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Low transfection efficiency in endothelial cells (EC) is still a bottleneck for the majority of siRNA-based vascular delivery approaches. In this work, we developed a lipid-based nanoparticle (LNP) formulation based on a combination of a permanently charged cationic lipid-DOTAP and a conditionally ionized cationic lipid-MC3 (DOTAP/MC3) for the enhanced delivery of siRNA into EC. Compared with a single DOTAP or MC3-based benchmark LNP, we demonstrated that the DOTAP/MC3 LNP formulation shows the best transfection efficiency both in primary EC in vitro and in endothelium in zebrafish. The high transfection activity of the DOTAP/MC3 LNP formulation is achieved by a combination of improved endothelial association mediated by DOTAP and MC3-triggered efficient siRNA intracellular release in EC. Furthermore, AbVCAM-1-coupled DOTAP/MC3 LNP-mediated siRNARelA transfection showed pronounced anti-inflammatory effects in inflammatory-activated primary EC by effectively blocking the NF-κB pathway. In conclusion, the combination of permanent and ionizable cationic lipids in LNP formulation provides an effective endothelial cell delivery of siRNA. [ABSTRACT FROM AUTHOR]

Vascular networks comprise endothelial cells and mural cells, which include pericytes and smooth muscle cells. To elucidate the mechanisms controlling mural cell recruitment during development and tissue regeneration, we studied zebrafish caudal fin arteries. Mural cells colonizing arteries proximal to the body wrapped around them, whereas those in more distal regions extended protrusions along the proximo-distal vascular axis. Both cell populations expressed platelet-derived growth factor receptor ß (pdgfrb) and the smooth muscle cell marker myosin heavy chain 11a (myh11a). Most wrapping cells in proximal locations additionally expressed actin alpha2, smooth muscle (acta2). Loss of Pdgfrb signalling specifically decreased mural cell numbers at the vascular front. Using lineage tracing, we demonstrate that precursor cells located in periarterial regions and expressing Pgdfrb can give rise to mural cells. Studying tissue regeneration, we did not find evidence that newly formed mural cells were derived from pre-existing cells. Together, our findings reveal conserved roles for Pdgfrb signalling in development and regeneration, and suggest a limited capacity of mural cells to selfrenew or contribute to other cell types during tissue regeneration. [ABSTRACT FROM AUTHOR]

Liposomes can efficiently deliver messenger RNA (mRNA) into cells. When mRNA cocktails encoding different proteins are needed, a considerable challenge is to efficiently deliver all mRNAs into the cytosol of each individual cell. In this work, two methods are explored to co‐deliver varying ratiometric doses of mRNA encoding red (R) or green (G) fluorescent proteins and it is found that packaging mRNAs into the same lipoplexes (mingle‐lipoplexes) is crucial to efficiently deliver multiple mRNA types into the cytosol of individual cells according to the pre‐defined ratio. A mixture of lipoplexes containing only one mRNA type (single‐lipoplexes), however, seem to follow the "first come – first serve" principle, resulting in a large variation of R/G uptake and expression levels for individual cells leading to ratiometric dosing only on the population level, but rarely on the single‐cell level. These experimental observations are quantitatively explained by a theoretical framework based on the stochasticity of mRNA uptake in cells and endosomal escape of mingle‐ and single‐lipoplexes, respectively. Furthermore, the findings are confirmed in 3D retinal organoids and zebrafish embryos, where mingle‐lipoplexes outperformed single‐lipoplexes to reliably bring both mRNA types into single cells. This benefits applications that require a strict control of protein expression in individual cells. [ABSTRACT FROM AUTHOR]

Liposomes can efficiently deliver messenger RNA (mRNA) into cells. When mRNA cocktails encoding different proteins are needed, a considerable challenge is to efficiently deliver all mRNAs into the cytosol of each individual cell. In this work, two methods are explored to co‐deliver varying ratiometric doses of mRNA encoding red (R) or green (G) fluorescent proteins and it is found that packaging mRNAs into the same lipoplexes (mingle‐lipoplexes) is crucial to efficiently deliver multiple mRNA types into the cytosol of individual cells according to the pre‐defined ratio. A mixture of lipoplexes containing only one mRNA type (single‐lipoplexes), however, seem to follow the "first come – first serve" principle, resulting in a large variation of R/G uptake and expression levels for individual cells leading to ratiometric dosing only on the population level, but rarely on the single‐cell level. These experimental observations are quantitatively explained by a theoretical framework based on the stochasticity of mRNA uptake in cells and endosomal escape of mingle‐ and single‐lipoplexes, respectively. Furthermore, the findings are confirmed in 3D retinal organoids and zebrafish embryos, where mingle‐lipoplexes outperformed single‐lipoplexes to reliably bring both mRNA types into single cells. This benefits applications that require a strict control of protein expression in individual cells. [ABSTRACT FROM AUTHOR]

Liposomes can efficiently deliver messenger RNA (mRNA) into cells. When mRNA cocktails encoding different proteins are needed, a considerable challenge is to efficiently deliver all mRNAs into the cytosol of each individual cell. In this work, two methods are explored to co‐deliver varying ratiometric doses of mRNA encoding red (R) or green (G) fluorescent proteins and it is found that packaging mRNAs into the same lipoplexes (mingle‐lipoplexes) is crucial to efficiently deliver multiple mRNA types into the cytosol of individual cells according to the pre‐defined ratio. A mixture of lipoplexes containing only one mRNA type (single‐lipoplexes), however, seem to follow the "first come – first serve" principle, resulting in a large variation of R/G uptake and expression levels for individual cells leading to ratiometric dosing only on the population level, but rarely on the single‐cell level. These experimental observations are quantitatively explained by a theoretical framework based on the stochasticity of mRNA uptake in cells and endosomal escape of mingle‐ and single‐lipoplexes, respectively. Furthermore, the findings are confirmed in 3D retinal organoids and zebrafish embryos, where mingle‐lipoplexes outperformed single‐lipoplexes to reliably bring both mRNA types into single cells. This benefits applications that require a strict control of protein expression in individual cells. [ABSTRACT FROM AUTHOR]

Chemokines are vertebrate-specific, structurally related proteins that function primarily in controlling cell movements by activating specific 7-transmembrane receptors. Chemokines play critical roles in a large number of biological processes and are also involved in a range of pathological conditions. For these reasons, chemokines are at the focus of studies in developmental biology and of clinically oriented research aimed at controlling cancer, inflammation, and immunological diseases. The small size of the zebrafish embryos, their rapid external development, and optical properties as well as the large number of eggs and the fast expansion in genetic tools available make this model an extremely useful one for studying the function of chemokines and chemokine receptors in an in vivo setting. Here, we review the findings relevant to the role that chemokines play in the context of directed single-cell migration, primarily in neutrophils and germ cells, and compare it to the collective cell migration of the zebrafish lateral line. We present the current knowledge concerning the formation of the chemokine gradient, its interpretation within the cell, and the molecular mechanisms underlying the cellular response to chemokine signals during directed migration. [ABSTRACT FROM AUTHOR]

SoxF family members have been linked to arterio-venous specification events and human pathological conditions, but in contrast to Sox17 and Sox18, a detailed in vivo analysis of a Sox7 mutant model is still lacking. In this study we generated zebrafish sox7 mutants to understand the role of Sox7 during vascular development. By in vivo imaging of transgenic zebrafish lines we show that sox7 mutants display a short circulatory loop around the heart as a result of aberrant connections between the lateral dorsal aorta (LDA) and either the venous primary head sinus (PHS) or the common cardinal vein (CCV). In situ hybridization and live observations in flt4:mCitrine transgenic embryos revealed increased expression levels of flt4 in arterial endothelial cells at the exact location of the aberrant vascular connections in sox7 mutants. An identical circulatory short loop could also be observed in newly generated mutants for hey2 and efnb2. By genetically modulating levels of sox7, hey2 and efnb2 we demonstrate a genetic interaction of sox7 with hey2 and efnb2. The specific spatially confined effect of loss of Sox7 function can be rescued by overexpressing the Notch intracellular domain (NICD) in arterial cells of sox7 mutants, placing Sox7 upstream of Notch in this aspect of arterial development. Hence, sox7 levels are crucial in arterial specification in conjunction with hey2 and efnb2 function, with mutants in all three genes displaying shunt formation and an arterial block. [ABSTRACT FROM AUTHOR]

Bone mineralization is an essential step during the embryonic development of vertebrates, and bone serves vital functions in human physiology. To systematically identify unique gene functions essential for osteogenesis, we performed a forward genetic screen in zebrafish and isolated a mutant, no bone (nob), that does not form any mineralized bone. Positional cloning of nob identified the causative gene to encode ectonucleoside triphosphate/diphosphohydrolase 5 (entpd5); analysis of its expression pattern demonstrates that entpd5 is specifically expressed in osteoblasts. An additional mutant, dragonfish (dgf), exhibits ectopic mineralization in the craniofacial and axial skeleton and encodes a loss-of-function allele of ectonucleotide pyrophosphatase phosphodiesterase 1 (enpp1). Intriguingly, generation of double-mutant nob/dgf embryos restored skeletal mineralization in nob mutants, indicating that mechanistically, Entpd5 and Enpp1 act as reciprocal regulators of phosphate/pyrophosphate homeostasis in vivo. Consistent with this, entpd5 mutant embryos can be rescued by high levels of inorganic phosphate, and phosphate-regulating factors, such as fgf23 and npt2a, are significantly affected in entpd5 mutant embryos. Our study demonstrates that Entpd5 represents a previously unappreciated essential player in phosphate homeostasis and skeletal mineralization. [ABSTRACT FROM AUTHOR]

The primitive heart tube is composed of an outer myocardial and an inner endocardial layer that will give rise to the cardiac valves and septa. Specification and differentiation of these two cell layers are among the earliest events in heart development, but the embryonic origins and genetic regulation of early endocardial development remain largely undefined. We have analyzed early endocardial development in the zebrafish using time-lapse confocal microscopy and show that the endocardium seems to originate from a region in the lateral plate mesoderm that will give rise to hematopoietic cells of the primitive myeloid lineage. Endocardial precursors appear to rapidly migrate to the site of heart tube formation, where they arrive prior to the bilateral myocardial primordia. Analysis of a newly discovered zebrafish Scl/Tal1 mutant showed an additional and previously undescribed role of this transcription factor during the development of the endocardium. In Scl/Tal1 mutant embryos, endocardial precursors are specified, but migration is severely defective and endocardial cells aggregate at the ventricular pole of the heart. We further show that the initial fusion of the bilateral myocardial precursor populations occurs independently of the endocardium and tal1 function. Our results suggest early separation of the two components of the primitive heart tube and imply Scl/Tal1 as an indispensable component of the molecular hierarchy that controls endocardium morphogenesis. [ABSTRACT FROM AUTHOR]

The only immunoglobulin heavy-chain classes known so far in teleosts have beenµandd. We identify here a previously unknown class, immunoglobulin?, expressed in zebrafish and other teleosts. In the zebrafish heavy-chain locus, variable (V) gene segments lie upstream of two tandem diversity, joining and constant (DJC) clusters, resembling the mouse T cell receptora(Tcra) andd(Tcrd) locus. V genes rearrange to (DJC)? or to (DJC)µ without evidence of switch rearrangement. The zebrafish immunoglobulin?gene (ighz) and mouse Tcrd, which are proximal to the V gene array, are expressed earlier in development. In adults, ighz was expressed only in kidney and thymus, which are primary lymphoid organs in teleosts. This additional class adds complexity to the immunoglobulin repertoire and raises questions concerning the evolution of immunoglobulins and the regulation of the differential expression of ighz and ighm. [ABSTRACT FROM AUTHOR]

Lymphatic vessels have important roles in fluid homeostasis, fat absorption, inflammation and cancer metastasis and develop in a dynamic process (called lymphangiogenesis) involving budding, migration and proliferation of lymphangioblasts. Using a genetic screen in zebrafish we identify ccbe1 (collagen and calcium-binding EGF domain-1) as indispensible for embryonic lymphangiogenesis. Ccbe1 acts at the same stage of development as Vegfc and is required for lymphangioblast budding and angiogenic sprouting from venous endothelium. [ABSTRACT FROM AUTHOR]

The article focuses on the research conducted by Jeroen Bussman and colleagues on the significance of the vascular endothelial growth factor (VEGF) Receptors to Zebrafish. It serves as a guideline in the nomenclature of various animal species. It mentions that in analysing of the VEGF receptor repertoire in diverging vertebrates, it identified a fourth representative of the gene family which was secondarily lost within the eutherian lineage but was found to be present in marsupials and platypus.

The endothelial cells of the vertebrate lymphatic system assemble into complex networks, but local cues that guide the migration of this distinct set of cells are currently unknown. As a model for lymphatic patterning, we have studied the simple vascular network of the zebrafish trunk consisting of three types of lymphatic vessels that develop in close connection with the blood vasculature. We have generated transgenic lines that allow us to distinguish between arterial, venous and lymphatic endothelial cells (LECs) within a single zebrafish embryo. We found that LECs migrate exclusively along arteries in a manner that suggests that arterial endothelial cells serve as the LEC migratory substrate. In the absence of intersegmental arteries, LEC migration in the trunk is blocked. Our data therefore demonstrate a crucial role for arteries in LEC guidance. [ABSTRACT FROM AUTHOR]