Main description:

This volume discusses the latest imaging and molecular techniques for studying microcephaly through neural progenitor proliferation, survival, and gene expression. All of the methods covered in this book use the mouse as a model system, and include cellular, metabolic, transcriptomic studies of neural progenitors, MR imaging studies of brain growth in 3-dimensions and developmentally-adapted studies of behavior in neonatal mice. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.

Cutting-edge and comprehensive, Microcephaly: Methods and Protocols is a valuable resource for all researchers who want to expand their knowledge and understanding of this important and developing field.

Contents:

Part I Cellular Analyses of Neural Progenitors

1. Dissociation of Cerebellar Granule Neuron Progenitors for Culture, FACS, Transcriptomics, and Molecular Biology
Jennifer Karin Ocasio

2. Maintaining Cerebellar Granule Neuron Progenitors in Cell Culture
Jennifer Karin Ocasio

3. Proliferation Analysis of Cerebellar Granule Neuron Progenitors for Microcephaly Research, Using Immunofluorescent Staining and Flow Cytometry
Jennifer Karin Ocasio

4. Conventional and Spectral Karyotyping of Murine Cerebellar Granule Neuron Progenitors
Gouri Nanjangud

Part II Analyses of Neural Progenitors In Situ

5. Processing of Neonatal Mouse Brains for Immunohistochemical Analysis, As Required for Including Spindle Orientation Analysis and Analysis of DNA Damage and Apoptosis
Gabriela De la Cruz, Nana Nikolaishvili-Feinberg, Scott E. Williams, and Timothy R. Gershon

6. Automated Immunofluorescence Staining for Analysis of DNA Damage and Apoptosis in Brain Sections
Gabriela De la Cruz, Nana Nikolaishvili-Feinberg, and Timothy R. Gershon

7. Automated Immunofluorescence Staining for Analysis of Mitotic Stages and Division Orientation in Brain Sections
Gabriela De la Cruz, Nana Nikolaishvili-Feinberg, and Scott E. Williams

Part III RNA-seq Analysis with Single-Cell Resolution (scRNA-seq)

8. scRNA-seq for Microcephaly Research [1]: Single-Cell Droplet Encapsulation, mRNA Capture, and cDNA Synthesis
Benjamin Babcock and Seth Weir

9. scRNA-seq for Microcephaly Research [2]: Preparation of Single-Cell cDNA Libraries
Benjamin Babcock and Seth Weir

10. scRNA-seq for Microcephaly Research [3]: Computational Analysis of scRNA-seq Data
Benjamin Babcock and Daniel Malawsky

11. scRNA-seq for Microcephaly Research [4]: Dirichlet Regression for Single Cell Population Differences
Daniel Malawsky and Timothy R. Gershon

Part IV MRI Analyses of the Mouse Brain

12. Mouse Brain MRI, Including In Vivo, Ex Vivo, and fcMRI for the Study of Microcephaly
Martin J. MacKinnon, Tzu-Wen W. Wang, and Yen-Yu I. Shih

13. Whole-Body Mouse Fluxomic Analysis to Detect Metabolic Disruptions Associated with Microcephaly, using 13C Isotopes
Andrey P. Tikunov

Part V Behavioral Phenotyping in Neonatal Mice

14. Neonatal Behavioral Screen for Mouse Models of Neurodevelopmental Disorders
Kathryn M. Harper, Viktoriya D. Nikolova, Monika E. Conrad, and Sheryl S. Moy