Sumio Isogai

Angiogenic network formation in the developing vertebrate trunk.

We have used time-lapse multiphoton microscopy of living Tg(fli1:EGFP)y1 zebrafish embryos to examine how a patterned, functional network of angiogenic blood vessels is generated in the early vertebrate trunk. Angiogenic vascular sprouts emerge from the longitudinal trunk axial vessels (the dorsal aorta and posterior cardinal vein) in two spatially and temporally distinct steps. Dorsal aorta-derived sprouts form an initial primary network of vascular segments, followed by emergence of vein-derived secondary vascular sprouts that interact and interconnect dynamically with the primary network to initiate vascular flow. Using transgenic silent heart mutant embryos, we show that the gross anatomical patterning of this network of vessels does not require blood circulation. However, our results suggest that circulatory flow dynamics play an important role in helping to determine the pattern of interconnections between the primary network and secondary sprouts, and thus the final arterial or venous identity of the vessels in the functional network. We discuss a model to explain our results combining genetic programming of overall vascular architecture with hemodynamic determination of circulatory flow patterns.

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.

Semaphorin3a1 regulates angioblast migration and vascular development in zebrafish embryos.

Semaphorins are a large family of secreted and cell surface molecules that guide neural growth cones to their targets during development. Some semaphorins are expressed in cells and tissues beyond the nervous system suggesting the possibility that they function in the development of non-neural tissues as well. In the trunk of zebrafish embryos endothelial precursors (angioblasts) are located ventral and lateral to the somites. The angioblasts migrate medially and dorsally along the medial surface of the somites to form the dorsal aorta just ventral to the notochord. Here we show that in zebrafish Sema3a1 is involved in angioblast migration in vivo. Expression of sema3a1 in somites and neuropilin 1, which encodes for a component of the Sema3a receptor, in angioblasts suggested that Sema3a1 regulates the pathway of the dorsally migrating angioblasts. Antisense knockdown of Sema3a1 inhibited the formation of the dorsal aorta. Induced ubiquitous expression of sema3a1 in hsp70:gfpsema3a1myc transgenic embryos inhibited migration of angioblasts ventral and lateral to the somites and retarded development of the dorsal aorta, resulting in severely reduced blood circulation. Furthermore, analysis of cells that express angioblast markers following induced expression of sema3a1 or in a mutant that changes the expression of sema3a1 in the somites confirmed these results. These data implicate Sema3a1, a guidance factor for neural growth cones, in the development of the vascular system.

Chemokine Signaling Directs Trunk Lymphatic Network Formation along the Preexisting Blood Vasculature

The lymphatic system is crucial for fluid homeostasis, immune responses, and numerous pathological processes. However, the molecular mechanisms responsible for establishing the anatomical form of the lymphatic vascular network remain largely unknown. Here, we show that chemokine signaling provides critical guidance cues directing early trunk lymphatic network assembly and patterning. The chemokine receptors Cxcr4a and Cxcr4b are expressed in lymphatic endothelium, whereas chemokine ligands Cxcl12a and Cxcl12b are expressed in adjacent tissues along which the developing lymphatics align. Loss- and gain-of-function studies in zebrafish demonstrate that chemokine signaling orchestrates the stepwise assembly of the trunk lymphatic network. In addition to providing evidence for a lymphatic vascular guidance mechanism, these results also suggest a molecular basis for the anatomical coalignment of lymphatic and blood vessels.

Filamin C plays an essential role in the maintenance of the structural integrity of cardiac and skeletal muscles, revealed by the medaka mutant zacro

Filamin C is an actin-crosslinking protein that is specifically expressed in cardiac and skeletal muscles. Although mutations in the filamin C gene cause human myopathy with cardiac involvement, the function of filamin C in vivo is not yet fully understood. Here we report a medaka mutant, zacro (zac), that displayed an enlarged heart, caused by rupture of the myocardiac wall, and progressive skeletal muscle degeneration in late embryonic stages. We identified zac to be a homozygous nonsense mutation in the filamin C (flnc) gene. The medaka filamin C protein was found to be localized at myotendinous junctions, sarcolemma, and Z-disks in skeletal muscle, and at intercalated disks in the heart. zac embryos showed prominent myofibrillar degeneration at myotendinous junctions, detachment of myofibrils from sarcolemma and intercalated disks, and focal Z-disk destruction. Importantly, the expression of γ-actin, which we observed to have a strong subcellular localization at myotendinous junctions, was specifically reduced in zac mutant myotomes. Inhibition of muscle contraction by anesthesia alleviated muscle degeneration in the zac mutant. These results suggest that filamin C plays an indispensable role in the maintenance of the structural integrity of cardiac and skeletal muscles for support against mechanical stress.

Imaging blood vessels in the zebrafish

Understanding on the mechanisms of vascular branching morphogenesis has become a subject of enormous scientific and clinical interest. Zebrafish, which have small, accessible, transparent embryos and larvae, provides a unique living animal model to facilitating high-resolution imaging on ubiquitous and deep localization of vessels within embryo development and also in adult tissues. In this chapter, we have summarized various methods for vessel imaging in zebrafish, including in situ hybridization for vascular-specific genes, resin injection- or dye injection-based vessel visualization, and alkaline phosphatase staining. We also described detail protocols for live imaging of vessels by microangiography or using various transgenic zebrafish lines.

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.

Vascular anatomy of the developing medaka, Oryzias latipes: a complementary fish model for cardiovascular research on vertebrates.

The zebrafish has become a very useful vertebrate model for cardiovascular research, but detailed morphogenetic studies have revealed that it differs from mammals in certain aspects of the primary circulatory system, in particular, the early vitelline circulation. We searched for another teleost species that might serve as a complementary model for the formation of these early primary vessels. Here (and online at http://www.shigen.nig.ac.jp/medaka/atlas/), we present a detailed characterization of the vascular anatomy of the developing medaka embryo from the stage 24 (1 day 20 hr) through stage 30 (3 days 10 hr). Three‐dimensional images using confocal microangiography show that the medaka, Oryzias latipes, follows the common embryonic circulatory pattern consisting of ventral aorta, aortic arches, dorsal aorta, transverse vessels, vitelline capillary plexus, and marginal veins. The medaka, thus, may serve as a valuable model system for genetic analysis of the primary vasculature of vertebrates. Developmental Dynamics 235:734–746, 2006.

Renal glomerulogenesis in medaka fish, Oryzias latipes

We provide an overview of glomerulogenesis in medaka from the embryo to the adult by means of in situ hybridization with the wt1 gene as a marker as well as histology and three‐dimensional images. The pronephric glomus starts to develop in the intermediate mesoderm during early somitogenesis, is completed before hatching, and persists throughout the lifetime of the fish. Within 5 days after hatching, mesonephric glomerulus formation begins in the caudomedial end of the pronephric sinus and duct area. The number of glomeruli reaches approximately 200–300 in each kidney within 2 months after hatching. wt1 expression during nephron maturation served as a marker for the formation of the mesenchymal condensate and the nephrogenic body. Existence of mesenchymal condensates and persistence of wt1 expression in the adult kidney suggest that the mesonephros retains precursor cells that may be capable of contributing to neoglomerulogenesis during adulthood. Developmental Dynamics 237:2342–2352, 2008.

Platelet demand modulates the type of intravascular protrusion of megakaryocytes in bone marrow

Megakaryocytes (MKs) generate platelets via intravascular protrusions termed proplatelets, which are tandem arrays of platelet-sized swellings with a beaded appearance. However, it remains unclear whether all intravascular protrusions in fact become proplatelets, and whether MKs generate platelets without forming proplatelets. Here, we visualised the sequential phases of intravascular MK protrusions and fragments in living mouse bone marrow (BM), using intravital microscopy, and examined their ultrastructure. The formation of intravascular protrusions was observed to be a highly dynamic process, in which the size and shape of the protrusions changed sequentially prior to the release of platelet progenitors. Among these intravascular protrusions, immature thick protrusions were distinguished from proplatelets by their size and the dynamic morphogenesis seen by time-lapse observation. In ultrastructural analyses, the thick protrusions and their fragments were characterised by a peripheral zone, abundant endoplasmic reticulum and demarcation membrane system, and random microtubule arrays. Proplatelets were predominant among BM sinusoids in the physiological state; however, during an acute thrombocytopenic period, thick protrusions increased markedly in the sinusoids. These results strongly suggested that BM MKs form and release two types of platelet progenitors via distinct intravascular protrusions, and that platelet demand modulates the type of intravascular protrusion that is formed in vivo.