Mouse Molecular Embryology

In situ hybridization is a powerful method for detecting endogenous mRNA sequences in morphologically preserved samples. We provide in situ hybridization methods, which are specifi cally optimized for mouse embryonic samples as whole mounts and section tissues. Additionally, β-Galactosidase (β-gal) is a popular reporter for detecting the expression of endogenous or exogenous genes. We reveal that 6-chloro-3indoxyl-βD -galactopyranoside (S-gal) is a more sensitive substrate for β-gal activity than 5-bromo-4chloro-3-indolyl-βD -galactoside (X-gal). S-gal is advantageous where β-gal activity is limited including early stage mouse embryos. As a result of the increased sensitivity as well as the color compatibility of S-gal, we successfully combined β-gal staining using S-gal with in situ hybridization using DIG-labeled probes in both whole mounts and sections.

[1]  E. S. Hunter,et al.  Apoptosis and morphology in mouse embryos by confocal laser scanning microscopy. , 1999, Methods.

[2]  T. Kanda,et al.  Histone–GFP fusion protein enables sensitive analysis of chromosome dynamics in living mammalian cells , 1998, Current Biology.

[3]  P. Kulesa,et al.  An in vivo comparison of photoactivatable fluorescent proteins in an avian embryo model , 2007, Developmental dynamics : an official publication of the American Association of Anatomists.

[4]  A. Hadjantonakis,et al.  In vivo imaging and differential localization of lipid‐modified GFP‐variant fusions in embryonic stem cells and mice , 2006, Genesis.

[5]  K. McCall,et al.  Cell-autonomous requirement for DNaseII in non-apoptotic cell death , 2009, Cell Death and Differentiation.

[6]  Fast and Precise Protein Tracking Using Repeated Reversible Photoactivation , 2006, Traffic.

[7]  S. Shi,et al.  Kaede-centrin1 labeling of mother and daughter centrosomes in mammalian neocortical neural progenitors. , 2010, Current protocols in stem cell biology.

[8]  Konstantin A Lukyanov,et al.  Photoswitchable cyan fluorescent protein for protein tracking , 2004, Nature Biotechnology.

[9]  G. Salvesen,et al.  The apoptosome: signalling platform of cell death , 2007, Nature Reviews Molecular Cell Biology.

[10]  B. Conradt.  Genetic control of programmed cell death during animal development. , 2009, Annual review of genetics.

[11]  M. Matsui,et al.  In vivo analysis of autophagy in response to nutrient starvation using transgenic mice expressing a fluorescent autophagosome marker. , 2003, Molecular biology of the cell.

[12]  Y. Tsujimoto,et al.  Another way to die: autophagic programmed cell death , 2005, Cell Death and Differentiation.

[13]  A. Miyawaki,et al.  Regulated Fast Nucleocytoplasmic Shuttling Observed by Reversible Protein Highlighting , 2004, Science.

[14]  M. Dickinson,et al.  Using a histone yellow fluorescent protein fusion for tagging and tracking endothelial cells in ES cells and mice , 2005, Genesis.

[15]  D. Green,et al.  How do BCL-2 proteins induce mitochondrial outer membrane permeabilization? , 2008, Trends in cell biology.

[16]  J. Visser,et al.  Involvement of a matrix metalloproteinase in MIS-induced cell death during urogenital development. , 2002, Development.

[17]  C. Reutelingsperger,et al.  Annexin V-affinity assay: a review on an apoptosis detection system based on phosphatidylserine exposure. , 1998, Cytometry.

[18]  J. Cunnick,et al.  Acridine orange differential staining of total DNA and RNA in normal and galactosemic lens epithelial cells in culture using flow cytometry. , 1995, Current eye research.

[19]  Peter Dedecker,et al.  Photo-induced protonation/deprotonation in the GFP-like fluorescent protein Dronpa: mechanism responsible for the reversible photoswitching , 2006, Photochemical & photobiological sciences : Official journal of the European Photochemistry Association and the European Society for Photobiology.

[20]  Michael Z. Lin,et al.  Autofluorescent proteins with excitation in the optical window for intravital imaging in mammals. , 2009, Chemistry & biology.

[21]  J. Segalen,et al.  Ultrastructure of mullerian and wolffian ducts of fetal rabbit in vivo and in organ culture. , 1994, Tissue & cell.

[22]  Takeshi Tokuhisa,et al.  The role of autophagy during the early neonatal starvation period , 2004, Nature.

[23]  S. Lukyanov,et al.  Natural Animal Coloration Can Be Determined by a Nonfluorescent Green Fluorescent Protein Homolog* , 2000, The Journal of Biological Chemistry.

[24]  T. P. Neufeld,et al.  The cell biology of autophagy in metazoans: a developing story , 2008, Development.

[25]  T. Riss,et al.  Caspase activity assays. , 2008, Methods in molecular biology.

[26]  D. Segretain,et al.  Gap junction turnover is achieved by the internalization of small endocytic double-membrane vesicles. , 2009, Molecular biology of the cell.

[27]  Z. Darżynkiewicz,et al.  Flow cytometric detection of apoptosis: comparison of the assays of in situ DNA degradation and chromatin changes. , 1994, Cytometry.

[28]  L. Luo,et al.  A global double‐fluorescent Cre reporter mouse , 2007, Genesis.

[29]  P. Donahoe,et al.  Programmed cell death in the Müllerian duct induced by Müllerian inhibiting substance. , 1977, The American journal of anatomy.

[30]  Vladislav V Verkhusha,et al.  Conversion of the monomeric red fluorescent protein into a photoactivatable probe. , 2005, Chemistry & biology.

[31]  A. Ting,et al.  Fluorescent probes for super-resolution imaging in living cells , 2008, Nature Reviews Molecular Cell Biology.

[32]  J. Wiedenmann,et al.  A photoactivatable marker protein for pulse-chase imaging with superresolution , 2010, Nature Methods.

[33]  M. S. Cooper,et al.  Visualizing morphogenesis in transgenic zebrafish embryos using BODIPY TR methyl ester dye as a vital counterstain for GFP , 2005, Developmental dynamics : an official publication of the American Association of Anatomists.

[34]  J. Rossant,et al.  Spatial and temporal patterns of ERK signaling during mouse embryogenesis , 2003, Development.

[35]  M. Affolter,et al.  Genetic Control of Cell Intercalation during Tracheal Morphogenesis in Drosophila , 2004, Current Biology.

[36]  Roger Y. Tsien,et al.  Seeing the Machinery of Live Cells , 1998, Science.

[37]  S. Lukyanov,et al.  Chromophore Environment Provides Clue to “Kindling Fluorescent Protein” Riddle* , 2003, The Journal of Biological Chemistry.

[38]  Masaaki Komatsu,et al.  Loss of autophagy in the central nervous system causes neurodegeneration in mice , 2006, Nature.

[39]  A. Miyawaki,et al.  An optical marker based on the UV-induced green-to-red photoconversion of a fluorescent protein , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[40]  H. Ichijo,et al.  Molecular mechanisms of the decision between life and death: regulation of apoptosis by apoptosis signal-regulating kinase 1. , 2001, Journal of biochemistry.

[41]  Atsushi Miyawaki,et al.  Monitoring cellular movement in vivo with photoconvertible fluorescence protein “Kaede” transgenic mice , 2008, Proceedings of the National Academy of Sciences.

[42]  Michael Brand,et al.  Lineage restriction maintains a stable organizer cell population at the zebrafish midbrain-hindbrain boundary , 2005, Development.

[43]  Paul M Kulesa,et al.  Neural crest invasion is a spatially-ordered progression into the head with higher cell proliferation at the migratory front as revealed by the photoactivatable protein, KikGR. , 2008, Developmental biology.

[44]  L. Penn,et al.  The Oscar-worthy role of Myc in apoptosis. , 2006, Seminars in cancer biology.

[45]  I. Weissman,et al.  Clonal analysis of mouse development reveals a polyclonal origin for yolk sac blood islands. , 2006, Developmental cell.

[46]  A. Hadjantonakis,et al.  Photomodulatable fluorescent proteins for imaging cell dynamics and cell fate , 2009, Organogenesis.

[47]  N. Mizushima,et al.  Methods in Mammalian Autophagy Research , 2010, Cell.

[48]  L. Covarrubias,et al.  Death is the major fate of medial edge epithelial cells and the cause of basal lamina degradation during palatogenesis , 2004, Development.

[49]  W. Garrard,et al.  Roles of the Major Apoptotic Nuclease-DNA Fragmentation Factor-in Biology and Disease , 2008, Cellular and Molecular Life Sciences.

[50]  Guido Kroemer,et al.  Caspase-independent cell death , 2005, Nature Medicine.

[51]  P. Clarke,et al.  Developmental cell death: morphological diversity and multiple mechanisms , 2004, Anatomy and Embryology.

[52]  Konstantin A Lukyanov,et al.  Near-infrared fluorescent proteins , 2010, Nature Methods.

[53]  I. Darby,et al.  Histochemical localization of apoptosis with in situ labeling of fragmented DNA. , 2006, Methods in molecular biology.

[54]  S. Ben‐Sasson,et al.  Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation , 1992, The Journal of cell biology.

[55]  K. Ravichandran,et al.  Engulfment of apoptotic cells: signals for a good meal , 2007, Nature Reviews Immunology.

[56]  Yoko Kurotaki,et al.  Blastocyst Axis Is Specified Independently of Early Cell Lineage But Aligns with the ZP Shape , 2007, Science.

[57]  R. W. Draft,et al.  Transgenic strategies for combinatorial expression of fluorescent proteins in the nervous system , 2007, Nature.

[58]  Sonja Nowotschin,et al.  Use of KikGR a photoconvertible green-to-red fluorescent protein for cell labeling and lineage analysis in ES cells and mouse embryos , 2009, BMC Developmental Biology.

[59]  J. Rodríguez-León,et al.  Retinoic acid regulates programmed cell death through BMP signalling , 1999, Nature Cell Biology.

[60]  Patrick R. Griffin,et al.  Identification and inhibition of the ICE/CED-3 protease necessary for mammalian apoptosis , 1995, Nature.

[61]  K. Roth,et al.  In situ immunodetection of activated caspase-3 in apoptotic neurons in the developing nervous system , 1998, Cell Death and Differentiation.

[62]  R. Lockshin,et al.  Delayed internucleosomal DNA fragmentation in programmed cell death , 1993, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[63]  Peter Dedecker,et al.  Reversible single-molecule photoswitching in the GFP-like fluorescent protein Dronpa. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[64]  George H. Patterson,et al.  A Photoactivatable GFP for Selective Photolabeling of Proteins and Cells , 2002, Science.

[65]  Paul Martin,et al.  Dynamic Analysis of Actin Cable Function during Drosophila Dorsal Closure , 2002, Current Biology.

[66]  S. Lukyanov,et al.  Tracking intracellular protein movements using photoswitchable fluorescent proteins PS-CFP2 and Dendra2 , 2007, Nature Protocols.

[67]  D. Vaux,et al.  The mitochondrial death squad: hardened killers or innocent bystanders? , 2005, Current opinion in cell biology.

[68]  D. Vaux.  Toward an understanding of the molecular mechanisms of physiological cell death. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[69]  Z. Darżynkiewicz.  Differential staining of DNA and RNA in intact cells and isolated cell nuclei with acridine orange. , 1990, Methods in cell biology.

[70]  J. Hurlé,et al.  Programmed cell death in the developing limb. , 2002, The International journal of developmental biology.

[71]  M. Field,et al.  Structural characterization of IrisFP, an optical highlighter undergoing multiple photo-induced transformations , 2008, Proceedings of the National Academy of Sciences.

[72]  Guido Kroemer,et al.  Self-eating and self-killing: crosstalk between autophagy and apoptosis , 2007, Nature Reviews Molecular Cell Biology.

[73]  Anna-Katerina Hadjantonakis,et al.  Dynamic in vivo imaging and cell tracking using a histone fluorescent protein fusion in mice , 2004, BMC biotechnology.

[74]  H. Lomelí,et al.  Reactive oxygen species participate in the control of mouse embryonic cell death. , 1998, Experimental cell research.

[75]  Frank Bradke,et al.  Growth cone‐like waves transport actin and promote axonogenesis and neurite branching , 2009, Developmental neurobiology.

[76]  F. Hughes,et al.  Biochemical identification of apoptosis (programmed cell death) in granulosa cells: evidence for a potential mechanism underlying follicular atresia. , 1991, Endocrinology.

[77]  Sharad Kumar,et al.  Caspase function in programmed cell death , 2007, Cell Death and Differentiation.

[78]  J. Briscoe,et al.  A fluorescent reporter of caspase activity for live imaging , 2008, Proceedings of the National Academy of Sciences.

[79]  Semi-rational engineering of a green-emitting coral fluorescent protein into an efficient highlighter. , 2005 .

[80]  Kristin L. Hazelwood,et al.  A bright and photostable photoconvertible fluorescent protein for fusion tags , 2009, Nature Methods.

[81]  S. Korsmeyer,et al.  Mouse models of cell death , 2001, Nature Genetics.

[82]  J. Lippincott-Schwartz,et al.  Erratum: Photoactivatable mCherry for high-resolution two-color fluorescence microscopy , 2009, Nature Methods.

[83]  R. Lansford,et al.  Four-color, 4-D time-lapse confocal imaging of chick embryos. , 2006, BioTechniques.

[84]  S. Castro-Obregón,et al.  Motoneuronal death during spinal cord development is mediated by oxidative stress , 2005, Cell Death and Differentiation.

[85]  Atsushi Miyawaki,et al.  mKikGR, a Monomeric Photoswitchable Fluorescent Protein , 2008, PloS one.

[86]  K. Finegan,et al.  Regulation of neuronal survival by the extracellular signal-regulated protein kinase 5 , 2008, Cell Death and Differentiation.

[87]  Y. Gañán,et al.  Internucleosomal DNA fragmentation and programmed cell death (apoptosis) in the interdigital tissue of the embryonic chick leg bud. , 1993, Journal of cell science.

[88]  Atsushi Miyawaki,et al.  Photo-induced peptide cleavage in the green-to-red conversion of a fluorescent protein. , 2003, Molecular cell.

[89]  Philippe Soriano,et al.  Evolutionary Divergence of Platelet-Derived Growth Factor Alpha Receptor Signaling Mechanisms , 2003, Molecular and Cellular Biology.

[90]  M. Kurokawa,et al.  Caspases and Kinases in a Death Grip , 2009, Cell.

[91]  J. Wiedenmann,et al.  A green to red photoconvertible protein as an analyzing tool for early vertebrate development , 2007, Developmental dynamics : an official publication of the American Association of Anatomists.

[92]  S. Castro-Obregón,et al.  Progressive interdigital cell death: regulation by the antagonistic interaction between fibroblast growth factor 8 and retinoic acid , 2009, Development.

[93]  M. Raff,et al.  Is programmed cell death required for neural tube closure? , 1997, Current Biology.

[94]  Y. Mishina,et al.  BMP signals control limb bud interdigital programmed cell death by regulating FGF signaling , 2007, Development.

[95]  D. Green,et al.  The BCL-2 family reunion. , 2010, Molecular cell.

[96]  V. Fadok,et al.  Appearance of Phosphatidylserine on Apoptotic Cells Requires Calcium-mediated Nonspecific Flip-Flop and Is Enhanced by Loss of the Aminophospholipid Translocase* , 1997, The Journal of Biological Chemistry.

[97]  J. M. Rogers,et al.  Confocal laser scanning microscopy of apoptosis in organogenesis-stage mouse embryos. , 1998, Cytometry.

[98]  Eric H. Baehrecke,et al.  How death shapes life during development , 2002, Nature Reviews Molecular Cell Biology.

[99]  Christian Eggeling,et al.  Breaking the diffraction barrier in fluorescence microscopy at low light intensities by using reversibly photoswitchable proteins. , 2005, Proceedings of the National Academy of Sciences of the United States of America.