Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Oligodendrocyte precursor cell specification is regulated by bidirectional neural progenitor–endothelial cell crosstalk

Abstract

Neural-derived signals are crucial regulators of CNS vascularization. However, whether the vasculature responds to these signals by means of elongating and branching or in addition by building a feedback response to modulate neurodevelopmental processes remains unknown. In this study, we identified bidirectional crosstalk between the neural and the vascular compartment of the developing CNS required for oligodendrocyte precursor cell specification. Mechanistically, we show that neural progenitor cells (NPCs) express angiopoietin-1 (Ang1) and that this expression is regulated by Sonic hedgehog. We demonstrate that NPC-derived Ang1 signals to its receptor, Tie2, on endothelial cells to induce the production of transforming growth factor beta 1 (TGFβ1). Endothelial-derived TGFβ1, in turn, acts as an angiocrine molecule and signals back to NPCs to induce their commitment toward oligodendrocyte precursor cells. This work demonstrates a true bidirectional collaboration between NPCs and the vasculature as a critical regulator of oligodendrogenesis.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Fig. 1: Ang1 is expressed in SC NPCs and regulates OPC specification.
Fig. 2: NPC-derived Ang1 regulates OPC specification.
Fig. 3: Exogenous Ang1 rescues OPC specification defects in Ang1 fl/flNestin:Cre and Ang1 fl/flOlig2:Cre embryos ex vivo.
Fig. 4: Reduced OPC specification in Ang1-deficient embryos cannot be explained by morphological vascular defects.
Fig. 5: Tie2 is exclusively expressed by blood vessels.
Fig. 6: Endothelial Tie2 signaling is required for OPC specification.
Fig. 7: EC-derived TGFβ1 is regulated by Tie2 signaling and required for proper OPC specification.

Data availability

The data that support the findings of this study are available in the manuscript or the Supplementary Information. All reagents and additional data from this study are available upon reasonable request from the corresponding author. Source data are provided with this paper.

Code availability

The modifications to the original code of Delile et al.18 are available upon reasonable request. Source data are provided with this paper.

References

  1. 1.

    Fancy, S. P., Chan, J. R., Baranzini, S. E., Franklin, R. J. & Rowitch, D. H. Myelin regeneration: a recapitulation of development? Annu Rev. Neurosci. 34, 21–43 (2011).

    CAS  PubMed  Article  Google Scholar 

  2. 2.

    Rowitch, D. H. & Kriegstein, A. R. Developmental genetics of vertebrate glial-cell specification. Nature 468, 214–222 (2010).

    CAS  PubMed  Article  Google Scholar 

  3. 3.

    Bergles, D. E. & Richardson, W. D. Oligodendrocyte development and plasticity. Cold Spring Harb. Perspect. Biol. 8, a020453 (2015).

    PubMed  Article  Google Scholar 

  4. 4.

    Yu, K., McGlynn, S. & Matise, M. P. Floor plate-derived Sonic hedgehog regulates glial and ependymal cell fates in the developing spinal cord. Development 140, 1594–1604 (2013).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  5. 5.

    Farreny, M. A. et al. FGF signaling controls Shh-dependent oligodendroglial fate specification in the ventral spinal cord. Neural Dev. 13, 3 (2018).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  6. 6.

    Dias, J. M., Alekseenko, Z., Applequist, J. M. & Ericson, J. Tgfβ signaling regulates temporal neurogenesis and potency of neural stem cells in the CNS. Neuron 84, 927–939 (2014).

    CAS  PubMed  Article  Google Scholar 

  7. 7.

    Dutta, D. J. et al. Combinatorial actions of Tgfβ and Activin ligands promote oligodendrocyte development and CNS myelination. Development 141, 2414–2428 (2014).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  8. 8.

    Rabadan, M. A. et al. Jagged2 controls the generation of motor neuron and oligodendrocyte progenitors in the ventral spinal cord. Cell Death Differ. 19, 209–219 (2012).

    CAS  PubMed  Article  Google Scholar 

  9. 9.

    Cui, X. Y. et al. NB-3/Notch1 pathway via Deltex1 promotes neural progenitor cell differentiation into oligodendrocytes. J. Biol. Chem. 279, 25858–25865 (2004).

    CAS  PubMed  Article  Google Scholar 

  10. 10.

    Paredes, I., Himmels, P. & Ruiz de Almodovar, C. Neurovascular communication during CNS development. Dev. Cell 45, 10–32 (2018).

    CAS  PubMed  Article  Google Scholar 

  11. 11.

    Himmels, P. et al. Motor neurons control blood vessel patterning in the developing spinal cord. Nat. Commun. 8, 14583 (2017).

    PubMed  PubMed Central  Article  Google Scholar 

  12. 12.

    Augustin, H. G. & Koh, G. Y. Organotypic vasculature: from descriptive heterogeneity to functional pathophysiology. Science 357, eeal2379 (2017).

    Article  CAS  Google Scholar 

  13. 13.

    Rafii, S., Butler, J. M. & Ding, B. S. Angiocrine functions of organ-specific endothelial cells. Nature 529, 316–325 (2016).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  14. 14.

    Ramasamy, S. K., Kusumbe, A. P. & Adams, R. H. Regulation of tissue morphogenesis by endothelial cell-derived signals. Trends Cell Biol. 25, 148–157 (2015).

    PubMed  Article  Google Scholar 

  15. 15.

    Lorenz, L. et al. Mechanosensing by β1 integrin induces angiocrine signals for liver growth and survival. Nature 562, 128–132 (2018).

    CAS  PubMed  Article  Google Scholar 

  16. 16.

    Hu, J. et al. Endothelial cell-derived angiopoietin-2 controls liver regeneration as a spatiotemporal rheostat. Science 343, 416–419 (2014).

    CAS  PubMed  Article  Google Scholar 

  17. 17.

    Augustin, H. G., Koh, G. Y., Thurston, G. & Alitalo, K. Control of vascular morphogenesis and homeostasis through the angiopoietin–Tie system. Nat. Rev. Mol. Cell Biol. 10, 165–177 (2009).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  18. 18.

    Delile, J. et al. Single cell transcriptomics reveals spatial and temporal dynamics of gene expression in the developing mouse spinal cord. Development 146, dev173807 (2019).

  19. 19.

    Tekki-Kessaris, N. et al. Hedgehog-dependent oligodendrocyte lineage specification in the telencephalon. Development 128, 2545–2554 (2001).

    CAS  PubMed  Google Scholar 

  20. 20.

    Li, Y. et al. Sonic hedgehog (Shh) regulates the expression of angiogenic growth factors in oxygen-glucose-deprived astrocytes by mediating the nuclear receptor NR2F2. Mol. Neurobiol. 47, 967–975 (2013).

    CAS  PubMed  Article  Google Scholar 

  21. 21.

    Pola, R. et al. The morphogen Sonic hedgehog is an indirect angiogenic agent upregulating two families of angiogenic growth factors. Nat. Med. 7, 706–711 (2001).

    CAS  PubMed  Article  Google Scholar 

  22. 22.

    Yam, P. T., Langlois, S. D., Morin, S. & Charron, F. Sonic hedgehog guides axons through a noncanonical, Src-family-kinase-dependent signaling pathway. Neuron 62, 349–362 (2009).

    CAS  PubMed  Article  Google Scholar 

  23. 23.

    Langlois, S. D., Morin, S., Yam, P. T. & Charron, F. Dissection and culture of commissural neurons from embryonic spinal cord. J. Vis. Exp. 39, 1773 (2010).

  24. 24.

    Agius, E. et al. Converse control of oligodendrocyte and astrocyte lineage development by Sonic hedgehog in the chick spinal cord. Dev. Biol. 270, 308–321 (2004).

    CAS  PubMed  Article  Google Scholar 

  25. 25.

    Touahri, Y. et al. Sulfatase 1 promotes the motor neuron-to-oligodendrocyte fate switch by activating Shh signaling in Olig2 progenitors of the embryonic ventral spinal cord. J. Neurosci. 32, 18018–18034 (2012).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  26. 26.

    Sato, T. N. et al. Distinct roles of the receptor tyrosine kinases Tie-1 and Tie-2 in blood vessel formation. Nature 376, 70–74 (1995).

    CAS  PubMed  Article  Google Scholar 

  27. 27.

    Jeansson, M. et al. Angiopoietin-1 is essential in mouse vasculature during development and in response to injury. J. Clin. Invest. 121, 2278–2289 (2011).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  28. 28.

    Kim, K. H., Nakaoka, Y., Augustin, H. G. & Koh, G. Y. Myocardial angiopoietin-1 controls atrial chamber morphogenesis by spatiotemporal degradation of cardiac jelly. Cell Rep. 23, 2455–2466 (2018).

    CAS  PubMed  Article  Google Scholar 

  29. 29.

    Tronche, F. et al. Disruption of the glucocorticoid receptor gene in the nervous system results in reduced anxiety. Nat. Genet. 23, 99–103 (1999).

    CAS  PubMed  Article  Google Scholar 

  30. 30.

    Pringle, N. P. & Richardson, W. D. A singularity of PDGF alpha-receptor expression in the dorsoventral axis of the neural tube may define the origin of the oligodendrocyte lineage. Development 117, 525–533 (1993).

    CAS  PubMed  Google Scholar 

  31. 31.

    Dessaud, E. et al. Interpretation of the Sonic hedgehog morphogen gradient by a temporal adaptation mechanism. Nature 450, 717–720 (2007).

    CAS  PubMed  Article  Google Scholar 

  32. 32.

    Simons, M. & Nave, K. A. Oligodendrocytes: myelination and axonal support. Cold Spring Harb. Perspect. Biol. 8, a020479 (2015).

    PubMed  Article  Google Scholar 

  33. 33.

    Motoike, T. et al. Universal GFP reporter for the study of vascular development. Genesis 28, 75–81 (2000).

    CAS  PubMed  Article  Google Scholar 

  34. 34.

    Savant, S. et al. The orphan receptor Tie1 controls angiogenesis and vascular remodeling by differentially regulating Tie2 in Tip and stalk cells. Cell Rep. 12, 1761–1773 (2015).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  35. 35.

    Chu, M. et al. Angiopoietin receptor Tie2 is required for vein specification and maintenance via regulating COUP-TFII. eLife 5, e21032 (2016).

  36. 36.

    Tsai, H. H. et al. Oligodendrocyte precursors migrate along vasculature in the developing nervous system. Science 351, 379–384 (2016).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  37. 37.

    Palazuelos, J., Klingener, M. & Aguirre, A. TGFβ signaling regulates the timing of CNS myelination by modulating oligodendrocyte progenitor cell cycle exit through SMAD3/4/FoxO1/Sp1. J. Neurosci. 34, 7917–7930 (2014).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  38. 38.

    Mecha, M. et al. Expression of TGF-βs in the embryonic nervous system: analysis of interbalance between isoforms. Dev. Dyn. 237, 1709–1717 (2008).

    CAS  PubMed  Article  Google Scholar 

  39. 39.

    Massague, J. TGFβ signalling in context. Nat. Rev. Mol. Cell Biol. 13, 616–630 (2012).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  40. 40.

    Marklund, U. et al. Domain-specific control of neurogenesis achieved through patterned regulation of Notch ligand expression. Development 137, 437–445 (2010).

    CAS  PubMed  Article  Google Scholar 

  41. 41.

    Barber, M. et al. Vascular-derived vegfa promotes cortical interneuron migration and proximity to the vasculature in the developing forebrain. Cereb. Cortex 28, 2577–2593 (2018).

    PubMed  PubMed Central  Article  Google Scholar 

  42. 42.

    Segarra, M., Aburto, M. R., Hefendehl, J. & Acker-Palmer, A. Neurovascular interactions in the nervous system. Annu. Rev. Cell Dev. Biol. 35, 615–635 (2019).

    CAS  PubMed  Article  Google Scholar 

  43. 43.

    Arita, Y. et al. Myocardium-derived angiopoietin-1 is essential for coronary vein formation in the developing heart. Nat. Commun. 5, 4552 (2014).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  44. 44.

    Kessaris, N. et al. Competing waves of oligodendrocytes in the forebrain and postnatal elimination of an embryonic lineage. Nat. Neurosci. 9, 173–179 (2006).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  45. 45.

    Yuen, T. J. et al. Oligodendrocyte-encoded HIF function couples postnatal myelination and white matter angiogenesis. Cell 158, 383–396 (2014).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  46. 46.

    Muzumdar, M. D., Tasic, B., Miyamichi, K., Li, L. & Luo, L. A global double-fluorescent Cre reporter mouse. Genesis 45, 593–605 (2007).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  47. 47.

    Claxton, S. et al. Efficient, inducible Cre-recombinase activation in vascular endothelium. Genesis 46, 74–80 (2008).

    CAS  PubMed  Article  Google Scholar 

  48. 48.

    Liu, H. The role of EphB/ephrinB in inflammation. PhD thesis, Universität Heidelberg https://archiv.ub.uni-heidelberg.de/volltextserver/15772/1/Hui%20Liu%20thesis%202611%20final.pdf (2013).

  49. 49.

    Hua, Z. L., Smallwood, P. M. & Nathans, J. Frizzled3 controls axonal development in distinct populations of cranial and spinal motor neurons. eLife 2, e01482 (2013).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank K. Armin-Nave and W. Möbius (Max Planck Institute of Experimental Medicine, Göttingen) for their expert advice during the study. We thank S. Quaggin (Northwestern University) for providing the Ang1 fl/fl mice; T.M. Jessell and T. Marquardt (University of Göttingen) for the Olig2:Cre mice; H. Monyer (DKFZ) for the Nestin:Cre mice; M. Fruttiger (University College London) for providing the Pdgfb:CreERT2 mice; and M. Hecker (Heidelberg University) for providing the Tie2Cre:ERT2 mice. We thank P. Carmeliet (VIB, KULeuven) and H. Marti (Heidelberg University) for providing the Shh and the Ang1 and Tie2 ISH probes, respectively. We also thank the Nikon Imaging Center at Heidelberg University for providing their imaging facility. We thank P. Himmels for her help with the mTmGNestin:Cre mice and the entire Ruiz de Almodovar’s group for helpful discussions. This work was supported by ERC-CoG (864875); by a grant of the Deutsche Forschungsgemeinschaft (DFG) from FOR2325 (‘Interactions at the neurovascular interface’ (to C.R.d.A.)); DFG grants from SFB1366 (‘Vascular control of organ function’, project number 39404578 (to H.G.A. and C.R.d.A.)); DFG grant of the SFB873 (‘Maintenance and differentiation of stem cells in health and disease’ (to C.R.d.A.)), DFG grant of the SFB1158 (‘From nociception to chronic pain: structure–function properties of neural pathways and their reorganization’ (to C.R.d.A.)), by funds from the Baden-Württemberg Stiftung special programme ‘Angioformatics single cell platform’ (to C.R.d.A and H.G.A), by the European Research Council 787181 ‘Angiomature’ (to H.G.A.), by the Hertie Foundation (medMS MyLab program, P1180016 (to L.S.)), by the National Multiple Sclerosis Society (FG-1902-33617 (to L.S.)) and by the National Institutes of Health (P30 DK114857 (PI-SEQ)). I.P. was supported by a PhD fellowship, Becas Chile (CONICYT).

Author information

Affiliations

Authors

Contributions

I.P. and C.R.d.A. designed the study and wrote the paper. C.R.d.A. supervised the study. I.P. performed the experimental work and most of the data collection and analysis. J.R.V., B.S., C.F.R. and J.D. performed experiments and participated in data analysis. H.A. and M.R. helped with the experiments and data analysis. E.G. helped by providing the Tie2 fl/flPdgfbCreERT2 mice. J.R.V. and G.S. performed bioinformatic analysis. I.P., C.R.d.A., J.R.V., B.S., C.F.R., J.D., L.S., H.G.A., E.G. and G.S. participated in data discussion and interpretation. All authors provided input and editing during manuscript preparation. C.R.d.A. acquired the funding.

Corresponding author

Correspondence to Carmen Ruiz de Almodóvar.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Peer review information Nature Neuroscience thanks Victoria Bautch, Benedikt Berninger, and Michelle Monje for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Ang1 is dynamically expressed in the embryonic developing SC and forebrain.

a, ISH for Ang1 in transverse sections from E10.5 to E13.5 in the anterior-posterior axis of the SC (cervical, thoracic and lumbar levels). Data is representative from at least 3 independent experiments. Scale bar 100μm. b, Combined Ang1 ISH (pseudo-coloured in red) with immunofluorescence for Isl1/2+ motor neurons (green). At E11.5 Ang1 is expressed only in a subset of brachial and lumbar motor neurons. At E12.5 and E13.5 Ang1 is expressed by a dorsal-thoracic MN population. Data is representative from at least 3 independent experiments. Scale bar 50μm (inset) and 100μm (upper panel). Open arrowhead points to Ang1 expression in NPCs. Filled arrowhead points to MN expressing Ang1. MN: motor neuron. c, Predicted expression of Ang1 in all neural progenitors between E9.5 to E13.5 using previously published single-cell RNAseq data18. Ang1 is continuously expressed in a group of ventral progenitor domains (pMN-p0) from E10.5 to E13.5, while its expression is continuously decreased in a group of dorsal progenitors (dp2-RP) from E9.5 to E13.5. A threshold considering that Ang1 was expressed if at least two cells of a particular domain express more than one Ang1 transcript was set to show Ang1 positive expression. d, Ang1 ISH in forebrain coronal hemi-section detected at E11.5 and E12.5. Green arrowhead points to Ang1 expression in the MGE. Data is representative from at least 3 independent experiments. Scale bar 100 μm. Cx: cortex. LGE: lateral ganglionic eminence. MGE: medial ganglionic eminence.

Extended Data Fig. 2 Shh-induced Ang1 is required for OPC specification.

a, Scheme showing the steps to obtain neural progenitor-derived neurospheres (NSPs) from a single-cell suspension collected from E11.5 SCs via ‘open book preparation’. After the second passage NSPs were stimulated for RNA extraction. b, Image of Secondary NSPs (NSP-2) used for all experiments. Data is representative from at least 3 independent experiments. Scale bar 100μm. c,d, NSPs (in first (NSP-1) or second passage (NSP-2)) express characteristic neural stem cell markers such as Sox2 and Nestin (c). NSPs express components of the Shh pathway (Shh, Ptch and Gli1), Ang1, but not its receptor Tie2, its co-receptor Tie1 nor the endothelial cell marker Cd31 (d). Data is representative from 3 independent experiments. e, Scheme illustrating the steps for preparation of flat-mount SC explants. The SC of E11.5 embryos was isolated by ‘open book preparation’. SC ‘open books’ were cultured for 24 h. The dashed line indicates the limit of the ventricular zone (VZ) and the mantle zone (MZ). V: ventral; D: dorsal; A: anterior; P: posterior. FP: floor plate. pMN: motor neuron progenitor domain. f, Example of 3D rendering of explants images (images are shown in 2D in main Fig. 1e). Scale bar 50μm. g, Transverse sections of wild-type SCs explants immunostained for Olig2 and Sox10 to detect OPCs in the VZ (open arrowheads) and MZ (filled arrowheads) after culturing with vehicle (control DMSO), Shh inhibitor Sant1 300 nM, and/or Ang1 300 ng/mL for 24 hours. Scale bar 100 µm. h,i, Relative expression of Ptch (h) and Ang1 (i) mRNA of control (vehicle) and Sant1 treated wild-type SC explants. Graphs show mean±SEM (Control n = 16, Sant1 n = 15 of 4 independent litters). Unpaired two-sided T-Test, Ptch ****p<0.0001, Ang1**p=0.0062. j, Quantification of the number of OPCs in the MZ of the different conditions shown in (g), normalized to control. Graph show mean±SEM (Control n = 12, Sant1 n = 11, Sant1+Ang1 n = 12, of 5 independent litters). One-way ANOVA, Control vs +Sant1 **p=0.0027; +Sant1 vs +Sant1+Ang1 *p=0.0155. Source data

Extended Data Fig. 3 Ang1 fl/flNestin:Cre embryos and SC gross morphology appear normal, but show OPC specification defects.

a, ISH for Cre recombinase in transverse sections of E11.0 and E12.5 Ang1 fl/fl and Ang1 fl/flNestin:Cre embryo showing Cre expression. Data is representative from 3 independent embryos per genotype, from 2 litters. Scale bar 100μm. b, Transverse section of mTmGNestin:Cre E11.5 reporter embryo, showing Nestin-driven recombination, and consequently GFP expression (green), in NPCs and differentiated cells. Tomato (red) is excluded from Nestin expressing tissue. Data is representative from 3 independent embryos from one litter. Scale bar 100 μm. c, ISH for Ang1 in transverse sections of E11.0 and E12.5 Ang1 fl/fl and Ang1 fl/flNestin:Cre embryo showing efficient deletion of Ang1 mRNA in Ang1 fl/flNestin:Cre embryos. Data is representative 3 independent embryos per genotype, from 2 litters. Scale bar 100μm. d, Relative Ang1 mRNA expression in the neural compartment (CD31- negative fraction from CD31+ MACS isolation) from Ang1 fl/fl and Ang1 fl/flNestin:Cre E12.5 SCs. Graph show mean±SEM (Ang1 fl/fl n = 17, Ang1 fl/flNestin:Cre n = 15, from 4 independent litters). Unpaired two-sided T-Test, ***p=0.0001. e, Images of Ang1 fl/fl (control) vs Ang1 fl/flNestin:Cre (CNS-Ang1 deleted) embryos from E11.5 to E13.5. Scale bar 1 mm. f, Transverse thick sections of Ang1 fl/fl and Ang1 fl/flNestin:Cre immunostained for Nestin to visualize neural progenitor projections. Scale bar 100μm. g,h, Analysis at brachial (g) and thoracic (h) levels of SC tissue total area. Graphs show mean±SEM (Ang1 fl/fl n=9, Ang1 fl/flNestin:Cre n=10). Unpaired two-sided T-Test. i, Images of transverse sections at thoracic level of Ang1 fl/fl and Ang1 fl/flNestin:Cre E12.5 embryos showing the pMN domain immunostained for Olig2 and Sox10. Bracket: pMN; Open arrowheads indicate OPCs in the pMN and filled arrowheads OPCs in the MZ. Scale bar 50 µm. j, Quantification of OPCs in the VZ and MZ of Ang1 fl/fl and Ang1 fl/flNestin:Cre, normalized to controls (Ang1fl/fl). k, Analysis of the number of Olig2+ progenitors in the pMN. l, Ratio of pMN Sox10+ cells within the total number of Olig2+ pMN progenitor cells. For j-l: Graphs show mean±SEM (Ang1 fl/fl n=7, Ang1 fl/flNestin:Cre n=8, of 3 independent litters). Unpaired two-sided T-test. j: VZ ***p=0.0006; MZ **p=0.0028; l: Sox10% * p=0.0180. m, ISH for Pdgfrα in 18 μm transverse sections of E12.5 Ang1 fl/fl and Ang1 fl/flNestin:Cre embryo at brachial and thoracic levels. Data is representative from Ang1 fl/fl n=7, Ang1 fl/flNestin:Cre n=8, from 3 independent litters. Arrowheads indicate Pdgfrα+ cells. Scale bar 100μm. n, Transverse sections at brachial level of Ang1 fl/fl and Ang1 fl/flNestin:Cre E16.5 embryos immunostained for Olig2. Scale bar 100μm. o, Quantification of Olig2+ OL lineage cells in E16.5 Ang1 fl/fl and Ang1 fl/flNestin:Cre embryos, normalized to control (Ang1 fl/fl) littermates. Graphs show mean±SEM (Ang1 fl/fl n=10, Ang1 fl/flNestin:Cre n=13, of 3 independent litters). Unpaired two-sided T-test. Source data

Extended Data Fig. 4 Ang1 fl/flOlig2:Cre embryos and SC gross morphology appear normal, but show OPC specification defects.

a, ISH for Cre recombinase in transverse sections of E12.5 Ang1 fl/fl and Ang1 fl/flOlig2:Cre embryo showing Cre expression only in the pMN domain. Data is representative from at least 3 independent embryos per genotype, from 2 litters. Scale bar 100μm. b, ISH for Ang1 (pseudo-coloured in red) combined with immunostaining for Olig2 and Isl1/2. Filled arrowheads point to the pMN and open arrowheads point to MNs in transverse sections of E11.5 Ang1 fl/fl and Ang1 fl/flOlig2:Cre embryos. Note the efficient deletion of Ang1 in the pMN and MNs of Ang1 fl/flOlig2:Cre embryos. Data is representative from at least 3 independent embryos per genotype, from 2 litters. Scale bar 50μm (inset) 100μm (upper panel). c, Images of Ang1 fl/fl (control) vs Ang1 fl/flOlig2:Cre embryos from E11.5 to E13.5. Scale bar 1 mm. d, Nissl staining in transverse sections of Ang1 fl/fl and Ang1 fl/flOlig2:Cre E12.5 embryos. Data is representative from at least 3 embryos per genotype, from 2 independent litters. Scale bar 100μm. e,f, Analysis at brachial (e) and thoracic (f) levels of SC total area. Graphs show mean±SEM (Ang1 fl/fl n=7, Ang1 fl/flOlig2:Cre n=14, from 3 independent litters). Unpaired two-sided T-Test. g, Images of transverse sections at thoracic level of Ang1 fl/fl (control) and Ang1 fl/flOlig2:Cre E12.5 embryos immunostained for Olig2 and Sox10. Bracket: pMN; Filled arrowhead point to MZ OPCs and open arrowheads to VZ OPCs. Scale bar 50μm. h, Quantification of OPCs in the VZ and MZ of Ang1 fl/fl and Ang1 fl/flOlig2:Cre, normalized to controls (Ang1fl/fl). i, Analysis of the number of Olig2+ progenitors in the pMN. j, Ratio of pMN Sox10+ cells within the total number of Olig2+ pMN progenitor cells. For h-j: Graphs show mean±SEM (Ang1 fl/fl n=10, Ang1 fl/flOlig2:Cre n=12, of 3 independent litters). Unpaired two-sided T-test. h: VZ *p=0.0110; MZ **p=0.0076; j: Sox10% **p=0.0033. k, ISH for Pdgfrα in 40 μm transverse sections of E12.5 Ang1 fl/fl and Ang1 fl/flOlig2:Cre embryo at brachial and thoracic levels. Data is representative from Ang1 fl/fl n=7 and Ang1 fl/flOlig2:Cre n=9, from 2 independent litters. Arrowheads indicate Pdgfrα positive cells. Scale bar 100μm. l, Transverse sections at brachial level of Ang1 fl/fl (control) and Ang1 fl/flOlig2:Cre E16.5 embryos immunostained for Olig2. Scale bar 100μm. m, Quantification of Olig2+ cells (OL lineage) in E16.5 Ang1 fl/fl and Ang1 fl/flOlig2:Cre embryos, normalized to control (Ang1 fl/fl) littermates. Graphs show mean±SEM (Ang1 fl/fl n=13, Ang1 fl/flOlig2:Cre n=8, of 3 independent litters). Unpaired two-sided T-test. Source data

Extended Data Fig. 5 Impaired OL differentiation and maturation in neural specific Ang1-deficient mice.

a, Transverse sections at brachial level of Ang1 fl/fl (control) and Ang1 fl/flNestin:Cre E16.5 embryos hybridized with an ISH probe for Plp. Arrowheads point to Plp+ pre-OLs lining the ventral funiculus. Scale bar 100μm. b, Quantification of Plp+ cell counts in E16.5 Ang1 fl/fl and Ang1 fl/flNestin:Cre embryos, normalized to control (Ang fl/fl) littermates. Graph show mean±SEM (Ang1 fl/fl n=7, Ang1 fl/flNestin:Cre n=7, of 3 independent litters). Unpaired two-sided T-test, *p=0.0413. c, Transverse sections at brachial level of Ang1 fl/fl (control) and Ang1 fl/flOlig2:Cre E16.5 embryos hybridized with an ISH probe for Plp. Arrowheads point to Plp+ pre-OLs lining the ventral funiculus. Scale bar 100μm. d, Quantification of Plp+ cell counts in E16.5 Ang1 fl/fl and Ang1 fl/flOlig2:Cre embryos, normalized to control (Ang fl/fl) littermates. Graph show mean±SEM (Ang1 fl/fl n=9, Ang1 fl/flOlig2:Cre n=6, of 2 independent litters). Unpaired two-sided T-test, ***p=0.0008. e, Transverse sections from Ang1 fl/fl and Ang1 fl/flNestin:Cre neonatal SC at postnatal day (P6) hybridized with an ISH probe for Plp. Blue arrowheads indicate white matter (WM). The rest of the SC is grey matter (GM). Scale bar 100μm. f-h, Plp+ density (percentage of the surface occupied by Plp signal) was measured in Ang1 fl/fl and Ang1 fl/flNestin:Cre in ventral white matter (WM) and shown as %Plp of ventral WM area (f). Plp+ density was also measured in the developing grey matter (GM) (g), and shown as %Plp of GM area, and in the entire SC shown as %Plp of total SC area (h). Graphs show mean±SEM (Ang1 fl/fl n=7, Ang1 fl/flOlig2:Cre n=5, of 2 independent litters). Unpaired two-sided T-test (WM *p=0.0359; GM *p=0.0333; SC **p=0.0064). i, Transverse sections from Ang1 fl/fl and Ang1 fl/flOlig2:Cre neonatal SC at postnatal day (P6) hybridized with an ISH probe for Plp. Blue arrowheads indicate white matter (WM). The rest of the SC is grey matter (GM). Scale bars 100μm. j–l, Plp+ density was measured in Ang1 fl/fl and Ang1 fl/flOlig2:Cre in ventral white matter (WM) and shown as %Plp of ventral WM area (j). Plp+ density was also measured in the developing grey matter (GM) (k), and shown as %Plp of GM area, and in the entire SC (l), and shown as %Plp of total SC area. Graphs show mean±SEM (Ang1 fl/fl n=6, Ang1 fl/flOlig2:Cre n=3, of 2 independent litters). Unpaired two-sided T-Test. m, 3D reconstruction (left panels) and serial slices spaced by approx. 5 μm distance (right panels) of ventral funiculus field of view showing MBP-myelinated NF+ axons of Ang1 fl/fl (upper panels) and Ang1 fl/flNestin:Cre (lower panels). Insets in dashed boxes in the right upper corner are shown for Slice 1 of both genotypes indicating an example of diameter measurements for NF+ axons (1-continuous line) and fibers NF+ axons + MBP+-myelin ring (2- dashed line). Dashed boxes in middle of the Slice 2 and 3 follow the same axon at increasing depths from Slice 1. Scale bar (left-3D reconstruction): 3 µm and right panels: 5 µm. n,o, Percentage of myelinated axons in a slice of field of view (n) and quantification of the total number of axons (o) in the ventral funiculus of Ang1 fl/fl and Ang1 fl/flNestin:Cre p6 SCs. Graphs show mean±SEM (Ang1 fl/fl n=5, Ang1 fl/flNestin:Cre n=4 pups, of 2 independent litters). Unpaired two-sided T-test. p, Analysis of MBP-based myelin thickness of NF+ axons from the ventral funiculus of Ang1 fl/fl and Ang1 fl/flNestin:Cre p6 SCs. Values were normalized to control (Ang1fl/fl) littermates. Graphs show mean±SEM (values for four images were averaged for total of Ang1 fl/fl n=5, Ang1 fl/fl Nestin:Cre n=4 pups, from 2 independent litters). Unpaired two-sided T-test, *p=0.0390. q, g-ratio measured in MBP-surrounded NF+ axons from the ventral funiculus classified in four bins of axon calibre of Ang1 fl/fl and Ang1 fl/flNestin:Cre P6 SCs. Graphs show mean±SEM (Bin 0.2–0.4: Ang1 fl/fl n=3, Ang1 fl/flNestin:Cre n=3 axons; Bin 0.4–0.6: Ang1 fl/fl n=53, Ang1 fl/flNestin:Cre n=44 axons; Bin 0.6–0.8: Ang1 fl/fl n=88, Ang1 fl/fl Nestin:Cre n=61 axons; Bin >0.8: Ang1 fl/fl n=58, Ang1 fl/fl Nestin:Cre n=63 axons). In total, Ang1 fl/fl =5 [total 191 axons], Ang1 fl/fl Nestin:Cre =4 [total:171 axons] pups, of 2 independent litters were analysed. Mann-Whitney two-sided U-test, ****p<0.0001. r, Analysis of MBP fluorescence intensity surrounding NF+ axons of a ventral funiculus field of view of Ang1 fl/fl and Ang1 fl/flNestin:Cre P6 SCs. Graphs show mean±SEM (Bin 0.2–0.4: Ang1 fl/fl n=58, Ang1 fl/fl Nestin:Cre n=60 axons; Bin 0.4–0.6: Ang1 fl/fl n=92, Ang1 fl/flNestin:Cre n=82 axons; Bin 0.6–0.8: Ang1 fl/fl n=55 Ang1 fl/fl Nestin:Cre n=39 axons; Bin >0.8: Ang1 fl/fl n=41, Ang1 fl/flNestin:Cre n=31 axons). In total Ang1 fl/fl =5 [246 total axons] and Ang1 fl/flNestin:Cre =4 [total 212 axons] pups, of 2 independent litters were analysed. Mann-Whitney two-sided U-test, Bin 0.2–0.4 *p=0.0367, Bin 0.4–0.6 **p=0.0021, Bin>0.8 *p=0.0319. Source data

Extended Data Fig. 6 NPC-derived Ang1 promotes OPC specification via a paracrine mechanism.

a,b, ISH for Shh in transverse sections of embryo SCs (a) and relative expression of Shh mRNA (b) in the neural compartment of E12.5 Ang1 fl/fl vs Ang1 fl/flNestin:Cre embryos. Graphs show mean±SEM (Ang1 fl/fl n=17, Ang1 fl/flNestin:Cre n=15, of 4 independent litters). Unpaired two-sided T-test. Scale bar 100μm (a). c,d, ISH for Shh in transverse sections of embryo SCs (c) and relative expression of Shh mRNA (d) in the neural compartment of E12.5 Ang1 fl/fl v/s Ang1 fl/flOlig2:Cre. Graphs show mean±SEM (Ang1 fl/fl n=19, Ang1 fl/flOlig2:Cre n=15, of 4 independent litters). Unpaired two-sided T-test. Scale bar 100μm (c). e, Representative images of neural-progenitors derived NSPs derived from E11.5 wild-type embryo SCs and cultured for 72 h in differentiation conditions and treated with vehicle (control), Ang1 (100 mg/mL) and/or Tie2/FC (2 µg/mL). In vitro cultures were immunostained for Olig2 (labelling neural progenitors and OL lineage cells) and PDFGRα (labelling committed OPCs, arrowheads). Scale bar 50μm. f, Quantification of Olig2+ cells per field of view (FOV) of NSP in vitro cultures treated as in (e), normalized to control conditions. g, Quantification of PDGFRα+ cells per field of view (FOV) of NSP in vitro cultures treated as in (e), normalized to control conditions. Graphs show mean±SEM (n= 6 (independent experiments) isolated and differentiated NSPs cell cultures), one-way ANOVA.

Extended Data Fig. 7 Tie2 fl/flPdgfb:CreERT2 embryos and SC gross morphology appear normal, but show OPC specification defects.

a, Images of E12.5 Tie2 fl/fl (control) and Tie2 fl/flPdgfb:CreERT2 embryos. Scale bar 1 mm. b, Tie2 fl/fl and Tie2 fl/flPdgfb:CreERT2 E12.5 transverse 40 μm sections stained with IsoB4 showing the SC. Scale bar 100μm. c, Blood vessel (BV) density quantification of Tie2 fl/fl (control) and Tie2 fl/flPdgfb:CreERT2 and normalized to control (Tie2 fl/fl) littermates (c). d, Analysis of SC total area in Tie2 fl/fl and Tie2 fl/flPdgfb:CreERT2 E12.5 embryos (d). For c,d: Graphs show mean±SEM (Tie2 fl/fl n=11, Tie2 fl/flPdgfb:CreERT2 n= 6, of 2 independent litters). Unpaired two-sided T-test. e, Images of thoracic transverse sections of Tie2 fl/fl and Tie2 fl/flPdgfb:CreERT2 E12.5 SCs. Left images: entire SC immunostained with Olig2. Right images: pMN domain immunostained for Olig2 and Sox10. Bracket: pMN; open arrowheads indicate OPCs in the pMN and filled arrowheads in the MZ. Scale bars 100μm (left panels) and 50 µm (right (pMN) panels). f, Quantification of OPCs in the VZ (Tie2 fl/fl n=8, Tie2 fl/flPdgfb:Cre n=4, of 2 independent litters) and MZ (Tie2 fl/fl n=11, Tie2 fl/flPdgfb:Cre n=6, of 2 independent litters) of Tie2 fl/fl and Tie2 fl/flPdgfb:CreERT2, normalized to controls (Tie2 fl/fl). Graphs show mean±SEM. Unpaired two-sided T-test (MZ *p=0.0338). g, Percentage of pMN Sox10+ cells within the total number of Olig2+ pMN progenitor cells. h, Analysis of Olig2+ progenitor number in the pMN. Graphs show mean±SEM. Unpaired two-sided T-test (Sox10% *p=0.0379). Graphs (g) and (h) show mean±SEM (Tie2 fl/fl n=8, Tie2 fl/flPdgfb:Cre n= 4, of 2 independent litters). Unpaired two-sided T-test. Source data

Extended Data Fig. 8 Cxcl12 and Tgfβ2 are unchanged in Ang1 fl/flNestin:Cre Tie2 fl/flPdgfb:CreERT2 embryo SCs.

a, Relative expression of Cxcl12 mRNA in CD31+ECs sorted from Ang1 fl/fl and Ang1 fl/flNestin:Cre E12.5 SCs. Graphs show mean±SEM (Ang1 fl/fl n=11, Ang1 fl/flNestin:Cre n=10, of 3 independent litters). Unpaired two-sided T-test. b, Relative expression of Cxcl12 mRNA in CD31+ECs sorted from Tie2 fl/fl and Tie2 fl/flPdgfb:CreERT2 SCs. Graphs show mean±SEM (Tie2 fl/fl n=4, Tie2 fl/flPdgfb:CreERT2 n=7, of 2 independent litters). Unpaired two-sided T-test. c,d, ISH for Tgfβ2 in transverse sections from E12.5 Ang1 fl/fl vs Ang1 fl/flNestin:Cre (c) and Ang1 fl/fl vs Ang1 fl/flOlig2:Cre (d) embryos. Scale bar 100μm. e, Relative expression of Tgfβ2 mRNA from CD31- negative selection (isolated neural compartment) from Ang1 fl/fl and Ang1 fl/flNestin:Cre E12.5 SCs. Graphs show mean±SEM (Ang1 fl/fl n=17, Ang1 fl/flNestin:Cre n=15, from 4 independent litters). Unpaired two-sided T-test. f, Relative expression of Tgfβ2 mRNA from CD31- negative selection (isolated neural compartment) from Tie2 fl/fl and Tie2 fl/flPdgfb:CreERT2 E12.5 SCs. Graphs show mean±SEM (Tie2 fl/fl n=11, Tie2 fl/fl Pdgfb:CreERT2 n=11, from 3 independent litters). Unpaired two-sided T-test. Source data

Extended Data Fig. 9 Model of bi-directional NPC-EC communication regulating OPC specification.

a, Step-by-step model of the identified NPC-EC bidirectional crosstalk required for OPC specification. 1 and 2. Floor plate/Notochord-derived Shh transcriptionally regulates Ang1 expression in NPCs. 3. NPC-derived Ang1 produced binds and activates Tie2 in ECs. 4 and 5. Tie2 activation triggers Tgfβ1 transcriptional upregulation in ECs. 6. TGFβ1, signals back to pMN NPCs inducing phosphorylation of its intracellular effector SMAD3. 7. NPCs are induced to specify their fate to OPCs.

Supplementary information

Supplementary Information

Supplementary Tables 1–14.

Reporting Summary

Source data

Source Data Fig. 1

Statistical Source Data

Source Data Fig. 2

Statistical Source Data

Source Data Fig. 3

Statistical Source Data

Source Data Fig. 4

Statistical Source Data

Source Data Fig. 5

Statistical Source Data

Source Data Fig. 7

Statistical Source Data

Source Data Extended Data Fig. 2

Statistical Source Data

Source Data Extended Data Fig. 3

Statistical Source Data

Source Data Extended Data Fig. 4

Statistical Source Data

Source Data Extended Data Fig. 5

Statistical Source Data

Source Data Extended Data Fig. 7

Statistical Source Data

Source Data Extended Data Fig. 8

Statistical Source Data

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Paredes, I., Vieira, J.R., Shah, B. et al. Oligodendrocyte precursor cell specification is regulated by bidirectional neural progenitor–endothelial cell crosstalk. Nat Neurosci (2021). https://doi.org/10.1038/s41593-020-00788-z

Download citation

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing