The pathways involved in embryonic development have been a rich resource for understanding disease in adults, as well as being critically important in tracing the effects of genetic lesions and environmental poisons in the fetus. Frog embryos have been particularly useful due to the large size of the frog egg and embryo. New tools we developed for measuring the expression of RNA at a single-cell level, and advances in protein and phosphopeptide measurement technologies, offer hope for dramatic progress in understanding how signals involved in the maturation of the embryo direct individual cells to adopt specific fates. Our first goal is to define cell types using single-cell transcriptomics, and to define the lineages that result in specific cell types using high resolution temporal mappings. Targeted transcriptomics and proteomics of important molecules involved in specifying cell fate, such as transcription factors, will provide an index of the levels of signaling activity in each individual cell. This will result in an unprecedentedly detailed molecular picture of the factors involved in producing the phenotypes, and their interconversions from the early cleavage stage to the middle of organogenesis. The Xenopus model system allows us to dissect out portions of the early embryo that differentiate to ectoderm if not disturbed, called the animal cap. In the context of the embryo the cells in the animal cap receive a number of developmental signals, including Nodal, BMP, and Wnt. Combinations of these three signals (in different proportions) are capable of generating many of the major tissues. We will expose animal caps to a matrix of these three signals and trace the differentiation pathways that result, using single-cell RNA sequencing. This study of the molecular roots of differentiation decisions will be used to develop a mathematical approach, based on machine learning, to predicting the results of an attempted perturbation of the development of Xenopus. We will ask whether cell types are carefully specified by tightly controlled combinations of ligands or whether there are default states that are hard to escape from (basins of attraction), that therefore form the majority of embryonic cell types. The answer to this question is central to our understanding of how the Xenopus embryo reliably develops into a frog, and will accelerate efforts to create computational methods to predict the behavior of other biological pathways such as those involved in cancer.

Public Health Relevance

The complexity of biology makes it hard to predict what effect a mutation or a drug will have. We will use new tools to measure when genes are expressed at the individual cell level throughout the course of development of a vertebrate embryo. This will give us new information on the cell types involved in tissue and organ formation, and will provide an unprecedentedly detailed dataset that we will use to develop a mathematical model of how the decision to become a specific cell type is made.

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
Research Project (R01)
Project #
5R01HD073104-08
Application #
9701256
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Coulombe, James N
Project Start
2012-09-01
Project End
2022-05-31
Budget Start
2019-06-01
Budget End
2020-05-31
Support Year
8
Fiscal Year
2019
Total Cost
Indirect Cost
Name
Harvard Medical School
Department
Biology
Type
Schools of Medicine
DUNS #
047006379
City
Boston
State
MA
Country
United States
Zip Code
02115
Briggs, James A; Weinreb, Caleb; Wagner, Daniel E et al. (2018) The dynamics of gene expression in vertebrate embryogenesis at single-cell resolution. Science 360:
Gujral, Taranjit S; Kirschner, Marc W (2017) Hippo pathway mediates resistance to cytotoxic drugs. Proc Natl Acad Sci U S A 114:E3729-E3738
Bryant, Donald M; Johnson, Kimberly; DiTommaso, Tia et al. (2017) A Tissue-Mapped Axolotl De Novo Transcriptome Enables Identification of Limb Regeneration Factors. Cell Rep 18:762-776
Presler, Marc; Van Itallie, Elizabeth; Klein, Allon M et al. (2017) Proteomics of phosphorylation and protein dynamics during fertilization and meiotic exit in the Xenopus egg. Proc Natl Acad Sci U S A 114:E10838-E10847
Savova, Virginia; Pearl, Esther J; Boke, Elvan et al. (2017) Transcriptomic insights into genetic diversity of protein-coding genes in X. laevis. Dev Biol 424:181-188
Briggs, James Alexander; Li, Victor C; Lee, Seungkyu et al. (2017) Mouse embryonic stem cells can differentiate via multiple paths to the same state. Elife 6:
Pierre, Anaëlle; Sallé, Jérémy; Wühr, Martin et al. (2016) Generic Theoretical Models to Predict Division Patterns of Cleaving Embryos. Dev Cell 39:667-682
Wühr, Martin; Güttler, Thomas; Peshkin, Leonid et al. (2015) The Nuclear Proteome of a Vertebrate. Curr Biol 25:2663-71
Peshkin, Leonid; Wühr, Martin; Pearl, Esther et al. (2015) On the Relationship of Protein and mRNA Dynamics in Vertebrate Embryonic Development. Dev Cell 35:383-94
Gladilin, Evgeny; Eils, Roland; Peshkin, Leonid (2015) On the embryonic cell division beyond the contractile ring mechanism: experimental and computational investigation of effects of vitelline confinement, temperature and egg size. PeerJ 3:e1490

Showing the most recent 10 out of 20 publications