Eph receptors and their ephrin ligands are key conserved regulators of axon guidance and can function in a variety of signaling modes. Here we analyze the genetic and cellular requirements for Eph signaling in a Caenorhabditis elegans axon guidance choice point, the ventral guidance of axons in the amphid commissure. The C. elegans Eph receptor EFN-1 has both kinase-dependent and kinase-independent roles in amphid ventral guidance. Of the four C. elegans ephrins, we find that only EFN-1 has a major role in amphid axon ventral guidance, and signals in both a receptor kinase-dependent and kinase-independent manner. Analysis of EFN-1 and EFN-1 expression and tissue-specific requirements is consistent with a model in which VAB-1 acts in amphid neurons, interacting with EFN-1 expressed on surrounding cells. Unexpectedly, left-hand neurons are more strongly affected than right-hand neurons by loss of Eph signaling, indicating a previously undetected left-right asymmetry in the requirement for Eph signaling. By screening candidate genes involved in Eph signaling, we find that the Eph kinase-independent pathway involves the ABL-1 nonreceptor tyrosine kinase and possibly the phosphatidylinositol 3-kinase pathway. Overexpression of ABL-1 is sufficient to rescue EFN-1 ventral guidance defects cell autonomously. Our results reveal new aspects of Eph signaling in a single axon guidance decision in vivo.E PHRINS and their cell surface receptors, the Eph receptor tyrosine kinases (EphR), play critical roles in many axon guidance processes, including midline guidance and growth cone collapse (Drescher et al. 1995;Cowan et al. 2000). In contrast to long-range guidance cues, Eph signaling involves short-range interactions between a transmembrane receptor and transmembrane (ephrin-B) or GPI-linked (ephrin-A) ligands. Eph signaling is complex and multifunctional, capable of mediating both repulsion and attraction depending on ephrin concentration even in the same neurons (Hansen et al. 2004). Many of the signaling pathways downstream of Eph receptors and ephrins regulate cell movement or cell adhesion (Kullander and Klein 2002;Pasquale 2005).Because Eph receptors and ephrins are cell surface molecules, they can operate in a variety of signaling modes (Kullander and Klein 2002;Egea and Klein 2007;Pasquale 2008). Eph receptors can generate kinase-dependent "forward" signals, in which ligand binding triggers receptor dimerization, activating the intrinsic kinase activity of the receptor, and initiating responses in the receptor-expressing cell. Kinase-dependent forward Eph signaling contributes to many processes including retinotopic mapping (Hindges et al. 2002), axonal midline avoidance after crossing (Yokoyama et al. 2001), neural crest cell migration (Smith et al. 1997), and migration of neural progenitors (Catchpole and Henkemeyer 2011). This regulation of diverse developmental processes occurs in part via kinase-dependent interactions with downstream effectors including Src-family kinases (Zisch et al. 1998;Knoll and Dr...

The use of CRISPR-based experiments in an undergraduate course is appealing because of the ease of editing, and the relevance of CRISPR to current research. Before the COVID-19 pandemic, we developed an in-person lab for a high-enrollment course that allowed students to design and conduct CRISPR editing experiments in budding yeast, Saccharomyces cerevisiae .