The chemotropic guidance cue netrin-1 promotes neurite outgrowth through its receptor Deleted in Colorectal Cancer (DCC) via activation of Rac1. The guanine nucleotide exchange factor (GEF) linking netrin-1/DCC to Rac1 activation has not yet been identified. Here, we show that the RhoGEF Trio mediates Rac1 activation in netrin-1 signaling. We found that Trio interacts with the netrin-1 receptor DCC in mouse embryonic brains and that netrin-1-induced Rac1 activation in brain is impaired in the absence of Trio. Trio(-/-) cortical neurons fail to extend neurites in response to netrin-1, while they are able to respond to glutamate. Accordingly, netrin-1-induced commissural axon outgrowth is reduced in Trio(-/-) spinal cord explants, and the guidance of commissural axons toward the floor plate is affected by the absence of Trio. The anterior commissure is absent in Trio-null embryos, and netrin-1/DCC-dependent axonal projections that form the internal capsule and the corpus callosum are defective in the mutants. Taken together, these findings establish Trio as a GEF that mediates netrin-1 signaling in axon outgrowth and guidance through its ability to activate Rac1.

Netrin-1 acts as a chemoattractant molecule to guide commissural neurons (CN) toward the floor plate by interacting with the receptor deleted in colorectal cancer (DCC). The molecular mechanisms underlying Netrin-1–DCC signaling are still poorly characterized. Here, we show that DCC is phosphorylated in vivo on tyrosine residues in response to Netrin-1 stimulation of CN and that the Src family kinase inhibitors PP2 and SU6656 block both Netrin-1–dependent phosphorylation of DCC and axon outgrowth. PP2 also blocks the reorientation of Xenopus laevis retinal ganglion cells that occurs in response to Netrin-1, which suggests an essential role of the Src kinases in Netrin-1–dependent orientation. Fyn, but not Src, is able to phosphorylate the intracellular domain of DCC in vitro, and we demonstrate that Y1418 is crucial for DCC axon outgrowth function. Both DCC phosphorylation and Netrin-1–induced axon outgrowth are impaired in Fyn^−/− CN and spinal cord explants. We propose that DCC is regulated by tyrosine phosphorylation and that Fyn is essential for the response of axons to Netrin-1.

The chemotropic guidance cue netrin-1 mediates attraction of migrating axons during central nervous system development through the receptor Deleted in Colorectal Cancer (DCC). Downstream of netrin-1, activated Rho GTPases Rac1 and Cdc42 induce cytoskeletal rearrangements within the growth cone. The Rho guanine nucleotide exchange factor (GEF) Trio is essential for Rac1 activation downstream of netrin-1/DCC, but the molecular mechanisms governing Trio activity remain elusive. Here, we demonstrate that Trio is phosphorylated by Src family kinases in the embryonic rat cortex in response to netrin-1. In vitro, Trio was predominantly phosphorylated at Tyr²⁶²² by the Src kinase Fyn. Though the phospho-null mutant Trio^Y2622F retained GEF activity toward Rac1, its expression impaired netrin-1-induced Rac1 activation and DCC-mediated neurite outgrowth in N1E-115 neuroblastoma cells. Trio^Y2622F impaired netrin-1-induced axonal extension in cultured cortical neurons and was unable to colocalize with DCC in growth cones, in contrast to wild-type Trio. Furthermore, depletion of Trio in cortical neurons reduced the level of cell surface DCC in growth cones, which could be restored by expression of wild-type Trio but not Trio^Y2622F. Together, these findings demonstrate that Trio^Y2622 phosphorylation is essential for the regulation of the DCC/Trio signaling complex in cortical neurons during netrin-1-mediated axon outgrowth.

In the last decade, several mouse models for RhoA, Rac1, and Cdc42 have emerged and have contributed a great deal to understanding the precise functions of Rho GTPases at early stages of development. This review summarizes our current knowledge of various mouse models of tissue-specific ablation of Cdc42, Rac1, and RhoA with emphasis on early embryogenesis, epithelial and skin morphogenesis, tubulogenesis, development of the central nervous system, and limb development.

One of the fundamental steps during development of the nervous system is the formation of proper connections between neurons and their target cells—a process called neural wiring, failure of which causes neurological disorders ranging from autism to Down’s syndrome. Axons navigate through the complex environment of a developing embryo toward their targets, which can be far away from their cell bodies. Successful implementation of neuronal wiring, which is crucial for fulfillment of all behavioral functions, is achieved through an intimate interplay between axon guidance and neural activity. In this review, our focus will be on axon pathfinding and the implication of some of its downstream molecular components in neurological disorders. More precisely, we will talk about axon guidance and the molecules implicated in this process. After, we will briefly review the Rho family of small GTPases, their regulators, and their involvement in downstream signaling pathways of the axon guidance cues/receptor complexes. We will then proceed to the final and main part of this review, where we will thoroughly comment on the implication of the regulators for Rho GTPases—GEFs (Guanine nucleotide Exchange Factors) and GAPs (GTPase-activating Proteins)—in neurological diseases and disorders.