Abstract
The capacity to visualize the lymphatic vasculature in three-dimensions has revolutionized our understanding of the morphogenetic mechanisms important for constructing the lymphatic vascular network during development. Two complementary approaches are commonly employed to assess the function of genes and signaling pathways important for development of the dermal lymphatic vasculature in the mouse embryo. The first of these is whole-mount immunostaining of embryonic skin to analyze dermal lymphatic vessel network patterning and morphology in two and three dimensions. The second is immunostaining of thin tissue sections to examine lymphatic vessel identity, lumen formation and protein localization within discrete lymphatic endothelial cells in a two-dimensional setting. Here we present detailed protocols for multicolor immunofluorescent immunostaining of embryonic dorsal skin and thin tissue cryosections. Each of these methods generates high-resolution images of the dermal lymphatic vasculature, yielding information integral to in-depth characterization of lymphatic vessel phenotypes in the developing mouse embryo.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Gerhardt H, Golding M, Fruttiger M, Ruhrberg C, Lundkvist A, Abramsson A, Jeltsch M, Mitchell C, Alitalo K, Shima D, Betsholtz C (2003) VEGF guides angiogenic sprouting utilizing endothelial tip cell filopodia. J Cell Biol 161(6):1163–1177. https://doi.org/10.1083/jcb.200302047
Norrmen C, Ivanov KI, Cheng J, Zangger N, Delorenzi M, Jaquet M, Miura N, Puolakkainen P, Horsley V, Hu J, Augustin HG, Yla-Herttuala S, Alitalo K, Petrova TV (2009) FOXC2 controls formation and maturation of lymphatic collecting vessels through cooperation with NFATc1. J Cell Biol 185(3):439–457. https://doi.org/10.1083/jcb.200901104
Bernier-Latmani J, Petrova TV (2016) High-resolution 3D analysis of mouse small-intestinal stroma. Nat Protoc 11(9):1617–1629. https://doi.org/10.1038/nprot.2016.092
Betterman KL, Paquet-Fifield S, Asselin-Labat ML, Visvader JE, Butler LM, Stacker SA, Achen MG, Harvey NL (2012) Remodeling of the lymphatic vasculature during mouse mammary gland morphogenesis is mediated via epithelial-derived lymphangiogenic stimuli. Am J Pathol 181(6):2225–2238. https://doi.org/10.1016/j.ajpath.2012.08.035
Gordon EJ, Rao S, Pollard JW, Nutt SL, Lang RA, Harvey NL (2010) Macrophages define dermal lymphatic vessel calibre during development by regulating lymphatic endothelial cell proliferation. Development 137(22):3899–3910. https://doi.org/10.1242/dev.050021
James JM, Nalbandian A, Mukouyama YS (2013) TGFbeta signaling is required for sprouting lymphangiogenesis during lymphatic network development in the skin. Development 140(18):3903–3914. https://doi.org/10.1242/dev.095026
Abràmoff MD, Magalhães PJ, Ram SJ (2004) Image processing with ImageJ. Biophoton Int 11(7):36–42
Shayan R, Karnezis T, Tsantikos E, Williams SP, Runting AS, Ashton MW, Achen MG, Hibbs ML, Stacker SA (2007) A system for quantifying the patterning of the lymphatic vasculature. Growth Factors 25(6):417–425. https://doi.org/10.1080/08977190801932550
Acknowledgments
This work was supported by grants from the NHMRC (APP1061365) and ARC (DP150103110). NLH is supported by an ARC Future Fellowship (FT130101254).
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2018 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Betterman, K.L., Harvey, N.L. (2018). Histological and Morphological Characterization of Developing Dermal Lymphatic Vessels. In: Oliver, G., Kahn, M. (eds) Lymphangiogenesis. Methods in Molecular Biology, vol 1846. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8712-2_2
Download citation
DOI: https://doi.org/10.1007/978-1-4939-8712-2_2
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-8711-5
Online ISBN: 978-1-4939-8712-2
eBook Packages: Springer Protocols