Comprehensive single cell mRNA profiling reveals a detailed roadmap for pancreatic endocrinogenesis.

Deciphering mechanisms of endocrine cell induction, specification and lineage allocation in vivo will provide valuable insights into how the islets of Langerhans are generated. Currently, it is ill defined how endocrine progenitors segregate into different endocrine subtypes during development. Here, we generated a novel neurogenin 3 (Ngn3)-Venus fusion (NVF) reporter mouse line, that closely mirrors the transient endogenous Ngn3 protein expression. To define an in vivo roadmap of endocrinogenesis, we performed single cell RNA sequencing of 36,351 pancreatic epithelial and NVF+ cells during secondary transition. This allowed Ngn3low endocrine progenitors, Ngn3high endocrine precursors, Fev+ endocrine lineage and hormone+ endocrine subtypes to be distinguished and time-resolved, and molecular programs during the step-wise lineage restriction steps to be delineated. Strikingly, we identified 58 novel signature genes that show the same transient expression dynamics as Ngn3 in the 7260 profiled Ngn3-expressing cells. The differential expression of these genes in endocrine precursors associated with their cell-fate allocation towards distinct endocrine cell types. Thus, the generation of an accurately regulated NVF reporter allowed us to temporally resolve endocrine lineage development to provide a fine-grained single cell molecular profile of endocrinogenesis in vivo.

Transgenic pigs expressing near infrared fluorescent protein-A novel tool for noninvasive imaging of islet xenotransplants.

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.

Mutations in the paxillin-binding site of integrin-linked kinase (ILK) destabilize the pseudokinase domain and cause embryonic lethality in mice.

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 Val(386) and Thr(387) 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 ILK-VT/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.

Graded inhibition of oncogenic Ras-signaling by multivalent Ras-binding domains.

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.

Delineating mouse ß-cell identity during lifetime and in diabetes with a single cell atlas.

Although multiple pancreatic islet single-cell RNA-sequencing (scRNA-seq) datasets have been generated, a consensus on pancreatic cell states in development, homeostasis and diabetes as well as the value of preclinical animal models is missing. Here, we present an scRNA-seq cross-condition mouse islet atlas (MIA), a curated resource for interactive exploration and computational querying. We integrate over 300,000 cells from nine scRNA-seq datasets consisting of 56 samples, varying in age, sex and diabetes models, including an autoimmune type 1 diabetes model (NOD), a glucotoxicity/lipotoxicity type 2 diabetes model (db/db) and a chemical streptozotocin ß-cell ablation model. The ß-cell landscape of MIA reveals new cell states during disease progression and cross-publication differences between previously suggested marker genes. We show that ß-cells in the streptozotocin model transcriptionally correlate with those in human type 2 diabetes and mouse db/db models, but are less similar to human type 1 diabetes and mouse NOD ß-cells. We also report pathways that are shared between ß-cells in immature, aged and diabetes models. MIA enables a comprehensive analysis of ß-cell responses to different stressors, providing a roadmap for the understanding of ß-cell plasticity, compensation and demise.

A transcriptional cross species map of pancreatic islet cells.

OBJECTIVE: Pancreatic islets of Langerhans secrete hormones to regulate systemic glucose levels. Emerging evidence suggests that islet cells are functionally heterogeneous to allow a fine-tuned and efficient endocrine response to physiological changes. A precise description of the molecular basis of this heterogeneity, in particular linking animal models to human islets, is an important step towards identifying the factors critical for endocrine cell function in physiological and pathophysiological conditions. METHODS: In this study, we used single-cell RNA sequencing to profile more than 50'000 endocrine cells isolated from healthy human, pig and mouse pancreatic islets and characterize transcriptional heterogeneity and evolutionary conservation of those cells across the three species. We systematically delineated endocrine cell types and α- and ß-cell heterogeneity through prior knowledge- and data-driven gene sets shared across species, which altogether capture common and differential cellular properties, transcriptional dynamics and putative driving factors of state transitions. RESULTS: We showed that global endocrine expression profiles correlate, and that critical identity and functional markers are shared between species, while only approximately 20% of cell type enriched expression is conserved. We resolved distinct human α- and ß-cell states that form continuous transcriptional landscapes. These states differentially activate maturation and hormone secretion programs, which are related to regulatory hormone receptor expression, signaling pathways and different types of cellular stress responses. Finally, we mapped mouse and pig cells to the human reference and observed that the spectrum of human α- and ß-cell heterogeneity and aspects of such functional gene expression are better recapitulated in the pig than mouse data. CONCLUSIONS: Here, we provide a high-resolution transcriptional map of healthy human islet cells and their murine and porcine counterparts, which is easily queryable via an online interface. This comprehensive resource informs future efforts that focus on pancreatic endocrine function, failure and regeneration, and enables to assess molecular conservation in islet biology across species for translational purposes.

Primary cilia and SHH signaling impairments in human and mouse models of Parkinson's disease.

Parkinson's disease (PD) as a progressive neurodegenerative disorder arises from multiple genetic and environmental factors. However, underlying pathological mechanisms remain poorly understood. Using multiplexed single-cell transcriptomics, we analyze human neural precursor cells (hNPCs) from sporadic PD (sPD) patients. Alterations in gene expression appear in pathways related to primary cilia (PC). Accordingly, in these hiPSC-derived hNPCs and neurons, we observe a shortening of PC. Additionally, we detect a shortening of PC in PINK1-deficient human cellular and mouse models of familial PD. Furthermore, in sPD models, the shortening of PC is accompanied by increased Sonic Hedgehog (SHH) signal transduction. Inhibition of this pathway rescues the alterations in PC morphology and mitochondrial dysfunction. Thus, increased SHH activity due to ciliary dysfunction may be required for the development of pathoetiological phenotypes observed in sPD like mitochondrial dysfunction. Inhibiting overactive SHH signaling may be a potential neuroprotective therapy for sPD.

scPower accelerates and optimizes the design of multi-sample single cell transcriptomic studies.

Single cell RNA-seq has revolutionized transcriptomics by providing cell type resolution for differential gene expression and expression quantitative trait loci (eQTL) analyses. However, efficient power analysis methods for single cell data and inter-individual comparisons are lacking. Here, we present scPower; a statistical framework for the design and power analysis of multi-sample single cell transcriptomic experiments. We modelled the relationship between sample size, the number of cells per individual, sequencing depth, and the power of detecting differentially expressed genes within cell types. We systematically evaluated these optimal parameter combinations for several single cell profiling platforms, and generated broad recommendations. In general, shallow sequencing of high numbers of cells leads to higher overall power than deep sequencing of fewer cells. The model, including priors, is implemented as an R package and is accessible as a web tool. scPower is a highly customizable tool that experimentalists can use to quickly compare a multitude of experimental designs and optimize for a limited budget.

Epithelial cell plasticity drives endoderm formation during gastrulation.

It is generally accepted that epiblast cells ingress into the primitive streak by epithelial-to-mesenchymal transition (EMT) to give rise to the mesoderm; however, it is less clear how the endoderm acquires an epithelial fate. Here, we used embryonic stem cell and mouse embryo knock-in reporter systems to combine time-resolved lineage labelling with high-resolution single-cell transcriptomics. This allowed us to resolve the morphogenetic programs that segregate the mesoderm from the endoderm germ layer. Strikingly, while the mesoderm is formed by classical EMT, the endoderm is formed independent of the key EMT transcription factor Snail1 by mechanisms of epithelial cell plasticity. Importantly, forkhead box transcription factor A2 (Foxa2) acts as an epithelial gatekeeper and EMT suppressor to shield the endoderm from undergoing a mesenchymal transition. Altogether, these results not only establish the morphogenetic details of germ layer formation, but also have broader implications for stem cell differentiation and cancer metastasis.

Vertical sleeve gastrectomy triggers fast ß-cell recovery upon overt diabetes.

OBJECTIVE: The effectiveness of bariatric surgery in restoring ß-cell function has been described in type-2 diabetes (T2D) patients and animal models for years, whereas the mechanistic underpinnings are largely unknown. The possibility of vertical sleeve gastrectomy (VSG) to rescue far-progressed, clinically-relevant T2D and to promote ß-cell recovery has not been investigated on a single-cell level. Nevertheless, characterization of the heterogeneity and functional states of ß-cells after VSG is a fundamental step to understand mechanisms of glycaemic recovery and to ultimately develop alternative, less-invasive therapies. METHODS: We performed VSG in late-stage diabetic db/db mice and analyzed the islet transcriptome using single-cell RNA sequencing (scRNA-seq). Immunohistochemical analyses and quantification of ß-cell area and proliferation complement our findings from scRNA-seq. RESULTS: We report that VSG was superior to calorie restriction in late-stage T2D and rapidly restored normoglycaemia in morbidly obese and overt diabetic db/db mice. Single-cell profiling of islets of Langerhans showed that VSG induced distinct, intrinsic changes in the ß-cell transcriptome, but not in that of α-, δ-, and PP-cells. VSG triggered fast ß-cell redifferentiation and functional improvement within only two weeks of intervention, which is not seen upon calorie restriction. Furthermore, VSG expanded ß-cell area by means of redifferentiation and by creating a proliferation competent ß-cell state. CONCLUSION: Collectively, our study reveals the superiority of VSG in the remission of far-progressed T2D and presents paths of ß-cell regeneration and molecular pathways underlying the glycaemic benefits of VSG.

CD81 marks immature and dedifferentiated pancreatic ß-cells.

OBJECTIVE: Islets of Langerhans contain heterogeneous populations of insulin-producing ß-cells. Surface markers and respective antibodies for isolation, tracking, and analysis are urgently needed to study ß-cell heterogeneity and explore the mechanisms to harness the regenerative potential of immature ß-cells. METHODS: We performed single-cell mRNA profiling of early postnatal mouse islets and re-analyzed several single-cell mRNA sequencing datasets from mouse and human pancreas and islets. We used mouse primary islets, iPSC-derived endocrine cells, Min6 insulinoma, and human EndoC-ßH1 ß-cell lines and performed FAC sorting, Western blotting, and imaging to support and complement the findings from the data analyses. RESULTS: We found that all endocrine cell types expressed the cluster of differentiation 81 (CD81) during pancreas development, but the expression levels of this protein were gradually reduced in ß-cells during postnatal maturation. Single-cell gene expression profiling and high-resolution imaging revealed an immature signature of ß-cells expressing high levels of CD81 (CD81high) compared to a more mature population expressing no or low levels of this protein (CD81low/-). Analysis of ß-cells from different diabetic mouse models and in vitro ß-cell stress assays indicated an upregulation of CD81 expression levels in stressed and dedifferentiated ß-cells. Similarly, CD81 was upregulated and marked stressed human ß-cells in vitro. CONCLUSIONS: We identified CD81 as a novel surface marker that labels immature, stressed, and dedifferentiated ß-cells in the adult mouse and human islets. This novel surface marker will allow us to better study ß-cell heterogeneity in healthy subjects and diabetes progression.

Diet-induced alteration of intestinal stem cell function underlies obesity and prediabetes in mice.

Excess nutrient uptake and altered hormone secretion in the gut contribute to a systemic energy imbalance, which causes obesity and an increased risk of type 2 diabetes and colorectal cancer. This functional maladaptation is thought to emerge at the level of the intestinal stem cells (ISCs). However, it is not clear how an obesogenic diet affects ISC identity and fate. Here we show that an obesogenic diet induces ISC and progenitor hyperproliferation, enhances ISC differentiation and cell turnover and changes the regional identities of ISCs and enterocytes in mice. Single-cell resolution of the enteroendocrine lineage reveals an increase in progenitors and peptidergic enteroendocrine cell types and a decrease in serotonergic enteroendocrine cell types. Mechanistically, we link increased fatty acid synthesis, Ppar signaling and the Insr-Igf1r-Akt pathway to mucosal changes. This study describes molecular mechanisms of diet-induced intestinal maladaptation that promote obesity and therefore underlie the pathogenesis of the metabolic syndrome and associated complications.

Non-canonical Wnt/PCP signalling regulates intestinal stem cell lineage priming towards enteroendocrine and Paneth cell fates.

A detailed understanding of intestinal stem cell (ISC) self-renewal and differentiation is required to treat chronic intestinal diseases. However, the different models of ISC lineage hierarchy1-6 and segregation7-12 are subject to debate. Here, we have discovered non-canonical Wnt/planar cell polarity (PCP)-activated ISCs that are primed towards the enteroendocrine or Paneth cell lineage. Strikingly, integration of time-resolved lineage labelling with single-cell gene expression analysis revealed that both lineages are directly recruited from ISCs via unipotent transition states, challenging the existence of formerly predicted bi- or multipotent secretory progenitors7-12. Transitory cells that mature into Paneth cells are quiescent and express both stem cell and secretory lineage genes, indicating that these cells are the previously described Lgr5+ label-retaining cells7. Finally, Wnt/PCP-activated Lgr5+ ISCs are molecularly indistinguishable from Wnt/ß-catenin-activated Lgr5+ ISCs, suggesting that lineage priming and cell-cycle exit is triggered at the post-transcriptional level by polarity cues and a switch from canonical to non-canonical Wnt/PCP signalling. Taken together, we redefine the mechanisms underlying ISC lineage hierarchy and identify the Wnt/PCP pathway as a new niche signal preceding lateral inhibition in ISC lineage priming and segregation.

Asc-1 regulates white versus beige adipocyte fate in a subcutaneous stromal cell population.

Adipose tissue expansion, as seen in obesity, is often metabolically detrimental causing insulin resistance and the metabolic syndrome. However, white adipose tissue expansion at early ages is essential to establish a functional metabolism. To understand the differences between adolescent and adult adipose tissue expansion, we studied the cellular composition of the stromal vascular fraction of subcutaneous adipose tissue of two and eight weeks old mice using single cell RNA sequencing. We identified a subset of adolescent preadipocytes expressing the mature white adipocyte marker Asc-1 that showed a low ability to differentiate into beige adipocytes compared to Asc-1 negative cells in vitro. Loss of Asc-1 in subcutaneous preadipocytes resulted in spontaneous differentiation of beige adipocytes in vitro and in vivo. Mechanistically, this was mediated by a function of the amino acid transporter ASC-1 specifically in proliferating preadipocytes involving the intracellular accumulation of the ASC-1 cargo D-serine.

Identification and characterization of distinct brown adipocyte subtypes in C57BL/6J mice.

Brown adipose tissue (BAT) plays an important role in the regulation of body weight and glucose homeostasis. Although increasing evidence supports white adipose tissue heterogeneity, little is known about heterogeneity within murine BAT. Recently, UCP1 high and low expressing brown adipocytes were identified, but a developmental origin of these subtypes has not been studied. To obtain more insights into brown preadipocyte heterogeneity, we use single-cell RNA sequencing of the BAT stromal vascular fraction of C57/BL6 mice and characterize brown preadipocyte and adipocyte clonal cell lines. Statistical analysis of gene expression profiles from brown preadipocyte and adipocyte clones identify markers distinguishing brown adipocyte subtypes. We confirm the presence of distinct brown adipocyte populations in vivo using the markers EIF5, TCF25, and BIN1. We also demonstrate that loss of Bin1 enhances UCP1 expression and mitochondrial respiration, suggesting that BIN1 marks dormant brown adipocytes. The existence of multiple brown adipocyte subtypes suggests distinct functional properties of BAT depending on its cellular composition, with potentially distinct functions in thermogenesis and the regulation of whole body energy homeostasis.

Epithelial Planar Bipolarity Emerges from Notch-Mediated Asymmetric Inhibition of Emx2.

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.

Targeted pharmacological therapy restores ß-cell function for diabetes remission.

Dedifferentiation of insulin-secreting ß cells in the islets of Langerhans has been proposed to be a major mechanism of ß-cell dysfunction. Whether dedifferentiated ß cells can be targeted by pharmacological intervention for diabetes remission, and ways in which this could be accomplished, are unknown as yet. Here we report the use of streptozotocin-induced diabetes to study ß-cell dedifferentiation in mice. Single-cell RNA sequencing (scRNA-seq) of islets identified markers and pathways associated with ß-cell dedifferentiation and dysfunction. Single and combinatorial pharmacology further show that insulin treatment triggers insulin receptor pathway activation in ß cells and restores maturation and function for diabetes remission. Additional ß-cell selective delivery of oestrogen by Glucagon-like peptide-1 (GLP-1-oestrogen conjugate) decreases daily insulin requirements by 60%, triggers oestrogen-specific activation of the endoplasmic-reticulum-associated protein degradation system, and further increases ß-cell survival and regeneration. GLP-1-oestrogen also protects human ß cells against cytokine-induced dysfunction. This study not only describes mechanisms of ß-cell dedifferentiation and regeneration, but also reveals pharmacological entry points to target dedifferentiated ß cells for diabetes remission.

Modelling the endocrine pancreas in health and disease.

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 ß-cells, but the pathomechanisms underlying ß-cell failure in type 1 diabetes mellitus and type 2 diabetes mellitus are still poorly defined. Regeneration of ß-cell mass from residual islet cells or replacement by ß-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 ß-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.

Point mutations in the PDX1 transactivation domain impair human ß-cell development and function.

OBJECTIVE: Hundreds of missense mutations in the coding region of PDX1 exist; however, if these mutations predispose to diabetes mellitus is unknown. METHODS: In this study, we screened a large cohort of subjects with increased risk for diabetes and identified two subjects with impaired glucose tolerance carrying common, heterozygous, missense mutations in the PDX1 coding region leading to single amino acid exchanges (P33T, C18R) in its transactivation domain. We generated iPSCs from patients with heterozygous PDX1P33T/+, PDX1C18R/+ mutations and engineered isogenic cell lines carrying homozygous PDX1P33T/P33T, PDX1C18R/C18R mutations and a heterozygous PDX1 loss-of-function mutation (PDX1+/-). RESULTS: Using an in vitro ß-cell differentiation protocol, we demonstrated that both, heterozygous PDX1P33T/+, PDX1C18R/+ and homozygous PDX1P33T/P33T, PDX1C18R/C18R mutations impair ß-cell differentiation and function. Furthermore, PDX1+/- and PDX1P33T/P33T mutations reduced differentiation efficiency of pancreatic progenitors (PPs), due to downregulation of PDX1-bound genes, including transcription factors MNX1 and PDX1 as well as insulin resistance gene CES1. Additionally, both PDX1P33T/+ and PDX1P33T/P33T mutations in PPs reduced the expression of PDX1-bound genes including the long-noncoding RNA, MEG3 and the imprinted gene NNAT, both involved in insulin synthesis and secretion. CONCLUSIONS: Our results reveal mechanistic details of how common coding mutations in PDX1 impair human pancreatic endocrine lineage formation and ß-cell function and contribute to the predisposition for diabetes.