Acute Drug Treatment in the Early C. elegans Embryo

Abstract

Genetic and genome-wide RNAi approaches available in C. elegans, combined with tools for visualizing subcellular events with high-resolution, have led to increasing adoption of the early C. elegans embryo as a model for mechanistic and functional genomic analysis of cellular processes. However, a limitation of this system has been the impermeability of the embryo eggshell, which has prevented the routine use of small molecule inhibitors. Here, we present a method to permeabilize and immobilize embryos for acute inhibitor treatment in conjunction with live imaging. To identify a means to permeabilize the eggshell, we used a dye uptake assay to screen a set of 310 candidate genes defined by a combination of bioinformatic criteria. This screen identified 20 genes whose inhibition resulted in >75% eggshell permeability, and 3 that permeabilized embryos with minimal deleterious effects on embryo production and early embryonic development. To mount permeabilized embryos for acute drug addition in conjunction with live imaging, we combined optimized inhibition of one of these genes with the use of a microfabricated chamber that we designed. We demonstrate that these two developments enable the temporally controlled introduction of inhibitors for mechanistic studies. This method should also open new avenues of investigation by allowing profiling and specificity-testing of inhibitors through comparison with genome-wide phenotypic datasets.

Extracellular leucine-rich repeat proteins are required to organize the apical extracellular matrix and maintain epithelial junction integrity inC. elegans

“…7E-J), indicating a defect in larval cuticle organization. A requirement for eLRRon proteins in apical ECM organization is further supported by the eggshell defects observed after depletion of maternal egg-6 (Carvalho et al, 2011;Sonnichsen et al, 2005) (A. Singson, personal communication). In summary, eLRRon single mutants show penetrant and early, but relatively mild, defects in apical ECM…”

“…2B,C). egg-6 was independently identified and named based on its eggshell-defective RNAi phenotype (A. Singson, personal communication) (Carvalho et al, 2011;Sonnichsen et al, 2005). cs67 mutants had a C to T…”

A novel small molecule that disrupts a key event during the oocyte-to-embryo transition in C. elegans

“…S5A). This phenotype often occurs if eggshell assembly or function is defective (Carvalho et al, 2011;Edgar, 1995;Johnston et al, 2006;Olson et al, 2006;Parry et al, 2009). We therefore examined eggshell integrity using DAPI incorporation, which is normally excluded from live embryos (Rappleye et al, 1999;Fig.…”

Maternal MEMI Promotes Female Meiosis II in Response to Fertilization in Caenorhabditis elegans

“…The middle chitin layer is assembled after fertilization (Maruyama et al 2007), and it provides mechanical strength and a block to polyspermy (Johnston et al 2010). The inner proteoglycan layer is deposited during anaphase I, and then a distinct permeability barrier that involves fatty acid biosynthesis and modification enzymes (Tagawa et al 2001;Rappleye et al 2003;Benenati et al 2009;Carvalho et al 2011) forms during anaphase II (Olson et al 2012). Because loss of memi-1/2/3 function resulted in a skipped meiosis II, we reasoned that these embryos might exhibit defects in eggshell permeability.…”