We study polarity establishment of early Caenorhabditis elegans embryos utilizing a novel drug delivery assay system coupled with micromanipulation systems. The project involves injecting cytoskeleton directed inhibitors using carbon nanopipettes and subsequent direct fluorescence live imaging of their effects on the distribution of proteins implicated in the polarity establishment pathway.
Establishment of cell polarity in C. elegans embryos
The protein cues that define cellular polarity are well conserved between organisms and play important roles in cellular development, differentiation, and motility. The single-celled C. elegans embryo is well established as a model system for studying cell polarity. During the first cell cycle a remarkable reorganization of the cytoskeletal and cytoplasmic components occurs, culminating in an asymmetric first division yielding daughter cells with different sizes, cell cycle rates and developmental potential. The anterior and posterior poles of the embryo are demarcated by the PAR (partitioning defective) proteins; PAR-2, in conjunction with PAR-1, localizes to the posterior cortex, while PAR-6, in complex with PAR-3 and aPKC (atypical protein kinase C), localizes to the anterior half. In early single cell embryo development PAR-6 is found throughout the cortex and as normal development progresses, PAR-6 regresses to the anterior cortex and PAR-2 localizes to the posterior cortex. PAR-6 and PAR-2 localize in a mutually exclusive manner, and the initial pattern formation coincides with dramatic reorganization of the cytoskeleton suggesting a relationship between cell polarization and cytoskeletal components. It is widely accepted that actomyosin contractility plays a significant role in polarity establishment and there is evidence that microtubules can also direct polarity establishment in the early embryo.
Model of polarity establishment.
(A) Immediately following meiosis II, the embryo exhibits uniform cortical ruffling. (B) centrosomal cue causes local destabilization of the actomyosin network and the posterior end smooths. (C) the actomyosin network recedes from the posterior (removing the anterior PAR complex, not shown), microtubules polymerize facilitating weak PAR-2 binding and a local increase in the cytoplasmic concentration of PAR-2, promoting its loading at the cortex through a diffusion process. (D) The maternal pronucleus starts migrating and PAR-2 domain expands in the wake of the actomyosin network until pseudo cleavage. (E) Pseudo cleavage relaxes, pronuclei meet, and (F) move to the center of the embryo while microtubules form extensive cortical contacts. (G) anaphase and (H) two-cell embryo. Actin filaments and microtubules are depicted as green and black rods respectively, cytoplasmic PAR-2 as a red gradient, cortical PAR-2 as a red line, and centrosomes and pronuclei/nuclei as light grey spots.
Direct injection of embryos using carbon nanopipettes
A number of experimental approaches have been used to address the role of the cytoskeleton in C. elegans embryos polarity, including RNAi knockdown of individual proteins, genetic analyses, and treatment with chemical inhibitors. Each system has inherent limitations over the induced perturbations of the system. The use of chemical inhibitors for such studies has been difficult due to the robust, unyielding eggshell that surrounds the C. elegans embryo. Permeablilization of the eggshell for exposure to specific drugs has been difficult to reproducibly control in very early embryos, and often requires subsequent fixation and staining of the embryos to visualization the result. Because many of the cytoskeletal components are essential, RNAi knockdown and genetic mutation can reduce viability and/or result in sterility resulting in few embryos for analysis, and these may have only partial elimination of protein activity. Genetic mutants or RNAi knockdown of genes to produce C. elegans embryos with more permeable eggshells have also been useful for exposing embryos to drugs, but it would be more useful for studies of polarity to directly introduce inhibitors into very early one-cell embryos of any genotype. A technique that allows for precise temporal control over drug treatments, as well as the ability to saturate the system, would be conducive to parsing out the distinct proteins and pathways necessary for cellular polarity. We have developed a microinjection technique, using carbon nanopipettes (CNPs) to directly introduce small molecules into early embryos, coupled with live imaging to visualize the effects.
This direct injection approach has allowed us to examine cytoskeletal components of polarity establishment at an extremely early developmental stage and, subsequently, offer new insights into their role in embryo polarization. We have investigated the roles of microtubules and microfilaments by injecting nocodazole and Latrunculin A (LatA), respectively, into an early one-cell embryo following the completion of meiosis metaphase II, prior to polarity establishment. By live imaging we determined the consequences of such perturbation on the distribution of two critical polarity proteins, the posterior protein PAR-2 (PAR-2::mCherry) and the anterior protein PAR-6 (PAR-6::mCherry). These experiments allowed us to distinguish the roles of microfilaments and microtubules in defining the anteroposterior axis.
Perturbation of PAR-2 localization by latrunculin A treatment.
(A) Control injection of 3.75% DMSO (N = 10). (B) injection of 90 μM Latrunculin A (N = 8). For each panel, columns from left to right: DIC; NMY2::GFP, H2B::GFP and Par2::mCherry.
Injection of YOYO-1 into multi-cell embryos.
(A) Two-cell embryo in which the P1 blastomere has been injected with 1 μM YOYO-1. (B) Four-cell embryo in which the ABa blastomere has been injected with 1 μM YOYO-1. (C) L1 larvae resulting from injected embryo in panel (A).