Craniofacial Development Methods and Protocols

This volume explores scientific methodologies currently employed to integrate observational developmental biology, tissue explant and cell-based approaches and genetic/molecular technologies to develop a holistic understanding of craniofacial development. Chapters guide readers through the use of disparate models to study formation of the head and face (c. elegans, zebrafish, mouse, alongside human imaging approaches), together with cell culture, tissue explant and in vivo cell imaging and analysis techniques. At the molecular level, chapters include analysing gene expression using in-situ hybridisation and single-cell RNA-Sequencing (scRNA-SEQ), as well as genetic modification techniques such as CRISPR/Cas9-mediated deletion. Written in the format of the highly successful Methods in Molecular Biology series, each chapter includes an introduction to the topic, lists necessary materials and reagents, includes tips on troubleshooting and known pitfalls, and step-by-step, readily reproducible protocols. 

1. Using Caenorhabditis elegans as a model for mechanistic insights of craniofacial development.- Wholemount In-situ Hybridisation (WISH) in Zebrafish Embryos to Analyse Craniofacial Developmen.- Elucidationof Gene Expression Patterns in The Craniofacial Tissues Of Mouse Embryos By Whole-MountIn Situ Hybridization.- Visualizationof the Cartilage and Bone Elements in The Craniofacial Structures by Alcian blue and Alizarin Red Staining.- Methodology for Free-Floating Organ Culture of Mid-Gestation Maxillary Primordial Tissue.- Methods of Palate Culture in Later Palatogenesis: Elevation, Horizontal Outgrowth, and Fusion.- Single cell RNA-seq: Cell Isolation and Data Analysis.- Generating Zebrafish RNA-less Mutant Alleles by Deleting Gene Promoters with CRISPR/Cas9.- Craniofacial Phenomics: Three-Dimensional Assessment of the Size and Shape of Cranial and Dentofacial Structures.- Micro-CT-based Bone Microarchitecture Analysis of the Murine Skull.- Characterization of mammalian in vivo enhancers using mouse transgenesis and CRISPR genome editing.- Histological Techniques for Sectioning Bones of the Vertebrate Craniofacial Skeleton.- Engineering Epithelial-Mesenchymal Microtissues to Study Cell-Cell Interactions in Development.- Ex Vivo Culture of Human Cranial Suture Cells.- Scaffolds for Use in Craniofacial Bone Regeneration.- Mandible Explant Assay for The Analysis of Meckel’s Cartilage Development.- High Resolution Histology for Craniofacial Studies On Zebrafish and Other Teleost Models.- Live Imaging the Dynamics Of Mammalian Neural Crest Cell Migration.- Salivary Gland Development In Culture.- Antenatal Ultrasound Imaging for Analysis of Human Craniosynostosis.