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Sloan Kettering Institute

3 ARTICLES PUBLISHED IN JoVE

Developmental Biology

Quantitative Analysis of Protein Expression to Study Lineage Specification in Mouse Preimplantation Embryos

Nestor Saiz¹, Minjung Kang¹, Nadine Schrode¹, Xinghua Lou¹, Anna-Katerina Hadjantonakis¹

¹Developmental Biology Program, Sloan Kettering Institute

This protocol presents a method to perform quantitative, single-cell in situ analysis of protein expression to study lineage specification in mouse preimplantation embryos. The procedures necessary for collection of blastocysts, whole-mount immunofluorescent detection of proteins, imaging of samples on a confocal microscope, and nuclear segmentation and image analysis are described.

Genetics

Genome Editing and Directed Differentiation of hPSCs for Interrogating Lineage Determinants in Human Pancreatic Development

Zhong-Dong Shi^*1, Chew-Li Soh^*1, Zengrong Zhu^*1, Danwei Huangfu¹

¹Developmental Biology Program, Sloan Kettering Institute

Protocols to generate hPSC mutant lines using the iCRISPR platform and to differentiate hPSCs into glucose-responsive β-like cells are described. Combining genome editing technology with hPSC-directed differentiation provides a powerful platform for the systematic analysis of the role of lineage determinants in human development and disease progression.

Developmental Biology

Isotropic Light-Sheet Microscopy and Automated Cell Lineage Analyses to Catalogue Caenorhabditis elegans Embryogenesis with Subcellular Resolution

Leighton H. Duncan^1,5, Mark W. Moyle^1,5, Lin Shao^1,5, Titas Sengupta^1,5, Richard Ikegami^1,5, Abhishek Kumar^4,5, Min Guo^4,5, Ryan Christensen^4,5, Anthony Santella^2,5, Zhirong Bao^2,5, Hari Shroff^4,5, William Mohler^3,5, Daniel A. Colón-Ramos^1,5,6

¹Department of Neuroscience and Department of Cell Biology, Yale University School of Medicine, ²Developmental Biology Program, Sloan Kettering Institute, ³Department of Genetics and Genome Sciences and Center for Cell Analysis and Modeling, University of Connecticut Health Center, ⁴Section on High Resolution Optical Imaging, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, ⁵WormGUIDES.org, ⁶Instituto de Neurobiología, Recinto de Ciencias Médicas, Universidad de Puerto Rico

Here, we present a combinatorial approach using high-resolution microscopy, computational tools, and single-cell labeling in living C. elegans embryos to understand single cell dynamics during neurodevelopment.