Biology subjects

Samasa, B.

Publications and source records attributed to Samasa, B..

4 recordsLinked to original sources

bioRxiv2022

mRNA-mediated induced regeneration of the corneal endothelium

Loss of vision due to corneal endothelial dysfunction affects millions worldwide. The development of new treatments is hampered by the incomplete knowledge of the regenerative capacity of corneal endothelial cells in vivo. Herein, we developed a mouse model to directly monitor corneal endothelial regeneration in real time, and at the single cell level, by two-photon microscopy. We show that the mouse corneal endothelium recapitulates the main features of human endothelial physiology, including complete cellular quiescence and a decline in cell density with aging. Critically, we demonstrate the endogenous regenerative potential of the tissue by capturing the proliferation of corneal endothelial cells during repair of large injuries. By single cell lineage tracing analysis, we provide evidence that corneal endothelial cells are equipotent in their ability to activate the cell cycle and contribute to tissue regeneration. Based on these findings we developed a feasible therapeutic approach to stimulate the regeneration of the corneal endothelium, using modified mRNA technology. To reprogram corneal endothelial cells in vivo and unlock their ability to escape quiescence, we combined five modified mRNAs encoding for proteins involved in cell cycle activation. Injection of the encapsulated mRNAs directly into the eye of older mice induced transient proliferation of corneal endothelial cells that led to an increase in endothelial cell density, effectively reversing the effect of aging. This therapeutic strategy offers a compelling paradigm for treating ocular disease and modulating tissue regeneration in organs with limited endogenous ability.

cell biology↗

bioRxiv2021

Long-Term Imaging of Living Adult Zebrafish

The zebrafish has become a widely used animal model due in large part to its accessibility to and usefulness for high-resolution optical imaging. Although zebrafish research has historically focused mostly on early development, in recent years the fish has increasingly been used to study regeneration, cancer metastasis, behavior, and other processes taking place in juvenile and adult animals. However, imaging of live adult zebrafish is extremely challenging, with survival of adult fish limited to a few tens of minutes using standard imaging methods developed for zebrafish embryos and larvae. Here, we describe a new method for imaging intubated adult zebrafish using a specially designed 3D printed chamber for long-term imaging of adult zebrafish on inverted microscope systems. We demonstrate the utility of this new system by nearly day-long observation of neutrophil recruitment to a wound area in living double-transgenic adult casper zebrafish with fluorescently labeled neutrophils and lymphatic vessels.

developmental biology↗

bioRxiv2021

The nanoscale organization of the Wnt signaling integrator Dishevelled in the development-essential vegetal cortex domain of an egg and early embryo

Wnt/{beta}-catenin (cWnt) signaling is a crucial regulator of development and Dishevelled (Dsh/Dvl) functions as an integral part of this pathway by linking Wnt binding to the frizzled:LRP5/6 receptor complex with {beta}-catenin-stimulated gene expression. In many cell types Dsh has been localized to ill-defined cytoplasmic puncta, however in sea urchin eggs and embryos confocal fluorescence microscopy has shown that Dsh is localized to puncta present in a novel and development-essential vegetal cortex domain (VCD). In the present study, we used super-resolution light microscopy and platinum replica TEM to provide the first views of the ultrastructural organization of Dsh within the sea urchin VCD. 3D-SIM imaging of isolated egg cortices demonstrated the concentration gradient-like distribution of Dsh in the VCD, whereas higher resolution STED imaging revealed that some individual Dsh puncta consisted of more than one fluorescent source. Platinum replica immuno-TEM localization showed that Dsh puncta on the cytoplasmic face of the plasma membrane consisted of aggregates of pedestal-like structures each individually labeled with the C-terminus specific Dsh antibody. These aggregates were resistant to detergent extraction and treatment with drugs that disrupt actin filaments or inhibit myosin II contraction, and coexisted with the first division actomyosin contractile ring. These results confirm and extend previous studies and reveal, for the first time in any cell type, the nanoscale organization of plasma membrane tethered Dsh. Our current working hypothesis is that these Dsh pedestals represent a prepositioned scaffold organization that is important for canonical Wnt pathway activation at the sea urchin vegetal organization and may also be relevant to the submembranous Dsh puncta present in other eggs and embryos.

cell biology↗

bioRxiv2020

Live Imaging of Intracranial Lymphatics in the Zebrafish

RationaleThe recent discovery of meningeal lymphatics in mammals is reshaping our understanding of fluid homeostasis and cellular waste management in the brain, but visualization and experimental analysis of these vessels is challenging in mammals. Although the optical clarity and experimental advantages of zebrafish have made this an essential model organism for studying lymphatic development, the existence of meningeal lymphatics has not yet been reported in this species. ObjectiveExamine the intracranial space of larval, juvenile, and adult zebrafish to determine whether and where intracranial lymphatic vessels are present. Methods and ResultsUsing high-resolution optical imaging of the meninges in living animals, we show that zebrafish possess a meningeal lymphatic network comparable to that found in mammals. We confirm that this network is separate from the blood vascular network and that it drains interstitial fluid from the brain. We document the developmental origins and growth of these vessels into a distinct network separated from the external lymphatics. Finally we show that these vessels contain immune cells and perform live imaging of immune cell trafficking and transmigration in meningeal lymphatics. ConclusionsThis discovery establishes the zebrafish as a important new model for experimental analysis of meningeal lymphatic development, and opens up new avenues for probing meningeal lymphatic function in health and disease.

developmental biology↗