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Germline epigenetic programming, including genomic imprinting, substantially influences offspring development. Polycomb Repressive Complex 2 (PRC2) plays an important role in Histone 3 Lysine 27 trimethylation (H3K27me3)-dependent imprinting, loss of which leads to placental hyperplasia in mammalian offspring generated by somatic cell nuclear transfer (SCNT). In this study, we show that offspring from mouse oocytes lacking the Polycomb protein Embryonic Ectoderm Development (EED) were initially growth restricted, characterised by low blastocyst cell counts and substantial mid-gestational developmental delay. This initial developmental delay was followed by striking late-gestational placental hyperplasia, fetal catch-up growth and extended gestational length that culminated in offspring overgrowth. This involved remodelling of the placenta, including expansion of fetal and maternal tissues and conspicuous expansion of the glycogen enriched cell population in the junctional zone that was associated with a delay in parturition. Despite this remodelling and offspring catchup growth, fetal/placental weight ratio and fetal blood glucose levels were low indicating low placental efficiency. Genome-wide analyses identified extensive transcriptional dysregulation in affected placentas, including a range of imprinted and non-imprinted genes and increased expression of the H3K27me3-imprinted geneSlc38a4,which regulates transport of essential amino acids in the placenta. Our data provide an explanation for apparently opposing observations of growth restriction and overgrowth of offspring derived fromEed-nulloocytes and demonstrate that PRC2-dependent programming in the oocyte regulates fetal and placental growth and developmental outcomes.

The regulation of higher order chromatin structure is complex and dynamic; however we do not yet understand the full suite of mechanisms governing architecture. Here we reveal the non-canonical SMC protein Smchd1 as a novel regulator of long-range chromatin interactions, and add it to the canon of epigenetic proteins required forHoxgene regulation. The effect of losing Smchd1-dependent chromatin interactions has varying outcomes dependent on chromatin context. At autosomal targets transcriptionally sensitive to Smchd1 deletion, we find increased short-range interactions and ectopic enhancer activation. By contrast, the inactive X chromosome is transcriptionally refractive to Smchd1 ablation, despite chromosome-wide increases in short-range interactions. There we observe spreading of H3K27me3 domains into regions not normally decorated by this mark. Together these data suggest Smchd1 has the capacity to insulate the chromatin, thereby limiting access to other chromatin modifying proteins.

INTRODUCTIONThe allocation of different progenitor populations to embryonic structures can be visualized by tracking the distribution of cells to specific tissues in the live embryo. A critical prerequisite for cell tracking is to identify unambiguously the progenitors and their descendants during morphogenesis. This can be achieved by using molecular markers that are expressed from transgenes integrated into the genome or as episomal DNA constructs, or by tagging the cells with exogenous markers that are incorporated into the cell membrane or cytoplasmic components of the cells. These labels can be introduced by dye-labeling the membrane, injecting marker enzyme into the cytoplasm, or integrating reporter constructs by transfection or electroporation. This protocol describes how to label cells in the endoderm (which, at this stage of development, is the superficial tissue layer) of live mouse embryos at 7.0-7.5 days post-coitum (dpc), using two carbocyanine dyes (DiI and DiO).

INTRODUCTIONAnalysis of the developmental fate of cell populations in different parts of the embryo enables the construction of fate maps. These reveal the organization of the body plan and can presage the expression of molecular characteristics of cell lineages and the formation of body parts. The efficacy of fate-mapping techniques is critically dependent on their ability to track the cells and all their descendants without compromising the development of the embryo. Cell grafting involves isolating a population of genetically tagged cells from a transgenic embryo and grafting them to a defined site in a nontransgenic host embryo. Tissue colonization is analyzed using a genetic tag (e.g., a fluorescent protein that can be visualized noninvasively) that allows tracking of the transplanted cells and their descendants in the host embryo throughout development in culture. Alternatively, a lacZ transgene can be used to localize graft-derived cells histologically. Differentiation of the graft-derived cells can be studied by examining the expression of molecular markers by in situ hybridization of gene transcripts or immunohistochemical detection of lineage-specific proteins. This protocol describes how to graft cells isolated from a donor embryo into the germ layer of a wild-type host mouse embryo at 7-7.5 days post-coitum (dpc).