Age-dependent effects of secreted Semaphorins 3A, 3F, and 3E on developing hippocampal axons: in vitro effects and phenotype of Semaphorin 3A (-/-) mice.

We studied the role of Semaphorins in the formation of hippocampal connections at embryonic and early postnatal stages. We show that the embryonic entorhinal cortex has a repulsive effect on embryonic hippocampal axons that disappears gradually at postnatal stages. Such chemorepulsion is blocked by Neuropilin-1 and -2 blocking antibodies. However, at perinatal stages, the inner layers of the entorhinal cortex attract CA1 axons. At these stages, Sema3A and Sema3F bind commissural and entorhinal axons. Sema3A and Sema3F repel hippocampal axons at E14-P2, but not at E13. A similar spatiotemporal pattern of chemorepulsion is observed for Sema3A on entorhinal axons, in contrast to Sema3F, which repels these axons only at postnatal ages. Sema3E also repels hippocampal axons but exclusively at E14. We show that Sema3A and Sema3F can induce the collapse of hippocampal growth cones and that membrane-bound Sema3A and Sema3F can guide hippocampal axons in the stripe assay. In sema3A (-/-) mice, the entorhinohippocampal projection is largely normal although single axons innervate aberrantly the stratum radiatum and the hilus. Thus, the chemorepulsion evoked by Sema3A, Sema3E, and Sema3F is dynamically regulated in the developing hippocampal formation.

Diversity and specificity of actions of Slit2 proteolytic fragments in axon guidance.

The Slits are secreted proteins that bind to Robo receptors and play a role in axon guidance and neuronal migration. In vertebrates, Slit2 is a major chemorepellent for developing axons and is involved in the control of midline crossing. In vivo, Slit2 is cleaved into 140 kDa N-terminal (Slit2-N) and 55-60 kDa C-terminal (Slit2-C) fragments, although the uncleaved/full-length form can also be isolated from brain extract. We explored the functional activities of Slit2 fragments by engineering mutant and truncated versions of Slit2 representing the N-, C-, and full/uncleavable (Slit2-U) fragments. Only Slit2-N and Slit2-U bind the Robo proteins. We found that in collagen gel, olfactory bulb (OB) but not dorsal root ganglia (DRG) axons are repelled by Slit2-N and Slit2-U. Moreover, only Slit2-N membranes or purified protein-induced OB growth cones collapse. Finally, we found that only recombinant Slit2-N could induce branching of DRG axons and that this effect was antagonized by Slit2-U. Therefore, different axons have distinct responses to Slit2 fragments, and these proteins have different growth-promoting capacities.

Sensory axon response to substrate-bound Slit2 is modulated by laminin and cyclic GMP.

In vertebrates, Slit2 is a chemorepellent for some developing axons but stimulates axonal elongation and branching of sensory axons. In vivo, Slit2 is cleaved into 140-kDa N-terminal (Slit2-N) and 55- to 60-kDa C-terminal fragments, but the uncleaved/full-length form can also be isolated from brain extracts. As Slit2-N and full-length Slit2 bind tightly to cell membranes, we decided to explore the response of rat dorsal root ganglia (DRG) axons to substrate-bound Slit2 fragments in the stripe assay. Slit2 fragments were avoided by DRG axons when expressed on membranes or coated as stripes on laminin. However, when the Slit2 stripes were coated on fibronectin, DRG axons still avoided full-length Slit2 but grew preferentially on Slit2-N. DRG axon response to Slit2 fragments could be modulated by cGMP and by a laminin-1 peptide. These results strongly support the idea that extracellular matrix proteins modulate the response of growth cones to chemotropic molecules by modulating cyclic nucleotide levels.

Netrin-1-mediated axon outgrowth and cAMP production requires interaction with adenosine A2b receptor.

The netrins, a family of laminin-related secreted proteins, are critical in controlling axon elongation and pathfinding. The DCC (for deleted in colorectal cancer) protein was proposed as a receptor for netrin-1 in the light of many observations including the inhibition of netrin-1-mediated axon outgrowth and attraction in the presence of an anti-DCC antiserum, the similitude of nervous system defects in DCC and netrin-1 knockout mice and the results of receptor swapping experiments. Previous studies have failed to show a direct interaction of DCC with netrin-1 (ref. 10), suggesting the possibility of an additional receptor or co-receptor. Here we show that DCC interacts with the membrane-associated adenosine A2b receptor, a G-protein-coupled receptor that induces cAMP accumulation on binding adenosine. We show that A2b is actually a netrin-1 receptor and induces cAMP accumulation on binding netrin-1. Finally, we show that netrin-1-dependent outgrowth of dorsal spinal cord axons directly involves A2b. Together our results indicate that the growth-promoting function of netrin-1 may require a receptor complex containing DCC and A2b.

Slit2-Mediated chemorepulsion and collapse of developing forebrain axons.

Diffusible chemorepellents play a major role in guiding developing axons toward their correct targets by preventing them from entering or steering them away from certain regions. Genetic studies in Drosophila revealed a novel repulsive guidance system that prevents inappropriate axons from crossing the CNS midline; this repulsive system is mediated by the Roundabout (Robo) receptor and its secreted ligand Slit. In rodents, Robo and Slit are expressed in the spinal cord and Slit can repel spinal motor axons in vitro. Here, we extend these findings into higher brain centers by showing that Robo1 and Robo2, as well as Slit1 and Slit2, are often expressed in complementary patterns in the developing forebrain. Furthermore, we show that human Slit2 can repel olfactory and hippocampal axons and collapse their growth cones.

The mouse homeodomain protein OTX2 regulates NCAM promoter activity.

The homeodomain transcription factor OTX2 is involved in defining regional identities in developing rostral brain. It appears to participate in morphogenetic processes leading to the formation of boundaries and substrates for early axon growth, processes which are in the end largely based on site-specific expression of cell adhesion molecules. Here, we present evidence that a candidate target of OTX2 is the gene encoding the neural cell adhesion molecule, NCAM. When Otx2 is transfected into NIH3T3 cells, NCAM protein expression is upregulated. Moreover, while mock-transfected cells display only the 140 kDa-isoform of NCAM, Otx2 transfected cells express also the two other major isoforms (NCAM-120 and -180), in agreement with the presence of the corresponding transcripts in Northern blots. In addition, transient expression of Otx2 in COS7 cells is able to dramatically enhance the transcriptional activity of the NCAM promoter. Taken together, our results argue for a regulation of NCAM expression by OTX2.

A potential role for the OTX2 homeoprotein in creating early 'highways' for axon extension in the rostral brain.

Brain pattern formation starts with a subdivision of the neuroepithelium through site-specific expression of regulatory genes and, subsequently, the boundaries between presumptive neuromeres may provide a scaffold for early formation of axon tracts. In the mouse forebrain, the transcription factor OTX2 is strongly expressed at several such boundaries. Combining dye tracing and staining for OTX2 protein, we show that a number of early fibre tracts develop within stripes of OTX2 expression. To analyse a putative influence of OTX2 on the expression of molecules involved in neurite growth, we generated several clones of NIH3T3 cells stably expressing OTX2 protein at varying levels. As shown by immunoblotting, Otx2 transfection affects the expression of a variety of cell and substratum adhesion molecules, rendering the cells a favourable substratum in neurite outgrowth assays. Among the molecules upregulated with increasing levels of OTX2 are NCAM, tenascin-C and DSD-1-PG, which also in situ colocalize with zones of OTX2 expression at boundaries. These data suggest that Otx2 might be involved in defining local substrata for axon extension in the forebrain.