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

Darren Z. L. Mok, Paul W. Sternberg, Takao Inoue

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. 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. 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.