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Embryologists Polarized Over Early Cell Fate Determination

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Science  06 May 2005:
Vol. 308, Issue 5723, pp. 782-783
DOI: 10.1126/science.308.5723.782

Scientists are trying to determine when the first asymmetry occurs in the mouse embryo, but the embryo has so far thwarted their efforts

Embryologist Hans Spemann famously pointed out 60 years ago that we are standing and walking with parts of our body that would have been used for thinking had they developed in another part of the embryo. Yet scientists still aren't sure when the cells in a mammalian embryo start to take on the individual identities that will determine their eventual fates in the organism.

Currently, a debate is raging over the answer to this question. Some embryologists think that even the earliest cells have, if not an immutable destiny, at least a tendency to form one part of the embryo or another. Not everyone is convinced, however, and in recent months researchers have published a flurry of papers laying out their evidence that the earliest embryonic cells do—or don't—carry inherent preferences that tilt them toward one destiny or another.

The studies address one of developmental biologists' most fundamental questions: How can a single cell—the fertilized egg—give rise to embryos and later animals with a distinct front, back, top, and bottom? In some species, the answers are well known. The unfertilized fly egg, for example, already contains concentrations of proteins in different regions that influence the eventual location of the fly's head and posterior. In frogs, one of the first events after fertilization is the development of a prominent “gray crescent” on the side of the egg opposite where the sperm has just entered, which contains key signals crucial for development.

But pinning down what happens in the mammalian embryo has always been much more difficult. In the first place, the eggs of mammals are tiny—less than one-thousandth of the volume of a frog egg. And their embryos inconveniently develop inside the mother's body, making direct observations in the embryo's natural environment extremely difficult. What is certain is that the cells of mammalian embryos are much more flexible than those of their amphibian or insect counterparts. Scientists can take a two-, four-, or even eight-cell mouse embryo, tease the cells apart, recombine them with cells from another embryo, and produce a healthy mouse. In frogs and fish, such tricks yield animals with two heads or other major abnormalities.

Predestined?

Scientists are debating whether the first two cells of a newly created mouse embryo have a tendency toward different fates in later development.

CREDIT: T. HIIRAGI AND D. SOLTER, NATURE 430, 360 (2004)

For decades, those experiments led most scientists to assume that the cells making up an early mouse embryo are equivalent, and that the first signs of embryonic polarity—having an up-down or left-right axis—appear in the blastocyst, a slightly oblong ball of a few dozen cells that forms about a week after fertilization. “The paradigm has been that [the mouse embryo] is a blank sheet until you start to make the blastocyst,” says developmental biologist Janet Rossant of the Samuel Lunenfeld Institute at the University of Toronto, Canada.

By that time, cells have developed into at least two types: those of the inner cell mass, which will form the fetus as well as parts of the placenta and surrounding tissues, and the trophoblast cells, which will form much of the placenta but will not contribute to the developing fetus. At this stage, the embryo has a clear polarity: The inner cell mass clusters at one end of the blastocyst, which developmental biologists call the embryonic side, and the other half, called the abembryonic side, contains a hollow cavity called the blastocoel.

But over the past decade, several groups probing the embryo's earliest stages have found evidence suggesting that the embryo's directionality might arise well before blastocyst formation. Some of the first hints came from Richard Gardner's lab at the University of Oxford in the United Kingdom. In 2001, he and his colleagues reported experiments in which they used tiny drops of oil to mark the two cells produced by the first division of the fertilized mouse egg. The researchers found evidence that the “equator” created by the first cell division tends to be roughly in the same plane as to the equator dividing the embryonic and abembryonic regions of the blastocyst, leading them to wonder if the egg itself might have north and south poles that influence the fate of cells derived from one hemisphere or the other. “It took 5 years to publish because I didn't believe it myself,” Gardner says.

At about the same time, Magdalena Zernicka-Goetz and her colleagues at the University of Cambridge, U.K., started to look at exactly where the progeny of the embryo's first two cells end up. In 2001, they reported in Development that by carefully marking the sister cells with different-colored dyes, they found that the descendents of one tend to form the embryonic side—whereas the other gave rise to the abembryonic side. That suggested, the authors said, that from the first division on, the embryo has a polarity of its own.

Later that year, the team reported results from a complex and marathonlike set of experiments to see if cells in the four-cell-stage embryo are distinguishable from one another. To identify the four cells reproducibly, the researchers took advantage of the fact that the mouse egg itself is not perfectly symmetrical. It remains attached to the so-called second polar body, which contains genetic material ejected during the egg's maturation. The side with the polar body is called the “animal” pole, whereas the opposite side is called the “vegetal” pole.

According to Zernicka-Goetz and her colleagues, the two cells formed by the first cell division almost always divide with their cleavage planes roughly perpendicular to each other. One division follows the longitude of the oocyte and the other the latitude, so that the four-cell embryo consists of one cell containing mainly animal cytoplasm, one with mostly vegetal cytoplasm, and two cells containing a mix of animal and vegetal cytoplasm.

In experiments that began at 6 a.m. and ran for nearly 20 hours, Zernicka-Goetz, Karolina Piotrowska-Nitsche, also at Cambridge, and their colleagues carefully tracked the divisions of early embryos, broke them apart at the four-cell stage, and created embryo chimeras with known compositions of the different cells. In previous experiments in which chimeric embryos were created by randomly mixing cells from four-cell embryos, nearly all the chimeras developed normally.

Early patterns.

The blastocyst-stage embryo has distinct embryonic and abembryonic poles.

CREDIT: ADAPTED FROM R. PEDERSEN, NATURE 409, 473 (2001), PHOTO FROM T. HIIRAGI

In contrast, the Cambridge team found that none of the chimeras made from cells that contained predominantly vegetal cytoplasm developed when implanted into foster mothers. Chimeras containing predominantly animal cytoplasm developed about 25% of the time, and those containing cytoplasm from both hemispheres developed 87% of the time—a result comparable to that of the random mixing experiments.

“The result shocked us,” says Zernicka-Goetz. “It certainly isn't what we expected.” But she says further studies support the result. Last month, she and Piotrowska-Nitsche reported in Mechanisms of Development that the predominantly “vegetal” cell of the four-cell embryo contributes almost nothing to the inner cell mass of the blastocyst.

None of the rules her team has identified are hard and fast, Zernicka-Goetz admits. And she acknowledges that many scientists continue to believe that because early cells are so flexible, there is no underlying pattern in the egg or early embryo. However, she says, “the other possibility is that there is a pattern, but not a determinant pattern—that you have a set of biases that push cells toward a certain path. We think we have evidence that this is closer to the truth.”

Davor Solter of the Max Planck Institute for Immunobiology in Freiburg, Germany, is one of those who is not yet convinced. Indeed, he disputes most of Zernicka-Goetz's conclusions. For one, he and his colleague Takashi Hiiragi, also at the Max Planck Institute for Immunobiology, find no tendency for the first cell division to occur on a plane perpendicular to the animal-vegetal equator. In contrast, they reported in Nature last year that in their time-lapse recordings of the first cell division, the angle of division is mostly influenced by the relative location of the sperm and oocyte pronuclei as they move toward the center of the cell. They claim that the polar body, which starts out marking the so-called animal pole, actually moves toward the cleavage plane in about half of the embryos they observed, which might have influenced the observations that Gardner and Zernicka-Goetz report.

And for another, Solter and Hiiragi don't see evidence that the first cell division correlates with the later axis of the blastocyst. One factor that might be complicating the experiments is the dynamic movements of the embryos as they develop. Solter says that “these embryos are like spinning yo-yos” in the time-lapse movies, which makes it nearly impossible to keep track of the angle of the original cell divisions.

Solter, Hiiragi, and several colleagues also report in the 1 May issue of Genes and Development that they can find no evidence that one sister cell contributes preferentially to one end of the blastocyst or the other. In these experiments, the team used dye techniques similar to those of Zernicka-Goetz to mark cells at the two-cell stage and then filmed embryos using time-lapse photography for 3 days as the two cells grew into blastocysts. But the results showed no clear pattern of daughter cells in the resulting blastocysts, Solter says.

Axis of disagreement.

In some experiments in which one cell of the two-cell embryo is stained red and the other blue, the progeny of one cell tends to form the embryonic region of the blastocyst, whereas the other gives rise to the abembryonic region (micrograph on left). In other embryos (middle and right), the line is more difficult to draw. (DNA is stained green.)

CREDIT: B. PLUSA ET AL, NATURE 434, 391 (2005)

Some of the blastocysts did show patterns similar to those Zernicka-Goetz reports, Solter acknowledges, but they were a minority. Only about 25% of the embryos they observed had blastocysts in which the daughter cells sorted predominantly into the embryonic or abembryonic part—far less than the nearly 70% reported by Zernicka-Goetz and other groups. Solter, Hiiragi, and their colleagues propose that mechanical forces on and within the developing embryo determine its eventual polarity. But Zernicka-Goetz says her team used a different method of measuring the boundary between the cell types, by painstakingly counting cells at different layers in the blastocyst. “I would be very interested to ask them to analyze their data in the same way,” she says. “Only then can we really compare our results.”

For his part, Gardner is taking something of a middle ground. “There is undeniable evidence that there is prepatterning” in the embryo, he says. But different techniques used in different labs—and the inherent flexibility of the embryo itself—make it very difficult to determine exactly how much. For example, he says, his lab, like that of Zernicka-Goetz, continues to see a consistent pattern between the plane of the first cell division and the shape of the blastocyst. But, Gardner adds, in his lab “we don't see a shred of evidence” that the cells of the four-cell embryo are different.

If there is one point on which all parties would agree, it's that the techniques used so far make clear answers extremely hard to come by. “When people observe embryos, there's always a lot of variability,” says the Lunenfeld Institute's Rossant. “If you're looking for a certain result, you'll see it, but there will always be some results that do not fit.” So far, she says, “you have to say that arguments on both sides are inconclusive.”

The definitive experiment, Rossant and others say, would be to identify a gene or protein, like those already identified in frog or fly embryos, that clearly marks the fate of different early embryonic cells. Zernicka-Goetz and her colleagues are searching for such a factor, she says, looking for differences in gene expression signatures, or in more subtle modifications of the cell's internal architecture. If the genetic search is successful, Solter says, he will be convinced. If someone could find a gene expressed in a specific region of the egg—or in one early cell and not others—and if removing that gene interrupts development, “then absolutely, prepatterning is proven,” Solter says. “If such a gene exists, it will be found.”

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