Zebrafish gbx1 refines the Midbrain-Hindbrain Boundary border and mediates the Wnt8 posteriorization signal

International audienceBACKGROUND: Studies in mouse, Xenopus and chicken have shown that Otx2 and Gbx2 expression domains are fundamental for positioning the midbrain-hindbrain boundary (MHB) organizer. Of the two zebrafish gbx genes, gbx1 is a likely candidate to participate in this event because its early expression is similar to that reported for Gbx2 in other species. Zebrafish gbx2, on the other hand, acts relatively late at the MHB. To investigate the function of zebrafish gbx1 within the early neural plate, we used a combination of gain- and loss-of-function experiments. RESULTS: We found that ectopic gbx1 expression in the anterior neural plate reduces forebrain and midbrain, represses otx2 expression and repositions the MHB to a more anterior position at the new gbx1/otx2 border. In the case of gbx1 loss-of-function, the initially robust otx2 domain shifts slightly posterior at a given stage (70% epiboly), as does MHB marker expression. We further found that ectopic juxtaposition of otx2 and gbx1 leads to ectopic activation of MHB markers fgf8, pax2.1 and eng2. This indicates that, in zebrafish, an interaction between otx2 and gbx1 determines the site of MHB development. Our work also highlights a novel requirement for gbx1 in hindbrain development. Using cell-tracing experiments, gbx1 was found to cell-autonomously transform anterior neural tissue into posterior. Previous studies have shown that gbx1 is a target of Wnt8 graded activity in the early neural plate. Consistent with this, we show that gbx1 can partially restore hindbrain patterning in cases of Wnt8 loss-of-function. We propose that in addition to its role at the MHB, gbx1 acts at the transcriptional level to mediate Wnt8 posteriorizing signals that pattern the developing hindbrain. CONCLUSION: Our results provide evidence that zebrafish gbx1 is involved in positioning the MHB in the early neural plate by refining the otx2 expression domain. In addition to its role in MHB formation, we have shown that gbx1 is a novel mediator of Wnt8 signaling during hindbrain patterning

Integrated and toolsupported teaching of testing, debugging, and verification

Abstract. This paper reports about the design, implementation, and experiences with a non-traditional formal methods course called “Testing, Debugging, and Verification”. The course aims at integrating formal and informal methods as much as possible. It is supposed to be accessible to participants without extensive mathematical training.

Verifying Object-Oriented Programs with KeY: A Tutorial

This paper is a tutorial on performing formal specification and semi-automatic verification of Java programs with the formal software development tool KeY. This tutorial aims to fill the gap between elementary introductions using toy examples and state-of-art case studies by going through a self-contained, yet non-trivial, example. It is hoped that this contributes to explain the problems encountered in verification of imperative, object-oriented programs to a readership outside the limited community of active researchers

Formal specification with the Java modeling language

This text is a general, self contained, and tool independent introduction into the Java Modeling Language, JML. It appears in a book about the KeY approach and tool, because JML is the dominating starting point of KeY style Java verification. However, this chapter does not depend on KeY, nor any other specific tool, nor on any specific verification methodology. With this text, the authors aim to provide, for the time being, the definitive, general JML tutorial

Verifying Object-Oriented Programs with KeY: A Tutorial

This paper is a tutorial on performing formal specification and semi-automatic verification of Java programs with the formal software development tool KeY. This tutorial aims to fill the gap between elementary introductions using toy examples and state-of-art case studies by going through a self-contained, yet non-trivial, example. It is hoped that this contributes to explain the problems encountered in verification of imperative, object-oriented programs to a readership outside the limited community of active researchers

Deductive Software Verification - The KeY Book

Static analysis of software with deductive methods is a highly dynamic field of research on the verge of becoming a mainstream technology in software engineering. It consists of a large portfolio of - mostly fully automated - analyses: formal verification, test generation, security analysis, visualization, and debugging. All of them are realized in the state-of-art deductive verification framework KeY. This book is the definitive guide to KeY that lets you explore the full potential of deductive software verification in practice. It contains the complete theory behind KeY for active researchers who want to understand it in depth or use it in their own work. But the book also features fully self-contained chapters on the Java Modeling Language and on Using KeY that require nothing else than familiarity with Java. All other chapters are accessible for graduate students (M.Sc. level and beyond). The KeY framework is free and open software, downloadable from the book companion website which contains also all code examples mentioned in this book