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

BACKGROUND: Lymphatic vessels are complex three-dimensional (3D) structures that facilitate tissue fluid clearance and regulate immune responses during health and inflammation. Recent advances in wholemount immunolabelling and 3D imaging have provided insights into organ-specific heterogeneity of lymphatic vessel 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 tissue sectioning or inadequate penetration of conventional antibodies into intact tissues. RESULTS: We generated and characterised nanobody clones targeting lymphatic vessel endothelial hyaluronan receptor 1 (LYVE1), a marker of lymphatic vessels. Compared with a conventional anti-LYVE1 polyclonal antibody, nanobodies were able to penetrate whole mouse organs more rapidly and at a greater depth, facilitating labelling of lymphatic vessel networks within intact mouse organs. Utilising this new tool, we found that lymphatics within the kidney, an organ in which labelling of these vessels is challenging, have spatially restricted LYVE1 expression compared with lymphatics of skin, heart, and lung. The appearance of LYVE1- kidney lymphatics coincided with the early postnatal period in mice, with single-cell RNA sequencing analysis revealing their transcriptome to be enriched for markers of either collecting vessels or lymphatic valves. CONCLUSIONS: Our findings highlight a characteristic feature of kidney lymphatic vessels, whilst providing a novel experimental tool for characterisation, isolation, or perturbation of lymphatic vessels in health and disease.