INTRODUCTION During pattern formation in vertebrate neural development regional gene expression is integrated and co-ordinated, such that different cell types can arise in specific spatial and temporal domains (Lumsden and Krumlauf, 1996). A key mechanism that governs restricted gene expression is the local transcriptional control by DNA-binding transcription factors. Together with secreted patterning molecules that mediate cell interactions at a distance, they are required to establish and maintain positional information along the axes of the developing embryo. Transcription factors act via cis-regulatory DNA elements on the control of target gene expression. Therefore, in order to advance from the analysis and manipulation of gene expression towards a general understanding of complex gene interactions and epistatic relationships, it is necessary to gain insight into the cis- regulation of the participating genes. Reporter genes such as the bacterial beta-galactosidase gene (lacZ) or green fluorescent protein (GFP) are useful tools to study promoter activity in transgenic animals, and the construction and analysis of transgenic lines is of prime importance for designing experimental tools. lacZ reporter gene technology has previously been used to study gene expression in zebrafish embryos (Culp et al., 1991; Amsterdam et al., 1995; Amsterdam et al., 1996). However, only a few cases of stable transgene expression driven by tissue-specific, zebrafish promoters have been reported so far (Long et al., 1997; Jessen et al., 1998; Higashijima et al., 2000), and to date, transgenesis has not been exploited during zebrafish mutant analysis. Here, we utilise comparative analysis of reporter gene expression in wild-type and mutant zebrafish embryos, and find 3227 Development 129, 3227-3239 (2002) Printed in Great Britain © The Company of Biologists Limited 2002 DEV1823 The pax2.1 gene encodes a paired-box transcription factor that is one of the earliest genes to be specifically activated in development of the midbrain and midbrain-hindbrain boundary (MHB), and is required for the development and organizer activity of this territory. To understand how this spatially restricted transcriptional activity of pax2.1 is achieved, we have isolated and characterized the pax2.1- promoter using a lacZ and a GFP reporter gene in transient injection assays and transgenic lines. Stable transgenic expression of this reporter gene shows that a 5.3-kb fragment of the 5′ region contains most, but not all, elements required for driving pax2.1 expression. The expressing tissues include the MHB, hindbrain, spinal cord, ear and pronephros. Transgene activation in the pronephros and developing ear suggests that these pax2.1- expressing tissues are composed of independently regulated subdomains. In addition, ectopic but spatially restricted activation of the reporter genes in rhombomeres 3 and 5 and in the forebrain, which do not normally express endogenous pax2.1, demonstrates the importance of negative regulation of pax2.1. Comparison of transgene expression in wild-type and homozygous pax2.1 mutant no isthmus (noi) embryos reveals that the transgene contains control element(s) for a novel, positive transcriptional feedback loop in MHB development. Transcription of endogenous pax2.1 at the MHB is known to be initially Pax2.1 independent, during activation in late gastrulation. In contrast, transgene expression requires the endogenous Pax2.1 function. Transplantations, mRNA injections and morpholino knock-down experiments show that this feedback regulation of pax2.1 transcription occurs cell- autonomously, and that it requires eng2 and eng3 as known targets for Pax2.1 regulation. We suggest that this novel feedback loop may allow continuation of pax2.1 expression, and hence development of the MHB organizer, to become independent of the patterning machinery of the gastrula embryo. Key words: Pax, Transgenesis, no isthmus, Fgf8, engrailed, Zebrafish, Danio rerio, Cell signaling, CNS, Midbrain, Hindbrain, Isthmus, Organizer, Pattern formation SUMMARY A novel positive transcriptional feedback loop in midbrain-hindbrain boundary development is revealed through analysis of the zebrafish pax2.1 promoter in transgenic lines Alexander Picker 1,† , Steffen Scholpp 1 , Heike Böhli 1 , Hiroyuki Takeda 2 and Michael Brand 1, * 1 Max-Planck-Institute of Molecular Cell Biology and Genetics (Dresden), Pfotenhauerstr. 108, 01307 Dresden, Germany 2 Division of Early Embryogenesis, National Institute of Genetics, Mishima 411-8540, Japan † Present address: Lion Bioscience, Waldhofer Strasse 98, 69123 Heidelberg, Germany *Author for correspondence (e-mail: brand@mpi-cbg.de) Accepted 8 April 2002