Combined activity of the two Gli2 genes of zebrafish play a major role in Hedgehog signaling during zebrafish neurodevelopment.

It has been proposed that the downstream mediator of the evolutionarily conserved Hedgehog pathway Gli2 plays a relatively minor role in neural development of zebrafish. The second gli2 of zebrafish, gli2b, is expressed in the neural plate and the central nervous system. Our comparative analysis of the developmental role of gli2/gli2b demonstrate a major role of the two Gli2s in mediating Hh signaling. The Gli2s play an early Hh-independent repressor role in the maintenance of neural progenitors and an Hh-dependent activating role during cell differentiation in the floor plate, branchial motor neurons, and sensory neurons. Our analysis of Gli2b loss-of-function using antisense morpholino oligonucleotides indicates that the functions of the two Gli2s diverged in evolution. Gli2b acts in cell proliferation and plays an early role in the hindbrain within a regulatory cascade involving Notch and Ngn1, as well as a role as specific activator in rhombomere 4.

Zebrafish transgenic Enhancer TRAP line database (ZETRAP).

BACKGROUND: The zebrafish, Danio rerio, is used as a model organism to study vertebrate genetics and development. An effective enhancer trap (ET) in zebrafish using the Tol2 transposon has been demonstrated. This approach could be used to study embryogenesis of a vertebrate species in real time and with high resolution. DESCRIPTION: The information gathered during the course of systematic investigation of many ET transgenic lines have been collected and compiled in the form of an online database--the Zebrafish Enhancer TRAP lines database (ZETRAP). CONCLUSION: ZETRAP is a web-based system that provides data and information to the scientific community about the developmental, genetic and genomic aspects of transgenic zebrafish lines obtained using Tol2 transposon-mediated transgenesis. The current version (version 1.0) contains description of 27 ET lines that express EGFP in various organs and tissues, for example, heart, brain, notochord, gut, etc. It also includes information on insertion sites of the Tol2 transposon in these lines.

Tol2 transposon-mediated enhancer trap to identify developmentally regulated zebrafish genes in vivo.

We have used the Tol2 transposable element to design and perform effective enhancer trapping in zebrafish. Modified transposon DNA and transposase RNA were delivered into zebrafish embryos by microinjection to produce heritable insertions in the zebrafish genome. The enhancer trap construct carries the EGFP gene controlled by a partial epithelial promoter from the keratin8 gene. Enhanced green fluorescent protein (EGFP) is used as a marker to select F1 transgenic fish and as a reporter to trap enhancers. We have isolated 28 transgenic lines that were derived from the 37 GFP-positive F0 founders and displayed various specific EGFP expression patterns in addition to basal expression from the modified keratin 8 promoter. Analyses of expression by whole-mount RNA in situ hybridization demonstrated that these patterns could recapitulate the expression of the tagged genes to a variable extent and, therefore, confirmed that our construct worked effectively as an enhancer trap. Transgenic offspring from the 37 F0 EGFP-positive founders have been genetically analyzed up to the F2 generation. Flanking sequences from 65 separate transposon insertion sites were identified by thermal asymmetric interlaced polymerase chain reaction. Injection of the transposase RNA into transgenic embryos induced remobilization of genomic Tol2 copies producing novel insertions including some in the germ line. The approach has great potential for developmental and anatomical studies of teleosts.