Intraperitoneal dye injection method for visualizing the functioning lymphatic vascular system in zebrafish and medaka.

A hierarchically organized lymphatic vascular system extends throughout the vertebrate body for tissue fluid homeostasis, immune trafficking, and the absorption of dietary fats. Intralymphatic dye injection and serial sectioning have been the main tools for visualizing lymphatic vessels. Specific markers for identifying the lymphatic vasculature in zebrafish and medaka have appeared as new tools that enable the study of lymphangiogenesis using genetic and experimental manipulation. Transgenic fishes have become excellent organisms for visualizing the lymphatic vasculature in living embryos, but this method has limited usefulness, especially in later developmental stages. The functional lymphatic endothelium predominantly takes up foreign particles in zebrafish and medaka. We utilized this physiological activity and lymph flow to label lymphatic vessels. Intraperitoneal injection of trypan blue is useful for temporal observations of the lymphatic ducts, which are essential for large-scale genetic screening, while cinnabar (HgS) injection allows identification of the lymphatic endothelium under electron microscopy, avoiding artefactual damage. This injection method, which is not high in cost and does not require high skill or special devices, is applicable to any live fish with functioning lymphatic vessels, even mutants, with high reproducibility for visualizing the entire lymphatic vascular system.

Development of the larval lymphatic system in zebrafish.

The lymphatic vascular system is a hierarchically organized complex network essential for tissue fluid homeostasis, immune trafficking and absorption of dietary fats in the human body. Despite its importance, the assembly of the lymphatic network is still not fully understood. The zebrafish is a powerful model organism that enables study of lymphatic vessel development using high-resolution imaging and sophisticated genetic and experimental manipulation. Although several studies have described early lymphatic development in the fish, lymphatic development at later stages has not been completely elucidated. In this study, we generated a new Tg(mrc1a:egfp)y251 transgenic zebrafish that uses a mannose receptor, C type 1 (mrc1a) promoter to drive strong EGFP expression in lymphatic vessels at all stages of development and in adult zebrafish. We used this line to describe the assembly of the major vessels of the trunk lymphatic vascular network, including the later-developing collateral cardinal, spinal, superficial lateral and superficial intersegmental lymphatics. Our results show that major trunk lymphatic vessels are conserved in the zebrafish, and provide a thorough and complete description of trunk lymphatic vessel assembly.

Integration of vascular systems between the brain and spinal cord in zebrafish.

Cerebral and spinal vascular systems are organized individually, and they then conjugate at their border, through the integration of basilar artery and vertebral arteries. Zebrafish (Danio rerio) is an ideal organism for studying early vascular development, and the precise procedure of cranial and truncal vascular formation has been previously demonstrated using this model. However, the stepwise process of the integration between the brain and spinal cord has not been clearly elucidated. In this study, we describe the integration of the independent vascular systems for the brain and spinal cord, using transgenic zebrafish expressing enhanced green fluorescent protein in endothelial cells. Initially, basilar artery and primordial hindbrain channels, into which internal carotid arteries supplied blood, were connected with dorsal longitudinal anastomose vessels, via the first intersegmental artery. This initial connection was not influenced by flow dynamics, suggesting that vascular integration in this region is controlled by genetic cues. Vertebral arteries were formed individually as longitudinal vessels beneath the spinal cord, and became integrated with the basilar artery during subsequent remodeling. Furthermore, we confirmed the basal vasculature was well conserved in adult zebrafish. Observations of vascular integration presented herein will contribute to an understanding of regulatory mechanisms behind this process.

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.

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.

Generation and application of endogenously floxed alleles for cell-specific knockout in zebrafish.

The zebrafish is amenable to a variety of genetic approaches. However, lack of conditional deletion alleles limits stage- or cell-specific gene knockout. Here, we applied an existing protocol to establish a floxed allele for gata2a but failed to do so due to off-target integration and incomplete knockin. To address these problems, we applied simultaneous co-targeting with Cas12a to insert loxP sites in cis, together with transgenic counterscreening and comprehensive molecular analysis, to identify off-target insertions and confirm targeted knockins. We subsequently used our approach to establish endogenously floxed alleles of foxc1a, rasa1a, and ruvbl1, each in a single generation. We demonstrate the utility of these alleles by verifying Cre-dependent deletion, which yielded expected phenotypes in each case. Finally, we used the floxed gata2a allele to demonstrate an endothelial autonomous requirement in lymphatic valve development. Together, our results provide a framework for routine generation and application of endogenously floxed alleles in zebrafish.

Lung evolution in vertebrates and the water-to-land transition.

A crucial evolutionary change in vertebrate history was the Palaeozoic (Devonian 419-359 million years ago) water-to-land transition, allowed by key morphological and physiological modifications including the acquisition of lungs. Nonetheless, the origin and early evolution of vertebrate lungs remain highly controversial, particularly whether the ancestral state was paired or unpaired. Due to the rarity of fossil soft tissue preservation, lung evolution can only be traced based on the extant phylogenetic bracket. Here we investigate, for the first time, lung morphology in extensive developmental series of key living lunged osteichthyans using synchrotron x-ray microtomography and histology. Our results shed light on the primitive state of vertebrate lungs as unpaired, evolving to be truly paired in the lineage towards the tetrapods. The water-to-land transition confronted profound physiological challenges and paired lungs were decisive for increasing the surface area and the pulmonary compliance and volume, especially during the air-breathing on land.

All life on Earth started out under water. However, around 400 million years ago some vertebrates, such as fish, started developing limbs and other characteristics that allowed them to explore life on land. One of the most pivotal features to evolve was the lungs, which gave vertebrates the ability to breathe above water. Most land-living vertebrates, including humans, have two lungs which sit on either side of their chest. The lungs extract oxygen from the atmosphere and transfer it to the bloodstream in exchange for carbon dioxide which then gets exhaled out in to the atmosphere. How this important organ first evolved is a hotly debated topic. This is largely because lung tissue does not preserve well in fossils, making it difficult to trace how the lungs of vertebrates changed over the course of evolution. To overcome this barrier, Cupello et al. compared the lungs of living species which are crucial to understand the early stages of the water-to-land transition. This included four species of lunged bony fish which breathe air at the water surface, and a four-legged salamander that lives on land. Cupello et al. used a range of techniques to examine how the lungs of the bony fish and salamander changed shape during development. The results suggested that the lungs of vertebrates started out as a single organ, which became truly paired later in evolution once vertebrates started developing limbs. This anatomical shift increased the surface area available for exchanging oxygen and carbon dioxide so that vertebrates could breathe more easily on land. These findings provide new insights in to how the lung evolved into the paired structure found in most vertebrates alive today. It likely that this transition allowed vertebrates to fully adapt to breathing above water, which may explain why this event only happened once over the course of evolution.

The para-aortic ridge plays a key role in the formation of the renal, adrenal and gonadal vascular systems.

Renal, adrenal, gonadal, ureteral and inferior phrenic arteries vary in their level of origin and in their calibre, number and precise anatomical relationship to other structures. Studies of the origin and early development of these arteries have evoked sharp disputes. The ladder theory of Felix, which states that 'All the mesonephric arteries may persist; from them are formed the phrenic, suprarenal, renal and internal spermatic arteries' has been generally quoted in the anatomical textbooks without rigorous verification for 100 years. In this study, we re-examined this theory by performing micro-injection of dye and resin into rat (Rattus norvegicus) embryos. Our results revealed that most of the mesonephric arteries had degenerated before the metanephros started its ascent. The definitive renal, adrenal, gonadal, ureteral and inferior phrenic arteries appeared as new branches from the gonadal artery and/or directly from the abdominal aorta to the para-aortic ridge. Coincidental to this, the anatomical architecture of the inter-renal vascular cage, which consists of the interlobar and arcuate arteries and their collateral veins, was completed within the developing metanephros. We demonstrated that the delicate renal vascular cage switched from the primary renal artery to the definitive renal artery and that the route of venous drainage changed from the posterior cardinal vein to the inferior (caudal) vena cava.

Lymphatic development.

The lymphatic system is essential for fluid homeostasis, immune responses, and fat absorption, and is involved in many pathological processes, including tumor metastasis and lymphedema. Despite its importance, progress in understanding the origins and early development of this system has been hampered by lack of defining molecular markers and difficulties in observing lymphatic cells in vivo and performing genetic and experimental manipulation of the lymphatic system. Recent identification of new molecular markers, new genes with important functional roles in lymphatic development, and new experimental models for studying lymphangiogenesis has begun to yield important insights into the emergence and assembly of this important tissue. This review focuses on the mechanisms regulating development of the lymphatic vasculature during embryogenesis.

Valves Are a Conserved Feature of the Zebrafish Lymphatic System.

The lymphatic system comprises blind-ended tubes that collect interstitial fluid and return it to the circulatory system. In mammals, unidirectional lymphatic flow is driven by muscle contraction working in conjunction with valves. Accordingly, defective lymphatic valve morphogenesis results in backflow leading to edema. In fish species, studies dating to the 18th century failed to identify lymphatic valves, a precedent that currently persists, raising the question of whether the zebrafish could be used to study the development of these structures. Here, we provide functional and morphological evidence of valves in the zebrafish lymphatic system. Electron microscopy revealed valve ultrastructure similar to mammals, while live imaging using transgenic lines identified the developmental origins of lymphatic valve progenitors. Zebrafish embryos bearing mutations in genes required for mammalian valve morphogenesis show defective lymphatic valve formation and edema. Together, our observations provide a foundation from which to further investigate lymphatic valve formation in zebrafish.

Anomalous inferior mesenteric artery supplying the ascending, transverse, descending, and sigmoid colons.

We have encountered in our anatomical practice the first case and an extremely rare second case in which the ascending, transverse, descending, and sigmoid colons were supplied by the inferior mesenteric artery. The causes of colic artery anomalies are generally explained in conjunction with the development of the superior mesenteric artery, which is intimately related to embryonic elongation and midgut rotation. However, this embryological model was inapplicable to both cases. This difficulty motivated us to seek possible relationships with reported anomalous inferior mesenteric arteries in adults as well as their embryological causes. We consider that the aberrant right colic artery found in 2009 is an "intermesenteric artery" which anastomoses the superior (or its middle colic branch) and inferior mesenteric artery, but secondarily lost its origin from the superior mesenteric artery. The aberrant colic artery found in 2010 is a "middle-inferior mesenteric artery" in which the inferior mesenteric artery formed a common trunk with remnant middle mesenteric artery.

Imaging the developing lymphatic system using the zebrafish.

The lymphatic system is essential for immune responses, fluid homeostasis, and fat absorption, and is involved in many pathological processes, including tumour metastasis and lymphoedema. Despite its importance, progress in understanding the origins and early development of this system has been hampered by difficulties in observing lymphatic cells in vivo and performing genetic and experimental manipulation of the lymphatic system. These difficulties stem in part from the lack of a model organism combining these features. The zebrafish is a genetically accessible vertebrate with an optically clear embryo permitting high-resolution in vivo imaging, but the existence of a lymphatic vascular system has not been previously reported in this model organism. Using a series of morphological, molecular and functional studies we have visualized and characterized lymphatic vessels in the developing zebrafish. Our studies show that the zebrafish possesses a lymphatic system that shares many of the characteristic features of lymphatic vessels found in other vertebrates. Using multiphoton time-lapse imaging we have carried out in vivo cell tracking experiments to trace the origins of lymphatic endothelial cells. Our data provide conclusive new evidence supporting a venous origin for primitive lymphatic endothelial cells.

Three-layered architecture of the popliteal fascia that acts as a kinetic retinaculum for the hamstring muscles.

When patients report pain in the popliteal fossa upon knee extension, the pain is usually localized in the lower region of the popliteal fossa. However, some patients complain of pain in the upper region of the popliteal fossa as the knee is flexed, which motivated us to examine the role of the popliteal fascia as the retinaculum of the hamstring muscles. Thirty-four thighs from 19 Japanese cadavers were dissected. The popliteal fascia was defined as the single aponeurotic sheet covering the popliteal fossa. We found that the fascia acted as a three-layered retinaculum for the flexor muscles of the thigh and provided a secure route for neurovascular structures to the lower leg in any kinetic position of the knee joint. The superficial layer of the popliteal fascia covering the thigh was strongly interwoven with the epimysium of biceps femoris along its lateral aspect and with that of the semimembranosus along its medial aspect, ensuring that the flexor muscles remained in their correct positions. The intermediate layer arose from the medial side of biceps femoris and merged medially with the superficial layer. The profound layer stretched transversely between the biceps femoris and the semimembranosus. Moreover, we investigated the nerve distribution in the popliteal fascia using Sihler's staining and whole-mount immunostaining for neurofilaments. The three-layered fascia was constantly innervated by branches from the posterior femoral cutaneous or saphenous nerve. The nerves were closely related and distributed to densely packed collagen fibers in the superficial layer as free or encapsulated nerve endings, suggesting that the fascia is involved in pain in the upper region of the popliteal fossa.

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.

Immunohistochemical demonstration of lymphatic vessels in adult zebrafish.

Morphological profiles of lymphatic vessels in adult zebrafish trunk and ovary were studied by immunohistochemistry and electron microscopy. The present immunohistochemistry for Prox1 was successful in demonstrating lymphatic vessels in zebrafish. The zebrafish trunk revealed two types of bilateral longitudinal lymphatic trunks draining lymph centripetally along dorsal aorta and posterior cardinal veins. Large honeycomb lymphatic sinus was further shown around common cardinal veins. In the zebrafish ovary, the lymphatic vessels, comprising endothelial cells only, encompassed arterioles in their lumen. This peculiar structure appeared to be conserved in vertebrates including mammals and might serve for control of blood temperature and tissue homeostasis. The present study is first to delineate lymphatic vessels in adult zebrafish by immunohistochemistry. Our immunohistochemical results showed usefulness of immunostaining for Prox1 not only for demonstration of lymphatic vessels in zebrafish, but also for examination of their function and dynamics in pathophysiological condition.

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.

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.

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.

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.