By Jennifer O'Brien

Researchers including Cambridge University’s Magdalena Zernicka-Goetz, PhD, and UC San Francisco’s Roger Pedersen, PhD, have made a finding in the mouse embryo that they say provides a fundamental insight into how the body forms in mammals. And this information, they say, might be useful in the future in regulating the differentiation of embryonic stem cells.

The investigators discovered that the tiny mass of cells that forms in the first days following fertilization of the egg has already taken on an organizational structure and begun to initiate events that predict the spatial patterning of the later embryo.

They made their discovery by tracing the fate and behavior of a key group of cells in the early embryo, known as the inner cell mass, from its origin in the so-called blastocyst stage—when the embryo is a free-floating, hollow ball of cells—through to its progression into the later embryo, implanted in the uterine wall.

The finding, which showed that the inner cell mass projected a clear and consistent pattern of organization from the preimplantation blastocyst to the implanted embryo—when the body forms—offers profound insight into the timing and process by which mammals begin to take shape.
The discovery, made by scientists at the Wellcome/CRC Institute, University of Cambridge and UCSF, is published in the current issue of Development.

The researchers conducted their study by focusing on the development of a type of cell known as visceral endoderm, which emerges in the embryo as it begins to implant in the uterine wall. 

They believe that the cell plays a role in the organization of the embryo at the next stage of development, gastrulation—a process that gives rise to the three primary tissues from which all organs and cells of the body emerge, including pancreas, beating heart and brain cells. 

These three primary tissues, endoderm, mesoderm and ectoderm, originate from the inner cell mass, which also gives rise to embryonic stem cells. Embryonic stem cells are developmentally flexible cells that can be grown in culture from early embryos, and researchers hope that, in the future, they could be induced, in culture, to differentiate into specific cell types, such as pancreatic and beating heart cells, for use as transplants in ailing people.

The embryo moves into high gear with the onset of gastrulation, initiating a major shift in the arrangement of its cells.  Cells begin migrating out along an invisible axis, forming two poles, signifying the future head and tail regions. From there, cell migration continues to form the front and back and ultimately to generate the various organs. It is this process that ultimately gives rise to the jumping legs of frogs, the wings of butterflies and the arms and legs of humans.

And the force that directs gastrulation is “the key,” says Pedersen, UCSF professor of obstetrics, gynecology and reproductive sciences, and a co-author of the study, “to understanding how the body forms.”

The researchers’ discovery that the pre-implantation mammalian embryo carries the genesis of the embryo’s body plan represents a profound shift in common wisdom. Researchers have known that, in most other organisms, including frogs, chicks and sea urchins, the newly fertilized egg immediately takes on an organization that predicts the future body plan.

Their fertilized eggs have axes that dictate a head-tail, front-back, and right-left polarity that gives rise to the same axes in the later organism.

But compelling evidence had suggested that the axis of the mammalian fertilized egg and early embryo could not predict the organization of the later embryo. If a fertilized mouse egg were cut to remove either of its poles, it could still develop normally; the same was true if two 8-cell embryos were combined with each other to form a single embryo.

“Those studies had led to the view that no organization in the egg or early embryo was likely to be absolutely essential for later development,” says Magdalena Zernicka-Goetz, PhD, a Senior Lister Fellow at the Wellcome/CRC Institute and Department of Genetics, University of Cambridge, and the senior author of the paper. “The question has remained, how does a mammal develop its body plan if it starts with something that doesn’t seem to have any organization? Knowing now that the early embryo does have organization that is important for later development leads us to ask, ‘what kind of system can be so flexible as to recover its patterning when it is experimentally perturbed?’ “

Building on the shoulders of a study published in 1997 that showed that the fertilized mouse egg contained organization that was predictive of organization at the blastocyst stage, before the embryo is implanted in the uterus, Zernicka-Goetz and Pedersen set out to examine whether the blastocyst might provide cues to the spatial patterning of the later embryo, when gastrulation had begun.

They focused their study on inner cell mass cells adjacent to a particular feature of the embryo known as the polar body.  This feature serves as a marker of the blastocyst axis of symmetry, as discovered in the 1997 study. They marked cells with green fluorescent protein, a marker for living cells, transferred the blastocyst to the uterus of foster mothers, and traced the cells’ movement into the post-implantation visceral endoderm, because studies had pointed to the role of this tissue in determining axial organization.

They allowed the blastocyst to develop until early gastrulation, first recognized by the accumulation of migrating cells in the location of the embryo’s future backbone. They then scrutinized the distribution and number of the descendants of the marked inner cell mass cells and discovered that visceral endoderm cells that arose near the polar body of the blastocyst were located at one end of the embryo, while those opposite the polar body became located at the other end.

The fact that the cells projected in a uniform and consistent way suggests, suggests, the researchers say, that the early blastocyst’s axis of bilateral symmetry predicts the spatial patterning of the post-implantation embryo.

“This is the first evidence that the polarity at post-implantation stages, when the body plan is established, can be traced back to events before implantation,” says Zernicka-Goetz.”

The fact that the fertilized egg of the mouse - and probably other mammals—share this principle of early organization with other organisms offers fuel for future studies.

“Knowing what we now do, the mouse embryo model will be that much more effective for these studies, and the other models studied may be informative of things we hadn’t anticipated; in other words, the finding may make frogs more germane to us,” says Zernicka-Goetz.

Researchers have already identified molecular systems in frogs that are strongly implicated in inducing gastrulation, and these systems could play an important role in animals with backbones, including mammals, the researchers say. It is possible that the as-yet-unidentified molecular mechanisms of body formation in the mouse embryo could be similar to those seen in the frog.

The new results, the researchers say, could also prove useful for gaining control over embryonic stem cells. “Looking at what’s going on molecularly in embryos at the time of gastrulation could provide insight into the molecular forces underlying embryonic stem cell differentiation,” says Pedersen.  “This could provide the missing clue as to how to control the differentiation of stem cells in vitro.”

The most likely way to get embryonic stem cells to differentiate into specific cell lines, he surmises, is to do what the embryo does - first make them differentiate into endoderm, mesoderm and ectoderm.  Once this has been achieved, scientists, theoretically, could expose the resulting cells to other signaling molecules that would cause them to give rise to more specialized cell types, such as pancreas, heart cells or neurons.

“With what we now know about the origin of specific parts of the embryo,” he says, “we can ask, ‘what is it about their history that makes them develop as they do?’ Now we know where and when to look in mammalian embryos for answers to this critical question.”

“Embryologists already have shown that if mouse embryonic stem cells are put in a blastocyst they develop normally and form all the tissues of the mouse. We want to understand how to bring about that differentiation in cell culture,” he says.

Additional co-authors of the study were Roberta J. Weber, B.S., who joined the study as a UCSF staff researcher, then continued as a graduate student in Dr. Zernicka-Goetz’ group in the Wellcome/CRC Institute and Department of Genetics at the University of Cambridge, Cambridge, England; Florence Wianny, Ph.D., a postdoctoral fellow in Dr. Zernicka-Goetz’ group, and Martin J. Evans, Ph.D., Professor in the Wellcome/CRC Institute and Department of Genetics, University of Cambridge, Cambridge.

Funding for the study was provided by Wellcome Trust Project Grants, the Lister Institute of Preventative Medicine, the Cancer Research Campaign and the National Institute of Child Health and Human Development.

1 Visceral endoderm, plays a role in the development of the early embryo but does not actually give rise to the embryo’s tissues or the organs of the body.)