Solei Cermenati

Engineering of a light-gated potassium channel

An optogenetic tool to silence neurons Potassium channels in the cell membrane open and close in response to molecular signals to alter the local membrane potential. Cosentino et al. linked a light-responsive module to the pore of a potassium channel to build a genetically encoded channel called BLINK1 that is closed in the dark and opens in response to low doses of blue light. Zebrafish embryos expressing BLINK1 in their neurons changed their behavior in response to blue light. Science, this issue p. 707 Blue light opens a channel to silence excitable neurons. The present palette of opsin-based optogenetic tools lacks a light-gated potassium (K+) channel desirable for silencing of excitable cells. Here, we describe the construction of a blue-light–induced K+ channel 1 (BLINK1) engineered by fusing the plant LOV2-Jα photosensory module to the small viral K+ channel Kcv. BLINK1 exhibits biophysical features of Kcv, including K+ selectivity and high single-channel conductance but reversibly photoactivates in blue light. Opening of BLINK1 channels hyperpolarizes the cell to the K+ equilibrium potential. Ectopic expression of BLINK1 reversibly inhibits the escape response in light-exposed zebrafish larvae. BLINK1 therefore provides a single-component optogenetic tool that can establish prolonged, physiological hyperpolarization of cells at low light intensities.

Sox18 Genetically Interacts With VegfC to Regulate Lymphangiogenesis in Zebrafish

Objective—Lymphangiogenesis is regulated by transcription factors and by growth factor pathways, but their interplay has not been extensively studied so far. We addressed this issue in zebrafish. Approach and Results—Mutations in the transcription factor–coding gene SOX18 and in VEGFR3 cause lymphedema, and the VEGFR3/Flt4 ligand VEGFC plays an evolutionarily conserved role in lymphangiogenesis. Here, we report a strong genetic interaction between Sox18 and VegfC in the early phases of lymphatic development in zebrafish. Knockdown of sox18 selectively impaired lymphatic sprouting from the cardinal vein and resulted in defective lymphatic thoracic duct formation. Sox18 and the related protein Sox7 play redundant roles in arteriovenous differentiation. We used a novel transgenic line that enables inducible expression of a dominant-negative mutant form of mouse Sox18 protein. Our data led us to conclude that Sox18 is crucially involved in lymphangiogenesis after arteriovenous differentiation. Combined partial knockdown of sox18 and vegfc, using subcritical doses of specific morpholinos, revealed a synergistic interaction in both venous and lymphatic sprouting from the cardinal vein and greatly impaired thoracic duct formation. Conclusions—This interaction suggests a previously unappreciated crosstalk between the growth factor and transcription factor pathways that regulate lymphangiogenesis in development and disease.

The HMGB protein gene family in zebrafish: Evolution and embryonic expression patterns.

The High-Mobility Group Box (HMGB) proteins are highly abundant proteins with both nuclear and extracellular roles in key biological processes. In mammals, three family members are present: HMGB1, HMGB2 and HMGB3. We characterized the HMGB family in zebrafish and report a detailed phylogenetic analysis of HMGB proteins. The B1, B2, and B3 subfamilies are present in cartilaginous fish, bony fish, and tetrapods, while jawless fish sequences emerge as basal to the gene family expansion. Two co-orthologs of each mammalian HMGB gene are present in zebrafish. All six zebrafish hmgb genes are maternally expressed, but huge differences in expression levels exist during embryonic development. The hmgb2a/hmgb2b genes are the most highly expressed, while hmgb3b is expressed at the lowest level. Remarkably, hmgb3 genes are not present in fugu, medaka, Tetraodon and stickleback. Our analysis highlights substantial overlaps, but also subtle differences and specificities in the expression patterns of the zebrafish hmgb genes.

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

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.

Nfix Induces a Switch in Sox6 Transcriptional Activity to Regulate MyHC-I Expression in Fetal Muscle

Summary Sox6 belongs to the Sox gene family and plays a pivotal role in fiber type differentiation, suppressing transcription of slow-fiber-specific genes during fetal development. Here, we show that Sox6 plays opposite roles in MyHC-I regulation, acting as a positive and negative regulator of MyHC-I expression during embryonic and fetal myogenesis, respectively. During embryonic myogenesis, Sox6 positively regulates MyHC-I via transcriptional activation of Mef2C, whereas during fetal myogenesis, Sox6 requires and cooperates with the transcription factor Nfix in repressing MyHC-I expression. Mechanistically, Nfix is necessary for Sox6 binding to the MyHC-I promoter and thus for Sox6 repressive function, revealing a key role for Nfix in driving Sox6 activity. This feature is evolutionarily conserved, since the orthologs Nfixa and Sox6 contribute to repression of the slow-twitch phenotype in zebrafish embryos. These data demonstrate functional cooperation between Sox6 and Nfix in regulating MyHC-I expression during prenatal muscle development.

Zebrafish Tmem230a cooperates with the Delta/Notch signaling pathway to modulate endothelial cell number in angiogenic vessels†

During embryonic development, new arteries, and veins form from preexisting vessels in response to specific angiogenic signals. Angiogenic signaling is complex since not all endothelial cells exposed to angiogenic signals respond equally. Some cells will be selected to become tip cells and acquire migration and proliferation capacity necessary for vessel growth while others, the stalk cells become trailer cells that stay connected with pre‐existing vessels and act as a linkage to new forming vessels. Additionally, stalk and tip cells have the capacity to interchange their roles. Stalk and tip cellular responses are mediated in part by the interactions of components of the Delta/Notch and Vegf signaling pathways. We have identified in zebrafish, that the transmembrane protein Tmem230a is a novel regulator of angiogenesis by its capacity to regulate the number of the endothelial cells in intersegmental vessels by co‐operating with the Delta/Notch signaling pathway. Modulation of Tmem230a expression by itself is sufficient to rescue improper number of endothelial cells induced by aberrant expression or inhibition of the activity of genes associated with the Dll4/Notch pathway in zebrafish. Therefore, Tmem230a may have a modulatory role in vessel‐network formation and growth. As the Tmem230 sequence is conserved in human, Tmem230 may represent a promising novel target for drug discovery and for disease therapy and regenerative medicine in promoting or restricting angiogenesis.

Sox18 and VegfC genetically interact in the early phases of lymphatic development in zebrafish

Abstracts from the NAVBO Workshops in Vascular Biologys from the NAVBO Workshops in Vascular Biology October 14–18, 2012 in Pacific Grove, CA Springer Science+Business Media Dordrecht 2012 Springer junior investigator award presentation PDGF signaling regulates smooth muscle differentiation and accelerates atherosclerosis