James A. Elkins, Jr. '41 Preceptor in Molecular Biology
Faculty AssistantJennifer Munko
- Ph.D., Genetics, Friedrich Miescher Institute, Basel, Switzerland
- M.Sc. Molecular Biology and Genetics, University of Szeged, Hungary
Research AreaCell Biology, Development & Cancer
Research FocusQuantitative approaches for understanding mammalian preimplantation embryo formation
Prospective graduate students and postdocs with interest in mechanobiology, biophysics, developmental and stem cell biology and/or imaging are encouraged to apply. Applicants with experience in light sheet imaging and data analysis are particularly welcome!
Quantitative approaches for understanding mammalian preimplantation embryo formation
We are interested in (1) understanding the molecular basis of how cells make fate decisions and pattern into an embryo and (2) exploring the minimal mechanistic requirements underlying the unlimited developmental potential of early embryonic cells prior to differentiation with the aim of translating this knowledge into advances in stem cell biology.
There are few better mammalian model systems than the preimplantation mouse embryo to study cell fate decisions and morphogenetic events at a single cell resolution. By the time the fertilized egg completes seven rounds of division, three different cell types have been formed as products of two consecutive cell fate decisions and are organized into a structure called the blastocyst embryo. Cells are set aside that will form the fetus (epiblast cells) and cells which will make extraembryonic tissues such as the placenta (trophectoderm cells) or the yolk sac endoderm (primitive endoderm cells). Formation of these cell lineages is instrumental for the successful outcome of pregnancy, yet they arise in a low complexity structure, which is also highly amenable to experimental manipulation. We have recently developed a highly efficient CRISPR/Cas9-based method for site-specific insertion of large DNA fragments into the mouse embryo genome, which has further expanded our genetic toolbox to interrogate early mouse development. We generate mouse models with fluorescent protein or RNA reporters, protein degron systems, optogenetic constructs and other synthetic biology tools and use advanced imaging techniques to measure the spatial and temporal dynamics of molecular events and to probe gene function. Our goal is to understand how cellular states are controlled in the embryo and to be able to stably reproduce these states in vitro, in form of novel embryo-derived stem cell types.
The molecular basis for generating cellular diversity
Embryonic patterning kicks off by initiating cellular divergence. The two cell fate decisions during preimplantation embryo development are each textbook examples of different heterogeneity-generating mechanisms, which are widely employed in nature. The first divergence arises from cell polarization followed by asymmetric cell division, yielding different daughter cell types. The second involves an initially uniform population of cells differentiating in a random “salt and pepper” pattern, followed by sorting out into distinct compartments. We are interested in the molecular mechanisms underlying the initiation of these events and seek to answer questions such as how polarization is initiated and inherited; and what the molecular determinants are for heterogeneous cellular response?
The role of mechanical forces in shaping cell fate decisions in the embryo
Cell fate-specific signaling networks and gene expression are not only determined by the biochemical signals a cell receives, but are also controlled by the mechanical inputs a cell experiences. Mechanical forces are obviously at play during preimplantation embryo morphogenesis – cells use the actomyosin cytoskeleton and adhesion molecules to generate and transmit forces, which result in embryo compaction, dynamic cell rearrangements and blastocyst cavity formation. We are specifically interested in how physical forces are relayed into cell fate decisions and how they complement known biochemical signaling activities.
Mechanisms of totipotency in vivo and in vitro
Totipotency – the unbiased ability to generate both embryonic and extraembryonic cell types - has only unequivocally been shown to exist during the first few cell divisions of early mouse development. Surprisingly, very little is known about the molecular mechanisms controlling this unique state in mammals. Our goal is to identify the minimal gene regulatory network responsible for conferring such limitless potential by using a combination of bioinformatical approaches and harnessing our advances in CRISPR-based methods to probe gene function during early development. Additionally, guided by lessons from our embryo studies, we aim to establish a novel stem cell type with unbiased differentiation potential. These stem cells will serve as a source for generating synthetic embryos, which will in turn allow us to gain crucial developmental insights with the use of only in vitro substrates and will be a starting point for learning how to engineer a group of cells into more complex multicellular structures, such as tissues or organs.
Imaging developmental cell cycles. Biophys J. 2021 ;120(19):4149-4161. .
New mouse models for high resolution and live imaging of planar cell polarity proteins in vivo. Development. 2021 ;148(18). .
All models are wrong, but some are useful: Establishing standards for stem cell-based embryo models. Stem Cell Reports. 2021 ;16(5):1117-1141. .
Evaluating totipotency using criteria of increasing stringency. Nat Cell Biol. 2021 ;23(1):49-60. .
Efficient Generation of Large-Fragment Knock-In Mouse Models Using 2-Cell (2C)-Homologous Recombination (HR)-CRISPR. Curr Protoc Mouse Biol. 2020 ;10(1):e67. .
Generation of Large Fragment Knock-In Mouse Models by Microinjecting into 2-Cell Stage Embryos. Methods Mol Biol. 2020 ;2066:89-100. .
Eszter Posfai started as an Assistant Professor of Molecular Biology at Princeton University in January 2019. Her laboratory focuses on understanding mammalian preimplantation embryo formation using quantitative approaches. Originally from Szeged, Hungary she received an MSc in Molecular Biology and Genetics from the University of Szeged and completed her PhD in Genetics at the Friedrich Miescher Institute in Basel, Switzerland. Intrigued by the earliest events of mammalian development, during her PhD she studied the role of epigenetic mechanisms in the germ line and how they impacted the development of the next generation. Joining the lab of Dr. Janet Rossant for her postdoctoral work at the Sickkids Research Institute in Toronto she explored how the earliest cell fate decisions are made in the mouse embryo and co-developed new genetic tools to query these processes at a single cell resolution.