Karina Yaniv

Live imaging of lymphatic development in the zebrafish.

The lymphatic system has become the subject of great interest in recent years because of its important role in normal and pathological processes. Progress in understanding the origins and early development of this system, however, has been hampered by difficulties in observing lymphatic cells in vivo and in performing defined genetic and experimental manipulation of the lymphatic system in currently available model organisms. Here, we show that the optically clear developing zebrafish provides a useful model for imaging and studying lymphatic development, with a lymphatic system that shares many of the morphological, molecular and functional characteristics of the lymphatic vessels found in other vertebrates. Using two-photon time-lapse imaging of transgenic zebrafish, we trace the migration and lineage of individual cells incorporating into the lymphatic endothelium. Our results show lymphatic endothelial cells of the thoracic duct arise from primitive veins through a novel and unexpected pathway.

The RNA-binding protein Vg1 RBP is required for cell migration during early neural development.

After mid-blastula transition, populations of cells within the Xenopus embryo become motile. Using antisense morpholino oligonucleotides, we find that Vg1 RBP, an RNA-binding protein implicated in RNA localization in oocytes, is required for the migration of cells forming the roof plate of the neural tube and, subsequently, for neural crest migration. These cells are properly determined but remain at their site of origin. Consistent with a possible role in cell movement, Vg1 RBP asymmetrically localizes to extended processes in migrating neural crest cells. Given that Vg1 RBP is a member of the conserved VICKZ family of proteins, expressed in embryonic and neoplastic cells, these data shed light on the likely role of these RNA-binding proteins in regulating cell movements during both development and metastasis.

ApoB-containing lipoproteins regulate angiogenesis by modulating expression of VEGF receptor 1.

Despite the clear major contribution of hyperlipidemia to the prevalence of cardiovascular disease in the developed world, the direct effects of lipoproteins on endothelial cells have remained obscure and are under debate. Here we report a previously uncharacterized mechanism of vessel growth modulation by lipoprotein availability. Using a genetic screen for vascular defects in zebrafish, we initially identified a mutation, stalactite (stl), in the gene encoding microsomal triglyceride transfer protein (mtp), which is involved in the biosynthesis of apolipoprotein B (ApoB)-containing lipoproteins. By manipulating lipoprotein concentrations in zebrafish, we found that ApoB negatively regulates angiogenesis and that it is the ApoB protein particle, rather than lipid moieties within ApoB-containing lipoproteins, that is primarily responsible for this effect. Mechanistically, we identified downregulation of vascular endothelial growth factor receptor 1 (VEGFR1), which acts as a decoy receptor for VEGF, as a key mediator of the endothelial response to lipoproteins, and we observed VEGFR1 downregulation in hyperlipidemic mice. These findings may open new avenues for the treatment of lipoprotein-related vascular disorders.

Lymphatic vessels arise from specialized angioblasts within a venous niche

How cells acquire their fate is a fundamental question in developmental and regenerative biology. Multipotent progenitors undergo cell-fate restriction in response to cues from the microenvironment, the nature of which is poorly understood. In the case of the lymphatic system, venous cells from the cardinal vein are thought to generate lymphatic vessels through trans-differentiation. Here we show that in zebrafish, lymphatic progenitors arise from a previously uncharacterized niche of specialized angioblasts within the cardinal vein, which also generates arterial and venous fates. We further identify Wnt5b as a novel lymphatic inductive signal and show that it also promotes the ‘angioblast-to-lymphatic’ transition in human embryonic stem cells, suggesting that this process is evolutionarily conserved. Our results uncover a novel mechanism of lymphatic specification, and provide the first characterization of the lymphatic inductive niche. More broadly, our findings highlight the cardinal vein as a heterogeneous structure, analogous to the haematopoietic niche in the aortic floor.

S1P1 inhibits sprouting angiogenesis during vascular development

Coordination between the vascular system and forming organs is essential for proper embryonic development. The vasculature expands by sprouting angiogenesis, during which tip cells form filopodia that incorporate into capillary loops. Although several molecules, such as vascular endothelial growth factor A (Vegfa), are known to induce sprouting, the mechanism that terminates this process to ensure neovessel stability is still unknown. Sphingosine-1-phosphate receptor 1 (S1P1) has been shown to mediate interaction between endothelial and mural cells during vascular maturation. In vitro studies have identified S1P1 as a pro-angiogenic factor. Here, we show that S1P1 acts as an endothelial cell (EC)-autonomous negative regulator of sprouting angiogenesis during vascular development. Severe aberrations in vessel size and excessive sprouting found in limbs of S1P1-null mouse embryos before vessel maturation imply a previously unknown, mural cell-independent role for S1P1 as an anti-angiogenic factor. A similar phenotype observed when S1P1 expression was blocked specifically in ECs indicates that the effect of S1P1 on sprouting is EC-autonomous. Comparable vascular abnormalities in S1p1 knockdown zebrafish embryos suggest cross-species evolutionary conservation of this mechanism. Finally, genetic interaction between S1P1 and Vegfa suggests that these factors interplay to regulate vascular development, as Vegfa promotes sprouting whereas S1P1 inhibits it to prevent excessive sprouting and fusion of neovessels. More broadly, because S1P, the ligand of S1P1, is blood-borne, our findings suggest a new mode of regulation of angiogenesis, whereby blood flow closes a negative feedback loop that inhibits sprouting angiogenesis once the vascular bed is established and functional.

The mid-developmental transition and the evolution of animal body plans

Animals are grouped into ~35 ‘phyla’ based upon the notion of distinct body plans. Morphological and molecular analyses have revealed that a stage in the middle of development—known as the phylotypic period—is conserved among species within some phyla. Although these analyses provide evidence for their existence, phyla have also been criticized as lacking an objective definition, and consequently based on arbitrary groupings of animals. Here we compare the developmental transcriptomes of ten species, each annotated to a different phylum, with a wide range of life histories and embryonic forms. We find that in all ten species, development comprises the coupling of early and late phases of conserved gene expression. These phases are linked by a divergent ‘mid-developmental transition’ that uses species-specific suites of signalling pathways and transcription factors. This mid-developmental transition overlaps with the phylotypic period that has been defined previously for three of the ten phyla, suggesting that transcriptional circuits and signalling mechanisms active during this transition are crucial for defining the phyletic body plan and that the mid-developmental transition may be used to define phylotypic periods in other phyla. Placing these observations alongside the reported conservation of mid-development within phyla, we propose that a phylum may be defined as a collection of species whose gene expression at the mid-developmental transition is both highly conserved among them, yet divergent relative to other species.

Endothelial cells promote migration and proliferation of enteric neural crest cells via β1 integrin signaling

Enteric neural crest-derived cells (ENCCs) migrate along the intestine to form a highly organized network of ganglia that comprises the enteric nervous system (ENS). The signals driving the migration and patterning of these cells are largely unknown. Examining the spatiotemporal development of the intestinal neurovasculature in avian embryos, we find endothelial cells (ECs) present in the gut prior to the arrival of migrating ENCCs. These ECs are patterned in concentric rings that are predictive of the positioning of later arriving crest-derived cells, leading us to hypothesize that blood vessels may serve as a substrate to guide ENCC migration. Immunohistochemistry at multiple stages during ENS development reveals that ENCCs are positioned adjacent to vessels as they colonize the gut. A similar close anatomic relationship between vessels and enteric neurons was observed in zebrafish larvae. When EC development is inhibited in cultured avian intestine, ENCC migration is arrested and distal aganglionosis results, suggesting that ENCCs require the presence of vessels to colonize the gut. Neural tube and avian midgut were explanted onto a variety of substrates, including components of the extracellular matrix and various cell types, such as fibroblasts, smooth muscle cells, and endothelial cells. We find that crest-derived cells from both the neural tube and the midgut migrate avidly onto cultured endothelial cells. This EC-induced migration is inhibited by the presence of CSAT antibody, which blocks binding to beta1 integrins expressed on the surface of crest-derived cells. These results demonstrate that ECs provide a substrate for the migration of ENCCs via an interaction between beta1 integrins on the ENCC surface and extracellular matrix proteins expressed by the intestinal vasculature. These interactions may play an important role in guiding migration and patterning in the developing ENS.

Zebrafish as a new animal model to study lymphangiogenesis

The lymphatic system is essential for fluid homeostasis, fat absorption and immune responses, and also plays key roles under pathological conditions, such as tumor metastasis, lymphoedema and inflammation. The main function of the lymphatic vascular system is to return excess interstitial fluid back to the blood vascular system. Lymph, including fluid, macromolecules, leukocytes and activated antigen-presenting cells, is transported from the blind-ended lymphatic capillaries toward the collecting lymphatic vessels; for there, it is returned to the blood circulation through lymphatico-venous junctions (Alitalo et al. in Nature 438:946–954, 2005). Despite its importance, lymphangiogenesis remains poorly understood. The lack of specific markers has complicated the identification of lymph vessels, and a small animal model that could be genetically manipulated to discover the function of novel lymphangiogenic candidates has only recently become available (Ny et al. in Nat Med 11(9):998–1004, 2005). Since 2004, we have worked to make the zebrafish a new genetic model for unraveling the function of candidate genes involved in lymphangiogenesis. We have demonstrated that zebrafish possess a lymphatic vascular system that shares the morphological, molecular and functional characteristics of the lymphatic vessels found in other vertebrates (Yaniv et al. in Nat Med 12(6):711–716, 2006). In this process, we realized that it was necessary to seek a common definition for the lymph system which would be applicable from fish to man. The aim of this article is to review classical, mainly morphological, studies in order to elucidate the nature of the lymphatic system.

Zebrafish as a Model for Monocarboxyl Transporter 8-Deficiency

Background: Mutations in the thyroid hormone transporter MCT8 are associated with the psychomotor retardation Allan-Herndon-Dudley syndrome (AHDS). Results: In zebrafish, as in humans, mct8 is expressed primarily in the nervous system. Elimination of MCT8 causes severe neural impairment. Conclusion: MCT8 is a crucial regulator during zebrafish embryonic development. Significance: Establishment of the first vertebrate model for MCT8 deficiency, which exhibits a neurological phenotype. Allan-Herndon-Dudley syndrome (AHDS) is a severe psychomotor retardation characterized by neurological impairment and abnormal thyroid hormone (TH) levels. Mutations in the TH transporter, monocarboxylate transporter 8 (MCT8), are associated with AHDS. MCT8 knock-out mice exhibit impaired TH levels; however, they lack neurological defects. Here, the zebrafish mct8 gene and promoter were isolated, and mct8 promoter-driven transgenic lines were used to show that, similar to humans, mct8 is primarily expressed in the nervous and vascular systems. Morpholino-based knockdown and rescue experiments revealed that MCT8 is strictly required for neural development in the brain and spinal cord. This study shows that MCT8 is a crucial regulator during embryonic development and establishes the first vertebrate model for MCT8 deficiency that exhibits a neurological phenotype.

On the pathway of mineral deposition in larval zebrafish caudal fin bone

A poorly understood aspect of bone biomineralization concerns the mechanisms whereby ions are sequestered from the environment, concentrated, and deposited in the extracellular matrix. In this study, we follow mineral deposition in the caudal fin of the zebrafish larva in vivo. Using fluorescence and cryo-SEM-microscopy, in combination with Raman and XRF spectroscopy, we detect the presence of intracellular mineral particles located between bones, and in close association with blood vessels. Calcium-rich particles are also located away from the mineralized bone, and these are also in close association with blood vessels. These observations challenge the view that mineral formation is restricted to osteoblast cells juxtaposed to bone, or to the extracellular matrix. Our results, derived from observations performed in living animals, contribute a new perspective to the comprehensive mechanism of bone formation in vertebrates, from the blood to the bone. More broadly, these findings may shed light on bone mineralization processes in other vertebrates, including humans.