a b s t r a c tRecent methodological advancements in microscopy and DNA sequencing-based methods provide unprecedented new insights into the spatio-temporal relationships between chromatin and nuclear machineries. We discuss a model of the underlying functional nuclear organization derived mostly from electron and super-resolved fluorescence microscopy studies. It is based on two spatially co-aligned, active and inactive nuclear compartments (ANC and INC). The INC comprises the compact, transcriptionally inactive core of chromatin domain clusters (CDCs). The ANC is formed by the transcriptionally active periphery of CDCs, called the perichromatin region (PR), and the interchromatin compartment (IC). The IC is connected to nuclear pores and serves nuclear import and export functions. The ANC is the major site of RNA synthesis. It is highly enriched in epigenetic marks for transcriptionally competent chromatin and RNA Polymerase II. Marks for silent chromatin are enriched in the INC. Multi-scale cross-correlation spectroscopy suggests that nuclear architecture resembles a random obstacle network for diffusing proteins. An increased dwell time of proteins and protein complexes within the ANC may help to limit genome scanning by factors or factor complexes to DNA exposed within the ANC.

BackgroundA Xist RNA decorated Barr body is the structural hallmark of the compacted inactive X territory in female mammals. Using super-resolution three-dimensional structured illumination microscopy (3D-SIM) and quantitative image analysis, we compared its ultrastructure with active chromosome territories (CTs) in human and mouse somatic cells, and explored the spatio-temporal process of Barr body formation at onset of inactivation in early differentiating mouse embryonic stem cells (ESCs).ResultsWe demonstrate that all CTs are composed of structurally linked chromatin domain clusters (CDCs). In active CTs the periphery of CDCs harbors low-density chromatin enriched with transcriptionally competent markers, called the perichromatin region (PR). The PR borders on a contiguous channel system, the interchromatin compartment (IC), which starts at nuclear pores and pervades CTs. We propose that the PR and macromolecular complexes in IC channels together form the transcriptionally permissive active nuclear compartment (ANC). The Barr body differs from active CTs by a partially collapsed ANC with CDCs coming significantly closer together, although a rudimentary IC channel system connected to nuclear pores is maintained. Distinct Xist RNA foci, closely adjacent to the nuclear matrix scaffold attachment factor-A (SAF-A) localize throughout Xi along the rudimentary ANC. In early differentiating ESCs initial Xist RNA spreading precedes Barr body formation, which occurs concurrent with the subsequent exclusion of RNA polymerase II (RNAP II). Induction of a transgenic autosomal Xist RNA in a male ESC triggers the formation of an ‘autosomal Barr body’ with less compacted chromatin and incomplete RNAP II exclusion.Conclusions3D-SIM provides experimental evidence for profound differences between the functional architecture of transcriptionally active CTs and the Barr body. Basic structural features of CT organization such as CDCs and IC channels are however still recognized, arguing against a uniform compaction of the Barr body at the nucleosome level. The localization of distinct Xist RNA foci at boundaries of the rudimentary ANC may be considered as snap-shots of a dynamic interaction with silenced genes. Enrichment of SAF-A within Xi territories and its close spatial association with Xist RNA suggests their cooperative function for structural organization of Xi.

Morphogen gradients pattern the endoderm and specify liver and pancreatic progenitors in vivo. However, if specified organ progenitors can be identified and isolated during human pluripotent stem cell (hPSC) differentiation is unknown. Here, we report the identification of two novel surface markers, CD177/NB1 glycoprotein and inducible T cell co-stimulatory ligand CD275/ICOSL, that isolate specified organ progenitors from seemingly homogenous endoderm differentiations in vitro. These markers allow assessing anterior definitive endoderm (ADE) patterning and specification in human revealing different morphogen requirements and induction efficiencies for the generation of specified pancreatic and liver progenitors using known and novel differentiation paradigms. Furthermore, molecular profiling and characterisation of CD177 + and CD275 + ADE subpopulations identified differential expression of signalling components and inverse activation of canonical and non-canonical WNT signalling. This signalling milieu specifies CD275 + ADE progenitors towards the liver fate. In contrast, CD177 + ADE progenitors express and synthesize the secreted WNT, NODAL and BMP antagonist CERBERUS1 and are specified towards the pancreatic fate. Strikingly, isolated CD177 + ADE progenitors differentiate more homogenously into pancreatic progenitors as well as into functionally, more mature and glucose-responsive β-like cells in vitro, when compared to bulk endoderm differentiations. Overall, the identification of novel surface markers allowed us to isolate, monitor and understand human organ progenitor formation for the improved differentiation of β-like cells from hPSC.

Insulin and insulin-like growth factor 1 (Igf1) resistance in pancreatic β-cells causes overt diabetes, thus, therapeutic improvement may protect from β-cell failure 1-3 . Here, we identified a novel inhibitor of insulin (Insr) and Igf1 receptor (Igf1r) signalling in β-cells, which we named insulin inhibitory receptor (Inceptor; Iir). Inceptor contains an extracellular cysteine-rich domain with similarities to the Insr and Igf1r 4 and a mannose-6-phosphate domain found in the Igf2r 5 . Inceptor knock-out (KO) mice die within the first hours after birth with signs of hyperinsulinemia and hypoglycaemia. Molecular and cellular analysis of the Iir -/embryonic and postnatal pancreas showed increased Insr/Igf1r activation, resulting in augmented β-cell proliferation and mass. Similarly, inducible β-cellspecific Iir -/-KO in adult mice and in ex vivo islets led to increased Insr/Igf1r activation and β-cell proliferation, resulting in improved glucose tolerance in vivo. Mechanistically, Inceptor interacts with Insr and Igf1r to facilitate clathrinmediated endocytosis for receptor desensitisation. Blocking this physical interaction using monoclonal antibodies against the extracellular domain of Inceptor retained Inceptor and Insr at the plasma membrane to sustain Insr/Igf1r activation in β-cells. Taken together, Inceptor shields insulin-producing β-cells from constitutive pathway activation and provides a molecular target for Insr/Igf1r sensitisation and potential diabetes therapy.

ObjectiveThe transcription factors (TF) Foxa2 and Pdx1 are key regulators of beta-cell (β-cell) development and function. Mutations of these TFs or their respective cis-regulatory consensus binding sites have been linked to maturity diabetes of the young (MODY), pancreas agenesis, or diabetes susceptibility in human. Although Foxa2 has been shown to directly regulate Pdx1 expression during mouse embryonic development, the impact of this gene regulatory interaction on postnatal β-cell maturation remains obscure.MethodsIn order to easily monitor the expression domains of Foxa2 and Pdx1 and analyze their functional interconnection, we generated a novel double knock-in homozygous (FVFPBFDHom) fluorescent reporter mouse model by crossing the previously described Foxa2-Venus fusion (FVF) with the newly generated Pdx1-BFP (blue fluorescent protein) fusion (PBF) mice.ResultsAlthough adult PBF homozygous animals exhibited a reduction in expression levels of Pdx1, they are normoglycemic. On the contrary, despite normal pancreas and endocrine development, the FVFPBFDHom reporter male animals developed hyperglycemia at weaning age and displayed a reduction in Pdx1 levels in islets, which coincided with alterations in β-cell number and islet architecture. The failure to establish mature β-cells resulted in loss of β-cell identity and trans-differentiation towards other endocrine cell fates. Further analysis suggested that Foxa2 and Pdx1 genetically and functionally cooperate to regulate maturation of adult β-cells.ConclusionsOur data show that the maturation of pancreatic β-cells requires the cooperative function of Foxa2 and Pdx1. Understanding the postnatal gene regulatory network of β-cell maturation will help to decipher pathomechanisms of diabetes and identify triggers to regenerate dedifferentiated β-cell mass.