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.

  • Technical Report
  • Published:

Rapid functional dissection of genetic networks via tissue-specific transduction and RNAi in mouse embryos

Abstract

Using ultrasound-guided in utero infections of fluorescently traceable lentiviruses carrying RNAi or Cre recombinase into mouse embryos, we have demonstrated noninvasive, highly efficient selective transduction of surface epithelium, in which progenitors stably incorporate and propagate the desired genetic alterations. We achieved epidermal-specific infection using small generic promoters of existing lentiviral short hairpin RNA libraries, thus enabling rapid assessment of gene function as well as complex genetic interactions in skin morphogenesis and disease in vivo. We adapted this technology to devise a new quantitative method for ascertaining whether a gene confers a growth advantage or disadvantage in skin tumorigenesis. Using α1-catenin as a model, we uncover new insights into its role as a widely expressed tumor suppressor and reveal physiological interactions between Ctnna1 and the Hras1-Mapk3 and Trp53 gene pathways in regulating skin cell proliferation and apoptosis. Our study illustrates the strategy and its broad applicability for investigations of tissue morphogenesis, lineage specification and cancers.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Intra-amniotic injection of lentivirus at E9.5 results in noninvasive, high-efficiency, stable and epidermally restricted transduction.
Figure 2: Epidermal infection depends on viral titer and permits delivery of multiple viral constructs.
Figure 3: Rapid assay for measuring an epidermal growth advantage or disadvantage conferred by a gene mutation reveals an unexpected growth disadvantage following α1-catenin loss despite hyperproliferation.
Figure 4: Efficient epidermal-specific lentivirus RNAi-mediated knockdown of Ctnna1 faithfully recapitulates phenotypic abnormalities shown by K14-Cre conditional and LV-Cre induced knockout counterparts.
Figure 5: Use of RNAi knockdown in vivo to functionally dissect why loss of α1-catenin results in hyperproliferation but a growth disadvantage to the epidermis.

Similar content being viewed by others

References

  1. Holzinger, A., Trapnell, B.C., Weaver, T.E., Whitsett, J.A. & Iwamoto, H.S. Intraamniotic administration of an adenoviral vector for gene transfer to fetal sheep and mouse tissues. Pediatr. Res. 38, 844–850 (1995).

    Article  CAS  Google Scholar 

  2. Lu, B., Federoff, H.J., Wang, Y., Goldsmith, L.A. & Scott, G. Topical application of viral vectors for epidermal gene transfer. J. Invest. Dermatol. 108, 803–808 (1997).

    Article  CAS  Google Scholar 

  3. Liu, A., Joyner, A.L. & Turnbull, D.H. Alteration of limb and brain patterning in early mouse embryos by ultrasound-guided injection of Shh-expressing cells. Mech. Dev. 75, 107–115 (1998).

    Article  CAS  Google Scholar 

  4. Slevin, J.C. et al. High resolution ultrasound-guided microinjection for interventional studies of early embryonic and placental development in vivo in mice. BMC Dev. Biol. 6, 10 (2006).

    Article  Google Scholar 

  5. Endo, M. et al. Efficient in vivo targeting of epidermal stem cells by early gestational intraamniotic injection of lentiviral vector driven by the keratin 5 promoter. Mol. Ther. 16, 131–137 (2008).

    Article  CAS  Google Scholar 

  6. Punzo, C. & Cepko, C.L. Ultrasound-guided in utero injections allow studies of the development and function of the eye. Dev. Dyn. 237, 1034–1042 (2008).

    Article  CAS  Google Scholar 

  7. Silva, J.M. et al. Second-generation shRNA libraries covering the mouse and human genomes. Nat. Genet. 37, 1281–1288 (2005).

    Article  CAS  Google Scholar 

  8. Moffat, J. et al. A lentiviral RNAi library for human and mouse genes applied to an arrayed viral high-content screen. Cell 124, 1283–1298 (2006).

    Article  CAS  Google Scholar 

  9. Kanda, T., Sullivan, K.F. & Wahl, G.M. Histone-GFP fusion protein enables sensitive analysis of chromosome dynamics in living mammalian cells. Curr. Biol. 8, 377–385 (1998).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  11. Srinivas, S. et al. Cre reporter strains produced by targeted insertion of EYFP and ECFP into the ROSA26 locus. BMC Dev. Biol. 1, 4 (2001).

    Article  CAS  Google Scholar 

  12. Clayton, E. et al. A single type of progenitor cell maintains normal epidermis. Nature 446, 185–189 (2007).

    Article  CAS  Google Scholar 

  13. Vaezi, A., Bauer, C., Vasioukhin, V. & Fuchs, E. Actin cable dynamics and Rho/Rock orchestrate a polarized cytoskeletal architecture in the early steps of assembling a stratified epithelium. Dev. Cell 3, 367–381 (2002).

    Article  CAS  Google Scholar 

  14. Follenzi, A., Santambrogio, L. & Annoni, A. Immune responses to lentiviral vectors. Curr. Gene Ther. 7, 306–315 (2007).

    Article  CAS  Google Scholar 

  15. Perez-Moreno, M. et al. p120-catenin mediates inflammatory responses in the skin. Cell 124, 631–644 (2006).

    Article  CAS  Google Scholar 

  16. Vasioukhin, V., Bauer, C., Degenstein, L., Wise, B. & Fuchs, E. Hyperproliferation and defects in epithelial polarity upon conditional ablation of alpha-catenin in skin. Cell 104, 605–617 (2001).

    Article  CAS  Google Scholar 

  17. Xiangming, C. et al. Cooccurrence of reduced expression of alpha-catenin and overexpression of p53 is a predictor of lymph node metastasis in early gastric cancer. Oncology 57, 131–137 (1999).

    Article  CAS  Google Scholar 

  18. Nozawa, N. et al. Immunohistochemical alpha- and beta-catenin and E-cadherin expression and their clinicopathological significance in human lung adenocarcinoma. Pathol. Res. Pract. 202, 639–650 (2006).

    Article  CAS  Google Scholar 

  19. Kobielak, A. & Fuchs, E. Links between alpha-catenin, NF-kappaB, and squamous cell carcinoma in skin. Proc. Natl. Acad. Sci. USA 103, 2322–2327 (2006).

    Article  CAS  Google Scholar 

  20. Benjamin, J.M. & Nelson, W.J. Bench to bedside and back again: molecular mechanisms of alpha-catenin function and roles in tumorigenesis. Semin. Cancer Biol. 18, 53–64 (2008).

    Article  CAS  Google Scholar 

  21. Vasioukhin, V., Bauer, C., Yin, M. & Fuchs, E. Directed actin polymerization is the driving force for epithelial cell-cell adhesion. Cell 100, 209–219 (2000).

    Article  CAS  Google Scholar 

  22. Quintanilla, M., Brown, K., Ramsden, M. & Balmain, A. Carcinogen-specific mutation and amplification of Ha-ras during mouse skin carcinogenesis. Nature 322, 78–80 (1986).

    Article  CAS  Google Scholar 

  23. Leon, J., Guerrero, I. & Pellicer, A. Differential expression of the ras gene family in mice. Mol. Cell. Biol. 7, 1535–1540 (1987).

    Article  CAS  Google Scholar 

  24. Khavari, T.A. & Rinn, J. Ras/Erk MAPK signaling in epidermal homeostasis and neoplasia. Cell Cycle 6, 2928–2931 (2007).

    Article  CAS  Google Scholar 

  25. Pagès, G. et al. Defective thymocyte maturation in p44 MAP kinase (Erk 1) knockout mice. Science 286, 1374–1377 (1999).

    Article  Google Scholar 

  26. Ise, K. et al. Targeted deletion of the H-ras gene decreases tumor formation in mouse skin carcinogenesis. Oncogene 19, 2951–2956 (2000).

    Article  CAS  Google Scholar 

  27. Dimri, G.P. et al. A biomarker that identifies senescent human cells in culture and in aging skin in vivo. Proc. Natl. Acad. Sci. USA 92, 9363–9367 (1995).

    Article  CAS  Google Scholar 

  28. Derksen, P.W. et al. Somatic inactivation of E-cadherin and p53 in mice leads to metastatic lobular mammary carcinoma through induction of anoikis resistance and angiogenesis. Cancer Cell 10, 437–449 (2006).

    Article  CAS  Google Scholar 

  29. Villunger, A. et al. p53- and drug-induced apoptotic responses mediated by BH3-only proteins puma and noxa. Science 302, 1036–1038 (2003).

    Article  CAS  Google Scholar 

  30. Reuter, J.A. et al. Modeling inducible human tissue neoplasia identifies an extracellular matrix interaction network involved in cancer progression. Cancer Cell 15, 477–488 (2009).

    Article  CAS  Google Scholar 

  31. Guasch, G. et al. Loss of TGFbeta signaling destabilizes homeostasis and promotes squamous cell carcinomas in stratified epithelia. Cancer Cell 12, 313–327 (2007).

    Article  CAS  Google Scholar 

  32. Devenport, D. & Fuchs, E. Planar polarization in embryonic epidermis orchestrates global asymmetric morphogenesis of hair follicles. Nat. Cell Biol. 10, 1257–1268 (2008).

    Article  CAS  Google Scholar 

  33. Nowak, J.A. & Fuchs, E. Isolation and culture of epithelial stem cells. Methods Mol. Biol. 482, 215–232 (2009).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank M. Takeichi (RIKEN CDB) for antibodies and reagents; N. Stokes and Rockefeller Comparative Bioscience Center staff for expert care of mice; M. Schober and M. Perez-Moreno for helpful discussions; A. North and Rockefeller Bioimaging Resource Center staff for assistance with image acquisition and analysis; and S. Mazel and Rockefeller Flow Cytometry Resource Center staff for assistance with FACS. S.B. is supported by the International Human Frontier Science Program Organization. S.W. is an American Cancer Society Postdoctoral Fellow. This work was supported by a grant from the US National Institutes of Health (R01-AR27883). E.F. is an investigator with the Howard Hughes Medical Institute.

Author information

Authors and Affiliations

Authors

Contributions

S.B., G.L. and S.W. designed and performed the experiments and analyzed the raw data. S.B. and E.F. wrote the manuscript. E.F. supervised the project.

Corresponding author

Correspondence to Elaine Fuchs.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Figures

Supplementary Figures 1–9 (PDF 5493 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Beronja, S., Livshits, G., Williams, S. et al. Rapid functional dissection of genetic networks via tissue-specific transduction and RNAi in mouse embryos. Nat Med 16, 821–827 (2010). https://doi.org/10.1038/nm.2167

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nm.2167

This article is cited by

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