The polycomb-group gene Ezh2 is required for early mouse development

Abstract

Polycomb-group (Pc-G) genes are required for the stable repression of the homeotic selector genes and other developmentally regulated genes, presumably through the modulation of chromatin domains. Among the Drosophila Pc-G genes, Enhancer of zeste [E(z)] merits special consideration since it represents one of the Pc-G genes most conserved through evolution. In addition, the E(Z) protein family contains the SET domain, which has recently been linked with histone methyltransferase (HMTase) activity. Although E(Z)-related proteins have not (yet) been directly associated with HMTase activity, mammalian Ezh2 is a member of a histone deacetylase complex. To investigate its in vivo function, we generated mice deficient for Ezh2. The Ezh2 null mutation results in lethality at early stages of mouse development. Ezh2 mutant mice either cease developing after implantation or initiate but fail to complete gastrulation. Moreover, Ezh2-deficient blastocysts display an impaired potential for outgrowth, preventing the establishment of Ezh2-null embryonic stem cells. Interestingly, Ezh2 is up-regulated upon fertilization and remains highly expressed at the preimplantation stages of mouse development. Together, these data suggest an essential role for Ezh2 during early mouse development and genetically link Ezh2 with eed and YY1, the only other early-acting Pc-G genes.

FIG. 1

Targeting and genotyping of Ezh2-deficient mice. (A) Diagrammatic representation of the Ezh2 genomic locus, the replacement vector, and the targeted Ezh2 allele. Exons are indicated by black boxes with numbers referring to the starting amino acid positions of the respective exons (23). Also shown are the diagnostic EcoRI restriction sites and the internal probe used for Southern blot analysis, as are the primer pairs (arrowheads) for PCR genotyping. pA indicates polyadenylation signals. (B) Southern blot analysis of EcoRI-restricted DNA isolated from offspring of heterozygous (het) intercrosses. wt, wild type. (C) PCR genotyping of genomic DNA isolated from embryos of heterozygous intercrosses. (D) Whole-mount RNA in situ hybridization with day 7.5 wild-type and Ezh2 null embryos with an Ezh2-specific antisense probe.

FIG. 2

Arrested development and gastrulation failure in presumptive Ezh2-deficient embryos. Deciduae at day 7.0 to day 7.5 were isolated, sectioned at 5-μm thickness, stained with hematoxylin-eosin, and scored for being normal or abnormal based on size and morphology. (A) An almost-sagittal section through a wild-type embryo at day 7.5. (B to D) Sections of presumptive Ezh2 mutants isolated at days 7.0 to 7.5. The arrow in panel C highlights excessive amounts of mesoderm aberrantly accumulating onto the abnormal embryo. The different magnifications of each photograph are indicated. Abbreviations: A, anterior; Al, allantois; Am, amnion; E, embryonic; EE, embryonic ectoderm; Ec, ectoplacental cone; Ex, extraembryonic; ExE, extraembryonic ectoderm; P, posterior; Ch, chorion; VE, visceral endoderm.

FIG. 3

Ezh2 is broadly expressed during early mouse embryogenesis. Whole-mount RNA in situ hybridization analysis of wild-type day 6.5 (A), day 7.5 (B), and day 9.5 (C) embryos with an Ezh2-specific antisense probe or, as a control, with an Ezh2-specific sense probe (D) is shown. Abbreviations: A, anterior; Al, allantois; Am, amnion; Ch, chorion; EE, embryonic ectoderm; M, mesoderm; P, posterior.

FIG. 4

Impaired ES cell derivation and outgrowth potential of Ezh2-deficient blastocysts. Blastocysts were isolated from heterozygous intercrosses at day 3.5 and processed for either ES cell derivation (A and B) or outgrowth experiments (C to F). In panel A, a wild-type (wt) ES cell colony is shown, whereas panel B depicts the highly vacuoled and short-lived mutant cells derived from an Ezh2 null blastocyst. (C to F) Blastocysts were cultured in vitro for 7 days and photographed. Panels C and D show successful outgrowths from a wild-type blastocyst and an Ezh2 null blastocyst. Arrows point to the ICM and to the trophoblast giant cells (GC). Each percentage indicates the proportion of blastocysts (n = 68) displaying the depicted phenotype (see Table 4).

FIG. 5

Preimplantation expression of Ezh1 and Ezh2 in the early mouse embryo. RT-PCR was used to detect Ezh1, Ezh2, and Gapdh transcripts in total RNA preparations from wild-type oocytes, fertilized oocytes, morulae, and blastocysts. As a control, RT-PCR was also performed on total RNA from spleen tissue.