TECHNOLOGY REPORT Targeted Gene Expression in the Zebrafish Prechordal Plate Adi Inbal, 1 Jacek Topczewski, 2 and Lilianna Solnica-Krezel 1 * 1 Department of Biological Sciences, Vanderbilt University, Nashville, Tennessee 2 Department of Pediatrics, Northwestern University Feinberg School of Medicine and Children’s Memorial Research Center, Chicago, Illinois Received 26 August 2006; Accepted 26 August 2006 Summary: Targeted gene expression is a powerful tool for understanding gene function in vivo. In zebrafish, overexpression of gene products is typically accom- plished ubiquitously, without temporal and spatial speci- ficity. However, the yeast Gal4/UAS system can be used for targeted gene expression in zebrafish. Here we describe the generation and characterization of Tg[gsc: Gal4-VP16] transgenic zebrafish lines that harbor a con- struct encoding Gal4-VP16 transcriptional activator under the control of a fragment of the goosecoid gene promoter. Tg[gsc:Gal4-VP16] embryos express Gal4-VP16 RNA in presumptive prechordal plate mesendoderm during late blastula and throughout gastrulation. By crossing these fish to Tg[UAS-GFP] transgenic fish, we show that the gsc:Gal4-VP16 transgene is capable of driving strong expression of a target gene in the prechordal plate and its derivatives during gastrulation and segmentation. Thus, the use of Tg[gsc:Gal4-VP16] fish can help in understand- ing gene function in the prechordal plate, an embryonic structure that is crucial for normal neural patterning. genesis 44:584–588, 2006. Published 2006 Wiley-Liss, Inc. y Key words: Gal4-VP16; UAS; goosecoid; GFP A commonly used method for analyzing the function of a particular gene product is observing the results of its gain-of-function via overexpression. In zebrafish this is usually accomplished by injection into early embryos of synthetic RNA encoding the gene product of interest, and results in nearly ubiquitous expression. While this method can be informative, the lack of spatial and tem- poral transcriptional control has several disadvantages: the effects of overexpression on early development could mask later effects, tissue-specific function of the gene product is difficult or even impossible to interpret, and since the injected RNA is degraded, this method is useful only for relatively early developmental stages. Temporal and spatial control of the overexpressed gene product can provide much needed information, such as what are the cells/tissues in which the gene function is required normally, or where should it be downregulated. The Gal4/UAS (Upstream Activating Sequences) system, which enables tissue-specific gene expression, has been used extensively in D. melanogaster (reviewed by Duffy, 2002; Phelps and Brand, 1998). In this system, the yeast Gal4 transcriptional activator is expressed under the reg- ulation of a specific promoter/enhancer, and activates the transcription of target genes whose promoters con- tain UAS elements. The Gal4/UAS system was recently applied to zebrafish (Ko ¨ster and Fraser, 2001; Scheer and Campos-Ortega, 1999). However, while a growing num- ber of publications report the identification of tissue-spe- cific promoters and their use for reporter gene expres- sion, to date, very few tissue-specific Gal4 transgenic zebrafish lines have been reported (Beis et al., 2005; Hans et al., 2004; Scheer et al., 2001). Here we describe the generation of a zebrafish transgenic line, that expresses the Gal4-VP16 fusion protein (see below) in the prechordal plate under the regulation of the goose- coid (gsc) gene promoter, and can therefore drive expression of target genes in this tissue. The prechordal plate, a mesendodermal tissue found in all vertebrate embryos, is implicated in induction and patterning of the forebrain (Reviewed by Kiecker and Niehrs, 2001). At the beginning of gastrulation, prospec- tive mesendodermal cells that become internalized in the axial (dorsal) side of the gastrula, form the mesendo- dermal components of the gastrula organizer. The first cells to internalize will subsequently form the prechor- dal mesendoderm, and are followed by precursors of the notochord (Gritsman et al., 2000; Warga and Nu ¨sslein- Volhard, 1999). During the course of gastrulation, prechordal mesendoderm cells migrate rostrally to form the prechordal plate, which underlies and patterns the developing forebrain. The mechanisms by which the prechordal plate patterns the overlying anterior neuro- ectoderm involve secretion of signaling molecules such as Sonic hedgehog (Shh), Nodal and Bone Morphoge- y This article is a US Government work and, as such, is in the public domain in the United States of America. * Correspondence to: Lilianna Solnica-Krezel, Department of Biological Sciences, Vanderbilt University, VU Station B 351634, Nashville, TN 37235- 1634. E-mail: lilianna.solnica-krezel@vanderbilt.edu Contract grant sponsor: National Institutes of Health (NIH) and Vander- bilt Zebrafish Initiative AVCF. Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/dvg.20253 Published 2006 Wiley-Liss, Inc. genesis 44:584–588 (2006)