A method for rapid gain-of-function studies in the mouse embryonic nervoussystem


We used ultrasound image-guided injections of high-titer retroviral vectorsto obtain widespread introduction of genes into the mouse nervous system in utero as early as embryonic day 8.5 (E8.5). The vectors used includedinternal promoters that substantially improved proviral gene expression inthe ventricular zone of the brain. To demonstrate the utility of this system,we extended our previous work in vitro by infecting the telencephalon in vivo as early as E8.5 with a virus expressing Sonic Hedgehog. Infectedembryos showed gross morphological brain defects, as well as ectopic expressionof ventral telencephalic markers characteristic of either the medial or lateralganglionic eminences.

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Figure 1: Schematic representations of the retroviral constructs.
Figure 2: P21 phenotype of cells infected with PLAP-expressing viruses at E9.5.
Figure 3: Internal promoters improved vector expression within the ventricularzone.
Figure 4: Comparison of neuron distribution in samples infected with CLIA(G)and CLC(G).
Figure 5: Injection of retroviral stocks at E8.5 resulted in widespread infection.
Figure 6: Morphological defects in embryos infected with a Shh-expressing virus(CLES).
Figure 7: Dorsolateral Shh expression resulted in ectopic expression of ventralmarkers.


  1. 1

    Yan, Y. L., Jowett, T. & Postlethwait, J. H. Ectopic expression of hoxb2 after retinoic acid treatmentor mRNA injection: disruption of hindbrain and craniofacial morphogenesisin zebrafish embryos. Dev. Dyn. 213, 370–385 (1998).

  2. 2

    Gaiano, N. & Hopkins, N. Introducing genes into zebrafish. Biochim. Biophys. Acta. 1288, O11–14 (1996).

  3. 3

    Slack, J. M. Inducing factors in Xenopus early embryos. Curr. Biol. 4, 116–126 (1994).

  4. 4

    Bell, E. J. & Brickell, P. M. Replication-competent retroviralvectors for expressing genes in avian cells in vitro and in vivo. Mol. Biotechnol. 7, 289–298 (1997).

  5. 5

    Muramatsu, T., Mizutani, Y., Ohmori, Y. & Okumura, J. Comparison ofthree nonviral transfection methods for foreign gene expression in early chickenembryos in ovo. Biochem. Biophys. Res. Commun. 230, 376–380 (1997).

  6. 6

    Davidson, B. L., Allen, E. D., Kozarsky, K.F., Wilson, J. M. & Roessler, B. J. A model system for in vivo genetransfer into the central nervous system using an adenoviral vector. Nat. Genet. 3, 219–223 (1993).

  7. 7

    Moriyoshi, K., Richards, L. J., Akazawa, C., O'Leary, D. D. & Nakanishi, S. Labeling neural cells using adenoviralgene transfer of membrane-targeted GFP. Neuron 16, 255–260 (1996).

  8. 8

    Price, J., Turner, D. & Cepko, C. Lineage analysis in the vertebrate nervous system by retrovirus-mediatedgene transfer. Proc. Natl. Acad. Sci. USA 84, 156–160 (1987).

  9. 9

    Sanes, J. R., Rubenstein, J. L. & Nicolas, J. F. Use of a recombinant retrovirus to study post-implantationcell lineage in mouse embryos. EMBO J. 5, 3133–3142 (1986).

  10. 10

    Roe, T., Reynolds, T. C., Yu, G. & Brown, P. O. Integration ofmurine leukemia virus DNA depends on mitosis. EMBO J. 12, 2099–2108 (1993).

  11. 11

    Cepko, C. L., Fields-Berry, S., Ryder, E., Austin, C. & Golden, J. Lineage analysis using retroviralvectors. Curr. Top. Dev. Biol. 36, 51–74 (1998).

  12. 12

    Turner, D. L. & Cepko, C. L. A common progenitor for neuronsand glia persists in rat retina late in development. Nature 328, 131–136 (1987).

  13. 13

    Walsh, C. & Cepko, C. L. Widespread dispersion of neuronalclones across functional regions of the cerebral cortex. Science 255, 434–440 (1992).

  14. 14

    Furukawa, T., Morrow, E. M. & Cepko, C. L. Crx, a novel otx-like homeobox gene, shows photoreceptor-specificexpression and regulates photoreceptor differentiation. Cell 91, 531–541 (1997).

  15. 15

    Ishibashi, M. et al. Persistent expression of helix-loop-helix factor HES-1 preventsmammalian neural differentiation in the central nervous system. EMBO J. 13, 1799–1805 (1994).

  16. 16

    Burrows, R. C., Wancio, D., Levitt, P. & Lillien, L. Response diversityand the timing of progenitor cell maturation are regulated by developmentalchanges in EGFR expression in the cortex. Neuron 19, 251–267 (1997).

  17. 17

    Bao, Z. Z. & Cepko, C. L. The expression and function ofNotch pathway genes in the developing rat eye. J. Neurosci. 17, 1425–1434 (1997).

  18. 18

    Olsson, M., Campbell, K. & Turnbull, D. H. Specification of mouse telencephalic and mid-hindbrainprogenitors following heterotopic ultrasound-guided embryonic transplantation. Neuron 19, 761–772(1997).

  19. 19

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

  20. 20

    Kohtz, J. D., Baker, D. P., Corte, G. & Fishell, G. Regionalizationwithin the mammalian telencephalon is mediated by changes in responsivenessto Sonic Hedgehog. Development 125, 5079–5089 (1998).

  21. 21

    Naviaux, R. K., Costanzi, E., Haas, M. & Verma, I. M. The pCL vectorsystem: rapid production of helper-free, high-titer, recombinant retroviruses. J. Virol. 70, 5701–5705 (1996).

  22. 22

    Yee, J. K., Friedmann, T. & Burns, J. C. Generation of high-titer pseudotyped retroviral vectorswith very broad host range. Methods Cell Biol. 43, 99–112 (1994).

  23. 23

    Eidelman, O., Schlegel, R., Tralka, T. S. & Blumenthal, R. pH-dependent fusion induced by vesicular stomatitis virus glycoprotein reconstitutedinto phospholipid vesicles. J. Biol. Chem. 259, 4622–4628 (1984).

  24. 24

    Albritton, L. M., Tseng, L., Scadden, D. & Cunningham, J. M. A putativemurine ecotropic retrovirus receptor gene encodes a multiple membrane-spanningprotein and confers susceptibility to virus infection. Cell 57, 659–666 (1989).

  25. 25

    Lange, C. & Blankenstein, T. Loss of retroviral gene expressionin bone marrow reconstituted mice correlates with down-regulation of geneexpression in long-term culture initiating cells. Gene Ther. 4, 303–308 (1997).

  26. 26

    Gorman, C. M., Rigby, P. W. & Lane, D. P. Negative regulation of viral enhancers in undifferentiatedembryonic stem cells. Cell 42, 519–526 (1985).

  27. 27

    Kempler, G., Freitag, B., Berwin, B., Nanassy, O. & Barklis, E. Characterization of the Moloney murine leukemia virusstem cell-specific repressor binding site. Virology 193, 690–699 (1993).

  28. 28

    Johansson, C. B. et al. Identification of a neural stem cell in the adult mammaliancentral nervous system. Cell 96, 25–34 (1999).

  29. 29

    Temple, S. & Alvarez-Buylla, A. Stem cells in the adult mammaliancentral nervous system. Curr. Opin. Neurobiol. 9, 135–141 (1999).

  30. 30

    Reynolds, B.A. & Weiss, S. Clonal and populationanalyses demonstrate that an EGF-responsive mammalian embryonic CNS precursoris a stem cell. Dev. Biol. 175, 1–13 (1996).

  31. 31

    Halliday, A. L. & Cepko, C. L. Generation andmigration of cells in the developing striatum. Neuron 9, 15–26 (1992).

  32. 32

    Takahashi, T., Nowakowski, R. S. & Caviness, V. S. Jr. The leaving or Q fraction of themurine cerebral proliferative epithelium: a general model of neocortical neuronogenesis. J. Neurosci. 16, 6183–6196 (1996).

  33. 33

    Johnson, A. D. & Krieg, P. A. pXeX, a vectorfor efficient expression of cloned sequences in Xenopus embryos. Gene 147, 223–226 (1994).

  34. 34

    Sawicki, J. A., Morris, R. J., Monks, B., Sakai, K. & Miyazaki, J. A composite CMV-IE enhancer/beta-actin promoter is ubiquitouslyexpressed in mouse cutaneous epithelium. Exp. Cell Res. 244, 367–369 (1998).

  35. 35

    Niwa, H., Yamamura, K. & Miyazaki, J. Efficient selection for high-expression transfectantswith a novel eukaryotic vector. Gene 108, 193–199 (1991).

  36. 36

    Tan, S. S. & Breen, S. Radial mosaicism and tangential celldispersion both contribute to mouse neocortical development. Nature 362, 638–640 (1993).

  37. 37

    Rakic, P. Radial versus tangential migration of neuronal clones in the developing cerebralcortex. Proc. Natl. Acad. Sci. USA 92, 11323–11327 (1995).

  38. 38

    Echelard, Y. et al. Sonic hedgehog, a member of a family of putative signalingmolecules, is implicated in the regulation of CNS polarity. Cell 75, 1417–1430 (1993).

  39. 39

    Ericson, J. et al. Sonic hedgehog induces the differentiation of ventral forebrainneurons: a common signal for ventral patterning within the neural tube. Cell 81, 747–756 (1995).

  40. 40

    Roelink, H. et al. Floor plate and motor neuron induction by vhh-1, a vertebratehomolog of hedgehog expressed by the notochord. Cell 76, 761–775 (1994).

  41. 41

    Bulfone, A. et al. The mouse Dlx-2 (Tes-1) gene is expressed in spatially restricteddomains of the forebrain, face and limbs in midgestation mouse embryos. Mech. Dev. 40, 129–140 (1993).

  42. 42

    Lazzaro, D., Price, M., de Felice, M. & Di Lauro, R. The transcriptionfactor TTF-1 is expressed at the onset of thyroid and lung morphogenesis andin restricted regions of the foetal brain. Development 113, 1093–1104 (1991).

  43. 43

    Shimamura, K. & Rubenstein, J. L. Inductive interactions directearly regionalization of the mouse forebrain. Development 124, 2709–2718 (1997).

  44. 44

    Toresson, H., Mata de Urquiza, A., Fagerstrom, C., Perlmann, T. & Campbell, K. Retinoids are produced by gliain the lateral ganglionic eminence and regulate striatal neuron differentiation. Development 126, 1317–1326 (1999).

  45. 45

    Chiang, C. et al. Cyclopia and defective axial patterning in mice lacking Sonichedgehog gene function. Nature 383, 407–413 (1996).

  46. 46

    Ruiz i Altaba, A. Catching a Gli-mpse of Hedgehog. Cell 90, 193–196 (1997).

  47. 47

    Schaeren-Wiemers, N. & Gerfin-Moser, A. A singleprotocol to detect transcripts of various types and expression levels in neuraltissue and cultured cells: in situ hybridization using digoxigenin-labelledcRNA probes. Histochemistry 100, 431–440 (1993).

  48. 48

    Mullen, R. J., Buck, C. R. & Smith, A. M. NeuN, a neuronal specific nuclear protein in vertebrates. Development 116, 201–211 (1992).

  49. 49

    Sprinkle, T. J. 2',3′-cyclic nucleotide 3′-phosphodiesterase, an oligodendrocyte-Schwanncell and myelin-associated enzyme of the nervous system. Crit.Rev. Neurobiol. 4, 235–301 (1989).

  50. 50

    Bignami, A. & Dahl, D. Astrocyte-specific protein and neuroglialdifferentiation. An immunofluorescence study with antibodies to the glialfibrillary acidic protein. J. Comp. Neurol. 153, 27–38 (1974).

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We thank Rusty Lansford for bringing the CA regulatory element to our attentionand Alex Langston and Robin Kimmel for input at the start of this work. Wealso thank Maria McCarthy and Connie Cepko for discussions, Michelle Starz-Gaianofor reading the manuscript, Ulf Eriksson for providing the anti-CRBP antibodyand K. Mahon for the dlx2 in situ probe. This work was supported by NIH grantsNS32993 (G.F.), NS38461 (D.H.T.) and HL62334 (D.H.T.). Additional supportwas provided by March of Dimes Grant 6-FY99-634 (G.F.). N.G. is supportedby a postdoctoral fellowship from the American Cancer Society (PF4473).

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Correspondence to Daniel H. Turnbull or Gord Fishell.

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Gaiano, N., Kohtz, J., Turnbull, D. et al. A method for rapid gain-of-function studies in the mouse embryonic nervoussystem. Nat Neurosci 2, 812–819 (1999). https://doi.org/10.1038/12186

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