Researchers including Cambridge University's Magdalena Zernicka-Goetz, PhD, andUC San Francisco's Roger Pedersen, PhD, have made a finding in the mouse embryothat they say provides a fundamental insight into how the body forms inmammals. And this information, they say, might be useful in the future inregulating the differentiation of embryonic stem cells.
The investigators discovered that the tiny mass of cells that forms in thefirst days following fertilization of the egg has already taken on anorganizational structure and begun to initiate events that predict the spatialpatterning of the later embryo.
They made their discovery by tracing the fate and behavior of a key group ofcells in the early embryo, known as the inner cell mass, from its origin in theso-called blastocyst stage -- when the embryo is a free-floating, hollow ballof cells -- through to its progression into the later embryo, implanted in theuterine wall.
The finding, which showed that the inner cell mass projected a clear andconsistent pattern of organization from the preimplantation blastocyst to theimplanted embryo -- when the body forms -- offers profound insight into thetiming and process by which mammals begin to take shape. The discovery, made byscientists at the Wellcome/CRC Institute, University of Cambridge and UCSF, ispublished in the current issue of Development.
The researchers conducted their study by focusing on the development of a typeof cell known as visceral endoderm, which emerges in the embryo as it begins toimplant in the uterine wall. They believe that the cell plays a role in theorganization of the embryo at the next stage of development, gastrulation -- aprocess that gives rise to the three primary tissues from which all organs andcells of the body emerge, including pancreas, beating heart and brain cells.
These three primary tissues, endoderm, mesoderm and ectoderm, originate fromthe inner cell mass, which also gives rise to embryonic stem cells. Embryonicstem cells are developmentally flexible cells that can be grown in culture fromearly embryos, and researchers hope that, in the future, they could be induced,in culture, to differentiate into specific cell types, such as pancreatic andbeating heart cells, for use as transplants in ailing people.
The embryo moves into high gear with the onset of gastrulation, initiating amajor shift in the arrangement of its cells. Cells begin migrating out alongan invisible axis, forming two poles, signifying the future head and tailregions. From there, cell migration continues to form the front and back andultimately to generate the various organs. It is this process that ultimatelygives rise to the jumping legs of frogs, the wings ofbutterflies and the arms and legs of humans.
And the force that directs gastrulation is "the key," says Pedersen, UCSFprofessor of obstetrics, gynecology and reproductive sciences, and a co-authorof the study, "to understanding how the body forms."
The researchers' discovery that the pre-implantation mammalian embryo carriesthe genesis of the embryo's body plan represents a profound shift in commonwisdom. Researchers have known that, in most other organisms, including frogs,chicks and sea urchins, the newly fertilized egg immediately takes on anorganization that predicts the future body plan. Their fertilized eggs haveaxes that dictate a head-tail, front-back, and right-left polarity that givesrise to the same axes in the later organism.
But compelling evidence had suggested that the axis of the mammalian fertilizedegg and early embryo could not predict the organization of the later embryo. Ifa fertilized mouse egg were cut to remove either of its poles, it could stilldevelop normally; the same was true if two 8-cell embryos were combined witheach other to form a single embryo.
"Those studies had led to the view that no organization in the egg or earlyembryo was likely to be absolutely essential for later development," saysMagdalena Zernicka-Goetz, PhD, a Senior Lister Fellow at the Wellcome/CRCInstitute and Department of Genetics, University of Cambridge, and the seniorauthor of the paper. "The question has remained, how does a mammal develop itsbody plan if it starts with something that doesn't seem to have anyorganization? Knowing now that the early embryo does have organization that isimportant for later development leads us to ask, 'what kind of system can be soflexible as to recover its patterning when it is experimentally perturbed?' "
Building on the shoulders of a study published in 1997 that showed that thefertilized mouse egg contained organization that was predictive of organizationat the blastocyst stage, before the embryo is implanted in the uterus,Zernicka-Goetz and Pedersen set out to examine whether the blastocyst mightprovide cues to the spatial patterning of the later embryo, when gastrulationhad begun.
They focused their study on inner cell mass cells adjacent to a particularfeature of the embryo known as the polar body. This feature serves as a markerof the blastocyst axis of symmetry, as discovered in the 1997 study. Theymarked cells with green fluorescent protein, a marker for living cells,transferred the blastocyst to the uterus of foster mothers, and traced thecells' movement into the post-implantation visceral endoderm, because studieshad pointed to the role of this tissue in determining axial organization.
They allowed the blastocyst to develop until early gastrulation, firstrecognized by the accumulation of migrating cells in the location of theembryo's future backbone. They then scrutinized the distribution and number ofthe descendants of the marked inner cell mass cells and discovered thatvisceral endoderm cells that arose near the polar body of the blastocyst werelocated at one end of the embryo, while those opposite the polar body becamelocated 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 bilateralsymmetry predicts the spatial patterning of the post-implantation embryo.
"This is the first evidence that the polarity at post-implantation stages, whenthe body plan is established, can be traced back to events beforeimplantation," 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 forfuture studies.
"Knowing what we now do, the mouse embryo model will be that much moreeffective for these studies, and the other models studied may be informative ofthings we hadn't anticipated; in other words, the finding may make frogs moregermane to us," says Zernicka-Goetz.
Researchers have already identified molecular systems in frogs that arestrongly implicated in inducing gastrulation, and these systems could play animportant role in animals with backbones, including mammals, the researcherssay. It is possible that the as-yet-unidentified molecular mechanisms of bodyformation in the mouse embryo could be similar to those seen in the frog.
The new results, the researchers say, could also prove useful for gainingcontrol over embryonic stem cells. "Looking at what's going on molecularly inembryos at the time of gastrulation could provide insight into the molecularforces underlying embryonic stem cell differentiation," says Pedersen. "Thiscould provide the missing clue as to how to control the differentiation of stemcells in vitro."
The most likely way to get embryonic stem cells to differentiate into specificcell lines, he surmises, is to do what the embryo does - first make themdifferentiate into endoderm, mesoderm and ectoderm. Once this has beenachieved, scientists, theoretically, could expose the resulting cells to othersignaling molecules that would cause them to give rise to more specialized celltypes, such as pancreas, heart cells or neurons.
"With what we now know about the origin of specific parts of the embryo," hesays, "we can ask, 'what is it about their history that makes them develop asthey do?' Now we know where and when to look in mammalian embryos for answersto this critical question."
"Embryologists already have shown that if mouse embryonic stem cells are put ina blastocyst they develop normally and form all the tissues of the mouse. Wewant to understand how to bring about that differentiation in cell culture," hesays.
Additional co-authors of the study were Roberta J. Weber, B.S., who joined thestudy as a UCSF staff researcher, then continued as a graduate student in Dr.Zernicka-Goetz' group in the Wellcome/CRC Institute and Department of Geneticsat the University of Cambridge, Cambridge, England; Florence Wianny, Ph.D., apostdoctoral fellow in Dr. Zernicka-Goetz' group, and Martin J. Evans, Ph.D.,Professor in the Wellcome/CRC Institute and Department of Genetics, Universityof Cambridge, Cambridge.
Funding for the study was provided by Wellcome Trust Project Grants, the ListerInstitute of Preventative Medicine, the Cancer Research Campaign and theNational Institute of Child Health and Human Development.
1 (Visceral endoderm, plays a role in the development of the early embryo butdoes not actually give rise to the embryo's tissues or the organs of the body.)
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