Biology subjects

Bartunek, P.

Publications and source records attributed to Bartunek, P..

5 recordsLinked to original sources

bioRxiv2022

Bioluminescent zebrafish transplantation model for drug discovery

In the last decade, zebrafish have accompanied the mouse as a robust animal model for cancer research. The possibility of screening small-molecule inhibitors in a large number of zebrafish embryos makes this model particularly valuable. However, the dynamic visualization of fluorescently labeled tumor cells needs to be complemented by a more sensitive, easy, and rapid mode for evaluating tumor growth in vivo to enable high-throughput screening of clinically relevant drugs. In this study we proposed and validated a pre-clinical screening model for drug discovery by utilizing bioluminescence as our readout for the determination of transplanted cancer cell growth and inhibition in zebrafish embryos. For this purpose, we used NanoLuc luciferase, which ensured rapid cancer cell growth quantification in vivo with high sensitivity and low background when compared to conventional fluorescence measurements. This allowed us large-scale evaluation of in vivo drug responses of 180 kinase inhibitors in zebrafish. Our bioluminescent screening platform could facilitate identification of new small-molecules for targeted cancer therapy as well as for drug repurposing.

pharmacology and toxicology↗

bioRxiv2021

Colony stimulating factor 1 signaling regulates myeloid fates in zebrafish via distinct action of its receptors and ligands

Macrophage colony-stimulating factor receptor (M-CSFR/CSF1R) signaling is crucial for the differentiation, proliferation, and survival of myeloid cells. Therapeutic targeting of the CSF1R pathway is a promising strategy in many human diseases, including neurological disorders or cancer. Zebrafish are commonly used for human disease modeling and preclinical therapeutic screening. Therefore, it is necessary to understand the proper function of cytokine signaling in zebrafish to reliably model human-related diseases. Here, we investigate the roles of zebrafish Csf1rs and their ligands - Csf1a, Csf1b and Il34, in embryonic and adult myelopoiesis. The proliferative effect of exogenous Csf1a on embryonic macrophages is connected to both receptors as it is diminished in both csf1ra{Delta}5bp and csf1rb{Delta}4bp mutants. There is no evident effect of Csf1b in zebrafish embryonic myelopoiesis. Further, we uncover an unknown role of Csf1rb in zebrafish granulopoiesis. Deregulation of Csf1rb signaling leads to failure in myeloid differentiation resulting in neutropenia throughout the whole lifespan. Surprisingly, Il34 signaling through Csf1rb seems to be of high importance as both csf1rb{Delta}4bp and il34{Delta}5bp deficient zebrafish larvae lack granulocytes. Our single-cell RNA sequencing analysis of adult whole kidney marrow (WKM) hematopoietic cells suggests that csf1rb is expressed mainly by blood and myeloid progenitors and that the expression of csf1ra and csf1rb is non-overlapping. We point out differentially expressed genes important in hematopoietic cell differentiation and immune response in selected WKM populations. Our findings could improve the understanding of myeloid cell function and lead to the further study of CSF1R pathway deregulation in disease, mostly in cancerogenesis. Key pointsO_LIcsf1ra and csf1rb are indispensable for macrophage differentiation and together with csf1a regulate embryonic macrophage fates in zebrafish C_LIO_LIcsf1rb is important for granulocyte differentiation and migration and together with il34 it regulates embryonic granulocytic fates in zebrafish C_LI

developmental biology↗

bioRxiv2020

Zebrafish Kit ligands cooperate with erythropoietin to promote erythroid cell expansion

Kit ligand (Kitlg) is pleiotropic cytokine with a prominent role in vertebrate erythropoiesis. Although the role of Kitlg in this process has not yet been reported in Danio rerio (zebrafish), in the present study, we show that its function is evolutionary conserved. Zebrafish possess two copies of Kitlg genes (Kitlga and Kitlgb) due to whole genome duplication. To determine the role of each ligand in zebrafish, we performed a series of ex vivo and in vivo gain- and loss-of-function experiments. First, we tested the biological activity of recombinant Kitlg proteins in suspension culture from zebrafish whole kidney marrow and we demonstrate that Kitlga is necessary for expansion of erythroid progenitors ex vivo. To further address the role of kitlga and kitlgb in hematopoietic development in vivo, we performed gain-of-function experiments in zebrafish embryos, showing that both ligands cooperate with erythropoietin (Epo) to promote erythroid cell expansion. Finally, using the kita mutant (kitab5/b5 or sparse), we show that Kita receptor is crucial for Kitlga/b cooperation with Epo in erythroid cells. In summary, using optimized suspension culture conditions with recombinant cytokines (Epo, Kitlga), we are reporting for the first time ex vivo suspension cultures of zebrafish hematopoietic progenitor cells, which can serve as an indispensable tool to study normal and aberrant hematopoiesis in zebrafish. Furthermore, we conclude that although partial functional diversification of Kit ligands has been described in other processes, in erythroid development, both paralogs play a similar role and their function is evolutionary conserved. Key points: O_LIKit signaling contributes to erythroid cell development and is conserved from fish to man C_LIO_LIEx vivo expansion and self-renewal of zebrafish erythroid progenitors requires addition of recombinant Kitlga C_LI

developmental biology↗

bioRxiv2018

Heterocyclic sterol probes for live monitoring of sterol trafficking and lysosomal storage disorders

The monitoring of intracellular cholesterol homeostasis and trafficking is of great importance because their imbalance leads to many pathologies. Reliable tools for cholesterol detection are in demand. This study presents the design and synthesis of fluorescent probes for cholesterol recognition and demonstrates their selectivity by a variety of methods. The construction of dedicated library of 14 probes was based on heterocyclic (pyridine)-sterol derivatives with various attached fluorophores. The most promising probe, a P1-BODIPY conjugate FP-5, was analyzed in detail and showed an intensive labeling of cellular membranes followed by intracellular redistribution into various cholesterol rich organelles and vesicles. FP-5 displayed a stronger signal, with faster kinetics, than the commercial TF-Chol probe. In addition, cells with pharmacologically disrupted cholesterol transport, or with a genetic mutation of cholesterol transporting protein NPC1, exhibited strong and fast FP-5 labeling in the endo/lysosomal compartment, co-localizing with filipin staining of cholesterol. Hence, FP-5 has high potential as a new probe for monitoring cholesterol trafficking and its disorders.\n\nSignificance statementCholesterol is a vital steroid molecule with many important functions in animal cells. Although its dysregulation is associated with an expanding list of clinically important pathologies, the study of its role is limited by a lack of reliable tools for live intracellular monitoring. This study demonstrates the applicability of a novel class of heterocyclic sterol probes. These probes exhibit fast cellular uptake with effective fluorescence labeling of sterol species in a variety of living cells, without a need for artificial carriers. When applied to Niemann-Pick disease type C1 cells, they identified massive accumulation of cholesterol in the endosome/lysosome compartment. Thus, several probes from the same series can also be used for visualizing lysosomal storage disorders and sterol transporting pathologies.

cell biology↗

bioRxiv2018

Zebrabase: An intuitive tracking solution for aquatic model organisms

Small fish species, like zebrafish or medaka, are constantly gaining popularity in basic research and disease modeling as a useful alternative to rodent model organisms. However, the tracking options for fish within a facility are rather limited. Here, we present an aquatic species tracking database, Zebrabase, developed in our zebrafish research and breeding facility that represents a practical and scalable solution and an intuitive platform for scientists, fish managers and caretakers, in both small and large facilities. Zebrabase is a scalable, crossplatform fish tracking database developed especially for research fish facilities but can be easily adapted for a wide variety of aquatic model organisms housed in tanks. It provides sophisticated tracking, reporting, and management functions that help keep the animalrelated records well-organized including a QR code functionality for tank labeling. The implementation of various user roles ensures a functional hierarchy and customized access to specific functions and data. In addition, Zebrabase enables easy personalization of rooms and racks, and its advanced statistics and reporting options make it an excellent tool for creating periodic reports of animal usage and productivity. The communication between the facility and researchers can be streamlined by the requesting capabilities of the database. Finally, Zebrabase also features an interactive breeding history and a smart interface with advanced visualizations and intuitive color coding allowing to speed up the processes.

developmental biology↗