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Diabetes mellitus is a multifactorial disease affecting increasing numbers of patients worldwide. Progression to insulin-dependent diabetes mellitus is characterized by the loss or dysfunction of pancreatic β-cells, but the pathomechanisms underlying β-cell failure in type 1 diabetes mellitus and type 2 diabetes mellitus are still poorly defined. Regeneration of β-cell mass from residual islet cells or replacement by β-like cells derived from stem cells holds great promise to stop or reverse disease progression. However, the development of new treatment options is hampered by our limited understanding of human pancreas organogenesis due to the restricted access to primary tissues. Therefore, the challenge is to translate results obtained from preclinical model systems to humans, which requires comparative modelling of β-cell biology in health and disease. Here, we discuss diverse modelling systems across different species that provide spatial and temporal resolution of cellular and molecular mechanisms to understand the evolutionary conserved genotype-phenotype relationship and translate them to humans. In addition, we summarize the latest knowledge on organoids, stem cell differentiation platforms, primary micro-islets and pseudo-islets, bioengineering and microfluidic systems for studying human pancreas development and homeostasis ex vivo. These new modelling systems and platforms have opened novel avenues for exploring the developmental trajectory, physiology, biology and pathology of the human pancreas. [ABSTRACT FROM AUTHOR]

Background Ras is a membrane-associated small G-protein that funnels growth and differentiation signals into downstream signal transduction pathways by cycling between an inactive, GDP-bound and an active, GTP-bound state. Aberrant Ras activity as a result of oncogenic mutations causes de novo cell transformation and promotes tumor growth and progression. Results Here, we describe a novel strategy to block deregulated Ras activity by means of oligomerized cognate protein modules derived from the Ras-binding domain of c-Raf (RBD), which we named MSOR for multivalent scavengers of oncogenic Ras. The introduction of well-characterized mutations into RBD was used to adjust the affinity and hence the blocking potency of MSOR towards activated Ras. MSOR inhibited several oncogenic Ras-stimulated processes including downstream activation of Erk1/2, induction of matrix-degrading enzymes, cell motility and invasiveness in a graded fashion depending on the oligomerization grade and the nature of the individual RBD-modules. The amenability to accurate experimental regulation was further improved by engineering an inducible MSOR-expression system to render the reversal of oncogenic Ras effects controllable. Conclusion MSOR represent a new tool for the experimental and possibly therapeutic selective blockade of oncogenic Ras signals. [ABSTRACT FROM AUTHOR]

Integrin-linked kinase (ILK) localizes to focal adhesions (FAs) where it regulates cell spreading, migration, and growth factor receptor signaling. Previous reports showed that overexpressed ILK in which Val386 and Thr387 were substituted with glycine residues (ILK-VT/GG) could neither interact with paxillin nor localize to FA in cells expressing endogenous wild-type ILK, implying that paxillin binding to ILK is required for its localization to FAs. Here, we show that introducing this mutation into the germ line of mice (ILK-VT/GG) caused vasculogenesis defects, resulting in a general developmental delay and death at around embryonic day 12.5. Fibroblasts isolated from ILKVT/ GG mice contained mutant ILK in FAs, showed normal adhesion to and spreading on extracellular matrix substrates but displayed impaired migration. Biochemical analysis revealed that VT/GG substitutions decreased ILK protein stability leading to decreased ILK levels and reduced binding to paxillin and α-parvin. Because paxillin depletion did not affect ILK localization to FAs, the embryonic lethality and the in vitro migration defects are likely due to the reduced levels of ILK-VT/GG and diminished binding to parvins. [ABSTRACT FROM AUTHOR]

Background: Islet xenotransplantation is a promising concept for beta‐cell replacement therapy. Reporter genes for noninvasive monitoring of islet engraftment, graft mass changes, long‐term survival, and graft failure support the optimization of transplantation strategies. Near‐infrared fluorescent protein (iRFP) is ideal for fluorescence imaging (FI) in tissue, but also for multispectral optoacoustic tomography (MSOT) with an even higher imaging depth. Therefore, we generated reporter pigs ubiquitously expressing iRFP. Methods: CAG‐iRPF720 transgenic reporter pigs were generated by somatic cell nuclear transfer from FACS‐selected stable transfected donor cells. Neonatal pig islets (NPIs) were transplanted into streptozotocin‐diabetic immunodeficient NOD‐scid IL2Rgnull (NSG) mice. FI and MSOT were performed to visualize different numbers of NPIs and to evaluate associations between signal intensity and glycemia. MSOT was also tested in a large animal model. Results: CAG‐iRFP transgenic NPIs were functionally equivalent with wild‐type NPIs. Four weeks after transplantation under the kidney capsule, FI revealed a twofold higher signal for 4000‐NPI compared to 1000‐NPI grafts. Ten weeks after transplantation, the fluorescence intensity of the 4000‐NPI graft was inversely correlated with glycemia. After intramuscular transplantation into diabetic NSG mice, MSOT revealed clear dose‐dependent signals for grafts of 750, 1500, and 3000 NPIs. Dose‐dependent MSOT signals were also revealed in a pig model, with stronger signals after subcutaneous (depth ∼6 mm) than after submuscular (depth ∼15 mm) placement of the NPIs. Conclusions: Islets from CAG‐iRFP transgenic pigs are fully functional and accessible to long‐term monitoring by state‐of‐the‐art imaging modalities. The novel reporter pigs will support the development and preclinical testing of novel matrices and engraftment strategies for porcine xeno‐islets. [ABSTRACT FROM AUTHOR]

Most plane-polarized tissues are formed by identically oriented cells [ 1 , 2 ]. A notable exception occurs in the vertebrate vestibular system and lateral-line neuromasts, where mechanosensory hair cells orient along a single axis but in opposite directions to generate bipolar epithelia [ 3–5 ]. In zebrafish neuromasts, pairs of hair cells arise from the division of a non-sensory progenitor [ 6 , 7 ] and acquire opposing planar polarity via the asymmetric expression of the polarity-determinant transcription factor Emx2 [ 8–11 ]. Here, we reveal the initial symmetry-breaking step by decrypting the developmental trajectory of hair cells using single-cell RNA sequencing (scRNA-seq), diffusion pseudotime analysis, lineage tracing, and mutagenesis. We show that Emx2 is absent in non-sensory epithelial cells, begins expression in hair-cell progenitors, and is downregulated in one of the sibling hair cells via signaling through the Notch1a receptor. Analysis of Emx2-deficient specimens, in which every hair cell adopts an identical direction, indicates that Emx2 asymmetry does not result from auto-regulatory feedback. These data reveal a two-tiered mechanism by which the symmetric monodirectional ground state of the epithelium is inverted by deterministic initiation of Emx2 expression in hair-cell progenitors and a subsequent stochastic repression of Emx2 in one of the sibling hair cells breaks directional symmetry to establish planar bipolarity. • Unipotent hair-cell progenitors (UHCPs) initiate Emx2 expression • Emx2 expression symmetry is broken between sibling hair cells • Notch1a-activity-mediated symmetry breaking is stochastic • Anatomical and genetic asymmetries are separable Kozak et al. combine single-cell RNA sequencing, pseudotime trajectory analysis, and mutagenesis to reveal the initial symmetry-breaking step that results in the establishment of planar bipolarity in a sensory epithelium. [ABSTRACT FROM AUTHOR]