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The embryonic mouse hindbrain as a qualitative and quantitative model for studying the molecular and cellular mechanisms of angiogenesis

Nature Protocols volume 8, pages 418–429 (2013)Cite this article

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Abstract

The mouse embryo hindbrain is a robust and adaptable model for studying sprouting angiogenesis. It permits the spatiotemporal analysis of organ vascularization in normal mice and in mouse strains with genetic mutations that result in late embryonic or perinatal lethality. Unlike postnatal models such as retinal angiogenesis or Matrigel implants, there is no requirement for the breeding of conditional knockout mice. The unique architecture of the hindbrain vasculature allows whole-mount immunolabeling of blood vessels and high-resolution imaging, as well as easy quantification of angiogenic sprouting, network density and vessel caliber. The hindbrain model also permits the visualization of ligand binding to blood vessels in situ and the analysis of blood vessel growth within a natural multicellular microenvironment in which endothelial cells (ECs) interact with non-ECs to refine the 3D organ architecture. The entire procedure, from embryo isolation to imaging and through to results analysis, takes approximately 4 d.

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Figure 1: Time course of blood vessel growth in the mouse embryo hindbrain.
Figure 2: Immunolabeling to visualize developing blood vessels in the mouse embryo hindbrain.
Figure 3: AP fusion protein binding to the mouse embryo hindbrain.
Figure 4: Dissection of an E12.5 mouse embryo hindbrain.
Figure 5: Examples of vascular defects in the hindbrain of mice from Vegfa+/120 intercrosses.

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Acknowledgements

We thank the staff of the Biological Resources Unit at the UCL Institute of Ophthalmology for help with mouse husbandry and the Imaging Facility of the UCL Institute of Ophthalmology for maintenance of the confocal microscopes. The protocol described in this manuscript was developed with funding from the Wellcome Trust (095623/Z/11/Z) and the Medical Research Council (MRC) (project grant G0601093) to C.R. and PhD studentships from the British Heart Foundation to A.F. (ref. 44626) and A.P. (ref. FS/10/54/28680), as well as a PhD studentship from the MRC to C.H.M. (G0700020).

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  1. Joaquim M Vieira

    Present address: Present address: Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, UK.,

Authors and Affiliations

  1. UCL Institute of Ophthalmology, University College London, London, UK

    Alessandro Fantin, Joaquim M Vieira, Alice Plein, Charlotte H Maden & Christiana Ruhrberg

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  1. Alessandro Fantin
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Contributions

A.F., A.P., C.H.M. and C.R. prepared the figures and Supplementary Videos 1 and 2. All authors contributed to the development of the protocol and wrote the manuscript.

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Correspondence to Christiana Ruhrberg.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Figure 1

Histochemical IB4 staining of an E12.5 mouse embryo hindbrain. An E12.5 hindbrain was labelled with biotinylated IB4 followed by HRP-conjugated streptavidin, flatmounted and imaged at the indicated magnifications. (a-c) Flatmounting the hindbrain with the pial side up allows visualisation of radial vessels entering the brain. (d-f) Flatmounting the hindbrain with the ventricular side up allows visualisation of the subventricular vascular plexus. The dotted boxes in (a,d) indicate the areas shown at higher magnification in (b,e), the dotted boxes in (b,e) those shown at higher magnification in (c,f), respectively; the size of each field in (c,f) is 500 μm x 500 μm, i.e. 0.25 mm2. (g,h) Counting of radial vessels and vascular intersections in the fields shown in (c,f); green dots were used to track vessels that have been counted. (i,j) A 100 μm transverse vibratome section through the E12.5 hindbrain shown in (a); radial vessels (rv) extend from the pial side of the hindbrain and form the SVP on the ventricular side; the boxed area in (i) is shown at a higher magnification in panel (j). Scale bars: (a,d) − 1 mm; (b,e,i) − 200 μm, (c,f) - 100 μm. All animal procedures were performed in accordance with institutional and UK Home Office guidelines. (PDF 1660 kb)

Supplementary Figure 2

Dissection of an E11.5 wild type mouse embryo hindbrain. Selected snapshots from supplemental movie 1 demonstrate key steps in hindbrains dissection. An E 11.5 embryo is decapitated roughly at shoulder height (a), and the anterior part of the head is removed (b). The hindbrain is revealed after peeling away the dorsal skin and hindbrain roof-plate membrane (c), allowing the hindbrain to unfurl (d-f). Next, the non-neural tissue underneath the hindbrain is peeled away (g,h). Finally, the hindbrain is trimmed to remove the spinal cord (i) and midbrain (j). The ventricular (k) and pial side (l) of the dissected hindbrain are shown. All animal procedures were performed in accordance with institutional and UK Home Office guidelines. (PDF 4927 kb)

Supplementary Video 1

Dissection of an E11.5 wild type mouse embryo hindbrain. This movie shows the procedure for dissecting an E11.5 hindbrain from a wild type mouse; the same procedure should be followed to dissect embryos at E12.5 or genetically altered mouse embryos at E11.5 or E12.5. Please turn on the sound to listen to an accompanying commentary explaining the dissection stages. All animal procedures were performed in accordance with institutional and UK Home Office guidelines. (MPG 26776 kb)

Supplementary Video 2

Dissection of an E11.5 Tie2-Cre;Nrp1fl/− mouse hindbrain. This movie shows the dissection of an E11.5 hindbrain from a Tie2-Cre;Nrp1fl/− mouse embryo that lacks NRP1 in endothelial cells. Note that vascular malformations can be observed already during the dissection procedure. All animal procedures were performed in accordance with institutional and UK Home Office guidelines. (MPG 29014 kb)

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Fantin, A., Vieira, J., Plein, A. et al. The embryonic mouse hindbrain as a qualitative and quantitative model for studying the molecular and cellular mechanisms of angiogenesis. Nat Protoc 8, 418–429 (2013). https://doi.org/10.1038/nprot.2013.015

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