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An RNA interference screen uncovers a new molecule in stem cell self-renewal and long-term regeneration

Abstract

Adult stem cells sustain tissue maintenance and regeneration throughout the lifetime of an animal1,2. These cells often reside in specific signalling niches that orchestrate the stem cell’s balancing act between quiescence and cell-cycle re-entry based on the demand for tissue regeneration2,3,4. How stem cells maintain their capacity to replenish themselves after tissue regeneration is poorly understood. Here we use RNA-interference-based loss-of-function screening as a powerful approach to uncover transcriptional regulators that govern the self-renewal capacity and regenerative potential of stem cells. Hair follicle stem cells provide an ideal model. These cells have been purified and characterized from their native niche in vivo and, in contrast to their rapidly dividing progeny, they can be maintained and passaged long-term in vitro5,6,7. Focusing on the nuclear proteins and/or transcription factors that are enriched in stem cells compared with their progeny5,6, we screened 2,000 short hairpin RNAs for their effect on long-term, but not short-term, stem cell self-renewal in vitro. To address the physiological relevance of our findings, we selected one candidate that was uncovered in the screen: TBX1. This transcription factor is expressed in many tissues but has not been studied in the context of stem cell biology. By conditionally ablating Tbx1 in vivo, we showed that during homeostasis, tissue regeneration occurs normally but is markedly delayed. We then devised an in vivo assay for stem cell replenishment and found that when challenged with repetitive rounds of regeneration, the Tbx1-deficient stem cell niche becomes progressively depleted. Addressing the mechanism of TBX1 action, we discovered that TBX1 acts as an intrinsic rheostat of BMP signalling: it is a gatekeeper that governs the transition between stem cell quiescence and proliferation in hair follicles. Our results validate the RNA interference screen and underscore its power in unearthing new molecules that govern stem cell self-renewal and tissue-regenerative potential.

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Figure 1: In vitro RNAi screen for genes involved in stem cell long-term self-renewal.
Figure 2: The transcription factor TBX1 is highly enriched in stem cells in vivo and in vitro.
Figure 3: Tbx1 -null stem cells fail in an in vivo assay for stem cell self-renewal and long-term tissue regeneration.
Figure 4: TBX1 controls stem cell proliferation in part by fine-tuning the response to BMP signalling.

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Primary accessions

Gene Expression Omnibus

Data deposits

Microarray data have been deposited in the Gene Expression Omnibus database under accession number GSE35575.

References

  1. 1

    Li, L. & Clevers, H. Coexistence of quiescent and active adult stem cells in mammals. Science 327, 542–545 (2010)

    ADS  CAS  Article  Google Scholar 

  2. 2

    Fuchs, E. The tortoise and the hair: slow-cycling cells in the stem cell race. Cell 137, 811–819 (2009)

    CAS  Article  Google Scholar 

  3. 3

    Zon, L. I. Intrinsic and extrinsic control of haematopoietic stem-cell self-renewal. Nature 453, 306–313 (2008)

    ADS  CAS  Article  Google Scholar 

  4. 4

    He, S., Nakada, D. & Morrison, S. J. Mechanisms of stem cell self-renewal. Annu. Rev. Cell Dev. Biol. 25, 377–406 (2009)

    CAS  Article  Google Scholar 

  5. 5

    Blanpain, C., Lowry, W. E., Geoghegan, A., Polak, L. & Fuchs, E. Self-renewal, multipotency, and the existence of two cell populations within an epithelial stem cell niche. Cell 118, 635–648 (2004)

    CAS  Article  Google Scholar 

  6. 6

    Greco, V. et al. A two-step mechanism for stem cell activation during hair regeneration. Cell Stem Cell 4, 155–169 (2009)

    CAS  Article  Google Scholar 

  7. 7

    Watt, F. M. & Jensen, K. B. Epidermal stem cell diversity and quiescence. EMBO Mol. Med. 1, 260–267 (2009)

    CAS  Article  Google Scholar 

  8. 8

    Ying, Q. L., Nichols, J., Chambers, I. & Smith, A. BMP induction of Id proteins suppresses differentiation and sustains embryonic stem cell self-renewal in collaboration with STAT3. Cell 115, 281–292 (2003)

    CAS  Article  Google Scholar 

  9. 9

    Molofsky, A. V. et al. Bmi-1 dependence distinguishes neural stem cell self-renewal from progenitor proliferation. Nature 425, 962–967 (2003)

    ADS  CAS  Article  Google Scholar 

  10. 10

    Nishino, J., Kim, I., Chada, K. & Morrison, S. J. Hmga2 promotes neural stem cell self-renewal in young but not old mice by reducing p16Ink4a and p19Arf Expression. Cell 135, 227–239 (2008)

    CAS  Article  Google Scholar 

  11. 11

    Topley, G. I., Okuyama, R., Gonzales, J. G., Conti, C. & Dotto, G. P. p21WAF1/Cip1 functions as a suppressor of malignant skin tumor formation and a determinant of keratinocyte stem-cell potential. Proc. Natl Acad. Sci. USA 96, 9089–9094 (1999)

    ADS  CAS  Article  Google Scholar 

  12. 12

    Kippin, T. E., Martens, D. J. & van der Kooy, D. p21 loss compromises the relative quiescence of forebrain stem cell proliferation leading to exhaustion of their proliferation capacity. Genes Dev. 19, 756–767 (2005)

    CAS  Article  Google Scholar 

  13. 13

    Tumbar, T. et al. Defining the epithelial stem cell niche in skin. Science 303, 359–363 (2004)

    ADS  CAS  Article  Google Scholar 

  14. 14

    Ito, M., Kizawa, K., Hamada, K. & Cotsarelis, G. Hair follicle stem cells in the lower bulge form the secondary germ, a biochemically distinct but functionally equivalent progenitor cell population, at the termination of catagen. Differentiation 72, 548–557 (2004)

    Article  Google Scholar 

  15. 15

    Hsu, Y. C., Pasolli, H. A. & Fuchs, E. Dynamics between stem cells, niche, and progeny in the hair follicle. Cell 144, 92–105 (2011)

    CAS  Article  Google Scholar 

  16. 16

    Osorio, K. M. et al. Runx1 modulates developmental, but not injury-driven, hair follicle stem cell activation. Development 135, 1059–1068 (2008)

    CAS  Article  Google Scholar 

  17. 17

    Aggarwal, V. S. et al. Mesodermal Tbx1 is required for patterning the proximal mandible in mice. Dev. Biol. 344, 669–681 (2010)

    CAS  Article  Google Scholar 

  18. 18

    Chen, L., Fulcoli, F. G., Tang, S. & Baldini, A. Tbx1 regulates proliferation and differentiation of multipotent heart progenitors. Circ. Res. 105, 842–851 (2009)

    CAS  Article  Google Scholar 

  19. 19

    Lien, W. H. et al. Genome-wide maps of histone modifications unwind in vivo chromatin states of the hair follicle lineage. Cell Stem Cell 9, 219–232 (2011)

    CAS  Article  Google Scholar 

  20. 20

    Xu, H. et al. Tbx1 has a dual role in the morphogenesis of the cardiac outflow tract. Development 131, 3217–3227 (2004)

    CAS  Article  Google Scholar 

  21. 21

    Vasioukhin, V., Degenstein, L., Wise, B. & Fuchs, E. The magical touch: genome targeting in epidermal stem cells induced by tamoxifen application to mouse skin. Proc. Natl Acad. Sci. USA 96, 8551–8556 (1999)

    ADS  CAS  Article  Google Scholar 

  22. 22

    Korchynskyi, O. & ten Dijke, P. Identification and functional characterization of distinct critically important bone morphogenetic protein-specific response elements in the Id1 promoter. J. Biol. Chem. 277, 4883–4891 (2002)

    CAS  Article  Google Scholar 

  23. 23

    Hollnagel, A., Oehlmann, V., Heymer, J., Ruther, U. & Nordheim, A. Id genes are direct targets of bone morphogenetic protein induction in embryonic stem cells. J. Biol. Chem. 274, 19838–19845 (1999)

    CAS  Article  Google Scholar 

  24. 24

    Fulcoli, F. G., Huynh, T., Scambler, P. J. & Baldini, A. Tbx1 regulates the BMP–Smad1 pathway in a transcription independent manner. PLoS ONE 4, e6049 (2009)

    ADS  Article  Google Scholar 

  25. 25

    Blessing, M., Nanney, L. B., King, L. E., Jones, C. M. & Hogan, B. L. Transgenic mice as a model to study the role of TGF-β-related molecules in hair follicles. Genes Dev. 7, 204–215 (1993)

    CAS  Article  Google Scholar 

  26. 26

    Botchkarev, V. A. et al. Noggin is a mesenchymally derived stimulator of hair-follicle induction. Nature Cell Biol. 1, 158–164 (1999)

    CAS  Article  Google Scholar 

  27. 27

    Kulessa, H., Turk, G. & Hogan, B. L. Inhibition of Bmp signaling affects growth and differentiation in the anagen hair follicle. EMBO J. 19, 6664–6674 (2000)

    CAS  Article  Google Scholar 

  28. 28

    Kobielak, K., Stokes, N., de la Cruz, J., Polak, L. & Fuchs, E. Loss of a quiescent niche but not follicle stem cells in the absence of bone morphogenetic protein signaling. Proc. Natl Acad. Sci. USA 104, 10063–10068 (2007)

    ADS  CAS  Article  Google Scholar 

  29. 29

    Andl, T. et al. Epithelial Bmpr1a regulates differentiation and proliferation in postnatal hair follicles and is essential for tooth development. Development 131, 2257–2268 (2004)

    CAS  Article  Google Scholar 

  30. 30

    Oshimori, N. & Fuchs, E. Paracrine TGF-β signaling counterbalances BMP-mediated repression in hair follicle stem cell activation. Cell Stem Cell 10, 63–75 (2012)

    CAS  Article  Google Scholar 

  31. 31

    R Foundation for Statistical Computing. The R Project for Statistical Computinghttp://www.r-project.org〉 (2011)

    Google Scholar 

  32. 32

    Beronja, S., Livshits, G., Williams, S. & Fuchs, E. Rapid functional dissection of genetic networks via tissue-specific transduction and RNAi in mouse embryos. Nature Med. 16, 821–827 (2010)

    CAS  Article  Google Scholar 

Download references

Acknowledgements

We are grateful to A. Baldini for the Tbx1fl/fl mice, the Comparative Biology Centre (AAALAC-accredited) for expert handling and care of the mice, The Rockefeller University FCRC for FACS sorting (supported by NYSTEM funds through NYSDOH, contract C023046), S. Dewell and The Rockefeller University Genomic Resource Centre for high-throughput sequencing, The Memorial Sloan Kettering Genomics Core Facility for RNA and microarray processing, Fuchs’ lab members Y. Hsu, B. Keyes, X. Wu, D. Devenport and L. Zhang for comments and suggestions, W. H. Lien for providing epigenetic ChIP-seq data for the Tbx1 gene in vivo and P. Janki for assisting in the production of the pooled virus for screening. This work was supported by grants from the National Institutes of Health (R01-AR050452; E.F.), the Empire State Stem Cell (NYSTEM N09G074; E.F.), a New York Stem Cell Foundation-Druckenmiller Fellowship (T.C.) and a NYSTEM Scholar Award (C026722; T.C.). E.F. is an Investigator of the Howard Hughes Medical Institute.

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T.C. and E.F. designed the study. T.C. carried out the experiments and analysed the data. E.H. analysed the sequencing data. S.B. contributed to optimizing and testing methods used in the screen. N.O. carried out the Smad1 shRNA-related experiments. N.S. participated in the experiments involving mouse handling. T.C. and E.F. co-wrote the paper. E.F. supervised the research.

Corresponding author

Correspondence to Elaine Fuchs.

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The authors declare no competing financial interests.

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Chen, T., Heller, E., Beronja, S. et al. An RNA interference screen uncovers a new molecule in stem cell self-renewal and long-term regeneration. Nature 485, 104–108 (2012). https://doi.org/10.1038/nature10940

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