Among the most recognizable characteristics that separate mammalian development from that of other organisms is the implantation of the embryo into the mother’s uterus. This implantation process is a complex series of interactions and decisions, both on a cellular and genetic level. While much research has been done in mice, not much is known about the specifics of embryonic implantation in primates; however, recent studies have attempted to bridge this information gap. In order to explore embryonic implantation more thoroughly, it is perhaps more prudent to first review the basics of mammalian, and consequentially primate, developmental biology.
Female mammals form oocytes in an organ known as the ovary. During ovulation, a mature oocyte is released from the ovary, and travels down the fallopian tube. If no sperm is present to fertilize the egg, it will continue through the fallopian tube to the uterus, where it ultimately fails to attach, resulting in menstruation. However, if sperm is present, and fertilization is successful, a series of division events is triggered. Figure 1.1 below illustrates this crucial process:
As the fertilized zygote progresses down the fallopian tube, the cells within divide. After the 8-cell compact morula (CM) phase, the cells encounter their first lineage decision. This checkpoint is important because it is the beginning of embryonic stem cell differentiation, the process which ultimately decides the cell fates of the organism. At this early blastocyst stage, various chemical, genetic, and cellular interactions determine if these earliest stem cells differentiate into the inner cellular mass (ICM) or the trophectoderm (TE). The trophectoderm is the outer covering of cells that eventually forms the placental interface between mother and offspring. In other words, the trophectoderm is necessary for successful embryonic development as it is integral in the transfer of nutrients from the mother to the child. The ICM is the bundle of cells that will eventually form the rest of the organism. Stem cells in the ICM will differentiate into either the endoderm, mesoderm, or ectoderm. Here is a neat video that further explains the journey to implantation:
Shortly before contact with the endometrium, the trophectoderm further differentiates into two regions: the syncytiotrophoblast and the cytotrophoblast .
- Syncytiotrophoblast -- non-boundaried, multi-nucleic cell outer layer that produces lytic enzymes and secreted factors that induce apoptosis of endothelial endometrial cells. Allows for embryo to penetrate and embed itself within the endometrium.
- Cytotrophoblast -- inner layer of single nucleated cells, source of extensive mitotic activity.
- This link gives a nice timeline for trophoblast development during implantation into the uterus.
- For more information on placental development, visit this sister page.
Research concerning trophectoderm differentiation from embryonic stem cells has boundless clinical applications, especially in the realms of infertility and uterine complications. Any understanding of placental development lends better understanding to those pathologies that involve this incredible, if temporary, organ. For instance, preeclampsia is a condition in which mothers experience dangerously high blood pressure in the late stages of pregnancy; complications from this condition often lead to premature birth, which can cause a myriad of problems for the newborn . As research about trophectoderm development continues, more horizons are available for advancements in both pre- and neonatal medicine. Another promising aspect of trophectoderm development is that of embryonic stem cell differentiation, renewal, and maintenance. Knowing what and when triggers ESC differentiation could provide therapies for countless diseases.
Key players in trophectoderm differentiation: the role of CDX2:
What factors determine whether cells differentiate into the trophectoderm (TE) or the inner cell mass (ICM)? Experiments in mice implicated that the caudal-type homeodomain protein CDX2 was important for TE formation and maintainence, and thus could be a candidate in primate TE function as well. Other transcription factors considered to be essential in mouse species that could also be potentially important in primates include Oct4, Nanog, and Sox2. This experiment examined the levels and effects of these transcription factors in the rhesus macaques oocytes . CDX2 and Oct4 expression were shown to be present in varying levels throughout primate embryology, illustrated in the following figure:
Figure 1.1 demonstrates that there are relatively low levels of CDX2 in the germinal vesicle, MI, and MII oocytes. The level then spikes in pro-nuclear zygotes, and is then diminished in cleavage stages. Levels of CDX2 also rise in blastocyst stages. In addition, when treated with protein specific antibodies, fluorescent imaging revealed that in monkey blastocysts, CDX2 expression was limited to the TE, while Oct2 expression was confined to the ICM.
So, CDX2 is present when trophectoderm development takes place and localizes to the outer TE layer. Could this then be a gene factor of importance in trophoblast differentiation?
In order to determine the role of CDX2 in primate oocytes, the protein was knocked-down using two morpholino anti-sense strands that targeted the protein sequence. In these down-regulated embryos, embryos failed to expand and differentiate (Figure 1.2A). Thus, without CDX2, embryos collapsed and development was arrested.
Quite interestingly, knocking-down expression of CDX2 did not affect expression of Oct4 (above, Figure 1.2C). While these genes may not depend on each other for expression, they certainly interact with each other when both expressed. Initially, both Oct4 and CDX2 are co-expressed in the ICM and the TE. As the divisions progress, Oct4 becomes localized to the ICM and CDX2 becomes localized to the TE. Studies in mice show that CDX2 actually down-regulates Oct4 expression, repressing it in the TE as development continues. The same is true for primate species. In fact, the ratio of CDX2:Oct4 plays a significant role in cell-fate determination:
- If Oct4:CDX2 ratio is large, the maintenance of pluripotent ICM is supported.
- If Oct4:CDX2 ratio is low, trophectoderm differentiation is promoted.
Therefore, the presence of CDX2 is an important transcription factor in driving cells into the trophectoderm lineage, as opposed to the inner cell mass direction.
Factors that affect embryonic stem cell differentiation: the role of Banf-1 in ESC self-renewal and maintenance:
Congratulations! The Oct4:CDX2 ratio is in your favor, and you are now part of the inner cell mass (ICM). Now what? In the earliest stages of development (before the 8-cell stage), ESC’s are totipotent, and may form any tissue type. After this stage, however, cells lose their totipotency and must commit to a more narrow cell fate, as diagramed below:
Embryonic stem cells in the ICM are pluripotent, categorized by their ability to differentiate into any of the three germ layers that will form the entirety of the organism. What signals them to give up their pluripotency?
To self-renew or not to self-renew: that is the question. Self-renewal is an important process in maintaining the pluripotent state of embryonic stem cells. Factors that affect the maintenance of pluripotency also affect whether a cell will differentiate into a more specialized subset; thus genes that regulate this maintenance may also regulate the decision to differentiate. In one study, the first known line of primate embryonic stem cells was isolated and maintained; amazingly, these cells were still pluripotent over a year later, and as such could differentiate into any of the three germ layers .
Certain genes have been implicated as master regulators of early stages of developmental differentiation, including Oct4, Nanog and Sox2. In their study, Cox et al. screened proteomic databases for protein targets of these regulators, eventually honing in on a Sox2-associated protein by the name of Banf-1 . Banf-1 is thought to be involved in cell cycle progression, in particular the formation of the nuclear envelope during telophase. In addition, previous studies in which Banf-1 was knocked-down in C. elegans and Drosophila resulted in arrested embryonic development. This particular study attempted to extend Banf-1 functionality research to mammalian species, in this case, both in mouse and human embryonic stem cells. Banf-1 was knocked-down using RNAi in both mouse and humans ESC. In mESC, knocking out Banf-1 significantly disrupted ESC self-renewal and survival, predominantly by altering the cell cycle. Cells were also observed to undergo increasing rates of apoptosis. In addition, knocking-out Banf-1 induced differentiation, suggesting that the gene is necessary for maintaining the pluripotency of mESC. In humans, down-regulating Banf-1 knockouts also resulted in decreased ESC survival and self-renewal, however, unlike their murine counterparts, there was no observed induction for differentiation. Banf-1 knock-out also had no significant effect on the localization of pluripotency markers such as Oct4, Nanog, and Sox2. Results from decreased survival in hESC are demonstrated in the figure below:
Thus, this study arrived at two important conclusions:
- Banf-1 is necessary for ESC maintenance and self-renewal in mouse, and more pertinently, human species.
- While Banf-1 induces differentiation in mice, it does not in humans, nor does it affect the localization of pluripotency markers.
Although these results were conclusive about cell proliferation, not much was confirmed about the role of Banf-1 in hESC differentiation. Furthermore, the reasons for choosing Banf-1 were not clearly mentioned in the paper, as many other important nuclear proteins surfaced when screening for Sox2 associated proteins. Overall, these conclusions highlight the differences in embryonic stem cell differentiation decisions between vertebrate species, and could perhaps provide insight into evolutionary processes. What lead to the development of differentials in ESC triggers in mice and primates? Further research may provide the answer. Additionally, the role of other proteins that associate with Oct4, Nanog, and Sox2, as identified through the use of proteomic database screening, may be useful in future ventures to determine what gene, or combination of genes, work to spur ESC differentiation in primates.
1. “Invasion of the trophoblast and embedding”, http://www.embryology.ch/anglais/gnidation/etape03.html
2. Maltepe, Emin, Anna I. Bakardjiev, and Susan J. Fisher. “The Placenta: Transcriptional, Epigenetic, and Physiological Integration during Development.” Journal of Clinical Investigation 120.4 (2010): 1016-025. Print.
3. Sritanaudomchai et al. “CDX2 in the Formation of the Trophectoderm Lineage in Primate Embryos.” Developmental Biology 335.1 (2009): 179-87. Print
4. Thomson, J. A. “Isolation of a Primate Embryonic Stem Cell Line.” Proceedings of the National Academy of Sciences 92.17 (1995): 7844-848. Print.
5. Cox et al. “Banf1 Is Required to Maintain the Self-renewal of Both Mouse and Human Embryonic Stem Cells.” Journal of Cell Science 124 (2011). Print
Image -- Primates: http://www.topnews.in/law/primates-more-resilient-other-animals-seasonal-ups-and-downs-240121
Image -- Embryo development: Nature Publishing Group, 2006
Image -- Cell potency diagram: http://scienceblogs.com/clock/2006/12/from_two_cells_to_many_cell_di.php