An in vivo method to quantify lymphangiogenesis in zebrafish

• Published in: PloS one Vol. 7; no. 9; p. e45240
• Main Authors: Hoffman, Scott J, Psaltis, Peter J, Clark, Karl J, Spoon, Daniel B, Chue, Colin D, Ekker, Stephen C, Simari, Robert D
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 13-09-2012; Public Library of Science (PLoS)
• Subjects: Angiogenesis; Animal models; Animals; Biochemistry; Biology; Cancer; Capillaries; Chemical inhibitors; Danio rerio; Embryo, Nonmammalian; Embryos; Endothelial Cells - transplantation; Endothelium; Experiments; Fertilization; Fluorescein Angiography - methods; Fluorescent Dyes; Humans; In vivo methods and tests; Inflammation; Inhibitors; Laboratory animals; Lymphangiogenesis - drug effects; Lymphangiogenesis - physiology; Lymphatic Vessels - anatomy & histology; Lymphatic Vessels - drug effects; Lymphatic Vessels - physiology; Lymphedema; Mammals; Medicine; Melanoma; Melanoma - pathology; Metastasis; Methods; Models, Biological; Modulators; Molecular biology; Morpholinos - pharmacology; Pathogenesis; Transparency; Transplantation; Tumor Cells, Cultured - transplantation; Vascular endothelial growth factor; Vascular Endothelial Growth Factor C - pharmacology; Zebrafish; Zebrafish - embryology; Zebrafish - physiology; United States > US; Minnesota
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0045240

Lymphangiogenesis is a highly regulated process involved in the pathogenesis of disease. Current in vivo models to assess lymphangiogenesis are largely unphysiologic. The zebrafish is a powerful model system for studying development, due to its rapid growth and transparency during early stages of life. Identification of a network of trunk lymphatic capillaries in zebrafish provides an opportunity to quantify lymphatic growth in vivo.
Late-phase microangiography was used to detect trunk lymphatic capillaries in zebrafish 2- and 3-days post-fertilization. Using this approach, real-time changes in lymphatic capillary development were measured in response to modulators of lymphangiogenesis. Recombinant human vascular endothelial growth factor (VEGF)-C added directly to the zebrafish aqueous environment as well as human endothelial and mouse melanoma cell transplantation resulted in increased lymphatic capillary growth, while morpholino-based knockdown of vegfc and chemical inhibitors of lymphangiogenesis added to the aqueous environment resulted in decreased lymphatic capillary growth.
Lymphatic capillaries in embryonic and larval zebrafish can be quantified using late-phase microangiography. Human activators and small molecule inhibitors of lymphangiogenesis, as well as transplanted human endothelial and mouse melanoma cells, alter lymphatic capillary development in zebrafish. The ability to rapidly quantify changes in lymphatic growth under physiologic conditions will allow for broad screening of lymphangiogenesis modulators, as well as help define cellular roles and elucidate pathways of lymphatic development.