lyve1 expression reveals novel lymphatic vessels and new mechanisms for lymphatic vessel development in zebrafish.

We have generated novel transgenic lines that brightly mark the lymphatic system of zebrafish using the lyve1 promoter. Facilitated by these new transgenic lines, we generated a map of zebrafish lymphatic development up to 15 days post-fertilisation and discovered three previously uncharacterised lymphatic vessel networks: the facial lymphatics, the lateral lymphatics and the intestinal lymphatics. We show that a facial lymphatic vessel, termed the lateral facial lymphatic, develops through a novel developmental mechanism, which initially involves vessel growth through a single vascular sprout followed by the recruitment of lymphangioblasts to the vascular tip. Unlike the lymphangioblasts that form the thoracic duct, the lymphangioblasts that contribute to the lateral facial lymphatic vessel originate from a number of different blood vessels. Our work highlights the additional complexity of lymphatic vessel development in the zebrafish that may increase its versatility as a model of lymphangiogenesis.

Next generation sequencing for diagnosis of hereditary anemia: Experience in a Spanish reference center.

BACKGROUND AND AIMS: Hereditary anemia (HA) encloses a wide group of rare inherited disorders with clinical and hematologic overlaps that complicate diagnosis. MATERIALS AND METHODS: A 48-gene panel was developed to diagnose HA by Next Generation Sequencing (NGS) in a large cohort of 165 patients from 160 unrelated families. RESULTS: Patients were divided in: A) patients who had a suspicion of a specific type of HA (n = 109), and B) patients who had a suspicion of HA but with no clear type (n = 56). Diagnostic performance was 83.5% in group A and a change of the initial diagnosis occurred in 11% of these patients. In group B, 35.7% of patients achieved a genetic diagnosis. NGS identified 6 cases of xerocytosis, 6 of pyruvate kinase (PK) deficiency, 4 of G6PD, and 1 case of phytosterolemia with no initial suspicion of these pathologies, which is clinically relevant since they have specific treatment. Five patients were found to carry variants associated to two different pathologies (4 of them combining a metabolic deficiency and a membrane defect), and 44 new variants were identified in 41 patients. CONCLUSION: The use of NGS is a sensitive technique to diagnose HA and it shows better performance when patients are better characterized.

An in vivo antilymphatic screen in zebrafish identifies novel inhibitors of mammalian lymphangiogenesis and lymphatic-mediated metastasis.

The growth of new lymphatic vessels (lymphangiogenesis) in tumors is an integral step in the metastatic spread of tumor cells, first to the sentinel lymph nodes that surround the tumor and then elsewhere in the body. Currently, no selective agents designed to prevent lymphatic vessel growth have been approved for clinical use, and there is an important potential clinical niche for antilymphangiogenic agents. Using a zebrafish phenotype-based chemical screen, we have identified drug compounds, previously approved for human use, that have antilymphatic activity. These include kaempferol, a natural product found in plants; leflunomide, an inhibitor of pyrimidine biosynthesis; and cinnarizine and flunarizine, members of the type IV class of calcium channel antagonists. Antilymphatic activity was confirmed in a murine in vivo lymphangiogenesis Matrigel plug assay, in which kaempferol, leflunomide, and flunarizine prevented lymphatic growth. We show that kaempferol is a novel inhibitor of VEGFR2/3 kinase activity and is able to reduce the density of tumor-associated lymphatic vessels as well as the incidence of lymph node metastases in a metastatic breast cancer xenograft model. However, in this model, kaempferol administration was also associated with tumor deposits in the pancreas and diaphragm, and flunarizine was found to be tumorigenic. Although this screen revealed that zebrafish is a viable platform for the identification and development of mammalian antilymphatic compounds, it also highlights the need for focused secondary screens to ensure appropriate efficacy of hits in a tumor context.