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The establishment of a single cell type regeneration paradigm in the zebrafish provides an opportunity to investigate the genetic mechanisms specific to regeneration processes. We previously demonstrated that regeneration melanocytes arise from cell division of the otherwise quiescent melanocyte precursors following larval melanocyte ablation with a small molecule, MoTP. The ease of ablating melanocytes by MoTP allows us to conduct a forward genetic screen for mechanisms specific to regeneration from such precursors or stem cells. Here, we reported the identification of two mutants, earthaj23e1 and juliej24e1 from a melanocyte ablation screen. Both mutants develop normal larval melanocytes, but upon melanocyte ablation, each mutation results in a distinct stage-specific defect in melanocyte regeneration. Positional cloning reveals that the earthaj23e1 mutation is a nonsense mutation in gfpt1 (glutamine:fructose-6-phosphate aminotransferase 1), the rate-limiting enzyme in glucosamine-6-phosphate biosynthesis. Our analyses reveal that a mutation in gfpt1 specifically affects melanocyte differentiation (marked by melanin production) at a late stage during regeneration and that gfpt1 acts cell autonomously in melanocytes to promote ontogenetic melanocyte darkening. We identified that the juliej24e1 mutation is a splice-site mutation in skiv2l2 (superkiller viralicidic activity 2-like 2), a predicted DEAD-box RNA helicase. Our in situ analysis reveals that the mutation in skiv2l2 causes defects in cell proliferation, suggesting that skiv2l2 plays a role in regulating melanoblast proliferation during early stages of melanocyte regeneration. This finding is consistent with previously described role for cell division during larval melanocyte regeneration. The analyses of these mutants reveal their stage-specific roles in melanocyte regeneration. Interestingly, these mutants identify regeneration-specific functions not only in early stages of the regeneration process, but also in late stages of differentiation of the regenerating melanocyte. We suggest that mechanisms of regeneration identified in this mutant screen may reveal fundamental differences between the mechanisms that establish differentiated cells during embryogenesis, and those involved in larval or adult growth.

Author Summary

Programs of ontogenetic development and regeneration share many components. Differences in genetic requirements between regeneration and development may identify mechanisms specific to the stem cells that maintain cell populations in postembryonic stages, or identify other regeneration-specific functions. Here, we utilize a forward genetic approach that takes advantage of single cell type ablation and regeneration to isolate mechanisms specific to regeneration of the zebrafish melanocyte. Upon chemical ablation of melanocytes, zebrafish larvae reconstitute their larval pigment pattern from undifferentiated precursors or stem cells. We isolated two zebrafish mutants that develop embryonic melanocytes normally but fail to regenerate their melanocytes upon ablation. This phenotype suggests the regeneration-specific roles of the mutated genes. We further identified the mutations in gfpt1 and skiv2l2 and show their stage-specific roles in melanocyte regeneration. Interestingly, these mutants identify regeneration-specific functions not only in early stages of the regeneration process (skiv2l2), but also in late stages of differentiation of the regenerating melanocyte (gfpt1). We suggest that mechanisms of regeneration identified in this mutant screen may reveal fundamental differences between the mechanisms that establish differentiated cells during embryogenesis and those involved in larval or adult growth.

Age-related macular degeneration (AMD) is the leading cause of blindness in the elderly in the developed world. We conducted a genome-wide association study in a series of families enriched for AMD and completed a meta-analysis of this new data with results from reanalysis of an existing study of a late-stage case/control cohort. We tested the top findings for replication in 1 896 cases and 1 866 controls and identified two novel genetic protective factors for AMD. In addition to the CFH (p=2.3×10−64) and ARMS2 (p=1.2×10−60) loci, we observed a protective effect at rs429608, an intronic SNP in SKIV2L (p=5.3×10−15), a gene near the C2/BF locus, that indicates the protective effect may be mediated by variants other than the C2/BF variants previously studied. Haplotype analysis at this locus identified three protective haplotypes defined by the rs429608 protective allele. We also identified a new potentially protective effect at rs2679798 in MYRIP (p=2.9×10−4), a gene involved in retinal pigment epithelium melanosome trafficking. Interestingly, MYRIP was initially identified in the family-based scan and was confirmed in the case-control set. From these efforts, we report the identification of two novel protective factors for AMD and confirm the previously known associations at CFH, ARMS2 and C3.

The formation of branchiomeric nerves (cranial nerves V, VII, IX and X) from their sensory, motor and glial components is poorly understood. The current model for cranial nerve formation is based on the Vth nerve, in which sensory afferents are formed first and must enter the hindbrain in order for the motor efferents to exit. Using transgenic zebrafish lines to discriminate between motor neurons, sensory neurons and peripheral glia, we show that this model does not apply to the remaining three branchiomeric nerves. For these nerves, the motor efferents form prior to the sensory afferents, and their pathfinding show no dependence on sensory axons, as ablation of cranial sensory neurons by ngn1 knockdown had no effect. In contrast, the sensory limbs of the IXth and Xth nerves (but not the Vth or VIIth) were misrouted in gli1 mutants, which lack hindbrain bmn, suggesting that the motor efferents are crucial for appropriate sensory axon projection in some branchiomeric nerves. For all four nerves, peripheral glia were the intermediate component added and had a critical role in nerve integrity but not in axon guidance, as foxd3 null mutants lacking peripheral glia exhibited defasciculation of gVII, gIX, and gX axons. The bmn efferents were unaffected in these mutants. These data demonstrate that multiple mechanisms underlie formation of the four branchiomeric nerves. For the Vth, sensory axons initiate nerve formation, for the VIIth the sensory and motor limbs are independent, and for the IXth/Xth motor axons initiate formation. In all cases the glia are patterned by the initiating set of axons and are needed to maintain axon fasciculation. These results reveal that coordinated interactions between the three neural cell types in branchiomeric nerves differ according to their axial position.

Prior studies with transgenic zebrafish confirmed the functionality of the transcription factor Gal4 to drive expression of other genes under the regulation of upstream activator sequences (UAS). However, widespread application of this powerful binary system has been limited, in part, by relatively inefficient techniques for establishing transgenic zebrafish and by the inadequacy of Gal4 to effect high levels of expression from UAS-regulated genes. We have used the Tol2 transposition system to distribute a self-reporting gene/enhancer trap vector efficiently throughout the zebrafish genome. The vector uses the potent, hybrid transcription factor Gal4-VP16 to activate expression from a UAS:eGFP reporter cassette. In a pilot screen, stable transgenic lines were established that express eGFP in reproducible patterns encompassing a wide variety of tissues, including the brain, spinal cord, retina, notochord, cranial skeleton and muscle, and can transactivate other UAS-regulated genes. We demonstrate the utility of this approach to track Gal4-VP16 expressing migratory cells in UAS:Kaede transgenic fish, and to induce tissue-specific cell death using a bacterial nitroreductase gene under UAS control. The Tol2-mediated gene/enhancer trapping system together with UAS transgenic lines provide valuable tools for regulated gene expression and for targeted labeling and ablation of specific cell types and tissues during early zebrafish development.

Background

The complex neuronal circuitry of the dorsal horn of the spinal cord is as yet poorly understood. However, defining the circuits underlying the transmission of information from primary afferents to higher levels is critical to our understanding of sensory processing. In this study, we have examined phosphodiesterase 1C (Pde1c) BAC transgenic mice in which a green fluorescent protein (GFP) reporter gene reflects Pde1c expression in sensory neuron subpopulations in the dorsal root ganglia and spinal cord.

Results

Using double labeling immunofluorescence, we demonstrate GFP expression in specific subpopulations of primary sensory neurons and a distinct neuronal expression pattern within the spinal cord dorsal horn. In the dorsal root ganglia, their distribution is restricted to those subpopulations of primary sensory neurons that give rise to unmyelinated C fibers (neurofilament 200 negative). A small proportion of both non-peptidergic (IB4-binding) and peptidergic (CGRP immunoreactive) subclasses expressed GFP. However, GFP expression was more common in the non-peptidergic than the peptidergic subclass. GFP was also expressed in a subpopulation of the primary sensory neurons immunoreactive for the vanilloid receptor TRPV1 and the ATP-gated ion channel P2X3. In the spinal cord dorsal horn, GFP positive neurons were largely restricted to lamina I and to a lesser extent lamina II, but surprisingly did not coexpress markers for key neuronal populations present in the superficial dorsal horn.

Conclusion

The expression of GFP in subclasses of nociceptors and also in dorsal horn regions densely innervated by nociceptors suggests that Pde1c marks a unique subpopulation of nociceptive sensory neurons.

Background

Optic nerve regeneration (ONR) following injury is a model for central nervous system regeneration. In zebrafish, ONR is rapid - neurites cross the lesion and enter the optic tectum within 7 days; in mammals regeneration does not take place unless astrocytic reactivity is suppressed. Glial fibrillary acidic protein (GFAP) is used as a marker for retinal and optic nerve astrocytes in both fish and mammals, even though it has long been known that astrocytes of optic nerves in many fish, including zebrafish, express cytokeratins and not GFAP. We used immunofluorescence to localize GFAP and cytokeratin in wild-type zebrafish and transgenic zebrafish expressing green fluorescent protein (GFP) under control of a GFAP promoter to determine the pattern of expression of intermediate filaments in retina and optic nerve.

Findings

GFAP labeling and GFAP gene expression as indicated by GFP fluorescence was found only in the Müller glial cells of the retina. Within Müller cells, GFP fluorescence filled the entire cell while GFAP labelling was more restricted in distribution. No GFAP expression was observed in optic nerves. Cytokeratin labeling of astrocytes was observed throughout the optic nerve and less intensely in cells in the retinal inner plexiform layer. The retinal inner limiting membrane was strongly labeled by anti-cytokeratin.

Conclusions

Studies of astrocyte function during ONR in zebrafish cannot solely rely on GFAP as an astrocyte marker or indicator of reactivity. Future studies of ONR in zebrafish should include evaluation of changes in cytokeratin expression and localization in the optic nerve.

Zebrafish have long been utilized to study the cellular and molecular mechanisms of development by time-lapse imaging of the living transparent embryo. Here we describe a method to mount zebrafish embryos for long-term imaging and demonstrate how to automate the capture of time-lapse images using a confocal microscope. We also describe a method to create controlled, precise damage to individual branches of peripheral sensory axons in zebrafish using the focused power of a femtosecond laser mounted on a two-photon microscope. The parameters for successful two-photon axotomy must be optimized for each microscope. We will demonstrate two-photon axotomy on both a custom built two-photon microscope and a Zeiss 510 confocal/two-photon to provide two examples.

Zebrafish trigeminal sensory neurons can be visualized in a transgenic line expressing GFP driven by a sensory neuron specific promoter 1. We have adapted this zebrafish trigeminal model to directly observe sensory axon regeneration in living zebrafish embryos. Embryos are anesthetized with tricaine and positioned within a drop of agarose as it solidifies. Immobilized embryos are sealed within an imaging chamber filled with phenylthiourea (PTU) Ringers. We have found that embryos can be continuously imaged in these chambers for 12-48 hours. A single confocal image is then captured to determine the desired site of axotomy. The region of interest is located on the two-photon microscope by imaging the sensory axons under low, non-damaging power. After zooming in on the desired site of axotomy, the power is increased and a single scan of that defined region is sufficient to sever the axon. Multiple location time-lapse imaging is then set up on a confocal microscope to directly observe axonal recovery from injury.

Background

Among functional elements of a metazoan gene, enhancers are particularly difficult to find and annotate. Pioneering experiments in Drosophila have demonstrated the value of enhancer "trapping" using an invertebrate to address this functional genomics problem.

Results

We modulated a Sleeping Beauty transposon-based transgenesis cassette to establish an enhancer trapping technique for use in a vertebrate model system, zebrafish Danio rerio. We established 9 lines of zebrafish with distinct tissue- or organ-specific GFP expression patterns from 90 founders that produced GFP-expressing progeny. We have molecularly characterized these lines and show that in each line, a specific GFP expression pattern is due to a single transposition event. Many of the insertions are into introns of zebrafish genes predicted in the current genome assembly. We have identified both previously characterized as well as novel expression patterns from this screen. For example, the ET7 line harbors a transposon insertion near the mkp3 locus and expresses GFP in the midbrain-hindbrain boundary, forebrain and the ventricle, matching a subset of the known FGF8-dependent mkp3 expression domain. The ET2 line, in contrast, expresses GFP specifically in caudal primary motoneurons due to an insertion into the poly(ADP-ribose) glycohydrolase (PARG) locus. This surprising expression pattern was confirmed using in situ hybridization techniques for the endogenous PARG mRNA, indicating the enhancer trap has replicated this unexpected and highly localized PARG expression with good fidelity. Finally, we show that it is possible to excise a Sleeping Beauty transposon from a genomic location in the zebrafish germline.

Conclusions

This genomics tool offers the opportunity for large-scale biological approaches combining both expression and genomic-level sequence analysis using as a template an entire vertebrate genome.

Background

We have developed genetic methods in zebrafish by using the Tol2 transposable element; namely, transgenesis, gene trapping, enhancer trapping and the Gal4FF-UAS system. Gene trap constructs contain a splice acceptor and the GFP or Gal4FF (a modified version of the yeast Gal4 transcription activator) gene, and enhancer trap constructs contain the zebrafish hsp70l promoter and the GFP or Gal4FF gene. By performing genetic screens using these constructs, we have generated transgenic zebrafish that express GFP and Gal4FF in specific cells, tissues and organs. Gal4FF expression is visualized by creating double transgenic fish carrying a Gal4FF transgene and the GFP reporter gene placed downstream of the Gal4-recognition sequence (UAS). Further, the Gal4FF-expressing cells can be manipulated by mating with UAS effector fish. For instance, when fish expressing Gal4FF in specific neurons are crossed with the UAS:TeTxLC fish carrying the tetanus neurotoxin gene downstream of UAS, the neuronal activities are inhibited in the double transgenic fish. Thus, these transgenic fish are useful to study developmental biology and neurobiology.

Description

To increase the usefulness of the transgenic fish resource, we developed a web-based database named zTrap http://kawakami.lab.nig.ac.jp/ztrap/. The zTrap database contains images of GFP and Gal4FF expression patterns, and genomic DNA sequences surrounding the integration sites of the gene trap and enhancer trap constructs. The integration sites are mapped onto the Ensembl zebrafish genome by in-house Blat analysis and can be viewed on the zTrap and Ensembl genome browsers. Furthermore, zTrap is equipped with the functionality to search these data for expression patterns and genomic loci of interest. zTrap contains the information about transgenic fish including UAS reporter and effector fish.

Conclusion

zTrap is a useful resource to find gene trap and enhancer trap fish lines that express GFP and Gal4FF in desired patterns, and to find insertions of the gene trap and enhancer trap constructs that are located within or near genes of interest. These transgenic fish can be utilized to observe specific cell types during embryogenesis, to manipulate their functions, and to discover novel genes and cis-regulatory elements. Therefore, zTrap should facilitate studies on genomics, developmental biology and neurobiology utilizing the transgenic zebrafish resource.

Transgenic technologies enable the manipulation and observation of circuits controlling behavior by permitting expression of genetically encoded reporter genes in neurons. Frequently though, neuronal expression is accompanied by transgene expression in non-neuronal tissues, which may preclude key experimental manipulations, including assessment of the contribution of neurons to behavior by ablation. To better restrict transgene expression to the nervous system in zebrafish larvae, we have used DNA sequences derived from the neuron-restrictive silencing element (NRSE). We find that one such sequence, REx2, when used in conjunction with several basal promoters, robustly suppresses transgene expression in non-neuronal tissues. Both in transient transgenic experiments and in stable enhancer trap lines, suppression is achieved without compromising expression within the nervous system. Furthermore, in REx2 enhancer trap lines non-neuronal expression can be de-repressed by knocking down expression of the NRSE binding protein RE1-silencing transcription factor (Rest). In one line, we show that the resulting pattern of reporter gene expression coincides with that of the adjacent endogenous gene, hapln3. We demonstrate that three common basal promoters are susceptible to the effects of the REx2 element, suggesting that this method may be useful for confining expression from many other promoters to the nervous system. This technique enables neural specific targeting of reporter genes and thus will facilitate the use of transgenic methods to manipulate circuit function in freely behaving larvae.

The matrix metalloproteinase (MMP) family of proteins degrades extracellular matrix (ECM) components as well as processes cytokines and growth factors. MMPs are involved in regulating ECM homeostasis in both normal physiology and disease pathophysiology. Here, we report the critical roles of mmp23b in normal zebrafish liver development. Mmp23b was initially identified as a gene linked to the genomic locus of an enhancer trap transgenic zebrafish line in which GFP expression was restricted to the developing liver. Follow-up analysis of mmp23b mRNA expression confirmed its liver-specific expression pattern. Morpholino (MO) knockdown of mmp23b resulted in defective hepatocyte proliferation, causing a reduction in liver size while maintaining relatively normal pancreas and gut development. Genetically, we showed that mmp23b functions through the tumor necrosis factor (TNF) signaling pathway. Antisense knockdown of tnfa or tnfb in zebrafish caused similar reductions of liver size whereas overexpression of tnfa or tnfb rescued liver defects in mmp23b morphants but not vice versa. Biochemically, MMP23B, the human ortholog of Mmp23b, directly interacts with TNF and mediates its release from the cell membrane in a cell culture system. Since mmp23b/MMP23B is highly conserved, our findings in zebrafish warrant further investigation of its role in regulating liver development in mammals.

The modular Gal4/UAS gene expression system has become an indispensable tool in modern biology. Several large-scale gene- and enhancer-trap screens in the zebrafish have generated hundreds of transgenic lines expressing Gal4 in unique patterns. However, the early embryonic expression of the Gal4 severely limits their use for studies on regeneration or behavior because UAS-driven effectors could disrupt normal organogenesis. To overcome this limitation, we explored the use of the Gal4 repressor Gal80 in transient assays and with stable transgenes to temporally control Gal4 activity. We also validated a strategy to delay Gal4-driven gene expression using a morpholino targeted to Gal4. The first approach is limited to transgenes expressing the native Gal4. The morphant approach can also be applied to transgenic lines expressing the Gal4-VP16 fusion protein. It promises to become a standard approach to delay Gal4-driven transgene expression and enhance the genetic toolkit for the zebrafish.

Summary

We report the expression pattern and construction of a transgenic zebrafish line for a transcription factor involved in otic vesicle formation and skeletogenesis. The zinc finger transcription factor sp7 (formerly called osterix) is reported as a marker of osteoblasts. Using bacterial artificial chromosome (BAC)-mediated transgenesis, we generated a zebrafish transgenic line for studying skeletal development, Tg(sp7:EGFP)b1212. Using a zebrafish BAC, EGFP was introduced downstream of the regulatory regions of sp7 and injected into 1 cell-stage embryos. In this transgenic line, GFP expression reproduces endogenous sp7 gene expression in the otic placode and vesicle, and in forming skeletal structures. GFP-positive cells were also detected in adult fish, and were found associated with regenerating fin rays post-amputation. This line provides an essential tool for the further study of zebrafish otic vesicle formation and the development and regeneration of the skeleton.

Background

Development of a functional retina depends on regulated differentiation of several types of neurons and generation of a highly complex network between the different types of neurons. In addition, each type of retinal neuron includes several distinct morphological types. Very little is known about the mechanisms responsible for generating this diversity of retinal neurons, which may also display specific patterns of regional distribution.

Results

In a screen in zebrafish, using a trapping vector carrying an engineered yeast Gal4 transcription activator and a UAS:eGFP reporter cassette, we have identified two transgenic lines of zebrafish co-expressing eGFP and Gal4 in specific subsets of retinal bipolar cells. The eGFP-labelling facilitated analysis of axon terminals within the inner plexiform layer of the adult retina and showed that the fluorescent bipolar cells correspond to previously defined morphological types. Strong regional restriction of eGFP-positive bipolar cells to the central part of the retina surrounding the optic nerve was observed in adult zebrafish. Furthermore, we achieved specific ablation of the labelled bipolar cells in 5 days old larvae, using a bacterial nitroreductase gene under Gal4-UAS control in combination with the prodrug metronidazole. Following prodrug treatment, nitroreductase expressing bipolar cells were efficiently ablated without affecting surrounding retina architecture, and recovery occurred within a few days due to increased generation of new bipolar cells.

Conclusion

This report shows that enhancer trapping can be applied to label distinct morphological types of bipolar cells in the zebrafish retina. The genetic labelling of these cells yielded co-expression of a modified Gal4 transcription activator and the fluorescent marker eGFP. Our work also demonstrates the potential utility of the Gal4-UAS system for induction of other transgenes, including a bacterial nitroreductase fusion gene, which can facilitate analysis of bipolar cell differentiation and how the retina recovers from specific ablation of these cells.

Background

Mutations in the LGI1 gene predispose to a rare, hereditary form of temporal epilepsy. Currently, little is known about the temporal and spatial expression pattern of Lgi1 during normal embryogenesis and so to define this more clearly we used a transgenic mouse line that expresses GFP under the control of Lgi1 cis-regulatory elements.

Results

During embryonic brain growth, high levels of Lgi1 expression were found in the surface ectoderm, the neuroepithelium, mesenchymal connective tissue, hippocampus, and sensory organs, such as eye, tongue, and the olfactory bulb. Lgi1 was also found in the cranial nerve nuclei and ganglia, such as vestibular, trigeminal, and dorsal ganglia. Expression of Lgi1 followed an orchestrated pattern during mouse development becoming more subdued in areas of the neocortex of the mid- and hind-brain in early postnatal animals, although high expression levels were retained in the choroid plexus and hippocampus. In late postnatal stages, Lgi1 expression continued to be detected in many areas in the brain including, hippocampus, paraventricular thalamic nuclei, inferior colliculus, and the cerebral aqueduct. We also showed that Lgi1-expressing cells co-express nestin, DCX, and beta-III tubulin suggesting that Lgi1-expressing cells are migratory neuroblasts.

Conclusion

These observations imply that Lgi1 may have a role in establishing normal brain architecture and neuronal functions during brain development suggesting that it may be involved in neurogenesis and neuronal plasticity, which become more specifically defined in the adult animal.

We have carried out a Gal4 enhancer trap screen in zebrafish, and have generated 184 stable transgenic lines with interesting expression patterns throughout the nervous system. Of these, three display clear expression in the tectum, each with a distinguishable and stereotyped distribution of Gal4 expressing cells. Detailed morphological analysis of single cells, using a genetic “Golgi-like” labelling method, revealed four common cell types (superficial, periventricular, shallow periventricular, and radial glial), along with a range of other less common neurons. The shallow periventricular (PV) and a subset of the PV neurons are tectal efferent neurons that target various parts of the reticular formation. We find that it is specifically PV neurons with dendrites in the deep tectal neuropil that target the reticular formation. This indicates that these neurons receive the tectum's highly processed visual information (which is fed from the superficial retinorecipient layers), and relay it to premotor regions. Our results show that the larval tectum, both broadly and at the single cell level, strongly resembles a miniature version of its adult counterpart, and that it has all of the necessary anatomical characteristics to inform motor responses based on sensory input. We also demonstrate that mosaic expression of GFP in Gal4 enhancer trap lines can be used to describe the types and abundance of cells in an expression pattern, including the architectures of individual neurons. Such detailed anatomical descriptions will be an important part of future efforts to describe the functions of discrete tectal circuits in the generation of behavior.

The conditional expression of transgenes at high levels in sparse and specific populations of neurons is important for high-resolution optogenetic analyses of neuronal circuits. We explored two complementary methods, viral gene delivery and the iTet-Off system, to express transgenes in the brain of zebrafish. High-level gene expression in neurons was achieved by Sindbis and Rabies viruses. The Tet system produced strong and specific gene expression that could be modulated conveniently by doxycycline. Moreover, transgenic lines showed expression in distinct, sparse and stable populations of neurons that appeared to be subsets of the neurons targeted by the promoter driving the Tet-activator. The Tet system therefore provides the opportunity to generate libraries of diverse expression patterns similar to gene trap approaches or the thy-1 promoter in mice, but with the additional possibility to pre-select cell types of interest. In transgenic lines expressing channelrhodopsin-2, action potential firing could be precisely controlled by two-photon stimulation at low laser power, presumably because the expression levels of the Tet-controlled genes were high even in adults. In channelrhodopsin-2-expressing larvae, optical stimulation with a single blue LED evoked distinct swimming behaviors including backward swimming. These approaches provide new opportunities for the optogenetic dissection of neuronal circuit structure and function.

The Gal4-UAS system provides powerful tools to analyze the function of genes and cells in vivo and has been extensively employed in Drosophila. The usefulness of this approach relies on the P element-mediated Gal4 enhancer trapping, which can efficiently generate transgenic fly lines expressing Gal4 in specific cells. Similar approaches, however, had not been developed in vertebrate systems due to the lack of an efficient transgenesis method. We have been developing transposon techniques by using the madaka fish Tol2 element. Taking advantage of its ability to generate genome-wide insertions, we developed the Gal4 gene trap and enhancer trap methods in zebrafish that enabled us to create various transgenic fish expressing Gal4 in specific cells. The Gal4-expressing cells can be visualized and manipulated in vivo by crossing the transgenic lines with transgenic fish lines carrying various reporter and effector genes downstream of UAS (upstream activating sequence). Thus, the Gal4 gene trap and enhancer trap methods together with UAS lines now make detailed analyses of genes and cells in zebrafish feasible. Here, we describe the protocols to perform Gal4 gene trap and enhancer trap screens in zebrafish and their application to the studies of vertebrate neural circuits.

Several transgenic zebrafish lines for liver development studies had been obtained in the first decade of this century, but not any transgenic GFP zebrafish lines that mark the through liver development and organogenesis were reported. In this study, we analyzed expression pattern of endogenous Apo-14 in zebrafish embryogenesis by whole-mount in situ hybridization, and revealed its expression in liver primordium and in the following liver development. Subsequently, we isolated zebrafish Apo-14 promoter of 1763 bp 5′-flanking sequence, and developed an Apo-14 promoter-driven transgenic zebrafish Tg(Apo14: GFP). And, maternal expression and post-fertilization translocation of Apo-14 promoter-driven GFP were observed in the transgenic zebrafish line. Moreover, we traced onset expression of Apo-14 promoter-driven GFP and developmental behavior of the expressed cells in early heterozygous embryos by out-crossing the Tg(Apo14: GFP) male to the wild type female. Significantly, the Apo-14 promoter-driven GFP is initially expressed around YSL beneath the embryo body at 10 hpf when the embryos develop to tail bud prominence. In about 14-somite embryos at 16–17 hpf, a typical “salt-and-pepper” expression pattern is clearly observed in YSL around the yolk sac. Then, a green fluorescence dot begins to appear between the notochord and the yolk sac adjacent to otic vesicle at about 20 hpf, which is later demonstrated to be liver primordium that gives rise to liver. Furthermore, we investigated dynamic progression of liver organogenesis in the Tg(Apo14: GFP) zebrafish, because the Apo-14 promoter-driven GFP is sustainably expressed from hepatoblasts and liver progenitor cells in liver primordium to hepatocytes in the larval and adult liver. Additionally, we observed similar morphology between the liver progenitor cells and the GFP-positive nuclei on the YSL, suggesting that they might originate from the same progenitor cells in early embryos. Overall, the current study provides a transgenic zebrafish line that marks the through liver organogenesis.

Cone photoreceptors in fish are typically arranged into a precise, reiterated pattern known as a ‘cone mosaic’. Cone mosaic patterns can vary in different fish species and in response to changes in habitat, yet their function and the mechanisms of their development remain speculative. Zebrafish (Danio rerio) have four cone subtypes arranged into precise rows in the adult retina. Here we describe larval zebrafish cone patterns and investigate a previously unrecognized transition between larval and adult cone mosaic patterns. Cone positions were determined in transgenic zebrafish, expressing green fluorescent protein (GFP) in their UV-sensitive cones, by the use of multiplex in situ hybridization labelling of various cone opsins. We developed a ‘mosaic metric’ statistical tool to measure local cone order. We found that ratios of the various cone subtypes in larval and adult zebrafish were statistically different. The cone photoreceptors in larvae form a regular heterotypic mosaic array, i.e. the position of any one cone spectral subtype relative to the other cone subtypes is statistically different from random. However, the cone spectral subtypes in larval zebrafish are not arranged in continuous rows as in the adult. We used cell birthdating to show that the larval cone mosaic pattern remains relatively disorganized, or perhaps is somewhat remodeled, within the adult retina. The abundance of cone subtypes relative to other subtypes is different in this larval remnant compared to that of larvae or canonical adult zebrafish retina. These observations provide baseline data for understanding the development of cone mosaics via comparative analysis of larval and adult cone development in a model species.

Purpose

Dopamine plays key roles in a variety of basic functions in the central nervous system. To study developmental and functional roles of dopaminergic cells in zebrafish, we have generated a transgenic line of zebrafish expressing green fluorescent protein (GFP) under the control of the tyrosine hydroxylase (th1) promoter.

Methods

A 12 kb gene fragment that contains the th1 promoter was isolated and ligated to the MmGFP coding sequence, linearized, microinjected into 1–2 cell stage embryos and the founders crossed with wild-type fish to screen for transgenic lines. Tg(−12th:MmGFP) embryos were visualized under fluorescence microscopy for GFP expression during development. Confocal microscopy was used to visualize GFP-labeled cells in the living whole mount retina and immunostained vertical sections of adult zebrafish retina. Single-cell reverse transcription polymerase chain reaction (RT–PCR) was performed on individual GFP+ cells collected from dispersed retinal cell cultures for th1 and dopamine transporter (dat). Loose-patch recordings of spike activity of GFP+ neurons were made in isolated whole mount retinas.

Results

th1 promoter-driven GFP exhibited robust expression in the brain and retina during zebrafish development. In juvenile and adult fish retinas, GFP was expressed in cells located in the inner nuclear layer. Immunocytochemistry with antibodies for GFP and TH showed that 29±2% of GFP-labeled cells also expressed TH. Two subpopulations of GFP-labeled cells were identified by fluorescent microscopy: bright GFP-expressing cells and dim GFP-expressing cells. Seminested single-cell RT–PCR showed that 71% of dim GFP-expressing cells expressed both th and dat mRNA. Loose-patch voltage-clamp recording from dim GFP-labeled cells in retinal whole mounts revealed that many of these dopaminergic neurons are spontaneously active in darkness.

Conclusions

Although this Tg(−12th:MmGFP) line is not a completely specific reporter for dopaminergic neurons, using relative GFP intensity we are able to enrich for the selection of retinal dopaminergic cells in vitro and in situ in molecular and electrophysiological experiments. This transgenic line provides a useful tool for studying retinal dopaminergic cells in the zebrafish.

In this paper we characterized expression of GATA1 and FLI1 gene promoters in thrombocytes of zebrafish transgenic lines, G1-GM2 and TG(fli1:EGFP)y1 that carry transgenes of GATA1 and FLI1 gene promoters driving GFP. We found two discrete populations of thrombocytes verified by morphology, labeled with GFP in both G1-GM2 and TG(fli1:EGFP)y1 lines: (1) the more intensely labeled GFP+ thrombocyte, and (2) the less intensely labeled GFP+ thrombocytes. The more intensely labeled GFP+ thrombocyte in G1-GM2 line and the less intensely labeled GFP+ thrombocytes in the TG(fli1:EGFP)y1 line corresponded to young thrombocytes. These results showed that young thrombocytes have higher GATA1 promoter activity, while mature thrombocytes have more FLI1 gene promoter transcription. This finding suggests that there is a gradual loss of GATA1 and gain of FLI1 expression as the thrombocytes mature, and this overexpression of FLI1 may help maintain the thrombocyte lineage. Furthermore, the presence of transcriptional factors similar to those found in megakaryocytes raises the possibility that vertebrate thrombocytes may be the forerunners of mammalian megakaryocytes and, therefore, could serve as a model to study megakaryocyte maturation.

Olfactory sensory neurons expressing particular olfactory receptors project to specific reproducible locations within the bulb. The axonal guidance cues that organize this precise projection pattern are only beginning to be identified. To aid in their identification and characterization, we generated a transgenic zebrafish line, OR111-7:IRES:Gal4, in which a small subset of olfactory sensory neurons is labeled. Most sensory neurons expressing the OR111-7 transgene project to a specific location within the bulb, the central zone protoglomerulus, while a smaller number project to the LG1 protoglomerulus. Inhibiting netrin/DCC signaling perturbs the ability of OR111-7 expressing axons to enter the olfactory bulb and alters their patterns of termination within the bulb. The netrin receptor DCC is expressed in olfactory sensory neurons around the time that they elaborate their axons, netrin1a is expressed near the medial-most margin of the olfactory bulb, and netrin1b is expressed within the ventral region of the bulb. Loss of netrin/DCC signaling components causes some OR111-7 expressing sensory axons to wander posteriorly after exiting the olfactory pit, away from netrin expressing areas in the bulb. OR111-7 expressing axons that enter the bulb target the central zone less precisely than normal, spreading away from netrin expressing regions. These pathfinding errors can be corrected by the re-expression of DCC within OR111-7 transgene expressing neurons in DCC morphant embryos. These findings implicate netrins as the only known attractants for olfactory sensory neurons, first drawing OR111-7 expressing axons into the bulb and then into the ventromedially positioned central zone protoglomerulus.

Background

The zebrafish, Danio rerio, is used as a model organism to study vertebrate genetics and development. An effective enhancer trap (ET) in zebrafish using the Tol2 transposon has been demonstrated. This approach could be used to study embryogenesis of a vertebrate species in real time and with high resolution.

Description

The information gathered during the course of systematic investigation of many ET transgenic lines have been collected and compiled in the form of an online database – the Zebrafish Enhancer TRAP lines database (ZETRAP).

Conclusion

ZETRAP is a web-based system that provides data and information to the scientific community about the developmental, genetic and genomic aspects of transgenic zebrafish lines obtained using Tol2 transposon-mediated transgenesis. The current version (version 1.0) contains description of 27 ET lines that express EGFP in various organs and tissues, for example, heart, brain, notochord, gut, etc. It also includes information on insertion sites of the Tol2 transposon in these lines.

Background

Rag1 (Recombination activation gene-1) mediates genomic rearrangement and is essential for adaptive immunity in vertebrates. This gene is also expressed in the olfactory epithelium, but its function there is unknown.

Results

Using a transgenic zebrafish line and immunofluorescence, we show that Rag1 is expressed and translated in a subset of olfactory sensory neurons (OSNs). Neurons expressing GFP under the Rag1 promoter project their axons to the lateral region of the olfactory bulb only, and axons with the highest levels of GFP terminate in a single glomerular structure. A subset of GFP-expressing neurons contain Gαo, a marker for microvillous neurons. None of the GFP-positive neurons express Gαolf, Gαq or the olfactory marker protein OMP. Depletion of RAG1, by morpholino-mediated knockdown or mutation, did not affect axon targeting. Calcium imaging indicates that amino acids evoke chemotopically organized glomerular activity patterns in a Rag1 mutant.

Conclusion

Rag1 expression is restricted to a subpopulation of zebrafish olfactory neurons projecting to the lateral olfactory bulb. RAG1 catalytic activity is not essential for axon targeting, nor is it likely to be required for regulation of odorant receptor expression or the response of OSNs to amino acids.