Transcriptional regulation in early embryo development

Bin GuDr. Bin Gu
Postdoctoral Fellow

Previously, I discovered the expression of Autoimmune Regulator (AIRE), a promiscuous transcription activator previously thought to be restrictively expressed in thymic medullary epithelium cells, where it promotes immune-tolerance, in embryonic stem (ES) cells and early embryos. I uncovered two unexpected functions of AIRE specific to ES cells and early embryonic cells: 1) AIRE sustains pluripotency by promoting a permissive expression profile1,2, and 2) AIRE maintains proliferative fidelity by sustaining mitotic spindle integrity3.  More recently, I developed 2C-HR-CRISPR4, which allows rapid generation of large fragment knock-in mouse models with high efficiency, in collaboration with Eszter Posfai.  With these, I am now dissecting the transcriptional regulatory mechanism during crucial processes in early embryo development such as zygotic genome activation and lineage segregation, using knock-in reporter mice, live imaging, live protein perturbation, and genomics technologies.

  1. Gu, al.Aire regulates the expression of differentiation-associated genes and self-renewal of embryonic stem cells. Biochem Biophys Res Commun 394, 418-423, doi:10.1016/j.bbrc.2010.03.042 (2010).
  2. Gu, al.Aire promotes the self-renewal of embryonic stem cells through Lin28. Stem Cells Dev 21, 2878-2890, doi:10.1089/scd.2012.0097 (2012).
  3. Gu, B., Lambert, J. P., Cockburn, K., Gingras, A. C. & Rossant, J. AIRE is a critical spindle-associated protein in embryonic stem cells. Elife 6, doi:10.7554/eLife.28131 (2017).
  4. Gu, B., Posfai, E. & Rossant, J. Efficient generation of targeted large insertions by microinjection into two-cell-stage mouse embryos. Nat Biotechnol 36, 632-637, doi:10.1038/nbt.4166 (2018).

Timing and commitment of epiblast and primitive endoderm cell fate

Postdoctoral Fellow, Brian BradshawDr. Brian Bradshaw
Postdoctoral Fellow

The second lineage decision, the separation of the Epiblast (EPI) and the Primitive Endoderm, (PE) is regulated through complex interactions centered on the gene regulatory network of NANOG-GATA6-FGF. Prior to separation, the cells of the ICM appear randomly distributed, expressing multiple genes that will later be restricted to either the EPI or PE lineages. FGF4 is the first gene to be differentially expressed within the ICM while the transcription factors NANOG and GATA6 are the first markers to exhibit restricted expression in EPI and PE biased ICM cells. Using the CRISPR/cas9 system I will create a number of reporters of these early lineage markers and study in vivo the timing and commitment of EPI and PE cell fate.

Investigating the master regulators that trigger the establishment of totipotency in human embryo

Dr. Tatsuya Yamakawa - Postdoctoral FellowDr. Tatsuya Yamakawa
Postdoctoral Fellow

In mammalian early development, only zygote and early stage blastomeres can fit the definition of totipotency when individual elastomers can late make all lineages including three germ layers and trophoblast layers. This developmental potential is progressively loss after first segregation of cell lineage to inner cell mass (ICM) and trophectoderm. Embryonic stem cells (ESCs) are derived from mammalian ICM when they are competent to make only embryonic lineages, which is considered as pluripotency. Unlike pluripotency network, the mechanisms required to totipotency are largely unknown. There are several reports showing totipotent-like property or conversion to totipotent stem cells from mouse ESCs. However, few reports showed such state in human ESCs. Therefore, the establishment of human totipotent stem cells is invaluable to elucidate the totipotent state during human early development.

In mouse model, Morgani S.M. showed that extraembryonic endoderm marker Hex marked totipotent-like population in mouse ESCs cultured in 2i/LIF condition. This population could contribute to both embryonic and extra-embryonic lineages. Further, I reported that human HHEX could enhance the reprogramming efficiency form human fibroblast toward pluripotent state during PhD research. I start this project from interesting the potential effect of HHEX on the differentiation ability of human cells.

Recent projects

Developmental timing and molecular characterization of commitment to inner cell mass and trophectoderm lineages

Eszter PosfaiDr. Eszter Posfai
Postdoctoral Fellow

Totipotency, the ability to generate all cell types of an organism, including its extra-embryonic supporting tissues, is a transient state during early mammalian development, persisting only for a few cleavage divisions after fertilization. Appearance of morphologically distinct cell types by the 16-cell stage denotes the start of the first lineage segregation. By the blastocyst stage, inner cells form the inner cell mass and outer cells give rise to the trophectoderm.

We are interested in the developmental timing and molecular mechanisms of how the cells of the early embryo with initially unlimited developmental potential gradually restrict their competence and commit to the first two lineages.

How is local FGF/ERK signaling read out at the single cell level to drive EPI/PE cell fate?

Marcelo GoissisDr. Marcelo Goissis
Former Postdoctoral Fellow

The second lineage decision in the early developing mouse embryo is the segregation of the epiblast and the hypoblast. The epiblast remains pluripotent and will go on to form the embryo proper, while the hypoblast will give rise to the extraembryonic endoderm lineages, including the yolk sac. There is compelling evidence that FGF signalling is responsible for inducing hypoblast cell formation in the developing embryo.

Utilizing live imaging techniques and a FRET reporter system we will investigate the response of FGF signalling in the developing embryo. These techniques will be utilized at the single cell level to manipulate cell signalling and assess changes in cell fate decisions and behaviour during tdifferentiation of the epiblast and hypoblast lineage in the mouse blastocyst.

Classification of species differences in early lineage specification

FlaviaDr. Flavia Barros
Former Postdoctoral Fellow

Since mammalian embryos share many physical similarities during development, it is not unreasonable to generalize observations across different species. However, this can be very misleading because embryos from various species develop at different rates. The disparities in timing can dramatically alter when and how cells in the embryo commit to become either the fetus or placenta. Several studies have reported species-specific differences in early cell decisions that dictate their eventual fate. In order to understand how these decisions are controlled in various mammalian embryos, we aim to study the role of signaling pathways in rat development, and compare and contrast to results from the developing mouse embryo.

Role of nitric oxide in pre-implantation embryos

Cheryl LeeDr. Cheryl Lee
Former Postdoctoral Fellow

Nitric oxide is a small signaling molecule with a short half-life released by cells. Its role in the vasculature has been well-studied. A lesser known function is its effects on embryonic development, as inhibition of NO production leads to developmental arrest by embryos in vitro. My work studies the function of nitric oxide in preimplantation embryos.

Characterization of Cdx2 function and regulation in the early mouse embryo

Eiichi OkamuraDr. Eiichi Okamura
Former Postdoctoral Fellow

Just before implantation into the uterine wall, the blastocyst stage embryo is composed of two types of cells, inner cell mass (ICM) and trophectoderm (TE). During later development, the ICM develops to fetus itself, whereas the TE becomes the trophoblast cells of the placenta. Embryonic stem (ES) cells and trophoblast stem (TS) cells can be established from these early lineages,and are used as a model cell lines of ICM and TE cells, respectively. It has been demonstrated that the Caudal-related homeobox protein 2 (Cdx2) plays a critical role in TE formation. Cdx2 functions as a “trigger” of TE formation and knock-out embryos lacking Cdx2 fail to specify the TE, resulting in early embryonic lethality. Interestingly, it is also known that ectopic expression of Cdx2 transforms ES cells to TS cells. However, the mechanisms of transcriptional regulation of Cdx2 and its detail functions during early lineage segregation are poorly understood. We are interested in understanding the function and regulation of Cdx2 in order to help elucidate the molecular mechanisms of TE formation using TS cells and genetically modified mice, taking advantage of molecular biology and live cell imaging technology.

Role of Nf2 in trophectoderm/inner cell mass specification

Katie CockburnDr. Katie Cockburn
Former PhD Student

The first two lineages to be specified during mammalian development are the trophectoderm (TE) and the inner cell mass (ICM). The TE forms from cells on the outside surface of the embryo, and these cells will give rise to the tissues of the placenta. In contrast, the ICM forms from the cells inside the embryo and will go on to form the embryonic body itself, as well as some additional extraembryonic tissues. Understanding how the cells of the early embryo interpret their position and use this information to adopt either a TE or ICM fate remains poorly understood.

We have identified a role for the upstream Hippo pathway member Nf2/Merlin in the process of TE/ICM specification. Nf2 is required to activate Hippo signaling specifically in inside cells of the embryo, and in its absence these cells fail to form ICM and instead take on a TE fate. Our aim is to understand on a molecular level what role Nf2 plays in Hippo signaling during TE/ICM specification and how this role is affected by cell position in the embryo.