Netrin-1 promotes naive pluripotency through Neo1 and Unc5b co-regulation of Wnt and MAPK signalling

Abstract

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...

The Netrin-1-Neogenin-1 signaling axis controls neuroblastoma cell migration via integrin-β1 and focal adhesion kinase activation

“…In this study, we found not only interaction between NEO1 and FAK but also the induction of FAK Y397 phosphorylation when cells are treated with exogenous NTN1. Importantly, our results are in line with recent data published by Huyghe et al, who indicated that NTN-1 signaling through NEO1 promotes FAK activation in mouse embryonic stem cells [16]. In addition, p-FAK Y397 inhibitor reduces cell migration of control cells to a similar extent when compared to NEO1 knock-down cells in a cell migration assay, indicating that FAK is downstream of the NEO1 signaling pathway.…”

“…The DCC/NEO1 receptors act as homodimers or form heterodimers with the UNC5 receptor family, sharing their binding to the Netrin ligands [15]. Netrin-1 (NTN1) is the best characterized Netrin ligand, and has been shown to interact with NEO1, leading to axonal guidance and cell migration, as well as cell-tocell adhesion and self reneval [11,16]. The binding between NTN1 and NEO1 involves FNIII domains 4 and 5 of NEO1 [17].…”

TSC1 binding to lysosomal PIPs is required for TSC complex translocation and mTORC1 regulation

“…For the generation of MEF lines stably expressing WT or mutant hTSC1, a modified sleeping-beauty-based vector, pITR-TTP2, was used. The original pITR-TTP vector, and the associated pCMV-Transposase vector, were a kind gift of Manuel Koch and Rolf Marschalek (described in (Huyghe et al, 2020;Kowarz et al, 2015;Wang et al, 2018)), in which the 3 0 multiple cloning site (MCS) was expanded by double-stranded oligo cloning to generate pITR-TTP2. The pITR-TTP2-hTSC1 WT and M1 mutant coding sequences were PCR amplified from pcDNA3-hTSC1 vectors and subcloned into pITR-TTP2.…”

Endothelial Unc5B controls blood-brain barrier integrity

“…Mechanistically, we identify the Unc5B intracellular UPA domain as a regulator of LRP6 interaction, suggesting that the UPA domain may induce LRP6 phosphorylation through recruitment of kinases or other mechanisms that remain to be determined. Recent studies in naïve pluripotent embryonic stem cells showed that Netrin1 binding to Unc5B induced FAK-mediated phosphorylation of GSK3α/β, a kinase implicated in LRP6 activation 38 , suggesting one possible mechanism. Finally, because Unc5B is expressed in arterial and capillary endothelium, but not in veins, it is likely to confer BBB integrity in a vessel-segment specific manner, underscoring heterogeneity of BBB regulation in different vascular segments 39 .…”