Netrin-1 is an evolutionarily conserved secreted extracellular matrix protein discovered using genetic and biochemical screens for its role in axon guidance at the central nervous system (CNS) midline1,2. Netrin-1 is expressed by cells localized at CNS midline, such as the floor plate in vertebrate embryos1,3. Growth cone turning assays and 3D gel diffusion assays showed that netrin-1 can attract commissural axons2,4–6. Loss-of-function experiments further demonstrated that commissural axon extension to the midline is severely impaired in absence of netrin-13,7–9. Together these data support a model in which commissural axons are attracted by a netrin-1 gradient diffusing from the midline. Here, we selectively ablated netrin-1 expression in floor plate cells using a Netrin-1 conditional mouse line. We found that hindbrain and spinal cord commissural axons develop normally in absence of floor plate-derived netrin-1. Furthermore, we show that netrin-1 is highly expressed by cells in the ventricular zone with the potential to release it at the pial surface where it binds to commissural axons. Importantly, netrin-1 deletion from the ventricular zone phenocopies commissural axon guidance defects previously described in Netrin-1 knockout mice. These results show that the classical textbook view that attraction of commissural axons is mediated by a gradient of floor plate-derived netrin-1 is inaccurate and that netrin-1 primarily acts locally by promoting growth cone adhesion.

In mouse embryonic stem cells (mESCs), chemical blockade of Gsk3α/β and Mek1/2 (2i) instructs a self-renewing ground state whose endogenous inducers are unknown. Here we show that the axon guidance cue Netrin-1 promotes naive pluripotency by triggering profound signalling, transcriptomic and epigenetic changes in mESCs. Furthermore, we demonstrate that Netrin-1 can substitute for blockade of Gsk3α/β and Mek1/2 to sustain self-renewal of mESCs in combination with leukaemia inhibitory factor and regulates the formation of the mouse pluripotent blastocyst. Mechanistically, we reveal how Netrin-1 and the balance of its receptors Neo1 and Unc5B co-regulate Wnt and MAPK pathways in both mouse and human ESCs. Netrin-1 induces Fak kinase to inactivate Gsk3α/β and stabilize β-catenin while increasing the phosphatase activity of a Ppp2r2c-containing Pp2a complex to reduce Erk1/2 activity. Collectively, this work identifies Netrin-1 as a regulator of pluripotency and reveals that it mediates different effects in mESCs depending on its receptor dosage, opening perspectives for balancing self-renewal and lineage commitment.Animal studies. Teratoma assays were performed with 7-week-old severe combined immunodeficient (SCID) male mice (CB17/SCID, Charles River). Ntn1 βgeo reporter and Netrin-1 conditional-KO mESCs were derived from C57/bl6 mixed-background pregnant females at 8-15 weeks of age. Blastocyst injections were done using BALB/cANRj embryos. Embryos were flushed with M2 medium (Sigma) and grown overnight in KSOM (Sigma) or sequential blast (Origio) medium. Genotyping of Ntn1 fl/fl embryos was performed as previously described 44 . X-gal was detected in blastocysts using secondary antibodies coupled with biotin, the Vectastain ABC kit and DAB (Vector System). Teratoma-formation assays were performed by injecting 1 × 10 6 mESCs in the testes of 7-week-old SCID mice. After 3-4 weeks, the mice were euthanized and lesions were surgically removed and fixed in formalin or in 4% paraformaldehyde for sections. For blastocyst injections, Ntn1 fl/fl , untreated or treated with TAM for 48 h, were injected into BALB/cANRj blastocysts. The day before injection, frozen BALB/cANRj morulas from Quickblasto (Janvier) were thawed according to the manufacturer's instructions and incubated overnight in KSOM medium (Millipore) at 37 °C with 5% CO 2 . Between 5 and 15 cells were injected into expanded blastocysts in M2 medium (Sigma) using standard blastocyst injection techniques. Blastocysts were then allowed to recover for a period of 1-3 h prior to being implanted into pseudopregnant females. All animal procedures were performed in accordance with institutional guidelines (French ceccapp project 01369.01).Cell culture. The following cell lines were used in the study. Cgr8 ES cells (ECACC 07032901) were provided by the B. Pain laboratory (SBRI, Bron, France). E14Tg2a ES cells (ATCC CRL 1821) were provided by the M. E. Torres Padilla laboratory (IES, Munchen, Germany). Ntn1 βgeo reporter 27 and Netrin-1 conditional-KO (Ntn1 fl/fl ) 44 mESC...