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 .

Many regulatory pathways are conserved in the zebrafish intestine compared to mammals, rendering it a strong model to study intestinal development. However, the (epi)genetic regulation of zebrafish intestinal development remains largely uncharacterized. We performed RNA-sequencing and chromatin immunoprecipitation (ChIP)-sequencing for activating (H3K4me3) and repressive (H3K27me3) chromatin marks on isolated intestines at 5, 7, and 9 days post-fertilization (dpf), during which zebrafish transit from yolk dependence to external feeding. RNA-sequencing showed the enrichment of metabolic maintenance genes at all time points and a significant increase in lipid metabolism between 5 and 9 dpf. A strong correlation was observed between gene expression and presence of chromatin marks on gene promoters; H3K4me3-marked genes were expressed higher than H3K27m3-marked genes. Next, we studied a key epigenetic player, Enhancer of zeste homolog 2 (Ezh2). Ezh2 places the repressive H3K27me3 mark on the genome and is highly conserved in vertebrates. We used the nonsense mutant allele ezh2(hu5670) to study the effect of ezh2 loss on intestinal development. These mutants survived gastrulation and died around 11 dpf, showing severe morphological defects in the intestine and liver, accompanied by decreased intestinal (fabp2) and hepatic (fabp10a) marker expressions. Our results suggest that Ezh2 is essential for proper intestinal tissue maintenance and overall survival.

Polycomb group (PcG) proteins are essential regulators of epigenetic gene silencing and development. The PcG protein enhancer of zeste homolog 2 (Ezh2) is a key component of the Polycomb Repressive Complex 2 and is responsible for placing the histone H3 lysine 27 trimethylation (H3K27me3) repressive mark on the genome through its methyltransferase domain. Ezh2 is highly conserved in vertebrates. We studied the role of ezh2 during development of zebrafish with the use of a mutant allele (ezh2(sa1199), R18STOP), which has a stop mutation in the second exon of the ezh2 gene. Two versions of the same line were used during this study. The first and original version of zygotic ezh2(sa1199) mutants unexpectedly retained ezh2 expression in brain, gut, branchial arches, and eyes at 3 days post-fertilization (dpf), as revealed by in-situ hybridization. Moreover, the expression pattern in homozygous mutants was identical to that of wild types, indicating that mutant ezh2 mRNA is not subject to nonsense mediated decay (NMD) as predicted. Both wild type and ezh2 mutant embryos presented edemas at 2 and 3 dpf. The line was renewed by selective breeding to counter select the non-specific phenotypes and survival was assessed. In contrast to earlier studies on ezh2 mutant zebrafish, ezh2(sa1199) mutants survived until adulthood. Interestingly, the ezh2 mRNA and Ezh2 protein were present during adulthood (70 dpf) in both wild type and ezh2(sa1199) mutant zebrafish. We conclude that the ezh2(sa1199) allele does not exhibit an ezh2 loss-of-function phenotype.