Highlights 3D-polymerized cell dispersions allow evaluation of tissue clearing protocols Protein fluorescence retention can be tested during tissue clearing A semi-automated random walker enables segmentation of memory exceeding image data

The liver is a major biosynthetic and detoxifying organ in vertebrates, but also generates 25%–50% of the lymph passing through the thoracic duct and is thereby the organ with the highest contribution to lymph flow. In contrast to its metabolic function, the role of the liver for lymph generation and composition is presently severely understudied. We took a rigorous, volume imaging-based approach to describe the microarchitecture and spatial composition of the hepatic lymphatic vasculature with cellular resolution in whole mount immune stained specimen ranging from thick sections up to entire mouse liver lobes. Here, we describe that in healthy adult livers, lymphatic vessels were exclusively located within the portal tracts, where they formed a unique, highly ramified tree. Ragged, spiky initials enmeshed the portal veins along their entire length and communicated with long lymphatic vessels that followed the path of the portal vein in close association with bile ducts. Together these lymphatic vessels formed a uniquely shaped vascular bed with a delicate architecture highly adapted to the histological structure of the liver. Unexpectedly, with the exception of short collector stretches at the porta hepatis, which we identified as exit point of the liver lymph vessels, the entire hepatic lymph vessel system was comprised of capillary lymphatic endothelial cells only. Functional experiments confirmed the space of Disse as the origin of the hepatic lymph and flow via the space of Mall to the portal lymph capillaries. After entry into the lymphatic initials, the lymph drained retrograde to the portal blood flow towards the exit at the liver hilum. Perinatally, the liver undergoes complex changes transforming from the main hematopoietic to the largest metabolic organ. We investigated the time course of lymphatic vessel development and identified the hepatic lymphatics to emerge postnatally in a process that relies on input from the VEGF-C/VERGFR-3 growth factor—receptor pair for formation of the fully articulate hepatic lymph vessel bed.

Lymphatic vessels are indispensable for tissue fluid homeostasis, transport of solutes and dietary lipids and immune cell trafficking. In contrast to blood vessels, which are easily visible by their erythrocyte cargo, lymphatic vessels are not readily detected in the tissue context. Their invisibility interferes with the analysis of the three-dimensional lymph vessel structure in large tissue volumes and hampers dynamic intravital studies on lymphatic function and pathofunction. An approach to overcome these limitations are mouse models, which express transgenic fluorescent proteins under the control of tissue-specific promotor elements. We introduce here the BAC-transgenic mouse reporter strain Vegfr3-tdTomato that expresses a membrane-tagged version of tdTomato under control of Flt4 regulatory elements. Vegfr3-tdTomato mice inherited the reporter in a mendelian fashion and showed selective and stable fluorescence in the lymphatic vessels of multiple organs tested, including lung, kidney, heart, diaphragm, intestine, mesentery, liver and dermis. In this model, tdTomato expression was sufficient for direct visualisation of lymphatic vessels by epifluorescence microscopy. Furthermore, lymph vessels were readily visualized using a number of microscopic modalities including confocal laser scanning, light sheet fluorescence and two-photon microscopy. Due to the early onset of VEGFR-3 expression in venous embryonic vessels and the short maturation time of tdTomato, this reporter offers an interesting alternative to Prox1-promoter driven lymphatic reporter mice for instance to study the developmental differentiation of venous to lymphatic endothelial cells.

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