Darius Balciunas

Harnessing a high cargo-capacity transposon for genetic applications in vertebrates.

Viruses and transposons are efficient tools for permanently delivering foreign DNA into vertebrate genomes but exhibit diminished activity when cargo exceeds 8 kilobases (kb). This size restriction limits their molecular genetic and biotechnological utility, such as numerous therapeutically relevant genes that exceed 8 kb in size. Furthermore, a greater payload capacity vector would accommodate more sophisticated cis cargo designs to modulate the expression and mutagenic risk of these molecular therapeutics. We show that the Tol2 transposon can efficiently integrate DNA sequences larger than 10 kb into human cells. We characterize minimal sequences necessary for transposition (miniTol2) in vivo in zebrafish and in vitro in human cells. Both the 8.5-kb Tol2 transposon and 5.8-kb miniTol2 engineered elements readily function to revert the deficiency of fumarylacetoacetate hydrolase in an animal model of hereditary tyrosinemia type 1. Together, Tol2 provides a novel nonviral vector for the delivery of large genetic payloads for gene therapy and other transgenic applications.

Nicotine response genetics in the zebrafish

Tobacco use is predicted to result in over 1 billion deaths worldwide by the end of the 21st century. How genetic variation contributes to the observed differential predisposition in the human population to drug dependence is unknown. The zebrafish (Danio rerio) is an emerging vertebrate model system for understanding the genetics of behavior. We developed a nicotine behavioral assay in zebrafish and applied it in a forward genetic screen using gene-breaking transposon mutagenesis. We used this method to molecularly characterize bdav/cct8 and hbog/gabbr1.2 as mutations with altered nicotine response. Each have a single human ortholog, identifying two points for potential scientific, diagnostic, and drug development for nicotine biology and cessation therapeutics. We show this insertional method generates mutant alleles that are reversible through Cre-mediated recombination, representing a conditional mutation system for the zebrafish. The combination of this reporter-tagged insertional mutagen approach and zebrafish provides a powerful platform for a rich array of questions amenable to genetic-based scientific inquiry, including the basis of behavior, epigenetics, plasticity, stress, memory, and learning.

The Structural and Functional Role of Med5 in the Yeast Mediator Tail Module

Med5 (Nut1) is identified here as a component of the Mediator tail region. Med5 is positioned peripherally to Med16 (Sin4) together with the three members of the putative Gal11 module, Med15 (Gal11), Med2, and Med3 (Pgd1). The biochemical analysis receives support from genetic interactions between med5Δ and med15Δ deletions. The med5Δ and med16Δ deletion strains share many phenotypes, including effects on mitochondrial function with enhanced growth on nonfermentable carbon sources, increased citrate synthase activity, and increased oxygen consumption. Deletion of the MED5 gene leads to increased transcription of nuclear genes encoding components of the oxidative phosphorylation machinery, whereas mitochondrial genes encoding components of the same machinery are down-regulated. We discuss a possible role for Med5 in coordinating nuclear and mitochondrial gene transcription.

SCORE Imaging: Specimen in a Corrected Optical Rotational Enclosure

Visual data collection is paramount for the majority of scientific research. The added transparency of the zebrafish (Danio rerio) allows for a greater detail of complex biological research that accompanies seemingly simple observational tools. We developed a visual data analysis and collection approach that takes advantage of the cylindrical nature of the zebrafish allowing for an efficient and effective method for image capture that we call Specimen in a Corrected Optical Rotational Enclosure imaging. To achieve a nondistorted image, zebrafish were placed in a fluorinated ethylene propylene tube with a surrounding optically corrected imaging solution (water). By similarly matching the refractive index of the housing (fluorinated ethylene propylene tubing) to that of the inner liquid and outer liquid (water), distortion was markedly reduced, producing a crisp imagable specimen that is able to be fully rotated 360 degrees. A similar procedure was established for fixed zebrafish embryos using convenient, readily available borosilicate capillaries surrounded by 75% glycerol. The method described here could be applied to chemical genetic screening and other related high-throughput methods within the fish community and among other scientific fields.

Development and Notch signaling requirements of the zebrafish choroid plexus.

Background 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. Methodology and Principal Findings Here we show that the transposon-mediated enhancer trap zebrafish line EtMn16 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. Conclusions and Significance 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.

Sleeping Beauty transposon for efficient gene delivery.

Publisher Summary This chapter focuses on the sleeping beauty transposon for efficient gene delivery. The core use of the Sleeping Beauty (SB) transposon in zebrafish has been previously published. The advantages of using SB over DNA injection methods include a higher rate of transgenesis, single-copy integrations with reproducible and long-term Mendelian expression of the transgene cassette, an increase in the total number of chromosomes modified within a particular founder animal strain, and an enzymatically precise engineering of the resulting host chromosome. SB is a potent and reliable gene transfer and expression method for zebrafish. SB is used as a two-component system in zebrafish embryos: a transposon DNA vector and transposase-encoding mRNA. The transposon consists of DNA cargo of choice flanked by palindromic sequences of inverted and direct repeats (IR/DRs). The transposase is delivered as synthetic mRNA, and the embryos potent early translational machinery processes the mRNA into the active transposase enzyme. Transposon vectors are injected along with transposase mRNA to achieve germline integration. The transposon DNA (8.3 ng/μl) and transposase mRNA (100 ng/μl) are mixed together on ice in an eppendorf tube just prior to injection. Combined with the capacity of the zebrafish model to accommodate forward genetic mutagenesis screens, the pu.1 transgenic lines described in the chapter can be very useful in identifying genes that influence myeloid-erythroid and lymphoid/progenitor cell development as well as the differentiation of other myeloid cell types, including granulocyte and monocyte/macrophage lineages.

Etv2 and fli1b function together as key regulators of vasculogenesis and angiogenesis.

Objective— The E26 transformation-specific domain transcription factor Etv2/Etsrp/ER71 is a master regulator of vascular endothelial differentiation during vasculogenesis, although its later role in sprouting angiogenesis remains unknown. Here, we investigated in the zebrafish model a role for Etv2 and related E26 transformation-specific factors, Fli1a and Fli1b in developmental angiogenesis. Approach and Results— Zebrafish fli1a and fli1b mutants were obtained using transposon-mediated gene trap approach. Individual fli1a and fli1b homozygous mutant embryos display normal vascular patterning, yet the angiogenic recovery observed in older etv2 mutant embryos does not occur in embryos lacking both etv2 and fli1b. Etv2 and fli1b double-deficient embryos fail to form any angiogenic sprouts and show greatly increased apoptosis throughout the axial vasculature. In contrast, fli1a mutation did not affect the recovery of etv2 mutant phenotype. Overexpression analyses indicate that both etv2 and fli1b, but not fli1a, induce the expression of multiple vascular markers and of each other. Temporal inhibition of Etv2 function using photoactivatable morpholinos indicates that the function of Etv2 and Fli1b during angiogenesis is independent from the early requirement of Etv2 during vasculogenesis. RNA-Seq analysis and chromatin immunoprecipitation suggest that Etv2 and Fli1b share the same transcriptional targets and bind to the same E26 transformation-specific sites. Conclusions— Our data argue that there are 2 phases of early vascular development with distinct requirements of E26 transformation-specific transcription factors. Etv2 alone is required for early vasculogenesis, whereas Etv2 and Fli1b function redundantly during late vasculogenesis and early embryonic angiogenesis.

Targeted transgene integration overcomes variability of position effects in zebrafish.

Zebrafish transgenesis is increasingly popular owing to the optical transparency and external development of embryos, which provide a scalable vertebrate model for in vivo experimentation. The ability to express transgenes in a tightly controlled spatio-temporal pattern is an important prerequisite for exploitation of zebrafish in a wide range of biomedical applications. However, conventional transgenesis methods are plagued by position effects: the regulatory environment of genomic integration sites leads to variation of expression patterns of transgenes driven by engineered cis-regulatory modules. This limitation represents a bottleneck when studying the precise function of cis-regulatory modules and their subtle variants or when various effector proteins are to be expressed for labelling and manipulation of defined sets of cells. Here, we provide evidence for the efficient elimination of variability of position effects by developing a PhiC31 integrase-based targeting method. To detect targeted integration events, a simple phenotype scoring of colour change in the lens of larvae is used. We compared PhiC31-based integration and Tol2 transgenesis in the analysis of the activity of a novel conserved enhancer from the developmentally regulated neural-specific esrrga gene. Reporter expression was highly variable among independent lines generated with Tol2, whereas all lines generated with PhiC31 into a single integration site displayed nearly identical, enhancer-specific reporter expression in brain nuclei. Moreover, we demonstrate that a modified integrase system can also be used for the detection of enhancer activity in transient transgenesis. These results demonstrate the power of the PhiC31-based transgene integration for the annotation and fine analysis of transcriptional regulatory elements and it promises to be a generally desirable tool for a range of applications, which rely on highly reproducible patterns of transgene activity in zebrafish.

Efficient Transposition of Tol2 in the Mouse Germline

Insertional mutagenesis screens play an integral part in the annotating of functional data for all sequenced genes in the postgenomic era. Chemical mutagenesis screens are highly efficient but identifying the causative gene can be a laborious task. Other mutagenesis platforms, such as transposable elements, have been successfully applied for insertional mutagenesis screens in both the mouse and rat. However, relatively low transposition efficiency has hampered their use as a high-throughput forward genetic mutagenesis screen. Here we report the first evidence of germline activity in the mouse using a naturally active DNA transposon derived from the medaka fish called Tol2, as an alternative system for high-throughput forward genetic mutagenesis screening tool.

The lineage-specific gene ponzr1 is essential for zebrafish pronephric and pharyngeal arch development

The Homeobox (Hox) and Paired box (Pax) gene families are key determinants of animal body plans and organ structure. In particular, they function within regulatory networks that control organogenesis. How these conserved genes elicit differences in organ form and function in response to evolutionary pressures is incompletely understood. We molecularly and functionally characterized one member of an evolutionarily dynamic gene family, plac8 onzin related protein 1 (ponzr1), in the zebrafish. ponzr1 mRNA is expressed early in the developing kidney and pharyngeal arches. Using ponzr1-targeting morpholinos, we show that ponzr1 is required for formation of the glomerulus. Loss of ponzr1 results in a nonfunctional glomerulus but retention of a functional pronephros, an arrangement similar to the aglomerular kidneys found in a subset of marine fish. ponzr1 is integrated into the pax2a pathway, with ponzr1 expression requiring pax2a gene function, and proper pax2a expression requiring normal ponzr1 expression. In addition to pronephric function, ponzr1 is required for pharyngeal arch formation. We functionally demonstrate that ponzr1 can act as a transcription factor or co-factor, providing the first molecular mode of action for this newly described gene family. Together, this work provides experimental evidence of an additional mechanism that incorporates evolutionarily dynamic, lineage-specific gene families into conserved regulatory gene networks to create functional organ diversity.