Mitchell-Riley syndrome iPSCs exhibit reduced pancreatic endoderm differentiation due to a mutation in RFX6.

Mitchell-Riley syndrome (MRS) is caused by recessive mutations in the regulatory factor X6 gene (RFX6) and is characterised by pancreatic hypoplasia and neonatal diabetes. To determine why individuals with MRS specifically lack pancreatic endocrine cells, we micro-CT imaged a 12-week-old foetus homozygous for the nonsense mutation RFX6 c.1129C>T, which revealed loss of the pancreas body and tail. From this foetus, we derived iPSCs and show that differentiation of these cells in vitro proceeds normally until generation of pancreatic endoderm, which is significantly reduced. We additionally generated an RFX6HA reporter allele by gene targeting in wild-type H9 cells to precisely define RFX6 expression and in parallel performed in situ hybridisation for RFX6 in the dorsal pancreatic bud of a Carnegie stage 14 human embryo. Both in vitro and in vivo, we find that RFX6 specifically labels a subset of PDX1-expressing pancreatic endoderm. In summary, RFX6 is essential for efficient differentiation of pancreatic endoderm, and its absence in individuals with MRS specifically impairs formation of endocrine cells of the pancreas head and tail.

A membrane-associated ß-catenin/Oct4 complex correlates with ground-state pluripotency in mouse embryonic stem cells.

The maintenance of pluripotency in mouse embryonic stem cells (mESCs) relies on the activity of a transcriptional network that is fuelled by the activity of three transcription factors (Nanog, Oct4 and Sox2) and balanced by the repressive activity of Tcf3. Extracellular signals modulate the activity of the network and regulate the differentiation capacity of the cells. Wnt/ß-catenin signaling has emerged as a significant potentiator of pluripotency: increases in the levels of ß-catenin regulate the activity of Oct4 and Nanog, and enhance pluripotency. A recent report shows that ß-catenin achieves some of these effects by modulating the activity of Tcf3, and that this effect does not require its transcriptional activation domain. Here, we show that during self-renewal there is negligible transcriptional activity of ß-catenin and that this is due to its tight association with membranes, where we find it in a complex with Oct4 and E-cadherin. Differentiation triggers a burst of Wnt/ß-catenin transcriptional activity that coincides with the disassembly of the complex. Our results establish that ß-catenin, but not its transcriptional activity, is central to pluripotency acting through a ß-catenin/Oct4 complex.

A systematic screen for protein-lipid interactions in Saccharomyces cerevisiae.

Protein-metabolite networks are central to biological systems, but are incompletely understood. Here, we report a screen to catalog protein-lipid interactions in yeast. We used arrays of 56 metabolites to measure lipid-binding fingerprints of 172 proteins, including 91 with predicted lipid-binding domains. We identified 530 protein-lipid associations, the majority of which are novel. To show the data set's biological value, we studied further several novel interactions with sphingolipids, a class of conserved bioactive lipids with an elusive mode of action. Integration of live-cell imaging suggests new cellular targets for these molecules, including several with pleckstrin homology (PH) domains. Validated interactions with Slm1, a regulator of actin polarization, show that PH domains can have unexpected lipid-binding specificities and can act as coincidence sensors for both phosphatidylinositol phosphates and phosphorylated sphingolipids.

Long-Term Culture of Self-renewing Pancreatic Progenitors Derived from Human Pluripotent Stem Cells.

Pluripotent stem cells have been proposed as an unlimited source of pancreatic ß cells for studying and treating diabetes. However, the long, multi-step differentiation protocols used to generate functional ß cells inevitably exhibit considerable variability, particularly when applied to pluripotent cells from diverse genetic backgrounds. We have developed culture conditions that support long-term self-renewal of human multipotent pancreatic progenitors, which are developmentally more proximal to the specialized cells of the adult pancreas. These cultured pancreatic progenitor (cPP) cells express key pancreatic transcription factors, including PDX1 and SOX9, and exhibit transcriptomes closely related to their in vivo counterparts. Upon exposure to differentiation cues, cPP cells give rise to pancreatic endocrine, acinar, and ductal lineages, indicating multilineage potency. Furthermore, cPP cells generate insulin+ ß-like cells in vitro and in vivo, suggesting that they offer a convenient alternative to pluripotent cells as a source of adult cell types for modeling pancreatic development and diabetes.

An interplay between extracellular signalling and the dynamics of the exit from pluripotency drives cell fate decisions in mouse ES cells.

Embryonic Stem cells derived from the epiblast tissue of the mammalian blastocyst retain the capability to differentiate into any adult cell type and are able to self-renew indefinitely under appropriate culture conditions. Despite the large amount of knowledge that we have accumulated to date about the regulation and control of self-renewal, efficient directed differentiation into specific tissues remains elusive. In this work, we have analysed in a systematic manner the interaction between the dynamics of loss of pluripotency and Activin/Nodal, BMP4 and Wnt signalling in fate assignment during the early stages of differentiation of mouse ES cells in culture. During the initial period of differentiation, cells exit from pluripotency and enter an Epi-like state. Following this transient stage, and under the influence of Activin/Nodal and BMP signalling, cells face a fate choice between differentiating into neuroectoderm and contributing to Primitive Streak fates. We find that Wnt signalling does not suppress neural development as previously thought and that it aids both fates in a context dependent manner. Our results suggest that as cells exit pluripotency they are endowed with a primary neuroectodermal fate and that the potency to become endomesodermal rises with time. We suggest that this situation translates into a "race for fates" in which the neuroectodermal fate has an advantage.

Single cell lineage analysis of mouse embryonic stem cells at the exit from pluripotency.

Understanding how interactions between extracellular signalling pathways and transcription factor networks influence cellular decision making will be crucial for understanding mammalian embryogenesis and for generating specialised cell types in vitro. To this end, pluripotent mouse Embryonic Stem (mES) cells have proven to be a useful model system. However, understanding how transcription factors and signalling pathways affect decisions made by individual cells is confounded by the fact that measurements are generally made on groups of cells, whilst individual mES cells differentiate at different rates and towards different lineages, even in conditions that favour a particular lineage. Here we have used single-cell measurements of transcription factor expression and Wnt/ß-catenin signalling activity to investigate their effects on lineage commitment decisions made by individual cells. We find that pluripotent mES cells exhibit differing degrees of heterogeneity in their expression of important regulators from pluripotency, depending on the signalling environment to which they are exposed. As mES cells differentiate, downregulation of Nanog and Oct4 primes cells for neural commitment, whilst loss of Sox2 expression primes cells for primitive streak commitment. Furthermore, we find that Wnt signalling acts through Nanog to direct cells towards a primitive streak fate, but that transcriptionally active ß-catenin is associated with both neural and primitive streak commitment. These observations confirm and extend previous suggestions that pluripotency genes influence lineage commitment and demonstrate how their dynamic expression affects the direction of lineage commitment, whilst illustrating two ways in which the Wnt signalling pathway acts on this network during cell fate assignment.

Dissecting ensemble networks in ES cell populations reveals micro-heterogeneity underlying pluripotency.

Analysis of transcription at the level of single cells in prokaryotes and eukaryotes has revealed the existence of heterogeneities in the expression of individual genes within genetically homogeneous populations. This variation is an emerging hallmark of populations of Embryonic Stem (ES) cells and has been ascribed to the stochasticity associated with the biochemical events that mediate gene expression. It has been suggested that these heterogeneities play a role in the maintenance of pluripotency. However, for the most part, studies have focused on individual genes in large cell populations. Here we use an existing dataset on the expression of eight genes involved in pluripotency in eighty-three ES cells to create Gene Regulatory Networks (GRNs) at the single cell level. We observe widespread heterogeneities in the expression of the eight genes, but analysis of correlations within individual cells reveals three distinct classes centered on the expression of Nanog, a marker of pluripotency, and Fgf5, a gene associated with differentiation: high levels of Nanog and low levels of Fgf5, low levels of Nanog and high levels of Fgf5, and low levels of both. Each of these classes is associated with a collection of active sub-networks, with differing degrees of connectivity between their elements, which define a cellular state: self-renewal, primed for differentiation or transition between the two. Though every cell should be governed by the same network, the active sub-networks may emerge due to considerations such as variation in (i) the expression level of active transcription factors (e.g. through post-translational modification or ligand/co-factor availability) or (ii) access to the target gene locus (e.g. via changes in chromatin status or epigenetic modifications). We conclude that heterogeneities in gene expression should not be interpreted as representing different states of a single unique network, but as a reflection of the activity of different sub-networks in sub-populations of cells.