Targeted gene expression is a powerful approach to study the function of genes and cells in vivo. In Drosophila, the P elementmediated 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.targeted gene expression ͉ Gal4-UAS ͉ tetanus toxin ͉ touch response ͉ interneuron

Boundary formation and epithelialization are crucial processes in the morphological segmentation of vertebrate somites. By a genetic screening procedure with zebrafish, we identified two genes, integrinalpha5 (itga5) and fibronectin (fn), required for these processes. Fibronectin proteins accumulate at somite boundaries in accordance with epithelialization of the somites. Both Fibronectin accumulation and the epithelialization are dependent on itga5, which is expressed in the most medial part of somites. Although somite boundaries are initially formed, but not maintained, in the anterior trunk of the mutant embryos deficient in either gene, their maintenance is defective at all axial levels of embryos deficient for both of these genes. Therefore, Integrinalpha5-directed assembly of Fibronectin appears critical for epithelialization and boundary maintenance of somites. Furthermore, with an additional deficiency in ephrin-B2a, the segmental defect in itga5 or fn mutant embryos is expanded posteriorly, indicating that both Integrin-Fibronectin and Eph-Ephrin systems function cooperatively in maintaining somite boundaries.

Members of the yeast polymerase‐associated factor 1 (Paf1) complex, which is composed of at least five components (Paf1, Rtf1, Cdc73, Leo1 and Ctr9), are conserved from yeast to humans. Although these proteins have been implicated in RNA polymerase II‐mediated transcription, their roles in vertebrate development have not been explained. Here, we show that a zebrafish mutant with a somite segmentation defect is deficient in rtf1. In addition, embryos deficient in rtf1 or ctr9 show abnormal development of the heart, ears and neural crest cells. rtf1 is required for correct RNA levels of the Notch‐regulated genes her1, her7 and deltaC, and also for Notch‐induced her1 expression in the presomitic mesoderm. Furthermore, the phenotype observed in rtf1‐deficient mutants is enhanced by an additional deficiency in mind bomb, which encodes an effector of Notch signalling. Therefore, zebrafish homologues of the yeast Paf1 complex seem to preferentially affect a subset of genes, including Notch‐regulated genes, during embryogenesis.

In Xenopus and zebrafish, BMP2, 4 and 7 have been implicated, after the onset of zygotic expression, in inducing and maintaining ventro-lateral cell fate during early development. We provide evidence here that a maternally expressed bone morphogenetic protein (BMP), Radar, may control early ventral specification in zebrafish. We show that Radar ventralizes zebrafish embryos and induces the early expression of bmp2b and bmp4. The analysis of Radar overexpression in both swirl/bmp2b mutants and embryos expressing truncated BMP receptors shows that Radar-induced ventralization is dependent on functional BMP2/4 pathways, and may initially rely on an Alk6-related signaling pathway. Finally, we show that while radar-injected swirl embryos still exhibit a strongly dorsalized phenotype, the overexpression of Radar into swirl/bmp2b mutant embryos restores ventral marker expression, including bmp4 expression. Our results suggest that a complex regulation of different BMP pathways controls dorso-ventral (DV) patterning from early cleavage stages until somitogenesis.

Early dorsoventral pattern formation in vertebrate embryos is regulated by opposing activities of ventralizing bone morphogenetic proteins (BMPs) and dorsal-specific BMP antagonists such as Chordin, Noggin and Follistatin. Specific defects in early dorsoventral patterning have been recently found in a number of zebrafish mutants, which exhibit either a ventralized or dorsalized phenotype. One of these, the ventralized mutant chordino (originally called dino) is caused by a mutation in the zebrafish chordin homologue and interacts genetically with the dorsalized mutant swirl. In swirl mutant embryos, dorsal structures such as notochord and somites are expanded while ventral structures such as blood and nephros are missing. Here we demonstrate that the swirl phenotype is caused by mutations in the zebrafish bmp2 gene (zbmp2). While injection of mRNAs encoded by the mutant alleles has no ventralizing effect, injection of wild-type zbmp2 mRNA leads to a complete rescue of the swirl mutant phenotype. Fertile adult mutant fish were obtained, showing that development after gastrulation is not dependent on zbmp2 function. In addition zBMP2 has no maternal role in mesoderm induction. Our analysis shows that swirl/BMP2, unlike mouse BMP2 but like mouse BMP4, is required for early dorsoventral patterning of the zebrafish embryo.