Early Endocardial Morphogenesis Requires Scl/Tal1

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

Zebrafish VEGF Receptors: A Guideline to Nomenclature

Stem Cell and Regenerative Biolog

Development of a Combined Lipid-Based Nanoparticle Formulation for Enhanced siRNA Delivery to Vascular Endothelial Cells

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

Development of curcumin-loaded zein nanoparticles for transport across the blood-brain barrier and inhibition of glioblastoma cell growth

Supramolecular & Biomaterials Chemistr

Entpd5 is essential for skeletal mineralization and regulates phosphate homeostasis in zebrafish

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

Together is Better: mRNA Co-Encapsulation in Lipoplexes is Required to Obtain Ratiometric Co-Delivery and Protein Expression on the Single Cell Level

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

ccm1 cell autonomously regulates endothelial cellular morphogenesis and vascular tubulogenesis in zebrafish

Cerebral cavernous malformations (CCMs) are a prevalent class of vascular anomalies characterized by thin-walled clusters of malformed blood vessels in the brain. Heritable forms are caused by mutations in CCM1, CCM2 and CCM3, but despite the importance of these factors in vascular biology, an understanding of their molecular and cellular functions remains elusive. Here we describe the characterization of a zebrafish embryonic model of CCM. Loss of ccm1 in zebrafish embryos leads to severe and progressive dilation of major vessels, despite normal endothelial cell fate and number. Vascular dilation in ccm1 mutants is accompanied by progressive spreading of endothelial cells and thinning of vessel walls despite ultrastructurally normal cell-cell contacts. Zebrafish ccm2 mutants display comparable vascular defects. Finally, we show that ccm1 function is cell autonomous, suggesting that it is endothelial cellular morphogenesis that is regulated by CCM proteins during development and pathogenesis