Evolutionary analysis and functional characterization of the forkhead transcription factor FoxG1

Includes bibliographical references (leaves 103-110).Forhead box G1 (FoxG1) is a winged-helix transcription factor that plays a crucial role in the development of the telecephalon, the most rostral region of the brain Here, FoxG1 acts as a transcriptional repressor and maintains the population of cortical progenitor cells by promoting their proliferation and inhibiting differrentiation. Vertebrate FoxG1 orthologs have highly conserved DNA-binding and corbosy-terminal domains that have functional roles. Conversely, no functional role has yet been assigned to the N-terminal domain which shows a high degree of variability across vertebrates, with a remarkable stretch of consecutive histidine, proline and glutamine (HPQ) residues in the mammalian orthologs. In this study it was tested whether differences in FoxG1 sequence amongst vertebrates might account for the increased cortex size of mammals compared to non-mammals. Furthermore, changes in the sub-cellular localization of FoxG1 in response to fibroblast growth factor 2 (FGF-2) were investigated in a neural precursor cell line

Towards consistent generation of pancreatic lineage progenitors from human pluripotent stem cells.

Human pluripotent stem cells can in principle be used as a source of any differentiated cell type for disease modelling, drug screening, toxicology testing or cell replacement therapy. Type I diabetes is considered a major target for stem cell applications due to the shortage of primary human beta cells. Several protocols have been reported for generating pancreatic progenitors by in vitro differentiation of human pluripotent stem cells. Here we first assessed one of these protocols on a panel of pluripotent stem cell lines for capacity to engender glucose sensitive insulin-producing cells after engraftment in immunocompromised mice. We observed variable outcomes with only one cell line showing a low level of glucose response. We, therefore, undertook a systematic comparison of different methods for inducing definitive endoderm and subsequently pancreatic differentiation. Of several protocols tested, we identified a combined approach that robustly generated pancreatic progenitors in vitro from both embryo-derived and induced pluripotent stem cells. These findings suggest that, although there are intrinsic differences in lineage specification propensity between pluripotent stem cell lines, optimal differentiation procedures may consistently direct a substantial fraction of cells into pancreatic specification.This research was supported by European Commission Grant agreement 241883, “BetaCellTherapy”, and by the United Kingdom Medical Research Council.This is the final version of the article. It first appeared from Royal Society Publishing via http://dx.doi.org/10.1098/rstb.2014.036

Prevention and reversal of thymus involution mediated by the transcription factor Foxn1

Central to the age-associated decrease in immune system function, characterised by the increase in the frequency and severity of infections and autoimmune diseases, is the decrease in production of naïve T cells by the thymus. This results from the targeted degeneration or involution of the thymus with age. One of the principal causes of involution is the loss of organisation and functionality of the thymic epithelium, which confers the primary function of the organ via interactive regulation of T cell development. Although the mechanisms that govern the deterioration of the thymic epithelium are poorly understood, a number of recent reports indicate that the transcription factor, Foxn1, is required to maintain this compartment in the postnatal thymus. Thus, the first aim of this study was to precisely profile Foxn1 expression levels in aging postnatal thymic epithelial cells. The second aim was to investigate the effects of upregulating Foxn1 in the aging thymus, which was achieved using a novel, regulatable Foxn1 mouse model generated during this study. In this study I show that Foxn1 is expressed at different levels in different postnatal thymic epithelial cell (TEC) sub-populations suggesting a dosage-dependent mode of action for Foxn1. Additionally, using two experimental approaches, I show that Foxn1 expression decreases with age in TECs, supporting the current data that implicate the loss of Foxn1 as a potential cause of thymus involution. Next, I generated a tissue-specific, regulatable Foxn1 mouse model that allowed me to modulate Foxn1 expression in the postnatal thymus. Firstly, using this model, I show that thymus involution can be prevented by the up-regulation and maintenance of Foxn1 expression from the onset of involution. Thymi that up-regulated Foxn1 were overtly larger and exhibited greater cellularity in both the thymocyte and epithelial compartments compared to age matched controls. Additionally, the larger TEC compartment contained a higher proportion of functional and proliferating TECs that upregulated a panel of genes involved in TEC development and function. Next, I show that Foxn1 up-regulation in aged, involuted thymi is sufficient to partially reverse involution, as shown by an increase in TEC organisation and intrathymic T cell numbers. While other strategies that promote thymic rebound or reversal have been reported, including cytokine treatment or sex steroid ablation, these approaches are complicated by side effects and toxicity. Hence, I propose a novel model for immune reconstitution through the regulation of Foxn1 expression in the postnatal thymus

The Cell-Surface Marker Sushi Containing Domain 2 Facilitates Establishment of Human Naive Pluripotent Stem Cells.

Recently naive human pluripotent stem cells (hPSCs) have been described that relate to an earlier stage of development than conventional hPSCs. Naive hPSCs remain challenging to generate and authenticate, however. Here we report that Sushi Containing Domain 2 (SUSD2) is a robust cell-surface marker of naive hPSCs in the embryo and in vitro. SUSD2 transcripts are enriched in the pre-implantation epiblast of human blastocysts and immunostaining shows localization of SUSD2 to KLF17-positive epiblast cells. SUSD2 mRNA is strongly expressed in naive hPSCs but is negligible in other hPSCs. SUSD2 immunostaining of live or fixed cells provides unambiguous discrimination of naive versus conventional hPSCs. SUSD2 staining or flow cytometry enable monitoring of naive hPSCs in maintenance culture, and their isolation and quantification during resetting of conventional hPSCs or somatic cell reprogramming. Thus SUSD2 is a powerful non-invasive tool for reliable identification and purification of the naive hPSC phenotype.This research was funded by the Medical Research Council of the United Kingdom (G1001028 and MR/P00072X/1) and European Commission Framework 7 (HEALTH-F4-2013-602423, PluriMes). The Cambridge Stem Cell Institute receives core support from the Wellcome Trust and the Medical Research Council. AS is a Medical Research Council Professor

Foxn1 Regulates Lineage Progression in Cortical and Medullary Thymic Epithelial Cells But Is Dispensable for Medullary Sublineage Divergence

The forkhead transcription factor Foxn1 is indispensable for thymus development, but the mechanisms by which it mediates thymic epithelial cell (TEC) development are poorly understood. To examine the cellular and molecular basis of Foxn1 function, we generated a novel and revertible hypomorphic allele of Foxn1. By varying levels of its expression, we identified a number of features of the Foxn1 system. Here we show that Foxn1 is a powerful regulator of TEC differentiation that is required at multiple intermediate stages of TE lineage development in the fetal and adult thymus. We find no evidence for a role for Foxn1 in TEC fate-choice. Rather, we show it is required for stable entry into both the cortical and medullary TEC differentiation programmes and subsequently is needed at increasing dosage for progression through successive differentiation states in both cortical and medullary TEC. We further demonstrate regulation by Foxn1 of a suite of genes with diverse roles in thymus development and/or function, suggesting it acts as a master regulator of the core thymic epithelial programme rather than regulating a particular aspect of TEC biology. Overall, our data establish a genetics-based model of cellular hierarchies in the TE lineage and provide mechanistic insight relating titration of a single transcription factor to control of lineage progression. Our novel revertible hypomorph system may be similarly applied to analyzing other regulators of development

Effect of Artemether-Lumefantrine Policy and Improved Vector Control on Malaria Burden in KwaZulu–Natal, South Africa

BACKGROUND: Between 1995 and 2000, KwaZulu–Natal province, South Africa, experienced a marked increase in Plasmodium falciparum malaria, fuelled by pyrethroid and sulfadoxine-pyrimethamine resistance. In response, vector control was strengthened and artemether-lumefantrine (AL) was deployed in the first Ministry of Health artemisinin-based combination treatment policy in Africa. In South Africa, effective vector and parasite control had historically ensured low-intensity malaria transmission. Malaria is diagnosed definitively and treatment is provided free of charge in reasonably accessible public-sector health-care facilities. METHODS AND FINDINGS: We reviewed four years of malaria morbidity and mortality data at four sentinel health-care facilities within KwaZulu–Natal's malaria-endemic area. In the year following improved vector control and implementation of AL treatment, malaria-related admissions and deaths both declined by 89%, and outpatient visits decreased by 85% at the sentinel facilities. By 2003, malaria-related outpatient cases and admissions had fallen by 99%, and malaria-related deaths had decreased by 97%. There was a concomitant marked and sustained decline in notified malaria throughout the province. No serious adverse events were associated causally with AL treatment in an active sentinel pharmacovigilance survey. In a prospective study with 42 d follow up, AL cured 97/98 (99%) and prevented gametocyte developing in all patients. Consistent with the findings of focus group discussions, a household survey found self-reported adherence to the six-dose AL regimen was 96%. CONCLUSION: Together with concurrent strengthening of vector control measures, the antimalarial treatment policy change to AL in KwaZulu–Natal contributed to a marked and sustained decrease in malaria cases, admissions, and deaths, by greatly improving clinical and parasitological cure rates and reducing gametocyte carriage

Foxn1 Is Dynamically Regulated in Thymic Epithelial Cells during Embryogenesis and at the Onset of Thymic Involution

Thymus function requires extensive cross-talk between developing T-cells and the thymic epithelium, which consists of cortical and medullary TEC. The transcription factor FOXN1 is the master regulator of TEC differentiation and function, and declining Foxn1 expression with age results in stereotypical thymic involution. Understanding of the dynamics of Foxn1 expression is, however, limited by a lack of single cell resolution data. We have generated a novel reporter of Foxn1 expression, Foxn1G, to monitor changes in Foxn1 expression during embryogenesis and involution. Our data reveal that early differentiation and maturation of cortical and medullary TEC coincides with precise sub-lineage-specific regulation of Foxn1 expression levels. We further show that initiation of thymic involution is associated with reduced cTEC functionality, and proportional expansion of FOXN1-negative TEC in both cortical and medullary sub-lineages. Cortex-specific down-regulation of Foxn1 between 1 and 3 months of age may therefore be a key driver of the early stages of age-related thymic involution

Wnt Inhibition Facilitates RNA-Mediated Reprogramming of Human Somatic Cells to Naive Pluripotency.

In contrast to conventional human pluripotent stem cells (hPSCs) that are related to post-implantation embryo stages, naive hPSCs exhibit features of pre-implantation epiblast. Naive hPSCs are established by resetting conventional hPSCs, or are derived from dissociated embryo inner cell masses. Here we investigate conditions for transgene-free reprogramming of human somatic cells to naive pluripotency. We find that Wnt inhibition promotes RNA-mediated induction of naive pluripotency. We demonstrate application to independent human fibroblast cultures and endothelial progenitor cells. We show that induced naive hPSCs can be clonally expanded with a diploid karyotype and undergo somatic lineage differentiation following formative transition. Induced naive hPSC lines exhibit distinctive surface marker, transcriptome, and methylome properties of naive epiblast identity. This system for efficient, facile, and reliable induction of transgene-free naive hPSCs offers a robust platform, both for delineation of human reprogramming trajectories and for evaluating the attributes of isogenic naive versus conventional hPSCs.This research was funded by the Medical Research Council of the United Kingdom (G1001028 and MR/P00072X/1) and European Commission Framework 7 (HEALTHF4-2013-602423, PluriMes). JY was supported by the Guangdong Provincial Key Laboratory, and FvM by a UKRI/MRC Rutherford Fund Fellowship. The Cambridge Stem Cell Institute receives core support from Wellcome and the Medical Research Council. AS is a Medical Research Council Professor