Abstract
In order to study basic mechanisms of sprouting angiogenesis, researchers worldwide rely on the use of model tissues such as rodent retina, which becomes vascularized postnatally, to study the growth of blood vessels. By definition, models have to be simple, recapitulating angiogenic processes in a stereotyped and relatively easy accessible manner, allowing the application of standardized analyses. These criteria also apply in an ideal manner to the embryonic mouse hindbrain, which becomes vascularized by sprouting angiogenesis from a preformed perineural vascular plexus, leading to the stereotypical formation of a capillary subventricular plexus. Similar to the retina model, between embryonic days 10.5 and 13.5, the hindbrain can be flat-mounted in an “open-book” preparation, allowing the analysis of the vascular bed in two-dimensional extension, of parameters like vessel density, morphology, and remodeling including branching and sprouting. In addition to sprouting angiogenesis, the hindbrain is a suitable model for investigating inductive mechanisms towards the blood–brain barrier phenotype of microvessels in the central nervous system. In this chapter, we describe how to fix, dissect, stain, and analyze the developing hindbrain vasculature.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Risau W (1997) Mechanisms of angiogenesis. Nature 386:671–674
Lee HS, Han J, Bai H-J et al (2009) Brain angiogenesis in developmental and pathological processes: regulation, molecular and cellular communication at the neurovascular interface. FEBS J 276:4622–4635
Fujita M, Cha YR, Pham VN et al (2011) Assembly and patterning of the vascular network of the vertebrate hindbrain. Development (Cambridge, England) 138:1705–1715
Quaegebeur A, Segura I, Carmeliet P (2010) Pericytes: blood–brain barrier safeguards against neurodegeneration? Neuron 68:321–323
Abbott NJ, Patabendige AAK, Dolman DEM et al (2010) Structure and function of the blood–brain barrier. Neurobiol Dis 37:13–25
Fantin A, Vieira JM, Plein A et al (2013) The embryonic mouse hindbrain as a qualitative and quantitative model for studying the molecular and cellular mechanisms of angiogenesis. Nat Protoc 8:418–429
Liebner S, Corada M, Bangsow T et al (2008) Wnt/beta-catenin signaling controls development of the blood–brain barrier. J Cell Biol 183:409–417
Maretto S, Cordenonsi M, Dupont S et al (2003) Mapping Wnt/beta-catenin signaling during mouse development and in colorectal tumors. Proc Natl Acad Sci U S A 100:3299–3304
Liebner S, Plate KH (2010) Differentiation of the brain vasculature: the answer came blowing by the Wnt. J Angiogenes Res 2:1
Cattelino A, Liebner S, Gallini R et al (2003) The conditional inactivation of the beta-catenin gene in endothelial cells causes a defective vascular pattern and increased vascular fragility. J Cell Biol 162:1111–1122
Stenman JM, Rajagopal J, Carroll TJ et al (2008) Canonical Wnt signaling regulates organ-specific assembly and differentiation of CNS vasculature. Science (New York, NY) 322:1247–1250
Daneman R, Agalliu D, Zhou L et al (2009) Wnt/beta-catenin signaling is required for CNS, but not non-CNS, angiogenesis. Proc Natl Acad Sci 106:641–646
Pitulescu ME, Schmidt I, Benedito R et al (2010) Inducible gene targeting in the neonatal vasculature and analysis of retinal angiogenesis in mice. Nat Protoc 5:1518–1534
Walantus W, Castaneda D, Elias L et al (2007) In Utero Intraventricular Injection and Electroporation of E15 Mouse Embryos. J Vis Exp 6:e239
Becker K, Jährling N, Saghafi S et al (2012) Chemical clearing and dehydration of GFP expressing mouse brains. PLoS ONE 7:e33916
Hama H, Kurokawa H, Kawano H et al (2011) Scale: a chemical approach for fluorescence imaging and reconstruction of transparent mouse brain. Nat Neurosci 14:1481–1488
Hägerling R, Pollmann C, Andreas M et al (2013) A novel multistep mechanism for initial lymphangiogenesis in mouse embryos based on ultramicroscopy. EMBO J 32:629–644
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2014 Springer Science+Business Media New York
About this protocol
Cite this protocol
Ziegler, N., Plate, K.H., Liebner, S. (2014). Analysis of Angiogenesis in the Developing Mouse Central Nervous System. In: Milner, R. (eds) Cerebral Angiogenesis. Methods in Molecular Biology, vol 1135. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-0320-7_5
Download citation
DOI: https://doi.org/10.1007/978-1-4939-0320-7_5
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-0319-1
Online ISBN: 978-1-4939-0320-7
eBook Packages: Springer Protocols