How cells acquire their fate is a fundamental question in developmental and regenerative biology. Multipotent progenitors undergo cell-fate restriction in response to cues from the microenvironment, the nature of which is poorly understood. In the case of the lymphatic system, venous cells from the cardinal vein are thought to generate lymphatic vessels through trans-differentiation. Here we show that in zebrafish, lymphatic progenitors arise from a previously uncharacterized niche of specialized angioblasts within the cardinal vein, which also generates arterial and venous fates. We further identify Wnt5b as a novel lymphatic inductive signal and show that it also promotes the ‘angioblast-to-lymphatic’ transition in human embryonic stem cells, suggesting that this process is evolutionarily conserved. Our results uncover a novel mechanism of lymphatic specification, and provide the first characterization of the lymphatic inductive niche. More broadly, our findings highlight the cardinal vein as a heterogeneous structure, analogous to the haematopoietic niche in the aortic floor.

Despite the clear major contribution of hyperlipidemia to the prevalence of cardiovascular disease in the developed world, the direct effects of lipoproteins on endothelial cells have remained obscure and are under debate. Here we report a previously uncharacterized mechanism of vessel growth modulation by lipoprotein availability. Using a genetic screen for vascular defects in zebrafish, we initially identified a mutation, stalactite (stl), in the gene encoding microsomal triglyceride transfer protein (mtp), which is involved in the biosynthesis of apolipoprotein B (ApoB)-containing lipoproteins. By manipulating lipoprotein concentrations in zebrafish, we found that ApoB negatively regulates angiogenesis and that it is the ApoB protein particle, rather than lipid moieties within ApoB-containing lipoproteins, that is primarily responsible for this effect. Mechanistically, we identified downregulation of vascular endothelial growth factor receptor 1 (VEGFR1), which acts as a decoy receptor for VEGF, as a key mediator of the endothelial response to lipoproteins, and we observed VEGFR1 downregulation in hyperlipidemic mice. These findings may open new avenues for the treatment of lipoprotein-related vascular disorders.

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SUMMARYCoordination between the vascular system and forming organs is essential for proper embryonic development. The vasculature expands by sprouting angiogenesis, during which tip cells form filopodia that incorporate into capillary loops. Although several molecules, such as vascular endothelial growth factor A (Vegfa), are known to induce sprouting, the mechanism that terminates this process to ensure neovessel stability is still unknown. Sphingosine-1-phosphate receptor 1 (S1P 1 ) has been shown to mediate interaction between endothelial and mural cells during vascular maturation. In vitro studies have identified S1P 1 as a pro-angiogenic factor. Here, we show that S1P 1 acts as an endothelial cell (EC)-autonomous negative regulator of sprouting angiogenesis during vascular development. Severe aberrations in vessel size and excessive sprouting found in limbs of S1P 1 -null mouse embryos before vessel maturation imply a previously unknown, mural cell-independent role for S1P 1 as an anti-angiogenic factor. A similar phenotype observed when S1P 1 expression was blocked specifically in ECs indicates that the effect of S1P 1 on sprouting is EC-autonomous. Comparable vascular abnormalities in S1p 1 knockdown zebrafish embryos suggest cross-species evolutionary conservation of this mechanism. Finally, genetic interaction between S1P 1 and Vegfa suggests that these factors interplay to regulate vascular development, as Vegfa promotes sprouting whereas S1P 1 inhibits it to prevent excessive sprouting and fusion of neovessels. More broadly, because S1P, the ligand of S1P 1 , is blood-borne, our findings suggest a new mode of regulation of angiogenesis, whereby blood flow closes a negative feedback loop that inhibits sprouting angiogenesis once the vascular bed is established and functional.

Microelectrode arrays increasingly serve to extracellularly record in parallel electrical activity from many excitable cells without inflicting damage to the cells by insertion of microelectrodes. Nevertheless, apart from rare cases they suffer from a low signal to noise ratio. The limiting factor for effective electrical coupling is the low seal resistance formed between the plasma membrane and the electronic device. Using transmission electron microscope analysis we recently reported that cultured Aplysia neurons engulf protruding micron size gold spines forming tight apposition which significantly improves the electrical coupling in comparison with flat electrodes (Hai et al 2009 Spine-shaped gold protrusions improve the adherence and electrical coupling of neurons with the surface of micro-electronic devices J. R. Soc. Interface 6 1153-65). However, the use of a transmission electron microscope to measure the extracellular cleft formed between the plasma membrane and the gold-spine surface may be inaccurate as chemical fixation may generate structural artifacts. Using live confocal microscope imaging we report here that cultured Aplysia neurons engulf protruding spine-shaped gold structures functionalized by an RGD-based peptide and to a significantly lesser extent by poly-l-lysine. The cytoskeletal elements actin and associated protein cortactin are shown to organize around the stalks of the engulfed gold spines in the form of rings. Neurons grown on the gold-spine matrix display varying growth patterns but maintain normal electrophysiological properties and form functioning synapses. It is concluded that the matrices of functionalized gold spines provide an improved substrate for the assembly of neuro-electronic hybrids.

Two-photon imaging of endogenous fluorescence can provide physiological and metabolic information from intact tissues. However, simultaneous imaging of multiple intrinsic fluorophores, such as nicotinamide adenine dinucleotide(phosphate) (NAD(P)H), flavin adenine dinucleotide (FAD) and retinoids in living systems is generally hampered by sequential multi-wavelength excitation resulting in motion artifacts. Here, we report on efficient and simultaneous multicolor two-photon excitation of endogenous fluorophores with absorption spectra spanning the 750-1040 nm range, using wavelength mixing. By using two synchronized pulse trains at 760 and 1041 nm, an additional equivalent two-photon excitation wavelength at 879 nm is generated, and achieves simultaneous excitation of blue, green and red intrinsic fluorophores. This method permits an efficient simultaneous imaging of the metabolic coenzymes NADH and FAD to be implemented with perfect image coregistration, overcoming the difficulties associated with differences in absorption spectra and disparity in concentration. We demonstrate ratiometric redox imaging free of motion artifacts and simultaneous two-photon fluorescence lifetime imaging (FLIM) of NADH and FAD in living tissues. The lifetime gradients of NADH and FAD associated with different cellular metabolic and differentiation states in reconstructed human skin and in the germline of live C. Elegans are thus simultaneously measured. Finally, we present multicolor imaging of endogenous fluorophores and second harmonic generation (SHG) signals during the early stages of Zebrafish embryo development, evidencing fluorescence spectral changes associated with development.Multiphoton microscopy is a powerful tool for label-free and non-invasive functional imaging in small organisms and tissues 1, 2 . Pulsed near infrared excitation light allows in-depth imaging based on contrasts such as endogenous fluorescence 2 , second harmonic generation (SHG) 3 and third harmonic generation (THG) 4 . Endogenous fluorescence in living tissues arises from several intrinsic biomarkers that play important roles in physiological processes 2 . The primary intracellular sources are NAD(P)H and FAD, the two major cofactors of redox reactions in the cell and central regulators of energy production and metabolism 5,6 . Their fluorescence reports on the metabolic activity of cells allowing tissue physiology and processes such as stem cell differentiation, cancer development and neurodegenerative diseases to being non-invasively monitored [7][8][9][10][11][12] . The fluorescence lifetimes of NADH and FAD are different upon binding to the protein during the electron transport chain. FLIM provides sensitive measurements of the free and protein-bound NAD(P)H ratio and of the redox states (NADH/NAD + ) of cells, and can be used to distinguish glycolytic and oxidative phosphorylation metabolic states [13][14][15][16][17] . Monitoring lifetime of free and protein-bound FAD has also been exploited to quantify redox ratio FAD/FADH 2 , and used as a b...

Background: Mutations in the thyroid hormone transporter MCT8 are associated with the psychomotor retardation Allan-Herndon-Dudley syndrome (AHDS). Results: In zebrafish, as in humans, mct8 is expressed primarily in the nervous system. Elimination of MCT8 causes severe neural impairment. Conclusion: MCT8 is a crucial regulator during zebrafish embryonic development. Significance: Establishment of the first vertebrate model for MCT8 deficiency, which exhibits a neurological phenotype.

Confocal Raman microspectroscopy and fluorescence imaging are two well-established methods providing functional insight into the extracellular matrix and into living cells and tissues, respectively, down to single molecule detection. In living tissues, however, cells and extracellular matrix coexist and interact. To acquire information on this cell-matrix interaction, we developed a technique for colocalized, correlative multispectral tissue analysis by implementing high-sensitivity, wide-field fluorescence imaging on a confocal Raman microscope. As a proof of principle, we study early stages of bone formation in the zebrafish (Danio rerio) larvae because the zebrafish has emerged as a model organism to study vertebrate development. The newly formed bones were stained using a calcium fluorescent marker and the maturation process was imaged and chemically characterized in vivo. Results obtained from early stages of mineral deposition in the zebrafish fin bone unequivocally show the presence of hydrogen phosphate containing mineral phases in addition to the carbonated apatite mineral. The approach developed here opens significant opportunities in molecular imaging of metabolic activities, intracellular sensing, and trafficking as well as in vivo exploration of cell-tissue interfaces under (patho-)physiological conditions.