Development of the zebrafish lymphatic system requires VEGFC signaling

Lymphangiogenesis results in the formation of a vascular network distinct from arteries and veins that serves to drain interstitial fluid from surrounding tissues and plays a pivotal role in the immune defense of vertebrates as well as in the progression of cancer and other diseases . In mammals, lymph vessels are lined by endothelial cells possibly sprouting from embryonic veins, and their development appears to be critically dependent on the function of PROX1 and VEGFC signaling . The existence of a lymphatic system in teleosts has been a matter of debate for decades. Here we show on the morphological, molecular, and functional levels that zebrafish embryos develop a lymphatic vasculature that serves to retrieve components of the interstitium to the lymph system. We demonstrate the existence of vessels that are molecularly and functionally distinct from blood vessels and show that the development of these vessels depends on Vegfc and VEGFR-3/Flt4 signaling. These findings imply that the molecular components controlling lymphangiogenesis in zebrafish and mammals are conserved and that the zebrafish lymphatic system develops early enough to allow in vivo observations, lineage tracing, and genetic as well as pharmacological screens.     

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.     

An In Vivo Method to Quantify Lymphangiogenesis in Zebrafish

Background

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.

Methods and Results

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.

Conclusion

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.     

Alk3/Alk3b and Smad5 Mediate BMP Signaling during Lymphatic Development in Zebrafish

Lymphatic vessels are essential to regulate interstitial fluid homeostasis and diverse immune responses. A number of crucial factors, such as VEGFC, SOX18, PROX1, FOX2C, and GJC2, have been implicated in differentiation and/or maintenance of lymphatic endothelial cells (LECs). In humans, dysregulation of these genes is known to cause lymphedema, a debilitating condition which adversely impacts the quality of life of affected individuals. However, there are no currently available pharmacological treatments for lymphedema, necessitating identification of additional factors modulating lymphatic development and function which can be targeted for therapy. In this report, we investigate the function of genes associated with Bone Morphogenetic Protein (BMP) signaling in lymphatic development using zebrafish embryos. The knock-down of BMP type II receptors, Bmpr2a and Bmpr2b, and type I receptors, Alk3 and Alk3b, as well as SMAD5, an essential cellular mediator of BMP signaling, led to distinct lymphatic defects in developing zebrafish. Therefore, it appears that each constituent of the BMP signaling pathway may have a unique function during lymphatic development. Taken together, our data demonstrate that BMP signaling is essential for normal lymphatic vessel development in zebrafish.     

prox1b Activity is essential in zebrafish lymphangiogenesis

Background

The lymphatic vascular system, draining interstitial fluids from most tissues and organs, exerts crucial functions in several physiological and pathological processes. Lymphatic system development depends on Prox1, the first marker to be expressed in the endothelial cells of the cardinal vein from where lymph vessels originate. Prox1 ortholog in the optically clear, easily manipulated zebrafish model has been previously isolated and its contribution to lymphangiogenesis has been clarified. Because of a round of genome duplication occurred at the base of teleosts radiation, several zebrafish genes have been retained in duplicate through evolution. We investigated for the presence of additional prox1 genes and determined their role in zebrafish lymphangiogenesis.

Methodology/Principal Findings

We isolated a second ortholog, named prox1b, and analyzed its expression during development by whole mount in situ hybridization (WISH). We detected strong prox1b expression in the endothelium of the posterior cardinal vein (PCV) from where lymphatic precursors originate. To analyze prox1b involvement in lymphangiogenesis we utilized the fli1:GFP transgenics and followed the formation of the toracic duct (TD), the primary lymph vessel in fish, after prox1b knockdown. Our findings clearly demonstrated that the absence of prox1b activity severely hampers the formation of the TD.

Conclusions/Significance

This work provides substantial progress toward the understanding of zebrafish lymphangiogenesis. In light of the features shared by the lymphatic systems of zebrafish and higher vertebrates, the establishment of such lymphatic model will provide a powerful tool to study, for instance, disorders of body fluid homeostasis, inflammation and cancer metastasis, and may ultimately contribute to novel therapies.     

Simultaneous Raman Microspectroscopy and Fluorescence Imaging of Bone Mineralization in Living Zebrafish Larvae

Confocal Raman microspectroscopy and fluorescence imaging are two well-established methods providing functional insight into the extracellular matrix and into living cells and tissues, respectively, down to single molecule detection. In living tissues, however, cells and extracellular matrix coexist and interact. To acquire information on this cell-matrix interaction, we developed a technique for colocalized, correlative multispectral tissue analysis by implementing high-sensitivity, wide-field fluorescence imaging on a confocal Raman microscope. As a proof of principle, we study early stages of bone formation in the zebrafish (Danio rerio) larvae because the zebrafish has emerged as a model organism to study vertebrate development. The newly formed bones were stained using a calcium fluorescent marker and the maturation process was imaged and chemically characterized in vivo. Results obtained from early stages of mineral deposition in the zebrafish fin bone unequivocally show the presence of hydrogen phosphate containing mineral phases in addition to the carbonated apatite mineral. The approach developed here opens significant opportunities in molecular imaging of metabolic activities, intracellular sensing, and trafficking as well as in vivo exploration of cell-tissue interfaces under (patho-)physiological conditions.     

High-throughput imaging of adult fluorescent zebrafish with an LED fluorescence macroscope

Zebrafish are a useful vertebrate model for the study of development, behavior, disease and cancer. A major advantage of zebrafish is that large numbers of animals can be economically used for experimentation; however, high-throughput methods for imaging live adult zebrafish had not been developed. Here, we describe protocols for building a light-emitting diode (LED) fluorescence macroscope and for using it to simultaneously image up to 30 adult animals that transgenically express a fluorescent protein, are transplanted with fluorescently labeled tumor cells or are tagged with fluorescent elastomers. These protocols show that the LED fluorescence macroscope is capable of distinguishing five fluorescent proteins and can image unanesthetized swimming adult zebrafish in multiple fluorescent channels simultaneously. The macroscope can be built and used for imaging within 1 day, whereas creating fluorescently labeled adult zebrafish requires 1 hour to several months, depending on the method chosen. The LED fluorescence macroscope provides a low-cost, high-throughput method to rapidly screen adult fluorescent zebrafish and it will be useful for imaging transgenic animals, screening for tumor engraftment, and tagging individual fish for long-term analysis.     

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.     

Zebrafish models in cardiac development and congenital heart birth defects

The zebrafish has become an ideal vertebrate animal system for investigating cardiac development due to its genetic tractability, external fertilization, early optical clarity and ability to survive without a functional cardiovascular system during development. In particular, recent advances in imaging techniques and the creation of zebrafish transgenics now permit the in vivo analysis of the dynamic cellular events that transpire during cardiac morphogenesis. As a result, the combination of these salient features provides detailed insight as to how specific genes may influence cardiac development at the cellular level. In this review, we will highlight how the zebrafish has been utilized to elucidate not only the underlying mechanisms of cardiac development and human congenital heart diseases (CHDs), but also potential pathways that may modulate cardiac regeneration. Thus, we have organized this review based on the major categories of CHDs-structural heart, functional heart, and vascular/great vessel defects, and will conclude with how the zebrafish may be further used to contribute to our understanding of specific human CHDs in the future.     

Inhibition of Plk1 induces mitotic infidelity and embryonic growth defects in developing zebrafish embryos

Polo-like kinase 1 (Plk1) is central to cell division. Here, we report that Plk1 is critical for mitosis in the embryonic development of zebrafish. Using a combination of several cell biology tools, including single-cell live imaging applied to whole embryos, we show that Plk1 is essential for progression into mitosis during embryonic development. Plk1 morphant cells displayed mitotic infidelity, such as abnormal centrosomes, irregular spindle assembly, hypercondensed chromosomes, and a failure of chromosome arm separation. Consequently, depletion of Plk1 resulted in mitotic arrest and finally death by 6 days post-fertilization. In comparison, Plk2 or Plk3 morphant embryos did not display any significant abnormalities. Treatment of embryos with the Plk1 inhibitor, BI 2536, caused a block in mitosis, which was more severe when used to treat plk1 morphants. Finally, using an assay to rescue the Plk1 morphant phenotype, we found that the kinase domain and PBD domains are both necessary for Plk1 function in zebrafish development. Our studies demonstrate that Plk1 is required for embryonic proliferation because its activity is crucial for mitotic integrity. Furthermore, our study suggests that zebrafish will be an efficient and economical in vivo system for the validation of anti-mitotic drugs.     

Notch-responsive cells initiate the secondary transition in larval zebrafish pancreas

Zebrafish provide a highly versatile model in which to study vertebrate development. Many recent studies have elucidated early events in the organogenesis of the zebrafish pancreas; however, several aspects of early endocrine pancreas formation in the zebrafish are not homologous to the mammalian system. To better identify mechanisms of islet formation in the zebrafish, with true homology to those observed in mammals, we have temporally and spatially characterized zebrafish secondary islet formation. As is the case in the mouse, we show that Notch inhibition leads to precocious differentiation of endocrine tissues. Furthermore, we have used transgenic fish expressing fluorescent markers under the control of a Notch-responsive element to observe the precursors of these induced endocrine cells. These pancreatic Notch-responsive cells represent a novel population of putative progenitors that are associated with larval pancreatic ductal epithelium, suggesting functional homology between secondary islet formation in zebrafish and the secondary transition in mammals. We also show that Notch-responsive cells persist in the adult pancreas and possess the classical characteristics of centroacinar cells, a cell type believed to be a multipotent progenitor cell in adult mammalian pancreas.     

Ret isoform function and marker gene expression in the enteric nervous system is conserved across diverse vertebrate species

The enteric nervous system (ENS) derives from migratory neural crest cells that colonize the developing gut tube, giving rise to an integrated network of neurons and glial cells, which together regulate important aspects of gut function, including coordinating the smooth muscle contractions of the gut wall. The absence of enteric neurons in portions of the gut (aganglionosis) is the defining feature of Hirschsprung’s disease (HSCR) and has been replicated in a number of mouse models. Mutations in the RET tyrosine kinase account for over half of familial cases of HSCR and mice mutant for Ret exhibit aganglionosis. RET exists in two main isoforms, RET9 and RET51 and studies in mouse have shown that RET9 is sufficient to allow normal development of the ENS. In the last several years, zebrafish has emerged as a model of vertebrate ENS development, having been supported by a number of demonstrations of conservation of gene function between zebrafish, mouse and human. In this study we further analyse the potential similarities and differences between ENS development in zebrafish, mouse and human. We demonstrate that zebrafish Ret is required in a dose-dependent manner to regulate colonization of the gut by neural crest derivatives, as in human. Additionally, we show that as in mouse and human, zebrafish ret is produced as two isoforms, ret9 and ret51. Moreover, we show that, as in mouse, the Ret9 isoform is sufficient to support colonization of the gut by enteric neurons. Finally, we identify zebrafish orthologues of genes previously identified to be expressed in the mouse ENS and demonstrate that these genes are expressed in the developing zebrafish ENS, thereby identifying useful ENS markers in this model organism. These studies reveal that the similarities between gene expression and gene function across vertebrate species is more extensive than previously appreciated, thus supporting the use of zebrafish as a general model for vertebrate ENS development and the use of zebrafish genetic screens as a way to identify candidate genes mutated in HSCR cases.     

Syk and Src Family Kinases Regulate C-type Lectin Receptor 2 (CLEC-2)-mediated Clustering of Podoplanin and Platelet Adhesion to Lymphatic Endothelial Cells

The interaction of C-type lectin receptor 2 (CLEC-2) on platelets with Podoplanin on lymphatic endothelial cells initiates platelet signaling events that are necessary for prevention of blood-lymph mixing during development. In the present study, we show that CLEC-2 signaling via Src family and Syk tyrosine kinases promotes platelet adhesion to primary mouse lymphatic endothelial cells at low shear. Using supported lipid bilayers containing mobile Podoplanin, we further show that activation of Src and Syk in platelets promotes clustering of CLEC-2 and Podoplanin. Clusters of CLEC-2-bound Podoplanin migrate rapidly to the center of the platelet to form a single structure. Fluorescence lifetime imaging demonstrates that molecules within these clusters are within 10 nm of one another and that the clusters are disrupted by inhibition of Src and Syk family kinases. CLEC-2 clusters are also seen in platelets adhered to immobilized Podoplanin using direct stochastic optical reconstruction microscopy. These findings provide mechanistic insight by which CLEC-2 signaling promotes adhesion to Podoplanin and regulation of Podoplanin signaling, thereby contributing to lymphatic vasculature development.     

Imaging circuit formation in zebrafish

The study of nervous system development has been greatly facilitated by recent advances in molecular biology and imaging techniques. These approaches are perfectly suited to young transparent zebrafish where they have allowed direct observation of neural circuit assembly in vivo. In this review we will highlight a number of key studies that have applied optical and genetic techniques in zebrafish to address questions relating to axonal and dendritic arbor development,synapse assembly and neural plasticity. These studies have revealed novel cellular phenomena and modes of growth that may reflect general principles governing the assembly of neural circuits.     

The zebrafish colourless gene regulates development of non-ectomesenchymal neural crest derivatives

Neural crest forms four major categories of derivatives: pigment cells, peripheral neurons, peripheral glia, and ectomesenchymal cells. Some early neural crest cells generate progeny of several fates. How specific cell fates become specified is still poorly understood. Here we show that zebrafish embryos with mutations in the colourless gene have severe defects in most crest-derived cell types, including pigment cells, neurons and specific glia. In contrast, craniofacial skeleton and medial fin mesenchyme are normal. These observations suggest that colourless has a key role in development of non-ectomesenchymal neural crest fates, but not in development of ectomesenchymal fates. Thus, the cls mutant phenotype reveals a segregation of ectomesenchymal and non-ectomesenchymal fates during zebrafish neural crest development. The combination of pigmentation and enteric nervous system defects makes colourless mutations a model for two human neurocristopathies, Waardenburg-Shah syndrome and Hirschsprung's disease.     

Approaches to measuring calcium in zebrafish: focus on neuronal development

Calcium ions are known to act as important cellular signals during nervous system development. In vitro studies have provided significant information on the role of calcium signals during neuronal development; however, the function of this messenger in nervous system maturation in vivo remains to be established. The zebrafish has emerged as a valuable model for the study of vertebrate embryogenesis. Fertilisation is external and the rapid growth of the transparent embryo, including development of internal organs, can be observed easily making it well suited for imaging studies. The developing nervous system is relatively simple and has been well characterised, allowing individual neurons to be identified. Using the zebrafish model, both intracellular and intercellular calcium signals throughout embryonic development have been characterised. This review summarises technical approaches to measure calcium signals in developing embryonic and larval zebrafish, and includes recent developments that will facilitate the study of calcium signalling in vivo. The application of calcium imaging techniques to investigate the action of this messenger during embryogenesis in intact zebrafish is illustrated by discussion of their contribution to our understanding of neuronal development in vivo.     

Unique and conserved aspects of gut development in zebrafish

Although the development of the digestive system of humans and vertebrate model organisms has been well characterized, relatively little is known about how the zebrafish digestive system forms. We define developmental milestones during organogenesis of the zebrafish digestive tract, liver, and pancreas and identify important differences in the way the digestive endoderm of zebrafish and amniotes is organized. Such differences account for the finding that the zebrafish digestive system is assembled from individual organ anlagen, whereas the digestive anlagen of amniotes arise from a primitive gut tube. Despite differences of organ morphogenesis, conserved molecular programs regulate pharynx, esophagus, liver, and pancreas development in teleosts and mammals. Specifically, we show that zebrafish faust/gata-5 is a functional ortholog of gata-4, a gene that is essential for the formation of the mammalian and avian foregut. Further, extraembryonic gata activity is required for this function in zebrafish as has been shown in other vertebrates. We also show that a loss-of-function mutation that perturbs sonic hedgehog causes defects in the development of the esophagus that parallel those associated with targeted disruption of this gene in mammals. Perturbation of sonic hedgehog also affects zebrafish liver and pancreas development, and these effects occur in a reciprocal fashion, as has been described during mammalian liver and ventral pancreas development. Together, these data define aspects of digestive system development necessary for the characterization of zebrafish mutants. Given the similarities of teleost and mammalian digestive physiology and anatomy, these findings have implications for developmental and evolutionary studies as well as research of human diseases, such as diabetes, liver cirrhosis, and cancer.