Marnie E. Halpern - US grants

DESCRIPTION (Taken directly from applicant's abstract); The fundamental strength of the zebrafish genetic system is phenotypic analysis. Embryos are accessible and optically clear, enabling defects that result from mutation to be readily scored. Thus, from the start, any large-scale genomic effort should exploit this unique property of a vertebrate genetic system. The overall goal of this proposal is to assemble a panel of deletions that uncovers the zebrafish genome. The deletion mapping panel will be a shared resource to assist zebrafish researchers in the identification of genes responsible for existing mutations, fulfilling the centraL objective of the Genomic Resources of the Zebrafish RFA. Previously identified and newly-induced gamma-ray mutagenized strains will be collected. Deficiency mutant phenotypes will be catalogued in an on- line database and mutations preserved as frozen sperm for later retrieval. Genomic DNA prepared from haploid mutants pre-selected for chromosomal deficiencies will be arrayed in multi-well plates for high-throughput PCR mapping of markers, cloned genes and ESTs, and for ease of distribution to the community. Cytological tools will also be developed to facilitate characterization of chromosomal rearrangements. As in non-vertebrate genetic systems, a zebrafish deletion panel is an essential resource, providing the necessary bridge between DNA sequence and gene function.

DESCRIPTION (provided by applicant): A wealth of tools to manipulate gene action in vivo has brought a new level of sophistication to studies of the invertebrate model Drosophila, largely through the use of a bipartite transcriptional regulatory system adapted from yeast. In this system, the transcription factor Gal4 binds to specific DNA sequences upstream of target genes to activate their transcription. By introducing these upstream activating sequences (UAS) next to a minimal promoter and any gene of interest, high levels of expression are obtained in the presence of Gal4. Other regulatory components include Gal80, a repressor that binds to Gal4 and limits its activity. By building both temporal and spatial control into the GAl4/UAS system for the fly, any gene can be induced in a given cell or tissue at a given time. This powerful approach has not only enhanced developmental studies and the generation of new disease models, but has provided insights into how neurons mediate complex behaviors in the adult brain. The goal of the proposed work is to expand upon and optimize the versatile Gal4/UAS system for the vertebrate model, the zebrafish. In initial studies, a new vector was produced that integrates throughout the zebrafish genome by transposition, thereby placing the Gal4 gene under the control of adjacent tissue-specific enhancers. Moreover, these gene/enhancer traps could activate other genes under UAS control, including fluorescent reporters of sub-cellular structure and effectors of cell death. Numerous researchers have requested plasmid constructs and the Gal4 driver and UAS reporter transgenic lines produced from this work. While robust, tissue-restricted expression is routinely achieved now in zebrafish, there is no reliable method to control gene expression temporally. Several approaches have been successfully used in Drosophila and will be tested for their efficacy to induce Gal4 activity in transgenic zebrafish. Some recovered transgenic lines show pronounced transcriptional silencing at the level of the UAS. Another aim of this study is to take advantage of these insertions to gain a greater understanding of how UAS regulated genes are silenced, with the goal of designing new transgenic vectors less susceptible to transcriptional repression. In addition, a battery of new tools under UAS control will be produced that will have broad utility in observing cellular morphology real-time, in long-term lineage studies and in mapping neuronal connectivity in the brain. The proposed collection of tools will bring a new and much needed versatility to zebrafish experimentation and, as with our first set of Gal4/UAS transgenic lines, will serve as a valuable resource for the research community. PUBLIC HEALTH RELEVANCE: The ability to manipulate gene expression in time and space using the Gal4/UAS transcriptional activation system of yeast revolutionized experimental approaches in the Drosophila model. Versatile Gal4-based methods allow researchers to visualize subsets of cells or sub-cellular structures with fluorescent markers, to monitor processes that underlie organ formation real-time, to asses protein function in selective tissues or cells, and to discover new genes acting in genetic pathways. The application of this methodology to zebrafish has unlimited potential and is sorely needed to address processes beyond early development, such as adult physiology and behavior. The proposed experiments are aimed at continuing a productive collaborative effort between three research groups to generate new Gal4/UAS transgenic tools for regulated gene expression in the zebrafish. An additional goal is to optimize the Gal4/UAS system by exploring the basis of transcriptional silencing of zebrafish transgenes and devising approaches to overcome it.

DESCRIPTION (provided by applicant): This is an application for support of a conference entitled Making and breaking the left-right axis: Laterality in development and disease to be held June 15-16, 2013 in Cancun, Mexico, just prior to the joint Society for Developmental Biology 72nd Annual Meeting and the 17th International Congress of Developmental Biology. The objective of this satellite symposium is to bring together researchers working on invertebrate and vertebrate models for an up-to-date presentation of the latest results on the development and disease relevance of left-right asymmetry. Although there have been many exciting recent developments in the field, a meeting on this specific subject has not been held since 2001, when a small workshop was sponsored by the Instituto Juan March in Madrid. Since then, numerous discoveries have been made on how sidedness is established and maintained in the embryo and the common disorders that can result from abnormal left-right patterning, such as ciliopathies, polycystic kidney and liver disease and complex congenital heart disease. New insights have also been obtained on the significance of laterality in the developing nervous system and its influence on behavior. The group of invited speakers will, for the first time, unite researchers working on left-right symmetry breaking in diverse developmental models and organ systems. The high degree of enthusiasm expressed by researchers who have already agreed to participate indicates the timeliness and level of interest for a symposium on this topic. We anticipate that this symposium will attract approximately 100 participants, with the size and format of the event encouraging maximal interaction among the attendees. The co-organizers will also inform the scientific community more broadly by serving as guest editors of a journal issue dedicated to the symposium topic on the development of L-R asymmetry.

DESCRIPTION (provided by applicant): Tools to control the expression of genes in vivo play an essential role in experimental biology and research on disease processes. Nowhere is this more apparent than in the transparent zebrafish embryo, where expression from transgenes such as those encoding fluorescent proteins allows discrete groups of cells or tissues to be labeled and followed over time, and with appropriate regulators, to be genetically modified or targeted for destruction. Binary transcriptional regulatory systems, such as the Gal4/UAS system of yeast, have revolutionized research in Drosophila by enabling precise temporal and spatial control of gene activation. However, this system has been less effective in zebrafish and other vertebrate models, in large part due to the progressive methylation and silencing of multicopy upstream activation sequences (UAS) needed to promote high levels of gene expression from integrated transgenes. In this project, we will adapt the Q regulatory system of Neurospora crassa for use in transgenic zebrafish. The Q system has many advantages, including 1. a higher level of transcriptional activation than achieved by Gal4, 2. a transcriptionl regulator, QF, that can be inactivated by a repressor, QS, 3, the ability to block repression and restore QF activity in the presence of quinic acid and, importantly, 4. a QF binding site (QUAS) that does not contain essential CpG dinucleotides, which are prone to DNA methylation and transcriptional silencing in zebrafish. Recently, the Q system was successfully applied to invertebrate models. Our preliminary data indicate that it also functions effectively to regulate transcription in zebrafish embryos. We propose to validate further the components of the Q system in zebrafish, to develop new methods for intersectional gene expression, to generate all reagents in Gateway compatible vectors for ease of use by other researchers, and to perform a large-scale, enhancer trap screen. We aim to establish a collection QF driver lines that activate reporter genes in unique, tissue-specific patterns. Of special interest is the identification of neural enhancer traps that will be of great value in future studies on brain development and behavior.