Zebrafish model

Transcriptome profiling of adult zebrafish at the late stage of chronic tuberculosis due to Mycobacterium marinum infection

by Jeroen Bussmann

The Mycobacterium marinum–zebrafish infection model was used in this study for analysis of a host transcriptome... more The Mycobacterium marinum–zebrafish infection model was used in this study for analysis of a host transcriptome response to mycobacterium infection at the organismal level. RNA isolated from adult zebrafish that showed typical signs of fish tuberculosis due to a chronic progressive infection with M. marinum was compared with RNA from healthy fish in microarray analyses. Spotted oligonucleotide sets (designed by Sigma-Compugen and MWG) and Affymetrix GeneChips were used, in total comprising 45,465 zebrafish transcript annotations. Based on a detailed comparative analysis and quantitative reverse transcriptase-PCR analysis, we present a validated reference set of 159 genes whose regulation is strongly affected by mycobacterial infection in the three types of microarrays analyzed. Furthermore, we analyzed the separate datasets of the microarrays with special emphasis on the expression profiles of immune-related genes. Upregulated genes include many known components of the inflammatory response and several genes that have previously been implicated in the response to mycobacterial infections in cell cultures of other organisms. Different marker genes of the myeloid lineage that have been characterized in zebrafish also showed increased expression. Furthermore, the zebrafish homologs of many signal transduction genes with relationship to the immune response were induced by M. marinum infection. Future functional analysis of these genes may contribute to understanding the mechanisms of mycobacterial pathogenesis. Since a large group of genes linked to immune responses did not show altered expression in the infected animals, these results suggest specific responses in mycobacterium-induced disease.

Zebrafish VEGF receptors: a guideline to nomenclature

by Jeroen Bussmann

To reflect that flk1/kdra is a prominent receptor in VEGF-A signaling in zebrafish, while at the same time indicating... more To reflect that flk1/kdra is a prominent receptor in VEGF-A signaling in zebrafish, while at the same time indicating that it represents a fourth class of vertebrate VEGF receptors (and is not the result of a teleost gene duplication), we propose to rename this gene kdr-like. As the zebrafish kdrb gene is clearly orthologous to mammalian KDR, we propose to rename this gene kdr.

von Hippel-Lindau tumor suppressor mutants faithfully model pathological hypoxia-driven angiogenesis and vascular retinopathies in zebrafish.

by Jeroen Bussmann

Biallelic inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene predisposes human patients to the... more Biallelic inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene predisposes human patients to the development of highly vascularized neoplasms in multiple organ systems. We show that zebrafish vhl mutants display a marked increase in blood vessel formation throughout the embryo, starting at 2 days post-fertilization. The most severe neovascularization is observed in distinct areas that overlap with high vegfa mRNA expression, including the vhl mutant brain and eye. Real-time quantitative PCR revealed increased expression of the duplicated VEGFA orthologs vegfaa and vegfab, and of vegfb and its receptors flt1, kdr and kdr-like, indicating increased vascular endothelial growth factor (Vegf) signaling in vhl mutants. Similar to VHL-associated retinal neoplasms, diabetic retinopathy and age-related macular degeneration, we show, by tetramethyl rhodamine-dextran angiography, that vascular abnormalities in the vhl(-/-) retina lead to vascular leakage, severe macular edema and retinal detachment. Significantly, vessels in the brain and eye express cxcr4a, a marker gene expressed by tumor and vascular cells in VHL-associated hemangioblastomas and renal cell carcinomas. VEGF receptor (VEGFR) tyrosine kinase inhibition (through exposure to sunitinib and 676475) blocked vhl(-/-)-induced angiogenesis in all affected tissues, demonstrating that Vegfaa, Vegfab and Vegfb are key effectors of the vhl(-/-) angiogenic phenotype through Flt1, Kdr and Kdr-like signaling. Since we show that the vhl(-/-) angiogenic phenotype shares distinct characteristics with VHL-associated vascular neoplasms, zebrafish vhl mutants provide a valuable in vivo vertebrate model to elucidate underlying mechanisms contributing to the development of these lesions. Furthermore, vhl mutant zebrafish embryos carrying blood vessel-specific transgenes represent a unique and clinically relevant model for tissue-specific, hypoxia-induced pathological angiogenesis and vascular retinopathies. Importantly, they will allow for a cost-effective, non-invasive and efficient way to screen for novel pharmacological agents and combinatorial treatments.

Ccbe1 is required for embryonic lymphangiogenesis and venous sprouting

by Jeroen Bussmann

Lymphatic vessels have important roles in fluid homeostasis, fat absorption, inflammation and cancer metastasis and... more Lymphatic vessels have important roles in fluid homeostasis, fat absorption, inflammation and cancer metastasis and develop in a dynamic process (called lymphangiogenesis) involving budding, migration and proliferation of lymphangioblasts. Using a genetic screen in zebrafish we identify ccbe1 (collagen and calcium-binding EGF domain-1) as indispensible for embryonic lymphangiogenesis. Ccbe1 acts at the same stage of development as Vegfc and is required for lymphangioblast budding and angiogenic sprouting from venous endothelium.

Role of Dll4/Notch in the formation and wiring of the lymphatic network in zebra fish

by Jeroen Bussmann

Objective— To study whether Notch signaling, which regulates cell fate decisions and vessel morphogenesis, controls... more Objective— To study whether Notch signaling, which regulates cell fate decisions and vessel morphogenesis, controls lymphatic development.

Methods and Results— In zebrafish embryos, sprouts from the axial vein have lymphangiogenic potential because they give rise to the first lymphatics. Knockdown of delta-like-4 (Dll4) or its receptors Notch-1b or Notch-6 in zebrafish impaired lymphangiogenesis. Dll4/Notch silencing reduced the number of sprouts producing the string of parchordal lymphangioblasts; instead, sprouts connecting to the intersomitic vessels were formed. At a later phase, Notch silencing impaired navigation of lymphatic intersomitic vessels along their arterial templates.

Conclusion— These studies imply critical roles for Notch signaling in the formation and wiring of the lymphatic network.

Arteries provide essential guidance cues for lymphatic endothelial cells in the zebrafish trunk

by Jeroen Bussmann

The endothelial cells of the vertebrate lymphatic system assemble into complex networks, but local cues that guide the... more The endothelial cells of the vertebrate lymphatic system assemble into complex networks, but local cues that guide the migration of this distinct set of cells are currently unknown. As a model for lymphatic patterning, we have studied the simple vascular network of the zebrafish trunk consisting of three types of lymphatic vessels that develop in close connection with the blood vasculature. We have generated transgenic lines that allow us to distinguish between arterial, venous and lymphatic endothelial cells (LECs) within a single zebrafish embryo. We found that LECs migrate exclusively along arteries in a manner that suggests that arterial endothelial cells serve as the LEC migratory substrate. In the absence of intersegmental arteries, LEC migration in the trunk is blocked. Our data therefore demonstrate a crucial role for arteries in LEC guidance.

Arterial-venous network formation during brain vascularization involves hemodynamic regulation of chemokine signaling

by Jeroen Bussmann

During angiogenic sprouting, newly forming blood vessels need to connect to the existing vasculature in order to... more During angiogenic sprouting, newly forming blood vessels need to connect to the existing vasculature in order to establish a functional circulatory loop. Previous studies have implicated genetic pathways, such as VEGF and Notch signaling, in controlling angiogenesis. We show here that both pathways similarly act during vascularization of the zebrafish central nervous system. In addition, we find that chemokine signaling specifically controls arterial-venous network formation in the brain. Zebrafish mutants for the chemokine receptor cxcr4a or its ligand cxcl12b establish a decreased number of arterial-venous connections, leading to the formation of an unperfused and interconnected blood vessel network. We further find that expression of cxcr4a in newly forming brain capillaries is negatively regulated by blood flow. Accordingly, unperfused vessels continue to express cxcr4a, whereas connection of these vessels to the arterial circulation leads to rapid downregulation of cxcr4a expression and loss of angiogenic characteristics in endothelial cells, such as filopodia formation. Together, our findings indicate that hemodynamics, in addition to genetic pathways, influence vascular morphogenesis by regulating the expression of a proangiogenic factor that is necessary for the correct pathfinding of sprouting brain capillaries.

Rotation and asymmetric development of the zebrafish heart requires directed migration of cardiac progenitor cells

by Jeroen Bussmann

We have used high-resolution 4D imaging of cardiac progenitor cells (CPCs) in zebrafish to investigate the earliest... more We have used high-resolution 4D imaging of cardiac progenitor cells (CPCs) in zebrafish to investigate the earliest left-right asymmetric movements during cardiac morphogenesis. Differential migratory behavior within the heart field was observed, resulting in a rotation of the heart tube. The leftward displacement and rotation of the tube requires hyaluronan synthase 2 expression within the CPCs. Furthermore, by reducing or ectopically activating BMP signaling or by implantation of BMP beads we could demonstrate that BMP signaling, which is asymmetrically activated in the lateral plate mesoderm and regulated by early left-right signals, is required to direct CPC migration and cardiac rotation. Together, these results support a model in which CPCs migrate toward a BMP source during development of the linear heart tube, providing a mechanism by which the left-right axis drives asymmetric development of the vertebrate heart.

Early endocardial morphogenesis requires Scl/Tal1

by Jeroen Bussmann

The primitive heart tube is composed of an outer myocardial and an inner endocardial layer that will give rise to the... more The primitive heart tube is composed of an outer myocardial and an inner endocardial layer that will give rise to the cardiac valves and septa. Specification and differentiation of these two cell layers are among the earliest events in heart development, but the embryonic origins and genetic regulation of early endocardial development remain largely undefined. We have analyzed early endocardial development in the zebrafish using time-lapse confocal microscopy and show that the endocardium seems to originate from a region in the lateral plate mesoderm that will give rise to hematopoietic cells of the primitive myeloid lineage. Endocardial precursors appear to rapidly migrate to the site of heart tube formation, where they arrive prior to the bilateral myocardial primordia. Analysis of a newly discovered zebrafish Scl/Tal1 mutant showed an additional and previously undescribed role of this transcription factor during the development of the endocardium. In Scl/Tal1 mutant embryos, endocardial precursors are specified, but migration is severely defective and endocardial cells aggregate at the ventricular pole of the heart. We further show that the initial fusion of the bilateral myocardial precursor populations occurs independently of the endocardium and tal1 function. Our results suggest early separation of the two components of the primitive heart tube and imply Scl/Tal1 as an indispensable component of the molecular hierarchy that controls endocardium morphogenesis.

ccm1 cell autonomously regulates endothelial cellular morphogenesis and vascular tubulogenesis in zebrafish

by Jeroen Bussmann

Cerebral cavernous malformations (CCMs) are a prevalent class of vascular anomalies characterized by thin-walled... more Cerebral cavernous malformations (CCMs) are a prevalent class of vascular anomalies characterized by thin-walled clusters of malformed blood vessels in the brain. Heritable forms are caused by mutations in CCM1, CCM2 and CCM3, but despite the importance of these factors in vascular biology, an understanding of their molecular and cellular functions remains elusive. Here we describe the characterization of a zebrafish embryonic model of CCM. Loss of ccm1 in zebrafish embryos leads to severe and progressive dilation of major vessels, despite normal endothelial cell fate and number. Vascular dilation in ccm1 mutants is accompanied by progressive spreading of endothelial cells and thinning of vessel walls despite ultrastructurally normal cell–cell contacts. Zebrafish ccm2 mutants display comparable vascular defects. Finally, we show that ccm1 function is cell autonomous, suggesting that it is endothelial cellular morphogenesis that is regulated by CCM proteins during development and pathogenesis.

The immunoglobulin heavy-chain locus in zebrafish: identification and expression of a previously unknown isotype, immunoglobulin Z

by Jeroen Bussmann

The only immunoglobulin heavy-chain classes known so far in teleosts have been mu and delta. We identify here a... more The only immunoglobulin heavy-chain classes known so far in teleosts have been mu and delta. We identify here a previously unknown class, immunoglobulin zeta, expressed in zebrafish and other teleosts. In the zebrafish heavy-chain locus, variable (V) gene segments lie upstream of two tandem diversity, joining and constant (DJC) clusters, resembling the mouse T cell receptor alpha (Tcra) and delta (Tcrd) locus. V genes rearrange to (DJC)(zeta) or to (DJC)(mu) without evidence of switch rearrangement. The zebrafish immunoglobulin zeta gene (ighz) and mouse Tcrd, which are proximal to the V gene array, are expressed earlier in development. In adults, ighz was expressed only in kidney and thymus, which are primary lymphoid organs in teleosts. This additional class adds complexity to the immunoglobulin repertoire and raises questions concerning the evolution of immunoglobulins and the regulation of the differential expression of ighz and ighm.

Expression analysis of the family of 14-3-3 proteins in zebrafish development

by Jeroen Bussmann

14-3-3 proteins comprise a family of dimeric multi-functional proteins present in all eukaryotes, that are important... more 14-3-3 proteins comprise a family of dimeric multi-functional proteins present in all eukaryotes, that are important in a whelm of ubiquitous biological processes. We have analyzed the genomic structure of all 14-3-3s from zebrafish comprising 11 genes and have analyzed their phylogeny. The gene family was cloned and its expression pattern in zebrafish embryogenesis was analyzed by whole mount in situ hybridization and microarray analysis with gene specific probes. We demonstrate that maternal mRNA of 14-3-3s is expressed evenly at the first cell division. At later stage all genes are expressed in a patterned way with, in most cases, intricate patterns in the developing brain. Our result shows distinct expression patterns of various genes. Microarray results show that differences in expression levels of highly similar 14-3-3 genes also occur in the adult stage.

Rapid BAC selection for tol2-mediated transgenesis in zebrafish.

by Jeroen Bussmann

The generation of zebrafish transgenic lines that express specific fluorophores in a cell- or tissue-specific manner... more The generation of zebrafish transgenic lines that express specific fluorophores in a cell- or tissue-specific manner is an important technique that takes full advantage of the optical clarity of the embryo. Identifying promoter fragments that faithfully recapitulate endogenous expression patterns and levels is often difficult and using large genomic DNA fragments, such as bacterial artificial chromosomes (BACs), makes the process of transgenesis less reliable. Here we provide a detailed protocol that allows for BAC selection and subsequent rapid modification through recombineering in Escherichia coli, resulting in BACs that can be injected into zebrafish embryos and, aided by tol2-mediated transgenesis, reliably yield stable transgenic lines. A number of BACs can be prepared in parallel, and injection of the BACs containing CFP/YFP/RFP or Gal4 cassettes allows for immediate testing of whether a particular BAC will yield the desired result. Furthermore, since injected embryos often show widespread expression, recombineered BACs provide an alternative to two-color in situ hybridizations: BACs injected into embryos of a different transgenic reporter line thus enable in vivo colocalization studies. Using this protocol, we have generated 66 stable lines for 23 different genes, with an average transgenesis rate above 10%. Importantly, we provide evidence that BAC size shows no apparent correlation to the transgenesis rate achieved and that there are no severe position effects.

Imaging of single light-responsive clock cells reveals fluctuating free-running periods

by Amanda-Jayne Carr

Light reaches the very heart of the zebrafish clock

by Amanda-Jayne Carr

Circuit Neuroscience in Zebrafish

by Gilad Jacobson

published in 'Current Biology', 2010

A central goal of modern neuroscience is to obtain a
mechanistic understanding of higher brain functions more A central goal of modern neuroscience is to obtain a
mechanistic understanding of higher brain functions
under healthy and diseased conditions. Addressing this
challenge requires rigorous experimental and theoretical
analysis of neuronal circuits. Recent advances in optogenetics,
high-resolution in vivo imaging, and reconstructions
of synaptic wiring diagrams have created new opportunities
to achieve this goal. To fully harness these
methods, model organisms should allow for a combination
of genetic and neurophysiological approaches in vivo.
Moreover, the brain should be small in terms of neuron
numbers and physical size. A promising vertebrate
organism is the zebrafish because it is small, it is transparent
at larval stages and it offers a wide range of genetic
tools and advantages for neurophysiological approaches.
Recent studies have highlighted the potential of zebrafish
for exhaustive measurements of neuronal activity patterns,
for manipulations of defined cell types in vivo and
for studies of causal relationships between circuit function
and behavior. In this article, we summarize background
information on the zebrafish as a model in modern systems
neuroscience and discuss recent results.

Cadherin 2(N-Cadherin) Plays An Essential Role In Zebrafish Cardiovascular Development

by Jessie Francl

Scototaxis As Anxiety-Like Behavior In Fish

by Caio Maximino

Measuring Anxiety In Zebrafish: A Critical Review

by Caio Maximino

Adenosine A1, but not A2, receptor blockade increases anxiety and arousal in zebrafish

by Caio Maximino

Adenosinergic systems have been implicated in anxiety-like states, as caffeine can induce a state of anxiety in human... more Adenosinergic systems have been implicated in anxiety-like states, as caffeine can induce a state of anxiety in human beings. Caffeine is an antagonist at A1 and A2 adenosine receptors but it remains unclear whether anxiety is mediated
by one or both of these. As the adenosinergic system is rather conserved, we opted to pursue these questions using zebrafish,
a widely used model organism in genetics and developmental biology. Zebrafish adenosine 1, 2A.1 and 2A.2 receptors conserve histidine residues in TM6 and TM7 that are responsible for affinity in bovine A1 receptor. We investigated the effects of
caffeine, PACPX (an A1 receptor antagonist) and 1,3-dimethyl-1-propargylxanthine (DMPX) (an A2 receptor antagonist) on
anxiety-like behaviour and locomotor activity of zebrafish in the scototaxis test as well as evaluated the effects of these drugs
on pigment aggregation. Caffeine increased anxiety at the dose of 100 mg ⁄ kg, while locomotion at the dose of 10 mg ⁄ kg was
increased. Both doses of 10 and 100 mg ⁄ kg induced pigment aggregation. PACPX, on the other hand, increased anxiety at a
dose of 6 mg ⁄ kg and induced pigment aggregation at the doses of 0.6 and 6 mg ⁄ kg, but did not produce a locomotor effect.
DMPX, in turn, increased locomotion at the dose of 6 mg ⁄ kg but did not produce any effect on pigment aggregation or anxiety-
like behaviour. These results indicate that blockade of A1-R, but not A2-R, induces anxiety and autonomic arousal, while
the blockade of A2-R induces hyperlocomotion. Thus, as in rodents, caffeine’s anxiogenic and arousing effects are probably
mediated by A1 receptors in zebrafish and its locomotor activating effect is probably mediated by A2 receptors.