At the American Society for Cell Biology’s annual meeting last month, Kat Hadjantonakis, Ph.D., an associate member of the Sloan-Kettering Institute, spoke about imaging live-cell dynamics in mouse embryos. She described imaging experiments designed to shed light on cell lineage differentiation and morphogenesis during mouse development in utero.
Researchers can study embryo maturation in situ with the use of optical probes and techniques such as gene targeting to introduce into a fertilized egg a gene that codes for a fluorescent fusion protein reporter.
Dr. Hadjantonakis’ group used a fusion reporter composed of the H2B histone protein and green fluorescent protein (GFP) to image blastocyst-stage embryos (the stage at which stem cells form), which are approximately 140 microns in diameter and contain about 150 cells. The H2B-GFP mouse strains express the reporter in endodermal cells.
The group found that the inner cell mass of the blastocyst comprises two cell lineages in a random fashion (the pluripotent epiblast and the primitive endoderm), “suggesting plasticity in lineage specification,” Dr. Hadjantonakis reported. The embryos will ultimately eliminate via apoptosis those cells that do not undergo proper lineage specification, she added.
Platelet-derived growth factor receptor alpha (PDGFRα) can be used as a marker of the primitive endoderm lineage and its derivatives. Dr. Hadjantonakis introduced the H2B-GFP fusion reporter into blastocysts under the control of PDGFRα regulatory elements. Live imaging of the embryos led to the conclusion that PDGF signaling is essential for the establishment and expansion of the primitive endoderm lineage.
Dr. Hadjantonakis also showed how laser confocal microscopy can be used to achieve single-cell resolution of an embryo. Combined with tissue-specific fluorescent-labeling techniques, it is possible to visualize and distinguish between cells of the endoderm, ectoderm, and mesoderm as the embryo develops, and to demonstrate tissue-specific expression and study cell signaling at the level of a single cell. “We are moving to single-cell resolution data,” she explained.
In addition, she described how photoconvertible probes can be used to visualize cell population dynamics and follow a group of cells over time, to study, for example, mouse embryo morphogenesis and determine cell fate. Dr. Hadjantonakis reported that her group is exploring the combination of photoconversion and subcellular localization to enable single-cell precision imaging.
Photoconversion protein monomers typically have a half-life in vivo of 48 to 52 hours, depending on the tissue in which they are expressed. Any sensor protein used for intravital imaging must be thoroughly evaluated to determine its potential for cytotoxic effects such as cell aggregation.
In the future, multiplexing techniques will rely on fluorescent reporters for differential staining of the nucleus and cytoplasm. Intravital imaging can then be used to gain additional insights into the pathways and changes associated with cell morphology, cell division, and cell death. The ability to study cell activity and viability at the single cell level in vivo could have important implications in drug discovery for understanding the effects of experimental compounds.