Abstract
Astrocytes communicate with the vascular endothelium via direct cell–cell contacts as well as a variety of secreted growth factors and extracellular matrix (ECM) proteins. Integrins are heterodimeric cell surface receptors for ECM protein ligands, and many integrin subunits are expressed in astrocytes. Here, we will discuss gene deletion strategies in mice that have deciphered functions for specific integrins in astrocyte-endothelial cell adhesion and signaling. Specifically, we will detail how Cre-lox molecular genetic techniques have revealed important roles for integrin αvβ8 in regulating cerebral blood vessel development and homeostasis. First, we will detail how to generate Cre-lox mutant mouse models that our group and others have used to study αvβ8 integrin in embryonic astroglial progenitors and postnatal astrocytes. Second, we will discuss how viral-delivered Cre can be used to acutely delete integrin genes in astrocytes within defined anatomic regions of the brain. Third, detailed in vivo methods to verify Cre-mediated gene recombination in astrocytes will be presented. Lastly, we will present one experimental strategy to determine how integrin gene deletion affects astrocyte-endothelial cell coupling in the CNS. While this review focuses on the generation and characterization of mice lacking αvβ8 integrin, these experimental strategies can be expanded to analyze other cell adhesion and signaling genes important for astroglial-mediated regulation of blood vessel development and homeostasis.
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References
McCarty, J. H. (2005) Cell biology of the neurovascular unit: implications for drug delivery across the blood-brain barrier, Assay Drug Dev Technol 3, 89–95.
McCarty, J. H. (2009) Integrin-mediated regulation of neurovascular development, physiology and disease, Cell Adh Migr 3, 211–215.
Milner, R., and Campbell, I. L. (2002) The integrin family of cell adhesion molecules has multiple functions within the CNS, J Neurosci Res 69, 286–291.
Abbott, N. J., Ronnback, L., and Hansson, E. (2006) Astrocyte-endothelial interactions at the blood-brain barrier, Nat Rev Neurosci 7, 41–53.
Sixt, M., Engelhardt, B., Pausch, F., Hallmann, R., Wendler, O., and Sorokin, L. M. (2001) Endothelial cell laminin isoforms, laminins 8 and 10, play decisive roles in T cell recruitment across the blood-brain barrier in experimental autoimmune encephalomyelitis, J Cell Biol 153, 933–946.
Wagner, S., Tagaya, M., Koziol, J. A., Quaranta, V., and del Zoppo, G. J. (1997) Rapid disruption of an astrocyte interaction with the extracellular matrix mediated by integrin alpha 6 beta 4 during focal cerebral ischemia/reperfusion, Stroke 28, 858–865.
Okada, Y., Copeland, B. R., Hamann, G. F., Koziol, J. A., Cheresh, D. A., and del Zoppo, G. J. (1996) Integrin alphavbeta3 is expressed in selected microvessels after focal cerebral ischemia, Am J Pathol 149, 37–44.
Shimamura, N., Matchett, G., Yatsushige, H., Calvert, J. W., Ohkuma, H., and Zhang, J. (2006) Inhibition of integrin alphavbeta3 ameliorates focal cerebral ischemic damage in the rat middle cerebral artery occlusion model, Stroke 37, 1902–1909.
McCarty, J. H. (2009) Cell adhesion and signaling networks in brain neurovascular units, Curr Opin Hematol 16, 209–214.
McCarty, J. H., Monahan-Earley, R. A., Brown, L. F., Keller, M., Gerhardt, H., Rubin, K., Shani, M., Dvorak, H. F., Wolburg, H., Bader, B. L., Dvorak, A. M., and Hynes, R. O. (2002) Defective associations between blood vessels and brain parenchyma lead to cerebral hemorrhage in mice lacking alphav integrins, Mol Cell Biol 22, 7667–7677.
Mobley, A. K., Tchaicha, J. H., Shin, J., Hossain, M. G., and McCarty, J. H. (2009) {beta}8 integrin regulates neurogenesis and neurovascular homeostasis in the adult brain, J Cell Sci 122, 1842–1851.
Zhu, J., Motejlek, K., Wang, D., Zang, K., Schmidt, A., and Reichardt, L. F. (2002) beta8 integrins are required for vascular morphogenesis in mouse embryos, Development 129, 2891–2903.
Milner, R., Huang, X., Wu, J., Nishimura, S., Pytela, R., Sheppard, D., and ffrench-Constant, C. (1999) Distinct roles for astrocyte alphavbeta5 and alphavbeta8 integrins in adhesion and migration, J Cell Sci 112 ( Pt 23), 4271–4279.
McCarty, J. H., Lacy-Hulbert, A., Charest, A., Bronson, R. T., Crowley, D., Housman, D., Savill, J., Roes, J., and Hynes, R. O. (2004) Selective ablation of {alpha}v integrins in the central nervous system leads to cerebral hemorrhage, seizures, axonal degeneration and premature death, Development.
Proctor, J. M., Zang, K., Wang, D., Wang, R., and Reichardt, L. F. (2005) Vascular development of the brain requires beta8 integrin expression in the neuroepithelium, J Neurosci 25, 9940–9948.
Cambier, S., Gline, S., Mu, D., Collins, R., Araya, J., Dolganov, G., Einheber, S., Boudreau, N., and Nishimura, S. L. (2005) Integrin alpha(v)beta8-mediated activation of transforming growth factor-beta by perivascular astrocytes: an angiogenic control switch, Am J Pathol 166, 1883–1894.
Mu, Z., Yang, Z., Yu, D., Zhao, Z., and Munger, J. S. (2008) TGFbeta1 and TGFbeta3 are partially redundant effectors in brain vascular morphogenesis, Mech Dev 125, 508–516.
Tchaicha, J. H., Mobley, A. K., Hossain, M. G., Aldape, K. D., and McCarty, J. H. A mosaic mouse model of astrocytoma identifies alphavbeta8 integrin as a negative regulator of tumor angiogenesis, Oncogene 29, 4460–4472.
Hirrlinger, P. G., Scheller, A., Braun, C., Hirrlinger, J., and Kirchhoff, F. (2006) Temporal control of gene recombination in astrocytes by transgenic expression of the tamoxifen-inducible DNA recombinase variant CreERT2, Glia 54, 11–20.
Bader, B. L., Rayburn, H., Crowley, D., and Hynes, R. O. (1998) Extensive vasculogenesis, angiogenesis, and organogenesis precede lethality in mice lacking all alpha v integrins, Cell 95, 507–519.
Soriano, P. (1999) Generalized lacZ expression with the ROSA26 Cre reporter strain, Nat Genet 21, 70–71.
Lewandoski, M. (2001) Conditional control of gene expression in the mouse, Nat Rev Genet 2, 743–755.
Mortensen, R. (2006) Overview of gene targeting by homologous recombination, Curr Protoc Mol Biol Chapter 23, Unit 23 21.
Mortensen, R. (2007) Overview of gene targeting by homologous recombination, Curr Protoc Neurosci Chapter 4, Unit 4 29.
Bajenaru, M. L., Zhu, Y., Hedrick, N. M., Donahoe, J., Parada, L. F., and Gutmann, D. H. (2002) Astrocyte-specific inactivation of the neurofibromatosis 1 gene (NF1) is insufficient for astrocytoma formation, Mol Cell Biol 22, 5100–5113.
Zhuo, L., Theis, M., Alvarez-Maya, I., Brenner, M., Willecke, K., and Messing, A. (2001) hGFAP-cre transgenic mice for manipulation of glial and neuronal function in vivo, Genesis 31, 85–94.
McLaughlin, M. E., Kruger, G. M., Slocum, K. L., Crowley, D., Michaud, N. A., Huang, J., Magendantz, M., and Jacks, T. (2007) The Nf2 tumor suppressor regulates cell-cell adhesion during tissue fusion, Proc Natl Acad Sci U S A 104, 3261–3266.
Tronche, F., Kellendonk, C., Kretz, O., Gass, P., Anlag, K., Orban, P. C., Bock, R., Klein, R., and Schutz, G. (1999) Disruption of the glucocorticoid receptor gene in the nervous system results in reduced anxiety, Nat Genet 23, 99–103.
Mori, T., Tanaka, K., Buffo, A., Wurst, W., Kuhn, R., and Gotz, M. (2006) Inducible gene deletion in astroglia and radial glia--a valuable tool for functional and lineage analysis, Glia 54, 21–34.
Branda, C. S., and Dymecki, S. M. (2004) Talking about a revolution: The impact of site-specific recombinases on genetic analyses in mice, Dev Cell 6, 7–28.
Zhu, H., Acquaviva, J., Ramachandran, P., Boskovitz, A., Woolfenden, S., Pfannl, R., Bronson, R. T., Chen, J. W., Weissleder, R., Housman, D. E., and Charest, A. (2009) Oncogenic EGFR signaling cooperates with loss of tumor suppressor gene functions in gliomagenesis, Proc Natl Acad Sci U S A 106, 2712–2716.
Forni, P. E., Scuoppo, C., Imayoshi, I., Taulli, R., Dastru, W., Sala, V., Betz, U. A., Muzzi, P., Martinuzzi, D., Vercelli, A. E., Kageyama, R., and Ponzetto, C. (2006) High levels of Cre expression in neuronal progenitors cause defects in brain development leading to microencephaly and hydrocephaly, J Neurosci 26, 9593–9602.
Naiche, L. A., and Papaioannou, V. E. (2007) Cre activity causes widespread apoptosis and lethal anemia during embryonic development, Genesis 45, 768–775.
Acknowledgments
This research was supported by grants awarded to J. H. M. from the National Institutes of Neurological Disease and Stroke (R01NS059876) and the Ellison Medical Foundation (AG-NS-0324-06).
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Mobley, A.K., McCarty, J.H. (2012). Use of Cre-Lox Technology to Analyze Integrin Functions in Astrocytes. In: Milner, R. (eds) Astrocytes. Methods in Molecular Biology, vol 814. Humana Press. https://doi.org/10.1007/978-1-61779-452-0_37
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DOI: https://doi.org/10.1007/978-1-61779-452-0_37
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