zfishbook: connecting you to a world of zebrafish revertible mutants

zfishbook is an internet-based openly accessible database of revertible protein trap gene-breaking transposon (GBT) insertional mutants in the zebrafish, Danio rerio. In these lines, a monomeric red fluorescent protein (mRFP) is encoded by an artificial 3′ exon, resulting in a translational fusion to endogenous loci. The natural transparency of the zebrafish embryo and larvae greatly facilitates the expression annotation of tagged loci using new capillary-based SCORE imaging methods. Molecular annotation of each line is facilitated by cloning methods such as 5′-Rapid Amplification of cDNA Ends (RACE) and inverse polymerase chain reaction (PCR). zfishbook (http://zfishbook.org) represents a central hub for molecular, expression and mutational information about GBT lines from the International Zebrafish Protein Trap Consortium (IZPTC) that includes researchers from around the globe. zfishbook is open to community-wide contributions including expression and functional annotation. zfishbook also represents a central location for information on how to obtain these lines from diverse members of the IZPTC and integration within other zebrafish community databases including Zebrafish Information Network (ZFIN), Ensembl and National Center for Biotechnology Information

An in vivo method to quantify lymphangiogenesis in zebrafish

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.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.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.Scott J. Hoffman, Peter J. Psaltis, Karl J. Clark, Daniel B. Spoon, Colin D. Chue, Stephen C. Ekker, Robert D. Simar

Fin development in a cartilaginous fish and the origin of vertebrate limbs

Recent fossil finds and experimental analysis of chick and mouse embryos highlighted the lateral fin fold theory, which suggests that two pairs of limbs in tetrapods evolved by subdivision of an elongated single fin1. Here we examine fin development in embryos of the primitive cartilaginous fish, Scyliorhinus canicula (dogfish) using scanning electron microscopy and investigate expression of genes known to be involved in limb positioning, identity and patterning in higher vertebrates. Although we did not detect lateral fin folds in dogfish embryos, Engrailed-1 expression suggests that the body is compartmentalized dorso-ventrally. Furthermore, specification of limb identity occurs through the Tbx4 and Tbx5 genes, as in higher vertebrates. In contrast, unlike higher vertebrates, we did not detect Shh transcripts in dogfish fin-buds, although dHand (a gene involved in establishing Shh) is expressed. In S. canicula, the main fin axis seems to lie parallel to the body axis. 'Freeing' fins from the body axis and establishing a separate 'limb' axis has been proposed to be a crucial step in evolution of tetrapod limbs2, 3. We suggest that Shh plays a critical role in this process

Recurrent Modification of a Conserved Cis-Regulatory Element Underlies Fruit Fly Pigmentation Diversity

The development of morphological traits occurs through the collective action of networks of genes connected at the level of gene expression. As any node in a network may be a target of evolutionary change, the recurrent targeting of the same node would indicate that the path of evolution is biased for the relevant trait and network. Although examples of parallel evolution have implicated recurrent modification of the same gene and cis-regulatory element (CRE), little is known about the mutational and molecular paths of parallel CRE evolution. In Drosophila melanogaster fruit flies, the Bric-à-brac (Bab) transcription factors control the development of a suite of sexually dimorphic traits on the posterior abdomen. Female-specific Bab expression is regulated by the dimorphic element, a CRE that possesses direct inputs from body plan (ABD-B) and sex-determination (DSX) transcription factors. Here, we find that the recurrent evolutionary modification of this CRE underlies both intraspecific and interspecific variation in female pigmentation in the melanogaster species group. By reconstructing the sequence and regulatory activity of the ancestral Drosophila melanogaster dimorphic element, we demonstrate that a handful of mutations were sufficient to create independent CRE alleles with differing activities. Moreover, intraspecific and interspecific dimorphic element evolution proceeded with little to no alterations to the known body plan and sex-determination regulatory linkages. Collectively, our findings represent an example where the paths of evolution appear biased to a specific CRE, and drastic changes in function were accompanied by deep conservation of key regulatory linkages. © 2013 Rogers et al

Fish-Specific Duplicated dmrt2b Contributes to a Divergent Function through Hedgehog Pathway and Maintains Left-Right Asymmetry Establishment Function

Gene duplication is thought to provide raw material for functional divergence and innovation. Fish-specific dmrt2b has been identified as a duplicated gene of the dmrt2a/terra in fish genomes, but its function has remained unclear. Here we reveal that Dmrt2b knockdown zebrafish embryos display a downward tail curvature and have U-shaped somites. Then, we demonstrate that Dmrt2b contributes to a divergent function in somitogenesis through Hedgehog pathway, because Dmrt2b knockdown reduces target gene expression of Hedgehog signaling, and also impairs slow muscle development and neural tube patterning through Hedgehog signaling. Moreover, the Dmrt2b morphants display defects in heart and visceral organ asymmetry, and, some lateral-plate mesoderm (LPM) markers expressed in left side are randomized. Together, these data indicate that fish-specific duplicated dmrt2b contributes to a divergent function in somitogenesis through Hedgehog pathway and maintains the common function for left-right asymmetry establishment

Expression Analysis of PAC1-R and PACAP Genes in Zebrafish Embryos

This study describes the expression of the pituitary adenylate cyclase-activating polypeptide (PACAP1 and PACAP2) and PAC1 receptor genes (PAC1a-R and PAC1b-R) in the brain of zebrafish (Danio rerio) during development. In situ hybridization of the 24- and 48-hpf embryos revealed that PACAP genes were expressed in the telencephalon, the diencephalon, the rhombencephalon, and the neurons in the dorsal part of the spinal cord. PACAP2 mRNA appears to be the most abundant form during brain development. The two PAC1-R subtypes showed a similar expression pattern: mRNAs were detected in the forebrain, the thalamus, and the rhombencephalon. However, in the tectum, only PAC1b-R gene was detected. These results suggest that, in fish, PACAP may play a role in brain development

Atoh8, a bHLH Transcription Factor, Is Required for the Development of Retina and Skeletal Muscle in Zebrafish

Math6/atoh8, a bHLH transcription factor, is thought to be indispensable for early embryonic development and likely has important roles in vertebrate tissue-specific differentiation. However, the function of Atoh8 during early development is not clear because homozygous knockout causes embryonic lethality in mice. We have examined the effects of the atoh8 gene on the differentiation of retina and skeletal muscle during early development in zebrafish.We isolated a Math6 homologue in zebrafish, designated as zebrafish atoh8. Whole -mount in situ hybridization analysis showed that zebrafish atoh8 is dynamically expressed mainly in developing retina and skeletal muscle. Atoh8-MO knock-down resulted in reduced eye size with disorganization of retinal lamination. The reduction of atoh8 function also affected the arrangement of paraxial cells and differentiated muscle fibers during somite morphogenesis.Our results show that Atoh8 is an important regulator for the development of both the retina and skeletal muscles necessary for neural retinal cell and myogenic differentiation during zebrafish embryogenesis

Modeling Neurodegeneration in Zebrafish

The zebrafish, Danio rerio, has been established as an excellent vertebrate model for the study of developmental biology and gene function. It also has proven to be a valuable model to study human diseases. Here, we reviewed recent publications using zebrafish to study the pathology of human neurodegenerative diseases including Parkinson’s, Huntington’s, and Alzheimer’s. These studies indicate that zebrafish genes and their human homologues have conserved functions with respect to the etiology of neurodegenerative diseases. The characteristics of the zebrafish and the experimental approaches to which it is amenable make this species a useful complement to other animal models for the study of pathologic mechanisms of neurodegenerative diseases and for the screening of compounds with therapeutic potential

Neuropilin-1 Modulates p53/Caspases Axis to Promote Endothelial Cell Survival

Vascular permeability factor/vascular endothelial growth factor (VPF/VEGF), one of the crucial pro-angiogenic factors, functions as a potent inhibitor of endothelial cell (EC) apoptosis. Previous progress has been made towards delineating the VPF/VEGF survival signaling downstream of the activation of VEGFR-2. Here, we seek to define the function of NRP-1 in VPF/VEGF-induced survival signaling in EC and to elucidate the concomitant molecular signaling events that are pivotal for our understanding of the signaling of VPF/VEGF. Utilizing two different in vitro cell culture systems and an in vivo zebrafish model, we demonstrate that NRP-1 mediates VPF/VEGF-induced EC survival independent of VEGFR-2. Furthermore, we show here a novel mechanism for NRP-1-specific control of the anti-apoptotic pathway in EC through involvement of the NRP-1-interacting protein (NIP/GIPC) in the activation of PI-3K/Akt and subsequent inactivation of p53 pathways and FoxOs, as well as activation of p21. This study, by elucidating the mechanisms that govern VPF/VEGF-induced EC survival signaling via NRP-1, contributes to a better understanding of molecular mechanisms of cardiovascular development and disease and widens the possibilities for better therapeutic targets

Development and Notch Signaling Requirements of the Zebrafish Choroid Plexus

The choroid plexus (CP) is an epithelial and vascular structure in the ventricular system of the brain that is a critical part of the blood-brain barrier. The CP has two primary functions, 1) to produce and regulate components of the cerebral spinal fluid, and 2) to inhibit entry into the brain of exogenous substances. Despite its importance in neurobiology, little is known about how this structure forms.Here we show that the transposon-mediated enhancer trap zebrafish line Et(Mn16) expresses green fluorescent protein within a population of cells that migrate toward the midline and coalesce to form the definitive CP. We further demonstrate the development of the integral vascular network of the definitive CP. Utilizing pharmacologic pan-notch inhibition and specific morpholino-mediated knockdown, we demonstrate a requirement for Notch signaling in choroid plexus development. We identify three Notch signaling pathway members as mediating this effect, notch1b, deltaA, and deltaD.This work is the first to identify the zebrafish choroid plexus and to characterize its epithelial and vasculature integration. This study, in the context of other comparative anatomical studies, strongly indicates a conserved mechanism for development of the CP. Finally, we characterize a requirement for Notch signaling in the developing CP. This establishes the zebrafish CP as an important new system for the determination of key signaling pathways in the formation of this essential component of the vertebrate brain