Netrin 1 and Dcc signalling are required for confinement of central axons within the central nervous system

Abstract

The establishment of anatomically stereotyped axonal projections is fundamental to neuronal function. While most neurons project their axons within the central nervous system (CNS), only axons of centrally born motoneurons and peripherally born sensory neurons link the CNS and peripheral nervous system (PNS) together by navigating through specialized CNS/PNS transition zones. Such selective restriction is of importance because inappropriate CNS axonal exit could lead to loss of correct connectivity and also to gain of erroneous functions. However, to date, surprisingly little is known about the molecular-genetic mechanisms that regulate how central axons are confined within the CNS during development. Here, we show that netrin 1/Dcc/Unc5 chemotropism contributes to axonal confinement within the CNS. In both Ntn1 and Dcc mutant mouse embryos, some spinal interneuronal axons exit the CNS by traversing the CNS/PNS transition zones normally reserved for motor and sensory axons. We provide evidence that netrin 1 signalling preserves CNS/PNS axonal integrity in three ways: (1) netrin 1/Dcc ventral attraction diverts axons away from potential exit points; (2) a Dcc/Unc5c-dependent netrin 1 chemoinhibitory barrier in the dorsolateral spinal cord prevents interneurons from being close to the dorsal CNS/PNS transition zone; and (3) a netrin 1/Dcc-dependent, Unc5c-independent mechanism that actively prevents exit from the CNS. Together, these findings provide insights into the molecular mechanisms that maintain CNS/PNS integrity and, to the best of our knowledge, present the first evidence that chemotropic signalling regulates interneuronal CNS axonal confinement in vertebrates.

Commissural axonal corridors instruct neuronal migration in the mouse spinal cord

“…DCC is a receptor of the ligand Netrin-1, yet the commissural axonal and migration phenotypes in Ntn-1 and Dcc mutant embryos are similar but not identical 25 . Several plausible explanations could account for this: first, several Netrin-1 receptors are known to be expressed in the developing spinal cord; therefore, Netrin-1 is modulating its effect via receptors in addition to DCC [46][47][48][49] . Second, Draxin, which is expressed in the developing spinal cord, was recently shown to be an alternative ligand for DCC 50 .…”

“…Genotyped Robo3, Robo3;AtoZ þ and Boc mutant mouse embryos were obtained from Professor Jane Dodd (Columbia University, New York, USA) and Professor Paola Bovolenta (CBM, Universidad Autonoma de Madrid, Spain), respectively. Mouse embryos were staged and processed as described previously 24,46,57 . Briefly, for genotyping genomic PCR, in-situ hybridization and/or epifluorescence (GFP) were used.…”

“…The following previously described oligonucleotide primers were used in genotyping: GFP (GFP forward 5 0 -gca agc tga ccc tga agt tca-3 0 and GFP reverse 5 0 -aac tcc agc agg acc atg tga-3 0 ); b-galactosidase (LacZ-80 5 0 -gtc aat ccg ccg ttt gtt ccc acg g-3 0 and LacZ-81 5 0 -gcg tgt acc aca gcg gat ggt tcg g-3 0 ); DCC (DCC WT forward 5 0 -ggc cat tga ggt tcc ttt-3 0 , DCC WT reverse 5 0 -aag acg acc aca cgc gag-3 0 and DCC mutant 5 0 -tcc tcg tgc ttt acg gta tc-3 0 ); Neomyscin (Neo forward 5 0 -gga gag gct att cgg cta tga-3 0 and Neo reverse 5 0 -ctt tct cgg cag gag caa ggt-3 0 ). Ntn-1 Lcaz/LacZ embryos were genotyped by a vastly reduced level of Netrin-1 transcript detected by in-situ hybridization with the Netrin-1 exon 3-7 riboprobe (see below) 46 . Animal and experiments were performed according to institutional guidelines and national laws, and approved by the Ethical Committee for Animal Experiments for Northern Sweden.…”

“…Immunohistochemistry was performed as described previously 46,60 . Briefly, sectioned samples were blocked for 30 min at room temperature in blocking buffer (2.5% v/v heat-inactivated fetal bovine serum, in PBS-with or without 0.1% v/v Triton X-100).…”

Livin’ On The Edge: glia shape nervous system transition zones

“…To identify the molecular mechanisms that mediate this selective permeability at TZs, studies have targeted essential axon pathfinding pathways, including Netrin, Semaphorins and Plexins (Table 1) [29,31–35]. …”