Long-term ex ovo culture of Caenorhabditis elegans embryos

The genetic tractability, transparency and invariant development of the C. elegans embryo have led to its broad adoption as a model system for the study of cell and developmental biology. Its impermeable eggshell has restricted the use of small-molecule interventions during embryogenesis. Existing genetic approaches for rendering the embryo permeable to acute small molecule treatment have increased the accessibility of early embryogenesis of pharmacological manipulation but compromise long-term viability for use in studies of later developmental processes or post-exposure physiology. Here, we describe the use of an optimized enzymatic eggshell digestion protocol coupled with a minimal, serum-free culture media that supports the survival and normal development of ex ovo embryos through larval maturation and adulthood. We show that this approach renders embryos permeable to a wide range of small molecules, enabling precise temporal manipulation of developmental processes previously inaccessible through conventional genetic or physical methods. We demonstrate the utility of this technique through the application of small molecule fluorescent dyes, the pharmacological modulation of key cytoskeletal components including microtubules and actin, as well as the minus-end-directed microtubule motor protein dynein, highlighting applications for study of cell division, morphogenesis, and neuronal cell biology, especially in later stages of embryogenesis. This approach expands the experimental toolkit available for labeling and manipulating developmental processes in C. elegans .