strains and facilitate rapid and high-throughput fluorescent protein::AID tagging of genes.