Efrem Foglia

Induced early expression of mrf4 but not myog rescues myogenesis in the myod/myf5 double-morphant zebrafish embryo

Muscle regulatory factors activate myogenesis in all vertebrates, but their role has been studied in great detail only in the mouse embryo, where all but myogenin – Myod, Myf5 and Mrf4 – are sufficient to activate (albeit not completely) skeletal myogenesis. In the zebrafish embryo, myod and myf5 are required for induction of myogenesis because their simultaneous ablation prevents muscle development. Here we show that mrf4 but not myog can fully rescue myogenesis in the myod/myf5 double morphant via a selective and robust activation of myod, in keeping with its chromatin-remodelling function in vitro. Rescue does not happen spontaneously, because the gene, unlike that in the mouse embryo, is expressed only at the onset of muscle differentiation, Moreover, because of the transient nature of morpholino inhibition, we were able to investigate how myogenesis occurs in the absence of a myotome. We report that in the complete absence of a myotome, subsequent myogenesis is abolished, whereas myogenesis does proceed, albeit abnormally, when the morpholino inhibition was not complete. Therefore our data also show that the early myotome is essential for subsequent skeletal muscle differentiation and patterning in the zebrafish.

Sphingosine-1-Phosphate Receptor-1 Controls Venous Endothelial Barrier Integrity in Zebrafish

Objective—Endothelial sphingosine-1-phosphate (S1P) receptor-1 (S1P1) affects different vascular functions, including blood vessel maturation and permeability. Here, we characterized the role of the zS1P1 ortholog in vascular development in zebrafish. Methods and Results—zS1P1 is expressed in dorsal aorta and posterior cardinal vein of zebrafish embryos at 24 to 30 hours postfertilization. zS1P1 downregulation by antisense morpholino oligonucleotide injection causes early pericardial edema, lack of blood circulation, alterations of posterior cardinal vein structure, and late generalized edema. Also, zS1P1 morphants are characterized by downregulation of vascular endothelial cadherin (VE-cadherin) and Eph receptor EphB4a expression and by disorganization of zonula occludens 1 junctions in posterior cardinal vein endothelium, with no alterations of dorsal aorta endothelium. VE-cadherin knockdown results in similar vascular alterations, whereas VE-cadherin overexpression is sufficient to rescue venous vascular integrity defects and EphB4a downregulation in zS1P1 morphants. Finally, S1P1 small interfering RNA transfection and the S1P1 antagonist (R)-3-amino-(3-hexylphenylamino)-4-oxobutylphosphonic acid (W146) cause EPHB4 receptor down-modulation in human umbilical vein endothelial cells and the assembly of zonula occludens 1 intercellular contacts is prevented by the EPHB4 antagonist TNYL-RAW peptide in these cells. Conclusion—The data demonstrate a nonredundant role of zS1P1 in the regulation of venous endothelial barrier in zebrafish and identify a S1P1/VE-cadherin/EphB4a genetic pathway that controls venous vascular integrity.

Time-Gated Optical Projection Tomography Allows Visualization of Adult Zebrafish Internal Structures

Optical imaging through biological samples is compromised by tissue scattering and currently various approaches aim to overcome this limitation. In this paper we demonstrate that an all optical technique, based on non-linear upconversion of infrared ultrashort laser pulses and on multiple view acquisition, allows the reduction of scattering effects in tomographic imaging. This technique, namely Time-Gated Optical Projection Tomography (TGOPT), is used to reconstruct three dimensionally the internal structure of adult zebrafish without staining or clearing agents. This method extends the use of Optical Projection Tomography to optically diffusive samples yielding reconstructions with reduced artifacts, increased contrast and improved resolution with respect to those obtained with non-gated techniques. The paper shows that TGOPT is particularly suited for imaging the skeletal system and nervous structures of adult zebrafish.

The Synaptic Proteins β-Neurexin and Neuroligin Synergize With Extracellular Matrix-Binding Vascular Endothelial Growth Factor A During Zebrafish Vascular Development

Objective—The goal of this study was to determine the in vivo functions of the synaptic proteins neurexins and neuroligins in embryonic vascular system development using zebrafish as animal model. Methods and Results—In the present study, we show that the knockdown of the &agr;-form of neurexin 1a induces balance defects and reduced locomotory activity, whereas &bgr;-neurexin 1a and neuroligin 1 morphants present defects in sprouting angiogenesis and vascular remodeling, in particular in the caudal plexus and subintestinal vessels. Coinjection of low doses of morpholinos for &bgr;-neurexin 1a and neuroligin 1 together or in combination with morpholinos targeting the heparin-binding isoforms of vascular endothelial growth factor A (encoded by the VEGFAb gene) recapitulates the observed abnormalities, suggesting synergistic activity of these molecules. Similar coinjection experiments with morpholinos, targeting the enzyme heparan sulfate 6-O-sulfotransferase 2, confirm the presence of a functional correlation between extracellular matrix maturation and &bgr;-neurexin 1a or neuroligin 1. Conclusion—Our data represent the first in vivo evidence of the role of neurexin and neuroligin in embryonic blood vessel formation and provide insights into their mechanism of action

Conserved and divergent functions of Nfix in skeletal muscle development during vertebrate evolution

During mouse skeletal muscle development, the Nfix gene has a pivotal role in regulating fetal-specific transcription. Zebrafish and mice share related programs for muscle development, although zebrafish develops at a much faster rate. In fact, although mouse fetal muscle fibers form after 15 days of development, in fish secondary muscle fibers form by 48 hours post-fertilization in a process that until now has been poorly characterized mechanically. In this work, we studied the zebrafish ortholog Nfix (nfixa) and its role in the proper switch to the secondary myogenic wave. This allowed us to highlight evolutionarily conserved and divergent functions of Nfix. In fact, the knock down of nfixa in zebrafish blocks secondary myogenesis, as in mouse, but also alters primary slow muscle fiber formation. Moreover, whereas Nfix mutant mice are motile, nfixa knockdown zebrafish display impaired motility that probably depends upon disruption of the sarcoplasmic reticulum. We conclude that, during vertebrate evolution, the transcription factor Nfix lost some specific functions, probably as a consequence of the different environment in which teleosts and mammals develop.

Ve-ptp modulates vascular integrity by promoting adherens junction maturation.

Background Endothelial cell junctions control blood vessel permeability. Altered permeability can be associated with vascular fragility that leads to vessel weakness and haemorrhage formation. In vivo studies on the function of genes involved in the maintenance of vascular integrity are essential to better understand the molecular basis of diseases linked to permeability defects. Ve-ptp (Vascular Endothelial-Protein Tyrosine Phosphatase) is a transmembrane protein present at endothelial adherens junctions (AJs). Methodology/Principal Findings We investigated the role of Ve-ptp in AJ maturation/stability and in the modulation of endothelial permeability using zebrafish (Danio rerio). Whole-mount in situ hybridizations revealed zve-ptp expression exclusively in the developing vascular system. Generation of altered zve-ptp transcripts, induced separately by two different splicing morpholinos, resulted in permeability defects closely linked to vascular wall fragility. The ultrastructural analysis revealed a statistically significant reduction of junction complexes and the presence of immature AJs in zve-ptp morphants but not in control embryos. Conclusions/Significance Here we show the first in vivo evidence of a potentially critical role played by Ve-ptp in AJ maturation, an important event for permeability modulation and for the development of a functional vascular system.

Zebrafish Numb and Numblike are involved in primitive erythrocyte differentiation

Background Notch signaling is an evolutionarily conserved regulatory circuitry implicated in cell fate determination in various developmental processes including hematopoietic stem cell self-renewal and differentiation of blood lineages. Known endogenous inhibitors of Notch activity are Numb-Nb and Numblike-Nbl, which play partially redundant functions in specifying and maintaining neuronal differentiation. Nb and Nbl are expressed in most tissues including embryonic and adult hematopoietic tissues in mice and humans, suggesting possible roles for these proteins in hematopoiesis. Methodology and Principal Findings We employed zebrafish to investigate the possible functional role of Numb and Numblike during hematopoiesis, as this system allows a detailed analysis even in embryos with severe defects that would be lethal in other organisms. Here we describe that nb/nbl knockdown results in severe reduction or absence of embryonic erythrocytes in zebrafish. Interestingly, nb/nbl knocked-down embryos present severe downregulation of the erythroid transcription factor gata1. This results in erythroblasts which fail to mature and undergo apoptosis. Our results indicate that Notch activity is increased in embryos injected with nb/nbl morpholino, and we show that inhibition of Notch activation can partially rescue the hematopoietic phenotype. Conclusions and Significance Our results provide the first in vivo evidence of an involvement of Numb and Numblike in zebrafish erythroid differentiation during primitive hematopoiesis. Furthermore, we found that, at least in part, the nb/nbl morphant phenotype is due to enhanced Notch activation within hematopoietic districts, which in turn results in primitive erythroid differentiation defects.

Quantitative measurement of blood velocity in zebrafish with optical vector field tomography

Microscopy techniques can readily visualize the finest details of embryo vasculature, but still lack to provide a complete three-dimensional representation of blood flow parameters. We present an in-vivo 3D imaging technique, able to reconstruct the blood cell velocity vector over a large volume of zebrafish embryos. This low cost and relatively simple technique is exploited to quantitatively assess blood velocity in the zebrafish tail at different stages of development.

Electron multiplying charge-coupled device-based fluorescence cross-correlation spectroscopy for blood velocimetry on zebrafish embryos

Abstract. Biomedical issues in vasculogenesis and cardiogenesis require methods to follow hemodynamics with high spatial (micrometers) and time (milliseconds) resolution. At the same time, we need to follow relevant morphogenetic processes on large fields of view. Fluorescence cross-correlation spectroscopy coupled to scanning or wide-field microscopy meets these needs but has limited flexibility in the excitation pattern. To overcome this limitation, we develop here a two-photon two-spots setup coupled to an all-reflective near-infrared (NIR) optimized scanning system and to an electron multiplying charge-coupled device. Two NIR laser spots are spaced at adjustable micron-size distances (1 to 50 μm) by means of a Twyman-Green interferometer and repeatedly scanned on the sample, allowing acquisition of information on flows at 4 ms–3 μm time-space resolution in parallel on an extended field of view. We analyze the effect of nonhomogeneous and variable flow on the cross-correlation function by numerical simulations and show exemplary application of this setup in studies of blood flow in zebrafish embryos in vivo. By coupling the interferometer with the scanning mirrors and by computing the cross-correlation function of fluorescent red blood cells, we are able to map speed patterns in embryos’ vessels.

Non invasive mapping of the blood velocity field in zebrafish with optical tomography

We present a non-invasive method to measure the 3D map of the velocity vector field of blood in zebrafish embryos. The method can be applied to study the vascular system at different development stages.