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Although increased cell-to-cell variability in transcription or epigenetic marks has been proposed to be a major hallmark of ageing, little is known about the molecular diversity of stem cells during ageing. Here we present a single cell multi-omics study of mouse muscle stem cells, combining single-cell transcriptome and DNA methylome profiling.

Aged cells show a global increase of uncoordinated transcriptional heterogeneity biased towards genes regulating cell-niche interactions. We find context-dependent alterations of DNA methylation in aged stem cells. Importantly, promoters with increased methylation heterogeneity are associated with increased transcriptional heterogeneity of the genes they drive. These results indicate that epigenetic drift, by accumulation of stochastic DNA methylation changes in promoters, is associated with the degradation of coherent transcriptional networks during stem cell ageing.

Furthermore, our observations also shed light on the mechanisms underlying the DNA methylation clock. Understanding how cell identity transitions occur and whether there are multiple paths between the same beginning and end states are questions of wide interest.

Here we show that acquisition of naive pluripotency can follow transcriptionally and mechanistically distinct routes. Starting from post-implantation epiblast stem cells EpiSCs , one route advances through a mesodermal state prior to naive pluripotency induction, whereas another transiently resembles the early inner cell mass and correspondingly gains greater developmental potency.

These routes utilize distinct signaling networks and transcription factors but subsequently converge on the same naive endpoint, showing surprising flexibility in mechanisms underlying identity transitions and suggesting that naive pluripotency is a multidimensional attractor state. These route differences are reconciled by precise expression of Oct4 as a unifying, essential, and sufficient feature.

We propose that fine-tuned regulation of this "transition factor" underpins multidimensional access to naive pluripotency, offering a conceptual framework for understanding cell identity transitions. Epigenetic clocks are mathematical models that predict the biological age of an individual using DNA methylation data and have emerged in the last few years as the most accurate biomarkers of the aging process. However, little is known about the molecular mechanisms that control the rate of such clocks.

Here, we have examined the human epigenetic clock in patients with a variety of developmental disorders, harboring mutations in proteins of the epigenetic machinery. We recently derived mouse expanded potential stem cells EPSCs from individual blastomeres by inhibiting the critical molecular pathways that predispose their differentiation.

EPSCs had enriched molecular signatures of blastomeres and possessed developmental potency for all embryonic and extra-embryonic cell lineages. Here, we report the derivation of porcine EPSCs, which express key pluripotency genes, are genetically stable, permit genome editing, differentiate to derivatives of the three germ layers in chimeras and produce primordial germ cell-like cells in vitro.

Under similar conditions, human embryonic stem cells and induced pluripotent stem cells can be converted, or somatic cells directly reprogrammed, to EPSCs that display the molecular and functional attributes reminiscent of porcine EPSCs. Importantly, trophoblast stem-cell-like cells can be generated from both human and porcine EPSCs. Our pathway-inhibition paradigm thus opens an avenue for generating mammalian pluripotent stem cells, and EPSCs present a unique cellular platform for translational research in biotechnology and regenerative medicine.

Remarkably, although hydroxymethylation levels are high in the mouse brain, the potential role of TET proteins in adult neurogenesis is unknown. We show here that a non-catalytic action of TET3 is essentially required for the maintenance of the neural stem cell NSC pool in the adult subventricular zone SVZ niche by preventing premature differentiation of NSCs into non-neurogenic astrocytes.

This occurs through direct binding of TET3 to the paternal transcribed allele of the imprinted gene Small nuclear ribonucleoprotein-associated polypeptide N Snrpn , contributing to transcriptional repression of the gene. Our work describes a novel mechanism of control of an imprinted gene in the regulation of adult neurogenesis through an unconventional role of TET3. Following publication of the original article [1], it was reported that the incorrect "Additional file 3" was published.

The correct additional file is given below. Across the animal kingdom, gastrulation represents a key developmental event during which embryonic pluripotent cells diversify into lineage-specific precursors that will generate the adult organism. Here we report the transcriptional profiles of , single cells from mouse embryos collected at nine sequential time points ranging from 6.

We construct a molecular map of cellular differentiation from pluripotency towards all major embryonic lineages, and explore the complex events involved in the convergence of visceral and primitive streak-derived endoderm. Furthermore, we use single-cell profiling to show that Tal1 chimeric embryos display defects in early mesoderm diversification, and we thus demonstrate how combining temporal and transcriptional information can illuminate gene function.

Together, this comprehensive delineation of mammalian cell differentiation trajectories in vivo represents a baseline for understanding the effects of gene mutations during development, as well as a roadmap for the optimization of in vitro differentiation protocols for regenerative medicine. Alternative splicing is a key regulatory mechanism in eukaryotic cells and increases the effective number of functionally distinct gene products.

Using bulk RNA sequencing, splicing variation has been studied across human tissues and in genetically diverse populations. This has identified disease-relevant splicing events, as well as associations between splicing and genomic features, including sequence composition and conservation. However, variability in splicing between single cells from the same tissue or cell type and its determinants remains poorly understood.

The molecular regulation of zygotic genome activation ZGA in mammals remains an exciting area of research. Primed mouse embryonic stem cells contain a rare subset of "2C-like" cells that are epigenetically and transcriptionally similar to the two-cell embryo and thus represent an in vitro approximation for studying ZGA transcription regulation.

However, it remains unknown what upstream maternal factors initiate ZGA in either a Dux-dependent or Dux-independent manner. Here we performed a candidate-based overexpression screen, identifying, among others, developmental pluripotency-associated 2 Dppa2 and Dppa4 as positive regulators of 2C-like cells and transcription of ZGA genes. In the germline, promoter DNA demethylation coincides with expression of Dppa2 and Dppa4, which remain expressed until embryonic day 7. Furthermore, Dppa2 and Dppa4 are also expressed during induced pluripotent stem cell iPSC reprogramming at the time that 2C-like transcription transiently peaks.

Through a combination of overexpression, knockdown, knockout, and rescue experiments together with transcriptional analyses, we show that Dppa2 and Dppa4 directly regulate the 2C-like cell population and associated transcripts, including Dux and the Zscan4 cluster. Importantly, we teased apart the molecular hierarchy in which the 2C-like transcriptional program is initiated and stabilized. Dppa2 and Dppa4 require Dux to initiate 2C-like transcription, suggesting that they act upstream by directly regulating Dux.

Supporting this, ChIP-seq chromatin immunoprecipitation [ChIP] combined with high-throughput sequencing analysis revealed that Dppa2 and Dppa4 bind to the Dux promoter and gene body and drive its expression. Conventional human embryonic stem cells are considered to be primed pluripotent but can be induced to enter a naive state. However, the transcriptional features associated with naive and primed pluripotency are still not fully understood.

Here we used single-cell RNA sequencing to characterize the differences between these conditions. We observed that both naive and primed populations were mostly homogeneous with no clear lineage-related structure and identified an intermediate subpopulation of naive cells with primed-like expression. We found that the naive-primed pluripotency axis is preserved across species, although the timing of the transition to a primed state is species specific.

We also identified markers for distinguishing human naive and primed pluripotency as well as strong co-regulatory relationships between lineage markers and epigenetic regulators that were exclusive to naive cells. Our data provide valuable insights into the transcriptional landscape of human pluripotency at a cellular and genome-wide resolution.

Here we demonstrate that the non-canonical SMC family protein, SmcHD1, which is important for gene silencing on Xi, contributes to this unique chromosome architecture. Specifically, allelic mapping of the transcriptome and epigenome in SmcHD1 mutant cells reveals the appearance of sub-megabase domains defined by gene activation, CpG hypermethylation and depletion of Polycomb-mediated H3K27me3.

Xi chromosome architecture changes also occurred following SmcHD1 knockout in a somatic cell model, but in this case, independent of Xi gene derepression. We conclude that SmcHD1 is a key factor in defining the unique chromosome architecture of Xi.

Dietary, pharmacological and genetic interventions can extend health- and lifespan in diverse mammalian species. DNA methylation has been implicated in mediating the beneficial effects of these interventions; methylation patterns deteriorate during ageing, and this is prevented by lifespan-extending interventions. However, whether these interventions also actively shape the epigenome, and whether such epigenetic reprogramming contributes to improved health at old age, remains underexplored.

We analysed published, whole-genome, BS-seq data sets from mouse liver to explore DNA methylation patterns in aged mice in response to three lifespan-extending interventions: dietary restriction DR , reduced TOR signaling rapamycin , and reduced growth Ames dwarf mice.

Dwarf mice show enhanced DNA hypermethylation in the body of key genes in lipid biosynthesis, cell proliferation and somatotropic signaling, which strongly correlates with the pattern of transcriptional repression. Remarkably, DR causes a similar hypermethylation in lipid biosynthesis genes, while rapamycin treatment increases methylation signatures in genes coding for growth factor and growth hormone receptors.

Shared changes of DNA methylation were restricted to hypermethylated regions, and they were not merely a consequence of slowed ageing, thus suggesting an active mechanism driving their formation. By comparing the overlap in ageing-independent hypermethylated patterns between all three interventions, we identified four regions, which, independent of genetic background or gender, may serve as novel biomarkers for longevity-extending interventions.

In summary, we identified gene body hypermethylation as a novel and partly conserved signature of lifespan-extending interventions in mouse, highlighting epigenetic reprogramming as a possible intervention to improve health at old age.

The mouse embryo is the canonical model for mammalian preimplantation development. Recent advances in single cell profiling allow detailed analysis of embryogenesis in other eutherian species, including human, to distinguish conserved from divergent regulatory programs and signalling pathways in the rodent paradigm. Here, we identify and compare transcriptional features of human, marmoset and mouse embryos by single cell RNA-seq. Zygotic genome activation correlates with the presence of polycomb repressive complexes in all three species, while ribosome biogenesis emerges as a predominant attribute in primate embryos, supporting prolonged translation of maternally deposited RNAs.

We find that transposable element expression signatures are species, stage and lineage specific. The pluripotency network in the primate epiblast lacks certain regulators that are operative in mouse, but encompasses WNT components and genes associated with trophoblast specification.

Unexpectedly, OTX2 is also associated with primitive endoderm specification in human and non-human primate blastocysts. Our cross-species analysis demarcates both conserved and primate-specific features of preimplantation development, and underscores the molecular adaptability of early mammalian embryogenesis. Nucleosomes are the basic unit of chromatin that help the packaging of genetic material while controlling access to the genetic information.

The underlying DNA sequence, together with transcription-associated proteins and chromatin remodelling complexes, are important factors that influence the organization of nucleosomes. Here, we show that the naturally occurring DNA modification, 5-formylcytosine 5fC is linked to tissue-specific nucleosome organization.

Our study reveals that 5fC is associated with increased nucleosome occupancy in vitro and in vivo. We demonstrate that 5fC-associated nucleosomes at enhancers in the mammalian hindbrain and heart are linked to elevated gene expression. Our study also reveals the formation of a reversible-covalent Schiff base linkage between lysines of histone proteins and 5fC within nucleosomes in a cellular environment. We define their specific genomic loci in mouse embryonic stem cells and look into the biological consequences of these DNA-histone Schiff base sites.

Collectively, our findings show that 5fC is a determinant of nucleosome organization and plays a role in establishing distinct regulatory regions that control transcription. Maternal overnutrition has been associated with increased susceptibility to develop obesity and neurological disorders later in life. Most epidemiological as well as experimental studies have focused on the metabolic consequences across generations following an early developmental nutritional insult.

Recently, it has been shown that maternal high-fat diet HFD affects third-generation female body mass via the paternal lineage. We showed here that the offspring born to HFD ancestors displayed addictive-like behaviors as well as obesity and insulin resistance up to the third generation in the absence of any further exposure to HFD. These findings, implicate that the male germ line is a major player in transferring phenotypic traits.

These behavioral and physiological alterations were paralleled by reduced striatal dopamine levels and increased dopamine 2 receptor density. Interestingly, by the third generation a clear gender segregation emerged, where females showed addictive-like behaviors while male HFD offspring showed an obesogenic phenotype. However, methylome profiling of F1 and F2 sperm revealed no significant difference between the offspring groups, suggesting that the sperm methylome might not be the major carrier for the transmission of the phenotypes observed in our mouse model.

Together, our study for the first time demonstrates that maternal HFD insult causes sustained alterations of the mesolimbic dopaminergic system suggestive of a predisposition to develop obesity and addictive-like behaviors across multiple generations. Exit from pluripotency and priming for differentiation into somatic lineages is associated with genome-wide de novo DNA methylation.

Using a combination of single-cell sequencing and quantitative biophysical modeling, we show that this variability is associated with coherent, genome-scale oscillations in DNA methylation with an amplitude dependent on CpG density. Analysis of parallel single-cell transcriptional and epigenetic profiling provides evidence for oscillatory dynamics both in vitro and in vivo.

These observations provide insights into the emergence of epigenetic heterogeneity during early embryo development, indicating that dynamic changes in DNA methylation might influence early cell fate decisions. Recent research has focused on environmental effects that control tissue functionality and systemic metabolism. However, whether such stimuli affect human thermogenesis and body mass index BMI has not been explored.

Here we show retrospectively that the presence of brown adipose tissue BAT and the season of conception are linked to BMI in humans. In mice, we demonstrate that cold exposure CE of males, but not females, before mating results in improved systemic metabolism and protection from diet-induced obesity of the male offspring. Integrated analyses of the DNA methylome and RNA sequencing of the sperm from male mice revealed several clusters of co-regulated differentially methylated regions DMRs and differentially expressed genes DEGs , suggesting that the improved metabolic health of the offspring was due to enhanced BAT formation and increased neurogenesis.

The conclusions are supported by cell-autonomous studies in the offspring that demonstrate an enhanced capacity to form mature active brown adipocytes, improved neuronal density and more norepinephrine release in BAT in response to cold stimulation. Taken together, our results indicate that in humans and in mice, seasonal or experimental CE induces an epigenetic programming of the sperm such that the offspring harbor hyperactive BAT and an improved adaptation to overnutrition and hypothermia.

Defective germline reprogramming in Piwil4 Miwi2 - and Dnmt3l-deficient mice results in the failure to reestablish transposon silencing, meiotic arrest and progressive loss of spermatogonia. Here we sought to understand the molecular basis for this spermatogonial dysfunction. Through a combination of imaging, conditional genetics and transcriptome analysis, we demonstrate that germ cell elimination in the respective mutants arises as a result of defective de novo genome methylation during reprogramming rather than because of a function for the respective factors within spermatogonia.

In both Miwi2 and Dnmt3l spermatogonia, the intracisternal-A particle IAP family of endogenous retroviruses is derepressed, but, in contrast to meiotic cells, DNA damage is not observed. Instead, we find that unmethylated IAP promoters rewire the spermatogonial transcriptome by driving expression of neighboring genes.

Finally, spermatogonial numbers, proliferation and differentiation are altered in Miwi2 and Dnmt3l mice. In summary, defective reprogramming deregulates the spermatogonial transcriptome and may underlie spermatogonial dysfunction. A remarkable epigenetic remodelling process occurs shortly after fertilization, which restores totipotency to the zygote.

This involves global DNA demethylation, chromatin remodelling, genome spatial reorganization and substantial transcriptional changes. Key to these changes is the transition from the maternal environment of the oocyte to an embryonic-driven developmental expression programme, a process termed the maternal-to-zygotic transition MZT. Zygotic genome activation occurs predominantly at the two-cell stage in mice and the eight-cell stage in humans, yet the dynamics of its control are still mostly obscure.

In recent years, partly due to single-cell and low-cell number epigenomic studies, our understanding of the epigenetic and chromatin landscape of preimplantation development has improved considerably. In this Review, we discuss the latest advances in the study of the MZT, focusing on DNA methylation, histone post-translational modifications, local chromatin structure and higher-order genome organization.

We also discuss key mechanistic studies that investigate the mode of action of chromatin regulators, transcription factors and non-coding RNAs during preimplantation development. Finally, we highlight areas requiring additional research, as well as new technological advances that could assist in eventually completing our understanding of the MZT. Whole-genome bisulfite sequencing WGBS is becoming an increasingly accessible technique, used widely for both fundamental and disease-oriented research.

Library preparation methods benefit from a variety of available kits, polymerases and bisulfite conversion protocols. Although some steps in the procedure, such as PCR amplification, are known to introduce biases, a systematic evaluation of biases in WGBS strategies is missing. Parallel single-cell sequencing protocols represent powerful methods for investigating regulatory relationships, including epigenome-transcriptome interactions.

Here, we report a single-cell method for parallel chromatin accessibility, DNA methylation and transcriptome profiling. We validate scNMT-seq by applying it to differentiating mouse embryonic stem cells, finding links between all three molecular layers and revealing dynamic coupling between epigenomic layers during differentiation.

The recent advent of methods for high-throughput single-cell molecular profiling has catalyzed a growing sense in the scientific community that the time is ripe to complete the year-old effort to identify all cell types in the human body. The Human Cell Atlas Project is an international collaborative effort that aims to define all human cell types in terms of distinctive molecular profiles such as gene expression profiles and to connect this information with classical cellular descriptions such as location and morphology.

An open comprehensive reference map of the molecular state of cells in healthy human tissues would propel the systematic study of physiological states, developmental trajectories, regulatory circuitry and interactions of cells, and also provide a framework for understanding cellular dysregulation in human disease.

Here we describe the idea, its potential utility, early proofs-of-concept, and some design considerations for the Human Cell Atlas, including a commitment to open data, code, and community. Reprogramming is a slow and inefficient process, suggesting the presence of safeguarding mechanisms that counteract cell fate conversion. One such mechanism is senescence.

To identify modulators of reprogramming-induced senescence, we performed a genome-wide shRNA screen in primary human fibroblasts expressing OSKM. Our data unveiled regulation of senescence as a novel way by which mechanistic target of rapamycin mTOR influences reprogramming. These contrasting actions contribute to explain the complex effect that mTOR has on reprogramming.

Overall, our study highlights the advantage of combining functional screens with scRNA-seq to accelerate the discovery of pathways controlling complex phenotypes. Erasure of DNA methylation and repressive chromatin marks in the mammalian germline leads to risk of transcriptional activation of transposable elements TEs. Inhibition of Dicer or Ago2 expression revealed that small RNAs are involved in an immediate response to demethylation-induced transposon activation, while the deposition of repressive histone marks follows as a chronic response.

Our results suggest that antisense TE transcription is a "trap" that elicits an endosiRNA response to restrain acute transposon activity during epigenetic reprogramming in the mammalian germline. DNA methylation is an important epigenetic modification in many species that is critical for development, and implicated in ageing and many complex diseases, such as cancer. Many cost-effective genome-wide analyses of DNA modifications rely on restriction enzymes capable of digesting genomic DNA at defined sequence motifs.

There are hundreds of restriction enzyme families but few are used to date, because no tool is available for the systematic evaluation of restriction enzyme combinations that can enrich for certain sites of interest in a genome. Herein, we present customised Reduced Representation Bisulfite Sequencing cuRRBS , a novel and easy-to-use computational method that solves this problem.

In addition, cuRRBS estimates the fold-reduction in sequencing costs and provides a robustness value for the personalised RRBS protocol, allowing users to tailor the protocol to their experimental needs. Finally, we have validated the accuracy of cuRRBS predictions for single and double enzyme digestions using two independent experimental datasets. Mouse embryonic stem cells derived from the epiblast contribute to the somatic lineages and the germline but are excluded from the extra-embryonic tissues that are derived from the trophectoderm and the primitive endoderm upon reintroduction to the blastocyst.

Here we report that cultures of expanded potential stem cells can be established from individual eight-cell blastomeres, and by direct conversion of mouse embryonic stem cells and induced pluripotent stem cells. Remarkably, a single expanded potential stem cell can contribute both to the embryo proper and to the trophectoderm lineages in a chimaera assay. Bona fide trophoblast stem cell lines and extra-embryonic endoderm stem cells can be directly derived from expanded potential stem cells in vitro.

Molecular analyses of the epigenome and single-cell transcriptome reveal enrichment for blastomere-specific signature and a dynamic DNA methylome in expanded potential stem cells. The generation of mouse expanded potential stem cells highlights the feasibility of establishing expanded potential stem cells for other mammalian species.

Single-cell multi-omics has recently emerged as a powerful technology by which different layers of genomic output-and hence cell identity and function-can be recorded simultaneously. Integrating various components of the epigenome into multi-omics measurements allows for studying cellular heterogeneity at different time scales and for discovering new layers of molecular connectivity between the genome and its functional output.

Measurements that are increasingly available range from those that identify transcription factor occupancy and initiation of transcription to long-lasting and heritable epigenetic marks such as DNA methylation. Together with techniques in which cell lineage is recorded, this multilayered information will provide insights into a cell's past history and its future potential. This will allow new levels of understanding of cell fate decisions, identity, and function in normal development, physiology, and disease.

The mouse inner cell mass ICM segregates into the epiblast and primitive endoderm PrE lineages coincident with implantation of the embryo. The epiblast subsequently undergoes considerable expansion of cell numbers prior to gastrulation.

To investigate underlying regulatory principles, we performed systematic single-cell RNA sequencing seq of conceptuses from E3. The epiblast shows reactivation and subsequent inactivation of the X chromosome, with Zfp57 expression associated with reactivation and inactivation together with other candidate regulators. At E6. Notably, our analyses suggest elevated transcriptional noise at E3.

By contrast, E6. Our study systematically charts transcriptional noise and uncovers molecular processes associated with early lineage decisions. Here we provide a method for resetting via transient histone deacetylase inhibition. The protocol is effective across multiple PSC lines and can proceed without karyotype change.

WNT inhibition stabilises the resetting process. The transcriptome of reset cells diverges markedly from that of primed PSCs and shares features with human inner cell mass ICM. Reset cells activate expression of primate-specific transposable elements. DNA methylation is globally reduced to a level equivalent to that in the ICM and is non-random, with gain of methylation at specific loci.

Methylation imprints are mostly lost, however. Reset cells can be re-primed to undergo tri-lineage differentiation and germline specification. In female reset cells, appearance of biallelic X-linked gene transcription indicates reactivation of the silenced X chromosome. On reconversion to primed status, XIST-induced silencing restores monoallelic gene expression. Aging of the hematopoietic stem cell HSC compartment is characterized by lineage bias and reduced stem cell function, the molecular basis of which is largely unknown.

DNA methylation changes at a discrete set of sites in the human genome are predictive of chronological and biological age. However, it is not known whether these changes are causative or a consequence of an underlying ageing process. It has also not been shown whether this epigenetic clock is unique to humans or conserved in the more experimentally tractable mouse.

Recent technological advances have enabled DNA methylation to be assayed at single-cell resolution. However, current protocols are limited by incomplete CpG coverage and hence methods to predict missing methylation states are critical to enable genome-wide analyses. We report DeepCpG, a computational approach based on deep neural networks to predict methylation states in single cells.

We evaluate DeepCpG on single-cell methylation data from five cell types generated using alternative sequencing protocols. DeepCpG yields substantially more accurate predictions than previous methods. Additionally, we show that the model parameters can be interpreted, thereby providing insights into how sequence composition affects methylation variability. Dietary restriction DR , a reduction in food intake without malnutrition, increases most aspects of health during aging and extends lifespan in diverse species, including rodents.

However, the mechanisms by which DR interacts with the aging process to improve health in old age are poorly understood. DNA methylation could play an important role in mediating the effects of DR because it is sensitive to the effects of nutrition and can affect gene expression memory over time. Single-cell RNA-seq enables the quantitative characterization of cell types based on global transcriptome profiles. We demonstrate that SC3 is capable of identifying subclones from the transcriptomes of neoplastic cells collected from patients.

The molecular pathways that regulate gain and loss of DNA methylation during mammalian development need to be tightly balanced to maintain a physiological equilibrium. Here we explore the relative contributions of the different pathways and enzymatic activities involved in methylation homeostasis in the context of genome-wide and locus-specific epigenetic reprogramming in mammals.

An adaptable epigenetic machinery allows global epigenetic reprogramming to concur with local maintenance of critical epigenetic memory in the genome, and appears to regulate the tempo of global reprogramming in different cell lineages and species. Base excision repair BER may become less effective with ageing resulting in accumulation of DNA lesions, genome instability and altered gene expression that contribute to age-related degenerative diseases.

The brain is particularly vulnerable to the accumulation of DNA lesions; hence, proper functioning of DNA repair mechanisms is important for neuronal survival. Although the mechanism of age-related decline in DNA repair capacity is unknown, growing evidence suggests that epigenetic events e. We hypothesize that epigenetic mechanisms are involved in mediating the age-related decline in BER in the brain.

Brains from male mice were isolated at months of age. Pyrosequencing analyses revealed significantly increased Ogg1 methylation with ageing, which correlated inversely with Ogg1 expression. The reduced Ogg1 expression correlated with enhanced expression of methyl-CpG binding protein 2 and ten-eleven translocation enzyme 2.

A significant inverse correlation between Neil1 methylation at CpG-site2 and expression was also observed. BER activity was significantly reduced and associated with increased 8-oxo-7,8-dihydro-2'-deoxyguanosine levels. These data indicate that Ogg1 and Neil1 expression can be epigenetically regulated, which may mediate the effects of ageing on DNA repair in the brain. Our current understanding of DNAme is based on measurements from bulk cell samples, which obscures intercellular differences and prevents analyses of rare cell types.

Thus, the ability to measure DNAme in single cells has the potential to make important contributions to the understanding of several key biological processes, such as embryonic development, disease progression and aging.

Here we present a detailed protocol for scBS-seq that includes our most recent developments to optimize recovery of CpGs, mapping efficiency and success rate; reduce hands-on time; and increase sample throughput with the option of using an automated liquid handler. We provide step-by-step instructions for each stage of the method, comprising cell lysis and bisulfite BS conversion, preamplification and adaptor tagging, library amplification, sequencing and, lastly, alignment and methylation calling.

An individual with relevant molecular biology expertise can complete library preparation within 3 d. Subsequent computational steps require d for someone with bioinformatics expertise. Mouse embryonic stem ES cells are locked into self-renewal by shielding from inductive cues. Release from this ground state in minimal conditions offers a system for delineating developmental progression from naive pluripotency. Here we examined the initial transition process.

The ES cell population behaves asynchronously. We therefore exploited a short-half-life RexGFP reporter to isolate cells either side of exit from naive status. Extinction of ES cell identity in single cells is acute. Cells newly departed from the ES cell state display features of early post-implantation epiblast and are distinct from primed epiblast. They also exhibit a genome-wide increase in DNA methylation, intermediate between early and late epiblast. These findings are consistent with the proposition that naive cells transition to a distinct formative phase of pluripotency preparatory to lineage priming.

Global DNA demethylation is an integral part of reprogramming processes in vivo and in vitro, but whether it occurs in the derivation of induced pluripotent stem cells iPSCs is not known. Here, we show that iPSC reprogramming involves both global and targeted demethylation, which are separable mechanistically and by their biological outcomes.

Cells at intermediate-late stages of reprogramming undergo transient genome-wide demethylation, which is more pronounced in female cells. Global demethylation requires activation-induced cytidine deaminase AID -mediated downregulation of UHRF1 protein, and abolishing demethylation leaves thousands of hypermethylated regions in the iPSC genome. Independently of AID and global demethylation, regulatory regions, particularly ESC enhancers and super-enhancers, are specifically targeted for hypomethylation in association with transcription of the pluripotency network.

Our results show that global and targeted DNA demethylation are conserved and distinct reprogramming processes, presumably because of their respective roles in epigenetic memory erasure and in the establishment of cell identity. DNA methylation plays a critical role in the regulation and maintenance of cell-type specific transcriptional programs. Targeted epigenome editing is an emerging technology to specifically regulate cellular gene expression in order to modulate cell phenotypes or dissect the epigenetic mechanisms involved in their control.

We show that targeting of these loci with single gRNAs leads to efficient and widespread methylation of the promoters. Multiplexing of several guide RNAs does not increase the efficiency of methylation. Peaks of targeted methylation were observed around 25 bp upstream and 40 bp downstream of the PAM site, while bp of the binding site itself are protected against methylation.

Furthermore, the introduced methylation causes transcriptional repression of the targeted genes. These new programmable epigenetic editors allow unprecedented control of the DNA methylation status in cells and will lead to further advances in the understanding of epigenetic signaling. Cardiac hypertrophic growth in response to pathological cues is associated with reexpression of fetal genes and decreased cardiac function and is often a precursor to heart failure.

In contrast, physiologically induced hypertrophy is adaptive, resulting in improved cardiac function. The processes that selectively induce these hypertrophic states are poorly understood. Here, we have profiled 2 repressive epigenetic marks, H3K9me2 and H3K27me3, which are involved in stable cellular differentiation, specifically in cardiomyocytes from physiologically and pathologically hypertrophied rat hearts, and correlated these marks with their associated transcriptomes. This analysis revealed the pervasive loss of euchromatic H3K9me2 as a conserved feature of pathological hypertrophy that was associated with reexpression of fetal genes.

Thus, we have established a conserved mechanism involving a departure of the cardiomyocyte epigenome from its adult cellular identity to a reprogrammed state that is accompanied by reexpression of fetal genes and pathological hypertrophy.

The ten-eleven translocation TET enzymes can catalyze the oxidation of 5-methylcytosine 5mC to 5-hydroxymethylcytosine 5hmC and further oxidized derivatives, thereby actively removing this memory. Nevertheless, the mechanism by which the TET enzymes are regulated, and the extent to which they can be manipulated, are poorly understood. We find that both ascorbate and RA or retinol promote the derivation of induced pluripotent stem cells synergistically and enhance the erasure of epigenetic memory.

This mechanistic insight has significance for the development of cell treatments for regenenerative medicine, and enhances our understanding of how intrinsic and extrinsic signals shape the epigenome. Primordial germ cell PGC development is characterized by global epigenetic remodeling, which resets genomic potential and establishes an epigenetic ground state.

Here we recapitulate PGC specification in vitro from naive embryonic stem cells and characterize the early events of epigenetic reprogramming during the formation of the human and mouse germline. Following rapid de novo DNA methylation during priming to epiblast-like cells, methylation is globally erased in PGC-like cells.

The dynamics of specification and epigenetic reprogramming show species-specific differences, in particular markedly slower reprogramming kinetics in the human germline. Differences in developmental kinetics may be explained by differential regulation of epigenetic modifiers. Our work establishes a robust and faithful experimental system of the early events of epigenetic reprogramming and regulation in the germline.

Mouse embryonic stem cells are dynamic and heterogeneous. For example, rare cells cycle through a state characterized by decondensed chromatin and expression of transcripts, including the Zscan4 cluster and MERVL endogenous retrovirus, which are usually restricted to preimplantation embryos. Here, we further characterize the dynamics and consequences of this transient cell state. Genome-wide DNA methylation and chromatin analyses revealed global DNA hypomethylation accompanying increased chromatin accessibility.

This transient DNA demethylation was driven by a loss of DNA methyltransferase proteins in the cells and occurred genome-wide. While methylation levels were restored once cells exit this state, genomic imprints remained hypomethylated, demonstrating a potential global and enduring influence of endogenous retroviral activation on the epigenome. TET is able to oxidise 5-methylcytosine 5mC to 5-hydroxymethylcytosine 5hmC , 5-formylcytosine 5fC and 5-carboxylcytosine 5caC.

TDG can excise the oxidative products 5fC and 5caC, initiating base excision repair. These modified bases are stable and detectable in the genome, suggesting that they could have epigenetic functions in their own right.

However, functional investigation of the genome-wide distribution of 5fC has been restricted to cell culture-based systems, while its in vivo profile remains unknown. Global demethylation is part of a conserved program of epigenetic reprogramming to naive pluripotency. The transition from primed hypermethylated embryonic stem cells ESCs to naive hypomethylated ones serum-to-2i is a valuable model system for epigenetic reprogramming.

We present a mathematical model, which accurately predicts global DNA demethylation kinetics. Experimentally, we show that the main drivers of global demethylation are neither active mechanisms Aicda, Tdg, and Tet nor the reduction of de novo methylation. UHRF1 protein, the essential targeting factor for DNMT1, is reduced upon transition to 2i, and so is recruitment of the maintenance methylation machinery to replication foci.

These mechanisms synergistically enforce global DNA hypomethylation in a replication-coupled fashion. Our observations establish the molecular mechanism for global demethylation in naive ESCs, which has key parallels with those operating in primordial germ cells and early embryos. Emerging single-cell epigenomic methods are being developed with the exciting potential to transform our knowledge of gene regulation. Here we review available techniques and future possibilities, arguing that the full potential of single-cell epigenetic studies will be realized through parallel profiling of genomic, transcriptional, and epigenetic information.

Conventional generation of stem cells from human blastocysts produces a developmentally advanced, or primed, stage of pluripotency. In vitro resetting to a more naive phenotype has been reported. However, whether the reset culture conditions of selective kinase inhibition can enable capture of naive epiblast cells directly from the embryo has not been determined. Here, we show that in these specific conditions individual inner cell mass cells grow into colonies that may then be expanded over multiple passages while retaining a diploid karyotype and naive properties.

The cells express hallmark naive pluripotency factors and additionally display features of mitochondrial respiration, global gene expression, and genome-wide hypomethylation distinct from primed cells. They transition through primed pluripotency into somatic lineage differentiation. Collectively these attributes suggest classification as human naive embryonic stem cells. Human counterparts of canonical mouse embryonic stem cells would argue for conservation in the phased progression of pluripotency in mammals.

Critical roles for DNA methylation in embryonic development are well established, but less is known about its roles during trophoblast development, the extraembryonic lineage that gives rise to the placenta. We find that oocyte-derived methylation plays a major role in regulating trophoblast development but that imprinting of the key placental regulator Ascl2 is only partially responsible for these effects. We have identified several methylation-regulated genes associated with trophoblast differentiation that are involved in cell adhesion and migration, potentially affecting trophoblast invasion.

Specifically, trophoblast-specific DNA methylation is linked to the silencing of Scml2, a Polycomb Repressive Complex 1 protein that drives loss of cell adhesion in methylation-deficient trophoblast. Our results reveal that maternal DNA methylation controls multiple differentiation-related and physiological processes in trophoblast via both imprinting-dependent and -independent mechanisms. Profiling of 61 mouse embryonic stem cells confirmed known links between DNA methylation and transcription.

Notably, the method revealed previously unrecognized associations between heterogeneously methylated distal regulatory elements and transcription of key pluripotency genes. Phenotypic plasticity is important in adaptation and shapes the evolution of organisms. However, we understand little about what aspects of the genome are important in facilitating plasticity.

Eusocial insect societies produce plastic phenotypes from the same genome, as reproductives queens and nonreproductives workers. The greatest plasticity is found in the simple eusocial insect societies in which individuals retain the ability to switch between reproductive and nonreproductive phenotypes as adults.

We lack comprehensive data on the molecular basis of plastic phenotypes. Here, we sequenced genomes, microRNAs miRNAs , and multiple transcriptomes and methylomes from individual brains in a wasp Polistes canadensis and an ant Dinoponera quadriceps that live in simple eusocial societies. In both species, we found few differences between phenotypes at the transcriptional level, with little functional specialization, and no evidence that phenotype-specific gene expression is driven by DNA methylation or miRNAs.

Instead, phenotypic differentiation was defined more subtly by nonrandom transcriptional network organization, with roles in these networks for both conserved and taxon-restricted genes. The general lack of highly methylated regions or methylome patterning in both species may be an important mechanism for achieving plasticity among phenotypes during adulthood.

These findings define previously unidentified hypotheses on the genomic processes that facilitate plasticity and suggest that the molecular hallmarks of social behavior are likely to differ with the level of social complexity. Here, we show that developmental dynamics of 5fC levels in mouse DNA differ from those of 5-hydroxymethylcytosine 5hmC , and using stable isotope labeling in vivo, we show that 5fC can be a stable DNA modification.

These results suggest that 5fC has functional roles in DNA that go beyond being a demethylation intermediate. Epigenetic reprogramming in the germline resets genomic potential and erases epigenetic memory. Three studies by Gkountela et al. Cellular senescence has been implicated in tumor suppression, development, and aging and is accompanied by large-scale chromatin rearrangements, forming senescence-associated heterochromatic foci SAHF.

However, how the chromatin is reorganized during SAHF formation is poorly understood. Furthermore, heterochromatin formation in senescence appears to contrast with loss of heterochromatin in Hutchinson-Gilford progeria. We mapped architectural changes in genome organization in cellular senescence using Hi-C.

Unexpectedly, we find a dramatic sequence- and lamin-dependent loss of local interactions in heterochromatin. This change in local connectivity resolves the paradox of opposing chromatin changes in senescence and progeria. In addition, we observe a senescence-specific spatial clustering of heterochromatic regions, suggesting a unique second step required for SAHF formation.

Comparison of embryonic stem cells ESCs , somatic cells, and senescent cells shows a unidirectional loss in local chromatin connectivity, suggesting that senescence is an endpoint of the continuous nuclear remodelling process during differentiation.

DNA methylomes are extensively reprogrammed during mouse pre-implantation and early germ cell development. The main feature of this reprogramming is a genome-wide decrease in 5-methylcytosine 5mC. Standard high-resolution single-stranded bisulfite sequencing techniques do not allow discrimination of the underlying passive replication-dependent or active enzymatic mechanisms of 5mC loss. We approached this problem by generating high-resolution deep hairpin bisulfite sequencing DHBS maps, allowing us to follow the patterns of symmetric DNA methylation at CpGs dyads on both DNA strands over single replications.

Fertilization triggers global erasure of paternal 5-methylcytosine as part of epigenetic reprogramming during the transition from gametic specialization to totipotency. This involves oxidation by TET3, but our understanding of its targets and the wider context of demethylation is limited to a small fraction of the genome.

We employed an optimized bisulfite strategy to generate genome-wide methylation profiles of control and TET3-deficient zygotes, using SNPs to access paternal alleles. This revealed that in addition to pervasive removal from intergenic sequences and most retrotransposons, gene bodies constitute a major target of zygotic demethylation. Methylation loss is associated with zygotic genome activation and at gene bodies is also linked to increased transcriptional noise in early development.

Our data map the primary contribution of oxidative demethylation to a subset of gene bodies and intergenic sequences and implicate redundant pathways at many loci. Unexpectedly, we demonstrate that TET3 activity also protects certain CpG islands against methylation buildup. The two chromatin types generally do not coexist at the same loci, suggesting mutual exclusivity. During development or in cancer, pericentromeric regions can adopt either epigenetic state, but the switching mechanism is unknown.

We used a quantitative locus purification method to characterize changes in pericentromeric chromatin-associated proteins in mouse embryonic stem cells deficient for either the methyltransferases required for DNA methylation or H3K9Me3. DNA methylation controls heterochromatin architecture and inhibits Polycomb recruitment. This suggests that BEND3 is a key factor in mediating a switch from constitutive to facultative heterochromatin. If it looks as though the adventurers will reach Black Peaks before the timeline has Herzen and Heidlemann setting out, you can use one or more of these delaying tactics.

Among its members are Etelka Herzen and Ernst Heidlemann. The patrol captain sees right away that the adventurers are not river traders, and interrogates them about their backgrounds, how they came to possess the boat, where they are going, and why. Each answer from the adventurers leads to several more questions, and by the time the patrol captain is satisfied or bribed sufficiently a whole day will have passed.

They have learned that there might be large chunks of warpstone in the Barren Hills. Ernst Heidlemann, whom the Characters encountered briefly in Enemy in Shadows page 22 , has been ordered to visit Grissenwald, near Nuln, and consult Etelka Herzen. The river is impassable until the blockage is cleared away. The river patrol are working with a gang of labourers and a couple of Dwarf engineers. It will be two days before a channel is clear unless someone helps with magic or gunpowder. The exact date is left blank, for you to fill in to fit your campaign.

Subsequent events have been numbered by day, according to the mode of transport used and the distance travelled. They set out for the Barren Hills on Day 9 rather than Day The events of Days 8—15 are ignored, and the rest of the timeline is moved up by six days. From then on, the adventurers should follow her trail until they eventually catch up with her. If the adventurers spend a day or two at the Signal 0 After the failure of their expedition to the Barren Hills, the cultists spend longer in Kemperbad than the day or two given in the timeline.

In the original campaign the pre-generated Characters came from the small town of Delberz. After the drama at the Red Barn, they had an opportunity to return there. The heroes in this version of the campaign are all Player-generated, and could be from anywhere: the chance of Delberz being their home is very remote. Of course, Characters who have an attachment to Delberz are welcome to go there, but the plot of this adventure does not require them to do so.

Hieronymus Blitzen, the mentor, can be found in the Appendix of this book. He can now be placed wherever you need. The Signal Tower page 27 is an ideal location for a confrontation with the Red Crown cultists. This happens when the adventurers return there with the sixth key after exploring the Barren Hills.

However, the adventurers may run across the cultists at some other point in time. Here are the most probable occasions. This attack, and the Watch inquiry that will follow, should delay the adventurers by anything from a few hours to a full day. There is a chance that a sharp-eyed adventurer will recognise Heidlemann riding along the bank.

He will be in the company of a lady and a couple of pack horses. After being recognised in Altdorf, though, Heidlemann is cautious, and has taken to wearing a broad-brimmed hat that hides much of his face. The River Stir is narrower than the Reik, but it is still a major waterway and traffic moves freely along it.

If necessary, you can use one of the ploys above to make sure the adventurers do not catch the cultists before they have reached the Barren Hills. The River Narn becomes considerably narrower and faster as it rises toward the Barren Hills. Working a boat upstream against the current is a slow business.

Half normal speed is good progress. The cultists will be able to travel faster on horseback. This could be a good time and place for a showdown, especially if the cultists have reached the conclusion that the meteorite is not here. If the Characters are strong combatants, the situation could turn into a three-sided fight between the adventurers, the cultists, and the Skaven.

Kemperbad, Day 15 or 16 If the adventurers waste no time in Grissenwald, there is a small chance that they might reach Kemperbad before the cultists have departed. Kemperbad is a small town, but it is possible for the two parties to miss each other. If Heidlemann saw the adventurers on the river above , he persuades Etelka that steps need to be taken.

She could scare off any hired thugs quite easily, leaving the adventurers to deal with the two wizards. For extra reinforcements and drama, she might clear the rock fall from The Cave of the Dead page 60 with an impressive gesture.

A normal ghost could not do such a thing with normal skeletons. The manner of their deaths, and the presence of warpstone, have given the undead strange and unexpected powers. If the party needs reinforcements, the conflict could take place near the village of Unterbaum page 57 or the Inn of the Roaring Falls page Some of the more able NPCs in either location could help out.

Between Kemperbad and the Signal Tower, Day 29—36 Again, an encounter during this period can work well, but it lacks the drama of a confrontation at the Signal Tower. For a really dramatic conclusion to the chase, it might be preferable to have the two groups miss each other in Kemperbad. The adventurers then reach the Signal Tower first and the cultists arrive at a suitable later time. Alternatively, some of the tricks mentioned above could be used to delay the adventurers so that they find the cultists in control of the Signal Tower, with the surviving Dwarfs held prisoner.

The Timeline The next part of the adventure revolves around the actions of two groups of people in different locations. It may not seem much like a race, as travel is only ever as fast as the nearest horse or riverboat, but keeping track of events in the timeline will make your life as a GM a bit easier. There are long distances to cover, secrets to reveal, and not a lot of time for the Players to do either. If the Players dawdle or delay too much, or get side-tracked by other events, you can inject a hint that they are wasting time by having a cultist or two watch them from a distance.

This monitoring becomes more overt as Heidlemann moves towards his goal. If the Players are sluggards, then you should feel free to rearrange matters so that Heidlemann has the sixth key as he arrives first, rather than second, at the Signal Tower. The adventurers are then in a chase to prevent him reaching the secret library. Heidlemann arrives at Grissenwald, and goes immediately to Black Peaks.

Herzen and Heidlemann leave Black Peaks on horseback. The two arrive at Kemperbad The expedition leaves Kemperbad on horseback. Once again, you are free to enliven the journey with hazards and encounters as desired. There are also some landmarks worth mentioning. As their boat rounds a river bend near the tower site, the adventurers see two Dwarfs waving frantically from a small wooden landingstage close to the construction works. Castle Reikguard As the adventurers travel up the Reik from Altdorf, they will pass the towering fortress of Castle Reikguard.

Give us a lift! We can pay! Commoners are not admitted within the castle. Enquiries at any of the nearby villages will reveal wildly contradictory rumours that the adventurers can pick up elsewhere. See News and Rumours, page One chain of signal towers runs between Altdorf and Nuln.

The adventurers pass a tower under construction a little way south of Castle Reikguard. From the river the characters can see the signal tower is being built on the remains of an older, partially ruined structure.

They are in a great hurry to leave and want to go to Altdorf or Nuln, or as close as they can get. Before the adventurers have a chance to question the two Dwarfs, another one comes running down to the bank. She is somewhat overweight and gasping for breath, her face almost as red as her voluminous breeches. Thingrim and Belegol fall silent as the newcomer arrives. Perched on top of the tower is wooden scaffolding, with stanchions, ropes, and pulleys. All over this confused-looking mess swarm a number of short, bearded, tool-wielding figures.

Clearly, work on the structure is not finished yet. Clearly the task has not been a pleasant one. Her eyes are red-rimmed with dark rings beneath, and a permanent frown furrows her brow. Aynjulls apologises for Thingrim and Belegol, whom she claims are simply 'overworked', but she does not look to be in any great hurry to get back to work herself.

Dagmar had his own reasons to keep his research away from prying eyes. The observatory was protected by various magical means, and its location was kept secret even from the rest of the family. One passage read as follows: This calls for wisdom.

The time shall come when the enemies of Chaos shall relax their guard; looking out from their fortress, they shall pay no heed to the shadow at their backs. Then shall the Great Mutator cause the warped moon to awaken, and the beloved of Morr shall clear his throat and spit upon the Empire. And where his spittle doth fall, there shall the weak fear to tread, but the possessor of the spittle shall wield great power.

Some years later, Dagmar stumbled across an ancient tome of astronomical records in which the following passage dated attracted his attention: Dagmar realised immediately that the meteorite mentioned in the astronomical records must actually be a piece of the moon, Morrslieb. Checking his histories, Dagmar learned that the hills around the headwaters of the River Narn had come to be known as the Barren Hills sometime around Concluding that the meteorite must possess great power, Dagmar set off for Kemperbad, where he raised a small expedition to journey into the Barren Hills.

I n this two thousandth, three hundredth and second year of our Empire, on the night of the ill-omened Hexenstag, Morrslieb did shine with a Greene and Unnatural Lighte, and its Shape was as if it bore a Grinning Countenance of Most Horryble Appearance.

According to legend, the expedition never returned. In fact, once he had located the meteorite, Dagmar slew the others and made his way home by a circuitous route to cover his tracks, with the chunk of warpstone safely stored in a lead-lined casket. T he Heavens were filled with a Greate Number of Shootinge Stars and some of these did seem to fall upon the Ground which groaned under their assault.

Unfortunately for Dagmar, he did not live to reap the fruits of his labours. One of his cousins, Hermann, known to chroniclers as Hermann the Mad, strangled Dagmar in a rage because his relative had refused to let him see the contents of a certain casket. In the customary manner of noble houses, the family covered up the murder.

Hermann was confined in an outer tower of the castle, and quietly erased from memory. The strange casket and its deadly contents were also never mentioned again. B eing Forewarned by the earlier events of Hexenstag, I was able to trace the course of a particularly large Shootinge Star, which seemed to issue from Morrslieb itself. According to my calculations, the Star must have fallen to earth in the Uplands of Talabeclande, near the Head-waters of the River Narne. The Ruinous Powers, however, have also recently discovered its existence.

Red Crown cultists and the Skaven ratmen are determined to possess it for their own terrible ends. They include a powerful ghoul and five zombies, which are responsible for the deaths, disappearances, and paralysis afflicting some of the Dwarfs working on the site. There is also a set of magical keys without which it is impossible to access the observatory. Others would have regarded this as an astronomical curiosity, but Dagmar had recently finished translating an ancient scroll written in the arcane language of daemons.

All good, solid dwarfs until this job. We were hired in Altdorf six weeks ago, with an Imperial Commission to build this signal tower as specified. Good visibility to the next towers up and down the chain, and this ruin is sound enough for a foundation.

These doors open of their own accord whenever the key comes within 1 yard of them, and close when it is more than 1 yard away. The key has no effect on any other doors. It is a rod of iron, 6 inches long and with a five-pointed star cross-section. There were accidents and injuries. Then they started falling sick, with no cause anyone can find.

Contracts have been signed, after all. But five days ago the disappearances started. Two more vanished only last night, leaving six of us, including me. Some think the place is an accursed elven burial mound. Or something worse. There are five other keys of the second type. These are carried by the five zombie guardians of the observatory see page These keys have a cross-section like a six-pointed star.

The third type of key needs to be recovered from the Barren Hills. This key, plus the five carried by the zombies, opens the trapdoor entrance to the secret library see page The library holds the vital information that will lead the adventurers to Castle Wittgenstein and the climax of this adventure!

But… I gave my word and the tower must be finished on time. What do you say? She will also pay the bonus if the mystery of what has been happening to her workers is solved. The Signal Tower The Dwarfs have managed to complete the bottom floor of the signal tower, which houses the signal fire and mirror.

The wooden pylon with its signalling arms has yet to be added. Both the tower and its foundation, which is the ground floor of the old observatory, are surrounded by wooden scaffolding. She has a diagram Handout 4 which she will show the adventurers to explain how the signal tower works.

If the adventurers decide to help, keep track of how much time they spend at the tower. This will affect the timeline page 24 which tracks their movements and those of the two Red Crown cultists. The floor of the tower is actually the ceiling of the observatory. It is from this room that the workers have disappeared. They were attacked by the ghoul which entered from the observatory below through a secret trapdoor. Breaching this wall is impossible without specialised siege or engineering equipment.

Aynjulls will reluctantly agree to create a hole if the adventurers insist. If the adventurers decide to spend the night here, the ghoul will attack in the small hours. The trapdoor opens noiselessly and the ghoul springs into the room. The creature is so swift that no one can attack it until it is clear of the trapdoor and fully in the room. The ghoul attempts to flee back through the trapdoor if it has 2 or fewer Wounds remaining.

In this case, the adventurers will have time to follow it, or to wedge the trapdoor open before it snaps shut. The secret doors are completely indistinguishable from other sections of the wall, even though there are obvious paths that come to a dead end at a blank wall where the doors are hidden. See The Magical Keys, page The place has a musty smell and is shrouded in dust and cobwebs. It has clearly not been used for a very long time. The Zombies There are five zombies in the observatory.

Three are in the library e , one in the laboratory c and the final one in the study d. The zombies are usually dormant, moving only to attack trespassers or to help one of their number who is under attack. A reinforcement zombie arrives on the second Round of combat, and another each Round until all five have appeared.

They were enchanted by Dagmar so that they would not need a controller, and they do not suffer from the Unstable Creature Trait while they are within the observatory. Original Observatory Stone Construction B. Strange geometric patterns adorn the floor, walls, and ceiling. Obscured by years of dust, these patterns lend the room a strange aspect, giving the appearance of impossible, disorienting angles.

All the doorways are covered in cobwebs, and dust falls from the ceiling as the adventurers pass through. When they first approach one of the doors, a mysterious and chilling wind will seem to rush past them. It only moves if someone enters the laboratory or if a fight starts involving one of its fellows. Secret Underground Library The laboratory is littered with alchemical apparatus.

Benches are piled with beakers, retort stands, odd-shaped jars, lumps of different ores, and so on. Various esoteric calculations have been chalked on most surfaces: the walls, ceiling, floor, and bench tops. A gargoyle-shaped lectern bearing a large, leather-bound book stands in one corner. The book is hand-written in Classical language and has copious marginalia and multiple revisions. An appropriately skilled Character would take days to read it thoroughly.

Studying it for a couple of hours reveals that it contains research into the summoning and control of skeletons, zombies, and ghouls. The information contained in the book can be studied in the same manner as a spell. It enables a wizard to control these types of undead, but not summon them.

A pinch of powdered warpstone is required for each undead creature, which the wizard may then control as if they had cast a Reanimate Spell WFRP, page The room contains a large desk, a plans chest, and a drawing board. Various ancient portraits hang on the walls. An adventurer smashing it open will get covered in ink. Inside there are quills, bottles of ink many dried up , and parchment. There is also a battered notebook with yellowing pages covered in complex calculations and notes written in a spidery hand.

Anyone with the Lore Magick Skill quickly works out that the calculations are to do with the orbit of the Chaos moon Morrslieb. No test is necessary as the moon is mentioned several times in the text. The plans chest has several antique maps showing various parts of the Empire, the Reikland, and the western marches of Talabecland in particular. Give your Players Handout 5 below. All the maps have a number of intersecting lines carefully drawn on them. One intersection in the Barren Hills has been roughly ringed.

There is, however, no clue as to what the ringed intersection means. Secret Library The drawing board itself is empty. The portraits depict various members of the von Wittgenstein family, though they are not labelled. The adventurers will not know this if they have not yet visited Castle Wittgenstein. However, the family resemblance between the portrait subjects is clear: all have aquiline noses, high foreheads, and thick, bushy eyebrows. The corridors radiating from the central hexagonal chamber are lined with shelves containing books.

These are on a variety of subjects, mostly written in Classical and Magickal, though some have marginalia in Reikspiel, Bretonnian, and Tilean. Propped in one corner of the room is an ornately carved staff. Anyone with the Detect Artefact Talent will feel a strong aura coming from the staff. The zombies will not attack anyone carrying it. Library 0 A section of false book fronts conceals a Potion of Healing. There are three zombies in this room, which is lined with bookshelves from floor to ceiling.

The books are written in a variety of mundane, secret, and arcane languages. Their musty condition means that each is worth 1D10 GCs. You can include a couple of historical or religious books if you want to pass on some background information to your Players. A table in the centre of the library bears three large books. The books are open at the pages shown in Handouts 6 and 7 page 28 , respectively. Inner Corridor The inner wall of this area is made of 2-inch-thick steel.

There are two inch-long bars attached to the wall at waist height, one at each end. If any two adventurers push on these bars, the entire wall rotates so that a gap in this wall will align with the central accessway area G, below. The pages of the third volume are filled with a spidery script in the Magick language.

The final entry is reproduced as Handout 8 below , and it is the most revealing. This provides the adventurers with the final piece of information they need to lead them to Castle Wittgenstein, and the climax of the adventure. Central Accessway Note: the entrance from the inner corridor area f can only be opened and closed by two people in that area. If that entrance is closed and the adventurers enter through the trapdoor, they can only leave the way they came in.

The floor here is inscribed with a glowing hexagram, or sixpointed star. These holes are star-shaped, and the right size to accept the keys carried by the five zombies. Of course, one key is missing unless it has been brought from the Barren Hills. If all six keys are inserted into the hexagram the secret trapdoor in the floor will open downwards.

Anyone standing on the trapdoor must make a Hard Initiative Test or fall 3 yards into the secret library beneath. You can make this location more challenging for them by making a few changes, like the following. The iron rods worn by the undead are completely inert. Instead, the head of the ghoul, or its whole body, is required to open the trapdoor. The same goes for the five zombies.

The sixth thumb key is a zombie that replaces of one of the Dagmar expedition skeletons see The Cave of the Dead, page Neither the ghoul nor the zombies carry anything like a key around their necks. Instead, the keys are stitched into their stomachs. The books with the handouts are on the shelves and it requires a thorough search to find them.

There are no required encounters or events for the rest of the journey to Kemperbad, although you can use the River Life of the Empire section of the Death on the Reik Companion as a source of ideas for enlivening the journey. A ferry service at the docks provides the safest way to cross the Reik between Nuln and Altdorf. The ferry costs 2d per passenger, and 4d for a horse. The locals joke that this is a foot tax. The Four Seasons Coaching Line maintains a large coaching inn and staging post at the nearby village of Jungbach.

The Town Kemperbad sits atop a foot cliff overlooking the Reik, just north of its confluence with the River Stir. The Stir originally plunged over a mighty waterfall into the Reik, but the river has cut a narrow gorge into the cliffs. The waterfall has slowly moved upstream to its present location at the confluence of the Stir and Narn. The gorge of the Stir is crossed by a narrow rope bridge, and this makes the town all but inaccessible to southern road traffic.

This means it is independent of the Reikland and answerable only to the Emperor. A Council of Thirteen governs the town. Its members are elected by the most influential guilds and families. The Emperor retains the right to appoint or dismiss any of the councillors, but has never done so. Fortunately for commerce, a dock has been built on the Reik at the base of the cliffs. This is connected to the town by an ingenious system of lifts. There are baskets and platforms of varying sizes, suspended from huge block and tackle sets on Elven ropes, and all raised and lowered by counterbalances.

The system is maintained and operated by Dwarven engineers. Remarkably, there has been only one fatal accident in the last fifty years. Some drunken young nobles tried to race each other to the bottom. The winner was too unwell to claim his prize.

Kemperbad has become very wealthy through trade, and all kinds of goods may be found here. In theory, the removal of any liege lord, and their accompanying tax demands, should reduce the cost of living. Therefore, Kemperbad is cleaner and better maintained than some other towns, as the council can afford to keep the streets in good order.

Use of the lifts costs 2d per person on the passenger platforms holding up to four average-sized individuals , and 2d per ten baskets or sacks of bulk cargo, or 30 encumbrance equivalent. Some merchants came from there a few weeks back and were insistent that Kemperbad used to be on the other bank of the river, and we must have up and moved it! City folk are all mad. The following information can be picked up in Kemperbad. As always, you can choose these, or pick something randomly.

Not all of the rumours are true. Everyone knows this is because she had an affair with a peasant, a Priest of Sigmar, or a noble of low standing each informant will confidently name a different paramour. He must have the same affliction as the Emperor! Each informant will give a different disease. He must be plotting to seize the throne!

Why does he love mutants all of a sudden? Has someone close to him developed a mutation? Rumours about Wittgendorf Use these rumours in response to questions about Castle Wittgenstein, Wittgendorf, or the Wittgenstein family. War could break out at any time. No one has gone near the place for years. Village mothers use the name of the place to frighten their children to sleep, though. Banditry and mutant attacks are threatening trade and the authorities have decided to act at last. Good thing too!

Arriving in 0 Castle Wittgenstein is a ruin. No one has lived there for years. No one comes, and no one goes. Kemperbad The Purple Hand The adventurers are most likely to arrive at Kemperbad for the first time as they travel south along the Reik, heading for Grissenwald and Black Peaks. They are also likely to stop here again on the way to the Barren Hills.

During the day, the docks are bustling. The adventurer feels a sharp tug at their hair. There is just time to notice a man dressed in purple disappearing into the crowd, but no chance to stop or follow him. In addition to trading, the adventurers may wish to find help in learning Skills or changing Careers. If the Players decide to take the time to do this, refer to Training and Mentors page Nothing obvious will happen as a result, but the adventurers will have plenty of time to worry.

For a typical cultist profile, see The Cultists, page These are very small and may easily be mistaken for potions. Etelka Herzen — Human Master Wizard On the surface, Etelka is a friendly but reserved noblewoman of medium build, with shoulder-length blonde hair, and blue eyes.

Underneath, she is ruthless in the pursuit of her aims, and deals harshly with any interference. In order to conceal her identity, she only uses magic when there are unlikely to be witnesses or survivors. If confronted, she usually waits a Round or two before deciding to cast spells.

Her skin exudes a very pungent odour, which she masks with strong perfumes. The fragrance sometimes lingers behind her for as much as 15 minutes, and anyone entering an area that she has recently left will not fail to notice it. Once per day, the wand may be used to turn a Major Miscast into a Minor Miscast instead.

Each time this occurs, roll a D If the result is less than or equal to the CN of the spell that produced the miscast, the wand shatters and is destroyed, dealing D10 Wounds to each Character within 2 yards. Although Shadow appears intelligent, with a disconcertingly knowing look in her eyes, she is an ordinary black cat. Ernst Heidlemann Ernst is a thin, lanky, weasel-faced individual with light-brown hair and a pasty complexion. He masquerades as a recently qualified physician in the employ of Lady Etelka, and maintains this deception by refusing to treat anyone but her.

Ernst is quiet and reserved, seldom speaking, but is often rude to those who are of no use to him. He is cruel and calculating, and while on the mission he will hire thugs to deal with any opposition, saving his powers until he can use them to the best effect. Aynjulls Isembeard Aynjulls usually wears an oil-stained leather jacket, brightred breeches, and great hobnailed boots.

Fiercely proud of her reputation for swift and reliable work, she can be a somewhat gruff and surly character. The continuing disappearance of her workforce has shaken her to the point where she is ready to try anything — even if it means asking for help from an Elf! He has been supplied by his masters with a preparation to offset this affliction, and he carries the vials with him at all times.

Without it, he loses 1 Toughness point every 12 hours, dying when his Toughness reaches zero. All are frightened and confused by the mysterious deaths of their colleagues, although they try not to let it show. Ghoul Champion One of the Wizard Dagmar's final experiments, this unfortunate creature has guarded its long dead creators home for centuries.

Preserved by foul sorcery and the occassional foolhardy rat, the Ghoul is ravenous for a more substantial meal. Trappings: Each Zombie carries a 6-inch-long iron key — resembling a 6-pointed star at the crosssection — on a leather thong about its neck. There is always a chance that the adventurers will cross paths with the Red Crown cultists see The Timeline, page 26 , but there will be little chance of a confrontation. Inquiring further along the river, the adventurers will hear similar stories from anyone who plies these waters regularly.

This gruesome body is not the first to be found in the river below the castle, and some of them were in a worse state. The point here is to create a sense of menace and foreshadowing. There are accounts to settle, but not quite yet. This impression of wrongness is magnified when they see it for the first time. It broods ominously over the river, and passing boats hug the opposite bank, as watermen tell tales of maimed and mutated corpses they have seen floating down this stretch of the river.

Its crew warn the adventurers to keep to the east bank, and direct their attention to something floating in the water. Grissenwald Map 7 is a small settlement at the confluence of the Rivers Grissen and Reik. It is a favourite stopping place for riverfolk and boasts a number of wharfside inns.

Asking about Black Peaks at any of the inns, the adventurers are told that it is a disused coal mine up in the hills, about five miles away. Once human, the floating corpse now resembles a bloated fish. If the adventurers try to drag it from the water decaying limbs come apart, and the skin sloughs off exposing hideously rotted flesh.

Anyone taking a close look at the body must make a Hard Cool Test to overcome a natural urge to vomit. She is described as a friendly, private woman who lives in a house near the mine and rarely visits the town. A Rude Interruption If the adventurers do fish the body out of the river, the passing vessel will head away as fast as it can. Its crew will shout to the adventurers to stay away from them, and to throw the body back: While the adventurers are making enquiries, two drunken Dwarfs enter the inn.

They are spoiling for a fight, and start insulting any strangers, including the adventurers. They will be particularly insulting to any Elf, but a Halfling or an obvious academic will do as a target. Who knows what diseases it has? And now you might have them, too! They smell worse dead than alive.

Undead, maybe. Them von Wittgensteins are lords and ladies, so do as they please! Folk say the castle is cursed, and the family along with it, and a plague will take anyone who goes near! The Dwarfs are relentless, and the adventurers can only escape their abuse by leaving. Grognard Players may decide to bypass the following chapters and head straight there. There are ways to prevent this. Another possibility is to have no visible way up the cliffs to the castle.

Perhaps there is a landing stage, but no road leads away from it. Close investigation reveals a secret doorway, but this can only be opened with a magical password that is found in the family history, hidden in the secret library. The first is to move Castle Wittgenstein and the village of Wittgendorf. When the party arrives at the location where the castle and village are marked on the map, they find a perfectly innocent village full of happy, healthy peasants, overlooked by a fine noble castle whose owners do not take kindly to being accused of consorting with Chaos.

You may think of other possibilities, but the key idea is to make sure that the adventurers cannot reach Wittgendorf without first going to the Barren Hills and then returning to the Signal Tower. If a fight does break out, the Dwarfs have no wish to kill anyone. They just want a decent recreational punch-up. If attacked, they pick up any improvised weapon that comes to hand: bottles, chairs and so on.

Killing one or both Dwarfs will land the adventurers in a blood feud see below with their clan. They ought to be rounded up, aye, and hung. Nasty natures, your dwarfs. Watch has done nothing. You can make the response time longer if your Players are enjoying the fight.

The patrol consists of four watchmen and a sergeant. They try to break up the fight and, if no one has been killed, they take the Dwarfs away to sleep off their drunkenness in the cells. If one or both of the Dwarfs have been killed, the Watch sergeant warns the adventurers to make themselves scarce. Other drinkers confirm the story. Three farms were recently attacked at night, leaving no survivors.

They came looking for a fight, picked the wrong folk. The patrol leaves, taking any Dwarf bodies with them. If the number of Dwarfs is reduced to six or fewer through fighting, the remainder mount a final, desperate, allout attack, led by Gorim Greathammer. The adventurers can end this feud by proving that the Dwarfs are not responsible for the raids.

If the adventurers killed either of the two Dwarfs in the inn, they are now involved in a blood feud. Every night, they are attacked by Dwarfs. If the adventurers are careless enough to split up or go out alone, the Dwarfs will take full advantage. These attacks take place at any time when the adventurers are at a location with good escape routes for their attackers. The Dwarfs have no wish to run into other humans or the Watch.

Khazid Slumbol A total of 15 Dwarfs live in this ramshackle collection of wooden huts. They were originally built well, but there has been little or no upkeep since. These Dwarfs are the last remaining miners, their money gone on drink. The adventurers will be in the greatest danger if they venture into Khazid Slumbol while the feud is in effect. The whole population of the shanty town surround them, trying to look nonchalant while clumsily hiding hammers and other improvised weapons behind their backs.

Only a complete idiot fails to notice the tension in the air. The adventurers have 3 or 4 Rounds to leave before a sudden cry rings out and the Dwarfs rush them. Even if there is no blood feud, any Elf visiting Khazid Slumbol will be heckled, pelted with rotten vegetables, and generally made unwelcome. Humans get a better reception, but nothing more than sullen stares. All they found was coal. They mined this for 27 years without finding so much as a speck of gold.

When Etelka Herzen arrived and offered them money for their mine, the Dwarfs accepted gladly. They even agreed to build her a house — partially in gratitude, partially for the money. Everyone in town suspects that the Dwarfs are behind the raids on several outlying homesteads. No one has any proof of this beyond the suspicious coincidence that the Dwarfs of Khazid Slumbol seem to have come into some money recently. All this is common knowledge in the town.

The Dwarfs will not talk about the source of their money because it is a deep and lasting embarrassment to the whole clan. Recently, their leader, Gorim Greathammer, sold his magical hammer to a travelling merchant and divided the money between the clan members.

It was a foolish act, born out of shame, but the sale was the only way of raising money for his people. Having taken the money the Dwarfs moved out of the mine, then began to wonder why Etelka wanted it so badly. They came to the completely erroneous conclusion that she knew there was gold in there, but had bewitched the mine to prevent them finding it.

Humiliated, they began to drink more than was good for them. The money soon went, and the half-built settlement of Khazid Slumbol degenerated into a filthy shanty town. A few Dwarfs abandoned their pride completely and begged in Grissenwald, spending what little money they were given on yet more drink. The test can be repeated every Round until it is successful, but any Character who fails the test cannot speak and will only recover in the open air.

If the adventurers manage to gain an audience with Gorim, they are led to a large wooden shack. Being Dwarf-built, the door is quite low. Any Human or Elf will have to stoop to enter, and any Elf will be firmly kicked up the backside as they bend over. From the other side of the room a deep, commanding voice sounds.

The floor is covered with moth-eaten wolf skins and a couple of large moths take flight as the adventurers enter. The smoke is unpleasant and makes it difficult to see. If asked about the mine, Gorim explains that they were tricked out of it by Etelka Herzen, who is an evil wizard. At last we had no choice but to sell. Little enough she gave us, to be sure! Etelka paid them a good price for an exhausted coal mine, although the Dwarfs refuse to see it.

We were robbed. Robbed blind. At all three farms the tracks are too old to follow for more than a couple of hundred yards. The Dwarfs believe that the rumours about the raids were started by the local Humans to discredit them, and they are greatly offended by the accusations.

If there is a Dwarf among the adventurers, that Character alone can make one Difficult Charm Test to calm the Dwarfs. If that fails the adventurers are ordered out of Khazid Slumbol, and every Dwarf in the shanty town gathers to chase them out.

If the adventurers visit any of the inhabited farms they are greeted by the frightened occupants as potential saviours. Answer that one! Dwarf adventurers get the same pointed greeting. Friends in Need If the adventurers offer to investigate the raids, or present Gorim with evidence that proves the Dwarfs are innocent, their mood will change. From now on the adventurers are regarded with a grudging friendship.

He will have up to five Dwarfs accompany the adventurers to Black Peaks, for a price. Initially he will ask for 2 shillings per Dwarf per day, but may be haggled down to 6d each. The Dwarfs have their own reasons for wanting to go to Black Peaks, so this is a limited, rather than full, alliance. They may be accompanying the adventurers, but they are not going to take orders. They always try to kill Goblins on sight, taking no prisoners, and possibly ruining any cunning plan of attack that the party have conceived.

Neither will they rush to the aid of an Elf in trouble during a fight with Greenskins. If the adventurers find only the sword and bring it back, their representative needs to make a Difficult Charm Test. If they have only seen Goblin tracks, then the representative must make a Hard Fellowship Test. Regardless of whether the Humans believe the adventurers, Gorim is grateful for their efforts. He may well offer the adventurers some Dwarven reinforcements. For the right remuneration, of course see Friends in Need, page There are 14 small farms around Grissenwald, all within eight miles of the town.

The three farms closest to Black Peaks are now in ruins from recent attacks. Anyone who searches the surrounding woods and makes a Difficult Intuition Test will find a Goblin corpse. Making a Difficult Perception Test reveals tracks made by many small humanoids and wolves. The next Goblin attack occurs on the night the adventurers arrive in Grissenwald.

Next day, the news is all over town. The fourth farm to be attacked was also not too far from Black Peaks, but no one has yet spotted a pattern. The map below shows a typical Grissenwald Farmhouse. The Grissenwald and Surrounds map page 45 shows the remaining farms.

Over the next half hour the noise comes closer, until a group of Goblin wolf riders can be seen at the edge of the forest. Some are carrying torches. It is possible that the adventurers might want to help the farmers defend themselves. They are welcome to try such a plan, but they cannot protect all the remaining farms at once. There is no guarantee that the farm they choose to protect will be the next one attacked. The Goblins are clever and will avoid any farms that show signs of strengthened defences.

Any group that includes a Dwarf will be unwelcome: In the first wave, eight mounted Goblins will leap the fences and throw their torches on to the thatched roofs of the wooden barn and outbuildings. Once these are alight, six more mounted Goblins and nine on foot attack the farmhouse. Their leader, a striking figure in a red robe, mounted on a large wolf, stays out of the attack, shouting orders, and occasionally waving his arms and chanting.

Mark my words! Depending on how you think your party are doing, you can either have the farmers help the adventurers or run around like panicked hens. The Goblins attack every second night, so another attack takes place on the third night after the adventurers arrive in Grissenwald.

The target will be the closest farm to Black Peaks that is still standing, and Goblin scouts can spot if a target is too strong to attack. During the day, the area appears deserted, but at night there are lights in the windows of the tower and, depending on the time, possibly some activity around the mine entrance. A rough trackway see the map above leads south out of the town toward Black Peaks, winding through the forest for about six and a half miles.

The mine at Black Peaks gets its name from the coal that it formerly produced. Etelka Herzen came here three years ago after leaving the university of Nuln. The Dwarfs had exhausted the mine, and accepted her offer to buy it along with the surrounding land. Etelka hired the Dwarfs to build her a tower, and after its completion they moved to Grissenwald and built Khazid Slumbol.

About a mile from the mine, the adventurers discover a Dwarf propped against a tree. He is ringed by four dead Goblins. The Dwarf is close to death himself, and has three Goblin arrows embedded in his chest.

The Dwarf is Durak, a prospector from Khazid Slumbol. The Goblins moved into the mine with their wolves, and performed various tasks for her. With Etelka away on a mission for the Red Crown, the Goblins have no one to stop them following their violent instincts, and Gutbag, their leader, has been organising raids on surrounding farmsteads. Durak passes out after delivering this message, and will bleed to death in 5 Rounds unless he receives treatment that restores at least 1 Wound.

It will be much more exciting if Gutbag is ready when the adventurers enter his room, however. Noisy or careless adventurers are likely to alert the Goblins: they will yell out warnings, and other Goblins will converge on their position over the next 2D10 Rounds. The nearest Goblins will be the first to arrive, followed by those in the tower, and finally the Goblins from the mine.

THE MINE Area Goblins Wolves 1 4 5 2 3 6 3 3 4 Nighttime The Goblin raids take place on alternate nights, so if there was a raid the previous night, the Goblins will be relaxing in the tower and mine same locations as during the day, but they are awake. Half an hour later they head down the main track towards Grissenwald. Any adventurers approaching the mine at this time will hear them coming and have time to hide before the Goblins reach them.

During a raid, 3—5 wounded Goblins are left at the tower, each with around half their total Wounds remaining. A guard is supposed to watch the winding approach from the mine to the tower, but is often asleep. For an added surprise, perhaps the adventurers find that removing one problem has created another. If Etelka was right and there is gold to be found, the Dwarfs will be ecstatic — and so will the people of Grissenwald, who can look forward to a new era of prosperity… so long as the monster can be defeated, that is.

On the other hand, there might be no Goblins at all — just a hidden shrine to Chaos where the Dwarfs practice nameless and long forbidden rites. They never returned to the Grey Mountains, and Etelka never betrayed them over the mine. Instead, they were her willing accomplices, searching for warpstone beneath the earth and suffering mutation as a result.

Etelka may actually be looking after them, but they plan to use the adventurers to destroy her so they can take over the area without fear of her magic. When Etelka Herzen bought the mine, she was convinced that the Dwarfs were right: there was gold there, somewhere beneath the stripped-out coal face.

To help locate it, she summoned a powerful monster an earth elemental, perhaps, or a powerful daemon. She ordered this creature not to eat her Goblin allies, and by camping in the mine they have effectively been keeping it contained there. Just when the adventurers are congratulating themselves on successfully completing this chapter, a wounded Dwarf flees the mine, with a terrible monster in pursuit. From the entrance, the smell of wolves and Goblins is very strong. It will take them at least two days to dig their way out.

When moving, its wheels squeak so loudly that every Wolf and Goblin in the mine is alerted. The three numbered chambers are used as sleeping quarters. They are extremely dirty and smelly, and have flea-ridden bedding on the floor. Ever since, they have been hiding in the forest, competing with mutants and Beastmen for food and territory.

They regard Etelka Herzen with mixed feelings: contempt because she is a Human, fear because of her magical powers, and jealousy because of her house and possessions, and her freedom to do as she pleases. In her absence, they have been exploring her house and enjoying themselves in the surrounds. Conditions in the mine are damp and dark with a pervasive smell. As the adventurers enter, dust and grit falls ominously from the ceiling. Although there is no immediate chance of a cave-in, any fighting or other violent activity may cause one.

This gets worse on the second Round, and on the third Round there is a loud, reverberating crack and a broken beam sags down from the roof. On the fourth Round the roof collapses, blocking a 2-yard stretch of passage. Closet — This contains cleaning equipment and a few Human and Halfling cloaks. It smells like a toilet, and has evidently been used as one by the Goblins. The two-storey tower house, below, is made from slabs of stone and is of Dwarven construction.

Tied up in a corner are Gurda and Hanse, taken from their farm by the Goblins. They have seen their family, friends, and neighbours slain and eaten, and if freed they will attack the Goblins with no regard for their own safety gaining the Frenzy Talent. If they survive, they will leave to rebuild their farm. Ground Floor The ground floor is paved with polished slabs of red stone. Light is provided by the window and by mirrors at the top of the stairwell. Each room has a two-way mirror which looks onto the stairwell.

The mirror is reflective on one side and transparent on the other, which makes it ideal for observing the stairwell and allows light into the rooms. From the stairwell, only the mirrors can be seen; it is impossible to look into the rooms without opening the doors. Reception Rooms — These rooms once contained fine, upholstered furniture. Subsequently, the Goblins destroyed every piece and turned the stuffing into crude, filthy piles of bedding in the corners of the rooms.

Whether there is a raid in progress or not, there is always a guard here at night. At that time, the guard is more alert and is likely to see anyone who approaches. There are four Goblins in each room unless the tribe is out on a raid. Dining Room — A large wooden table stands in the middle of the room, surrounded by eight high-backed wooden chairs. Cabinets around the walls once contained silver and other dining ware, but their doors hang open and they are gouged and befouled.

Staircase — A stone staircase leads up to the first floor. Any Character on the stairs is clearly visible from any adjoining room through the two-way mirrors. Kitchen — By contrast to the rest of the house, this room is still tidy. The height of the table and work benches are suitable for a Halfling. If the adventurers have entered the building undetected, Dumpling Hayfoot page 53 will be working in here during the day.

Otherwise, she is in her bedroom.

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