Maximiliano L. Suster

Genetic dissection of neural circuits by Tol2 transposon-mediated Gal4 gene and enhancer trapping in zebrafish.

Targeted gene expression is a powerful approach to study the function of genes and cells in vivo. In Drosophila, the P element-mediated Gal4-UAS method has been successfully used for this purpose. However, similar methods have not been established in vertebrates. Here we report the development of a targeted gene expression methodology in zebrafish based on the Tol2 transposable element and its application to the functional study of neural circuits. First, we developed gene trap and enhancer trap constructs carrying an engineered yeast Gal4 transcription activator (Gal4FF) and transgenic reporter fish carrying the GFP or the RFP gene downstream of the Gal4 recognition sequence (UAS) and showed that the Gal4FF can activate transcription through UAS in zebrafish. Second, by using this Gal4FF-UAS system, we performed large-scale screens and generated a large collection of fish lines that expressed Gal4FF in specific tissues, cells, and organs. Finally, we developed transgenic effector fish carrying the tetanus toxin light chain (TeTxLC) gene downstream of UAS, which is known to block synaptic transmission. We crossed the Gal4FF fish with the UAS:TeTxLC fish and analyzed double transgenic embryos for defects in touch response. From this analysis, we discovered that targeted expression of TeTxLC in distinct populations of neurons in the brain and the spinal cord caused distinct abnormalities in the touch response behavior. These studies illustrate that our Gal4FF gene trap and enhancer trap methods should be an important resource for genetic analysis of neuronal functions and behavior in vertebrates.

Embryonic assembly of a central pattern generator without sensory input

Locomotion depends on the integration of sensory information with the activity of central circuitry, which generates patterned discharges in motor nerves to appropriate muscles. Isolated central networks generate fictive locomotor rhythms (recorded in the absence of movement), indicating that the fundamental pattern of motor output depends on the intrinsic connectivity and electrical properties of these central circuits. Sensory inputs are required to modify the pattern of motor activity in response to the actual circumstances of real movement. A central issue for our understanding of how locomotor circuits are specified and assembled is the extent to which sensory inputs are required as such systems develop. Here we describe the effects of eliminating sensory function and structure on the development of the peristaltic motor pattern of Drosophila embryos and larvae. We infer that the circuitry for peristaltic crawling develops in the complete absence of sensory input; however, the integration of this circuitry into actual patterns of locomotion requires additional information from the sensory system. In the absence of sensory inputs, the polarity of movement is deranged, and backward peristaltic waves predominate at the expense of forward peristalsis.

Transposon-mediated BAC transgenesis in zebrafish and mice

BackgroundBacterial artificial chromosomes (BACs) are among the most widely used tools for studies of gene regulation and function in model vertebrates, yet methods for predictable delivery of BAC transgenes to the genome are currently limited. This is because BAC transgenes are usually microinjected as naked DNA into fertilized eggs and are known to integrate as multi-copy concatamers in the genome. Although conventional methods for BAC transgenesis have been very fruitful, complementary methods for generating single copy BAC integrations would be desirable for many applications.ResultsWe took advantage of the precise cut-and-paste behavior of a natural transposon, Tol2, to develop a new method for BAC transgenesis. In this new method, the minimal sequences of the Tol2 transposon were used to deliver precisely single copies of a ~70 kb BAC transgene to the zebrafish and mouse genomes. We mapped the BAC insertion sites in the genome by standard PCR methods and confirmed transposase-mediated integrations.ConclusionThe Tol2 transposon has a surprisingly large cargo capacity that can be harnessed for BAC transgenesis. The precise delivery of single-copy BAC transgenes by Tol2 represents a useful complement to conventional BAC transgenesis, and could aid greatly in the production of transgenic fish and mice for genomics projects, especially those in which single-copy integrations are desired.

Natural variation in Drosophila melanogaster diapause due to the insulin-regulated PI3-kinase

This study links natural variation in a Drosophila melanogaster overwintering strategy, diapause, to the insulin-regulated phosphatidylinositol 3-kinase (PI3-kinase) gene, Dp110. Variation in diapause, a reproductive arrest, was associated with Dp110 by using Dp110 deletions and genomic rescue fragments in transgenic flies. Deletions of Dp110 increased the proportion of individuals in diapause, whereas expression of Dp110 in the nervous system, but not including the visual system, decreased it. The roles of phosphatidylinositol 3-kinase for both diapause in D. melanogaster and dauer formation in Caenorhabditis elegans suggest a conserved role for this kinase in both reproductive and developmental arrests in response to environmental stresses.

Transposon-mediated BAC transgenesis in zebrafish

Bacterial artificial chromosomes (BACs) are widely used in studies of vertebrate gene regulation and function because they often closely recapitulate the expression patterns of endogenous genes. Here we report a step-by-step protocol for efficient BAC transgenesis in zebrafish using the medaka Tol2 transposon. Using recombineering in Escherichia coli, we introduce the iTol2 cassette in the BAC plasmid backbone, which contains the inverted minimal cis-sequences required for Tol2 transposition, and a reporter gene to replace a target locus in the BAC. Microinjection of the Tol2-BAC and a codon-optimized transposase mRNA into fertilized eggs results in clean integrations in the genome and transmission to the germline at a rate of ∼15%. A single person can prepare a dozen constructs within 3 weeks, and obtain transgenic fish within approximately 3–4 months. Our protocol drastically reduces the labor involved in BAC transgenesis and will greatly facilitate biological and biomedical studies in model vertebrates.

Transgenesis in Zebrafish with the Tol2 Transposon System

The zebrafish (Danio rerio) is a useful model for genetic studies of vertebrate development. Its embryos are transparent and develop rapidly outside the mother, making it feasible to visualize and manipulate specific cell types in the living animal. Zebrafish is well suited for transgenic manipulation since it is relatively easy to collect large numbers of embryos from adult fish. Several approaches have been developed for introducing transgenes into the zebrafish germline, from the injection of naked DNA to transposon-mediated integration. In particular, the Tol2 transposable element has been shown to create insertions in the zebrafish genome very efficiently. By using Tol2, gene trap and enhancer trap vectors containing the GFP reporter gene or yeast transcription activator Gal4 gene have been developed. Here we outline methodology for creating transgenic zebrafish using Tol2 vectors, and their applications to visualization and manipulation of specific tissues or cells in vivo and for functional studies of vertebrate neural circuits.

Co-option of Sox3 as the male-determining factor on the Y chromosome in the fish Oryzias dancena

Sex chromosomes harbour a primary sex-determining signal that triggers sexual development of the organism. However, diverse sex chromosome systems have been evolved in vertebrates. Here we use positional cloning to identify the sex-determining locus of a medaka-related fish, Oryzias dancena, and find that the locus on the Y chromosome contains a cis-regulatory element that upregulates neighbouring Sox3 expression in developing gonad. Sex-reversed phenotypes in Sox3(Y) transgenic fish, and Sox3(Y) loss-of-function mutants all point to its critical role in sex determination. Furthermore, we demonstrate that Sox3 initiates testicular differentiation by upregulating expression of downstream Gsdf, which is highly conserved in fish sex differentiation pathways. Our results not only provide strong evidence for the independent recruitment of Sox3 to male determination in distantly related vertebrates, but also provide direct evidence that a novel sex determination pathway has evolved through co-option of a transcriptional regulator potentially interacted with a conserved downstream component.

Transgenic tools to characterize neuronal properties of discrete populations of zebrafish neurons

The developing nervous system consists of a variety of cell types. Transgenic animals expressing reporter genes in specific classes of neuronal cells are powerful tools for the study of neuronal network formation. We generated a wide variety of transgenic zebrafish that expressed reporter genes in specific classes of neurons or neuronal progenitors. These include lines in which neurons of specific neurotransmitter phenotypes expressed fluorescent proteins or Gal4, and lines in which specific subsets of the dorsal progenitor domain in the spinal cord expressed fluorescent proteins. Using these, we examined domain organization in the developing dorsal spinal cord, and found that there are six progenitor domains in zebrafish, which is similar to the domain organization in mice. We also systematically characterized neurotransmitter properties of the neurons that are produced from each domain. Given that reporter gene expressions occurs in a wide area of the nervous system in the lines generated, these transgenic fish should serve as powerful tools for the investigation of not only the neurons in the dorsal spinal cord but also neuronal structures and functions in many other regions of the nervous system.

A novel conserved evx1 enhancer links spinal interneuron morphology and cis-regulation from fish to mammals.

Spinal interneurons are key components of locomotor circuits, driving such diverse behaviors as swimming in fish and walking in mammals. Recent work has linked the expression of evolutionarily conserved transcription factors to key features of interneurons in diverse species, raising the possibility that these interneurons are functionally related. Consequently, the determinants of interneuron subtypes are predicted to share conserved cis-regulation in vertebrates with very different spinal cords. Here, we establish a link between cis-regulation and morphology of spinal interneurons that express the Evx1 homeodomain transcription factor from fish to mammals. Using comparative genomics, and complementary transgenic approaches, we have identified a novel enhancer of evx1, that includes two non-coding elements conserved in vertebrates. We show that pufferfish evx1 transgenes containing this enhancer direct reporter expression to a subset of spinal commissural interneurons in zebrafish embryos. Pufferfish, zebrafish and mouse evx1 downstream genomic enhancers label selectively Evx1(+) V0 commissural interneurons in chick and rat embryos. By dissecting the zebrafish evx1 enhancer, we identify a role for a 25 bp conserved cis-element in V0-specific gene expression. Our findings support the notion that spinal interneurons shared between distantly related vertebrates, have been maintained in part via the preservation of highly conserved cis-regulatory modules.

The medaka zic1/zic4 mutant provides molecular insights into teleost caudal fin evolution.

Teleosts have an asymmetrical caudal fin skeleton formed by the upward bending of the caudal-most portion of the body axis, the ural region. This homocercal type of caudal fin ensures powerful and complex locomotion and is regarded as one of the most important innovations for teleosts during adaptive radiation in an aquatic environment. However, the mechanisms that create asymmetric caudal fin remain largely unknown. The spontaneous medaka (teleost fish) mutant, Double anal fin (Da), exhibits a unique symmetrical caudal skeleton that resembles the diphycercal type seen in Polypterus and Coelacanth. We performed a detailed analysis of the Da mutant to obtain molecular insight into caudal fin morphogenesis. We first demonstrate that a large transposon, inserted into the enhancer region of the zic1 and zic4 genes (zic1/zic4) in Da, is associated with the mesoderm-specific loss of their transcription. We then show that zic1/zic4 are strongly expressed in the dorsal part of the ural mesenchyme and thereby induce asymmetric caudal fin development in wild-type embryos, whereas their expression is lost in Da. Comparative analysis further indicates that the dorsal mesoderm expression of zic1/zic4 is conserved in teleosts, highlighting the crucial role of zic1/zic4 in caudal fin morphogenesis.