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Growth cone response to the bifunctional guidance cue netrin-1 is regulated by the activity of intracellular signaling intermediates such as protein kinase C-alpha (PKCalpha) and adenylyl cyclase. Among the diverse cellular events these enzymes regulate is receptor trafficking. Netrin-1, itself, may govern the activity of these signaling intermediates, thereby regulating axonal responses to itself. Alternatively, other ligands, such as activators of G protein-coupled receptors, may regulate responses to netrin-1 by governing these signaling intermediates. Here, we investigate the mechanisms controlling activation of PKCalpha and the subsequent downstream regulation of cell surface UNC5A receptors. We report that activation of adenosine receptors by adenosine analogs, or activation of the putative netrin-1 receptor, the G protein-coupled receptor adenosine A2b receptor (A2bR) results in PKCalpha-dependent removal of UNC5A from the cell surface. This decrease in cell surface UNC5A reduces the number of growth cones that collapse in response to netrin-1 and converts repulsion to attraction. We show these A2bR-mediated alterations in axonal response are not because of netrin-1 because netrin-1 neither binds A2bR, as assayed by protein overlay, nor stimulates PKCalpha-dependent UNC5A surface loss. Our results demonstrate that netrin-1-independent A2bR signaling governs the responsiveness of a neuron to netrin-1 by regulating the levels of cell surface UNC5A receptor.

In addition to their role as chemorepellent netrin-1 receptors, UNC5 proteins may mediate cell death because they induce apoptosis in cultured cells. To test this in vivo, we generated Unc5a (formerly Unc5h1) knockout mice and found that this deletion decreased apoptosis and increased the number of neurons in the spinal cord. In contrast, loss of netrin-1 (Ntn1) did not affect the amount of apoptosis, suggesting that NTN1 is not required for neuronal apoptosis in vivo.

In vertebrates, the receptor families deleted in colorectal cancer (DCC) and UNC5 mediate responses to the bifunctional guidance cue netrin-1. DCC mediates attraction, whereas a complex of DCC and UNC5 mediates repulsion. Thus, a primary determinant of the responsiveness of an axon to netrin-1 is the presence or absence of UNC5 family members on the cell surface. Currently, little is known about the role of receptor trafficking in regulating neuronal responses to netrin-1. We show that protein interacting with C-kinase 1 (PICK1) recruits activated protein kinase Calpha (PKCalpha) to MycUNC5A at the plasma membrane, stimulating its endocytosis. We identify two PKCalpha phosphorylation sites at serines 408 and 587, as well as dileucine internalization motifs, which are required for this endocytosis. We find that PKCalpha-stimulated internalization of UNC5A alters the functional response of developing hippocampal axons to netrin-1, preventing UNC5A-mediated growth cone collapse and converting netrin-1-stimulated chemorepulsion to attraction. To address whether this conversion in axonal response occurs in neurons expressing endogenous levels of UNC5, we show that mouse cerebellar granule axons exhibit chemorepulsion in a netrin-1 gradient and that this chemorepulsion is converted to chemoattraction after PKCalpha activation. We demonstrate that this repulsion depends on UNC5A because Unc5a-/- axons are not repelled and show this conversion depends on PICK1 because PICK1-/- axons are not converted to chemoattraction after PKCalpha activation. Together, these data provide a potential mechanism to explain how developing neurons alter their responsiveness to netrin-1 at intermediate choice points as they navigate to their targets.

Development of many organs, including the mammary gland, involves ductal morphogenesis. Mammary ducts are bi-layered tubular structures comprising an outer layer of cap/myoepithelial cells (MECs) and an inner layer of luminal epithelial cells (LECs). Slit2 is expressed by cells in both layers, with secreted SLIT2 broadly distributed throughout the epithelial compartment. By contrast, Robo1 is expressed specifically by cap/MECs. Loss-of-function mutations in Slit2 and Robo1 yield similar phenotypes, characterized by disorganized end buds (EBs) reminiscent of those present in Ntn1(-/-) glands, suggesting that SLIT2 and NTN1 function in concert during mammary development. Analysis of Slit2(-/-);Ntn1(-/-) glands demonstrates an enhanced phenotype that extends through the ducts and is characterized by separated cell layers and occluded lumens. Aggregation assays show that Slit2(-/-);Ntn1(-/-) cells, in contrast to wild-type cells, do not form bi-layered organoids, a defect rescued by addition of SLIT2. NTN1 has no effect alone, but synergistically enhances this rescue. Thus, our data establish a novel role for SLIT2 as an adhesive cue, acting in parallel with NTN1 to generate cell boundaries along ducts during bi-layered tube formation.

The neuronal guidance molecule, Netrin-1, has been suggested to play a role in the adhesion and migration of the mammary gland epithelium. Human and mouse Cripto-1 induce proliferation, migration, invasion and colony formation by epithelial cells in 3D matrices. Here we investigate whether Netrin-1 affects these Cripto-1-dependent activities in mouse mammary epithelial cells. Overexpression of Cripto-1 in EpH4 and HC-11 cells (EpH4/Cripto-1 or HC-11/Cripto-1) was associated with low expression of Netrin-1 and increased expression of its receptor Neogenin compared to that of wild-type cells. No change was observed in the expression of the other Netrin-1 receptor, UNC5H1. Treating EpH4/Cripto-1 or HC-11/Cripto-1 mammary cells with exogenous soluble Netrin-1 resulted in increased expression of E-cadherin and UNC5H1, decreased expression of vimentin and decreased activation of Akt as determined by western blotting. Colony formation by Eph4/Cripto-1 cells in 3D gels was significantly reduced in proximity to a Netrin-1 source, and mammary glands of transgenic mice overexpressing human Cripto-1 showed altered ductal growth in proximity to implanted Netrin-1-releasing pellets. Terminal end buds in the treated transgenic mice mammary glands also showed increased expression of E-cadherin and UNC5H1 and decreased expression of active Akt determined by immunohistochemistry. Together, these results suggest that regulation of Netrin-1 expression is important in regulating Cripto-1-dependent invasion and migration of mammary epithelial cells.

Netrin-1 and its receptors play an essential role patterning the nervous system by guiding neurons and axons to their targets. To explore whether netrin-1 organizes nonneural tissues, we examined its role in mammary gland morphogenesis. Netrin-1 is expressed in prelumenal cells, and its receptor neogenin is expressed in a complementary pattern in adjacent cap cells of terminal end buds (TEBs). We discovered that loss of either gene results in disorganized TEBs characterized by exaggerated subcapsular spaces, breaks in basal lamina, dissociated cap cells, and an increased influx of cap cells into the prelumenal compartment. Cell aggregation assays demonstrate that neogenin mediates netrin-1-dependent cell clustering. Thus, netrin-1 appears to act locally through neogenin to stabilize the multipotent progenitor (cap) cell layer during mammary gland development. Our results suggest that netrin-1 and its receptor neogenin provide an adhesive, rather than a guidance, function during nonneural organogenesis.

The guidance of developing axons in the nervous system is mediated partly by diffusible chemoattractants secreted by axonal target cells. Netrins are chemoattractants for commissural axons in the vertebrate spinal cord, but the mechanisms through which they produce their effects are unknown. We show that Deleted in Colorectal Cancer (DCC), a transmembrane protein of the immunoglobulin superfamily, is expressed on spinal commissural axons and possesses netrin-1-binding activity. Moreover, an antibody to DCC selectively blocks the netrin-1-dependent outgrowth of commissural axons in vitro. These results indicate that DCC is a receptor or a component of a receptor that mediates the effects of netrin-1 on commissural axons, and they complement genetic evidence for interactions between DCC and netrin homologs in C. elegans and Drosophila.