A new approach to manipulate the fate of single neural stem cells in tissue

A challenge in the field of neural stem cell biology is the mechanistic dissection of single stem cell behavior in tissue. Although such behavior can be tracked by sophisticated imaging techniques, current methods of genetic manipulation do not allow researchers to change the level of a defined gene product on a truly acute time scale and are limited to very few genes at a time. To overcome these limitations, we established microinjection of neuroepithelial/radial glial cells (apical progenitors) in organotypic slice culture of embryonic mouse brain. Microinjected apical progenitors showed cell cycle parameters that were indistinguishable to apical progenitors in utero, underwent self-renewing divisions and generated neurons. Microinjection of single genes, recombinant proteins or complex mixtures of RNA was found to elicit acute and defined changes in apical progenitor behavior and progeny fate. Thus, apical progenitor microinjection provides a new approach to acutely manipulating single neural stem and progenitor cells in tissue.

[1]  R. Pepperkok,et al.  An antibody against secretogranin I (chromogranin B) is packaged into secretory granules , 1989, The Journal of cell biology.

[2]  Shiaoching Gong,et al.  A gene expression atlas of the central nervous system based on bacterial artificial chromosomes , 2003, Nature.

[3]  W. Huttner,et al.  Cortical progenitor expansion, self-renewal and neurogenesis—a polarized perspective , 2011, Current Opinion in Neurobiology.

[4]  S. Pollard,et al.  Neural stem cells, neurons, and glia. , 2006, Methods in enzymology.

[5]  Federico Calegari,et al.  Live Imaging at the Onset of Cortical Neurogenesis Reveals Differential Appearance of the Neuronal Phenotype in Apical versus Basal Progenitor Progeny , 2008, PloS one.

[6]  W. Huttner,et al.  Asymmetric distribution of the apical plasma membrane during neurogenic divisions of mammalian neuroepithelial cells , 2004, The EMBO journal.

[7]  H. Okano,et al.  Asymmetric Inheritance of Radial Glial Fibers by Cortical Neurons , 2001, Neuron.

[8]  Arnold Kriegstein,et al.  The glial nature of embryonic and adult neural stem cells. , 2009, Annual review of neuroscience.

[9]  C. Englund,et al.  Intermediate neuronal progenitors (basal progenitors) produce pyramidal-projection neurons for all layers of cerebral cortex. , 2009, Cerebral cortex.

[10]  R. Pepperkok,et al.  Performance of an automated system for capillary microinjection into living cells. , 1988, Journal of biochemical and biophysical methods.

[11]  W. Denk,et al.  Targeted patch-clamp recordings and single-cell electroporation of unlabeled neurons in vivo , 2008, Nature Methods.

[12]  A. Kriegstein,et al.  Neurogenic radial glia in the outer subventricular zone of human neocortex , 2010, Nature.

[13]  Austin G Smith,et al.  Niche-Independent Symmetrical Self-Renewal of a Mammalian Tissue Stem Cell , 2005, PLoS biology.

[14]  A. Hadjantonakis,et al.  Tbr2 Directs Conversion of Radial Glia into Basal Precursors and Guides Neuronal Amplification by Indirect Neurogenesis in the Developing Neocortex , 2008, Neuron.

[15]  W. Huttner,et al.  Brca1 is required for embryonic development of the mouse cerebral cortex to normal size by preventing apoptosis of early neural progenitors , 2009, Development.

[16]  V. Margotta,et al.  Hyaluronate receptor CD44 is expressed by astrocytes in the adult chicken and in astrocyte cell precursors in early development of the chick spinal cord. , 1999, European journal of histochemistry : EJH.

[17]  Federico Calegari,et al.  Neural stem and progenitor cells shorten S-phase on commitment to neuron production , 2011, Nature communications.

[18]  Winfried Denk,et al.  Neurons arise in the basal neuroepithelium of the early mammalian telencephalon: a major site of neurogenesis. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[19]  Elly M. Tanaka,et al.  Cells keep a memory of their tissue origin during axolotl limb regeneration , 2009, Nature.

[20]  R. Pepperkok,et al.  Computer-Automated Capillary Microinjection of Macromolecules into Living Cells , 1994 .

[21]  A. Kriegstein,et al.  Development and Evolution of the Human Neocortex , 2011, Cell.

[22]  H. Ueda,et al.  Single-cell gene profiling defines differential progenitor subclasses in mammalian neurogenesis , 2008, Development.

[23]  J. Clarke,et al.  Monitoring neural progenitor fate through multiple rounds of division in an intact vertebrate brain , 2003, Development.

[24]  T. Weissman,et al.  Neurons derived from radial glial cells establish radial units in neocortex , 2001, Nature.

[25]  J. Wolff,et al.  The effect of cell division on the cellular dynamics of microinjected DNA and dextran. , 2002, Molecular therapy : the journal of the American Society of Gene Therapy.

[26]  J. Clarke,et al.  Neurons derive from the more apical daughter in asymmetric divisions in the zebrafish neural tube , 2010, Nature Neuroscience.

[27]  David J Cappelleri,et al.  Transcriptome transfer produces a predictable cellular phenotype , 2009, Proceedings of the National Academy of Sciences.

[28]  M. Ogawa,et al.  Periventricular notch activation and asymmetric Ngn2 and Tbr2 expression in pair-generated neocortical daughter cells , 2009, Molecular and Cellular Neuroscience.

[29]  M. A. García-Cabezas,et al.  A role for intermediate radial glia in the tangential expansion of the mammalian cerebral cortex. , 2011, Cerebral cortex.

[30]  Tomoko Nakanishi,et al.  ‘Green mice’ as a source of ubiquitous green cells , 1997, FEBS letters.

[31]  M. Götz,et al.  The cell biology of neurogenesis , 2006, International Journal of Developmental Neuroscience.

[32]  W. Huttner,et al.  Basolateral rather than apical primary cilia on neuroepithelial cells committed to delamination , 2012, Development.

[33]  W. Huttner,et al.  The cell biology of neural stem and progenitor cells and its significance for their proliferation versus differentiation during mammalian brain development. , 2008, Current opinion in cell biology.

[34]  Ying Liu,et al.  CD44 expression identifies astrocyte-restricted precursor cells. , 2004, Developmental biology.

[35]  S. Pääbo,et al.  Insulinoma-Associated 1 Has a Panneurogenic Role and Promotes the Generation and Expansion of Basal Progenitors in the Developing Mouse Neocortex , 2008, Neuron.

[36]  A. Hadjantonakis,et al.  Eomes::GFP—a tool for live imaging cells of the trophoblast, primitive streak, and telencephalon in the mouse embryo , 2007, Genesis.

[37]  W. Huttner,et al.  Selective Lengthening of the Cell Cycle in the Neurogenic Subpopulation of Neural Progenitor Cells during Mouse Brain Development , 2005, The Journal of Neuroscience.

[38]  V. Caviness,et al.  The cell cycle of the pseudostratified ventricular epithelium of the embryonic murine cerebral wall , 1995, The Journal of neuroscience : the official journal of the Society for Neuroscience.

[39]  U. Heinzmann,et al.  Effects of Wnt1 signaling on proliferation in the developing mid-/hindbrain region , 2004, Molecular and Cellular Neuroscience.

[40]  Elena Taverna,et al.  Neural Progenitor Nuclei IN Motion , 2010, Neuron.

[41]  R. Pepperkok,et al.  Automatic microinjection system facilitates detection of growth inhibitory mRNA. , 1988, Proceedings of the National Academy of Sciences of the United States of America.

[42]  T. Inoue,et al.  Gene transfer into cultured mammalian embryos by electroporation. , 2001, Methods.

[43]  S. Itohara,et al.  The Rho-GTPase cdc42 regulates neural progenitor fate at the apical surface , 2006, Nature Neuroscience.

[44]  Masaharu Ogawa,et al.  Morphological asymmetry in dividing retinal progenitor cells , 2003, Development, growth & differentiation.

[45]  M. Götz,et al.  Prospective isolation of functionally distinct radial glial subtypes—Lineage and transcriptome analysis , 2008, Molecular and Cellular Neuroscience.

[46]  Magdalena Götz,et al.  In vivo fate mapping and expression analysis reveals molecular hallmarks of prospectively isolated adult neural stem cells. , 2010, Cell stem cell.

[47]  S. Narumiya,et al.  Rho GTPases in animal cell mitosis. , 2006, Current opinion in cell biology.

[48]  J. Fish,et al.  OSVZ progenitors of human and ferret neocortex are epithelial-like and expand by integrin signaling , 2010, Nature Neuroscience.

[49]  C. Englund,et al.  Pax6, Tbr2, and Tbr1 Are Expressed Sequentially by Radial Glia, Intermediate Progenitor Cells, and Postmitotic Neurons in Developing Neocortex , 2005, The Journal of Neuroscience.

[50]  Andreas T Schaefer,et al.  Transfection via whole-cell recording in vivo: bridging single-cell physiology, genetics and connectomics , 2011, Nature Neuroscience.