The Guide to Investigation of Mouse Pregnancy

The Guide to Investigation of Mouse Pregnancy

1st Edition - December 9, 2013

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  • Editors: Anne Croy, Aureo Yamada, Francesco DeMayo, S. Lee Adamson
  • Hardcover ISBN: 9780123944450
  • eBook ISBN: 9780123947949

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Description

The Guide to Investigation of Mouse Pregnancy is the first publication to cover the mouse placenta or the angiogenic tree the mother develops to support the placenta. This much-needed resource covers monitoring of the cardiovascular system, gestational programming of chronic adult disease, epigenetic regulation, gene imprinting, and stem cells. Offering detailed and integrated information on how drugs, biologics, stress, and manipulations impact pregnancy in the mouse model, this reference highlights techniques used to analyze mouse pregnancy. Joining the ranks of much referenced mouse resources, The Guide to Investigation of Mouse Pregnancy is the only manual providing needed content on pregnancy in animal models for translational medicine and research.

Key Features

  • Provides instruction on how to collect pre-clinical data on pregnancy in mouse models for eventual use in human applications
  • Describes the angiogenic tree the mother’s uterus develops to support pregnancy and the monitoring of pregnancy-induced cardiovascular changes
  • Educates readers on placental cell lineages, decidual development including immune cells, epigenetic regulation, gene imprinting, stem cells, birth and lactation
  • Discusses how stress, environmental toxicants and other manipulations impact upon placental function and pregnancy success

Readership

This book is intended for basic scientists and clinical investigators researching reproduction, immunology, developmental biology, cardiology, gynecology, animal care, and veterinary medicine; graduate students; postdocs; veterinary technologists; and laboratory technologists.

Table of Contents

  • Biographies

    Preface

    Conception of This Publication

    Goals

    The Many Thank Yous Over the Course of This Project

    Contributors

    Part I: Atlas

    1. The Cycling and Pregnant Mouse: Gross Anatomy

    Chapter Summary

    Abbreviations

    Introduction

    Anatomic Changes in the Appearance of the Cycling and Pregnant Uterus

    Death of a Conceptus Does not Mean Pregnancy Failure

    Postpartum Uterine Involution

    Illustrative Plates

    References

    2. Implant Site Dissections

    Chapter Summary

    Abbreviations

    Introduction

    Dissection of Gestation Day 6.5–9.5 Uteri and Implant Sites

    Collection of the MLAp

    Dissection of Uteri and Implant Sites between Gestation Day 10.5 and Term

    Illustrative Plates

    References

    3. Microscopic Anatomy of the Pregnant Mouse Uterus Throughout Gestation

    Chapter Summary

    Abbreviations

    Introduction

    4. Anatomy of the Mouse Placenta Throughout Gestation

    Chapter Summary

    Abbreviations

    5. Whole-Mount Immunohistochemistry of Early Implantation Sites

    Chapter Summary

    Abbreviations

    Introduction

    Benefits and Limitations

    Whole-Mount Atlas Plates

    References

    6. Magnetic Resonance Imaging of the Mouse Placenta

    Chapter Summary

    Introduction

    Methods

    Future Perspectives

    References

    Part II: Basic Biology

    Interval A. Estrus, Early Development, Implantation and Uterine Receptivity

    7. Reproductive Tract Changes During the Mouse Estrous Cycle

    Chapter Summary

    Acknowledgments

    Introduction

    Female Mouse Reproductive Tract Anatomy

    The Mouse Estrous Cycle

    References

    8. First Cell Fate Decisions in Early Development: Towards Establishment of the Trophoblast Lineage

    Chapter Summary

    Abbreviations

    Introduction

    Cell Fate Decisions in Early Development

    Transcription Factors Regulating Early Cell Fate Decisions

    Epigenetic Modifications Underpin the Establishment of Stably Committed Cell Lineages

    Stem Cells to Model the First Cell Lineages: Deconstructing the Early Embryo

    Trophoblast Stem Cells as Critical Tool to Study Placental Development

    References

    9. Tetraploid Complementation Assay

    Chapter Summary

    Abbreviations

    Review

    References

    10. Biology of the Ectoplacental Cone

    Chapter Summary

    Abbreviations

    Chapter Contents

    The Blastocyst at the Uterine Cavity: Preparing for Embryo Implantation

    Embryo Implantation and the Mural Trophoblast

    Growth Characteristics and Fate of the Ectoplacental Cone Cells

    Summary

    References

    11. Decidualization and Endometrial Extracellular Matrix Remodeling

    Chapter Summary

    Abbreviations

    Establishment of the Maternal–Fetal Interface

    Endometrial Compartmentalization

    Endometrial Modifications Preceding Decidualization

    Decidualization of the Endometrium

    Dynamics of Decidual Development

    Control of Decidualization

    Mesenchymal and Epithelial Aspects in Decidualization

    Endometrial Extracellular Matrix Remodeling

    Regulation of Endometrial ECM Remodeling

    References

    Interval B. The Maternal-Fetal Interface - Primitive Streak to Term

    12. Postimplantation Development of the Chorioallantoic Placenta

    Chapter Summary

    Abbreviations

    Morphology of the Chorioallantoic Placenta

    Morphogenesis of the P-TGC Layer and Junctional Zone

    Morphogenesis of the Labyrinth Zone

    Studying Placental Phenotypes

    References

    13. Unique Features of Endometrial Dynamics During Pregnancy

    Chapter Summary

    Abbreviations

    Overview

    Pregnancy-Associated Uterine Tissue Remodeling

    Luminal Epithelium: Receptivity, Regression and Restoration

    Biology of the Endometrial Stroma

    References

    14. Pathology Analysis of the Placenta

    Chapter Summary

    Abbreviations

    Considerations in the Structural Analysis of Mouse Placental Specimens

    Major Lesion Patterns in Mouse Placenta and Their Causes

    References

    15. Changes in Structures and Relationships of Trophoblasts, Leukocytes, and Maternal Vessels in Implantation Sites to Midpregnancy

    Chapter Summary

    Abbreviations

    Introduction

    Orientation Landmarks: Features of the Uterine Wall Visualized by Whole Mount In Situ Immunohistochemistry

    Whole-Mount Features of Gd 4.5–5.5 Implant Sites

    Whole-Mount Features of Gd 6.5–9.5 Implant Sites

    Roles of Decidual Lymphocytes Defined by Whole-Mount Studies

    Whole-Mount Definition of Implant Site Relationships in Mice Lacking NK Cell Activation Receptors

    Whole-Mount Identification of Changes in Implant Sites in Mice Genetically Ablated for PGF

    Summary and Future Perspectives

    References

    16. The Uteroplacental, Fetoplacental, and Yolk Sac Circulations in the Mouse

    Chapter Summary

    Abbreviations

    Introduction

    The Yolk Sac Circulation

    The Uteroplacental Circulation

    The Umbilical Cord and Fetoplacental Circulation

    Strain Differences in Placental Circulation

    Summary and Future Perspectives

    References

    17. Physiological Exchange across Mouse Placenta

    Chapter Summary

    Abbreviations

    Introduction

    Flow and Membrane Limited Transfer

    Transporter Mediated Transfer

    Endocytosis/Exocytosis

    Summary and Future Perspectives

    References

    Further Reading

    18. Hypoxia-Associated Regulation within Implantation Sites

    Chapter Summary

    Abbreviations

    Introduction

    Oxygen and Pregnancy

    Hypoxia-Inducible Factors

    Gene Knockout Models

    High-Altitude Pregnancies

    Hypoxic Chambers

    Pharmacological Models

    Surgical Models

    Summary and Future Perspectives

    References

    19. Leukocyte Population Dynamics and Functions at the Maternal–Fetal Interface

    Chapter Summary

    Abbreviations

    Acknowledgments

    Introduction

    General Considerations

    NK Cells

    Macrophages

    Dendritic Cells

    Monocytes and Other Myeloid Precursors

    T Cells

    Summary and Future Perspectives

    References

    20. In Vivo Tracking of Mononuclear Cells in the Virgin Uterus and in Implantation Sites

    Chapter Summary

    Abbreviations

    Acknowledgments

    Introduction

    Two-Photon Microscopy and Its Use in Immunology

    Intravital Imaging of Uterus and Placentas

    Advantages and Limits of New Uterus/Placenta Intravital Imaging Techniques

    Image Processing

    Concluding Remarks about Intravital Imaging of Uterus and Placenta

    References

    21. Microchimerism in Mouse Pregnancy

    Chapter Summary

    Abbreviations

    Introduction to Microchimerism

    Fetal Microchimerism

    Conclusion and Perspectives

    References

    22. Systems Biology Analyses of the Placenta

    Chapter Summary

    What is Systems Biology?

    Genome Wide vs Targeted Techniques

    Measuring Gene Expression

    Sample Generation

    Software

    Statistics

    Bioinformatics

    Examples

    Conclusion

    References

    23. Contribution of Placental Genomic Imprinting and Identification of Imprinted Genes

    Chapter Summary

    Genomic Imprinting: Lessons from Mice

    Why Imprinting?

    Imprinting in the Placenta: A Special Role?

    Concluding Remarks

    References

    24. Epigenetics of Placental Development and Function

    Chapter Summary

    Abbreviations

    Introduction

    The Use of Epigenetics and Epigenomics to Describe Mammalian Cells and Tissues

    DNA Methylation

    Histone Modifications

    Epigenetic Control of Placental Development and Functions

    Epigenetic Abnormality in Developmental Defects and Chronic Diseases: The Influence of Drug and Environmental Chemicals and Nutrition on Epigenetic Systems

    Evolutionary Aspects of Placenta Viewed through Epigenetics

    Summary and Future Perspectives

    References

    25. Genome-Wide Analysis of the Mouse Deciduoma

    Chapter Summary

    Abbreviations

    Introduction

    Mouse Deciduoma Models: Historical and Current Perspectives

    Importance of Choosing a Suitable Model and Sample Type

    Transcriptome Analysis Using Microarrays

    New Frontiers in Genome-Wide Analyses of Mouse Deciduoma

    Perspectives on Future Genome-Wide Deciduoma Research

    References

    26. Conditional Gene Deletion in the Placenta Using the Cre-loxP System

    Chapter Summary

    Abbreviations

    Overview

    Validation of Cre-loxP Conditional Gene Deletion Mouse Models

    Summary

    References

    27. Genetic Engineering of Mice to Investigate Uterine Function in Early Pregnancy

    Chapter Summary

    Abbreviations

    Introduction

    Transgenic Models to Assess Uterine Function

    Use of Homologous Recombination and Targeting in ES Cells to Generate Efficient Models to Elucidate Uterine Function

    Summary

    References

    28. Lentivirus-Mediated Transduction of Optical Reporter Genes in Blastocysts for Placental Studies

    Chapter Summary

    Abbreviations

    Acknowledgment

    Introduction

    Modulation of Placental Functions by Generation of Chimeric Placentas

    Placental Gene Manipulation Based on Trophoblast Lineage-Specific Promoters

    Virus-Mediated Placenta-Specific Gene Delivery

    Summary and Future Perspectives

    References

    29. Blood Pressure Regulation during Mouse Pregnancy

    Chapter Summary

    Abbreviations

    Introduction

    Cardiovascular Changes in Mammalian Pregnancy (Human vs Mouse)

    Choices between Blood Pressure Recording Methodologies

    Normal Mouse Pregnancy

    Hypertensive Mouse Models

    Other Experimental Models Involving Reproduction

    Advantages and Limitations of Radiotelemetry

    References

    30. Intravital Imaging of Vasoactivity in the Uterine Arterial Tree during Pregnancy

    Chapter Summary

    Abbreviations

    Introduction

    Blood Flow Control

    Intravital Microscopy Preparation

    Imaging of the Endometrial Vasculature

    Branch Order Classification of the Endometrial Vasculature

    Vascular Reactivity of Intravital Preparation

    Summary and Future Perspectives

    References

    31. Multimodal Imaging of the Mouse Placenta

    Chapter Summary

    Abbreviations

    Acknowledgments

    Introduction

    Ultrasound Imaging of the Mouse Placenta

    Intravital Fluorescence Microscopy of the Mouse Placenta

    Photoacoustic Imaging of the Mouse Placenta

    Utilizing Bioluminescence Reporter Genes for Placenta Imaging (Luciferin–Luciferase)

    MRI Imaging of the Mouse Placenta

    Future Perspectives

    References

    Interval C. Parturition, Lactation and Postnatal Outcomes

    32. Regulation of Parturition

    Chapter Summary

    Uterine Activation during Labor

    Myometrial Stimulation

    Physiologic Uterine Inflammation and Labor Onset

    The Problem of Preterm Birth

    Mouse Models of PTB

    Genetic Manipulation of Labor Pathway in the Mouse

    Prevention of PTB

    Conclusions

    References

    33. Cervical Changes Accompanying Birth

    Chapter Summary

    Abbreviations

    Acknowledgments

    Introduction to the Pregnant Cervix

    Anatomy and Histology of the Mouse Cervix

    ECM Components of the Pregnant Cervix

    Phases of Cervical Remodeling

    Premature Cervical Ripening in Preterm Birth

    Summary and Future Perspectives

    References

    34. Peri-Partum Changes to Mouse Pubic Symphysis

    Chapter Summary

    Abbreviations

    Acknowledgments

    Introduction

    Nongravid Pubic Symphysis

    Phases of PS Remodeling

    Relevance of Postpartum PS Recovery

    Conclusions

    References

    35. Mammary Gland Morphogenesis and Function during Pregnancy

    Chapter Summary

    Abbreviations

    Introduction

    Mammary Gland Structure

    Postnatal Mammary Gland Development

    The Intersection of Progesterone and Prolactin Signals Specifies Mammary Epithelial Proliferation and Differentiation during Pregnancy Progression

    Summary and Future Perspectives

    References

    36. Gestational Programming of the Cardiovascular System

    Chapter Summary

    Abbreviations

    Introduction

    Hemodynamic and Biochemical Origins of Cardiovascular Disease

    Embryonic and Fetal Cardiac Assessments

    3D Cardiac Imaging

    Assessment of Cardiac Performance Using Ultrasound Biomicroscopy In Utero

    Concluding Remarks

    References

    37. Maternal Stress-Mediated Gene Expression Changes in the Mouse Placenta: A Mosaic Hypothesis of Epigenesis

    Chapter Summary

    Abbreviations

    Acknowledgment

    Introduction

    What is the Role of Epigenetics in Stress-Induced and Other Placental Gene Expression?

    To What Extent is Murine Placental Gene Expression Altered during the Course of Gestation?

    To What Extent is Placental Gene Expression Altered by Maternal Protein/Caloric Deprivation?

    What are the Correlates of Dietary Deprivation in Placentas of Other Mammals?

    To What Extent is Placental Gene Expression Altered by Maternal Caloric Excess?

    To What Extent is Placental Gene Expression Altered by Maternal Hypoxia?

    What are the Correlates in the Human Placenta?

    What are the Overall Perspectives and Critical Questions to Explore?

    Summary

    References

    38. Pregnancy Alterations from Environmental Pollutants

    Chapter Summary

    Abbreviations

    Introduction

    Reproductive and Developmental Toxicity Testing

    Environmental Air Pollution

    Reproductive Toxicity of Air Pollution

    Conclusions

    References

    Part III: Protocols

    39. Monitoring Mouse Estrous Cycles

    Chapter Summary

    Protocol: Collecting the Vaginal Epithelial Cells

    Protocol: Staining Vaginal Smears with May-Grünwald Giemsa stain

    References

    40. Lentivirus Transduction of Gene Tags in Blastocysts

    Chapter Summary

    Abbreviations

    Acknowledgment

    Overview

    Protocol

    Major Technical Limitations

    Troubleshooting

    References

    41. Tetraploid Complementation Assay

    Chapter Summary

    Abbreviations

    Protocol

    Materials

    Reagents

    Method

    References

    42. Pseudopregnant Bead-Induced Mouse Deciduoma Model

    Chapter Summary

    Abbreviations

    Overview

    Materials

    Solutions

    Protocol

    Alternate Proven Ways to Do Bead Transfer

    References

    43. Ectoplacental Cone Isolation, Culture and Assessment

    Chapter Summary

    Abbreviations

    Culturing Blastocysts to Study Trophoblast Cells

    Isolation and Culture of Ectoplacental Cone

    Coculture Systems

    The Ectoplacental Cone as a Xenograft

    Summary and Perspectives

    References

    44. Layer-Enriched Tissue Dissection of the Mouse Placenta in Late Gestation

    Chapter Summary

    Abbreviations

    Overview

    Protocol

    References

    45. Protocols for Placental Histology

    Chapter Summary

    Processing Sequence for Mouse Placental Specimens

    Histotechnological Practices for Preparing Mouse Placenta

    Final Thoughts

    References

    46. Best Practice for Quantifying the Microscopic Structure of Mouse Placenta: The Stereological Approach

    Chapter Summary

    Abbreviations

    Overview

    Protocol

    Concluding Remarks

    References

    47. Electron Microscopy and Immunoelectromicroscopy Protocols

    Chapter Summary

    Abbreviations

    Overview of Microscope Images

    The Physics of Electrons and the Chemistry of Biological Molecules in Electron Microscopy

    Handling of Mouse Reproductive Organs for Ultrastructural Analyses

    Protocols for Sample Processing for TEM Ultrastructural and Immunocytochemical Analyses

    Further Reading

    48. Laser Capture Microdissection

    Chapter Summary

    Abbreviations

    Introduction

    Advantages of LCM

    Disadvantages of LCM

    LCM Procedure

    Sample Preparation for LCM

    Slide Preparation

    Staining

    Microdissection Procedure

    RNA Extraction

    Antisense RNA Amplification (Optional)

    cDNA Synthesis

    Tips for Good Sample Processing

    Important Precautions while Dealing with RNA

    Common Problems Encountered in LCM

    References

    49. Immunofluorescence and Immunohistochemistry

    Chapter Summary

    Abbreviations

    Overview

    Protocol

    Troubleshooting

    Double Staining

    Reference

    50. Nonradioactive In Situ Hybridization: Optimization for Tissue Sections from Pregnant Uteri and Placenta during the First Half of Pregnancy

    Chapter Summary

    Abbreviations

    Overview

    Probe Preparation

    Tissue and Slide Preparation

    Hybridization

    Posthybridization Wash

    Color Development

    Fluorescent In Situ Hybridization

    Potential Optimization Steps

    Kits, Reagents, and Recipes

    References

    51. Whole Mount In Situ Immunohistochemistry

    Chapter Summary

    Abbreviations

    Overview

    Protocol

    Orientation of Stained Specimens

    Technical Limitations

    References

    52. Guidelines for Evaluation of Reproductive and Developmental Endpoints

    Chapter Summary

    Abbreviations

    Overview

    Initial Considerations

    Study Protocol

    Reporting

    References

    53. Flow Cytometric Analysis of Myometrial and Decidual Cell Suspensions

    Chapter Summary

    Abbreviations

    Overview

    Protocol

    Subset Analysis

    Technical Limitations

    References

    54. Uterine Leukocyte Isolation and Culture

    Chapter Summary

    Abbreviations

    Overview

    Uterine Leukocyte Isolation

    Uterine Leukocyte Culture

    Protocols

    References

    55. Scanning Electron Microscopy and Micro-Computed Tomography Imaging of the Utero- and Fetoplacental Circulations

    Chapter Summary

    Abbreviations

    Acknowledgments

    Overview

    Perfusion and Casting of the Uteroplacental Vascular Tree

    Perfusion and Casting of the Fetoplacental Vascular Tree

    Scanning Electron Microscope Imaging

    Micro-Computed Tomography Imaging

    References

    56. Radiotelemetry in Mouse Pregnacy

    Chapter Summary

    Abbreviations

    Overview

    Required/Suggested Equipment

    Surgical Protocol

    Postoperative Care

    Data Acquisition and Analysis

    Summary

    References

    57. Ex vivo Perfusion of the Mouse Placenta for Maternal–Fetal Interaction Studies during Pregnancy

    Chapter Summary

    Abbreviations

    Acknowledgments

    Overview

    Equipment and Reagents

    Protocol

    Critical Steps

    Anticipated Results

    References

    58. Evaluation of Placental Transport Function

    Chapter Summary

    Abbreviations

    Isolated Mouse Placental Plasma Membrane Vesicles

    Mouse Placental Fragments

    Measurement of Unidirectional Maternofetal Solute Clearance across the Intact Mouse Placenta

    Advantages, Limitations, and Technical Challenges of These Procedures

    References

    59. Hypoxia-Associated Regulation within Implantation Sites

    Chapter Summary

    Abbreviations

    Overview

    Fiber-Optic Oxygen Microsensor

    Conclusions

    References

    60. Method to Locate the Uterine Artery in Mice for Micro-Ultrasound Doppler Blood Velocity Examination

    Chapter Summary

    Abbreviations

    Overview

    Protocol

    References

    61. Two-Photon Intravital Microscopy of the Virgin and Pregnant Mouse Uterus

    Chapter Summary

    Abbreviations

    Overview

    Protocols

    Final Considerations about These Techniques

    References

    62. Intravital Imaging of Vasoactivity in the Uterine Arterial Vasculature Tree during Pregnancy

    Chapter Summary

    Abbreviations

    Overview

    Protocol

    Tissue Orientation

    Technical Limitations

    References

    63. MRI, Intra-vital, and Ex-vivo Fluorescence Microscopy of the Mouse Uterine Vasculature and Placenta

    Chapter Summary

    Abbreviations

    Acknowledgment

    Overview

    Magnetic Resonance Imaging

    Optical Imaging

    References

    64. Assessment of Changes in the Peripartum Cervix

    Chapter Summary

    Abbreviations

    Acknowledgments

    Assessment of Cervical Collagen Structure by Second-Harmonic Generation

    Analysis of Cervical Glycosaminoglycans

    Identification and Quantification of Immune Cell Populations by Flow Cytometry

    Preterm Birth Mouse Models

    References

    65. Pubic Symphysis Evaluation

    Chapter Summary

    Abbreviations

    Acknowledgments

    Introduction

    Animals and Interpubic Tissue Collection

    Classical Histological Fixation and Embedding

    Classical Histological Staining

    Morphometric Determination of the Length Profiles of Elastic Fibers

    Hyaluronic Acid Histochemical Analysis with Hyaluronic Acid Probes

    TUNEL Analysis for Evaluation of Cell Death in Interpubic Tissue

    Immunohistochemical/Immunofluorescence Protocols for Interpubic Tissues

    Transmission Electron Microscopy

    Scanning Electron Microscopy

    Explant Culture of Interpubic Tissues

    Biochemical and Molecular Processing

    Conclusion

    References

    66. Labor and Preterm Labor

    Chapter Summary

    Abbreviations

    Monitoring of Labor and Delivery

    Detection of Term and Preterm Parturition

    67. Epithelial Transplantation into the Cleared Mammary Fat Pad

    Chapter Summary

    Abbreviations

    Overview

    Protocol

    Technical Problems and Limitations

    References

    68. Dietary and Hypoxic Protocols That Alter Placental Gene Expression in Response to Maternal Stress

    Animals, Breeding, and Tissue Collection

    Antenatal Maternal Protein Restriction

    Antenatal Maternal Caloric Excess

    Antenatal Maternal Hypoxia

    Biological Significance

    References

    69. Parental-Specific Gene Expression and Epigenetic Analyses of Imprinted Genes in Mouse Placenta

    Chapter Summary

    Abbreviations

    Overview

    Protocol

    Technical Limitations

    References

    70. A Method for Obtaining Epigenomic Data

    Chapter Summary

    Bisulfite Reaction

    Chromatin Immunoprecipitation

    Bioinformatics Tools for Genome-wide Studies

    References

    71. Methods for Genetic Engineering in Mice

    Chapter Summary

    Abbreviations

    Overview

    Germline Gene Ablation Targeting Vector

    Conditional Gene Ablation Targeting Vector

    Conditional Overexpression Targeting Vector

    ES Cell Targeting

    Picking ES Cell Colonies and Injection Preparation

    Generation of Chimeric Mice

    Identification of Genetically Engineered Mice

    Equipment

    Reagents

    Summary and Future Perspectives

    References

    Appendix. Reproductive Parameters of Common, Commercially Available Mouse Strains

    Index

Product details

  • No. of pages: 832
  • Language: English
  • Copyright: © Academic Press 2014
  • Published: December 9, 2013
  • Imprint: Academic Press
  • Hardcover ISBN: 9780123944450
  • eBook ISBN: 9780123947949

About the Editors

Anne Croy

Affiliations and Expertise

Dept. of Biomedical and Molecular Sciences, School of Medicine, Queen's University, ON, Canada

Aureo Yamada

Affiliations and Expertise

Instituto de Biologia, Universidade Estadual de Campinas, Campinas, Brazil

Francesco DeMayo

Affiliations and Expertise

Developmental Biology, Baylor College of Medicine,TX, U.S.A.

S. Lee Adamson

Affiliations and Expertise

Samuel Lunenfeld Research Institute of Mount Sinai Hospital,ON,Canada

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