Abstract
This chapter provides information on imaging tools that can be employed to visualise and study lymphoid organ development. We focus on the use of genetically modified mouse models that take advantage of fluorescent protein expression in discrete cell populations, thus allowing live cell imaging during lymphoid organogenesis. We discuss approaches that allow characterisation of the cell types involved in the formation of lymphoid organs, including (i) functional assays in explant organ cultures and (ii) high-resolution whole-mount immunostaining methods, which are useful for the characterisation of specific cell populations in the context of the whole developing organ.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Owen JJ, Ritter MA. (1969) Tissue interaction in the development of thymus lymphocytes. J Exp Med 129, 431–42.
Petrie HT. (2002) Role of thymic organ structure and stromal composition in steady-state postnatal T-cell production. Immunol Rev 189, 8–19.
Le Douarin NM, Jotereau FV. (1975) Tracing of cells of the avian thymus through embryonic life in interspecific chimeras. J Exp Med 142, 17–40.
Foster K, Sheridan J, Veiga-Fernandes H, Roderick K, Pachnis V, Adams R, Blackburn C, Kioussis D, Coles M. (2008) Contribution of neural crest-derived cells in the embryonic and adult thymus. J Immunol 180, 3183–9.
Muller SM, Stolt CC, Terszowski G, Blum C, Amagai T, Kessaris N, Iannarelli P, Richardson WD, Wegner M, Rodewald HR. (2008) Neural crest origin of perivascular mesenchyme in the adult thymus. J Immunol 180, 5344–51.
Mebius RE. (2003) Organogenesis of lymphoid tissues. Nat Rev Immunol 3, 292–303.
Coles MC, Veiga-Fernandes H, Foster KE, Norton T, Pagakis SN, Seddon B, Kioussis D. (2006) Role of T and NK cells and IL7/IL7r interactions during neonatal maturation of lymph nodes. Proc Natl Acad Sci USA 103, 13457–62.
Mebius RE. (2007) Lymphoid organogenesis: educating stroma. Immunol Cell Biol 85, 79–80.
Veiga-Fernandes H, Coles MC, Foster KE, Patel A, Williams A, Natarajan D, Barlow A, Pachnis V, Kioussis D. (2007) Tyrosine kinase receptor RET is a key regulator of Peyer’s Patch organogenesis. Nature 446, 547–51.
Vondenhoff MF, Kraal G, Mebius RE. (2007) Lymphoid organogenesis in brief. Eur J Immunol 37(Suppl 1), S46–52.
Yoshida H, Kawamoto H, Santee SM, Hashi H, Honda K, Nishikawa S, Ware CF, Katsura Y, Nishikawa SI. (2001) Expression of alpha(4)beta(7) integrin defines a distinct pathway of lymphoid progenitors committed to T cells, fetal intestinal lymphotoxin producer, NK, and dendritic cells. J Immunol 167, 2511–21.
Fukuyama S, Kiyono H. (2007) Neuroregulator RET initiates Peyer’s-patch tissue genesis. Immunity 26, 393–5.
Festenstein R, Tolaini M, Corbella P, Mamalaki C, Parrington J, Fox M, Miliou A, Jones M, Kioussis D. (1996) Locus control region function and heterochromatin-induced position effect variegation. Science 271, 1123–5.
Kioussis D, Festenstein R. (1997) Locus control regions: overcoming heterochromatin-induced gene inactivation in mammals. Curr Opin Genet Dev 7, 614–9.
Zambrowicz BP, Imamoto A, Fiering S, Herzenberg LA, Kerr WG, Soriano P. (1997) Disruption of overlapping transcripts in the ROSA beta geo 26 gene trap strain leads to widespread expression of beta-galactosidase in mouse embryos and hematopoietic cells. Proc Natl Acad Sci USA 94, 3789–94.
Srinivas S, Watanabe T, Lin CS, William CM, Tanabe Y, Jessell TM, Costantini F. (2001) Cre reporter strains produced by targeted insertion of EYFP and ECFP into the ROSA26 locus. BMC Dev Biol 1, 4.
de Boer J, Williams A, Skavdis G, Harker N, Coles M, Tolaini M, Norton T, Williams K, Roderick K, Potocnik AJ, Kioussis D. (2003) Transgenic mice with hematopoietic and lymphoid specific expression of Cre. Eur J Immunol 33, 314–25.
Hoffman RM. (2005) The multiple uses of fluorescent proteins to visualize cancer in vivo. Nat Rev Cancer 5, 796–806.
Kurebayashi S, Ueda E, Sakaue M, Patel DD, Medvedev A, Zhang F, Jetten AM. (2000) Retinoid-related orphan receptor gamma (RORgamma) is essential for lymphoid organogenesis and controls apoptosis during thymopoiesis. Proc Natl Acad Sci USA 97, 10132–7.
Sun Z, Unutmaz D, Zou YR, Sunshine MJ, Pierani A, Brenner-Morton S, Mebius RE, Littman DR. (2000) Requirement for RORgamma in thymocyte survival and lymphoid organ development. Science 288, 2369–73.
Eberl G, Marmon S, Sunshine MJ, Rennert PD, Choi Y, Littman DR. (2004) An essential function for the nuclear receptor RORgamma(t) in the generation of fetal lymphoid tissue inducer cells. Nat Immunol 5, 64–73.
Danielian PS, Muccino D, Rowitch DH, Michael SK, McMahon AP. (1998) Modification of gene activity in mouse embryos in utero by a tamoxifen-inducible form of Cre recombinase. Curr Biol 8, 1323–6.
Gordon J, Xiao S, Hughes B 3rd, Su DM, Navarre SP, Condie BG, Manley NR. (2007) Specific expression of lacZ and Cre recombinase in fetal thymic epithelial cells by multiplex gene targeting at the Foxn1 locus. BMC Dev Biol 7, 69.
Kisanuki YY, Hammer RE, Miyazaki J, Williams SC, Richardson JA, Yanagisawa M. (2001) Tie2-Cre transgenic mice: a new model for endothelial cell-lineage analysis in vivo. Dev Biol 230, 230–42.
Bajenoff M, Egen JG, Qi H, Huang AY, Castellino F, Germain RN. (2007) Highways, byways and breadcrumbs: directing lymphocyte traffic in the lymph node. Trends Immunol 28, 346–52.
Bajenoff M, Germain RN. (2007) Seeing is believing: a focus on the contribution of microscopic imaging to our understanding of immune system function. Eur J Immunol 37(Suppl 1), S18–33.
Acknowledgments
Work in this chapter was funded by the Medical Research Council (MRC), UK. We wish to thank Vassilis Pachnis for helpful discussion; T. Norton and K. Williams for technical assistance. We also wish to thank Prof. D. Vestweber for kindly providing endomucin antibody. H.V.-F. and K.F. were supported by a grant from the European Union: Molecular Imaging LSHG-CT-2003-503259
Supplementary Video 1. Time-lapse video showing mobility of GFP cells in the wall of the gut (low magnification). This video shows a time-lapse sequence of E15.5 intestines. Time-lapse images were taken for 90Â min.
Supplementary Video 2. Three-dimensional reconstitution of an embryo section of E13.5. This sample was immunostained with anti-GFP (green) and the embryo structure is depicted in grey color. This video shows the developing lymph nodes and thymus. Magnification ×10.
Supplementary Video 3. Three-dimensional reconstitution of an adult thymus section. This sample was immunostained with anti-endomucin (red). This video shows the vessel and capillary endomucin-positive network within the thymus. Magnification ×40.
Paraformaldehyde fixation times vary according to size of tissues or organs. As guidance, E15.5 intestine will require 15Â min fixation at room temperature. These conditions may however vary according to the tissue and antibodies being used.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2010 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Veiga-Fernandes, H., Foster, K., Patel, A., Coles, M., Kioussis, D. (2010). Visualisation of Lymphoid Organ Development. In: Marelli-Berg, F., Nourshargh, S. (eds) T-Cell Trafficking. Methods in Molecular Biology, vol 616. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60761-461-6_11
Download citation
DOI: https://doi.org/10.1007/978-1-60761-461-6_11
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-60761-460-9
Online ISBN: 978-1-60761-461-6
eBook Packages: Springer Protocols