Transcriptional control of mesoderm patterning in zebrafish

In early vertebrate embryogenesis, TGF-beta (Transforming Growth Factor beta) signaling factors of the Nodal and BMP families have essential roles during formation and patterning of the three germ layers endoderm, mesoderm and ectoderm. In this project we will characterize the crucial interactions of the transcription factors Ntl, FoxH and Mixer in mediating mesoderm patterning downstream of TGF-beta signals. Previous studies identified Nodal and BMP proteins as morphogens that induce distinct fates in a dose-dependent manner. Currently it is not known how the stem cells of the embryo read these signals and how they translate them into distinct differentiation programs. Using genetic approaches in zebrafish we previously showed that formation of trunk, heart or head mesoderm is regulated by the interaction of the conserved genes foxH1, ntl and mixer. Our data suggest previously not analyzed combinatorial Ntl/FoxH1/Mixer functions for the conversion of TGF-beta signals into distinct differentiation programs. The aim of this project is to determine the targets of the FoxH1/Ntl/Mixer regulatory network and to characterize the molecular control of their regulation. For this project we will use the zebrafish as a unique vertebrate model organism in which complex genetic and molecular embryology approaches can be combined with genome-covering microarray-based analyses.

FoxH1 and Mixer are conserved transcription factors with central functions in vertebrate early cell specification downstream of the Nodal/TGFbeta signaling pathway. Genetic studies revealed gene specific requirements during formation of endoderm (Mixer) and axial mesoderm (FoxH1), as well as synergistic interactions during heart formation and early brain patterning. How these factors regulate fate specification is barely known. In this study the first genome wide description of the embryonic target genes of these factors was established. Target genes were characterized in the model organism zebrafish as based on a combination of micro-array based transcriptome comparison and ChIP sequencing. Comparison of these data with the embryonic genome occupancy by RNA-polymerase and Histone variants revealed a primarily transcription activating function of FoxH1, and context dependent functions of Mixer either as a transcriptional activator or repressor. In total over 600 potentially direct FoxH1 were identified, also including most of the known targets. Our data confirm known function of FoxH1 in the auto-regulation of the Nodal-signaling pathways and activation of mesendodermal transcription factors. In addition several genes with functions in other signaling pathways were identified, among them factors involved in Wnt, retinoic acid, FGF and chemokine signaling. Enhancer elements of selected genes were isolated and regulation by FoxH1 was confirmed in reporter assays. The analyses revealed a central role of FoxH1 in connecting or coordinating early embryonic signaling pathways. In case of Mixer this study is the first describing embryonic targets at all. Among the over 400 potentially direct targets 80 genes were found that showed co-regulation by Mixer and FoxH1. This included the established FoxH1 target genes lhx1a and pitx2, for which a co-activation by Foxh1 and Mixer was confirmed in genetic analyses and in molecular enhancer studies. In addition co-regulation was found for several fgf genes. Two fgf genes showed increased expression in mixer-mutants and reduced expression in foxH1-mutants. In the mixer mutants the upregulation of these fgfs correlates with the premature onset and ectopic expression of mesoderm markers. As FGF signaling blocks endoderm induction, the Mixer-dependent repression of fgfs suggests an indirect function of Mixer in endoderm formation. In summary, this study revealed new aspects of the function and interaction of Mixer and FoxH1 during embryonic cell specification and in regulating other early embryonic signaling pathways.