Phenotypic Mapping of Pathologic Cross-Talk between Glioblastoma and Innate Immune Cells by Synthetic Genetic Tracing.

Glioblastoma is a lethal brain tumor that exhibits heterogeneity and resistance to therapy. Our understanding of tumor homeostasis is limited by a lack of genetic tools to selectively identify tumor states and fate transitions. Here, we use glioblastoma subtype signatures to construct synthetic genetic tracing cassettes and investigate tumor heterogeneity at cellular and molecular levels, in vitro and in vivo. Through synthetic locus control regions, we demonstrate that proneural glioblastoma is a hardwired identity, whereas mesenchymal glioblastoma is an adaptive and metastable cell state driven by proinflammatory and differentiation cues and DNA damage, but not hypoxia. Importantly, we discovered that innate immune cells divert glioblastoma cells to a proneural-to-mesenchymal transition that confers therapeutic resistance. Our synthetic genetic tracing methodology is simple, scalable, and widely applicable to study homeostasis in development and diseases. In glioblastoma, the method causally links distinct (micro)environmental, genetic, and pharmacologic perturbations and mesenchymal commitment. SIGNIFICANCE: Glioblastoma is heterogeneous and incurable. Here, we designed synthetic reporters to reflect the transcriptional output of tumor cell states and signaling pathways' activity. This method is generally applicable to study homeostasis in normal tissues and diseases. In glioblastoma, synthetic genetic tracing causally connects cellular and molecular heterogeneity to therapeutic responses.This article is highlighted in the In This Issue feature, p. 521.

Corrigendum to "Development of event-specific qPCR detection methods for genetically modified alfalfa events J101, J163 and KK179" [Biomol. Detect. Quantif. 17 (March) (2019) 100076].

[This corrects the article DOI: 10.1016/j.bdq.2018.12.001.].

Development of event-specific qPCR detection methods for genetically modified alfalfa events J101, J163 and KK179.

Genetically modified alfalfa is authorized for cultivation in several countries since 2005. On the other hand, cultivation in or export to the European Union is not allowed and thus neither certified reference material nor official event-specific detection methods are available. Therefore, based on patent sequence information, event-specific real-time PCR detection methods targeting the junction sequence of the alfalfa genome and the transgenic insert of the respective events J101, J163 and KK179 were developed. Newly developed plasmids were used as reference material for assay optimization and in-house validation. Plasmid standards were quantified using digital droplet PCR and LOD95%, PCR efficiency, robustness and specificity of the assays were determined using real-time PCR. A LOD95% of 10 copies per PCR reaction was observed and PCR efficiencies of 95-97 % were achieved. Different real-time PCR instruments and PCR conditions were applied to test for robustness of the assays using DNA at a concentration of 30 copies per µL for each gm alfalfa event. All replicates were positive independent of the instrument or the PCR condition. DNA from certified reference material of different genetically modified crops as well as reference materials of the three events was used to experimentally test for specificity. No unspecific amplification signal was observed for any of the assays. Validation results were in line with the "Minimum Performance Requirements for Analytical Methods of GMO Testing" of the European Network of GMO Laboratories. Furthermore, an inter-laboratory comparison study was conducted to show the transferability and applicability of the methods and to verify the assay performance parameters.

Robo signalling controls pancreatic progenitor identity by regulating Tead transcription factors.

A complex interplay of intrinsic factors and extrinsic signalling pathways controls both cell lineage commitment and maintenance of cell identity. Loss of defined cellular states is the cause of many different cancers, including pancreatic cancer. Recent findings suggest a clinical role for the conserved SLIT/ROBO signalling pathway in pancreatic cancer. However, whilst this pathway has been extensively studied in many processes, a role for Slit and Robo genes in pancreas cell identity and plasticity has not been established yet. Here, we identify Slit/Robo signalling as a key regulator of pancreatic progenitor identity. We find that Robo1 and Robo2 are required for preserving pancreatic cell identity shortly after fate induction and, subsequently, for expansion of the pancreatic progenitor pool in the mouse. Furthermore, we show that Robo receptors control the expression of Tead transcription factors as well as its downstream transcriptional activity. Our work identifies an interplay between Slit/Robo pathway and Tead intrinsic regulators, functioning as gatekeeper of pancreatic cell identity.

The RhoGAP Stard13 controls insulin secretion through F-actin remodeling.

OBJECTIVE: Actin cytoskeleton remodeling is necessary for glucose-stimulated insulin secretion in pancreatic ß-cells. A mechanistic understanding of actin dynamics in the islet is paramount to a better comprehension of ß-cell dysfunction in diabetes. Here, we investigate the Rho GTPase regulator Stard13 and its role in F-actin cytoskeleton organization and islet function in adult mice. METHODS: We used Lifeact-EGFP transgenic animals to visualize actin cytoskeleton organization and dynamics in vivo in the mouse islets. Furthermore, we applied this model to study actin cytoskeleton and insulin secretion in mutant mice deleted for Stard13 selectively in pancreatic cells. We isolated transgenic islets for 3D-imaging and perifusion studies to measure insulin secretion dynamics. In parallel, we performed histological and morphometric analyses of the pancreas and used in vivo approaches to study glucose metabolism in the mouse. RESULTS: In this study, we provide the first genetic evidence that Stard13 regulates insulin secretion in response to glucose. Postnatally, Stard13 expression became restricted to the mouse pancreatic islets. We showed that Stard13 deletion results in a marked increase in actin polymerization in islet cells, which is accompanied by severe reduction of insulin secretion in perifusion experiments. Consistently, Stard13-deleted mice displayed impaired glucose tolerance and reduced glucose-stimulated insulin secretion. CONCLUSIONS: Taken together, our results suggest a previously unappreciated role for the RhoGAP protein Stard13 in the interplay between actin cytoskeletal remodeling and insulin secretion.

Stepwise reprogramming of liver cells to a pancreas progenitor state by the transcriptional regulator Tgif2.

The development of a successful lineage reprogramming strategy of liver to pancreas holds promises for the treatment and potential cure of diabetes. The liver is an ideal tissue source for generating pancreatic cells, because of its close developmental origin with the pancreas and its regenerative ability. Yet, the molecular bases of hepatic and pancreatic cellular plasticity are still poorly understood. Here, we report that the TALE homeoprotein TGIF2 acts as a developmental regulator of the pancreas versus liver fate decision and is sufficient to elicit liver-to-pancreas fate conversion both ex vivo and in vivo. Hepatocytes expressing Tgif2 undergo extensive transcriptional remodelling, which represses the original hepatic identity and, over time, induces a pancreatic progenitor-like phenotype. Consistently, in vivo forced expression of Tgif2 activates pancreatic progenitor genes in adult mouse hepatocytes. This study uncovers the reprogramming activity of TGIF2 and suggests a stepwise reprogramming paradigm, whereby a 'lineage-restricted' dedifferentiation step precedes the identity switch.

Mutually exclusive signaling signatures define the hepatic and pancreatic progenitor cell lineage divergence.

Understanding how distinct cell types arise from multipotent progenitor cells is a major quest in stem cell biology. The liver and pancreas share many aspects of their early development and possibly originate from a common progenitor. However, how liver and pancreas cells diverge from a common endoderm progenitor population and adopt specific fates remains elusive. Using RNA sequencing (RNA-seq), we defined the molecular identity of liver and pancreas progenitors that were isolated from the mouse embryo at two time points, spanning the period when the lineage decision is made. The integration of temporal and spatial gene expression profiles unveiled mutually exclusive signaling signatures in hepatic and pancreatic progenitors. Importantly, we identified the noncanonical Wnt pathway as a potential developmental regulator of this fate decision and capable of inducing the pancreas program in endoderm and liver cells. Our study offers an unprecedented view of gene expression programs in liver and pancreas progenitors and forms the basis for formulating lineage-reprogramming strategies to convert adult hepatic cells into pancreatic cells.

Rho signalling restriction by the RhoGAP Stard13 integrates growth and morphogenesis in the pancreas.

The development of functional organ architecture relies on coordinated morphogenesis and growth. In the developing pancreas, the branching epithelium is organised in discrete domains, delineating one specific domain of progenitor cells at the tip of the branches. The molecular mechanisms underlying the coordinated action of branching and proliferation in organ formation are largely unknown. Here, we identify the RhoGAP protein Stard13 as an essential regulator of pancreas tissue architecture in the mammalian embryo. Conditional ablation of Stard13 expression in the pancreas disrupts epithelial morphogenesis and tip-domain organisation, resulting in hampered proliferation of tip progenitors and subsequent organ hypoplasia. Stard13 acts by regulating Rho signalling spatially and temporally during pancreas development. Our findings provide new insights into the mechanisms that shape pancreatic epithelium to create a mature organ and establish a functional link between Rho-mediated control of epithelial remodelling and organ size determination, involving reciprocal interaction of actin-MAL/SRF and MAPK signalling pathways.

Combinations of genetic mutations in the adult neural stem cell compartment determine brain tumour phenotypes.

It has been suggested that intrinsic brain tumours originate from a neural stem/progenitor cell population in the subventricular zone of the post-natal brain. However, the influence of the initial genetic mutation on the phenotype as well as the contribution of mature astrocytes to the formation of brain tumours is still not understood. We deleted Rb/p53, Rb/p53/PTEN or PTEN/p53 in adult subventricular stem cells; in ectopically neurografted stem cells; in mature parenchymal astrocytes and in transplanted astrocytes. We found that only stem cells, but not astrocytes, gave rise to brain tumours, independent of their location. This suggests a cell autonomous mechanism that enables stem cells to generate brain tumours, whereas mature astrocytes do not form brain tumours in adults. Recombination of PTEN/p53 gave rise to gliomas whereas deletion of Rb/p53 or Rb/p53/PTEN generated primitive neuroectodermal tumours (PNET), indicating an important role of an initial Rb loss in driving the PNET phenotype. Our study underlines an important role of stem cells and the relevance of initial genetic mutations in the pathogenesis and phenotype of brain tumours.

ERK activation causes epilepsy by stimulating NMDA receptor activity.

The ERK MAPK signalling pathway is a highly conserved kinase cascade linking transmembrane receptors to downstream effector mechanisms. To investigate the function of ERK in neurons, a constitutively active form of MEK1 (caMEK1) was conditionally expressed in the murine brain, which resulted in ERK activation and caused spontaneous epileptic seizures. ERK activation stimulated phosphorylation of eukaryotic translation initiation factor 4E (eIF4E) and augmented NMDA receptor 2B (NR2B) protein levels. Pharmacological inhibition of NR2B function impaired synaptic facilitation in area cornus ammonicus region 3 (CA3) in acute hippocampal slices derived from caMEK1-expressing mice and abrogated epilepsy in vivo. In addition, expression of caMEK1 caused phosphorylation of the transcription factor, cAMP response element-binding protein (CREB) and increased transcription of ephrinB2. EphrinB2 overexpression resulted in increased NR2B tyrosine phosphorylation, which was essential for caMEK1-induced epilepsy in vivo, since conditional inactivation of ephrinB2 greatly reduced seizure frequency in caMEK1 transgenic mice. Therefore, our study identifies a mechanism of epileptogenesis that links MAP kinase to Eph/Ephrin and NMDA receptor signalling.

Disease-associated prion protein oligomers inhibit the 26S proteasome.

The mechanism of cell death in prion disease is unknown but is associated with the production of a misfolded conformer of the prion protein. We report that disease-associated prion protein specifically inhibits the proteolytic beta subunits of the 26S proteasome. Using reporter substrates, fluorogenic peptides, and an activity probe for the beta subunits, this inhibitory effect was demonstrated in pure 26S proteasome and three different cell lines. By challenge with recombinant prion and other amyloidogenic proteins, we demonstrate that only the prion protein in a nonnative beta sheet conformation inhibits the 26S proteasome at stoichiometric concentrations. Preincubation with an antibody specific for aggregation intermediates abrogates this inhibition, consistent with an oligomeric species mediating this effect. We also present evidence for a direct relationship between prion neuropathology and impairment of the ubiquitin-proteasome system (UPS) in prion-infected UPS-reporter mice. Together, these data suggest a mechanism for intracellular neurotoxicity mediated by oligomers of misfolded prion protein.

Hyperphosphorylation of tau and neurofilaments and activation of CDK5 and ERK1/2 in PTEN-deficient cerebella.

Inherited mutations to the tumor suppressor PTEN sporadically lead to cerebellar gangliocytoma characterized by migration defects. This has been modeled by CNS-specific PTEN ablation in mice, but the underlying mechanism cannot be explained by the known role of PTEN in Akt/PKB inactivation. Here we show that the loss of PTEN in mouse cerebellar neurons causes neurodegeneration by hyperphosphorylation of tau and neurofilaments, and activation of Cdk5 and pERK1/2, suggesting that dysregulation of the PTEN/pAkt pathway can mediate neurodegeneration.

Wnt signalling inhibits neural differentiation of embryonic stem cells by controlling bone morphogenetic protein expression.

Wnt signalling plays an important role in both embryonic development and in tumourigenesis. Activation of the signalling cascade by wnt, but also mutations of the adenomatous polyposis coli (APC) protein and of the phosphorylation domain of beta-catenin, result in accumulation of active beta-catenin in the nucleus, where it binds to TCF/LEF transcription factors. We studied the effect of wnt signalling in embryonic stem cells by either inactivating APC or by introducing a dominant active form of beta-catenin. Both resulted in inhibition of neural differentiation in vitro and after brain grafting and in activation of downstream targets of wnt signalling, such as cyclins, c-myc, and bone morphogenetic proteins (BMP). Neural differentiation could be partially restored by the addition of the BMP antagonist noggin. This suggests a mechanism regulating the fate of differentiating embryonic stem cells.

Absence of the prion protein homologue Doppel causes male sterility.

The agent that causes prion diseases is thought to be identical with PrP(Sc), a conformer of the normal prion protein PrP(C). PrP(C)-deficient mice do not exhibit major pathologies, perhaps because they express a protein termed Dpl, which shares significant biochemical and structural homology with PrP(C). To investigate the physiological function of Dpl, we generated mice harbouring a homozygous disruption of the Prnd gene that encodes Dpl. Dpl deficiency did not interfere with embryonic and postnatal development, but resulted in male sterility. Dpl protein was expressed at late stages of spermiogenesis, and spermatids of Dpl mutants were reduced in numbers, immobile, malformed and unable to fertilize oocytes in vitro. Mechanical dissection of the zona pellucida partially restored in vitro fertilization. We conclude that Dpl regulates male fertility by controlling several aspects of male gametogenesis and sperm-egg interaction.