As described in the goals and objectives section of this report, this project consists of four specific aims: Developing Tools for Experimental Analysis of Vascular Development in the Zebrafish The development of new tools to facilitate vascular studies in the zebrafish has been an important ongoing aim of this project. In previous work we (i) developed a widely used confocal microangiography method (ii) compiled an atlas of the anatomy of the developing zebrafish vasculature, (iii) generated a variety of transgenic zebrafish lines expressing different fluorescent proteins within vascular or lymphatic endothelial cells, making it possible for us to visualize vessel formation in intact, living embryos, and (iv) developed methodologies for long-term multiphoton confocal timelapse imaging of vascular development in transgenic fish. We are currently developing many new transgenic lines useful for in vivo vascular imaging as well as for in vivo blood or lymphatic endothelial-specific functional manipulation of signaling pathways involved in vascular specification, patterning, and morphogenesis. We are also developing new transgenic tools to facilitate high-throughput whole genome profiling of gene expression in selected cell types, in particular blood and lymphatic vascular endothelial and smooth muscle cells. Genetic Analysis of Vascular Development Previously, we have used forward-genetic ENU mutagenesis screens in transgenic zebrafish to generate, identify, and characterize many new zebrafish mutants affecting the formation of the developing vasculature. We identified and positionally cloned mutants with phenotypes including loss of most vessels or subsets of vessels, increased sprouting/branching, and vessel mispatterning. These mutants have resulted in numerous important dscoveries related to endothelial specification, arterial differentiation, vascular patterning, and VEGF signalling, to mention only a few. In our current studies we are focusing on several mutants affecting VEGF-dependent and VEGF-independent vascular signaling pathways. We are also carrying out additional genetic screens focusing on vascular smooth muscle. As part of separate project we recently developed a SNP map for the zebrafish and web-based tools that allow us to use whole-genome and whole-exome sequencing to greatly accelerate discovery of the defective genes from identified mutants. Analysis of Vascular Specification, Patterning, and Morphogenesis We have previously used multiphoton time-lapse imaging to characterize patterns of vessel assembly throughout the developing zebrafish, and used molecular and experimental analysis understand how this pattern arises and what cues guide vascular specification, differentiation, and network assembly during development. Our discoveries have included evidence that neuronal guidance factors play an important previously unknown role in vascular guidance and vascular patterning. Our current work includes projects aimed at (a) studying the specification, differentiation, and patterning of vascular smooth muscle in the zebrafish, making use of newlty developed transgenic tools, (b) understanding the role of intracellular signalling substrates in regulating vascular endothelial signalling, (c) exploring the role of BMP family ligands in modulating vessel growth and vascular integrity, (d) analyzing additional pro- or anti-angiogenic factors. Emergence of Hematopoietic Stem and Progenitor Cells from the Endothelium We have recently developed an additional aim based on our identification of a novel epigenetic mechanism regulating the emergence of hematopoietic stem and progenitor cells (HSPC) from the endothelium. HSPC are critical cells that emerge during early vertebrate development and subsequently seed the development of all blood cell lineages for the life of the animal. We have discovered a novel epigenetic mechanism intersecting with genetic pathways regulating the specification of these cells from endothelium. We are currently characterizing this epigenetic mechanism in detail.
| Gore, Aniket V; Pillay, Laura M; Venero Galanternik, Marina et al. (2018) The zebrafish: A fintastic model for hematopoietic development and disease. Wiley Interdiscip Rev Dev Biol 7:e312 |
| Nowak-Sliwinska, Patrycja; Alitalo, Kari; Allen, Elizabeth et al. (2018) Consensus guidelines for the use and interpretation of angiogenesis assays. Angiogenesis 21:425-532 |
| Muntifering, Michael; Castranova, Daniel; Gibson, Gregory A et al. (2018) Clearing for Deep Tissue Imaging. Curr Protoc Cytom 86:e38 |
| Stratman, Amber N; Pezoa, Sofia A; Farrelly, Olivia M et al. (2017) Interactions between mural cells and endothelial cells stabilize the developing zebrafish dorsal aorta. Development 144:115-127 |
| Stainier, Didier Y R; Raz, Erez; Lawson, Nathan D et al. (2017) Guidelines for morpholino use in zebrafish. PLoS Genet 13:e1007000 |
| Jung, Hyun Min; Castranova, Daniel; Swift, Matthew R et al. (2017) Development of the larval lymphatic system in zebrafish. Development 144:2070-2081 |
| Venero Galanternik, Marina; Castranova, Daniel; Gore, Aniket V et al. (2017) A novel perivascular cell population in the zebrafish brain. Elife 6: |
| Ulrich, Florian; Carretero-Ortega, Jorge; Menéndez, Javier et al. (2016) Reck enables cerebrovascular development by promoting canonical Wnt signaling. Development 143:147-59 |
| Moore, John C; Mulligan, Timothy S; Torres Yordán, Nora et al. (2016) T cell immune deficiency in zap70 mutant zebrafish. Mol Cell Biol : |
| Nohata, Nijiro; Uchida, Yutaka; Stratman, Amber N et al. (2016) Temporal-specific roles of Rac1 during vascular development and retinal angiogenesis. Dev Biol 411:183-194 |
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