Vectors based on adeno-associated virus type 2 (AAV2) are powerful tools for gene transfer and genome editing applications. The level of interest in this system has recently surged in response to ...reports of therapeutic efficacy in human clinical trials, most notably for those in patients with hemophilia B (ref. 3). Understandably, a recent report drawing an association between AAV2 integration events and human hepatocellular carcinoma (HCC) has generated controversy about the causal or incidental nature of this association and the implications for AAV vector safety. Here we describe and functionally characterize a previously unknown liver-specific enhancer-promoter element in the wild-type AAV2 genome that is found between the stop codon of the cap gene, which encodes proteins that form the capsid, and the right-hand inverted terminal repeat. This 124-nt sequence is within the 163-nt common insertion region of the AAV genome, which has been implicated in the dysregulation of known HCC driver genes and thus offers added insight into the possible link between AAV integration events and the multifactorial pathogenesis of HCC.
During embryogenesis, the stringent regulation of Wnt activity is crucial for the morphogenesis of the head and brain. The loss of function of the Wnt inhibitor Dkk1 results in elevated Wnt activity, ...loss of ectoderm lineage attributes from the anterior epiblast, and the posteriorisation of anterior germ layer tissue towards the mesendoderm. The modulation of Wnt signalling may therefore be crucial for the allocation of epiblast cells to ectoderm progenitors during gastrulation. To test this hypothesis, we examined the lineage characteristics of epiblast stem cells (EpiSCs) that were derived and maintained under different signalling conditions. We showed that suppression of Wnt activity enhanced the ectoderm propensity of the EpiSCs. Neuroectoderm differentiation of these EpiSCs was further empowered by the robust re-activation of Wnt activity. Therefore, during gastrulation, the tuning of the signalling activities that mediate mesendoderm differentiation is instrumental for the acquisition of ectoderm potency in the epiblast.
TWIST1, a basic helix-loop-helix transcription factor is essential for the development of cranial mesoderm and cranial neural crest-derived craniofacial structures. We have previously shown that, in ...the absence of TWIST1, cells within the cranial mesoderm adopt an abnormal epithelial configuration via a process reminiscent of a mesenchymal to epithelial transition (MET). Here, we show by gene expression analysis that loss of TWIST1 in the cranial mesoderm is accompanied by a reduction in the expression of genes that are associated with cell-extracellular matrix interactions and the acquisition of mesenchymal characteristics. By comparing the transcriptional profiles of cranial mesoderm-specific Twist1 loss-of-function mutant and control mouse embryos, we identified a set of genes that are both TWIST1-dependent and predominantly expressed in the mesoderm. ChIP-seq was used to identify TWIST1-binding sites in an in vitro model of a TWIST1-dependent mesenchymal cell state, and the data were combined with the transcriptome data to identify potential target genes. Three direct transcriptional targets of TWIST1 (Ddr2, Pcolce and Tgfbi) were validated by ChIP-PCR using mouse embryonic tissues and by luciferase assays. Our findings reveal that the mesenchymal properties of the cranial mesoderm are likely to be regulated by a network of TWIST1 targets that influences the extracellular matrix and cell-matrix interactions, and collectively they are required for the morphogenesis of the craniofacial structures.
•TWIST1 controls cell-ECM interaction and mesenchyme genes in cranial mesoderm.•We identified genes that are TWIST1-dependent and predominantly expressed in the mesoderm.•ChIP-seq identified potential TWIST1-targets in a TWIST1-dependent mesenchymal cell model.•Three transcriptional targets of TWIST1 (Ddr2, Pcolce and Tgfbi) were validated.
The developmental potential of cells, termed pluripotency, is highly dynamic and progresses through a continuum of naive, formative and primed states. Pluripotency progression of mouse embryonic stem ...cells (ESCs) from naive to formative and primed state is governed by transcription factors (TFs) and their target genes. Genomic techniques have uncovered a multitude of TF binding sites in ESCs, yet a major challenge lies in identifying target genes from functional binding sites and reconstructing dynamic transcriptional networks underlying pluripotency progression. Here, we integrated time-resolved 'trans-omic' datasets together with TF binding profiles and chromatin conformation data to identify target genes of a panel of TFs. Our analyses revealed that naive TF target genes are more likely to be TFs themselves than those of formative TFs, suggesting denser hierarchies among naive TFs. We also discovered that formative TF target genes are marked by permissive epigenomic signatures in the naive state, indicating that they are poised for expression prior to the initiation of pluripotency transition to the formative state. Finally, our reconstructed transcriptional networks pinpointed the precise timing from naive to formative pluripotency progression and enabled the spatiotemporal mapping of differentiating ESCs to their in vivo counterparts in developing embryos.
The Zero Childhood Cancer Program is a precision medicine program to benefit children with poor-outcome, rare, relapsed or refractory cancer. Using tumor and germline whole genome sequencing (WGS) ...and RNA sequencing (RNAseq) across 252 tumors from high-risk pediatric patients with cancer, we identified 968 reportable molecular aberrations (39.9% in WGS and RNAseq, 35.1% in WGS only and 25.0% in RNAseq only). Of these patients, 93.7% had at least one germline or somatic aberration, 71.4% had therapeutic targets and 5.2% had a change in diagnosis. WGS identified pathogenic cancer-predisposing variants in 16.2% of patients. In 76 central nervous system tumors, methylome analysis confirmed diagnosis in 71.1% of patients and contributed to a change of diagnosis in two patients (2.6%). To date, 43 patients have received a recommended therapy, 38 of whom could be evaluated, with 31% showing objective evidence of clinical benefit. Comprehensive molecular profiling resolved the molecular basis of virtually all high-risk cancers, leading to clinical benefit in some patients.
This article contains data related to the research article entitled “Transcriptional targets of TWIST1 in the cranial mesoderm regulate cell-matrix interactions and mesenchyme maintenance” by Bildsoe ...et al. (2016) 1. The data presented here are derived from: (1) a microarray-based comparison of sorted cranial mesoderm (CM) and cranial neural crest (CNC) cells from E9.5 mouse embryos; (2) comparisons of transcription profiles of head tissues from mouse embryos with a CM-specific loss-of-function of Twist1 and control mouse embryos collected at E8.5 and E9.5; (3) ChIP-seq using a TWIST1-specific monoclonal antibody with chromatin extracts from TWIST1-expressing MDCK cells, a model for a TWIST1-dependent mesenchymal state.
Abstract
The cranial neural crest and the cranial mesoderm are the source of tissues from which the bone and cartilage of the skull, face and jaws are constructed. The development of the cranial ...mesoderm is not well studied, which is inconsistent with its importance in craniofacial morphogenesis as a source of precursor tissue of the chondrocranium, muscles, vasculature and connective tissues, mechanical support for tissue morphogenesis, and the signaling activity that mediate interactions with the cranial neural crest. Phenotypic analysis of conditional knockout mouse mutants, complemented by the transcriptome analysis of differentially enriched genes in the cranial mesoderm and cranial neural crest, have identified signaling pathways that may mediate cross-talk between the two tissues. In the cranial mesenchyme,
Bmp4
is expressed in the mesoderm cells while its signaling activity could impact on both the mesoderm and the neural crest cells. In contrast,
Fgf8
is predominantly expressed in the cranial neural crest cells and it influences skeletal development and myogenesis in the cranial mesoderm. WNT signaling, which emanates from the cranial neural crest cells, interacts with BMP and FGF signaling in monitoring the switch between tissue progenitor expansion and differentiation. The transcription factor
Twist1
, a critical molecular regulator of many aspects of craniofacial development, coordinates the activity of the above pathways in cranial mesoderm and cranial neural crest tissue compartments.
Measures of socio-economic impacts of conservation interventions have largely been restricted to externally defined indicators focused on income, which do not reflect people's priorities. Using a ...holistic, locally grounded conceptualization of human well-being instead provides a way to understand the multi-faceted impacts of conservation on aspects of people's lives that they value. Conservationists are engaging with well-being for both pragmatic and ethical reasons, yet current guidance on how to operationalize the concept is limited. We present nine guiding principles based around a well-being framework incorporating material, relational and subjective components, and focused on gaining knowledge needed for decision-making. The principles relate to four key components of an impact evaluation: (i) defining well-being indicators, giving primacy to the perceptions of those most impacted by interventions through qualitative research, and considering subjective well-being, which can affect engagement with conservation; (ii) attributing impacts to interventions through quasi-experimental designs, or alternative methods such as theory-based, case study and participatory approaches, depending on the setting and evidence required; (iii) understanding the processes of change including evidence of causal linkages, and consideration of trajectories of change and institutional processes; and (iv) data collection with methods selected and applied with sensitivity to research context, consideration of heterogeneity of impacts along relevant societal divisions, and conducted by evaluators with local expertise and independence from the intervention.
Measures of socio-economic impacts of conservation interventions have largely been restricted to externally defined indicators focused on income, which do not reflect people's priorities. Using a ...holistic, locally grounded conceptualization of human well-being instead provides a way to understand the multi-faceted impacts of conservation on aspects of people's lives that they value. Conservationists are engaging with well-being for both pragmatic and ethical reasons, yet current guidance on how to operationalize the concept is limited. We present nine guiding principles based around a well-being framework incorporating material, relational and subjective components, and focused on gaining knowledge needed for decision-making. The principles relate to four key components of an impact evaluation: (i) defining well-being indicators, giving primacy to the perceptions of those most impacted by interventions through qualitative research, and considering subjective well-being, which can affect engagement with conservation; (ii) attributing impacts to interventions through quasi-experimental designs, or alternative methods such as theory-based, case study and participatory approaches, depending on the setting and evidence required; (iii) understanding the processes of change including evidence of causal linkages, and consideration of trajectories of change and institutional processes; and (iv) data collection with methods selected and applied with sensitivity to research context, consideration of heterogeneity of impacts along relevant societal divisions, and conducted by evaluators with local expertise and independence from the intervention.
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