Publications by authors named "Juergen A Knoblich"

83 Publications

Oxidative Metabolism Drives Immortalization of Neural Stem Cells during Tumorigenesis.

Cell 2020 Sep 10;182(6):1490-1507.e19. Epub 2020 Sep 10.

Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), 1030 Vienna, Austria. Electronic address:

Metabolic reprogramming is a key feature of many cancers, but how and when it contributes to tumorigenesis remains unclear. Here we demonstrate that metabolic reprogramming induced by mitochondrial fusion can be rate-limiting for immortalization of tumor-initiating cells (TICs) and trigger their irreversible dedication to tumorigenesis. Using single-cell transcriptomics, we find that Drosophila brain tumors contain a rapidly dividing stem cell population defined by upregulation of oxidative phosphorylation (OxPhos). We combine targeted metabolomics and in vivo genetic screening to demonstrate that OxPhos is required for tumor cell immortalization but dispensable in neural stem cells (NSCs) giving rise to tumors. Employing an in vivo NADH/NAD sensor, we show that NSCs precisely increase OxPhos during immortalization. Blocking OxPhos or mitochondrial fusion stalls TICs in quiescence and prevents tumorigenesis through impaired NAD regeneration. Our work establishes a unique connection between cellular metabolism and immortalization of tumor-initiating cells.
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http://dx.doi.org/10.1016/j.cell.2020.07.039DOI Listing
September 2020

Human organoids: model systems for human biology and medicine.

Nat Rev Mol Cell Biol 2020 10 7;21(10):571-584. Epub 2020 Jul 7.

Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna Biocenter (VBC), Vienna, Austria.

The historical reliance of biological research on the use of animal models has sometimes made it challenging to address questions that are specific to the understanding of human biology and disease. But with the advent of human organoids - which are stem cell-derived 3D culture systems - it is now possible to re-create the architecture and physiology of human organs in remarkable detail. Human organoids provide unique opportunities for the study of human disease and complement animal models. Human organoids have been used to study infectious diseases, genetic disorders and cancers through the genetic engineering of human stem cells, as well as directly when organoids are generated from patient biopsy samples. This Review discusses the applications, advantages and disadvantages of human organoids as models of development and disease and outlines the challenges that have to be overcome for organoids to be able to substantially reduce the need for animal experiments.
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http://dx.doi.org/10.1038/s41580-020-0259-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7339799PMC
October 2020

Prospero Phase-Separating the Way to Neuronal Differentiation.

Dev Cell 2020 02;52(3):251-252

Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna BioCenter (VBC), Vienna, Austria; Medical University of Vienna, Vienna, Austria. Electronic address:

Drosophila neural progenitors require the transcriptional repressor Prospero to promptly establish the neuronal fate of their daughter cells to avoid tumorigenesis. In this issue of Developmental Cell, Liu et al. (2020) find that Prospero is mitotically implanted and forms liquid-like droplets mediating HP1a condensation to permanently repress its targets.
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http://dx.doi.org/10.1016/j.devcel.2020.01.022DOI Listing
February 2020

Human blood vessel organoids as a model of diabetic vasculopathy.

Nature 2019 01 16;565(7740):505-510. Epub 2019 Jan 16.

Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna, Austria.

The increasing prevalence of diabetes has resulted in a global epidemic. Diabetes is a major cause of blindness, kidney failure, heart attacks, stroke and amputation of lower limbs. These are often caused by changes in blood vessels, such as the expansion of the basement membrane and a loss of vascular cells. Diabetes also impairs the functions of endothelial cells and disturbs the communication between endothelial cells and pericytes. How dysfunction of endothelial cells and/or pericytes leads to diabetic vasculopathy remains largely unknown. Here we report the development of self-organizing three-dimensional human blood vessel organoids from pluripotent stem cells. These human blood vessel organoids contain endothelial cells and pericytes that self-assemble into capillary networks that are enveloped by a basement membrane. Human blood vessel organoids transplanted into mice form a stable, perfused vascular tree, including arteries, arterioles and venules. Exposure of blood vessel organoids to hyperglycaemia and inflammatory cytokines in vitro induces thickening of the vascular basement membrane. Human blood vessels, exposed in vivo to a diabetic milieu in mice, also mimic the microvascular changes found in patients with diabetes. DLL4 and NOTCH3 were identified as key drivers of diabetic vasculopathy in human blood vessels. Therefore, organoids derived from human stem cells faithfully recapitulate the structure and function of human blood vessels and are amenable systems for modelling and identifying the regulators of diabetic vasculopathy, a disease that affects hundreds of millions of patients worldwide.
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http://dx.doi.org/10.1038/s41586-018-0858-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7116578PMC
January 2019

Time-resolved transcriptomics in neural stem cells identifies a v-ATPase/Notch regulatory loop.

J Cell Biol 2018 09 29;217(9):3285-3300. Epub 2018 Jun 29.

Institute of Molecular Biotechnology of the Austrian Academy of Sciences, Vienna, Austria

neural stem cells (neuroblasts [NBs]) divide asymmetrically by differentially segregating protein determinants into their daughter cells. Although the machinery for asymmetric protein segregation is well understood, the events that reprogram one of the two daughter cells toward terminal differentiation are less clear. In this study, we use time-resolved transcriptional profiling to identify the earliest transcriptional differences between the daughter cells on their way toward distinct fates. By screening for coregulated protein complexes, we identify vacuolar-type H-ATPase (v-ATPase) among the first and most significantly down-regulated complexes in differentiating daughter cells. We show that v-ATPase is essential for NB growth and persistent activity of the Notch signaling pathway. Our data suggest that v-ATPase and Notch form a regulatory loop that acts in multiple stem cell lineages both during nervous system development and in the adult gut. We provide a unique resource for investigating neural stem cell biology and demonstrate that cell fate changes can be induced by transcriptional regulation of basic, cell-essential pathways.
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http://dx.doi.org/10.1083/jcb.201711167DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6123005PMC
September 2018

Coordinated Control of mRNA and rRNA Processing Controls Embryonic Stem Cell Pluripotency and Differentiation.

Cell Stem Cell 2018 04;22(4):543-558.e12

Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna Biocenter (VBC), Dr. Bohr-Gasse 3, 1030 Vienna, Austria. Electronic address:

Stem cell-specific transcriptional networks are well known to control pluripotency, but constitutive cellular processes such as mRNA splicing and protein synthesis can add complex layers of regulation with poorly understood effects on cell-fate decisions. Here, we show that the RNA binding protein HTATSF1 controls embryonic stem cell differentiation by regulating multiple aspects of RNA processing during ribosome biogenesis. HTATSF1, in a complex with splicing factor SF3B1, controls intron removal from ribosomal protein transcripts and regulates ribosomal RNA transcription and processing, thereby controlling 60S ribosomal abundance and protein synthesis. HTATSF1-dependent protein synthesis is essential for naive pre-implantation epiblast to transition into post-implantation epiblast, a stage with transiently low protein synthesis, and further differentiation toward neuroectoderm. Together, these results identify coordinated regulation of ribosomal RNA and protein synthesis by HTATSF1 and show that this essential mechanism controls protein synthesis during early mammalian embryogenesis.
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http://dx.doi.org/10.1016/j.stem.2018.03.002DOI Listing
April 2018

Tracing Stem Cell Division in Adult Neurogenesis.

Cell Stem Cell 2018 02;22(2):143-145

Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna Biocenter (VBC), 1030 Vienna, Austria. Electronic address:

Neural stem cells in the ventricular-subventricular zone of the adult brain continuously generate differentiated neurons without depleting the stem cell pool. In this issue of Cell Stem Cell, Obernier et al. (2018) present the surprising finding that this occurs through mostly symmetric divisions that either generate two differentiating or two self-renewing daughter cells.
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http://dx.doi.org/10.1016/j.stem.2018.01.012DOI Listing
February 2018

The tumor suppressor Brat controls neuronal stem cell lineages by inhibiting Deadpan and Zelda.

EMBO Rep 2018 01 30;19(1):102-117. Epub 2017 Nov 30.

Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna, Austria

The TRIM-NHL protein Brain tumor (Brat) acts as a tumor suppressor in the brain, but how it suppresses tumor formation is not completely understood. Here, we combine temperature-controlled RNAi with transcriptome analysis to identify the immediate Brat targets in neuroblasts. Besides the known target Deadpan (Dpn), our experiments identify the transcription factor Zelda (Zld) as a critical target of Brat. Our data show that Zld is expressed in neuroblasts and required to allow re-expression of Dpn in transit-amplifying intermediate neural progenitors. Upon neuroblast division, Brat is enriched in one daughter cell where its NHL domain directly binds to specific motifs in the 3'UTR of and mRNA to mediate their degradation. In mutants, both Dpn and Zld continue to be expressed, but inhibition of either transcription factor prevents tumorigenesis. Our genetic and biochemical data indicate that Dpn inhibition requires higher Brat levels than Zld inhibition and suggest a model where stepwise post-transcriptional inhibition of distinct factors ensures sequential generation of fates in a stem cell lineage.
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http://dx.doi.org/10.15252/embr.201744188DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5757284PMC
January 2018

The splicing co-factor Barricade/Tat-SF1 is required for cell cycle and lineage progression in neural stem cells.

Development 2017 11 21;144(21):3932-3945. Epub 2017 Sep 21.

Institute of Molecular Biotechnology of the Austrian Academy of Science (IMBA), Dr. Bohr-Gasse 3, 1030 Vienna, Austria

Stem cells need to balance self-renewal and differentiation for correct tissue development and homeostasis. Defects in this balance can lead to developmental defects or tumor formation. In recent years, mRNA splicing has emerged as an important mechanism regulating cell fate decisions. Here we address the role of the evolutionarily conserved splicing co-factor Barricade (Barc)/Tat-SF1/CUS2 in neural stem cell (neuroblast) lineage formation. We show that Barc is required for the generation of neurons during brain development by ensuring correct neural progenitor proliferation and differentiation. Barc associates with components of the U2 small nuclear ribonucleoprotein (snRNP) complex, and its depletion causes alternative splicing in the form of intron retention in a subset of genes. Using bioinformatics analysis and a cell culture-based splicing assay, we found that Barc-dependent introns share three major traits: they are short, GC rich and have weak 3' splice sites. Our results show that Barc, together with the U2 snRNP complex, plays an important role in regulating neural stem cell lineage progression during brain development and facilitates correct splicing of a subset of introns.
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http://dx.doi.org/10.1242/dev.152199DOI Listing
November 2017

Guided self-organization and cortical plate formation in human brain organoids.

Nat Biotechnol 2017 Jul 31;35(7):659-666. Epub 2017 May 31.

IMBA-Institute of Molecular Biotechnology of the Austrian Academy of Science, Vienna, Austria.

Three-dimensional cell culture models have either relied on the self-organizing properties of mammalian cells or used bioengineered constructs to arrange cells in an organ-like configuration. While self-organizing organoids excel at recapitulating early developmental events, bioengineered constructs reproducibly generate desired tissue architectures. Here, we combine these two approaches to reproducibly generate human forebrain tissue while maintaining its self-organizing capacity. We use poly(lactide-co-glycolide) copolymer (PLGA) fiber microfilaments as a floating scaffold to generate elongated embryoid bodies. Microfilament-engineered cerebral organoids (enCORs) display enhanced neuroectoderm formation and improved cortical development. Furthermore, reconstitution of the basement membrane leads to characteristic cortical tissue architecture, including formation of a polarized cortical plate and radial units. Thus, enCORs model the distinctive radial organization of the cerebral cortex and allow for the study of neuronal migration. Our data demonstrate that combining 3D cell culture with bioengineering can increase reproducibility and improve tissue architecture.
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http://dx.doi.org/10.1038/nbt.3906DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5824977PMC
July 2017

Fused cerebral organoids model interactions between brain regions.

Nat Methods 2017 Jul 10;14(7):743-751. Epub 2017 May 10.

Institute of Molecular Biotechnology of the Austrian Academy of Science (IMBA), Vienna, Austria.

Human brain development involves complex interactions between different regions, including long-distance neuronal migration or formation of major axonal tracts. Different brain regions can be cultured in vitro within 3D cerebral organoids, but the random arrangement of regional identities limits the reliable analysis of complex phenotypes. Here, we describe a coculture method combining brain regions of choice within one organoid tissue. By fusing organoids of dorsal and ventral forebrain identities, we generate a dorsal-ventral axis. Using fluorescent reporters, we demonstrate CXCR4-dependent GABAergic interneuron migration from ventral to dorsal forebrain and describe methodology for time-lapse imaging of human interneuron migration. Our results demonstrate that cerebral organoid fusion cultures can model complex interactions between different brain regions. Combined with reprogramming technology, fusions should offer researchers the possibility to analyze complex neurodevelopmental defects using cells from neurological disease patients and to test potential therapeutic compounds.
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http://dx.doi.org/10.1038/nmeth.4304DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5540177PMC
July 2017

The hope and the hype of organoid research.

Development 2017 03;144(6):938-941

Department of Genetics, University of Cambridge, Downing Site, Cambridge CB2 3EH, UK

The recent increase in organoid research has been met with great enthusiasm, as well as expectation, from the scientific community and the public alike. There is no doubt that this technology opens up a world of possibilities for scientific discovery in developmental biology as well as in translational research, but whether organoids can truly live up to this challenge is, for some, still an open question. In this Spotlight article, Meritxell Huch and Juergen Knoblich begin by discussing the exciting promise of organoid technology and give concrete examples of how this promise is starting to be realised. In the second part, Matthias Lutolf and Alfonso Martinez-Arias offer a careful and considered view of the state of the organoid field and its current limitations, and lay out the approach they feel is necessary to maximise the potential of organoid technology.
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http://dx.doi.org/10.1242/dev.150201DOI Listing
March 2017

Self-organized developmental patterning and differentiation in cerebral organoids.

EMBO J 2017 05 10;36(10):1316-1329. Epub 2017 Mar 10.

Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna Biocenter (VBC), Vienna, Austria

Cerebral organoids recapitulate human brain development at a considerable level of detail, even in the absence of externally added signaling factors. The patterning events driving this self-organization are currently unknown. Here, we examine the developmental and differentiative capacity of cerebral organoids. Focusing on forebrain regions, we demonstrate the presence of a variety of discrete ventral and dorsal regions. Clearing and subsequent 3D reconstruction of entire organoids reveal that many of these regions are interconnected, suggesting that the entire range of dorso-ventral identities can be generated within continuous neuroepithelia. Consistent with this, we demonstrate the presence of forebrain organizing centers that express secreted growth factors, which may be involved in dorso-ventral patterning within organoids. Furthermore, we demonstrate the timed generation of neurons with mature morphologies, as well as the subsequent generation of astrocytes and oligodendrocytes. Our work provides the methodology and quality criteria for phenotypic analysis of brain organoids and shows that the spatial and temporal patterning events governing human brain development can be recapitulated .
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http://dx.doi.org/10.15252/embj.201694700DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5430225PMC
May 2017

Human tissues in a dish: The research and ethical implications of organoid technology.

Science 2017 01;355(6322)

IMBA (Institute of Molecular Biotechnology of the Austrian Academy of Science), 1030 Vienna, Austria.

The ability to generate human tissues in vitro from stem cells has raised enormous expectations among the biomedical research community, patients, and the general public. These organoids enable studies of normal development and disease and allow the testing of compounds directly on human tissue. Organoids hold the promise to influence the entire innovation cycle in biomedical research. They affect fields that have been subjects of intense ethical debate, ranging from animal experiments and the use of embryonic or fetal human tissues to precision medicine, organoid transplantation, and gene therapy. However, organoid research also raises additional ethical questions that require reexamination and potential recalibration of ethical and legal policies. In this Review, we describe the current state of research and discuss the ethical implications of organoid technology.
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http://dx.doi.org/10.1126/science.aaf9414DOI Listing
January 2017

Induction of Expansion and Folding in Human Cerebral Organoids.

Cell Stem Cell 2017 03 29;20(3):385-396.e3. Epub 2016 Dec 29.

The Whitehead Institute for Biomedical Research, 9 Cambridge Center, Cambridge, MA 02142, USA; Department of Biology, Massachusetts Institute of Technology, 31 Ames Street, Cambridge, MA 02139, USA. Electronic address:

An expansion of the cerebral neocortex is thought to be the foundation for the unique intellectual abilities of humans. It has been suggested that an increase in the proliferative potential of neural progenitors (NPs) underlies the expansion of the cortex and its convoluted appearance. Here we show that increasing NP proliferation induces expansion and folding in an in vitro model of human corticogenesis. Deletion of PTEN stimulates proliferation and generates significantly larger and substantially folded cerebral organoids. This genetic modification allows sustained cell cycle re-entry, expansion of the progenitor population, and delayed neuronal differentiation, all key features of the developing human cortex. In contrast, Pten deletion in mouse organoids does not lead to folding. Finally, we utilized the expanded cerebral organoids to show that infection with Zika virus impairs cortical growth and folding. Our study provides new insights into the mechanisms regulating the structure and organization of the human cortex.
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http://dx.doi.org/10.1016/j.stem.2016.11.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6461394PMC
March 2017

Cerebral Organoids Recapitulate Epigenomic Signatures of the Human Fetal Brain.

Cell Rep 2016 12;17(12):3369-3384

Genomic Analysis Laboratory, The Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, CA 92037, USA; Howard Hughes Medical Institute, The Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, CA 92037, USA. Electronic address:

Organoids derived from human pluripotent stem cells recapitulate the early three-dimensional organization of the human brain, but whether they establish the epigenomic and transcriptional programs essential for brain development is unknown. We compared epigenomic and regulatory features in cerebral organoids and human fetal brain, using genome-wide, base resolution DNA methylome and transcriptome sequencing. Transcriptomic dynamics in organoids faithfully modeled gene expression trajectories in early-to-mid human fetal brains. We found that early non-CG methylation accumulation at super-enhancers in both fetal brain and organoids marks forthcoming transcriptional repression in the fully developed brain. Demethylated regions (74% of 35,627) identified during organoid differentiation overlapped with fetal brain regulatory elements. Interestingly, pericentromeric repeats showed widespread demethylation in multiple types of in vitro human neural differentiation models but not in fetal brain. Our study reveals that organoids recapitulate many epigenomic features of mid-fetal human brain and also identified novel non-CG methylation signatures of brain development.
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http://dx.doi.org/10.1016/j.celrep.2016.12.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5495578PMC
December 2016

Lab-Built Brains.

Sci Am 2016 12;316(1):26-31

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http://dx.doi.org/10.1038/scientificamerican0117-26DOI Listing
December 2016

MicroRNA-34/449 controls mitotic spindle orientation during mammalian cortex development.

EMBO J 2016 11 5;35(22):2386-2398. Epub 2016 Oct 5.

Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA) Vienna Biocenter (VBC), Vienna, Austria

Correct orientation of the mitotic spindle determines the plane of cellular cleavage and is crucial for organ development. In the developing cerebral cortex, spindle orientation defects result in severe neurodevelopmental disorders, but the precise mechanisms that control this important event are not fully understood. Here, we use a combination of high-content screening and mouse genetics to identify the miR-34/449 family as key regulators of mitotic spindle orientation in the developing cerebral cortex. By screening through all cortically expressed miRNAs in HeLa cells, we show that several members of the miR-34/449 family control mitotic duration and spindle rotation. Analysis of miR-34/449 knockout (KO) mouse embryos demonstrates significant spindle misorientation phenotypes in cortical progenitors, resulting in an excess of radial glia cells at the expense of intermediate progenitors and a significant delay in neurogenesis. We identify the junction adhesion molecule-A (JAM-A) as a key target for miR-34/449 in the developing cortex that might be responsible for those defects. Our data indicate that miRNA-dependent regulation of mitotic spindle orientation is crucial for cell fate specification during mammalian neurogenesis.
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http://dx.doi.org/10.15252/embj.201694056DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5109238PMC
November 2016

You Are What You Eat: Linking Metabolic Asymmetry and Cell Fate Choice.

Dev Cell 2016 May;37(3):206-8

Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), 1030 Vienna, Austria. Electronic address:

To defend against pathogens, activated immune cells must rapidly produce diverse lymphocyte subtypes. In a recent report in Nature, Verbist et al. (2016) describe how a regulatory loop acting between metabolic and transcriptional programs, centered around the asymmetric cell division machinery and the proto-oncogene c-Myc, establishes distinct T cell fates.
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http://dx.doi.org/10.1016/j.devcel.2016.04.017DOI Listing
May 2016

Human cerebral organoids recapitulate gene expression programs of fetal neocortex development.

Proc Natl Acad Sci U S A 2015 Dec 7;112(51):15672-7. Epub 2015 Dec 7.

Max Planck Institute for Evolutionary Anthropology, Department of Evolutionary Genetics, 04103 Leipzig, Germany; Max Planck Institute of Molecular Cell Biology and Genetics, 01307 Dresden, Germany;

Cerebral organoids-3D cultures of human cerebral tissue derived from pluripotent stem cells-have emerged as models of human cortical development. However, the extent to which in vitro organoid systems recapitulate neural progenitor cell proliferation and neuronal differentiation programs observed in vivo remains unclear. Here we use single-cell RNA sequencing (scRNA-seq) to dissect and compare cell composition and progenitor-to-neuron lineage relationships in human cerebral organoids and fetal neocortex. Covariation network analysis using the fetal neocortex data reveals known and previously unidentified interactions among genes central to neural progenitor proliferation and neuronal differentiation. In the organoid, we detect diverse progenitors and differentiated cell types of neuronal and mesenchymal lineages and identify cells that derived from regions resembling the fetal neocortex. We find that these organoid cortical cells use gene expression programs remarkably similar to those of the fetal tissue to organize into cerebral cortex-like regions. Our comparison of in vivo and in vitro cortical single-cell transcriptomes illuminates the genetic features underlying human cortical development that can be studied in organoid cultures.
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http://dx.doi.org/10.1073/pnas.1520760112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4697386PMC
December 2015

Mammary Stem Cell Self-Renewal Is Regulated by Slit2/Robo1 Signaling through SNAI1 and mINSC.

Cell Rep 2015 Oct 1;13(2):290-301. Epub 2015 Oct 1.

Department of Molecular, Cell and Developmental Biology, University of California, Santa Cruz, Santa Cruz, CA 95064, USA. Electronic address:

Tissue homeostasis requires somatic stem cell maintenance; however, mechanisms regulating this process during organogenesis are not well understood. Here, we identify asymmetrically renewing basal and luminal stem cells in the mammary end bud. We demonstrate that SLIT2/ROBO1 signaling regulates the choice between self-renewing asymmetric cell divisions (ACDs) and expansive symmetric cell divisions (SCDs) by governing Inscuteable (mInsc), a key member of the spindle orientation machinery, through the transcription factor Snail (SNAI1). Loss of SLIT2/ROBO1 signaling increases SNAI1 in the nucleus. Overexpression of SNAI1 increases mInsc expression, an effect that is inhibited by SLIT2 treatment. Increased mInsc does not change cell proliferation in the mammary gland (MG) but instead causes more basal cap cells to divide via SCD, at the expense of ACD, leading to more stem cells and larger outgrowths. Together, our studies provide insight into how the number of mammary stem cells is regulated by the extracellular cue SLIT2.
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http://dx.doi.org/10.1016/j.celrep.2015.09.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4606466PMC
October 2015

The TRIM-NHL protein Brat promotes axon maintenance by repressing src64B expression.

J Neurosci 2014 Oct;34(41):13855-64

Institute of Biology Valrose, University of Nice Sophia Antipolis, CNRS UMR7277, INSERM U1091, 06108 Nice Cedex 2, France and

The morphology and the connectivity of neuronal structures formed during early development must be actively maintained as the brain matures. Although impaired axon stability is associated with the progression of various neurological diseases, relatively little is known about the factors controlling this process. We identified Brain tumor (Brat), a conserved member of the TRIM-NHL family of proteins, as a new regulator of axon maintenance in Drosophila CNS. Brat function is dispensable for the initial growth of Mushroom Body axons, but is required for the stabilization of axon bundles. We found that Brat represses the translation of src64B, an upstream regulator of a conserved Rho-dependent pathway previously shown to promote axon retraction. Furthermore, brat phenotypes are phenocopied by src64B overexpression, and partially suppressed by reducing the levels of src64B or components of the Rho pathway, suggesting that brat promotes axon maintenance by downregulating the levels of Src64B. Finally, Brat regulates brain connectivity via its NHL domain, but independently of its previously described partners Nanos, Pumilio, and d4EHP. Thus, our results uncover a novel post-transcriptional regulatory mechanism that controls the maintenance of neuronal architecture by tuning the levels of a conserved rho-dependent signaling pathway.
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http://dx.doi.org/10.1523/JNEUROSCI.3285-13.2014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6608379PMC
October 2014

Generation of cerebral organoids from human pluripotent stem cells.

Nat Protoc 2014 Oct 4;9(10):2329-40. Epub 2014 Sep 4.

Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna, Austria.

Human brain development exhibits several unique aspects, such as increased complexity and expansion of neuronal output, that have proven difficult to study in model organisms. As a result, in vitro approaches to model human brain development and disease are an intense area of research. Here we describe a recently established protocol for generating 3D brain tissue, so-called cerebral organoids, which closely mimics the endogenous developmental program. This method can easily be implemented in a standard tissue culture room and can give rise to developing cerebral cortex, ventral telencephalon, choroid plexus and retinal identities, among others, within 1-2 months. This straightforward protocol can be applied to developmental studies, as well as to the study of a variety of human brain diseases. Furthermore, as organoids can be maintained for more than 1 year in long-term culture, they also have the potential to model later events such as neuronal maturation and survival.
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http://dx.doi.org/10.1038/nprot.2014.158DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4160653PMC
October 2014

Ecdysone and mediator change energy metabolism to terminate proliferation in Drosophila neural stem cells.

Cell 2014 Aug;158(4):874-888

Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), 1030 Vienna, Austria. Electronic address:

Stem cells are highly abundant during early development but become a rare population in most adult organs. The molecular mechanisms causing stem cells to exit proliferation at a specific time are not well understood. Here, we show that changes in energy metabolism induced by the steroid hormone ecdysone and the Mediator initiate an irreversible cascade of events leading to cell-cycle exit in Drosophila neural stem cells. We show that the timely induction of oxidative phosphorylation and the mitochondrial respiratory chain are required in neuroblasts to uncouple the cell cycle from cell growth. This results in a progressive reduction in neuroblast cell size and ultimately in terminal differentiation. Brain tumor mutant neuroblasts fail to undergo this shrinkage process and continue to proliferate until adulthood. Our findings show that cell size control can be modified by systemic hormonal signaling and reveal a unique connection between metabolism and proliferation in stem cells.
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http://dx.doi.org/10.1016/j.cell.2014.06.024DOI Listing
August 2014

The conserved discs-large binding partner Banderuola regulates asymmetric cell division in Drosophila.

Curr Biol 2014 Aug 31;24(16):1811-25. Epub 2014 Jul 31.

Institute of Molecular Biotechnology of the Austrian Academy of Sciences, Vienna 1030, Austria. Electronic address:

Background: Asymmetric cell division (ACD) is a key process that allows different cell types to be generated at precisely defined times and positions. In Drosophila, neural precursor cells rely heavily on ACD to generate the different cell types in the nervous system. A conserved protein machinery that regulates ACD has been identified in Drosophila, but how this machinery acts to allow the establishment of differential cell fates is not entirely understood.

Results: To identify additional proteins required for ACD, we have carried out an in vivo live imaging RNAi screen for genes affecting the asymmetric segregation of Numb in Drosophila sensory organ precursor cells. We identify Banderuola (Bnd), an essential regulator of cell polarization, spindle orientation, and asymmetric protein localization in Drosophila neural precursor cells. Genetic and biochemical experiments show that Bnd acts together with the membrane-associated tumor suppressor Discs-large (Dlg) to establish antagonistic cortical domains during ACD. Inhibiting Bnd strongly enhances the dlg phenotype, causing massive brain tumors upon knockdown of both genes. Because the mammalian homologs of Bnd and Dlg are interacting as well, Bnd function might be conserved in vertebrates, and it might also regulate cell polarity in higher organisms.

Conclusions: Bnd is a novel regulator of ACD in different types of cells. Our data place Bnd at the top of the hierarchy of the factors involved in ACD, suggesting that its main function is to mediate the localization and function of the Dlg tumor suppressor. Bnd has an antioncogenic function that is redundant with Dlg, and the physical interaction between the two proteins is conserved in evolution.
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http://dx.doi.org/10.1016/j.cub.2014.06.059DOI Listing
August 2014

Organogenesis in a dish: modeling development and disease using organoid technologies.

Science 2014 Jul 17;345(6194):1247125. Epub 2014 Jul 17.

IMBA-Institute of Molecular Biotechnology of the Austrian Academy of Science Vienna 1030, Austria.

Classical experiments performed half a century ago demonstrated the immense self-organizing capacity of vertebrate cells. Even after complete dissociation, cells can reaggregate and reconstruct the original architecture of an organ. More recently, this outstanding feature was used to rebuild organ parts or even complete organs from tissue or embryonic stem cells. Such stem cell-derived three-dimensional cultures are called organoids. Because organoids can be grown from human stem cells and from patient-derived induced pluripotent stem cells, they have the potential to model human development and disease. Furthermore, they have potential for drug testing and even future organ replacement strategies. Here, we summarize this rapidly evolving field and outline the potential of organoid technology for future biomedical research.
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http://dx.doi.org/10.1126/science.1247125DOI Listing
July 2014

Par3-mInsc and Gαi3 cooperate to promote oriented epidermal cell divisions through LGN.

Nat Cell Biol 2014 Aug 13;16(8):758-69. Epub 2014 Jul 13.

Howard Hughes Medical Institute, Laboratory of Mammalian Cell Biology &Development, The Rockefeller University, 1230 York Avenue, Box 300, New York, New York 10065, USA.

Asymmetric cell divisions allow stem cells to balance proliferation and differentiation. During embryogenesis, murine epidermis expands rapidly from a single layer of unspecified basal layer progenitors to a stratified, differentiated epithelium. Morphogenesis involves perpendicular (asymmetric) divisions and the spindle orientation protein LGN, but little is known about how the apical localization of LGN is regulated. Here, we combine conventional genetics and lentiviral-mediated in vivo RNAi to explore the functions of the LGN-interacting proteins Par3, mInsc and Gαi3. Whereas loss of each gene alone leads to randomized division angles, combined loss of Gnai3 and mInsc causes a phenotype of mostly planar divisions, akin to loss of LGN. These findings lend experimental support for the hitherto untested model that Par3-mInsc and Gαi3 act cooperatively to polarize LGN and promote perpendicular divisions. Finally, we uncover a developmental switch between delamination-driven early stratification and spindle-orientation-dependent differentiation that occurs around E15, revealing a two-step mechanism underlying epidermal maturation.
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http://dx.doi.org/10.1038/ncb3001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4159251PMC
August 2014

Dachsous-dependent asymmetric localization of spiny-legs determines planar cell polarity orientation in Drosophila.

Cell Rep 2014 Jul 3;8(2):610-21. Epub 2014 Jul 3.

Research Center for Biosignal, Akita University, Akita 010-8543, Japan; Department of Cell Biology and Morphology, Akita University Graduate School of Medicine, Akita 010-8543, Japan; Global COE program, Gunma University and Akita University, Akita 010-8543, Japan. Electronic address:

In Drosophila, planar cell polarity (PCP) molecules such as Dachsous (Ds) may function as global directional cues directing the asymmetrical localization of PCP core proteins such as Frizzled (Fz). However, the relationship between Ds asymmetry and Fz localization in the eye is opposite to that in the wing, thereby causing controversy regarding how these two systems are connected. Here, we show that this relationship is determined by the ratio of two Prickle (Pk) isoforms, Pk and Spiny-legs (Sple). Pk and Sple form different complexes with distinct subcellular localizations. When the amount of Sple is increased in the wing, Sple induces a reversal of PCP using the Ds-Ft system. A mathematical model demonstrates that Sple is the key regulator connecting Ds and the core proteins. Our model explains the previously noted discrepancies in terms of the differing relative amounts of Sple in the eye and wing.
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http://dx.doi.org/10.1016/j.celrep.2014.06.009DOI Listing
July 2014