This blog post delves into how cells at the early embryonic stage choose different fates based on interactions between key factors like OCT4 and CDX2, as well as differences in the distribution of bipolarity-determining substances.
The countless cells in our bodies originate from the zygote, a single cell formed by the fertilization of sperm and egg, through repeated cell division. In mammals, during the early blastocyst stage formed by zygote cell division, the trophoectoderm cells—which later become part of the placenta—separate from the inner cell mass (ICM) they surround. This ICM possesses pluripotency, the ability to differentiate into all cell types of the fetus. So, how is this inner cell mass formed?
The zygote undergoes approximately three rounds of cell division to reach the 8-cell stage, consisting of eight spherical cells. It then undergoes a process of compaction, accompanied by morphological changes, becoming an 8-cell morula. In the next stage, the 8-cell blastula becomes a 16-cell blastula through both mitotic division and differential division. Mitotic division refers to the division where the two resulting cells are identical, while differential division refers to the division where the two resulting cells become different. Some cells of the 8-cell blastocyst become the cells forming the outer layer of the 16-cell blastocyst through conservation division. The remaining cells undergo differentiation division, splitting into one cell for the outer layer and one cell for the interior, filling the inside. Thus, the 16-cell blastocyst first acquires a form distinguished by outer layer cells and inner cells.
Meanwhile, this dual cell division pattern repeats within the 16-cell blastocyst, leading to the formation of a 32-cell blastocyst. At this stage, the outer layer cells differentiate into the trophoectoderm cells that will later form the early blastocyst, while the inner cells differentiate into the cells that will constitute the inner cell mass. The key question here is how cells differentiate into distinct cell types at the 16-cell and 32-cell blastocyst stages.
Two major hypotheses have been proposed. First, the ‘inner–outer hypothesis’ explains that a single cell differentiates in different ways based on the degree of contact with neighboring cells and differences in exposure to the external environment. That is, cells deep within the blastocyst have greater contact with neighboring cells than surface cells and are not directly exposed to the external environment. This difference is thought to cause the deep cells and surface cells to differentiate into distinct cell types.
However, the discovery that specific substances become asymmetrically distributed within cells during the compaction process at the 8-cell blastocyst stage led to the proposal of a new ‘bipolar hypothesis’. Substances initially distributed evenly within the cells become redistributed toward the outer or inner regions after compaction. Consequently, each cell in the 8-cell blastocyst acquires a bipolar structure. These substances are termed ‘bipolar determination substances,’ and the core of the bipolar hypothesis is that cells differentiate into distinct types based on the distribution pattern of these substances. According to this hypothesis, when cells in the 8-cell blastocyst undergo mitotic division, the surface cells retain the distribution of polarizing material they possessed before division. However, the newly formed inner cells lack the polarizing material that was originally concentrated on the outer side. This difference between surface and inner cells is explained as the cause leading to their differentiation into distinct cell types.
Scientists focused on OCT4, involved in maintaining pluripotency, and CDX2, involved in neural crest formation, during the differentiation process of the outer cells and inner cells of the blastocyst. In the 8-cell stage blastocyst, OCT4 is evenly distributed throughout all cells, but CDX2 is not. This is because some of the bipolar crystalline material exists only in the outer regions of the cells, concentrating CDX2 towards the outer side. Subsequently, by the 16-cell stage, OCT4 gradually disappears from the outer cells, while the residual CDX2 in the inner cells gradually vanishes. This occurs because CDX2 suppresses OCT4 expression in the outer cells, and OCT4 suppresses CDX2 expression in the inner cells.
Meanwhile, the ‘Hippo’ signaling pathway, which suppresses the function of substances promoting CDX2 expression, has also been studied as a crucial phenomenon related to this process. According to this mechanism, Hippo signaling, present in all cells of the 16-cell stage embryo, activates when contact area with surrounding cells increases, thereby reducing CDX2 levels. These findings demonstrate that the interaction between CDX2 and OCT4 is a key factor determining the divergent fates of the two cells generated by differentiation and division. Recent studies have revealed that this complex mechanism is also organically linked to differences in intracellular mechanical forces, epigenetic regulation, and protein phosphorylation signaling. This confirms that the process of cell fate determination during early embryonic development is the product of highly sophisticated interactions.
