How fast do cells divide

From egg to embryo: chance sets the first course

Research Report 2007 - Max Planck Institute for Molecular Biomedicine

Dietrich, Jens-Erik; Hiiragi, Takashi
Nature has not made it easy for mammals. Like every vertebrate, they emerge from a fertilized egg cell. But unlike fish or frogs, the embryo cannot thrive on its own. Only if, after a few divisions, it succeeds in implanting its outer cells in the uterus, a fetus grows out of the inner ones. For a long time it was unclear when the embryo cells would first take different paths. Researchers at the MPI for Molecular Biomedicine, however, have come a good deal closer to the answer.
Nature hasn't made things easy for mammals. Admittedly, as any other vertebrate - they develop from a fertilized egg, but unlike fish or frogs, the embryo cannot prosper by itself. Only if it succeeds, after having divided a couple of times, in implanting with its outer cells in the womb, its inner cells will create a fetus. It has long been unclear as to when and how the cells of an embryo pursue various lineages. Scientists of the MPI for Molecular Biomedicine in Münster have now advanced a great deal towards unraveling this mystery.

introduction

The start of a person's life begins with a fusion: shortly after a sperm has penetrated a mature egg cell, the chromosomes of the egg and sperm cells meet. About 30 hours later, the fertilized egg divides for the first time - a process that is repeated about every 20 hours from now on. Two become four, eight and finally 16 cells (Fig. 1). The cell ball has barely grown compared to the egg cell, which measures just a tenth of a millimeter. But three to four days after fertilization, the embryo reaches the uterus: a stream of fluid and hairs in the fallopian tube have carried it there.

At this stage there is movement in the ball: liquid penetrates its interior and pushes the embryo cells apart. The embryo is now called a blastocyst and already consists of around 64 cells. They become flatter and more compact and eventually form a hollow sphere made up of two cell layers: the outer trophoblast and the inner embryoblast. This is the first decisive change in the course in the embryo. The tasks are at least roughly distributed among the cells [1]: the trophoblast becomes the outer embryonic shell and later part of the placenta that surrounds the germ and supplies it with oxygen and nutrients, and part of the inner one The child develops cells from which embryonic stem cells can be derived. These cells have a fascinating property called pluripotency - the ability to make any of the body's more than 200 different cell types.

After about five days, the blastocyst consists of a good 100 cells. In order to continue to grow and thrive, it has to implant itself in the uterus, the uterus, and make contact with the mother's bloodstream. To do this, the trophoblast cells secrete enzymes that dissolve some cells in the top layer of the uterine lining. The embryo can now push itself into the connective tissue of the mucous membrane. New skin cells grow over it and close the "wound".

During this implantation, the embryo absorbs proteins, sugars, fats and remnants of the destroyed mucous membrane. This has consequences: its diameter more than doubles. Small cavities (lacunae) filled with blood develop in the trophoblast. Eventually, the cells of the trophoblast partially dissolve the maternal blood vessels in the lining of the uterus. At the end of the second week, maternal blood can flow into the lacunae and leave them again through small blood vessels. In this way, the mother supplies the embryo with all vital resources for around nine months.

The crucial layer

What sounds so sophisticated and perfectly coordinated is actually a masterpiece of evolution. The trophoblast, a structure that only occurs in higher mammals and is therefore a key feature of this class of animals, plays a decisive role in this. This layer of cells not only enables the embryo to implant in the uterus. The placenta that emerges from it also forms an immunological barrier, thereby allowing the offspring to grow for a long time in the womb.

But how do the first differences between the cells arise? How do you know which of them should migrate inward and form the organism and which should form the placenta? The researchers in Takashi Hiiragi's junior research group are investigating precisely these questions in studies on fertilized egg cells from mice. At first glance, rodents do not have much in common with humans. Nevertheless, their genes, organs and cells are so similar that many of the knowledge gained from them can at least partially also be transferred to humans.

In order to track down the answers to their questions, Hiiragi and Jens-Erik Dietrich analyzed in numerous experiments when the first differences between the cells appear in the early mouse embryo. First, the scientists followed the properties and position of the individual cells in the early embryo as the cells divide.

It's the amount that counts

To this end, Hiiragi and Dietrich investigated the proportions of three proteins called Oct4, Cdx2 and Nanog in the individual cells. All three factors are known to be important regulators of embryonic development [2-4]. In addition, it was already known that the proteins in the 8-cell stage, when the fertilized egg cell has divided three times, can still be found in all cells [1]. Just a few divisions later, when the blastocyst already consists of 64 to 128 cells and has formed two clearly distinguishable cell layers, the proteins are only in the inner cell mass (Oct4 and Nanog), or exclusively in the outer cells (Cdx2), the trophoblast. The researchers made use of this striking difference to elucidate the molecular mechanisms that determine the fate of cells in the embryo in the first days of life (Fig. 2).

In one of their experiments, the Max Planck researchers isolated individual cells from mouse embryos that were currently in the 8-cell stage [5, 6]. The researchers then made the isolated cells in the culture dish divide one or two more times. Some of the cells produced two daughter cells of the same size. So they had split symmetrically. The other cells, on the other hand, had divided asymmetrically and formed two daughter cells of different sizes.

Unequal daughters

It is unclear which mechanism decides whether a cell divides symmetrically or asymmetrically. What is certain, however, is that a decisive biochemical change occurs with the asymmetrical division inside the cells [5]. As the researchers' protein analyzes showed, daughter cells - whether symmetrically or asymmetrically divided - contained roughly the same amount of Nanog. However, there was a clear difference in the amounts of Cdx2 contained in the cells: If a cell had divided asymmetrically, the larger daughter cell always had a higher concentration of Cdx2 than the smaller one [6].

In addition, after dividing twice in the culture dish, the cells formed mini-blastocysts of four cells. The cells that were on the outside always showed more Cdx2 than the inner ones (Fig. 3). The researchers conclude from this observation that the type of cell division initially determines how high the Cdx2 concentration in the cells is. The resulting protein pattern then determines whether the cell moves inside or on the outer surface of the growing blastocyst ball.

Amazingly, the researchers found, the number of cells in an embryo that undergo asymmetrical division is very variable. Apparently, the researchers suspect, this variability in the type of cell division is irrelevant for the formation of the blastocyst. The processes that determine the protein pattern in the cells are therefore extremely flexible in their regulation.

Chance sets the course

Hiiragi's and Dietrich's findings refute an assumption made by many scientists that in every egg cell an axis of division and thus also all further division steps are defined in their geometry at the time of fertilization [7]. According to the Münster researchers, the latest results suggest that the cells of the early embryo receive their respective “molecular profile” at random [8].

In fact, it is coincidence that sets the course for further development. At first, the differences in concentration of the relevant proteins are still small, soon they get bigger and bigger until a clear polarity emerges at the end: Those cells that drift outward in the course of blastocyst formation develop into trophoblasts, which become part of the placenta and afterwards Birth goes down with her [6]. The embryonic stem cells that can be derived from inside the blastocyst are also very interesting.

It has long been known that any of these stem cells can produce any of the more than 200 different cell types in the body. However, it is still largely unclear on which molecular factors this so-called pluripotency is based. Finding out is not only interesting for basic researchers. If, for example, it were possible to turn mature body cells back into such all-rounders, there would also be enormous opportunities for medicine. For the first time, it could then be possible to treat previously incurable diseases such as Parkinson's or diabetes with the aid of the patient's own, healthy replacement cells.

Cell stealing with unclear consequences

However, the investigations by the Max Planck researchers in Münster may soon also provide answers to a completely different question. In many countries, reproductive medicine has been performing genetic tests on embryos as part of artificial insemination for years. In this so-called pre-implantation diagnosis (PGD), which is banned in Germany, a single cell is removed from the few days old embryo in the laboratory for genetic analysis. If the test gives an unfavorable result, the seedling is not inserted into the woman's uterus and is allowed to die. If there are no defects in the genome of the embryo, it is inserted into the uterus. So far it has been assumed that cell stealing does not usually harm the embryo. Because quite a few PGD children have already been born. So far, however, nobody can know for sure. Because reliable statistics on miscarriages and failures In vitro-Fertilizations have not yet existed [9].

It is also questionable what the situation is about that cell that has to be removed for the genetic test and inevitably destroyed for analysis: Until now, no one can know from which division step the individual cells of the embryo lose their totipotency [9]. This is understood to mean the ability to grow into a complete individual in a suitable environment.

Several studies indicate that each of the four or eight cells that arose after the second and third division is still totipotent [8, 10]. However, further investigations such as those by Hiiragi and Dietrich will have to show when the fate of the cells is finally determined.

Original publications

Y. Yamanaka, A. Ralston, R. O. Stephenson, J. Rossant:
Cell and molecular regulation of the mouse blastocyst.
Developmental Dynamics 235, 2301-2314 (2006).
J. Nichols, B. Zevnik, K. Anastassiadis, H. Niwa, D. Klewe-Nebenius, I. Chambers, H. Schöler, A. Smith:
Formation of pluripotent stem cells in the mammalian embryo depends on the POU transcription factor Oct4.
D. Strumpf, C. A. Mao, Y. Yamanaka, A. Ralston, K. Chawengsaksophak, F. Beck, J. Rossant:
Cdx2 is required for correct cell fate specification and differentiation of trophectoderm in the mouse blastocyst.
Development 132, 2093-2102 (2005).
K. Mitsui, Y. Tokuzawa, H. Itoh, K. Segawa, M. Murakami, K. Takahashi, M. Maruyama, M. Maeda, S. Yamanaka:
The homeoprotein Nanog is required for maintenance of pluripotency in mouse epiblast and ES cells.
Cell 113, 631-642 (2003).
M. H. Johnson, C. A. Ziomek:
The foundation of two distinct cell lineages within the mouse morula.
J.-E. Dietrich, T. Hiiragi:
Stochastic patterning in the mouse pre-implantation embryo.
Development 134, 4219-4231 (2007).
Embryology. Embryologists polarized over early cell fate determination.
Science 308, 782-783 (2005).
V. B. Alarcon, Y. Marikawa:
Spatial alignment of the mouse blastocyst axis across the first cleavage plane is caused by mechanical constraint rather than developmental bias among blastomeres.
Molecular Reproduction and Development, Jan 14, Epub ahead of print (2008).
PGD, PND, research on embryos.
Articles, reports, contributions to discussions, comments in the Deutsches Ärzteblatt. Contributions from the years 2000 to 2003. 3rd, extended edition of the documentation.
www.aerzteblatt.de/dossiers/embryonenforschung
M. H. Johnson, J.M. McConnell:
Lineage allocation and cell polarity during mouse embryogenesis.
Seminars in Cell & Developmental Biology 15, 583-597 (2004).