Biology subjects

Hansmeier, N. R.

Publications and source records attributed to Hansmeier, N. R..

4 recordsLinked to original sources

bioRxiv2024

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

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 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.

immunology↗

bioRxiv2023

Mesenchymal Osr1+ cells regulate embryonic lymphatic vessel formation

The lymphatic system is formed during embryonic development by the commitment of specialized lymphatic endothelial cells (LECs) and their subsequent assembly in primary lymphatic vessels. While lymphatic cells are in continuous contact with mesenchymal cells during development and in adult tissues, the role of mesenchymal cells in lymphatic vasculature development remains poorly characterized. Here, we show that a subpopulation of mesenchymal cells expressing the transcription factor Osr1 are in close association with migrating LECs and established lymphatic vessels in mice. Lineage tracing experiments revealed that Osr1+ cells precede LEC arrival during lymphatic vasculature assembly in the back of the embryo. Using Osr1-deficient embryos and functional in vitro assays, we show that Osr1 acts in a non-cell autonomous manner controlling proliferation and early migration of LECs to peripheral tissues. Thereby, mesenchymal Osr1+ cells control in a bimodal manner the production of extracellular matrix scaffold components and signal ligands critical for lymphatic vessels formation.

developmental biology↗

bioRxiv2022

Cold-induced expression of a truncated Adenylyl Cyclase 3 acts as rheostat to brown fat function.

Promoting brown adipose tissue (BAT) activity has been recognized as innovative therapeutic approach to improve obesity and metabolic disease. Whilst the molecular circuitry underlying thermogenic activation of BAT is well understood, the processes underlying rheostatic regulation of BAT to maintain homeostasis and avoid excessive energy dissipation remain ill-defined. Increasing cyclic AMP (cAMP) biosynthesis is key for BAT activation. Here, we demonstrate that ADCY3, an adenylyl cyclase whose expression is induced during cold exposure and regulates cAMP homeostasis in thermogenic fat, is dispensable for BAT function in lean mice, but becomes critical during obesity. Furthermore, by combining RNA-seq with epigenomic H3K4me3 profiling, we detected a novel, cold-inducible promoter that generates a 5 truncated Adcy3-at mRNA isoform, Adcy3-at. Mice lacking only Adcy3-at, but not full-length Adcy3, displayed increased energy expenditure already under lean conditions and were protected against obesity and ensuing metabolic imbalances. Subcellularly, translated ADCY3-AT proteins are retained in the endoplasmic reticulum (ER), did not translocate to the cell membrane, and lacked enzymatic activity. By interacting with ADCY3, ADCY3-AT retained ADCY3 in the ER and, thereby, reduced the plasma membrane pool of ADCYs available for G-protein mediated cAMP synthesis. Thereby, ADCY3-AT acts as a signaling rheostat in BAT, limiting adverse consequences of uncurbed cAMP activity after long-term BAT activation. Adcy3-at induction was driven by a cold-induced, truncated isoform of the transcriptional cofactor PPARGC1A (PPARG Coactivator 1 Alpha, PPARGC1A-AT). Expression of Ppargc1a-at and Adcy3-at are evolutionary conserved, indicating that transcriptional rewiring by commissioning of alternative promoters is key for thermogenic fat function.

cell biology↗

bioRxiv2021

Vegfr3-tdTomato, a reporter mouse for microscopic visualization of lymphatic vessel by multiple modalities

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 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.

cell biology↗