Induced epigenetic changes memorized across generations in mice.

Transgenerational epigenetic inheritance in mammals has long been debatable. In this issue of Cell, Takahashi et al. induce DNA methylation at promoter-associated CpG islands (CGIs) of two metabolism-related genes and show that the acquired epigenetic changes and associated metabolic phenotypes are stably propagated across several generations in transgenic mice.

Time-aligned hourglass gastrulation models in rabbit and mouse.

The hourglass model describes the convergence of species within the same phylum to a similar body plan during development; however, the molecular mechanisms underlying this phenomenon in mammals remain poorly described. Here, we compare rabbit and mouse time-resolved differentiation trajectories to revisit this model at single-cell resolution. We modeled gastrulation dynamics using hundreds of embryos sampled between gestation days 6.0 and 8.5 and compared the species using a framework for time-resolved single-cell differentiation-flows analysis. We find convergence toward similar cell-state compositions at E7.5, supported by the quantitatively conserved expression of 76 transcription factors, despite divergence in surrounding trophoblast and hypoblast signaling. However, we observed noticeable changes in specification timing of some lineages and divergence of primordial germ cell programs, which in the rabbit do not activate mesoderm genes. Comparative analysis of temporal differentiation models provides a basis for studying the evolution of gastrulation dynamics across mammals.

The intrinsic and extrinsic effects of TET proteins during gastrulation.

Mice deficient for all ten-eleven translocation (TET) genes exhibit early gastrulation lethality. However, separating cause and effect in such embryonic failure is challenging. To isolate cell-autonomous effects of TET loss, we used temporal single-cell atlases from embryos with partial or complete mutant contributions. Strikingly, when developing within a wild-type embryo, Tet-mutant cells retain near-complete differentiation potential, whereas embryos solely comprising mutant cells are defective in epiblast to ectoderm transition with degenerated mesoderm potential. We map de-repressions of early epiblast factors (e.g., Dppa4 and Gdf3) and failure to activate multiple signaling from nascent mesoderm (Lefty, FGF, and Notch) as likely cell-intrinsic drivers of TET loss phenotypes. We further suggest loss of enhancer demethylation as the underlying mechanism. Collectively, our work demonstrates an unbiased approach for defining intrinsic and extrinsic embryonic gene function based on temporal differentiation atlases and disentangles the intracellular effects of the demethylation machinery from its broader tissue-level ramifications.

A single-embryo, single-cell time-resolved model for mouse gastrulation.

Mouse embryonic development is a canonical model system for studying mammalian cell fate acquisition. Recently, single-cell atlases comprehensively charted embryonic transcriptional landscapes, yet inference of the coordinated dynamics of cells over such atlases remains challenging. Here, we introduce a temporal model for mouse gastrulation, consisting of data from 153 individually sampled embryos spanning 36 h of molecular diversification. Using algorithms and precise timing, we infer differentiation flows and lineage specification dynamics over the embryonic transcriptional manifold. Rapid transcriptional bifurcations characterize the commitment of early specialized node and blood cells. However, for most lineages, we observe combinatorial multi-furcation dynamics rather than hierarchical transcriptional transitions. In the mesoderm, dozens of transcription factors combinatorially regulate multifurcations, as we exemplify using time-matched chimeric embryos of Foxc1/Foxc2 mutants. Our study rejects the notion of differentiation being governed by a series of binary choices, providing an alternative quantitative model for cell fate acquisition.

Ex utero mouse embryogenesis from pre-gastrulation to late organogenesis.

The mammalian body plan is established shortly after the embryo implants into the maternal uterus, and our understanding of post-implantation developmental processes remains limited. Although pre- and peri-implantation mouse embryos are routinely cultured in vitro1,2, approaches for the robust culture of post-implantation embryos from egg cylinder stages until advanced organogenesis remain to be established. Here we present highly effective platforms for the ex utero culture of post-implantation mouse embryos, which enable the appropriate development of embryos from before gastrulation (embryonic day (E) 5.5) until the hindlimb formation stage (E11). Late gastrulating embryos (E7.5) are grown in three-dimensional rotating bottles, whereas extended culture from pre-gastrulation stages (E5.5 or E6.5) requires a combination of static and rotating bottle culture platforms. Histological, molecular and single-cell RNA sequencing analyses confirm that the ex utero cultured embryos recapitulate in utero development precisely. This culture system is amenable to the introduction of a variety of embryonic perturbations and micro-manipulations, the results of which can be followed ex utero for up to six days. The establishment of a system for robustly growing normal mouse embryos ex utero from pre-gastrulation to advanced organogenesis represents a valuable tool for investigating embryogenesis, as it eliminates the uterine barrier and allows researchers to mechanistically interrogate post-implantation morphogenesis and artificial embryogenesis in mammals.

A Non-Canonical MITE in the WOX11 Promoter Is Associated with Robust Crown Root development in Rice.

The formation of tissues and organs in multicellular organisms is tightly controlled by transcriptional programs determined by temporal and spatial patterns of gene expression. As an important regulator of rice crown root development, WOX11 is essential for crown root formation and its transcript level is positively correlated with crown root biomass. However, how WOX11 is regulated during crown root primordium emergence and outgrowth still remains unknown. In this study, variations of the WOX11 genomic sequence were analyzed, and the highest genetic diversity was found within its promoter, which contained a non-canonical miniature inverted-repeat transposable element (ncMITE) sequence. Analysis of the WOX11 promoter-driven reporter gene GUS (ß-glucuronidase) transgenic plants pWOX11(ncMITE+):GUS and pWOX11(ncMITE-):GUS uncovered higher GUS expression levels in crown roots of pWOX11(ncMITE+):GUS plants. Furthermore, pWOX11(ncMITE+):WOX11-FLAG in wox11 background could complement the crown root number and length compared to those of the wild type, while pWOX11(ncMITE-):WOX11-FLAG could not. These results suggested that the ncMITE was positively associated with WOX11 transcripts in rice crown roots. In addition, DNA methylation nearby the ncMITE region attenuated the activation effect of the ncMITE on WOX11 expression, which might also be the cause conferred to the root-specific expression of WOX11. This work provides novel insight into WOX11 expression regulation and reveals a promising target for genetic improvement of root architecture in rice.

Rice Homeodomain Protein WOX11 Recruits a Histone Acetyltransferase Complex to Establish Programs of Cell Proliferation of Crown Root Meristem.

Shoot-borne crown roots are the major root system in cereals. Previous work has shown that the Wuschel-related homeobox gene WOX11 is necessary and sufficient to promote rice (Oryza sativa) crown root emergence and elongation. Here, we show that WOX11 recruits the ADA2-GCN5 histone acetyltransferase module to activate downstream target genes in crown root meristem. Rice ADA2 and GCN5 genes are highly expressed in root meristem and are shown to be essential for cell division and growth. WOX11 and ADA2-GCN5 commonly target and regulate a set of root-specific genes involved in energy metabolism, cell wall biosynthesis, and hormone response, some of which are known to be important for root development. The results indicate that the recruitment of ADA2-GCN5 by WOX11 establishes gene expression programs of crown root meristem cell division and suggest that permissive chromatin modification involving histone acetylation is a strategy for WOX11 to stimulate root meristem development.

WOX11 recruits a histone H3K27me3 demethylase to promote gene expression during shoot development in rice.

WUSCHEL-related homeobox (WOX) genes are key regulators of meristem activity and plant development, the chromatin mechanism of which to reprogram gene expression remains unclear. Histone H3K27me3 is a chromatin mark of developmentally repressed genes. How the repressive mark is removed from specific genes during plant development is largely unknown. Here, we show that WOX11 interacts with the H3K27me3 demethylase JMJ705 to activate gene expression during shoot development in rice. Genetic analysis indicates that WOX11 and JMJ705 cooperatively control shoot growth and commonly regulate the expression of a set of genes involved in meristem identity, chloroplast biogenesis, and energy metabolism in the shoot apex. Loss of WOX11 led to increased H3K27me3 and overexpression of JMJ705 decreased the methylation levels at a subset of common targets. JMJ705 is associated with most of the WOX11-binding sites found in the tested common targets in vivo, regardless of presence or absence of the JMJ705-binding motif. Furthermore, wox11 mutation reduced JMJ705-binding to many targets genome-wide. The results suggest that recruitment of JMJ705 to specific developmental pathway genes is promoted by DNA-binding transcription factors and that WOX11 functions to stimulate shoot growth through epigenetic reprogramming of genes involved in meristem development and energy-generating pathways.

The Interaction between Rice ERF3 and WOX11 Promotes Crown Root Development by Regulating Gene Expression Involved in Cytokinin Signaling.

Crown roots are the main components of the fibrous root system in rice (Oryza sativa). WOX11, a WUSCHEL-related homeobox gene specifically expressed in the emerging crown root meristem, is a key regulator in crown root development. However, the nature of WOX11 function in crown root development has remained elusive. Here, we identified a rice AP2/ERF protein, ERF3, which interacts with WOX11 and was expressed in crown root initials and during crown root growth. Functional analysis revealed that ERF3 was essential for crown root development and acts in auxin- and cytokinin-responsive gene expression. Downregulation of ERF3 in wox11 mutants produced a more severe root phenotype. Also, increased expression of ERF3 could partially complement wox11, indicating that the two genes functioned cooperatively to regulate crown root development. ERF3 and WOX11 shared a common target, the cytokinin-responsive gene RR2. The expression of ERF3 and WOX11 only partially overlapped, underlining a spatio-temporal control of RR2 expression and crown root development. Furthermore, ERF3-regulated RR2 expression was involved in crown root initiation, while the ERF3/WOX11 interaction likely repressed RR2 during crown root elongation. These results define a mechanism regulating gene expression involved in cytokinin signaling during different stages of crown root development in rice.

Regulation of histone methylation and reprogramming of gene expression in the rice inflorescence meristem.

Rice inflorescence meristem (IM) activity is essential for panicle development and grain production. How chromatin and epigenetic mechanisms regulate IM activity remains unclear. Genome-wide analysis revealed that in addition to genes involved in the vegetative to reproductive transition, many metabolic and protein synthetic genes were activated in IM compared with shoot apical meristem and that a change in the H3K27me3/H3K4me3 ratio was an important factor for the differential expression of many genes. Thousands of genes gained or lost H3K27me3 in IM, and downregulation of the H3K27 methyltransferase gene SET DOMAIN GROUP 711 (SDG711) or mutation of the H3K4 demethylase gene JMJ703 eliminated the increase of H3K27me3 in many genes. SDG711-mediated H3K27me3 repressed several important genes involved in IM activity and many genes that are silent in the IM but activated during floral organogenesis or other developmental stages. SDG711 overexpression augmented IM activity and increased panicle size; suppression of SDG711 by RNA interference had the opposite effect. Double knockdown/knockout of SDG711 and JMJ703 further reduced panicle size. These results suggest that SDG711 and JMJ703 have agonistic functions in reprogramming the H3K27me3/H3K4me3 ratio and modulating gene expression in the IM.

Jumonji C domain protein JMJ705-mediated removal of histone H3 lysine 27 trimethylation is involved in defense-related gene activation in rice.

Histone methylation is an important epigenetic modification in chromatin function, genome activity, and gene regulation. Dimethylated or trimethylated histone H3 lysine 27 (H3K27me2/3) marks silent or repressed genes involved in developmental processes and stress responses in plants. However, the role and the mechanism of the dynamic removal of H3K27me2/3 during gene activation remain unclear. Here, we show that the rice (Oryza sativa) Jumonji C (jmjC) protein gene JMJ705 encodes a histone lysine demethylase that specifically reverses H3K27me2/3. The expression of JMJ705 is induced by stress signals and during pathogen infection. Overexpression of the gene reduces the resting level of H3K27me2/3 resulting in preferential activation of H3K27me3-marked biotic stress-responsive genes and enhances rice resistance to the bacterial blight disease pathogen Xanthomonas oryzae pathovar oryzae. Mutation of the gene reduces plant resistance to the pathogen. Further analysis revealed that JMJ705 is involved in methyl jasmonate-induced dynamic removal of H3K27me3 and gene activation. The results suggest that JMJ705 is a biotic stress-responsive H3K27me2/3 demethylase that may remove H3K27me3 from marked defense-related genes and increase their basal and induced expression during pathogen infection.

Histone exchange sensors reveal variant specific dynamics in mouse embryonic stem cells.

Eviction of histones from nucleosomes and their exchange with newly synthesized or alternative variants is a central epigenetic determinant. Here, we define the genome-wide occupancy and exchange pattern of canonical and non-canonical histone variants in mouse embryonic stem cells by genetically encoded exchange sensors. While exchange of all measured variants scales with transcription, we describe variant-specific associations with transcription elongation and Polycomb binding. We found considerable exchange of H3.1 and H2B variants in heterochromatin and repeat elements, contrasting the occupancy and little exchange of H3.3 in these regions. This unexpected association between H3.3 occupancy and exchange of canonical variants is also evident in active promoters and enhancers, and further validated by reduced H3.1 dynamics following depletion of H3.3-specific chaperone, HIRA. Finally, analyzing transgenic mice harboring H3.1 or H3.3 sensors demonstrates the vast potential of this system for studying histone exchange and its impact on gene expression regulation in vivo.

WUSCHEL-related homeobox gene WOX11 increases rice drought resistance by controlling root hair formation and root system development.

Roots are essential organs for anchoring plants, exploring and exploiting soil resources, and establishing plant-microorganisms communities in vascular plants. Rice has a complex root system architecture consisting of several root types, including primary roots, lateral roots, and crown roots. Crown roots constitute the major part of the rice root system and play important roles during the growing period. Recently, we have refined a mechanism that involves ERF3/WOX11 interaction is required to regulate the expression of genes in the cytokinin signaling pathway during the different stages of crown roots development in rice. In this study, we further analyzed the root phenotypes of WOX11 transgenic plants and revealed that WOX11 also acts in controlling root hair development and enhancing rice drought resistance, in addition to its roles in regulating crown root and lateral root development. Based on this new finding, we proposed the mechanism of that WOX11 is involved in drought resistance by modulating rice root system development.

The R2R3-type MYB gene OsMYB91 has a function in coordinating plant growth and salt stress tolerance in rice.

Plants have evolved a number of different mechanisms to respond and to adapt to abiotic stress for their survival. However, the regulatory mechanisms involved in coordinating abiotic stress tolerance and plant growth are not fully understood. Here, the function of OsMYB91, an R2R3-type MYB transcription factor of rice was explored. OsMYB91 was induced by abiotic stress, especially by salt stress. Analysis of chromatin structure of the gene revealed that salt stress led to rapid removal of DNA methylation from the promoter region and rapid changes of histone modifications in the locus. Plants over-expressing OsMYB91 showed reduced plant growth and accumulation of endogenous ABA under control conditions. Under salt stress, the over-expression plants showed enhanced tolerance with significant increases of proline levels and a highly enhanced capacity to scavenge active oxygen as well as the increased induction of OsP5CS1 and LOC_Os03g44130 compared to wild type, while RNAi plants were less sensitive. In addition, expression of OsMYB91 was also induced by other abiotic stresses and hormone treatment. More interestingly, SLR1, the rice homolog of Arabidopsis DELLA genes that have been shown to integrate endogenous developmental signals with adverse environmental conditions, was highly induced by OsMYB91 over-expression, while the salt-induction of SLR1 expression was impaired in the RNAi plants. These results suggested that OsMYB91 was a stress-responsive gene that might be involved in coordinating rice tolerance to abiotic stress and plant growth by regulating SLR1 expression.

The rice WUSCHEL-related homeobox genes are involved in reproductive organ development, hormone signaling and abiotic stress response.

The WUSCHEL-related homeobox (WOX) genes are important transcription regulators participated in plant development processes. Rice (Oryza sativa L.) genome encodes at least 13 WOX members. In this study, a systematic microarray-based gene expression profiling of eleven WOX genes was performed for the whole life cycle of rice at 16 different tissues/organs of MH63 (rice indica cultivar), which included eight reproductive organs and eight vegetative tissues. The results demonstrated that four genes (OsWUS, OsNS1/OsNS2, OsWOX3 and OsWOX9A) were specifically expressed in panicle and endosperm development, and six genes (OsWOX5, OsWOX9B, OsWOX9D, OsWOX11, OsWOX12A and OsWOX12B) were preferentially expressed in seeds (72h after imbibitions) during root emergence or growth. In situ hybridization analysis revealed differential transcript levels of OsWOX4, OsWOX5, OsWOX9A and OsWOX12B during panicle development and embryogenesis. Results of qRT-PCR showed that expression of four rice WOX genes (OsWOX5, OsWOX11, OsWOX12B and OsWOX12A) was up- or down-regulated by plant hormones (auxin, cytokinin and gibberellin). More interestingly, most WOX genes were responsive to abiotic stress stimuli of drought, salt and cold. The molecular studies presented here will further provide insight in understanding the functions of rice WOX gene family in rice development, hormone signaling, and abiotic stress response.