Polycomb group (PcG) proteins are transcriptional repressors of numerous genes, many of which regulate cell cycle progression or developmental processes. We used zebrafish to study Enhancer of zeste homolog 2 (Ezh2), the PcG protein responsible for placing the transcriptional repressive H3K27me3 mark. We identified a nonsense mutant of ezh2 and generated maternal zygotic (MZ) ezh2 mutant embryos. In contrast to knockout mice for PcG proteins, MZezh2 mutant embryos gastrulate seemingly normal, but die around 2 days post fertilization displaying pleiotropic phenotypes. Expression analyses indicated that genes important for early development are not turned off properly, revealing a regulatory role for Ezh2 during zygotic gene expression. In addition, we suggest that Ezh2 regulates maternal mRNA loading of zygotes. Analyses of tissues arising later in development, such as heart, liver, and pancreas, indicated that Ezh2 is required for maintenance of differentiated cell fates. Our data imply that the primary role of Ezh2 is to maintain tissues after tissue specification. Furthermore, our work indicates that Ezh2 is essential to sustain tissue integrity and to set up proper maternal mRNA contribution, and presents a novel and powerful tool to study how PcG proteins contribute to early vertebrate development.Early development of multi-cellular organisms is a highly dynamic process requiring an exquisite and tight control over establishment and maintenance of cellular identity. Deregulation of these processes can lead to malformations or disease. Hence, a proper understanding of both cellular differentiation and maintenance of cell fate is relevant in many different settings.To enable proper cellular specification, expression profiles have to become spatially and temporally restricted during development. Because every cell in theory has the same DNA content gene expression has to be determined at a higher order of regulation. This is in part achieved by chromatin: the complex of DNA wrapped around an octamer of histones plus associated proteins. The histone-octamer contains histones H2A, H2B, H3, and H4, which can be post-translationally modified 1 . In addition, DNA itself can be modified by methylation 2 . The combination of modifications, sometimes also referred to as the epigenome, is thought to determine the accessibility and transcriptional activity of DNA.One of the protein complexes affecting chromatin modifications is the well-conserved Polycomb group (PcG) complex that was first identified in Drosophila. PcG proteins repress gene expression by depositing repressive histone marks, H3K27me3 and H2AK119Ub 3 . Well-known targets of PcG proteins are Hox genes 4 . Pioneering work established that PcG proteins are essential for proper patterning during early embryogenesis. In addition, it is proposed that PcG proteins are essential to balance pluripotency and differentiation potential of stem cells [5][6][7][8] . Besides a role in early embryogenesis, PcG proteins are important for tissue-specific development 9-12 .

The Polycomb group (PcG) protein family is a well-known group of epigenetic modifiers. We used zebrafish to investigate the role of Rnf2, the enzymatic subunit of PRC1. We found a positive correlation between loss of Rnf2 and upregulation of genes, especially of those whose promoter is normally bound by Rnf2. The heart of rnf2 mutants shows a tubular shaped morphology and to further understand the underlying mechanism, we studied gene expression of single wildtype and rnf2 mutant hearts. We detected the most pronounced differences at 3 dpf, including upregulation of heart transcription factors, such as tbx2a, tbx2b, and tbx3a. These tbx genes were decorated by broad PcG domains in wildtype whole embryo lysates. Chamber specific genes such as vmhc, myh6, and nppa showed downregulation in rnf2 mutant hearts. The marker of the working myocard, nppa, is negatively regulated by Tbx2 and Tbx3. Based on our findings and literature we postulate that loss of Rnf2-mediated repression results in upregulation and ectopic expression of tbx2/3, whose expression is normally restricted to the cardiac conductive system. This could lead to repression of chamber specific gene expression, a misbalance in cardiac cell types, and thereby to cardiac defects observed in rnf2 mutants.

Polycomb Group (PcG) genes are transcriptional repressors that are described to be important during development and differentiation. There is significant interest in PcGs proteins because of their role in stem cell biology and tumorigenesis. In this study we characterize the expression of a selection of PcG genes in the adult germline of zebrafish and during embryogenesis. In adults, expression of selected PcG genes is found to be enriched in germ line over somatic tissues. Therefore, the germ line of adult zebrafish was analyzed for the expression pattern of a selection of PcG genes by whole mount in situ hybridization. We detected presence of the tested PcG gene transcripts at early stages of both oogenesis and spermatogenesis. This enriched expression for early stages of gametogenesis is also observed in developing gonads at 4 and 5 weeks post fertilization. Additionally, zebrafish embryos were used to study the spatio-temporal expression patterns of a selection of PcG genes during development. The PcG genes that we tested are maternally loaded and ubiquitously expressed at early developmental stages, except of ezh1. The expression of the PcG genes that were assessed becomes enriched anteriorly and is more defined during tissue specification. The data shown here is an important resource for functional PcG gene studies in vivo.

Polycomb group (PcG) proteins are transcriptional repressors that are important regulators of cell fate during embryonic development. Among them, Ezh2 is responsible for catalyzing the epigenetic repressive mark H3K27me3 and is essential for animal development. The ability of zebrafish embryos lacking both maternal and zygotic ezh2 to form a normal body plan provides a unique model to comprehensively study Ezh2 function during early development in vertebrates. By using a multi-omics approach, we found that Ezh2 is required for the deposition of H3K27me3 and is essential for the recruitment of Polycomb group protein Rnf2. However, and despite the complete absence of PcG-associated epigenetic mark and proteins, only minor changes in H3K4me3 deposition and gene and protein expression occurred. These changes were mainly due to local deregulation of transcription factors outside their normal expression boundaries. Altogether, our results in zebrafish show that Polycomb-mediated gene repression is important right after the body plan is formed to maintain spatially restricted expression profiles of transcription factors and highlight the differences that exist in the timing of PcG protein action between vertebrate species.Summary statementOur unique zebrafish model of maternal and zygotic mutant for the Polycomb group gene ezh2 reveals major conserved and divergent mechanisms in epigenetic gene repression during vertebrate development.

Polycomb group (PcG) proteins are transcriptional repressors that are important regulators of cell fate during embryonic development. Among them, Ezh2 is responsible for catalyzing the epigenetic repressive mark H3K27me3 and is essential for animal development. The ability of zebrafish embryos lacking both maternal and zygotic ezh2 to form a normal body plan provides a unique model for comprehensively studying Ezh2 function during early development in vertebrates. By using a multi-omics approach, we found that Ezh2 is required for the deposition of H3K27me3 and is essential for proper recruitment of Polycomb group protein Rnf2. However, despite the complete absence of PcG-associated epigenetic mark and proteins, only minor changes in H3K4me3 deposition and gene and protein expression occur. These changes were mainly due to local dysregulation of transcription factors outside their normal expression boundaries. Altogether, our results in zebrafish show that Polycomb-mediated gene repression is important immediately after the body plan is formed to maintain spatially restricted expression profiles of transcription factors, and we highlight the differences that exist in the timing of PcG protein action between vertebrate species.