Skip to main content
SpringerLink
Log in

DLK1 Promotes Neurogenesis of Human and Mouse Pluripotent Stem Cell-Derived Neural Progenitors Via Modulating Notch and BMP Signalling

  • Published:
Stem Cell Reviews and Reports Aims and scope Submit manuscript

Abstract

A better understanding of the control of stem cell maintenance and differentiation fate choice is fundamental to effectively realising the potential of human pluripotent stem cells in disease modelling, drug screening and cell therapy. Dlk1 is a Notch related transmembrane protein that has been reportedly expressed in several neurogenic regions in the developing brain. In this study, we investigated the ability of Dlk1 in modulating the maintenance and differentiation of human and mouse ESC-derived neural progenitors. We found that DLK1, either employed as an extrinsic factor, or via transgene expression, consistently promoted the generation of neurons in both the mouse and human ESC-derived neural progenitors. DLK1 exerts this function by inducing cell cycle exit of the progenitors, as evidenced by an increase in the number of young neurons retaining BrdU labelling and cells expressing the cycling inhibitor P57Kip2. DLK1 antagonised the cell proliferation activity of Notch ligands Delta 1 and Jagged and inhibited Hes1-mediated Notch signaling as demonstrated by a luciferase reporter assay. Interestingly, we found that DLK1 promotes the neurogenic potential of human neural progenitor cells via suppression of Smad activation when they are challenged with BMP. Together, our data demonstrate for the first time a regulatory role for DLK1 in human and mouse neural progenitor differentiation and establish an interaction between DLK1 and Hes1-mediated Notch signaling in these cells. Furthermore, this study identifies DLK1 as a novel modulator of BMP/Smad signalling.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  1. Bachmann, E., Krogh, T. N., Hojrup, P., Skjodt, K., & Teisner, B. (1996). Mouse fetal antigen 1 (mFA1), the circulating gene product of mdlk, pref-1 and SCP-1: isolation, characterization and biology. Journal of Reproduction and Fertility, 107, 279–285.

    Article  PubMed  CAS  Google Scholar 

  2. Baladron, V., Ruiz-Hidalgo, M. J., Nueda, M. L., Diaz-Guerra, M. J., Garcia-Ramirez, J. J., Bonvini, E., et al. (2005). dlk acts as a negative regulator of Notch1 activation through interactions with specific EGF-like repeats. Experimental Cell Research, 303, 343–359.

    Article  PubMed  CAS  Google Scholar 

  3. Bauer, M., Szulc, J., Meyer, M., Jensen, C. H., Terki, T. A., Meixner, A., et al. (2008). Delta-like 1 participates in the specification of ventral midbrain progenitor derived dopaminergic neurons. Journal of Neurochemistry, 104, 1101–1115.

    Article  PubMed  CAS  Google Scholar 

  4. Bolos, V., Grego-Bessa, J., & de la Pompa, J. L. (2007). Notch signaling in development and cancer. Endocrine Reviews, 28, 339–363.

    Article  PubMed  CAS  Google Scholar 

  5. Bonaguidi, M. A., McGuire, T., Hu, M., Kan, L., Samanta, J., & Kessler, J. A. (2005). LIF and BMP signaling generate separate and discrete types of GFAP-expressing cells. Development, 132, 5503–5514.

    Article  PubMed  CAS  Google Scholar 

  6. Borghese, L., Dolezalova, D., Opitz, T., Haupt, S., Leinhaas, A., Steinfarz, B., et al. (2010). Inhibition of notch signaling in human embryonic stem cell-derived neural stem cells delays G1/S phase transition and accelerates neuronal differentiation in vitro and in vivo. Stem Cells, 28, 955–964.

    Article  PubMed  CAS  Google Scholar 

  7. Chambers, S. M., Fasano, C. A., Papapetrou, E. P., Tomishima, M., Sadelain, M., & Studer, L. (2009). Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling. Nature Biotechnology, 27, 275–280.

    Article  PubMed  CAS  Google Scholar 

  8. Chellappan, S. P., Giordano, A., & Fisher, P. B. (1998). Role of cyclin-dependent kinases and their inhibitors in cellular differentiation and development. Current Topics in Microbiology and Immunology, 227, 57–103.

    Article  PubMed  CAS  Google Scholar 

  9. Chiba, S. (2006). Notch signaling in stem cell systems. Stem Cells, 24, 2437–2447.

    Article  PubMed  CAS  Google Scholar 

  10. Christophersen, N. S., Gronborg, M., Petersen, T. N., Fjord-Larsen, L., Jorgensen, J. R., Juliusson, B., et al. (2007). Midbrain expression of Delta-like 1 homologue is regulated by GDNF and is associated with dopaminergic differentiation. Experimental Neurology, 204, 791–801.

    Article  PubMed  CAS  Google Scholar 

  11. da Rocha, S. T., Tevendale, M., Knowles, E., Takada, S., Watkins, M., & Ferguson-Smith, A. C. (2007). Restricted co-expression of Dlk1 and the reciprocally imprinted non-coding RNA, Gtl2: implications for cis-acting control. Developmental Biology, 306, 810–823.

    Article  PubMed  Google Scholar 

  12. Desai, A. R., & McConnell, S. K. (2000). Progressive restriction in fate potential by neural progenitors during cerebral cortical development. Development, 127, 2863–2872.

    PubMed  CAS  Google Scholar 

  13. Elkabetz, Y., Panagiotakos, G., Al Shamy, G., Socci, N. D., Tabar, V., & Studer, L. (2008). Human ES cell-derived neural rosettes reveal a functionally distinct early neural stem cell stage. Genes & Development, 22, 152–165.

    Article  CAS  Google Scholar 

  14. Gearhart, J. (1998). New potential for human embryonic stem cells. Science, 282, 1061–1062.

    Article  PubMed  CAS  Google Scholar 

  15. Gerrard, L., Rodgers, L., & Cui, W. (2005). Differentiation of human embryonic stem cells to neural lineages in adherent culture by blocking bone morphogenetic protein signaling. Stem Cells, 23, 1234–1241.

    Article  PubMed  CAS  Google Scholar 

  16. Goffredo, D., Conti, L., Di Febo, F., Biella, G., Tosoni, A., Vago, G., et al. (2008). Setting the conditions for efficient, robust and reproducible generation of functionally active neurons from adult subventricular zone-derived neural stem cells. Cell Death and Differentiation, 15, 1847–1856.

    Article  PubMed  CAS  Google Scholar 

  17. Gross, R. E., Mehler, M. F., Mabie, P. C., Zang, Z., Santschi, L., & Kessler, J. A. (1996). Bone morphogenetic proteins promote astroglial lineage commitment by mammalian subventricular zone progenitor cells. Neuron, 17, 595–606.

    Article  PubMed  CAS  Google Scholar 

  18. Ishibashi, M., Moriyoshi, K., Sasai, Y., Shiota, K., Nahanishi, S., & Kageyama, R. (1994). Persistent expression of helix-loop-helix factor HES-1 prevents mammalian neural differentiation in the central nervous system. The EMBO Journal, 13, 1799–1805.

    PubMed  CAS  Google Scholar 

  19. Jacobs, F. M., van der Linden, A. J., Wang, Y., von Oerthel, L., Sul, H. S., Burbach, J. P., et al. (2009). Identification of Dlk1, Ptpru and Klhl1 as novel Nurr1 target genes in meso-diencephalic dopamine neurons. Development, 136, 2363–2373.

    Article  PubMed  CAS  Google Scholar 

  20. Jensen, C. H., Krogh, T. N., Hojrup, P., Clausen, P. P., Skjodt, K., Larsson, L. I., et al. (1994). Protein structure of fetal antigen 1 (FA1). A novel circulating human epidermal-growth-factor-like protein expressed in neuroendocrine tumors and its relation to the gene products of dlk and pG2. European Journal of Biochemistry, 225, 83–92.

    Article  PubMed  CAS  Google Scholar 

  21. Jensen, C. H., Meyer, M., Schroder, H. D., Kliem, A., Zimmer, J., & Teisner, B. (2001). Neurons in the monoaminergic nuclei of the rat and human central nervous system express FA1/dlk. Neuroreport, 12, 3959–3963.

    Article  PubMed  CAS  Google Scholar 

  22. Kaneta, M., Osawa, M., Sudo, K., Nakauchi, H., Farr, A. G., & Takahama, Y. (2000). A role for pref-1 and HES-1 in thymocyte development. The Journal of Immunology, 164, 256–264.

    PubMed  CAS  Google Scholar 

  23. Kasai, M., Satoh, K., & Akiyama, T. (2005). Wnt signaling regulates the sequential onset of neurogenesis and gliogenesis via induction of BMPs. Genes to Cells, 10, 777–783.

    Article  PubMed  CAS  Google Scholar 

  24. Konstantoulas, C. J., Parmar, M., & Li, M. (2010). FoxP1 promotes midbrain identity in embryonic stem cell-derived dopamine neurons by regulating Pitx3. Journal of Neurochemistry, 113, 836–847.

    Article  PubMed  CAS  Google Scholar 

  25. Laborda, J., Sausville, E. A., Hoffman, T., & Notario, V. (1993). dlk, a putative mammalian homeotic gene differentially expressed in small cell lung carcinoma and neuroendocrine tumor cell line. The Journal of Biological Chemistry, 268, 3817–3820.

    PubMed  CAS  Google Scholar 

  26. Mabie, P. C., Mehler, M. F., & Kessler, J. A. (1999). Multiple roles of bone morphogenetic protein signaling in the regulation of cortical cell number and phenotype. The Journal of Neuroscience, 19, 7077–7088.

    PubMed  CAS  Google Scholar 

  27. Masamizu, Y., Ohtsuka, T., Takashima, Y., Nagahara, H., Takenaka, Y., Yoshikawa, K., et al. (2006). Real-time imaging of the somite segmentation clock: revelation of unstable oscillators in the individual presomitic mesoderm cells. Proceedings of the National Academy of Sciences of the United States of America, 103, 1313–1318.

    Article  PubMed  CAS  Google Scholar 

  28. Mathieu, C., Sii-Felice, K., Fouchet, P., Etienne, O., Haton, C., Mabondzo, A., et al. (2008). Endothelial cell-derived bone morphogenetic proteins control proliferation of neural stem/progenitor cells. Molecular and Cellular Neuroscience, 38, 569–577.

    Article  PubMed  CAS  Google Scholar 

  29. Miller, F. D., & Gauthier, A. S. (2007). Timing is everything: making neurons versus glia in the developing cortex. Neuron, 54, 357–369.

    Article  PubMed  CAS  Google Scholar 

  30. Nakamura, Y., Sakakibara, S., Miyata, T., Ogawa, M., Shimazaki, T., Weiss, S., et al. (2000). The bHLH gene hes1 as a repressor of the neuronal commitment of CNS stem cells. The Journal of Neuroscience, 20, 283–293.

    PubMed  CAS  Google Scholar 

  31. Nakashima, K., Takizawa, T., Ochiai, W., Yanagisawa, M., Hisatsune, T., Nakafuku, M., et al. (2001). BMP2-mediated alteration in the developmental pathway of fetal mouse brain cells from neurogenesis to astrocytogenesis. Proceedings of the National Academy of Sciences of the United States of America, 98, 5868–5873.

    Article  PubMed  CAS  Google Scholar 

  32. Nakashima, K., Wiese, S., Yanagisawa, M., Arakawa, H., Kimura, N., Hisatsune, T., et al. (1999). Developmental requirement of gp130 signaling in neuronal survival and astrocyte differentiation. J Neurosci., 19, 5429–5434.

    PubMed  CAS  Google Scholar 

  33. Nakashima, K., Yanagisawa, M., Arakawa, H., Kimura, N., Hisatsune, T., Kawabata, M., et al. (1999). Synergistic signaling in fetal brain by STAT3-Smad1 complex bridged by p300. Science, 284, 479–482.

    Article  PubMed  CAS  Google Scholar 

  34. Nueda, M. L., Baladron, V., Sanchez-Solana, B., Ballesteros, M. A., & Laborda, J. (2007). The EGF-like protein dlk1 inhibits notch signaling and potentiates adipogenesis of mesenchymal cells. Journal of Molecular Biology, 367, 1281–1293.

    Article  PubMed  CAS  Google Scholar 

  35. Ohno, N., Izawa, A., Hattori, M., Kageyama, R., & Sudo, T. (2001). dlk inhibits stem cell factor-induced colony formation of murine hematopoietic progenitors: Hes-1-independent effect. Stem Cells, 19, 71–79.

    Article  PubMed  CAS  Google Scholar 

  36. Park, I. H., Arora, N., Huo, H., Maherali, N., Ahfeldt, T., Shimamura, A., et al. (2008). Disease-specific induced pluripotent stem cells. Cell, 134, 877–886.

    Article  PubMed  CAS  Google Scholar 

  37. Pollard, S., Conti, L., & Smith, A. (2006). Exploitation of adherent neural stem cells in basic and applied neurobiology. Regenerative Medicine, 1, 111–118.

    Article  PubMed  CAS  Google Scholar 

  38. Qian, X., Shen, Q., Goderie, S. K., He, W., Capela, A., Davis, A. A., et al. (2000). Timing of CNS cell generation: a programmed sequence of neuron and glial cell production from isolated murine cortical stem cells. Neuron, 28, 69–80.

    Article  PubMed  CAS  Google Scholar 

  39. Schmidt, J. V., Matteson, P. G., Jones, B. K., Guan, X. J., & Tilghman, S. M. (2000). The Dlk1 and Gtl2 genes are linked and reciprocally imprinted. Genes & Development, 14, 1997–2002.

    CAS  Google Scholar 

  40. Sherr, C. J., & Roberts, J. M. (1999). CDK inhibitors: positive and negative regulators of G1-phase progression. Genes & Development, 13, 1501–1512.

    Article  CAS  Google Scholar 

  41. Shi, Y., & Massague, J. (2003). Mechanisms of TGF-beta signaling from cell membrane to the nucleus. Cell, 113, 685–700.

    Article  PubMed  CAS  Google Scholar 

  42. Smas, C. M., Chen, L., & Sul, H. S. (1997). Cleavage of membrane-associated pref-1 generates a soluble inhibitor of adipocyte differentiation. Molecular and Cellular Biology, 17, 977–988.

    PubMed  CAS  Google Scholar 

  43. Smas, C. M., & Sul, H. S. (1993). Pref-1, a protein containing EGF-like repeats, inhibits adipocyte differentiation. Cell, 73, 725–734.

    Article  PubMed  CAS  Google Scholar 

  44. Sul, H. S. (2009). Minireview: Pref-1: role in adipogenesis and mesenchymal cell fate. Molecular Endocrinology, 23, 1717–1725.

    Article  PubMed  CAS  Google Scholar 

  45. Sun, Y., Pollard, S., Conti, L., Toselli, M., Biella, G., Parkin, G., et al. (2008). Long-term tripotent differentiation capacity of human neural stem (NS) cells in adherent culture. Mol Cell Neurosci., 38, 245–258.

    Article  PubMed  CAS  Google Scholar 

  46. Takada, S., Paulsen, M., Tevendale, M., Tsai, C. E., Kelsey, G., Cattanach, B. M., et al. (2002). Epigenetic analysis of the Dlk1-Gtl2 imprinted domain on mouse chromosome 12: implications for imprinting control from comparison with Igf2-H19. Human Molecular Genetics, 11, 77–86.

    Article  PubMed  CAS  Google Scholar 

  47. Tanigaki, K., Nogaki, F., Takahashi, J., Tashiro, K., Kurooka, H., & Honjo, T. (2001). Notch1 and Notch3 Instructively Restrict bFGF-Responsive Multipotent Neural Progenitor Cells to an Astroglial Fate. Neuron, 29, 45–55.

    Article  PubMed  CAS  Google Scholar 

  48. Thomson, J. A., Itskovitz-Eldor, J., Shapiro, S. S., Waknitz, M. A., Swiergiel, J. J., Marshall, V. S., et al. (1998). Embryonic stem cell lines derived from human blastocysts. Science, 282, 1145–1147.

    Article  PubMed  CAS  Google Scholar 

  49. Wernig, M., Zhao, J. P., Pruszak, J., Hedlund, E., Fu, D., Soldner, F., et al. (2008). Neurons derived from reprogrammed fibroblasts functionally integrate into the fetal brain and improve symptoms of rats with Parkinson’s disease. Proceedings of the National Academy of Sciences of the United States of America, 105, 5856–5861.

    Article  PubMed  CAS  Google Scholar 

  50. Wylie, A. A., Murphy, S. K., Orton, T. C., & Jirtle, R. L. (2000). Novel imprinted DLK1/GTL2 domain on human chromosome 14 contains motifs that mimic those implicated in IGF2/H19 regulation. Genome Research, 10, 1711–1718.

    Article  PubMed  CAS  Google Scholar 

  51. Ying, Q. L., Stavridis, M., Griffiths, D., Li, M., & Smith, A. (2003). Conversion of embryonic stem cells into neuroectodermal precursors in adherent monoculture. Nature Biotechnology, 21, 183–186.

    Article  PubMed  CAS  Google Scholar 

  52. Yoon, K., & Gaiano, N. (2005). Notch signaling in the mammalian central nervous system: insights from mouse mutants. Nature Neuroscience, 8, 709–715.

    Article  PubMed  CAS  Google Scholar 

  53. Zhao, S., Nichols, J., Smith, A. G., & Li, M. (2004). SoxB transcription factors specify neuroectodermal lineage choice in ES cells. Molecular and Cellular Neuroscience, 27, 332–342.

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgement

We thank Drs. Sudo for pMIGhDLK1 and Kageyama for Hes1-luc reporter plasmids. This work was supported by EU (LSHG-CT-20060018739. ESTOOLS; FP7-HEALTH-2007-B-22943-NeurStemcell), Parkinson’s UK (4069) and the UK Medical Research Council. BS and PN were funded by a PhD studentship from the SPRING/Parkinson’s UK (Ref 5001) and Thai Government, respectively.

Conflicts of Interest

The authors declare no potential conflicts of interest.

Author information

Author notes

  1. Parinya Noisa

    Present address: Embryo Technology and Stem Cell Research Center, School of Biotechnology, Institute of Agricultural Technology, Suranaree University of Technology, 111 University Ave., Nakhon Ratchasima, 30000, Thailand

Authors and Affiliations

  1. Stem Cell Neurogenesis, MRC Clinical Sciences Centre, Faculty of Medicine, Imperial College London, London, W12 0NN, UK

    Beata Surmacz, Jessica R. Risner-Janiczek, Kailyn Hui & Meng Li

  2. Neurophysiology, MRC Clinical Sciences Centre, Faculty of Medicine, Imperial College London, London, W12 0NN, UK

    Mark Ungless

  3. Institute of Reproduction and Developmental Biology, Faculty of Medicine, Imperial College London, London, W12 0NN, UK

    Parinya Noisa & Wei Cui

Authors
  1. Beata Surmacz
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Parinya Noisa
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jessica R. Risner-Janiczek
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Kailyn Hui
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Mark Ungless
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Wei Cui
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Meng Li
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Meng Li.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Fig. S1

Characterisation of DLK1 engineered ESC and PA6 cells. A, Cells from 10 independent ESC clones were treated overnight with 1.2 μM tamoxifen before being processed for qRT-PCR using primers specific for hDLK1. B, Western blot analysis to detect DLK1 expression in the parental PA6 cells (C), and three pools of stable hDLK1 transfectants (P1-P3). (JPEG 8 kb)

High resolution image (TIFF 864 kb)

Fig. S2

Effects of recombinant hDLK1 on neural progenitor differentiation. Passage 4 hNPCs were cultured for 5 days with or without DLK1-Fc followed by staining for beta3-tubulin. (JPEG 14 kb)

High resolution image (TIFF 572 kb)

Fig. S3

Tmoxifen does not affect cell proliferation and differentiation. The parental R26CT2 mouse ES cells were induced to undergo monolayer differentiation. Notch ligands and tamoxifen were added at day 7 and cultures were fixed at day 12 followed by antibody staining against beta-tubulin3, Map2, Nestin, phosphor H3 and p57. Data presented was averaged from one experiment performed in triplicates. (JPEG 131 kb)

Fig. S4

Effect of DLK1 on Hes1-luciferase activity. Day 7 monolayer cultures of E14TG2a ES cells were transfected with Hes1-luc together with NICD, DLK1 or combined. Luciferase assay were performed 2 days later. DLK1 alone transfected sister cultures were processed for qPCR for hDLK1, and mouse Hes1, Ngn2 and Mash1. (JPEG 14 kb)

High resolution image (TIFF 1037 kb)

DOC 37.0 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Surmacz, B., Noisa, P., Risner-Janiczek, J.R. et al. DLK1 Promotes Neurogenesis of Human and Mouse Pluripotent Stem Cell-Derived Neural Progenitors Via Modulating Notch and BMP Signalling. Stem Cell Rev and Rep 8, 459–471 (2012). https://doi.org/10.1007/s12015-011-9298-7

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12015-011-9298-7

Keywords

Search

Navigation