Normal formation of a vertebrate body plan and loss of tissue maintenance in the absence of ezh2

Abstract

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 .

Histone demethylases Kdm6ba and Kdm6bb redundantly promote cardiomyocyte proliferation during zebrafish heart ventricle maturation

“…In contrast, our kdm6b genetic studies are consistent with an opposing view that this bivalency model incompletely represents in vivo H3K4me3/H3K27me3 observations and the effects of disrupting either PRC2 or MLL/COMPASS activity (reviewed in Piunti and Shilatifard, 2016). In further support, genetic loss of combined maternal and zygotic ezh2 and, resultantly, all H3K27me3 does not disrupt gastrulation or overall body plan establishment in zebrafish (San et al, 2016). Nevertheless, ezh2 -null fish do show pleiotropic embryonic defects, including in heart development, that are consistent with widespread poorly maintained cell identities (San et al, 2016).…”

“…In further support, genetic loss of combined maternal and zygotic ezh2 and, resultantly, all H3K27me3 does not disrupt gastrulation or overall body plan establishment in zebrafish (San et al, 2016). Nevertheless, ezh2 -null fish do show pleiotropic embryonic defects, including in heart development, that are consistent with widespread poorly maintained cell identities (San et al, 2016). …”

“…Li et al, 2014), implicating Kdm6b in cardiovascular development. In further support, H3K27me3 marks are conspicuously lost from key cardiogenic genes during in vitro cardiac differentiation of ES cells (Paige et al, 2012; Wamstad et al, 2012) and PRC2 is required to maintain cardiomyocyte identity (Delgado-Olguín et al, 2012; He et al, 2012; San et al, 2016). Additionally, Kdm6b −/− ESCs induced to undergo cardiomyocyte differentiation misexpress many genes implicated in early heart development, including decreased levels of the cardiac progenitor factors Mesp1 , Pdgfra , and Mef2c (Ohtani et al, 2011).…”

Maintenance of spatial gene expression by Polycomb-mediated repression after formation of a vertebrate body plan

“…Lately, the zebrafish embryo has emerged as a model of choice to study developmental epigenetics in vertebrates (Chrispijn et al, 2019, Lindeman et al, 2011, Murphy et al, 2018, Potok et al, 2013, Vastenhouw et al, 2010). Others and we previously used loss-of-function mutants to show that ezh2 is essential for zebrafish development (Dupret et al, 2017, San et al, 2016, San et al, 2018, Zhong et al, 2018). More particularly, our unique vertebrate model of zebrafish embryos mutant for both maternal and zygotic ezh2, referred as MZezh2 mutant embryos, develop seemingly normal until 1 dpf, forming a proper body plan.…”

“…More particularly, our unique vertebrate model of zebrafish embryos mutant for both maternal and zygotic ezh2, referred as MZezh2 mutant embryos, develop seemingly normal until 1 dpf, forming a proper body plan. These mutants ultimately die at 2 dpf, exhibiting a 100% penetrant pleiotropic phenotype associated with a loss of tissue maintenance (San et al, 2016). This makes zebrafish MZezh2 mutant embryos a unique model to study the function of Ezh2 during early development, from fertilization to tissue specification, in the unique context of a vertebrate embryo in which trimethylation of H3K27 has never occurred, unlike cell culture, conditional, or zygotic mutant models.…”