Multisite Assembly of Gateway Induced Clones (MAGIC): a flexible cloning toolbox with diverse applications in vertebrate model systems.

Here we present the Multisite Assembly of Gateway Induced Clones (MAGIC) system, which harnesses site-specific recombination-based cloning via Gateway technology for rapid, modular assembly of between 1 and 3 "Entry" vector components, all into a fourth, standard high copy "Destination" plasmid backbone. The MAGIC toolkit spans a range of in vitro and in vivo uses, from directing tunable gene expression, to driving simultaneous expression of microRNAs and fluorescent reporters, to enabling site-specific recombinase-dependent gene expression. All MAGIC system components are directly compatible with existing multisite gateway Tol2 systems currently used in zebrafish, as well as existing eukaryotic cell culture expression Destination plasmids, and available mammalian lentiviral and adenoviral Destination vectors, allowing rapid cross-species experimentation. Moreover, herein we describe novel vectors with flanking piggyBac transposon elements for stable genomic integration in vitro or in vivo when used with piggyBac transposase. Collectively, the MAGIC system facilitates transgenesis in cultured mammalian cells, electroporated mouse and chick embryos, as well as in injected zebrafish embryos, enabling the rapid generation of innovative DNA constructs for biological research due to a shared, common plasmid platform.

High-resolution analysis of central nervous system expression patterns in zebrafish Gal4 enhancer-trap lines.

BACKGROUND: The application of the Gal4/UAS system to enhancer and gene trapping screens in zebrafish has greatly increased the ability to label and manipulate cell populations in multiple tissues, including the central nervous system (CNS). However the ability to select existing lines for specific applications has been limited by the lack of detailed expression analysis. RESULTS: We describe a Gal4 enhancer trap screen in which we used advanced image analysis, including three-dimensional confocal reconstructions and documentation of expression patterns at multiple developmental time points. In all, we have created and annotated 98 lines exhibiting a wide range of expression patterns, most of which include CNS expression. Expression was also observed in nonneural tissues such as muscle, skin epithelium, vasculature, and neural crest derivatives. All lines and data are publicly available from the Zebrafish International Research Center (ZIRC) from the Zebrafish Model Organism Database (ZFIN). CONCLUSIONS: Our detailed documentation of expression patterns, combined with the public availability of images and fish lines, provides a valuable resource for researchers wishing to study CNS development and function in zebrafish. Our data also suggest that many existing enhancer trap lines may have previously uncharacterized expression in multiple tissues and cell types.

Zebrafish foxP2 zinc finger nuclease mutant has normal axon pathfinding.

foxP2, a forkhead-domain transcription factor, is critical for speech and language development in humans, but its role in the establishment of CNS connectivity is unclear. While in vitro studies have identified axon guidance molecules as targets of foxP2 regulation, and cell culture assays suggest a role for foxP2 in neurite outgrowth, in vivo studies have been lacking regarding a role for foxP2 in axon pathfinding. We used a modified zinc finger nuclease methodology to generate mutations in the zebrafish foxP2 gene. Using PCR-based high resolution melt curve analysis (HRMA) of G0 founder animals, we screened and identified three mutants carrying nonsense mutations in the 2(nd) coding exon: a 17 base-pair (bp) deletion, an 8bp deletion, and a 4bp insertion. Sequence analysis of cDNA confirmed that these were frameshift mutations with predicted early protein truncations. Homozygous mutant fish were viable and fertile, with unchanged body morphology, and no apparent differences in CNS apoptosis, proliferation, or patterning at embryonic stages. There was a reduction in expression of the known foxP2 target gene cntnap2 that was rescued by injection of wild-type foxP2 transcript. When we examined axon pathfinding using a pan-axonal marker or transgenic lines, including a foxP2-neuron-specific enhancer, we did not observe any axon guidance errors. Our findings suggest that foxP2 is not necessary for axon pathfinding during development.

Hypoxia disruption of vertebrate CNS pathfinding through ephrinB2 Is rescued by magnesium.

The mechanisms of hypoxic injury to the developing human brain are poorly understood, despite being a major cause of chronic neurodevelopmental impairments. Recent work in the invertebrate Caenorhabditis elegans has shown that hypoxia causes discrete axon pathfinding errors in certain interneurons and motorneurons. However, it is unknown whether developmental hypoxia would have similar effects in a vertebrate nervous system. We have found that developmental hypoxic injury disrupts pathfinding of forebrain neurons in zebrafish (Danio rerio), leading to errors in which commissural axons fail to cross the midline. The pathfinding defects result from activation of the hypoxia-inducible transcription factor (hif1) pathway and are mimicked by chemical inducers of the hif1 pathway or by expression of constitutively active hif1α. Further, we found that blocking transcriptional activation by hif1α helped prevent the guidance defects. We identified ephrinB2a as a target of hif1 pathway activation, showed that knock-down of ephrinB2a rescued the guidance errors, and showed that the receptor ephA4a is expressed in a pattern complementary to the misrouting axons. By targeting a constitutively active form of ephrinB2a to specific neurons, we found that ephrinB2a mediates the pathfinding errors via a reverse-signaling mechanism. Finally, magnesium sulfate, used to improve neurodevelopmental outcomes in preterm births, protects against pathfinding errors by preventing upregulation of ephrinB2a. These results demonstrate that evolutionarily conserved genetic pathways regulate connectivity changes in the CNS in response to hypoxia, and they support a potential neuroprotective role for magnesium.

Netrin/DCC signaling guides olfactory sensory axons to their correct location in the olfactory bulb.

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 lateral glomerulus 1 protoglomerulus. Inhibiting Netrin/DCC (deleted in colorectal cancer) 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 reexpression 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.

Gal80 intersectional regulation of cell-type specific expression in vertebrates.

Characterization and functional manipulation of specific groups of neurons in the vertebrate central nervous system (CNS) remains a major hurdle for understanding complex circuitry and functions. In zebrafish, the Gal4/UAS system has permitted expression of transgenes and enhancer trap screens, but is often limited by broad expression domains. We have developed a method for cell-type specific expression using Gal80 inhibition of Gal4-dependent expression. We show that native Gal4 is able to drive strong expression, that Gal80 can inhibit this expression, and that overlapping Gal4 and Gal80 expression can achieve "intersectional" expression in spatially and genetically defined subsets of neurons. We also optimize Gal80 for expression in vertebrates, track Gal80 expression with a co-expressed fluorescent marker, and use a temperature-sensitive allele of Gal80 to temporally regulate its function. These data demonstrate that Gal80 is a powerful addition to the genetic techniques available to map and manipulate neural circuits in zebrafish.

Identification of a dopaminergic enhancer indicates complexity in vertebrate dopamine neuron phenotype specification.

The dopaminergic neurons of the basal ganglia play critical roles in CNS function and human disease, but specification of dopamine neuron phenotype is poorly understood in vertebrates. We performed an in vivo screen in zebrafish to identify dopaminergic neuron enhancers, in order to facilitate studies on the specification of neuronal identity, connectivity, and function in the basal ganglia. Based primarily on identification of conserved non-coding elements, we tested 54 DNA elements from four species (zebrafish, pufferfish, mouse, and rat), that included 21 genes with known or putative roles in dopaminergic neuron specification or function. Most elements failed to drive CNS expression or did not express specifically in dopaminergic neurons. However, we did isolate a discrete enhancer from the otpb gene that drove specific expression in diencephalic dopaminergic neurons, although it did not share sequence conservation with regulatory regions of otpa or other dopamine-specific genes. For the otpb enhancer, regulation of expression in dopamine neurons requires multiple elements spread across a large genomic area. In addition, we compared our in vivo testing with in silico analysis of genomic regions for genes involved in dopamine neuron function, but failed to find conserved regions that functioned as enhancers. We conclude that regulation of dopaminergic neuron phenotype in vertebrates is regulated by dispersed regulatory elements.

Domain-specific regulation of foxP2 CNS expression by lef1.

BACKGROUND: FOXP2 is a forkhead transcription factor critical for normal development of language in humans, but little is known of its broader function and regulation during central nervous system (CNS) development. We report here that lef1, a member of the Lef/Tcf family of transcription factors activated by Wnt signaling, regulates foxP2 during embryogenesis, and we isolate novel foxP2 enhancers which are lef1-dependent. RESULTS: Loss, knock down, or inhibition of lef1 led to loss of foxP2 expression. We isolated DNA fragments from the foxP2 genomic region that function as enhancers to drive GFP expression in the CNS during development, including in the telencephalon, diencephalon, eye, tectum, and hindbrain. Three of these enhancers, foxP2-enhancerA.1, foxP2-enhancerB, and foxP2-enhancerD, contain putative Lef1 binding sites, and are regulated by lef1. However, two other genomic fragments containing Lef1 sites failed to function in vivo as enhancers. Chromatin immunoprecipitation confirmed that Lef1 binds to sites in foxP2-enhancerA.1 and foxP2-enhancerB. CONCLUSION: This work shows that lef1 is necessary for expression of foxP2 in the tectum, mid-hindbrain boundary, and hindbrain during CNS development, and is the first insight into the upstream regulation of foxP2 during development. We also demonstrate that in silico prediction of potential lef1 binding sites poorly predicts their ability to function in vivo as enhancers. The foxP2 enhancers we identified will allow dissection of foxP2's role during CNS development.

The Tol2kit: a multisite gateway-based construction kit for Tol2 transposon transgenesis constructs.

Transgenesis is an important tool for assessing gene function. In zebrafish, transgenesis has suffered from three problems: the labor of building complex expression constructs using conventional subcloning; low transgenesis efficiency, leading to mosaicism in transient transgenics and infrequent germline incorporation; and difficulty in identifying germline integrations unless using a fluorescent marker transgene. The Tol2kit system uses site-specific recombination-based cloning (multisite Gateway technology) to allow quick, modular assembly of [promoter]-[coding sequence]-[3' tag] constructs in a Tol2 transposon backbone. It includes a destination vector with a cmlc2:EGFP (enhanced green fluorescent protein) transgenesis marker and a variety of widely useful entry clones, including hsp70 and beta-actin promoters; cytoplasmic, nuclear, and membrane-localized fluorescent proteins; and internal ribosome entry sequence-driven EGFP cassettes for bicistronic expression. The Tol2kit greatly facilitates zebrafish transgenesis, simplifies the sharing of clones, and enables large-scale projects testing the functions of libraries of regulatory or coding sequences.

Charge immobilization of skeletal muscle Na+ channels: role of residues in the inactivation linker.

We investigated structural determinants of fast inactivation and deactivation in sodium channels by comparing ionic flux and charge movement in skeletal muscle channels, using mutations of DIII-DIV linker charges. Charge altering and substituting mutations at K-1317, K-1318 depolarized the g(V) curve but hyperpolarized the h(infinity) curve. Charge reversal and substitution at this locus reduced the apparent voltage sensitivity of open- and closed-state fast inactivation. These effects were not observed with charge reversal at E-1314, E-1315. Mutations swapping or neutralizing the negative cluster at 1314, 1315 and the positive cluster at 1317, 1318 indicated that local interactions dictate the coupling of activation to fast inactivation. Gating charge was immobilized before channel entry into fast inactivation in hNa(V)1.4 but to a lesser extent in mutations at K-1317, K-1318. These results suggest that charge is preferentially immobilized in channels inactivating from the open state. Recovery of gating charge proceeded with a single, fast phase in the double mutation K-1317R, K-1318R. This mutation also partially uncoupled recovery from deactivation. Our findings indicate that charged residues near the fast inactivation "particle" allosterically interact with voltage sensors to control aspects of gating in sodium channels.

Central charged residues in DIIIS4 regulate deactivation gating in skeletal muscle sodium channels.

1. Mutations in the S4 segment of domain III in the voltage gated skeletal muscle sodium channel hNa(V)1.4 were constructed to test the roles of each charged residue in deactivation gating. Mutations comprised charge reversals at K1-R6, charge neutralization, and substitution at R4 and R5. 2. Charge-reversing mutations at R4 and R5 produced the greatest alteration of activation parameters compared to hNa(V)1.4. Effects included depolarization of the conductance/voltage (g/V) curve, decreased valence and slowing of kinetics. 3. Reversal of charge at R2 to R4 hyperpolarized, and reversal at R5 or R6 depolarized the h (infinity) curve. Most DIIIS4 mutations slowed inactivation from the open state. R4E slowed closed state fast inactivation and R5E inhibited its completion .4. Deactivation from the open and/or inactivated state was prolonged in mutations reversing charge at R2 to R4 but accelerated by reversal of charge at R5 or R6. Effects were most pronounced at central charges R4 and R5. 5. Charge and structure each contribute to effects of mutations at R4 and R5 on channel gating. Effects of mutations on activation and deactivation at R4 and, to a lesser extent R5, were primarily owing to charge alteration, whereas effects on fast inactivation were charge independent.

Evolutionary diversification of TTX-resistant sodium channels in a predator-prey interaction.

Understanding the molecular genetic basis of adaptations provides incomparable insight into the genetic mechanisms by which evolutionary diversification takes place. Whether the evolution of common traits in different lineages proceeds by similar or unique mutations, and the degree to which phenotypic evolution is controlled by changes in gene regulation as opposed to gene function, are fundamental questions in evolutionary biology that require such an understanding of genetic mechanisms. Here we identify novel changes in the molecular structure of a sodium channel expressed in snake skeletal muscle, tsNa(V)1.4, that are responsible for differences in tetrodotoxin (TTX) resistance among garter snake populations coevolving with toxic newts. By the functional expression of tsNa(V)1.4, we show how differences in the amino-acid sequence of the channel affect TTX binding and impart different levels of resistance in four snake populations. These results indicate that the evolution of a physiological trait has occurred through a series of unique functional changes in a gene that is otherwise highly conserved among vertebrates.

K-aggravated myotonia mutations at residue G1306 differentially alter deactivation gating of human skeletal muscle sodium channels.

Fast inactivation and deactivation gating were compared between wild-type human voltage-gated skeletal muscle sodium channel (hNaV1.4) and potassium-aggravated myotonia (PAM) mutations G1306A, G1306E, and G1306V. Cell-attached macropatches were used to compare wild-type and PAM-gating properties in normal extracellular K+ (4 mM), decreased K+ (1 mM), and increased K+ (10 mM). G1306E/A increased the apparent valence of the conductance (g(V)) curve. Compared to hNaV1.4, the steady-state inactivation (h infinity) curve was depolarized for G1306E/A but hyperpolarized by G1306V, and this mutation increased apparent valence. G1306A/E slowed the rate of current rise towards peak activation. G1306V slowed open-state deactivation, inactivated-state deactivation, and recovery from fast inactivation. G1306A/E abbreviated open-state deactivation at negative commands. These mutants slowed open-state deactivation at more positive commands, at voltages for which fast inactivation might influence tail current decay. G1306E abbreviated recovery delay without affecting recovery rate. Low K+ increased peak current in hNaV1.4 and in G1306V. For G1306E, low K+ increased the rate of entry into fast inactivation, hyperpolarized the g(V) and h(infinity) curves, and increased recovery delay. Biophysical underpinnings of PAM caused by mutations of G1306 thus vary with the specific mutation, and hyperkalemic exacerbation of effects of mutations at this residue are not direct.

Temperature-sensitive defects in paramyotonia congenita mutants R1448C and T1313M.

The biophysical origins of paramyotonia congenita and its exacerbation in cold temperatures were examined. Human skeletal muscle voltage-gated sodium channels were expressed in Xenopus oocytes and macroscopic currents were recorded from cell-attached patches. Wild-type (hNaV1.4) channels were compared to two mutant channel isoforms, T1313M and R1448C. The voltage dependence and temperature sensitivity of activation, fast-inactivation onset and recovery, and deactivation were studied. Although activation and the onset of fast-inactivation were temperature sensitive in all three isoforms, and although these properties in mutant channels differed from those in wild-type channels, they did not account for cold-exacerbation. Deactivation, however, was disproportionately slower in R1448C, but not in T1313M, than in hNaV1.4. These defects may, at least in part, account for the clinical symptoms of paramyotonia congenita and its exacerbation by cold, and provide a basis for studies into the therapeutic alleviation of these symptoms.

Negatively charged residues adjacent to IFM motif in the DIII-DIV linker of hNa(V)1.4 differentially affect slow inactivation.

The effects on slow inactivation (SI) of charge substitutions, neutralizations, and reversals were studied for the negatively charged residues D1309 and EE1314,15 surrounding the IFM motif in the DIII-DIV cytoplasmic linker - the putative fast inactivation particle - of human skeletal muscle voltage-gated sodium channel (hNa(V)1.4). Changing aspartate (D) at position 1309 to glutamate (E) (substitution) did not strongly affect SI, whereas charge neutralization to glutamine (Q) and charge reversal to arginine (R) right-shifted the midpoint of the steady-state SI curve. Charge neutralization (D-->Q) at position 1309 also reduced the apparent valence associated with SI. Glutamates (E) at positions 1314 and 1315 were similarly mutated. Charge reversal (EE-->RR) right-shifted the steady-state SI curve and both reversal and substitution (EE-->DD) reduced its apparent valence. Charge neutralization (EE-->QQ) and reversal decreased the maximum probability of SI. These mutations also had differential effects on the rate of SI onset and recovery. These results suggest that charged residues in the DIII-DIV linker may interact with structures that control SI.

Negative charges in the DIII-DIV linker of human skeletal muscle Na+ channels regulate deactivation gating.

Charge reversing, neutralizing and substituting mutations at D1309 and EE1314,15 in the DIII-DIV linker of the human skeletal muscle sodium channel hNav1.4 were constructed and expressed in Xenopus oocytes. The effects of these mutations on conductance, inactivation and deactivation were determined using on-cell macropatches. D1309R caused a depolarizing shift of the conductance-voltage (g(V)) curve and increased the apparent valency of activation. D1309R and EE1314,15RR increased time to peak activation. D1309R caused a depolarizing shift of the steady-state fast inactivation curve, whereas EE1314,15RR produced a hyperpolarizing shift and decreased the apparent valency. Charge reversal at either D1309 or EE1314,15 slowed open-state fast inactivation and accelerated closed-state fast inactivation. D1309R accelerated recovery from fast inactivation, whereas EE1314,15RR and EE1314,15QQ slowed recovery. Deactivation from the inactivated state was determined by the delay in the onset to recovery from fast inactivation. Recovery delay was abbreviated for D1309R but was prolonged for EE1314,15RR and EE1314,15QQ. Open-state deactivation was determined from the time constant of the decay (tau D) of tail currents. tau D was slowed by D1309R, D1309E, EE1314,15RR and EE1314,15QQ. Our findings suggest an important role in deactivation gating in hNav1.4 for the negative cluster of charge at EE1314,15. These and previous findings suggest that clusters of negatively and positively charged residues in the hNav1.4 DIII-DIV linker differentially regulate the kinetics of fast inactivation.

Outer and central charged residues in DIVS4 of skeletal muscle sodium channels have differing roles in deactivation.

We tested the effects of charge-neutralizing mutations of the eight arginine residues in DIVS4 of the rat skeletal muscle sodium channel (rNa(V)1.4) on deactivation gating from the open and inactivated states. We hypothesized that neutralization of outer or central charges would accelerate the I-to-C transition as measured by recovery delay because these represent a portion of the immobilizable charge. R1Q abbreviated recovery delay as a consequence of reduced charge content. R4Q increased delay, whereas R5Q abbreviated delay, and charge-substitutions at these residues indicated that each effect was allosteric. We also hypothesized that neutralization of any residue in DIVS4 would slow the O-to-C transition with reduction in positive charge. Reduction in charge at R1, and to a lesser extent at R5, slowed open-state deactivation, while charge neutralizations at R2, R3, R4, R6, and R7 accelerated open-state deactivation. Our findings suggest that arginine residues in DIVS4 in rNa(V)1.4 have differing roles in channel closure from open and inactivated states. Furthermore, they suggest that deactivation in DIVS4 is regulated by charge interaction between the electrical field with the outermost residue, and by local allosteric interactions imparted by central charges.