PgmNr W4162: Fluorescent beads are a versatile tool for staging C. elegans in different life histories.

Authors:
l. nika; T. Gibson; R. Konkus; X. Karp


Institutes
Central Michigan University, Mount Pleasant, MI.


Keyword: Timing of developmental events, and circadian rhythms

Abstract:

C. elegans life history is dependent on environmental cues transduced through several signaling pathways. In favorable environmental conditions C. elegans develops continuously through four larval stages before molting into the adult stage. In contrast, adverse environmental conditions promote entry into the stress resistant dauer larva stage immediately following the second larval molt. Dauer larvae possess distinct properties including cellular and developmental arrest, extended lifespan, and discontinued feeding. If environmental conditions improve, dauer larvae recover to post-dauer L3 larvae, which are developmentally identical to continuously developing L3 larvae. Precise staging of larvae developing through either continuous or dauer life histories is essential for the study of development and/or dauer-related processes. Here, we describe a staging method taking advantage of the inability of dauer larvae and molting larvae to feed. Fluorescent beads are added to the bacterial food source, and dauer or molting larvae are identified by a lack of beads within their digestive tract. Bead-lacking worms are sorted using a dissecting microscope or a wormsorter. We show that lack of beads correlates with two molting markers: lack of pharyngeal pumping, and expression of mlt-10::GFP-pest. We then describe how this assay can be used to isolate dauer larvae formed by any of three common methods: starved plates, exogenous pheromone, and dauer-constitutive mutations. Lack of beads correlates well with other known markers of dauer formation, including greatly reduced pumping rate, presence of dauer alae, and SDS resistance. We find that using beads rather than SDS-resistance to identify dauer larvae enables the recovery of SDS-sensitive mutants, including cuticle mutants and partial dauer larvae formed by mutations within the dauer formation pathway. This method therefore provides a simple, transgene-free way to distinguish particular stages during development through continuous or dauer life histories.