Adoption of the Q transcriptional regulatory system for zebrafish transgenesis.

The Gal4-UAS regulatory system of yeast is widely used to modulate gene expression in Drosophila; however, there are limitations to its usefulness in transgenic zebrafish, owing to progressive methylation and silencing of the CpG-rich multicopy upstream activation sequence. Although a modified, less repetitive UAS construct may overcome this problem, it is highly desirable to have additional transcriptional regulatory systems that can be applied independently or in combination with the Gal4/UAS system for intersectional gene expression. The Q transcriptional regulatory system of Neurospora crassa functions similarly to Gal4/UAS. QF is a transcriptional activator that binds to the QUAS upstream regulatory sequence to drive reporter gene expression. Unlike Gal4, the QF binding site does not contain essential CpG dinucleotide sequences that are subject to DNA methylation. The QS protein is a repressor of QF mediated transcriptional activation akin to Gal80. The functionality of the Q system has been demonstrated in Drosophila and Caenorhabditis elegans and we now report its successful application to a vertebrate model, the zebrafish, Danio rerio. Several tissue-specific promoters were used to drive QF expression in stable transgenic lines, as assessed by activation of a QUAS:GFP transgene. The QS repressor was found to dramatically reduce QF activity in injected zebrafish embryos; however, a similar repression has not yet been achieved in transgenic animals expressing QS under the control of ubiquitous promoters. A dual reporter construct containing both QUAS and UAS, each upstream of different fluorescent proteins was also generated and tested in transient assays, demonstrating that the two systems can work in parallel within the same cell. The adoption of the Q system should greatly increase the versatility and power of transgenic approaches for regulating gene expression in zebrafish.

Genetically defined nucleus incertus neurons differ in connectivity and function.

The nucleus incertus (NI), a conserved hindbrain structure implicated in the stress response, arousal, and memory, is a major site for production of the neuropeptide relaxin-3. On the basis of goosecoid homeobox 2 (gsc2) expression, we identified a neuronal cluster that lies adjacent to relaxin 3a (rln3a) neurons in the zebrafish analogue of the NI. To delineate the characteristics of the gsc2 and rln3a NI neurons, we used CRISPR/Cas9 targeted integration to drive gene expression specifically in each neuronal group, and found that they differ in their efferent and afferent connectivity, spontaneous activity, and functional properties. gsc2 and rln3a NI neurons have widely divergent projection patterns and innervate distinct subregions of the midbrain interpeduncular nucleus (IPN). Whereas gsc2 neurons are activated more robustly by electric shock, rln3a neurons exhibit spontaneous fluctuations in calcium signaling and regulate locomotor activity. Our findings define heterogeneous neurons in the NI and provide new tools to probe its diverse functions.

Transgenerational analysis of transcriptional silencing in zebrafish.

The yeast Gal4/UAS transcriptional activation system is a powerful tool for regulating gene expression in Drosophila and has been increasing in popularity for developmental studies in zebrafish. It is also useful for studying the basis of de novo transcriptional silencing. Fluorescent reporter genes under the control of multiple tandem copies of the upstream activator sequence (UAS) often show evidence of variegated expression and DNA methylation in transgenic zebrafish embryos. To characterize this systematically, we monitored the progression of transcriptional silencing of UAS-regulated transgenes that differ in their integration sites and in the repetitive nature of the UAS. Transgenic larvae were examined in three generations for tissue-specific expression of a green fluorescent protein (GFP) reporter and DNA methylation at the UAS. Single insertions containing four distinct upstream activator sequences were far less susceptible to methylation than insertions containing fourteen copies of the same UAS. In addition, transgenes that integrated in or adjacent to transposon sequence exhibited silencing regardless of the number of UAS sites included in the transgene. Placement of promoter-driven Gal4 upstream of UAS-regulated responder genes in a single bicistronic construct also appeared to accelerate silencing and methylation. The results demonstrate the utility of the zebrafish for efficient tracking of gene silencing mechanisms across several generations, as well as provide useful guidelines for optimal Gal4-regulated gene expression in organisms subject to DNA methylation.

Specialized neurons in the right habenula mediate response to aversive olfactory cues.

Hemispheric specializations are well studied at the functional level but less is known about the underlying neural mechanisms. We identified a small cluster of cholinergic neurons in the dorsal habenula (dHb) of zebrafish, defined by their expression of the lecithin retinol acyltransferase domain containing 2 a (lratd2a) gene and their efferent connections with a subregion of the ventral interpeduncular nucleus (vIPN). The lratd2a-expressing neurons in the right dHb are innervated by a subset of mitral cells from both the left and right olfactory bulb and are activated upon exposure to the odorant cadaverine that is repellent to adult zebrafish. Using an intersectional strategy to drive expression of the botulinum neurotoxin specifically in these neurons, we find that adults no longer show aversion to cadaverine. Mutants with left-isomerized dHb that lack these neurons are also less repelled by cadaverine and their behavioral response to alarm substance, a potent aversive cue, is diminished. However, mutants in which both dHb have right identity appear more reactive to alarm substance. The results implicate an asymmetric dHb-vIPN neural circuit in the processing of repulsive olfactory cues and in modulating the resultant behavioral response.

A versatile aquatics facility inventory system with real-time barcode scan entry.

Research involving model organisms necessitates recording and archiving many types of animal maintenance and use data. We developed a comprehensive inventory system using FileMaker Pro® to incorporate, record, and archive data on zebrafish stocks, tank organization, husbandry, and fish usage. Our relational database is constructed of tables containing detailed information on fish identity, parents of origin, tank location, mutant phenotypes, caretakers, natural mating and in vitro fertilization experiments, and fish mortality. In addition to its basic annotation and reporting capabilities, the database allows barcode scan entry of several actions, for example, moving a tank of fish, mating or performing in vitro fertilization with specific fish, and recording dead fish. All data are input in real time using either barcode scanning or manual entry. The database provides several types of preformatted reports, as well as printed labels for tank location and stock identification. In summary, we have created a versatile, multipurpose inventory system that can be personalized and enhanced for any zebrafish facility and can be further adapted to organize data and archival information for other model systems or applications.

Directional asymmetry of the zebrafish epithalamus guides dorsoventral innervation of the midbrain target.

The zebrafish epithalamus, consisting of the pineal complex and flanking dorsal habenular nuclei, provides a valuable model for exploring how left-right differences could arise in the vertebrate brain. The parapineal lies to the left of the pineal and the left habenula is larger, has expanded dense neuropil, and distinct patterns of gene expression from the right habenula. Under the influence of Nodal signaling, positioning of the parapineal sets the direction of habenular asymmetry and thereby determines the left-right origin of habenular projections onto the midbrain target, the interpeduncular nucleus (IPN). In zebrafish with parapineal reversal, neurons from the left habenula project to a more limited ventral IPN region where right habenular axons would normally project. Conversely, efferents from the right habenula adopt a more extensive dorsoventral IPN projection pattern typical of left habenular neurons. Three members of the leftover-related KCTD (potassium channel tetramerization domain containing) gene family are expressed differently by the left and right habenula, in patterns that define asymmetric subnuclei. Molecular asymmetry extends to protein levels in habenular efferents, providing additional evidence that left and right axons terminate within different dorsoventral regions of the midbrain target. Laser-mediated ablation of the parapineal disrupts habenular asymmetry and consequently alters the dorsoventral distribution of innervating axons. The results demonstrate that laterality of the dorsal forebrain influences the formation of midbrain connections and their molecular properties.