Emilie Wilkie

Identification of liver-specific enhancer-promoter activity in the 3[prime] untranslated region of the wild-type AAV2 genome

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.

Differential response of epiblast stem cells to Nodal and Activin signalling: a paradigm of early endoderm development in the embryo

Mouse epiblast stem cells (EpiSCs) display temporal differences in the upregulation of Mixl1 expression during the initial steps of in vitro differentiation, which can be correlated with their propensity for endoderm differentiation. EpiSCs that upregulated Mixl1 rapidly during differentiation responded robustly to both Activin A and Nodal in generating foregut endoderm and precursors of pancreatic and hepatic tissues. By contrast, EpiSCs that delayed Mixl1 upregulation responded less effectively to Nodal and showed an overall suboptimal outcome of directed differentiation. The enhancement in endoderm potency in Mixl1-early cells may be accounted for by a rapid exit from the progenitor state and the efficient response to the induction of differentiation by Nodal. EpiSCs that readily differentiate into the endoderm cells are marked by a distinctive expression fingerprint of transforming growth factor (TGF)-β signalling pathway genes and genes related to the endoderm lineage. Nodal appears to elicit responses that are associated with transition to a mesenchymal phenotype, whereas Activin A promotes gene expression associated with maintenance of an epithelial phenotype. We postulate that the formation of definitive endoderm (DE) in embryoid bodies follows a similar process to germ layer formation from the epiblast, requiring an initial de-epithelialization event and subsequent re-epithelialization. Our results show that priming EpiSCs with the appropriate form of TGF-β signalling at the formative phase of endoderm differentiation impacts on the further progression into mature DE-derived lineages, and that this is influenced by the initial characteristics of the cell population. Our study also highlights that Activin A, which is commonly used as an in vitro surrogate for Nodal in differentiation protocols, does not elicit the same downstream effects as Nodal, and therefore may not effectively mimic events that take place in the mouse embryo.

Transcriptional targets of TWIST1 in the cranial mesoderm regulate cell-matrix interactions and mesenchyme maintenance

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.

Generation of genome-edited mouse epiblast stem cells via a detour through ES cell-chimeras.

Conventionally, mouse epiblast stem cells (EpiSCs) are derived directly from the epiblast or ectoderm germ layer of the post-implantation embryo. Self-renewing and multipotent EpiSC-like stem cells can also be derived by the conversion of embryonic stem cells (ESCs) via the provision of culture conditions that enable the maintenance of the EpiSCs. Here, we outline an experimental procedure for deriving EpiSCs from post-implantation chimeric embryos that are generated using genome-edited ESCs. This strategy enables the production of EpiSCs where (i) no genetically modified animals or ESCs are available, (ii) the impact of the genetic modification on post-implantation development, which may influence the property of the EpiSCs, is requisite knowledge for using the EpiSC for a specific investigation, and (iii) multiple editing of the genome is desirable to modify the biological attributes of the EpiSCs for studying, for example, the gene network activity on the trajectory of lineage differentiation and tissue morphogenesis.

Suppressing Nodal Signaling Activity Predisposes Ectodermal Differentiation of Epiblast Stem Cells

Summary The molecular mechanism underpinning the specification of the ectoderm, a transient germ-layer tissue, during mouse gastrulation was examined here in a stem cell-based model. We captured a self-renewing cell population with enhanced ectoderm potency from mouse epiblast stem cells (EpiSCs) by suppressing Nodal signaling activity. The transcriptome of the Nodal-inhibited EpiSCs resembles that of the anterior epiblast of embryonic day (E)7.0 and E7.5 mouse embryo, which is accompanied by chromatin modifications that reflect the priming of ectoderm lineage-related genes for expression. Nodal-inhibited EpiSCs show enhanced ectoderm differentiation in vitro and contribute to the neuroectoderm and the surface ectoderm in postimplantation chimeras but lose the propensity for mesendoderm differentiation in vitro and in chimeras. Our findings show that specification of the ectoderm progenitors is enhanced by the repression of Nodal signaling activity, and the ectoderm-like stem cells provide an experimental model to investigate the molecular characters of the epiblast-derived ectoderm.

A gene regulatory network anchored by LIM homeobox 1 for embryonic head development

Development of the embryonic head is driven by the activity of gene regulatory networks of transcription factors. LHX1 is a homeobox transcription factor that plays an essential role in the formation of the embryonic head. The loss of LHX1 function results in anterior truncation of the embryo caused by the disruption of morphogenetic movement of tissue precursors and the dysregulation of WNT signaling activity. Profiling the gene expression pattern in the Lhx1 mutant embryo revealed that tissues in anterior germ layers acquire posterior tissue characteristics, suggesting LHX1 activity is required for the allocation and patterning of head precursor tissues. Here, we used LHX1 as an entry point to delineate its transcriptional targets and interactors and construct a LHX1‐anchored gene regulatory network. Using a gain‐of‐function approach, we identified genes that immediately respond to Lhx1 activation. Meta‐analysis of the datasets of LHX1‐responsive genes and genes expressed in the anterior tissues of mouse embryos at head‐fold stage, in conjunction with published Xenopus embryonic LHX1 (Xlim1) ChIP‐seq data, has pinpointed the putative transcriptional targets of LHX1 and an array of genetic determinants functioning together in the formation of the mouse embryonic head.

Modulation of Epiblast Stem Cell fates by WNT-Mixl1 activity

This study aims to elucidate the molecular activity that influences lineage propensity in the early mouse embryo. To this aim, we used the embryo-derived epiblast stem cells (EpiSCs), which are developmentally similar to the epiblast of the gastrulating mouse embryo. WNT signalling activity can be modulated during the derivation and maintenance of the EpiSCs through blocking the release of WNT ligands by the chemical inhibitor IWP2. The blocking of WNT signalling activity leads to a bias of differentiation of the EpiSCs towards ectoderm derivatives, in contrast to the propensity of mesendoderm differentiation of classical EpiSCs. Analysis of the gene expression profiles revealed differences in the transcriptome between both EpiSCs. In the gastrulating embryo, the allocation of the epiblast cells to the mesendoderm lineage is accompanied by the expression of genes including Mixl1, a homeodomain transcription factor. Mixl1 is down regulated in the EpiSCs when WNT activity is inhibited, consistent with the bias of these EpiSCs toward ectodermal cells. This shift in cell differentiation trajectory implicates the plasticity of cell fates of EpiSC derived in WNT-free condition and that the lineage propensity can be re-set by changes in WNT activity. Interestingly, the mesendoderm propensity of the EpiSCs is correlated with the expediency and magnitude of activation of Mixl1. Further evidences gleaned from EpiSCs studies, place Mixl1 as a major regulator transcription factor for EMT – MET process during gastrulation in the mouse embryo. Given that Mixl1 activity may be regulated by WNT signalling, this raise the possibility that the activity of WNT-Mixl1 cascade is key to mesendoderm differentiation of the EpiSCs. Modulation of WNT activity and Mixl1 therefore have a convergent function in controlling the differentiation of the progenitor of germ layer tissues.

Identification of the TWIST1 interactome in craniofacial development using bioID

Nitric oxide plays an important role in stem cell differentiation and in endothelial cells it is expressed by endothelial nitric oxide synthase (eNOS) which is located within membrane caveolae through scaffolding with caveolin-1 (CAV-1WT) which inhibits NO production. In this study, we modified rat bone marrow mesenchymal stem cells (rBMSCs) with lentiviral vectors to co-express eNOS and mutant caveolin-1 (CAV-1F92A) to enhance NO generation and investigated endothelial reprogramming. A lenti-eNOS-GFP fusion vector which allowed FACS selection of eNOS positive cells increased NO production (6.9 ± 0.8 μM) compared to GFP alone (1.04 ± 0.22 μM) and un-transduced cells (0.91± 0.35 μM) (pb0.05). eNOSGFP and GFP cells were further re-transduced with CAV-1F92A or CAV-1WT. rBMSCeNOS+CAV-1F92A showed enhanced in vitro capillary tubule formation, upregulation of endothelial specific CD31, V-cadherin gene expression which was reversed by treatment with the eNOS inhibitor L-NAME. Activity of an endothelial specific FLT1eGFP lentiviral-reporter was also increased compared to controls. Furthermore, arterial specific Notch1, Hey 1 and DII4 gene expression was increased in rBMSCeNOS+CAV-1F92A whereas venous specific COUP-TFII and lymphatic specific Prox1 expression was significantly reduced. Canonical Wnt3A gene expression was increased in rBMSCeNOS+CAV-1F92A and treatment with L-NAME, resulted in reduced CD31 and Wnt3A expression, and the Wnt inhibitor Dkk1 also decreased CD31 expression. Western blot analysis showed increased β-catenin expression in rBMSCeNOS+CAV-1F92A confirming the relationship between NO and Wnt/β-catenin signaling. Furthermore rBMSCeNOS+CAV-1F92A cells showed decreased histone deacytaylase SIRT6 and DNA methyltransferase DNMT1 expression which was reversed with L-NAME treatment highlighting that NO may drive epigenetic modification during reprogramming. Finally, subcutaneous implantation of the rBMSCeNOS+CAV-1F92A cells seeded in polyurethane scaffolds in rats, resulted in formation of blood vessels. In summary, our data suggest that nitric oxide activates Wnt/-β-catenin signaling to promote endothelial reprogramming of BMSCs and may provide a novel platform for vascular repair.

Deciphering the gene regulatory network necessary for head formation

Development of the embryonic head is determined by the activity of gene regulatory networks (GRN) driven by transcription factors such as LHX1 and OTX2. Analysis of genetic mutants has revealed that loss of Lhx1 function at sequential steps of embryonic head formation leads to the truncation of head structures. We have implemented Lhx1-expressing embryonic stem cells (ESCs) to generate embryos for the identification of the in vivo ensemble of LHX1 downstream target genes. We have generated embryos from ESCs harbouring a conditional Lhx1 transgene and have confirmed inducible activation of Lhx1 in the embryos that are rendered amenable for RNA-seq and ChIP-seq analyses. Meta-analysis of gene expression profiles of differentiating Lhx1-expressing ESCs and the anterior germ layers of mouse gastrula-staged embryos, and ChIPseq data on the transcriptional targets of the LHX1 orthologue in Xenopus embryos has revealed a Head GRN, consisting of 92 genes as putative targets of LHX1 in the mouse. Among the putative targets, the functional attribute of nine that have no known phenotype are being studied by analysis of the chimeras derived from genome-edited ESCs. Preliminary findings of these studies will be presented.

Dataset of TWIST1-regulated genes in the cranial mesoderm and a transcriptome comparison of cranial mesoderm and cranial neural crest.

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.