Biology subjects

Weischer, S.

Publications and source records attributed to Weischer, S..

5 recordsLinked to original sources

bioRxiv2025

Optical tissue clearing and 3D imaging of intact primate testicular tissue: a novel technology development

Classical histology struggles to preserve three-dimensional spatial context, prompting the emergence of optical tissue clearing techniques that enable imaging of intact specimens at cellular or subcellular resolution. These techniques have revolutionised fields like cell biology, developmental biology, and neuroscience. However, their application in reproductive biology remains unexplored - particularly in studying the complexities of testicular development. We developed a novel, efficient and affordable toolbox for studying intact testicular tissues, PT-CLEAR3D, that stands for primate testis - whole mount staining, tissue clearing and three-dimensional imaging. Intact testicular tissues from humans (transgender model), common marmosets and macaques underwent antibody labelling, clearing with organic solvents, and three-dimensional imaging using light sheet fluorescence microscopy. Marker specificity was confirmed by immunofluorescence staining of 3 and 25 {micro}m testicular sections, followed by imaging with confocal. The testicular structure was evaluated using several markers: spermatogonia (melanoma-associated antigen 4), least differentiated spermatogonia (Piwi-like protein 4), Sertoli cells (vimentin and SRY-Box transcription Factor 9), peritubular myoid cells and vasculature (alpha-smooth muscle actin), and NucSpot as a nuclear dye. PT-CLEAR3D efficiently achieved optical transparency while a commercial kit that was ran in parallel was inefficient. This study presents a pioneering three-dimensional visualization of intact testicular samples of up to 50 mm3 in size and imaging depth of up to 4.5 mm across three primate species. Remarkably, PT-CLEAR3D revealed critical details at both tissue and cellular levels such as the spatial distribution of germ and somatic cells, cellular bridges, and vasculature. Furthermore, PT-CLEAR3D enabled three-dimensional reconstructions that effectively reduce confirmation bias enhancing our observation of spermatogonial clones organized as single cells, pairs, and quartets. Importantly, it adeptly identified testicular pathology and the persistence of germ cell clones in select tubules within the transgender testis following hormonal suppression of spermatogenesis. This technological development offers a versatile toolbox with benefits such as applicability across multiple species, fluorophore multiplexing, compatibility with different fixatives and deep tissue volumetric imaging with cellular resolution. Overall, PT-CLEAR3D establishes a foundation for spatial evaluation of testicular development, presenting substantial potential for advancing our understanding of the intricate kinetics of spermatogenesis in health and disease.

developmental biology↗

bioRxiv2024

Membrane tension regulation is required for wound repair

Disruptions of the eukaryotic plasma membrane due to chemical and mechanical challenges are frequent and detrimental, and thus need to be repaired to maintain proper cell function and avoid cell death. However, the cellular mechanisms involved in wound resealing and restoration of homeostasis are diverse and contended. Here, we show that clathrin-mediated endocytosis is induced at later stages of plasma membrane wound repair following the actual resealing of the wound. This compensatory endocytosis occurs near the wound, predominantly at sites of previous early endosome exocytosis which is required in the initial stage of membrane resealing, suggesting a spatio-temporal co-ordination of exo- and endocytosis during wound repair. Using cytoskeletal alterations and modulation of membrane tension and membrane area, we identify membrane tension as a major regulator of the wounding-associated exo- and endocytic events that mediate efficient wound repair. Thus, membrane tension changes are a universal trigger for plasma membrane wound repair modulating the exocytosis of early endosomes required for resealing and subsequent clathrin-mediated endocytosis acting at later stages to restore cell homeostasis and function.

cell biology↗

bioRxiv2022

svep1 and tie1 genetically interact and affect aspects of facial lymphatic development in a Vegfc-independent manner

Multiple factors are required to form functional lymphatic vessels. Here, we uncover an essential role for the secreted protein Svep1 and the transmembrane receptor Tie1 during the development of subpopulations of the zebrafish facial lymphatic network. This specific aspect of the facial network forms independently of Vegfc signalling, which otherwise is the most prominent signalling axis in all other lymphatic beds. Additionally, we find that multiple specific and newly uncovered phenotypic hallmarks of svep1 mutants are also present in tie1, but not in tie2 or vegfc mutants. These phenotypes are observed in the lymphatic vasculature of both head and trunk, as well as in the development of the dorsal longitudinal anastomotic vessel (DLAV) under reduced flow conditions. Furthermore, we show genetic interaction between svep1 and tie1 during the migration of parachordal lymphangioblasts (PLs). Therefore, our study demonstrates an important function for Tie1 signalling during lymphangiogenesis as well as blood vessel development in zebrafish and provides the first in vivo evidence for zebrafish Svep1 and Tie1 interaction. Since compound heterozygous mutations for SVEP1 and TIE2 have recently been reported in human glaucoma patients, our data have clinical relevance in demonstrating a role for Svep1 in Tie signalling in an in vivo setting.

developmental biology↗

bioRxiv2022

Lymphatic endothelium arises directly from specialized angioblasts

The lymphatic vasculature is essential for tissue fluid homeostasis, immune cell surveillance and dietary lipid absorption, and has emerged as a key regulator of organ growth and repair1. Despite significant advances in our understanding of lymphatic function, the precise developmental origin of lymphatic endothelial cells (LECs) has remained a point of debate for over a century2-5. It is currently widely accepted that most LECs are derived from venous endothelium4,6, although other sources have been described, including mesenchymal cells3, hemogenic endothelium7 and musculoendothelial progenitors8,9. Here we show that the initial expansion of mammalian LECs is driven primarily by the in situ differentiation of specialized angioblasts and not migration from venous endothelium. Single-cell RNA sequencing and genetic lineage tracing experiments in mouse revealed a population of Etv2+Prox1+ lymphangioblasts that arise directly from paraxial mesoderm-derived progenitors. Conditional lineage labelling and morphological analyses showed that these specialized angioblasts emerge within a tight spatiotemporal window, and give rise to LECs in numerous tissues. Analysis of early LEC proliferation and migration supported these findings, suggesting that emergence of LECs from venous endothelium is limited. Collectively, our data reconcile discrepancies between previous studies and indicate that LECs form through both de novo specification from lymphangioblasts and transdifferentiation from venous endothelium.

developmental biology↗

bioRxiv2020

Intravital Deep-Tumor Single-Beam 2-, 3- and 4-Photon Microscopy

Three-photon excitation has recently been introduced to perform intravital microscopy in deep, previously inaccessible layers of the brain. The applicability of deep-tissue three-photon excitation in more heterogeneously structured, dense tissue types remains, however, unclear. Here we show that in tumors and bone, high-pulse-energy low-duty-cycle infrared excitation near 1300 and 1700 nm enables two-up to fourfold increased tissue penetration compared to conventional 2-photon excitation. Using a single laser line, simultaneous 2-, 3- and 4-photon processes are effectively induced, enabling the simultaneous detection of blue to far-red fluorescence together with second and third harmonic generation. This enables subcellular resolution at power densities in the focus that are not phototoxic to live cells and without color aberration. Thus, infrared high-pulse-energy low-duty-cycle excitation advances deep intravital microscopy in strongly scattering tissue and, in a single scan, delivers rich multi-parameter datasets from cells and complex organ structures.

cancer biology↗