Morphologically defined sub-stages of C. elegans vulval development in the fourth larval stage

Background: During the fourth larval (L4) stage, vulval cells of C. elegans undergo extensive morphogenesis accompanied by changes in gene expression. This phase of vulval development, occurring after the well-studied induction of vulval cells, is not well understood but is potentially a useful context in which to study how a complex temporal sequence of events is regulated during development. However, a system for precisely describing different phases of vulval development in the L4 stage has been lacking. Results: We defined ten sub-stages of L4 based on morphological criteria as observed using Nomarski microscopy (L4.0 ~ L4.9). Precise timing of each sub-stage at 20 °C was determined. We also re-examined the timing of expression for several gene expression markers, and correlated the sub-stages with the timing of other developmental events in the vulva and the uterus. Conclusions: This scheme allows the developmental timing of an L4 individual to be determined at approximately one-hour resolution without the need to resort to time course experiments. These well-defined developmental stages will enable more precise description of gene expression and other developmental events