Preimplantation development - What, when and where?

Preimplantation development comprises the initial stages of mammalian development, before the embryo implants into the mother’s uterus. It begins with the fertilisation of the egg by the sperm, and takes place as the embryo travels along the oviduct towards the uterus. It lasts 4.5 days in the mouse, the best studied model, and 6 days in humans. The fertilised egg (the zygote) divides sequentially to form a 2-, 4-, 8-cell embryo and so on (Figure 1). When the embryo has 8-16 cells (or blastomeres), its shape resembles that of a blackberry and is therefore called morula, after the latin word for mulberry (morus). The preimplantation embryo is covered by a protein “shell” called zona pellucida, that prevents the embryo from attaching to the oviduct wall and causing an ectopic pregnancy. The cells of the morula then become tightly attached and the morula adopts a more spherical, smoother shape - this process is known as compaction. When it has about 32 cells, some of the blastomeres accumulate fluid in the intercellular spaces between them to form a cavity, in the process of cavitation. The embryo is now called the blastocyst (see figures 1 and 2). The blastocyst grows as the cells divide and the cavity expands until it arrives at the uterus, where it “hatches” from the zona pellucida to implant into the uterine wall (figure 2).

Figure 1. Stages of preimplantation development

Timeline: days since fertilisation (not to scale)

Figure 2. Blastocysts hatching

Yellow arrows: blastocysts breaking through the zona

Fine, but why?!

Mammals, like the rest of amniotes, do not develop free in water (unlike fish or amphibians), where oxygen is readily available. This is allowed by the development of a series of foetal or extraembryonic membranes (chorion, allantois and yolk sac) that mediate the exchange of gases, nutrients and waste products to and from the embryo. In mammals, the chorion and the allantois form the placenta, which allows this exchange to happen between the mother and the embryo. These membranes are produced by the embryo in parallel to the development of the foetus. Whereas fish or frog embryos start forming the embryo itself immediately after fertilisation, the first days of mammalian development are spent generating some of these membranes. During the preimplantation period, three cell populations are produced, that make up the blastocyst: the cells that will become the foetus (called the epiblast) and two epithelia that will form most of the extraembryonic membranes (the trophectoderm and the primitive endoderm) (see figure 1). The trophectoderm mediates the implantation of the embryo into the uterus and the initiation of the placenta. The primitive endoderm produces the tissues that determine the antero-posterior axis of the foetus and that form the yolk sac.

The differentiation of the trophectoderm and the inner cell mass

As the morula grows into the blastocyst, some of its cells remain on the outside of the embryo, while others are located on the inside of the morula forming an inner cell mass (ICM) (figure 3). The cells on the surface develop asymmetrical regions on their plasma membrane, the first step towards epithelialisation. The surface exposed to the outside medium is the apical surface, whereas the lateral and basal (or basolateral) surfaces are in contact with the neighbour cells. The border between the apical and basolateral surfaces is the intercellular junctions (tight junctions and adherens junctions) that hold the cells together. The apical membrane is covered in microvilli and has associated proteins, such as ezrin (which is associated with the cytoskeletal protein actin) or atypical protein kinase C (aPKC, an intracellular signalling molecule). The basolateral membranes have E-cadherin, the main adhesion protein at this stage. These cells are therefore polarised

The outside cells express regulatory transcription factors, such as TEAD4, CDX2 or GATA3, which in turn direct the expression of genes necessary for the functions of the trophectoderm (figure 3). The expression of these regulatory transcription factors is thought to be induced by some of the proteins on the apical surface of the outside cells. The position of the cells in the morula therefore would be the trigger for the differentiation of the cells into trophectoderm or ICM.

The cells of the ICM do not have asymmetrical cell surfaces, (are not polarised) and do not express trophectoderm genes. These cells remain totipotent, since they can differentiate into any of the cells in the embryo and even form trophectoderm if their position in the embryo is changed experimentally.

Figure 3. Trophectoderm and ICM differentiation

TE: trophectoderm. TEAD4, GATA3, CDX2 and OCT4 are transcription factors

The differentiation of the primitive endoderm and the epiblast

The cells of the ICM differentiate into the epiblast - which will form the foetus and, later on, other extraembryonic membranes - and the extraembryonic primitive endoderm. Some ICM cells secrete fibroblast growth factor-4 (FGF4) very early on, a signalling molecule necessary for the differentiation of the primitive endoderm. The ICM cells that perceive this signal suffer a change in their gene expression and differentiate into primitive endoderm (Figure 4). The cells that produce FGF4 bear very few molecules of FGF receptor on their surface, which makes them insensitive to it, and therefore do not differentiate. These cells form the epiblast. The epiblast is considered a pluripotent population because it can (and will) produce all the cells in the adult animal.

Epiblast and primitive endoderm cells make up the ICM of the early and mid blastocyst (around 3.5 days old in the mouse) (Figure 4). These two populations are intermixed in the ICM in a “salt and pepper” pattern. These cells move around the ICM as the blastocyst matures to become separated into two populations in the late blastocyst (around the time of implantation): primitive endoderm cells forming an epithelium between the epiblast and the blastocyst cavity, and the epiblast encapsulated between the trophectoderm and the primitive endoderm (Figures 1 and 4). The mechanisms and molecules behind this segregation remain unclear, although the ability of primitive endoderm cells to polarise when they are in contact with the blastocyst cavity seems to be the key to maintain these cells in position.

Figure 4. Primitive and epiblast segregation

Green arrows: cell migration. Orange arrows: FGF4

Epiblast and ES cells

When cultured in the right conditions, epiblast cells can be kept in this pluripotent state indefinitely as embryonic stem (ES) cells. These cells can self-renew (this is, produce daughter cells which also remain pluripotent) and, when exposed to the right stimuli, can differentiate into any cell of the organism. These properties not only allow us to study the differentiation mechanisms of these cells in vitro but also to modify their genome to produce genetically modified ES cells for research. When introduced again in a blastocyst, modified ES cells can integrate in the ICM and become part of the embryo, thus generating a chimera (after the mythological chimera). These chimeric animals can be bred to generate genetically modified mice for research purposes.

Glossary

  • Morula - Mammalian embryo composed of 8 or more cells, before it cavitates.
  • Blastocyst - Mammalian embryo in the preimplantation stage, characterised by the presence of a fluid-filled cavity and an inner cell mass enclosed by an epithelium, the trophectoderm.
  • Compaction - The process whereby the cells of a morula develop extensive intercellular junctions and adopt a spherical shape with no obvious cell borders.
  • Cavitation - Formation of a cavity within the mammalian preimplantation embryo through the accumulation of fluid in the intercellular spaces.
  • Polarisation - Asymmetry within a cell, whereby one area of the cell is molecularly (and/or morphologically) different from the rest.
  • Totipotency - The ability of a cell to differentiate into any cell of the organism.
  • Pluripotency - The ability of a cell to differentiate into any cell of the embryo and adult body, but not into trophectoderm or primitive endoderm.

References and other resources

Reviews

Rossant J and Tam PPL (2009). Blastocyst lineage formation, early embryonic asymmetries and axis patterning in the mouse. Development 136, 701-713. Article linkPubmed

Yamanaka Y, Ralston A, Stephenson RO and Rossant J (2006). Cell and molecular regulation of the mouse blastocyst. Dev Dyn 235, 2301-2314. Article linkPubmed

Nichols, J., and Smith, A. (2011). The origin and identity of embryonic stem cells. Development 138, 3–8. Article linkPubmed

 

Online resources

DevBio - an online companion to Scott F Gilbert’s Developmental Biology textbook (9th edition, Sinauer Associates, Sunderland, MA, 2010) - has  a section on compaction and ICM formation

UC Berkeley has a vast amount of lectures on different topics available on their YouTube channel. The biology course has lectures on reproduction and early development (lectures 3233 and 34).

 

Some key articles

Niwa, H., Toyooka, Y., Shimosato, D., Strumpf, D., Takahashi, K., Yagi, R. and Rossant, J. (2005). Interaction between Oct3/4 and Cdx2 determines trophectoderm differentiation. Cell 123, 917–929. Article link Pubmed

Chazaud, C., Yamanaka, Y., Pawson, T. and Rossant, J. (2006). Early lineage segregation between epiblast and primitive endoderm in mouse blastocysts through the Grb2-MAPK pathway. Dev Cell 10, 615–624. Article link Pubmed

Plusa, B., Piliszek, A., Frankenberg, S., Artus, J. and Hadjantonakis, A.-K. (2008). Distinct sequential cell behaviours direct primitive endoderm formation in the mouse blastocyst. Development 135, 3081–3091. Article linkPubmed

Nishioka, N., Inoue, K.-I., Adachi, K., Kiyonari, H., Ota, M., Ralston, A., Yabuta, N., Hirahara, S., Stephenson, R. O., Ogonuki, N., et al. (2009). The Hippo signaling pathway components Lats and Yap pattern Tead4 activity to distinguish mouse trophectoderm from inner cell mass. Dev Cell 16, 398–410. Article linkPubmed

Nichols, J., Silva, J., Roode, M. and Smith, A. (2009). Suppression of Erk signalling promotes ground state pluripotency in the mouse embryo. Development 136, 3215–3222. Article linkPubmed

 

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