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Hox genes play key roles in the anterior-posterior (AP) specification of all 3 germ layers during different developmental stages. It is only partially understood how they function in widely different developmental contexts, particularly with regards to extracellular signaling, and to what extent their function can be harnessed to guide cell specification in vitro. Here, we addressed the role of Hoxb1 in 2 distinct developmental contexts; in mouse embryonic stem cells (mES)-derived neuromesodermal progenitors (NMPs) and hindbrain neural progenitors. We found that Hoxb1 promotes NMP survival through the upregulation of Fgf8, Fgf17, and other components of Fgf signaling as well as the repression of components of the apoptotic pathway. Additionally, it upregulates other anterior Hox genes suggesting that it plays an active role in the early steps of AP specification. In neural progenitors, Hoxb1 synergizes with shh to repress anterior and dorsal neural markers, promote the expression of ventral neural markers and direct the specification of facial branchiomotorneuron (FBM)-like progenitors. Hoxb1 and shh synergize in regulating the expression of diverse signals and signaling molecules, including the Ret tyrosine kinase receptor. Finally, Hoxb1 synergizes with exogenous Glial cell line-derived neurotrophic factor (GDNF) to strengthen Ret expression and further promote the generation of FBM-like progenitors. Facial branchiomotorneuron-like progenitors survived for at least 6 months and differentiated into postmitotic neurons after orthotopic transplantation near the facial nucleus of adult mice. These results suggested that the patterning activity of Hox genes in combination with downstream signaling molecules can be harnessed for the generation of defined neural populations and transplantations with implications for neurodegenerative diseases.

The decline of brain function during aging is associated with epigenetic changes, including DNA methylation. Lifestyle interventions can improve brain function during aging, but their influence on age-related epigenetic changes is unknown. Using genome-wide DNA methylation sequencing, we here show that experiencing a stimulus-rich environment counteracts age-related DNA methylation changes in the hippocampal dentate gyrus of mice. Specifically, environmental enrichment prevented the aging-induced CpG hypomethylation at target sites of the methyl-CpG-binding protein Mecp2, which is critical to neuronal function. The genes at which environmental enrichment counteracted aging effects have described roles in neuronal plasticity, neuronal cell communication and adult hippocampal neurogenesis and are dysregulated with age-related cognitive decline in the human brain. Our results highlight the stimulating effects of environmental enrichment on hippocampal plasticity at the level of DNA methylation and give molecular insights into the specific aspects of brain aging that can be counteracted by lifestyle interventions., Decline of brain function during aging is associated with epigenetic changes, including DNA methylation. Here the authors provide evidence that environmental enrichment delays age-related DNA methylation alterations in the mouse hippocampus.

Cell-intrinsic response patterns control risks arising from genome-damage, preventing malignant transformation. The DNA sensor cyclic-GMP-AMP synthase (cGAS) has emerged as a new principle detecting genome damage, as it can be triggered by aberrant self-DNA. Stimulator of interferon genes (STING)-activation downstream of cGAS can drive cells into senescence or cell death and induces antiproliferative type I interferon (IFN) and pro-apoptotic tumor necrosis factor responses. Herein, we investigated how DNA damage-driven activation of cGAS/STING signaling impacts on hematopoiesis. Defective ribonucleotide excision repair (RER) in the hematopoietic system caused chromosomal instability as well as robust activation of the cGAS/STING/IFN axis, and compromised hematopoietic stem cell function, resulting in cytopenia and ultimately leukemia. Whereas loss of p53 largely rescued RER-deficient hematopoiesis at the cost of further accelerated leukemogenesis, the additional inactivation of cGAS, STING or type I IFN signaling had no detectable effect on blood cell generation and leukemia development. Moreover, cGAS-deficient hematopoiesis showed unaltered responses to spontaneous or acute DNA damage. Our data demonstrate that the cGAS/STING pathway is dispensable for the hematopoietic system coping with chronic or acute DNA damage and does not protect against leukemic transformation in the absence of RER.

BACKGROUND AND AIMS: NAFLD is initiated by steatosis and can progress through fibrosis and cirrhosis to HCC. The RNA binding protein human antigen R (HuR) controls RNAs at the posttranscriptional level; hepatocyte HuR has been implicated in the regulation of diet-induced hepatic steatosis. The present study aimed to understand the role of hepatocyte HuR in NAFLD development and progression to fibrosis and HCC. APPROACH AND RESULTS: Hepatocyte-specific, HuR-deficient mice and control HuR-sufficient mice were fed either a normal diet or an NAFLD-inducing diet. Hepatic lipid accumulation, inflammation, fibrosis, and HCC development were studied by histology, flow cytometry, quantitative PCR, and RNA sequencing. The liver lipidome was characterized by lipidomics analysis, and the HuR-RNA interactions in the liver were mapped by RNA immunoprecipitation sequencing. Hepatocyte-specific, HuR-deficient mice displayed spontaneous hepatic steatosis and fibrosis predisposition compared to control HuR-sufficient mice. On an NAFLD-inducing diet, hepatocyte-specific HuR deficiency resulted in exacerbated inflammation, fibrosis, and HCC-like tumor development. A multi-omic approach, including lipidomics, transcriptomics, and RNA immunoprecipitation sequencing revealed that HuR orchestrates a protective network of hepatic-metabolic and lipid homeostasis-maintaining pathways. Consistently, HuR-deficient livers accumulated, already at steady state, a triglyceride signature resembling that of NAFLD livers. Moreover, up-regulation of secreted phosphoprotein 1 expression mediated, at least partially, fibrosis development in hepatocyte-specific HuR deficiency on an NAFLD-inducing diet, as shown by experiments using antibody blockade of osteopontin. CONCLUSIONS: HuR is a gatekeeper of liver homeostasis, preventing NAFLD-related fibrosis and HCC, suggesting that the HuR-dependent network could be exploited therapeutically., Hepatology, 75 (4), ISSN:0270-9139, ISSN:1527-3350

Resting conventional T cells (Tconv) can be distinguished from T regulatory cells (Treg) by the canonical markers FOXP3, CD25 and CD127. However, the expression of these proteins alters after T-cell activation leading to overlap between Tconv and Treg. The objective of this study was to distinguish resting and antigen-responsive T effector (Tconv) and Treg using single-cell technologies. CD4+ Treg and Tconv cells were stimulated with antigen and responsive and non-responsive populations processed for targeted and non-targeted single-cell RNAseq. Machine learning was used to generate a limited set of genes that could distinguish responding and non-responding Treg and Tconv cells and which was used for single-cell multiplex qPCR and to design a flow cytometry panel. Targeted scRNAseq clearly distinguished the four-cell populations. A minimal set of 27genes was identified by machine learning algorithms to provide discrimination of the four populations at >95% accuracy. In all, 15 of the genes were validated to be differentially expressed by single-cell multiplex qPCR. Discrimination of responding Treg from responding Tconv could be achieved by a flow cytometry strategy that included staining for CD25, CD127, FOXP3, IKZF2, ITGA4, and the novel marker TRIM which was strongly expressed in Tconv and weakly expressed in both responding and non-responding Treg. A minimal set of genes was identified that discriminates responding and non-responding CD4+ Treg and Tconv cells and, which have identified TRIM as a marker to distinguish Treg by flow cytometry.

Most research on human pancreatic islets is conducted on samples obtained from normoglycaemic or diseased brain-dead donors and thus cannot accurately describe the molecular changes of pancreatic islet beta cells as they progress towards a state of deficient insulin secretion in type 2 diabetes (T2D). Here, we conduct a comprehensive multi-omics analysis of pancreatic islets obtained from metabolically profiled pancreatectomized living human donors stratified along the glycemic continuum, from normoglycemia to T2D. We find that islet pools isolated from surgical samples by laser-capture microdissection display remarkably more heterogeneous transcriptomic and proteomic profiles in patients with diabetes than in non-diabetic controls. The differential regulation of islet gene expression is already observed in prediabetic individuals with impaired glucose tolerance. Our findings demonstrate a progressive, but disharmonic, remodelling of mature beta cells, challenging current hypotheses of linear trajectories toward precursor or transdifferentiation stages in T2D. Furthermore, through integration of islet transcriptomics with preoperative blood plasma lipidomics, we define the relative importance of gene coexpression modules and lipids that are positively or negatively associated with HbA1c levels, pointing to potential prognostic markers. Wigger, Barovic and Brunner et al. perform a multidimensional analysis of islets from metabolically characterized patients who had undergone pancreatectomy, observing remarkable heterogeneity between samples from individuals with type 2 diabetes, thus arguing against models of linear beta-cell dedifferentiation in diabetes.

Cancer cells can switch between signaling pathways to regulate growth under different conditions. In the tumor microenvironment, this likely helps them evade therapies that target specific pathways. We must identify all possible states and utilize them in drug screening programs. One such state is characterized by expression of the transcription factor Hairy and Enhancer of Split 3 (HES3) and sensitivity to HES3 knockdown, and it can be modeled in vitro. Here, we cultured 3 primary human brain cancer cell lines under 3 different culture conditions that maintain low, medium, and high HES3 expression and characterized gene regulation and mechanical phenotype in these states. We assessed gene expression regulation following HES3 knockdown in the HES3-high conditions. We then employed a commonly used human brain tumor cell line to screen Food and Drug Administration (FDA)-approved compounds that specifically target the HES3-high state. We report that cells from multiple patients behave similarly when placed under distinct culture conditions. We identified 37 FDA-approved compounds that specifically kill cancer cells in the high-HES3-expression conditions. Our work reveals a novel signaling state in cancer, biomarkers, a strategy to identify treatments against it, and a set of putative drugs for potential repurposing.-Poser, S. W., Otto, O., Arps-Forker, C., Ge, Y., Herbig, M., Andree, C., Gruetzmann, K., Adasme, M. F., Stodolak, S., Nikolakopoulou, P., Park, D. M., Mcintyre, A., Lesche, M., Dahl, A., Lennig, P., Bornstein, S. R., Schroeck, E., Klink, B., Leker, R. R., Bickle, M., Chrousos, G. P., Schroeder, M., Cannistraci, C. V., Guck, J., Androutsellis-Theotokis, A. Controlling distinct signaling states in cultured cancer cells provides a new platform for drug discovery.

The adrenal gland is a key regulator during stress, and a high degree of plasticity is critical for sustaining homeostasis. Previously, we have shown that this is achieved in part through adult Nestin(+) progenitors located in both the adrenal cortex and medulla. To obtain a comprehensive characterization of these cells, we performed transcriptomics on isolated Nestin(+) cells from the two adrenal tissues. This analysis revealed that both populations displayed Schwann cell precursor properties suggesting a common neural crest origin, though the two populations showed distinct characteristics. Adrenomedullary Nestin(+) cells showed a commitment to the neural/glial lineage, while adrenocortical Nestin(+) cells showed a predestination to become steroid-producing cells. The data suggest that adult adrenal Schwann cell precursors might fulfill a role of coordinated structural tissue remodeling under stress answering the question why two completely different organs are united under one organ capsule.

Existing studies do not sufficiently describe the molecular changes of pancreatic islet beta cells leading to their deficient insulin secretion in type 2 diabetes (T2D). Here we address this deficiency with a comprehensive multi-omics analysis of metabolically profiled pancreatectomized living human donors stratified along the glycemic continuum from normoglycemia to T2D. Islet pools isolated from surgical samples by laser-capture microdissection had remarkably heterogeneous transcriptomic and proteomic profiles in diabetics, but not in non-diabetic controls. Transcriptomics analysis of this unique cohort revealed islet genes already dysregulated in prediabetic individuals with impaired glucose tolerance. Our findings demonstrate a progressive but disharmonic remodeling of mature beta cells, challenging current hypotheses of linear trajectories toward precursor or trans-differentiation stages in T2D. Further, integration of islet transcriptomics and pre-operative blood plasma lipidomics data enabled us to define the relative importance of gene co-expression modules and lipids positively or negatively associated with HbA1c levels, pointing to potential prognostic markers.

Selective isolation of DNA is crucial for applications in biology, bionanotechnology, clinical diagnostics and forensics. We herein report a smart methanol-responsive polymer (MeRPy) that can be programmed to bind and separate single- as well as double-stranded DNA targets. Captured targets are quickly isolated and released back into solution by denaturation (sequence-agnostic) or toehold-mediated strand displacement (sequence-selective). The latter mode allows 99.8% efficient removal of unwanted sequences and 79% recovery of highly pure target sequences. We applied MeRPy for the depletion of insulin, glucagon, and transthyretin cDNA from clinical next-generation sequencing (NGS) libraries. This step improved the data quality for low-abundance transcripts in expression profiles of pancreatic tissues. Its low cost, scalability, high stability and ease of use make MeRPy suitable for diverse applications in research and clinical laboratories, including enhancement of NGS libraries, extraction of DNA from biological samples, preparative-scale DNA isolations, and sorting of DNA-labeled non-nucleic acid targets., Krieg et al. describe a methanol responsive polymer that can capture complementary DNA using grafted oligonucleotides. They successfully demonstrate its efficacy with simultaneous and sequence-specific isolation of three target genes (cDNA) from clinical NGS libraries with high efficiency. This method is fast, effective, scalable, modular, and versatile.

In mammals, perivascular cell-derived scarring after spinal cord injury impedes axonal regrowth. In contrast, the extracellular matrix (ECM) in the spinal lesion site of zebrafish is permissive and required for axon regeneration. However, the cellular mechanisms underlying this interspecies difference have not been investigated. Here, we show that an injury to the zebrafish spinal cord triggers recruitment of pdgfrb

Objective The intestinal epithelium is a rapidly renewing tissue which plays central roles in nutrient uptake, barrier function and the prevention of intestinal inflammation. Control of epithelial differentiation is essential to these processes and is dependent on cell type-specific activity of transcription factors which bind to accessible chromatin. Here, we studied the role of SET Domain Bifurcated Histone Lysine Methyltransferase 1, also known as ESET (SETDB1), a histone H3K9 methyltransferase, in intestinal epithelial homeostasis and IBD. Design We investigated mice with constitutive and inducible intestinal epithelial deletion of Setdb1, studied the expression of SETDB1 in patients with IBD and mouse models of IBD, and investigated the abundance of SETDB1 variants in healthy individuals and patients with IBD. Results Deletion of intestinal epithelial Setdb1 in mice was associated with defects in intestinal epithelial differentiation, barrier disruption, inflammation and mortality. Mechanistic studies showed that loss of SETDB1 leads to de-silencing of endogenous retroviruses, DNA damage and intestinal epithelial cell death. Predicted loss-of-function variants in human SETDB1 were considerably less frequently observed than expected, consistent with a critical role of SETDB1 in human biology. While the vast majority of patients with IBD showed unimpaired mucosal SETDB1 expression, comparison of IBD and non-IBD exomes revealed over-representation of individual rare missense variants in SETDB1 in IBD, some of which are predicted to be associated with loss of function and may contribute to the pathogenesis of intestinal inflammation. Conclusion SETDB1 plays an essential role in intestinal epithelial homeostasis. Future work is required to investigate whether rare variants in SETDB1 contribute to the pathogenesis of IBD.

MicroRNAs (miRNAs) are short (∼22 nt) single-stranded non-coding RNAs that regulate gene expression at the post-transcriptional level. Over recent years, many studies have extensively characterized the involvement of miRNA-mediated regulation in neurogenesis and brain development. However, a comprehensive catalog of cortical miRNAs expressed in a cell-specific manner in progenitor types of the developing mammalian cortex is still missing. Overcoming this limitation, here we exploited a double reporter mouse line previously validated by our group to allow the identification of the transcriptional signature of neurogenic commitment and provide the field with the complete atlas of miRNA expression in proliferating neural stem cells, neurogenic progenitors and newborn neurons during corticogenesis. By extending the currently known list of miRNAs expressed in the mouse brain by over twofold, our study highlights the power of cell type-specific analyses for the detection of transcripts that would otherwise be diluted out when studying bulk tissues. We further exploited our data by predicting putative miRNAs and validated the power of our approach by providing evidence for the involvement of miR-486 in brain development.

Summary Trained innate immunity, induced via modulation of mature myeloid cells or their bone marrow progenitors, mediates sustained increased responsiveness to secondary challenges. Here, we investigated whether anti-tumor immunity can be enhanced through induction of trained immunity. Pre-treatment of mice with β-glucan, a fungal-derived prototypical agonist of trained immunity, resulted in diminished tumor growth. The anti-tumor effect of β-glucan-induced trained immunity was associated with transcriptomic and epigenetic rewiring of granulopoiesis and neutrophil reprogramming toward an anti-tumor phenotype; this process required type I interferon signaling irrespective of adaptive immunity in the host. Adoptive transfer of neutrophils from β-glucan-trained mice to naive recipients suppressed tumor growth in the latter in a ROS-dependent manner. Moreover, the anti-tumor effect of β-glucan-induced trained granulopoiesis was transmissible by bone marrow transplantation to recipient naive mice. Our findings identify a novel and therapeutically relevant anti-tumor facet of trained immunity involving appropriate rewiring of granulopoiesis., Graphical Abstract, Highlights • Trained innate immunity (TII) promotes anti-tumor activity • TII is linked to transcriptomic and epigenetic rewiring of granulopoiesis • Trained granulopoiesis promotes an anti-tumor phenotype in neutrophils • Trained granulopoiesis might bear potential for cancer immunotherapy, Pre-treatment of mice with β-glucan, an agonist of trained immunity responses, leads to epigenetic changes in granulopoiesis and neutrophil function to drive anti-tumor immune responses.

MicroRNAs (miRNAs) are short (∼22 nt) single-stranded non-coding RNAs that regulate gene expression at the post-transcriptional level. Over the past years, many studies have extensively characterized the involvement of miRNA-mediated regulation in neurogenesis and brain development. However, a comprehensive catalog of cortical miRNAs cell-specifically expressed in progenitor types of the developing mammalian cortex is still missing. Overcoming this limitation, here we exploited a double reporter mouse line previously validated by our group to allow the identification of the transcriptional signature to neurogenic commitment and provide the field with the complete atlas of miRNAs expression in proliferating neural stem cells, neurogenic progenitors and newborn neurons during corticogenesis. By extending the currently known list of miRNAs expressed in the mouse brain by over two fold, our study highlights the power of cell type-specific analyses for the detection of transcripts that would otherwise be diluted out when studying bulk tissues. We further exploited our data by predicting putative novel miRNAs and validated the power of our approach by providing novel evidence for the involvement of miR-486 as a novel player in brain development.

The presence of progenitor or stem cells in the adult pancreas and their potential involvement in homeostasis and cancer development remain unresolved issues. Here, we show that mouse centroacinar cells can be identified and isolated by virtue of the mitochondrial enzyme Aldh1b1 that they uniquely express. These cells are necessary and sufficient for the formation of self-renewing adult pancreatic organoids in an Aldh1b1-dependent manner. Aldh1b1-expressing centroacinar cells are largely quiescent, self-renew, and, as shown by genetic lineage tracing, contribute to all 3 pancreatic lineages in the adult organ under homeostatic conditions. Single-cell RNA sequencing analysis of these cells identified a progenitor cell population, established its molecular signature, and determined distinct differentiation pathways to early progenitors. A distinct feature of these progenitor cells is the preferential expression of small GTPases, including Kras, suggesting that they might be susceptible to Kras-driven oncogenic transformation. This finding and the overexpression of Aldh1b1 in human and mouse pancreatic cancers, driven by activated Kras, prompted us to examine the involvement of Aldh1b1 in oncogenesis. We demonstrated genetically that ablation of Aldh1b1 completely abrogates tumor development in a mouse model of Kras G12D -induced pancreatic cancer.

The decline of brain function during aging is associated with epigenetic changes, including DNA methylation. Lifestyle interventions can improve brain function during aging, but their influence on age-related epigenetic changes is unknown. Using genome-wide DNA methylation sequencing, we here show that environmental enrichment counteracted age-related DNA methylation changes in the hippocampal dentate gyrus of mice. Specifically, environmental enrichment prevented the aging-induced CpG hypomethylation at target sites of the methyl-CpG-binding protein Mecp2, which is known to control neuronal functions. The genes at which environmental enrichment counteracted aging effects have described roles in neuronal plasticity, neuronal cell communication and adult hippocampal neurogenesis and are dysregulated with age-related cognitive decline in the human brain. Our results highlight the rejuvenating effects of environmental enrichment at the level of DNA methylation and give molecular insights into the specific aspects of brain aging that can be counteracted by lifestyle interventions.

Synthesis of renin in renal renin-producing cells (RPCs) is controlled via the intracellular messenger cAMP. Interference with cAMP-mediated signaling by inducible knockout of Gs-alpha (Gsα) in RPCs of adult mice resulted in a complex adverse kidney phenotype. Therein, glomerular endothelial damage was most striking. In this study, we investigated whether Gsα knockout leads to a loss of RPCs, which itself may contribute to the endothelial injury. We compared the kidney phenotype of three RPC-specific conditional mouse lines during continuous induction of recombination. Mice expressing red fluorescent reporter protein tdTomato (tdT) in RPCs served as controls. tdT was also expressed in RPCs of the other two strains used, namely with RPC-specific Gsα knockout (Gsα mice) or with RPC-specific diphtheria toxin A expression (DTA mice, in which the RPCs should be diminished). Using immunohistological analysis, we found that RPCs decreased by 82% in the kidneys of Gsα mice as compared with controls. However, the number of tdT-positive cells was similar in the two strains, demonstrating that after Gsα knockout, the RPCs persist as renin-negative descendants. In contrast, both renin-positive and tdT-labeled cells decreased by 80% in DTA mice suggesting effective RPC ablation. Only Gsα mice displayed dysregulated endothelial cell marker expression indicating glomerular endothelial damage. In addition, a robust induction of genes involved in tissue remodelling with microvascular damage was identified in tdT-labeled RPCs isolated from Gsα mice. We concluded that Gsα/renin double-negative RPC progeny essentially contributes for the development of glomerular endothelial damage in our Gsα-deficient mice.

Understanding the mechanisms that promote the specification of pancreas progenitors and regulate their self‐renewal and differentiation will help to maintain and expand pancreas progenitor cells derived from human pluripotent stem (hPS) cells. This will improve the efficiency of current differentiation protocols of hPS cells into β‐cells and bring such cells closer to clinical applications for the therapy of diabetes. Aldehyde dehydrogenase 1b1 (Aldh1b1) is a mitochondrial enzyme expressed specifically in progenitor cells during mouse pancreas development, and we have shown that its functional inactivation leads to accelerated differentiation and deficient β‐cells. In this report, we aimed to identify small molecule inducers of Aldh1b1 expression taking advantage of a mouse embryonic stem (mES) cell Aldh1b1 lacZ reporter line and a pancreas differentiation protocol directing mES cells into pancreatic progenitors. We identified AMI‐5, a protein methyltransferase inhibitor, as an Aldh1b1 inducer and showed that it can maintain Aldh1b1 expression in embryonic pancreas explants. This led to a selective reduction in endocrine specification. This effect was due to a downregulation of Ngn3, and it was mediated through Aldh1b1 since the effect was abolished in Aldh1b1 null pancreata. The findings implicated methyltransferase activity in the regulation of endocrine differentiation and showed that methyltransferases can act through specific regulators during pancreas differentiation. Stem Cells 2019;37:640–651, A screen using small molecules and mouse embryonic stem cell derived pancreatic progenitors reveals that protein methyltransferases inhibit aldehyde dehydrogenase 1b1 expression. Aldehyde dehydrogenase 1b1 maintains pancreas progenitors and maintenance of its expression through protein methyltransferase inhibition delays selectively endocrine differentiation.

Summary Trained innate immunity fosters a sustained favorable response of myeloid cells to a secondary challenge, despite their short lifespan in circulation. We thus hypothesized that trained immunity acts via modulation of hematopoietic stem and progenitor cells (HSPCs). Administration of β-glucan (prototypical trained-immunity-inducing agonist) to mice induced expansion of progenitors of the myeloid lineage, which was associated with elevated signaling by innate immune mediators, such as IL-1β and granulocyte-macrophage colony-stimulating factor (GM-CSF), and with adaptations in glucose metabolism and cholesterol biosynthesis. The trained-immunity-related increase in myelopoiesis resulted in a beneficial response to secondary LPS challenge and protection from chemotherapy-induced myelosuppression in mice. Therefore, modulation of myeloid progenitors in the bone marrow is an integral component of trained immunity, which to date, was considered to involve functional changes of mature myeloid cells in the periphery., Graphical Abstract, Highlights • Trained immunity (TI) modulates hematopoietic progenitors in bone marrow • TI is associated with adaptations in cell metabolism in progenitors • TI increases expansion of hematopoietic progenitors and myelopoiesis • TI promotes beneficial responses to systemic inflammation and chemotherapy, Modulation of hematopoietic stem and progenitor cells during trained immunity allows a sustained response of myeloid cells to a secondary challenge despite their short lifespan in circulation.

Aims/hypothesis Pancreatic islet beta cell failure causes type 2 diabetes in humans. To identify transcriptomic changes in type 2 diabetic islets, the Innovative Medicines Initiative for Diabetes: Improving beta-cell function and identification of diagnostic biomarkers for treatment monitoring in Diabetes (IMIDIA) consortium (www.imidia.org) established a comprehensive, unique multicentre biobank of human islets and pancreas tissues from organ donors and metabolically phenotyped pancreatectomised patients (PPP). Methods Affymetrix microarrays were used to assess the islet transcriptome of islets isolated either by enzymatic digestion from 103 organ donors (OD), including 84 non-diabetic and 19 type 2 diabetic individuals, or by laser capture microdissection (LCM) from surgical specimens of 103 PPP, including 32 non-diabetic, 36 with type 2 diabetes, 15 with impaired glucose tolerance (IGT) and 20 with recent-onset diabetes (

Summary The hematopoietic stem cell (HSC) compartment consists of a small pool of cells capable of replenishing all blood cells. Although it is established that the hematopoietic system is assembled as a hierarchical organization under steady-state conditions, emerging evidence suggests that distinct differentiation pathways may exist in response to acute stress. However, it remains unclear how different hematopoietic stem and progenitor cell subpopulations behave under sustained chronic stress. Here, by using adult transgenic mice overexpressing erythropoietin (EPO; Tg6) and a combination of in vivo, in vitro, and deep-sequencing approaches, we found that HSCs respond differentially to chronic erythroid stress compared with their closely related multipotent progenitors (MPPs). Specifically, HSCs exhibit a vastly committed erythroid progenitor profile with enhanced cell division, while MPPs display erythroid and myeloid cell signatures and an accumulation of uncommitted cells. Thus, our results identify HSCs as master regulators of chronic stress erythropoiesis, potentially circumventing the hierarchical differentiation-detour., Highlights • Chronic high EPO drives only HSCs to exhibit an exclusive erythroid progenitor profile • MPPs accumulate as uncommitted cells and display differential signatures, In this article, Wielockx and colleagues reveal that chronic erythropoietic stress induces distinct differences in the behavior of HSCs and their closely related MPPs. In particular, HSCs show greater commitment to an erythroid progenitor profile with heightened cell division, whereas MPPs are characterized by erythroid and immune signatures and an accumulation of uncommitted cells.

During development, progenitor expansion, lineage allocation, and implementation of differentiation programs need to be tightly coordinated so that different cell types are generated in the correct numbers for appropriate tissue size and function. Pancreatic dysfunction results in some of the most debilitating and fatal diseases, including pancreatic cancer and diabetes. Several transcription factors regulating pancreas lineage specification have been identified, and Notch signalling has been implicated in lineage allocation, but it remains unclear how these processes are coordinated. Using a combination of genetic approaches, organotypic cultures of embryonic pancreata, and genomics, we found that sphingosine-1-phosphate (S1p), signalling through the G protein coupled receptor (GPCR) S1pr2, plays a key role in pancreas development linking lineage allocation and specification. S1pr2 signalling promotes progenitor survival as well as acinar and endocrine specification. S1pr2-mediated stabilisation of the yes-associated protein (YAP) is essential for endocrine specification, thus linking a regulator of progenitor growth with specification. YAP stabilisation and endocrine cell specification rely on Gαi subunits, revealing an unexpected specificity of selected GPCR intracellular signalling components. Finally, we found that S1pr2 signalling posttranscriptionally attenuates Notch signalling levels, thus regulating lineage allocation. Both S1pr2-mediated YAP stabilisation and Notch attenuation are necessary for the specification of the endocrine lineage. These findings identify S1p signalling as a novel key pathway coordinating cell survival, lineage allocation, and specification and linking these processes by regulating YAP levels and Notch signalling. Understanding lineage allocation and specification in the pancreas will shed light in the origins of pancreatic diseases and may suggest novel therapeutic approaches., Author summary The pancreas develops from a field of progenitor cells localised in a restricted region of the embryonic endoderm. These progenitor cells proliferate and eventually differentiate to generate the three distinct lineages comprising the endocrine (which include insulin-producing β cells), acinar, and ductal cells. The molecular pathways implicated in the early generation of pancreas progenitors and their proliferation are well understood. It is also known that the Notch signalling pathway is implicated in sequential binary cell fate decisions that generate the three lineages; however, other signals that may regulate this process remain unknown. Here, we show that a phospholipid, sphingosine-1-phosphate (S1p), generated by the progenitor cells themselves, acts as a signal necessary to define the acinar and endocrine lineage. We observe that in the absence of S1p only duct cells are generated and the survival of pancreas progenitors is compromised. The function of this signalling pathway in the generation of the endocrine cells is two-fold. Firstly, it stabilises yes-associated protein (YAP), a transcriptional gene coactivator known to regulate pancreatic progenitor proliferation, and we show that this stability is necessary for the activation of the endocrine specification program. Secondly, it attenuates Notch signalling, allowing the generation of endocrine and acinar cells. Notch attenuation is necessary for the stabilisation of the transcription factor Ngn3, which is required for the generation of endocrine cells. We conclude that S1p acts as an autocrine signal regulating YAP stabilisation and Notch attenuation to mediate pancreas specification. Understanding lineage allocation and specification in the pancreas will shed light on the origins of pancreatic diseases and may suggest novel therapeutic approaches.

This study identifies circular RNAs (circRNAs) in specific cell types of the developing mammalian cortex. This resource addresses the complexity and potential significance of this elusive class of transcripts., Circular (circ) RNAs have recently emerged as a novel class of transcripts whose identification and function remain elusive. Among many tissues and species, the mammalian brain is the organ in which circRNAs are more abundant and first evidence of their functional significance started to emerge. Yet, even within this well-studied organ, annotation of circRNAs remains fragmentary, their sequence is unknown, and their expression in specific cell types was never investigated. Overcoming these limitations, here we provide the first comprehensive identification of circRNAs and assessment of their expression patterns in proliferating neural stem cells, neurogenic progenitors, and newborn neurons of the developing mouse cortex. Extending the current knowledge about the diversity of this class of transcripts by the identification of nearly 4,000 new circRNAs, our study is the first to provide the full sequence information and expression patterns of circRNAs in cell types representing the lineage of neurogenic commitment. We further exploited our data by evaluating the coding potential, evolutionary conservation, and biogenesis of circRNAs that we found to arise from a specific subclass of linear mRNAs. Our study provides the arising field of circRNA biology with a powerful new resource to address the complexity and potential biological significance of this new class of transcripts.

Transcriptome analysis of somatic stem cells and their progeny is fundamental to identify new factors controlling proliferation versus differentiation during tissue formation. Here, we generated a combinatorial, fluorescent reporter mouse line to isolate proliferating neural stem cells, differentiating progenitors and newborn neurons that coexist as intermingled cell populations during brain development. Transcriptome sequencing revealed numerous novel long non-coding (lnc)RNAs and uncharacterized protein-coding transcripts identifying the signature of neurogenic commitment. Importantly, most lncRNAs overlapped neurogenic genes and shared with them a nearly identical expression pattern suggesting that lncRNAs control corticogenesis by tuning the expression of nearby cell fate determinants. We assessed the power of our approach by manipulating lncRNAs and protein-coding transcripts with no function in corticogenesis reported to date. This led to several evident phenotypes in neurogenic commitment and neuronal survival, indicating that our study provides a remarkably high number of uncharacterized transcripts with hitherto unsuspected roles in brain development. Finally, we focussed on one lncRNA, Miat, whose manipulation was found to trigger pleiotropic effects on brain development and aberrant splicing of Wnt7b. Hence, our study suggests that lncRNA-mediated alternative splicing of cell fate determinants controls stem-cell commitment during neurogenesis.

Defects of the intracellular enzyme 3′ repair exonuclease 1 (Trex1) cause the rare autoimmune condition Aicardi–Goutières syndrome and are associated with systemic lupus erythematosus. Trex1−/− mice develop type I IFN–driven autoimmunity, resulting from activation of the cytoplasmic DNA sensor cyclic GMP–AMP synthase by a nucleic acid substrate of Trex1 that remains unknown. To identify cell types responsible for initiation of autoimmunity, we generated conditional Trex1 knockout mice. Loss of Trex1 in dendritic cells was sufficient to cause IFN release and autoimmunity, whereas Trex1-deficient keratinocytes and microglia produced IFN but did not induce inflammation. In contrast, B cells, cardiomyocytes, neurons, and astrocytes did not show any detectable response to the inactivation of Trex1. Thus, individual cell types differentially respond to the loss of Trex1, and Trex1 expression in dendritic cells is essential to prevent breakdown of self-tolerance ensuing from aberrant detection of endogenous DNA.

Long non-coding (lnc)RNAs play key roles in many biological processes. Elucidating the function of lncRNAs in cell type specification during organ development requires knowledge about their expression in individual progenitor types rather than in whole tissues. To achieve this during cortical development, we used a dual-reporter mouse line to isolate coexisting proliferating neural stem cells, differentiating neurogenic progenitors and newborn neurons and assessed the expression of lncRNAs by paired-end, high-throughput sequencing. We identified 379 genomic loci encoding novel lncRNAs and performed a comprehensive assessment of cell-specific expression patterns for all, annotated and novel, lncRNAs described to date. Our study provides a powerful new resource for studying these elusive transcripts during stem cell commitment and neurogenesis.

Chromatoid bodies (CBs) are spermiogenesis-specific organelles of largely unknown function. CBs harbor various RNA species, RNA-associated proteins and proteins of the tudor domain family like TDRD6, which is required for a proper CB architecture. Proteome analysis of purified CBs revealed components of the nonsense-mediated mRNA decay (NMD) machinery including UPF1. TDRD6 is essential for UPF1 localization to CBs, for UPF1-UPF2 and UPF1-MVH interactions. Upon removal of TDRD6, the association of several mRNAs with UPF1 and UPF2 is disturbed, and the long 3’ UTR-stimulated but not the downstream exon-exon junction triggered pathway of NMD is impaired. Reduced association of the long 3’ UTR mRNAs with UPF1 and UPF2 correlates with increased stability and enhanced translational activity. Thus, we identified TDRD6 within CBs as required for mRNA degradation, specifically the extended 3’ UTR-triggered NMD pathway, and provide evidence for the requirement of NMD in spermiogenesis. This function depends on TDRD6-promoted assembly of mRNA and decay enzymes in CBs., Author Summary Tudor-domain containing protein 6 (TDRD6) is a central component of the chromatoid body (CB) in male germ cells. Chromatoid bodies, which are present in spermatids, contain RNA and protein, are not enclosed by membranes, and typically reside close to the nucleus. Without TDRD6, a much distorted CB structure is observed, and this work asked for the functional contribution of TDRD6 to spermatids. We found that TDRD6 is required for localization of an RNA degradation machinery to the CB. This so-called nonsense mediated decay (NMD) machinery, known from somatic cells, destroys mRNAs that feature premature stop codons. Absence of TDRD6 significantly impairs one specific mechanism of NMD, which depends on long 3’ untranslated regions of the transcripts. Thus, the CB component TDRD6 acts in the assembly of the NMD machinery in the CB.

Maintenance of stem cell properties is associated with reduced proliferation but it is unknown whether the transition kinetics through distinct cell cycle phases influences the function of HSCs. Mende et al examine the effects of increasing two cell cycle complexes CCND1–CDK4 and CCNE1–CDK2 on the transition kinetics of human HSCs and their maintenance and functional alterations in vivo., Maintenance of stem cell properties is associated with reduced proliferation. However, in mouse hematopoietic stem cells (HSCs), loss of quiescence results in a wide range of phenotypes, ranging from functional failure to extensive self-renewal. It remains unknown whether the function of human HSCs is controlled by the kinetics of cell cycle progression. Using human HSCs and human progenitor cells (HSPCs), we report here that elevated levels of CCND1–CDK4 complexes promoted the transit from G0 to G1 and shortened the G1 cell cycle phase, resulting in protection from differentiation-inducing signals in vitro and increasing human leukocyte engraftment in vivo. Further, CCND1–CDK4 overexpression conferred a competitive advantage without impacting HSPC numbers. In contrast, accelerated cell cycle progression mediated by elevated levels of CCNE1–CDK2 led to the loss of functional HSPCs in vivo. Collectively, these data suggest that the transition kinetics through the early cell cycle phases are key regulators of human HSPC function and important for lifelong hematopoiesis.

In mammals, perivascular cell-derived scarring after spinal cord injury impedes axonal regrowth. In contrast, the extracellular matrix (ECM) in the spinal lesion site of zebrafish is permissive and required for axon regeneration. However, the cellular mechanisms underlying this interspecies difference have not been investigated. Here, we show that an injury to the zebrafish spinal cord triggers recruitment of pdgfrb+ myoseptal and perivascular cells in a PDGFR signaling-dependent manner. Interference with pdgfrb+ cell recruitment or depletion of pdgfrb+ cells inhibits axonal regrowth and recovery of locomotor function. Transcriptional profiling and functional experiments reveal that pdgfrb+ cells upregulate expression of axon growth-promoting ECM genes (cthrc1a and col12a1a/b) and concomitantly reduce synthesis of matrix molecules that are detrimental to regeneration (lum and mfap2). Our data demonstrate that a switch in ECM composition is critical for axon regeneration after spinal cord injury and identify the cellular source and components of the growth-promoting lesion ECM.