A major goal of systems developmental biology is to create datasets and models that describe, simulate and predict the full complement of gene regulatory interactions during embryogenesis. Such datasets and models are essential to fully understand how genomic information is translated into anatomical structure. Reductionist approaches have identified interactions among dozens of genes, but technical limitations have hindered the systems-wide elucidation of regulatory relationships at both high spatial resolution and whole-genome scale. This project will address this challenge and use novel technologies that enable large-scale mutagenesis and genome-wide expression profiling of single cells to generate gene regulatory network models. The zebrafish blastula will be used as a vertebrate model system, because of its similarities to mammalian embryos and the applicability of powerful genetic, imaging and genomic approaches. The project builds on a long-standing collaboration that combines the Schier lab's expertise in developmental biology, imaging and genetics with the Regev's lab expertise in computational biology, genomics and systems biology. Optimized one-generation CRISPR/Cas9 genome editing will be used to generate mutants for dozens of transcription regulators expressed during early embryogenesis. Mutants will be characterized by generating whole-genome high-resolution gene expression atlases using a novel technology called Seurat. Seurat combines single-cell RNA sequencing with computational mapping of cells to specific regions and cell types in the embryo. The resulting transcriptome maps serve as the inputs to generate models for gene regulatory network activity using clustering-based and Bayesian modeling approaches. Regulatory interactions predicted in silico will be tested in vivo by analyzing gene expression upon perturbation of transcription regulators. The project fulfills the stated purpose of PAR-15-020 to complement the reductionist focus of modern developmental biology and provide a more comprehensive understanding of the causal relationships leading to normal and abnormal embryogenesis. The gene regulatory interactions discovered in this project will help inform programming and reprogramming approaches and will identify candidate interactions that might be involved in the development of birth defects. The project will generate extensive high-quality datasets and atlases for developmental and systems biologists and provide a framework to dissect gene regulatory networks in other systems.

Public Health Relevance

The development of animals is controlled by the differential expression of the genome in space and time. This project identifies the regulatory interactions that control gene expression during embryogenesis and will help understand how birth defects arise and how cells can be programmed to form specific cell types.

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
Research Project (R01)
Project #
5R01HD085905-02
Application #
9180711
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Mukhopadhyay, Mahua
Project Start
2015-12-01
Project End
2020-11-30
Budget Start
2016-12-01
Budget End
2017-11-30
Support Year
2
Fiscal Year
2017
Total Cost
Indirect Cost
Name
Harvard University
Department
Microbiology/Immun/Virology
Type
Schools of Arts and Sciences
DUNS #
082359691
City
Cambridge
State
MA
Country
United States
Zip Code
02138
McKenna, Aaron; Findlay, Gregory M; Gagnon, James A et al. (2016) Whole-organism lineage tracing by combinatorial and cumulative genome editing. Science 353:aaf7907