SummaryNetrins are secreted proteins that were first identified as guidance cues, directing cell and axon migration during neural development. Subsequent findings have demonstrated that netrins can influence the formation of multiple tissues, including the vasculature, lung, pancreas, muscle and mammary gland, by mediating cell migration, cell-cell interactions and cell-extracellular matrix adhesion. Recent evidence also implicates the ongoing expression of netrins and netrin receptors in the maintenance of cell-cell organisation in mature tissues. Here, we review the mechanisms involved in netrin signalling in vertebrate and invertebrate systems and discuss the functions of netrin signalling during the development of neural and non-neural tissues.Key words: DCC, Adhesion, Axon, Neogenin, Netrin, UNC5 IntroductionNetrins are a family of extracellular, laminin-related (see Glossary, Box 1) proteins that function as chemotropic guidance cues for migrating cells and axons during neural development. They act as chemoattractants for some cell types and chemorepellents for others. Loss-of-function mutations in netrin 1 or in certain netrin receptors are lethal in mice, highlighting the importance of netrin signalling during development. Insights into the functions of netrins have arisen from studies across a wide range of animal species, including invertebrates such as the nematode worm Caenorhabditis elegans and the fruit fly Drosophila melanogaster, non-mammalian vertebrates such as the frog Xenopus laevis, and mammals including rats, mice and humans.Since its discovery in the early 1990s, it is now becoming clear that the netrin gene family exhibits a rich biology, with significance beyond neural development, and contributes to the organisation of multiple tissues. Along with a number of other identified axon guidance cues (Hinck, 2004), secreted netrins influence organogenesis outside the central nervous system (CNS), directing cell migration and mediating cell-cell adhesion in the lung, pancreas, mammary gland, vasculature and muscle (Kang et al., 2004; Lejmi et al., 2008;Liu et al., 2004;Lu et al., 2004;Srinivasan et al., 2003;Yebra et al., 2003). Here, we discuss the cell biology of netrin and netrin receptor functions and review the downstream signal transduction mechanisms that they activate. We also provide an overview of netrin function during development, both within the nervous system and within other developing organs and tissues. Netrin family membersThe first reported member of the netrin family, uncoordinated-6 (UNC-6), was identified in a search for gene products that regulate neural development in C. elegans (Ishii et al., 1992). Netrins have since been identified and studied in multiple vertebrate and invertebrate species (Table 1), including X. laevis (de la Torre et al., 1997), D. melanogaster (Harris et al., 1996;Mitchell et al., 1996) and the sea anemone Nematostella vectensis (Matus et al., 2006), an animal that exhibits early hallmarks of the origins of bilateral symmetry. In mammals, three...

Netrin-1 regulates cell migration and adhesion during the development of the nervous system, vasculature, lung, pancreas, muscle, and mammary gland. It is also proposed to function as a dependence ligand that inhibits apoptosis; however, studies disagree regarding whether netrin-1 loss-of-function mice exhibit increased cell death. Furthermore, previously studied netrin-1 loss-of-function gene-trap mice express a netrin-1-β-galactosidase protein chimera with potential for toxic gain-of-function effects, as well as a small amount of wild-type netrin-1 protein. To unambiguously assess loss of function, we generated netrin-1 floxed and netrin-1 null mouse lines. Netrin-1(-/-) mice die earlier and exhibit more severe axon guidance defects than netrin-1 gene-trap mice, revealing that complete loss of function is more severe than previously reported. Netrin-1(-/-) embryos also exhibit increased expression of the netrin receptors DCC and neogenin that are proposed dependence receptors; however, increased apoptosis was not detected, inconsistent with netrin-1 being an essential dependence receptor ligand in the embryonic spinal cord.

Molecular cues, such as netrin 1, guide axons by influencing growth cone motility. Rho GTPases are a family of intracellular proteins that regulate the cytoskeleton, substrate adhesion and vesicle trafficking. Activation of the RhoA subfamily of Rho GTPases is essential for chemorepellent axon guidance; however, their role during axonal chemoattraction is unclear. Here, we show that netrin 1, through its receptor DCC, inhibits RhoA in embryonic spinal commissural neurons. To determine whether netrin 1-mediated chemoattraction requires Rho function, we inhibited Rho signaling and assayed axon outgrowth and turning towards netrin 1. Additionally, we examined two important mechanisms that influence the guidance of axons to netrin 1: substrate adhesion and transport of the netrin receptor DCC to the plasma membrane. We found that inhibiting Rho signaling increased plasma membrane DCC and adhesion to substrate-bound netrin 1, and also enhanced netrin 1-mediated axon outgrowth and chemoattractive axon turning. Conversely, overexpression of RhoA or constitutively active RhoA inhibited axonal responses to netrin 1. These findings provide evidence that Rho signaling reduces axonal chemoattraction to netrin 1 by limiting the amount of plasma membrane DCC at the growth cone, and suggest that netrin 1-mediated inhibition of RhoA activates a positive-feedback mechanism that facilitates chemoattraction to netrin 1. Notably, these findings also have relevance for CNS regeneration research. Inhibiting RhoA promotes axon regeneration by disrupting inhibitory responses to myelin and the glial scar. By contrast, we demonstrate that axon chemoattraction to netrin 1 is not only maintained but enhanced, suggesting that this might facilitate directing regenerating axons to appropriate targets.