Nanobody immunolabelling and three-dimensional imaging reveals spatially restricted LYVE1 expression by kidney lymphatic vessels in mice

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Abstract
Lymphatic vessels are complex three-dimensional (3D) structures that facilitate tissue fluid clearance and regulate immune responses in health and inflammatory contexts. Recent advances in wholemount immunolabelling and 3D imaging have provided insights into organ-specific heterogeneity of lymphatic structure and function. However, the visualisation of lymphatic vessels deep within an intact organ remains a challenge. We hypothesised that nanobodies, single-domain antibodies raised in camelid species, would result in improved labelling of lymphatics in intact mouse organs, without loss of information due to organ sectioning or inadequate penetration. We generated and characterised nanobody clones targeting lymphatic vessel endothelial hyaluronan receptor 1 (LYVE1), a marker of lymphatic capillaries. Nanobodies were superior at penetrating whole mouse organs and enhanced labelling of lymphatics compared with a conventional anti-LYVE1 polyclonal antibody. Utilising this new tool, we found that kidney lymphatics; an organ in which labelling of lymphatics is challenging, have spatially restricted LYVE1 expression compared with lymphatics of skin, heart, and lung. The timing of this LYVE1 spatial restriction coincides with the the early postnatal period in mice. Our findings highlight an unexpected, organ-specific characteristic of kidney lymphatic vessels, whilst providing a novel experimental tool for characterisation, isolation, or perturbation of lymphatic vessels in health and disease.

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