Publications by authors named "Wolfgang Enard"

72 Publications

TAMEP are brain tumor parenchymal cells controlling neoplastic angiogenesis and progression.

Cell Syst 2021 Mar 15;12(3):248-262.e7. Epub 2021 Feb 15.

Neurosurgical Research, University Hospital, LMU Munich, 81377 Munich, Germany; Walter-Brendel-Centre of Experimental Medicine, University Hospital, LMU Munich, 81377 Munich, Germany; German Cancer Research Center (DKFZ), German Cancer Consortium (DKTK), Partner Site Munich, 69120 Heidelberg, Germany. Electronic address:

Aggressive brain tumors like glioblastoma depend on support by their local environment and subsets of tumor parenchymal cells may promote specific phases of disease progression. We investigated the glioblastoma microenvironment with transgenic lineage-tracing models, intravital imaging, single-cell transcriptomics, immunofluorescence analysis as well as histopathology and characterized a previously unacknowledged population of tumor-associated cells with a myeloid-like expression profile (TAMEP) that transiently appeared during glioblastoma growth. TAMEP of mice and humans were identified with specific markers. Notably, TAMEP did not derive from microglia or peripheral monocytes but were generated by a fraction of CNS-resident, SOX2-positive progenitors. Abrogation of this progenitor cell population, by conditional Sox2-knockout, drastically reduced glioblastoma vascularization and size. Hence, TAMEP emerge as a tumor parenchymal component with a strong impact on glioblastoma progression.
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http://dx.doi.org/10.1016/j.cels.2021.01.002DOI Listing
March 2021

A non-invasive method to generate induced pluripotent stem cells from primate urine.

Sci Rep 2021 Feb 10;11(1):3516. Epub 2021 Feb 10.

Anthropology and Human Genomics, Department of Biology II, Ludwig-Maximilians-University, Großhaderner Straße 2, 82152, Martinsried, Germany.

Comparing the molecular and cellular properties among primates is crucial to better understand human evolution and biology. However, it is difficult or ethically impossible to collect matched tissues from many primates, especially during development. An alternative is to model different cell types and their development using induced pluripotent stem cells (iPSCs). These can be generated from many tissue sources, but non-invasive sampling would decisively broaden the spectrum of non-human primates that can be investigated. Here, we report the generation of primate iPSCs from urine samples. We first validate and optimize the procedure using human urine samples and show that suspension- Sendai Virus transduction of reprogramming factors into urinary cells efficiently generates integration-free iPSCs, which maintain their pluripotency under feeder-free culture conditions. We demonstrate that this method is also applicable to gorilla and orangutan urinary cells isolated from a non-sterile zoo floor. We characterize the urinary cells, iPSCs and derived neural progenitor cells using karyotyping, immunohistochemistry, differentiation assays and RNA-sequencing. We show that the urine-derived human iPSCs are indistinguishable from well characterized PBMC-derived human iPSCs and that the gorilla and orangutan iPSCs are well comparable to the human iPSCs. In summary, this study introduces a novel and efficient approach to non-invasively generate iPSCs from primate urine. This will extend the zoo of species available for a comparative approach to molecular and cellular phenotypes.
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http://dx.doi.org/10.1038/s41598-021-82883-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7876031PMC
February 2021

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

Nat Cell Biol 2021 01 4;23(1):23-31. Epub 2021 Jan 4.

Institute of Diabetes and Regeneration Research, Helmholtz Diabetes Center, Helmholtz Center Munich, Neuherberg, Germany.

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 hierarchy and segregation 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 progenitors. 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 cells. Finally, Wnt/PCP-activated Lgr5 ISCs are molecularly indistinguishable from Wnt/β-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.
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http://dx.doi.org/10.1038/s41556-020-00617-2DOI Listing
January 2021

Recent evolution of a TET-controlled and DPPA3/STELLA-driven pathway of passive DNA demethylation in mammals.

Nat Commun 2020 11 24;11(1):5972. Epub 2020 Nov 24.

Department of Biology II and Center for Integrated Protein Science Munich (CIPSM), Human Biology and BioImaging, Ludwig-Maximilians-Universität München, Planegg-Martinsried, Germany.

Genome-wide DNA demethylation is a unique feature of mammalian development and naïve pluripotent stem cells. Here, we describe a recently evolved pathway in which global hypomethylation is achieved by the coupling of active and passive demethylation. TET activity is required, albeit indirectly, for global demethylation, which mostly occurs at sites devoid of TET binding. Instead, TET-mediated active demethylation is locus-specific and necessary for activating a subset of genes, including the naïve pluripotency and germline marker Dppa3 (Stella, Pgc7). DPPA3 in turn drives large-scale passive demethylation by directly binding and displacing UHRF1 from chromatin, thereby inhibiting maintenance DNA methylation. Although unique to mammals, we show that DPPA3 alone is capable of inducing global DNA demethylation in non-mammalian species (Xenopus and medaka) despite their evolutionary divergence from mammals more than 300 million years ago. Our findings suggest that the evolution of Dppa3 facilitated the emergence of global DNA demethylation in mammals.
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http://dx.doi.org/10.1038/s41467-020-19603-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7686362PMC
November 2020

Continued Bcl6 Expression Prevents the Transdifferentiation of Established Tfh Cells into Th1 Cells during Acute Viral Infection.

Cell Rep 2020 10;33(1):108232

Institute for Immunology, Biomedical Center, Faculty of Medicine, LMU Munich, Grosshaderner Str. 9, 82152 Planegg-Martinsried, Germany; Medical Clinic III for Oncology, Hematology, Immuno-Oncology, and Rheumatology, University Hospital Bonn, University of Bonn, Venusberg-Campus 1, 53127 Bonn, Germany. Electronic address:

T follicular helper (Tfh) cells are crucial for the establishment of germinal centers (GCs) and potent antibody responses. Nevertheless, the T cell-intrinsic factors that are required for the maintenance of already-established Tfh cells and GCs remain largely unknown. Here, we use temporally guided gene ablation in CD4 T cells to dissect the contributions of the Tfh-associated chemokine receptor CXCR5 and the transcription factor Bcl6. Induced ablation of Cxcr5 has minor effects on the function of established Tfh cells, and Cxcr5-ablated cells still exhibit most of the features of CXCR5 Tfh cells. In contrast, continued Bcl6 expression is critical to maintain the GC Tfh cell phenotype and also the GC reaction. Importantly, Bcl6 ablation during acute viral infection results in the transdifferentiation of established Tfh into Th1 cells, thus highlighting the plasticity of Tfh cells. These findings have implications for strategies that boost or restrain Tfh cells and GCs in health and disease.
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http://dx.doi.org/10.1016/j.celrep.2020.108232DOI Listing
October 2020

Congenic expression of poly-GA but not poly-PR in mice triggers selective neuron loss and interferon responses found in C9orf72 ALS.

Acta Neuropathol 2020 08 19;140(2):121-142. Epub 2020 Jun 19.

German Center for Neurodegenerative Diseases (DZNE), Munich, 81377, Munich, Germany.

Expansion of a (GC) repeat in C9orf72 causes amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), but the link of the five repeat-encoded dipeptide repeat (DPR) proteins to neuroinflammation, TDP-43 pathology, and neurodegeneration is unclear. Poly-PR is most toxic in vitro, but poly-GA is far more abundant in patients. To directly compare these in vivo, we created congenic poly-GA and poly-PR mice. 40% of poly-PR mice were affected with ataxia and seizures, requiring euthanasia by 6 weeks of age. The remaining poly-PR mice were asymptomatic at 14 months of age, likely due to an 80% reduction of the transgene mRNA in this subgroup. In contrast, all poly-GA mice showed selective neuron loss, inflammation, as well as muscle denervation and wasting requiring euthanasia before 7 weeks of age. In-depth analysis of peripheral organs and blood samples suggests that peripheral organ failure does not drive these phenotypes. Although transgene mRNA levels were similar between poly-GA and affected poly-PR mice, poly-GA aggregated far more abundantly than poly-PR in the CNS and was also found in skeletal muscle. In addition, TDP-43 and other disease-linked RNA-binding proteins co-aggregated in rare nuclear inclusions in the hippocampus and frontal cortex only in poly-GA mice. Transcriptome analysis revealed activation of an interferon-responsive pro-inflammatory microglial signature in end-stage poly-GA but not poly-PR mice. This signature was also found in all ALS patients and enriched in C9orf72 cases. In summary, our rigorous comparison of poly-GA and poly-PR toxicity in vivo indicates that poly-GA, but not poly-PR at the same mRNA expression level, promotes interferon responses in C9orf72 disease and contributes to TDP-43 abnormalities and neuron loss selectively in disease-relevant regions.
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http://dx.doi.org/10.1007/s00401-020-02176-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7360660PMC
August 2020

Benchmarking single-cell RNA-sequencing protocols for cell atlas projects.

Nat Biotechnol 2020 06 6;38(6):747-755. Epub 2020 Apr 6.

CNAG-CRG, Centre for Genomic Regulation, Barcelona Institute of Science and Technology, Barcelona, Spain.

Single-cell RNA sequencing (scRNA-seq) is the leading technique for characterizing the transcriptomes of individual cells in a sample. The latest protocols are scalable to thousands of cells and are being used to compile cell atlases of tissues, organs and organisms. However, the protocols differ substantially with respect to their RNA capture efficiency, bias, scale and costs, and their relative advantages for different applications are unclear. In the present study, we generated benchmark datasets to systematically evaluate protocols in terms of their power to comprehensively describe cell types and states. We performed a multicenter study comparing 13 commonly used scRNA-seq and single-nucleus RNA-seq protocols applied to a heterogeneous reference sample resource. Comparative analysis revealed marked differences in protocol performance. The protocols differed in library complexity and their ability to detect cell-type markers, impacting their predictive value and suitability for integration into reference cell atlases. These results provide guidance both for individual researchers and for consortium projects such as the Human Cell Atlas.
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http://dx.doi.org/10.1038/s41587-020-0469-4DOI Listing
June 2020

c-Rel gain in B cells drives germinal center reactions and autoantibody production.

J Clin Invest 2020 06;130(6):3270-3286

Institute of Experimental Hematology, School of Medicine, Technical University of Munich, Munich, Germany.

Single-nucleotide polymorphisms and locus amplification link the NF-κB transcription factor c-Rel to human autoimmune diseases and B cell lymphomas, respectively. However, the functional consequences of enhanced c-Rel levels remain enigmatic. Here, we overexpressed c-Rel specifically in mouse B cells from BAC-transgenic gene loci and demonstrate that c-Rel protein levels linearly dictated expansion of germinal center B (GCB) cells and isotype-switched plasma cells. c-Rel expression in B cells of otherwise c-Rel-deficient mice fully rescued terminal B cell differentiation, underscoring its critical B cell-intrinsic roles. Unexpectedly, in GCB cells transcription-independent regulation produced the highest c-Rel protein levels among B cell subsets. In c-Rel-overexpressing GCB cells this caused enhanced nuclear translocation, a profoundly altered transcriptional program, and increased proliferation. Finally, we provide a link between c-Rel gain and autoimmunity by showing that c-Rel overexpression in B cells caused autoantibody production and renal immune complex deposition.
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http://dx.doi.org/10.1172/JCI124382DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7260018PMC
June 2020

ZBTB7A prevents RUNX1-RUNX1T1-dependent clonal expansion of human hematopoietic stem and progenitor cells.

Oncogene 2020 04 2;39(15):3195-3205. Epub 2020 Mar 2.

Department of Medicine III, University Hospital, LMU Munich, 81377, Munich, Germany.

ZBTB7A is frequently mutated in acute myeloid leukemia (AML) with t(8;21) translocation. However, the oncogenic collaboration between mutated ZBTB7A and the RUNX1-RUNX1T1 fusion gene in AML t(8;21) remains unclear. Here, we investigate the role of ZBTB7A and its mutations in the context of normal and malignant hematopoiesis. We demonstrate that clinically relevant ZBTB7A mutations in AML t(8;21) lead to loss of function and result in perturbed myeloid differentiation with block of the granulocytic lineage in favor of monocytic commitment. In addition, loss of ZBTB7A increases glycolysis and hence sensitizes leukemic blasts to metabolic inhibition with 2-deoxy-D-glucose. We observed that ectopic expression of wild-type ZBTB7A prevents RUNX1-RUNX1T1-mediated clonal expansion of human CD34+ cells, whereas the outgrowth of progenitors is enabled by ZBTB7A mutation. Finally, ZBTB7A expression in t(8;21) cells lead to a cell cycle arrest that could be mimicked by inhibition of glycolysis. Our findings suggest that loss of ZBTB7A may facilitate the onset of AML t(8;21), and that RUNX1-RUNX1T1-rearranged leukemia might be treated with glycolytic inhibitors.
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http://dx.doi.org/10.1038/s41388-020-1209-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7142018PMC
April 2020

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

Curr Biol 2020 03 27;30(6):1142-1151.e6. Epub 2020 Feb 27.

Research Unit of Sensory Biology & Organogenesis, Helmholtz Zentrum München, Ingolstädter Landstraße 1, 85764 Neuherberg, Germany. Electronic address:

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.
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http://dx.doi.org/10.1016/j.cub.2020.01.027DOI Listing
March 2020

A truncating Aspm allele leads to a complex cognitive phenotype and region-specific reductions in parvalbuminergic neurons.

Transl Psychiatry 2020 02 13;10(1):66. Epub 2020 Feb 13.

Institute of Developmental Genetics, Helmholtz Zentrum München, Neuherberg, Germany.

Neurodevelopmental disorders are heterogeneous and identifying shared genetic aetiologies and converging signalling pathways affected could improve disease diagnosis and treatment. Truncating mutations of the abnormal spindle-like microcephaly associated (ASPM) gene cause autosomal recessive primary microcephaly (MCPH) in humans. ASPM is a positive regulator of Wnt/β-Catenin signalling and controls symmetric to asymmetric cell division. This process balances neural progenitor proliferation with differentiation during embryogenesis, the malfunction of which could interfere with normal brain development. ASPM mutations may play a role also in other neurodevelopmental disorders, nevertheless, we lack the details of how or to what extent. We therefore assessed neurodevelopmental disease and circuit endophenotypes in mice with a truncating Aspm mutation. Aspm mice exhibited impaired short- and long-term object recognition memory and markedly enhanced place learning in the IntelliCage®. This behaviour pattern is reminiscent of a cognitive phenotype seen in mouse models and patients with a rare form of autism spectrum disorder (ASD) as well as in mouse models of altered Wnt signalling. These alterations were accompanied by ventriculomegaly, corpus callosum dysgenesis and decreased parvalbumin (PV)+ interneuron numbers in the hippocampal Cornu Ammonis (CA) region and thalamic reticular nucleus (TRN). PV+ cell number correlated to object recognition (CA and TRN) and place learning (TRN). This opens the possibility that, as well as causing MCPH, mutant ASPM potentially contributes to other neurodevelopmental disorders such as ASD through altered parvalbuminergic interneuron development affecting cognitive behaviour. These findings provide important information for understanding the genetic overlap and improved treatment of neurodevelopmental disorders associated with ASPM.
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http://dx.doi.org/10.1038/s41398-020-0686-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7026184PMC
February 2020

A systematic evaluation of single cell RNA-seq analysis pipelines.

Nat Commun 2019 10 11;10(1):4667. Epub 2019 Oct 11.

Anthropology and Human Genomics, Department of Biology II, Ludwig-Maximilians University, Munich, Germany.

The recent rapid spread of single cell RNA sequencing (scRNA-seq) methods has created a large variety of experimental and computational pipelines for which best practices have not yet been established. Here, we use simulations based on five scRNA-seq library protocols in combination with nine realistic differential expression (DE) setups to systematically evaluate three mapping, four imputation, seven normalisation and four differential expression testing approaches resulting in ~3000 pipelines, allowing us to also assess interactions among pipeline steps. We find that choices of normalisation and library preparation protocols have the biggest impact on scRNA-seq analyses. Specifically, we find that library preparation determines the ability to detect symmetric expression differences, while normalisation dominates pipeline performance in asymmetric DE-setups. Finally, we illustrate the importance of informed choices by showing that a good scRNA-seq pipeline can have the same impact on detecting a biological signal as quadrupling the sample size.
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http://dx.doi.org/10.1038/s41467-019-12266-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6789098PMC
October 2019

Actin stabilizing compounds show specific biological effects due to their binding mode.

Sci Rep 2019 07 5;9(1):9731. Epub 2019 Jul 5.

Department of Pharmacy, Ludwig-Maximilians-Universität, 81377, Munich, Germany.

Actin binding compounds are widely used tools in cell biology. We compare the biological and biochemical effects of miuraenamide A and jasplakinolide, a structurally related prototypic actin stabilizer. Though both compounds have similar effects on cytoskeletal morphology and proliferation, they affect migration and transcription in a distinctive manner, as shown by a transcriptome approach in endothelial cells. In vitro, miuraenamide A acts as an actin nucleating, F-actin polymerizing and stabilizing compound, just like described for jasplakinolide. However, in contrast to jasplakinolide, miuraenamide A competes with cofilin, but not gelsolin or Arp2/3 for binding to F-actin. We propose a binding mode of miuraenamide A, explaining both its similarities and its differences to jasplakinolide. Molecular dynamics simulations suggest that the bromophenol group of miurenamide A interacts with residues Tyr133, Tyr143, and Phe352 of actin. This shifts the D-loop of the neighboring actin, creating tighter packing of the monomers, and occluding the binding site of cofilin. Since relatively small changes in the molecular structure give rise to this selectivity, actin binding compounds surprisingly are promising scaffolds for creating actin binders with specific functionality instead of just "stabilizers".
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http://dx.doi.org/10.1038/s41598-019-46282-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6611809PMC
July 2019

Chivosazole A Modulates Protein-Protein Interactions of Actin.

J Nat Prod 2019 07 1;82(7):1961-1970. Epub 2019 Jul 1.

Department of Pharmacy , Ludwig-Maximilians-University , 81377 Munich , Germany.

Actin is a protein of central importance for many cellular key processes. It is regulated by local interactions with a large number of actin binding proteins (ABPs). Various compounds are known to either increase or decrease the polymerization dynamics of actin. However, no actin binding compound has been developed for clinical applications yet because of selectivity issues. We provide a crystal structure of the natural product chivosazole A (ChivoA) bound to actin and show that-in addition to inhibiting nucleation, polymerization, and severing of F-actin filaments-it selectively modulates binding of ABPs to G-actin: Although unphysiological actin dimers are induced by ChivoA, interaction with gelsolin, profilin, cofilin, and thymosin-β4 is inhibited. Moreover, ChivoA causes transcriptional effects differing from latrunculin B, an actin binder with a different binding site. Our data show that ChivoA and related compounds could serve as scaffolds for the development of actin binding molecules selectively targeting specific actin functions.
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http://dx.doi.org/10.1021/acs.jnatprod.9b00335DOI Listing
July 2019

Studying Cancer Heterogeneity by Single-Cell RNA Sequencing.

Methods Mol Biol 2019 ;1956:305-319

Anthropology & Human Genomics, Department of Biology II, Ludwig-Maximilians-University, Martinsried, Germany.

A major hurdle for the treatment of cancer is the incomplete understanding of its evolution through the course of its emergence, dispersal, and relapse. Genetic and epigenetic changes in combination with external cues and selective forces are the driving factors behind tumor heterogeneity. Understanding this variability within and across patients may partly explain the unpredictable outcomes of cancer treatments. Measuring the variation of gene expression levels within cells of the same tumor is a crucial part of this endeavor. Hence, the recently developed single-cell RNA-sequencing (scRNA-seq) technologies have become a valuable tool for cancer research. In practice, however, this is still challenging, especially for clinical samples. Here, we describe mcSCRB-seq (molecular crowding single-cell RNA barcoding and sequencing), a highly sensitive and powerful plate-based scRNA-seq method, which shows great capability to generate transcriptome data for cancer cells. mcSCRB-seq is not only characterized by high sensitivity due to molecular crowding and the use of unique molecular identifiers (UMIs) but also features an easy workflow and a low per-cell cost and does not require specialized equipment.
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http://dx.doi.org/10.1007/978-1-4939-9151-8_14DOI Listing
July 2019

Neandertal Introgression Sheds Light on Modern Human Endocranial Globularity.

Curr Biol 2019 01 13;29(1):120-127.e5. Epub 2018 Dec 13.

Language and Genetics Department, Max Planck Institute for Psycholinguistics, P.O. Box 310, 6500 AH, Nijmegen, the Netherlands; Donders Institute for Brain, Cognition and Behaviour, Radboud University, P.O. Box 9101, 6500 HB, Nijmegen, the Netherlands. Electronic address:

One of the features that distinguishes modern humans from our extinct relatives and ancestors is a globular shape of the braincase [1-4]. As the endocranium closely mirrors the outer shape of the brain, these differences might reflect altered neural architecture [4, 5]. However, in the absence of fossil brain tissue, the underlying neuroanatomical changes as well as their genetic bases remain elusive. To better understand the biological foundations of modern human endocranial shape, we turn to our closest extinct relatives: the Neandertals. Interbreeding between modern humans and Neandertals has resulted in introgressed fragments of Neandertal DNA in the genomes of present-day non-Africans [6, 7]. Based on shape analyses of fossil skull endocasts, we derive a measure of endocranial globularity from structural MRI scans of thousands of modern humans and study the effects of introgressed fragments of Neandertal DNA on this phenotype. We find that Neandertal alleles on chromosomes 1 and 18 are associated with reduced endocranial globularity. These alleles influence expression of two nearby genes, UBR4 and PHLPP1, which are involved in neurogenesis and myelination, respectively. Our findings show how integration of fossil skull data with archaic genomics and neuroimaging can suggest developmental mechanisms that may contribute to the unique modern human endocranial shape.
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http://dx.doi.org/10.1016/j.cub.2018.10.065DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6380688PMC
January 2019

Altered social behavior in mice carrying a cortical Foxp2 deletion.

Hum Mol Genet 2019 03;28(5):701-717

Inserm, Institut du Fer à Moulin, Sorbonne Université, Paris, France.

Genetic disruptions of the forkhead box transcription factor FOXP2 in humans cause an autosomal-dominant speech and language disorder. While FOXP2 expression pattern are highly conserved, its role in specific brain areas for mammalian social behaviors remains largely unknown. Here we studied mice carrying a homozygous cortical Foxp2 deletion. The postnatal development and gross morphological architecture of mutant mice was indistinguishable from wildtype (WT) littermates. Unbiased behavioral profiling of adult mice revealed abnormalities in approach behavior towards conspecifics as well as in the reciprocal responses of WT interaction partners. Furthermore mutant mice showed alterations in acoustical parameters of ultrasonic vocalizations, which also differed in function of the social context. Cell type-specific gene expression profiling of cortical pyramidal neurons revealed aberrant regulation of genes involved in social behavior. In particular Foxp2 mutants showed the downregulation of Mint2 (Apba2), a gene involved in approach behavior in mice and autism spectrum disorder in humans. Taken together these data demonstrate that cortical Foxp2 is required for normal social behaviors in mice.
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http://dx.doi.org/10.1093/hmg/ddy372DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6381386PMC
March 2019

Transcriptional effects of actin-binding compounds: the cytoplasm sets the tone.

Cell Mol Life Sci 2018 Dec 11;75(24):4539-4555. Epub 2018 Sep 11.

Department of Pharmacy, Center for Drug Research, Ludwig-Maximilians-University Munich, 81377, Munich, Germany.

Actin has emerged as a versatile regulator of gene transcription. Cytoplasmatic actin regulates mechanosensitive-signaling pathways such as MRTF-SRF and Hippo-YAP/TAZ. In the nucleus, both polymerized and monomeric actin directly interfere with transcription-associated molecular machineries. Natural actin-binding compounds are frequently used tools to study actin-related processes in cell biology. However, their influence on transcriptional regulation and intranuclear actin polymerization is poorly understood to date. Here, we analyze the effects of two representative actin-binding compounds, Miuraenamide A (polymerizing properties) and Latrunculin B (depolymerizing properties), on transcriptional regulation in primary cells. We find that actin stabilizing and destabilizing compounds inversely shift nuclear actin levels without a direct influence on polymerization state and intranuclear aspects of transcriptional regulation. Furthermore, we identify Miuraenamide A as a potent inducer of G-actin-dependent SRF target gene expression. In contrast, the F-actin-regulated Hippo-YAP/TAZ axis remains largely unaffected by compound-induced actin aggregation. This is due to the inability of AMOTp130 to bind to the amorphous actin aggregates resulting from treatment with miuraenamide. We conclude that actin-binding compounds predominantly regulate transcription via their influence on cytoplasmatic G-actin levels, while transcriptional processes relying on intranuclear actin polymerization or functional F-actin networks are not targeted by these compounds at tolerable doses.
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http://dx.doi.org/10.1007/s00018-018-2919-4DOI Listing
December 2018

Derivation of induced pluripotent stem cells in Japanese macaque (Macaca fuscata).

Sci Rep 2018 08 15;8(1):12187. Epub 2018 Aug 15.

Molecular Biology Section, Department of Cellular and Molecular Biology, Primate Research Institute, Kyoto University, Inuyama, Aichi, 484-8506, Japan.

Non-human primates are our closest relatives and are of special interest for ecological, evolutionary and biomedical research. The Japanese macaque (Macaca fuscata) has contributed to the progress of primatology and neurosciences over 60 years. Despite this importance, the molecular and cellular basis of the Japanese macaque remains unexplored since useful cellular tools are lacking. Here we generated induced pluripotent stem cells (iPSCs) from skin fibroblasts of the Japanese macaque with Sendai virus or plasmid vectors. The Japanese macaque iPSCs (jm-iPSCs) were established under feeder-free culture conditions, but feeder cells turned out to be essential for their maintenance. The jm-iPSCs formed human iPSC-like flat colonies which were positive for pluripotent antigens including alkaline phosphatase, SSEA4, and TRA-1-81. They also expressed endogenous OCT3/4, SOX2, L-MYC, and KLF4 and other pluripotent marker genes. The potential to differentiate into all three germ layers and neural stem cells was confirmed by embryoid body and neurosphere formation, respectively. The jm-iPSCs will provide a robust in vitro tool for investigating the underlying mechanisms of development and physiology studies with the Japanese macaque.
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http://dx.doi.org/10.1038/s41598-018-30734-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6093926PMC
August 2018

Sensitive and powerful single-cell RNA sequencing using mcSCRB-seq.

Nat Commun 2018 07 26;9(1):2937. Epub 2018 Jul 26.

Anthropology & Human Genomics, Department of Biology II, Ludwig-Maximilians-University, Großhaderner Straße 2, 82152, Martinsried, Germany.

Single-cell RNA sequencing (scRNA-seq) has emerged as a central genome-wide method to characterize cellular identities and processes. Consequently, improving its sensitivity, flexibility, and cost-efficiency can advance many research questions. Among the flexible plate-based methods, single-cell RNA barcoding and sequencing (SCRB-seq) is highly sensitive and efficient. Here, we systematically evaluate experimental conditions of this protocol and find that adding polyethylene glycol considerably increases sensitivity by enhancing cDNA synthesis. Furthermore, using Terra polymerase increases efficiency due to a more even cDNA amplification that requires less sequencing of libraries. We combined these and other improvements to develop a scRNA-seq library protocol we call molecular crowding SCRB-seq (mcSCRB-seq), which we show to be one of the most sensitive, efficient, and flexible scRNA-seq methods to date.
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http://dx.doi.org/10.1038/s41467-018-05347-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6062574PMC
July 2018

zUMIs - A fast and flexible pipeline to process RNA sequencing data with UMIs.

Gigascience 2018 06;7(6)

Anthropology and Human Genomics, Department of Biology II, Ludwig-Maximilians University, Grosshaderner Str. 2, 82152 Martinsried, Germany.

Background: Single-cell RNA-sequencing (scRNA-seq) experiments typically analyze hundreds or thousands of cells after amplification of the cDNA. The high throughput is made possible by the early introduction of sample-specific bar codes (BCs), and the amplification bias is alleviated by unique molecular identifiers (UMIs). Thus, the ideal analysis pipeline for scRNA-seq data needs to efficiently tabulate reads according to both BC and UMI.

Findings: zUMIs is a pipeline that can handle both known and random BCs and also efficiently collapse UMIs, either just for exon mapping reads or for both exon and intron mapping reads. If BC annotation is missing, zUMIs can accurately detect intact cells from the distribution of sequencing reads. Another unique feature of zUMIs is the adaptive downsampling function that facilitates dealing with hugely varying library sizes but also allows the user to evaluate whether the library has been sequenced to saturation. To illustrate the utility of zUMIs, we analyzed a single-nucleus RNA-seq dataset and show that more than 35% of all reads map to introns. Also, we show that these intronic reads are informative about expression levels, significantly increasing the number of detected genes and improving the cluster resolution.

Conclusions: zUMIs flexibility makes if possible to accommodate data generated with any of the major scRNA-seq protocols that use BCs and UMIs and is the most feature-rich, fast, and user-friendly pipeline to process such scRNA-seq data.
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http://dx.doi.org/10.1093/gigascience/giy059DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6007394PMC
June 2018

Micropatterning as a tool to identify regulatory triggers and kinetics of actin-mediated endothelial mechanosensing.

J Cell Sci 2018 05 29;131(10). Epub 2018 May 29.

Ludwig-Maximilians-University Munich, Department of Pharmacy, Center for Drug Research, 81377 Munich, Germany

Developmental processes, such as angiogenesis, are associated with a constant remodeling of the actin cytoskeleton in response to different mechanical stimuli. The mechanosensitive transcription factors MRTF-A (MKL1) and YAP (also known as YAP1) are important mediators of this challenging adaptation process. However, it is as yet unknown whether both pathways respond in an identical or in a divergent manner to a given microenvironmental guidance cue. Here, we use a micropatterning approach to dissect single aspects of cellular behavior in a spatiotemporally controllable setting. Using the exemplary process of angiogenesis, we show that cell-cell contacts and adhesive surface area are shared regulatory parameters of MRTF and YAP on rigid 2D surfaces. By analyzing MRTF and YAP under laminar flow conditions and during cell migration on dumbbell-shaped microstructures, we demonstrate that they exhibit different translocation kinetics. In conclusion, our work promotes the application of micropatterning techniques as a cell biological tool to study mechanosensitive signaling in the context of angiogenesis.
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http://dx.doi.org/10.1242/jcs.212886DOI Listing
May 2018

Quantitative single-cell transcriptomics.

Brief Funct Genomics 2018 07;17(4):220-232

Anthropology and Human Genomics, Department of Biology II, Ludwig-Maximilians University, Großhaderner Str. 2, Martinsried, Germany.

Single-cell RNA sequencing (scRNA-seq) is currently transforming our understanding of biology, as it is a powerful tool to resolve cellular heterogeneity and molecular networks. Over 50 protocols have been developed in recent years and also data processing and analyzes tools are evolving fast. Here, we review the basic principles underlying the different experimental protocols and how to benchmark them. We also review and compare the essential methods to process scRNA-seq data from mapping, filtering, normalization and batch corrections to basic differential expression analysis. We hope that this helps to choose appropriate experimental and computational methods for the research question at hand.
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http://dx.doi.org/10.1093/bfgp/ely009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6063296PMC
July 2018

Evolutionary routes and KRAS dosage define pancreatic cancer phenotypes.

Nature 2018 02 24;554(7690):62-68. Epub 2018 Jan 24.

Center for Translational Cancer Research (TranslaTUM), Technische Universität München, 81675 Munich, Germany.

The poor correlation of mutational landscapes with phenotypes limits our understanding of the pathogenesis and metastasis of pancreatic ductal adenocarcinoma (PDAC). Here we show that oncogenic dosage-variation has a critical role in PDAC biology and phenotypic diversification. We find an increase in gene dosage of mutant KRAS in human PDAC precursors, which drives both early tumorigenesis and metastasis and thus rationalizes early PDAC dissemination. To overcome the limitations posed to gene dosage studies by the stromal richness of PDAC, we have developed large cell culture resources of metastatic mouse PDAC. Integration of cell culture genomes, transcriptomes and tumour phenotypes with functional studies and human data reveals additional widespread effects of oncogenic dosage variation on cell morphology and plasticity, histopathology and clinical outcome, with the highest Kras levels underlying aggressive undifferentiated phenotypes. We also identify alternative oncogenic gains (Myc, Yap1 or Nfkb2), which collaborate with heterozygous Kras in driving tumorigenesis, but have lower metastatic potential. Mechanistically, different oncogenic gains and dosages evolve along distinct evolutionary routes, licensed by defined allelic states and/or combinations of hallmark tumour suppressor alterations (Cdkn2a, Trp53, Tgfβ-pathway). Thus, evolutionary constraints and contingencies direct oncogenic dosage gain and variation along defined routes to drive the early progression of PDAC and shape its downstream biology. Our study uncovers universal principles of Ras-driven oncogenesis that have potential relevance beyond pancreatic cancer.
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http://dx.doi.org/10.1038/nature25459DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6097607PMC
February 2018

The Human Cell Atlas.

Elife 2017 12 5;6. Epub 2017 Dec 5.

Ragon Institute of MGH, MIT and Harvard, Cambridge, United States.

The recent advent of methods for high-throughput single-cell molecular profiling has catalyzed a growing sense in the scientific community that the time is ripe to complete the 150-year-old effort to identify all cell types in the human body. The Human Cell Atlas Project is an international collaborative effort that aims to define all human cell types in terms of distinctive molecular profiles (such as gene expression profiles) and to connect this information with classical cellular descriptions (such as location and morphology). An open comprehensive reference map of the molecular state of cells in healthy human tissues would propel the systematic study of physiological states, developmental trajectories, regulatory circuitry and interactions of cells, and also provide a framework for understanding cellular dysregulation in human disease. Here we describe the idea, its potential utility, early proofs-of-concept, and some design considerations for the Human Cell Atlas, including a commitment to open data, code, and community.
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http://dx.doi.org/10.7554/eLife.27041DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5762154PMC
December 2017

GATA2/3-TFAP2A/C transcription factor network couples human pluripotent stem cell differentiation to trophectoderm with repression of pluripotency.

Proc Natl Acad Sci U S A 2017 11 25;114(45):E9579-E9588. Epub 2017 Oct 25.

Institute of Stem Cell Research, Helmholtz Center Munich, 85764 Neuherberg, Germany;

To elucidate the molecular basis of BMP4-induced differentiation of human pluripotent stem cells (PSCs) toward progeny with trophectoderm characteristics, we produced transcriptome, epigenome H3K4me3, H3K27me3, and CpG methylation maps of trophoblast progenitors, purified using the surface marker APA. We combined them with the temporally resolved transcriptome of the preprogenitor phase and of single APA+ cells. This revealed a circuit of bivalent TFAP2A, TFAP2C, GATA2, and GATA3 transcription factors, coined collectively the "trophectoderm four" (TEtra), which are also present in human trophectoderm in vivo. At the onset of differentiation, the TEtra factors occupy multiple sites in epigenetically inactive placental genes and in Functional manipulation of and indicated that they directly couple trophoblast-specific gene induction with suppression of pluripotency. In accordance, knocking down in primate embryos resulted in a failure to form trophectoderm. The discovery of the TEtra circuit indicates how trophectoderm commitment is regulated in human embryogenesis.
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http://dx.doi.org/10.1073/pnas.1708341114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5692555PMC
November 2017