Live imaging combined with the power of mouse genetics represents the essential next step forward towards unraveling the mechanisms regulating mammalian embryonic development. Our research program is committed to exploiting imaging methods in mammalian systems. Our ongoing and proposed experiments address not only how local interactions between cells and its immediate neighbors give rise to an emergent, higher-level of organization, but also how this process is regulated mechanistically by specific genes or gene networks. Our approach synergizes the fields of cell, developmental and computational biology. The long-term goal of this project is to elucidate the cell behaviors, lineage relationships and molecular mechanisms regulating gastrulation in mammals, using the mouse as an experimentaly tractable model. An immediate goal is to provide a detailed picture of the dynamic events operating within the primitive streak and emergent mesoderm. Specifically, we are focusing on one mesoderm subtype, the paraxial mesoderm, which gives rise to morphologically-distinct somites through a reiterative evolutionarily-conserved process amenable to genetic dissection. Despite extensive genetic analysis, the cellular dynamics underlying the morphogenesis of paraxial mesoderm, the tissue that gives rise to axial musculature of the body, are complex and not well understood. We hypothesize that the specification, proliferation and patterning of the paraxial mesoderm involves a carefully orchestrated stereotypical sequence of cell behaviors. Using live imaging combined with genetic labeling and the analysis of mutants which disrupt this process to varying degrees, we have begun to investigate the cell dynamics driving paraxial mesoderm specification and morphogenesis in the early postimplantation mouse embryo. Our observations have already revealed unexpected cell behaviors and challenged established lineage relationships. This line of research will be further explored in the three Specific Aims that constitute this proposal.
In Specific Aim 1 we investigate the fate of cells emerging from the mouse primitive streak. Using genetic inducible and photomodulatable fate mapping approaches, we will determine the fate of cells of the primitive streak, investigate the existence of a bipotential mesendoderm progenitor population, confirm the presence and identify the location of self-renewing paraxial mesoderm progenitors.
In Specific Aim 2 we will define the cell behaviors at the primitive streak leading to emergence of mesoderm. Live imaging and a panel of novel reporter strains represent a unique platform developed by our laboratory for acquiring quantitative information on cellular dynamics in mouse embryos. We will use these tools to define the cell behaviors (for example, movement and division) integral to the emergence of mesoderm. Then, we will test how these are misregulated in mutants affecting mesoderm formation.
In Specific Aim 3 we will define the cell behaviors operating within the paraxial mesoderm leading to somitogenesis. Using live imaging, we will determine the cell dynamics coincident with somite formation, and test how this morphogenetic process is misregulated at a cellular level in mutants.

Public Health Relevance

Live imaging combined with the power of mouse genetics represents the essential next step forward towards unraveling the mechanisms regulating embryonic development, homeostasis and disease progression. Our research program is committed to exploiting imaging methods in mammalian systems. Our ongoing and proposed experiments address not only how local interactions between a cell and its immediate neighbors give rise to an emergent, higher-level of organization, but also how this process is regulated mechanistically by specific genes or gene networks. Our approach synergizes the fields of cell, developmental and computational biology. The broad aim of this project is to elucidate the cell behaviors, lineage relationships and molecular mechanisms regulating embryonic development in mammals, using the mouse as an experimentaly tractable model. An immediate goal is to provide a detailed picture of the dynamic events operating at gastrulation, a critical process in which the three germ layers (mesoderm, endoderm and ectoderm) are generated. Specifically, we will probe the fate and dynamics of cells within the primitive streak, a transient structure directing gastrulation, and serving as the source of mesoderm and endoderm in the mouse embryo. We will then focus on the emergent paraxial mesoderm, a mesoderm subtype and the tissue that gives rise to the axial musculature of the body. After emerging from the primitive streak the paraxial mesoderm is segmented and sculpted into pairs of somites through a reiterative evolutionarily-conserved process amenable to genetic dissection. Despite the extensive genetic analysis that has been performed in mice, little is known about the intrinsic cell behaviors driving this key morphogenetic process. To understand the cellular dynamics underlying the paraxial mesoderm segmentation, we will use a combination of molecular, genetic, embryological and live imaging techniques. We hypothesize that the specification, proliferation and patterning of the paraxial mesoderm involves a carefully orchestrated stereotypical sequence of cell behaviors under the control of specific gene regulatory networks. Indeed, a rigorous understanding of normal paraxial mesoderm morphogenesis, including knowledge of the origin, commitment, specification and differentiation of cells generating paraxial mesoderm and its derivative tissues, should underpin logical efforts to understand cellular reprogramming, disease progression and design new therapeutic strategies for its derivative vital organ systems. In Aim 1, we will construct a high-resolution fate map of the mouse primitive streak. We will use genetic inducible and optical fate mapping techniques to label individual or small cohorts of cells and determine their destinations. This will allow us to investigate the presence of progenitors giving rise to mesoderm and endoderm, as well as self-renewing paraxial mesoderm progenitors. In Aim 2, we will use live imaging and a novel panel of reporter strains available in our laboratory to define the dynamic cell behaviors at the primitive streak leading to the emergence of mesoderm. Then, we will determine how these behaviors are affected in mutants that exhibit defects in mesoderm formation. Finally, in Aim 3, we will determine the cellular dynamics underlying somite formation within the paraxial mesoderm. This will be achieved using live imaging of wild type embryos followed by the analysis of mutants that exhibit various defects within the paraxial mesoderm, ranging in severity from a complete absence, to milder defects resulting in aberrant somite border formation.

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
Research Project (R01)
Project #
5R01HD052115-08
Application #
8446371
Study Section
Development - 2 Study Section (DEV2)
Program Officer
Mukhopadhyay, Mahua
Project Start
2005-12-01
Project End
2016-02-28
Budget Start
2013-04-01
Budget End
2014-03-31
Support Year
8
Fiscal Year
2013
Total Cost
$429,652
Indirect Cost
$194,740
Name
Sloan-Kettering Institute for Cancer Research
Department
Type
DUNS #
064931884
City
New York
State
NY
Country
United States
Zip Code
10065
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