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Wellcome Trust/CR UK Gurdon Institute, Tennis Court Road, Cambridge CB2 1QN, UK
Centre for Trophoblast Research, Department of Physiology, Development and Neuroscience Physiological Laboratory, Downing Street, Cambridge CB2 3EG, UK
Early mammalian development consists of two distinct phases separated by the event of implantation. Whereas much has been discovered about developmental mechanisms prior to implantation, the inability to culture and follow in real time cell behaviour over the period of implantation means that the second phase has not been explored in as much detail. Recently, a novel in-vitro culture system was described that permits continuous culture and time-lapse observations through the peri- and early post-implantation stages. This system has already delivered detailed information on the cellular processes accompanying early morphogenesis and allowed direct connections to be established between events occurring at the two developmental phases. This review discusses the potential of this novel technology and its possible applications that could have not only impact on basic science but also practical implications for human medicine.
Early mammalian development consists of two distinct phases separated by the event of implantation (preimplantation and post-implantation). Whereas much has been discovered about developmental mechanisms prior to implantation, the inability to culture and follow in real time cell behaviour over the period of implantation has resulted in the second phase remaining explored in much less detail. Recently, a novel in-vitro culture system was described that permits continuous culture and time-lapse observations through the peri- and early post-implantation stages. This system has already delivered detailed information on the cellular processes accompanying early morphogenesis and allowed direct connections to be established between events occurring at the two developmental phases. This review discusses the potential of this novel technology and its possible applications that could have not only impact on basic science but also practical implications for human medicine.
Mammalian embryo development and time-lapse technology
A key feature of early mammalian embryo development is the presence of two distinct phases, pre- and post-implantation – also called embryogenic and embryonic (
). The first phase leads to the separation of two types of tissue: the epiblast, from which the future body of the embryo will be built, and the tropho- and hypoblast, from which extra-embryonic tissues derive to provide support for development in the uterus. The second phase centres on differentiation within both types of tissue, notably the emergence of embryonic cells from the epiblast that leads to formation of the three embryonic germ layers that will form the structures and organs of the embryo and then the fetus (
Formation of the embryonic-abembryonic axis of the mouse blastocyst: relationships between orientation of early cleavage divisions and pattern of symmetric/asymmetric divisions.
), the detailed description of implantation and processes accompanying transition of the preimplantation blastocyst into the post-implantation embryo are still missing. The major obstacle limiting the ability to achieve greater understanding of the dynamics of this transition is the process of implantation and the further dependence of embryo wellbeing on maternal support. Despite several reports of attempts to culture embryos continuously throughout the pre- and post-implantation phases (
), they have not been widely adopted in the study of mammalian embryology. One reason for this may have been the inability to combine these culture approaches with time-lapse video microscopy, which limited the dynamic picture of developmental processes attained and so did not provide much new information in comparison with static pictures resulting from analysing embryos recovered at successive stages following implantation.
Recently, the Cambridge group led by Magdalena Zernicka-Goetz reported the development of a novel in-vitro culture system that allows the transition between pre- and post-implantation phases in the mouse embryo to be followed in real time through the egg cylinder stage as far as the onset of gastrulation (
). In this system, embryos develop on the surface of glass bottom dishes coated with hydrogel and a layer of extracellular matrix proteins. Importantly, application of imaging compatible dishes enabled this method to combine culture with time-lapse confocal microscopy, thereby providing insight into early embryonic processes at the level of cellular resolution. This approach allowed the Cambridge group to provide a precise description of anterior visceral endoderm development, a tissue essential for establishment of the first axis of polarity in the post-implantation embryo (
). Importantly, the study showed that embryos grown in this in-vitro system are morphologically and molecularly equivalent to embryos grown within the mouse uterus (Figure 1) (
). Thus, both show appropriate spatiotemporal expression patterns of key anterior and posterior marker genes such as Cerl, Lefty1 or Brachyury, indicating that a proper organization of tissues is occurring within the ‘post-implantation’ in-vitro embryo. In addition, embryonic stem cells, aggregated with mouse preimplantation embryos, became incorporated into the appropriate tissues of the egg cylinder. Together, these observations indicate that this novel culture system provides an ideal platform for detailed investigation of the mechanisms governing early morphogenesis.
Figure 1Schematic representation of mouse egg cylinder developed in vitro (left panel) and in vivo (right panel). In-vitro embryos developed on glass bottom dishes with hydrogel bonded to the glass and overlaid with layer of extracellular matrix (ECM) proteins. In-vitro and in-vivo egg cylinders are morphologically similar and consist of distinct embryonic and extra-embryonic parts. They both have a well-developed pro-amniotic cavity and a similar expression pattern of marker genes critical for anterior–posterior axis formation such as Cerl and Lefty (green, anterior marker) or Brachyury (red, posterior marker). The epiblast that will form the future body of the fetus is shown in yellow. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
The new culture system and understanding early morphogenesis
There are many processes in early post-implantation development that are not fully understood and would benefit from a comprehensive description at the cellular and molecular levels. One advantage of the new culture system is the ease with which it can utilize available transgenic mouse lines, such as those in which particular cellular components, such as chromatin or cell membranes, are labelled (
). Thus, use of endogenous vital labels allows characterization of dynamic processes to provide accurate and detailed pictures of development. For example, the ability to trace cellular components in live pre-implantation embryos has provided comprehensive databases on cell positions, shapes, division patterns and migratory tracks. These data have allowed a quantitative assessment to be made of the relative significance of lineage and microenvironment (or position) to a cell’s developmental fate and have proved invaluable for understanding preimplantation events (Figure 2) (
Formation of the embryonic-abembryonic axis of the mouse blastocyst: relationships between orientation of early cleavage divisions and pattern of symmetric/asymmetric divisions.
). The new in-vitro culture platform could deliver a similar understanding for the peri- and post-implantation phases, facilitating the construction of morphogenetic blueprints for particular tissues and structures. In addition to the characterization of normal development, the culture system also enables the observation in real time of the development of mutant or experimentally manipulated embryos by measuring such cell-level behaviours as mitosis, cell polarity and lineage mapping during complex tissue movements.
Figure 2Lineage tracing technology has proved essential for understanding preimplantation development. Following cell behaviour by tracing in real time the positions of cell structures such as the nucleus allows the construction of lineage trees which incorporate both the history and fate of individual cells within the embryo. In the example shown here, clones of each cell from a 2-cell-stage embryo are shown in either red or dark blue. As progeny of these clones acquires an inside position, their colours change to yellow and light blue, respectively. Inside cells are progenitors of the epiblast, which will build the body of the fetus, and the hypoblast, which will form the yolk sac; outside cells differentiate into trophectoderm that will give rise to placenta. During normal development, lineages originate in comparable proportions from both clones (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.).
Understanding development that fails to form tissues or structures correctly in the context of an embryo aids identification of key transcription factors, signalling molecules or other proteins that are required for advancement from one stage to the next. The dynamic character of these studies is likely to uncover novel components of the organizational blueprints for the subsequent patterning of the fetal body. Such studies assume particular significance for developmental events that span the period of implantation. Thus,
not only described how the anterior visceral endoderm forms after implantation but also traced back the precursors of this lineage into the blastocyst stage. This example shows that continuity of observations across pre- and post-implantation phases allows discovery of associations that would otherwise be difficult to identify. The ability to link pre- and post-implantation events is invaluable.
Study of environmental impacts on development
In recent years, an increasing number of studies has shown that the environment at the time of conception and during early preimplantation development can have a direct impact on the health of the fetus and adult organism (
). In a range of animal models, it was demonstrated that manipulation of the preimplantation environment in vivo (diet) or in vitro (culture conditions) can adversely affect the post-natal phenotype (
Maternal undernutrition during the preimplantation period of rat development causes blastocyst abnormalities and programming of postnatal hypertension.
). Astonishingly, it also resulted in an increased incidence of cardiovascular disease in adult mice, even when exposure to such diet was applied exclusively during early pre-implantation stages (
). This finding suggests the existence of early ‘sensing’ mechanisms within the embryo that adaptively optimize fetal growth and post-natal fitness to anticipated environmental conditions. The mechanism by which this adaptation occurs is not yet understood. However, the novel in-vitro culture platform could prove useful in shedding more light on the mechanisms underlying this phenomenon.
The potential of the novel culture system for regenerative medicine
Recently, much hope and attention has been given to the potential use of pluripotent cells in regenerative medicine (
). Many human diseases, such as neurodegenerative disorders, diabetes and certain types of liver and heart failure, are caused by defects in the quantity or functionality of particular cell types. Pluripotent cells, including embryonic stem cells and induced pluripotent stem cells are noted for their ability to become converted to cells with a particular fate, and as such seem to be suitable for use in cell replacement therapies. However, the ability to control the process of acquiring a particularly desired cell fate is limited. It has been estimated that, even for highly optimized protocols, the efficiency of differentiation/reprogramming to a preferred tissue type does not exceed 30% (
). In order to be able to use pluripotent cells in human medical treatments this efficiency has to increase dramatically. One way to achieve that increase is to gain more understanding about how a particular cell type is produced during early morphogenesis. As discussed above, this goal should be greatly facilitated by use of the novel culture platform. However, there is one important aspect that cannot be overlooked. Most of the knowledge and protocols relating to pluripotent cells were established in mouse, and between-species differences are becoming evident (
). For example, in striking contrast to what is observed for mouse, primate embryonic stem cells make poor contributions to full development after their injection into a host embryo (
Interspecies chimera between primate embryonic stem cells and mouse embryos: monkey ESCs engraft into mouse embryos, but not post-implantation fetuses.
), which suggest that they are not as pluripotent as their mouse counterparts. Given the medical potential of primate embryonic stem cells, it is crucial to understand how pluripotent these cells really are and to what extent their fate can be modulated. Study of these cells in the new culture system with respect to lineage commitment and within-embryo cell interactions, could form an initial but critical step towards progress in regenerative medicine.
Cell replacement therapy is not the only area of interest in regenerative medicine. An additional important aspect is the production of organs for transplantation. We live in era desperate for ethically acceptable and safe sources of human organs (
). Recently, significant progress has been made in production of functional organs in vivo. Using rodents as model organisms, researchers were able to ‘produce’ pancreas (
) via a ‘blastocyst complementation’ technique in which pluripotent cells were introduced into an embryo that was incapable itself of producing certain tissue types or organs (
). In this way, the empty developmental niche provided by the mutant embryo could be populated by pluripotent cells and the missing organ or tissue successfully produced. One can imagine that a similar system could be applied in the clinic, though human organs would have to be produced in xenogeneic systems, such as chimeric pigs or other large animals (
). However, achieving viable interspecific chimeras has proved to be difficult. Even in the best-studied rodent species, full-term development of rat–mice chimeras is very low, with a high contribution of xenogeneic cells being associated with morphological abnormalities and embryonic lethality (
). The nature of this xenogeneic barrier is unclear. Thus, understanding early morphogenesis with the aid of the novel culture platform may clarify this issue by identifying mechanisms responsible for failed development and in turn, help to establish future reliable organ ‘factories’.
In-vitro culture system and developmental toxicology
Finally, an in-vitro culture platform can aid assessment of safety of drugs and other chemicals for normal fetal development during pregnancy. Currently, developmental toxicity testing is based on traditional animal studies where the risk to prenatal development is assessed based on high-dosage oral administration of the test substance at distinct stages of pregnancy (
). This method of assessment is costly and low-throughput, and the adverse embryonic outcome is difficult to translate into a particular concentration of chemical compound (
). Screening developmental toxicity in vitro should greatly improve this situation. Additional to economic benefits, an in-vitro system should expand the range of data obtained from toxicological assessments. For example, by comparison with the substantial body of data acquired from in-vitro culture of control embryos, it should not only be possible to estimate the toxic dosage range of the tested chemical, but should also facilitate understanding of the cellular mechanisms involved in developmental toxicity. The tracking of movements and history of individual cells in the growing embryo that is facilitated by this in-vitro platform will allow assessment of early biological consequences of exposure to the chemical compound and hopefully will facilitate development of preventive strategies to avoid adverse morphological phenotypes.
Conclusion
In summary, the novel in-vitro culture platform provides a powerful tool to study early morphogenesis and understand processes underlying normal as well as abnormal development. As presented above, this system promises to elucidate the consequences for embryonic cells and therapeutically potent pluripotent cells of complex aspects of lineage commitment and interactions. It should be noted that this approach is not free from challenges especially, when considering its implementation for human embryos/tissues. From a technical point of view, this system was developed using mouse embryos and it will be necessary to adapt and optimize it to support human tissue development. Also, ‘implanting’ human embryos in vitro can lead to ethical and legal concerns such as the issues of to when legally the human embryo can be cultured and what procedures can ethically and legally be performed on them in different jurisdictions. These are matters for wide debate across the whole of society. Notwithstanding, implementation of this in-vitro system has the potential to uncover mechanisms that underlie early morphogenesis, which in turn could have a direct impact on human health.
Acknowledgement
This research was founded by the Wellcome Trust (senior fellowship to Magdalena Zernicka-Goetz).
References
Arnold S.J.
Robertson E.J.
Making a commitment: cell lineage allocation and axis patterning in the early mouse embryo.
Formation of the embryonic-abembryonic axis of the mouse blastocyst: relationships between orientation of early cleavage divisions and pattern of symmetric/asymmetric divisions.
Maternal undernutrition during the preimplantation period of rat development causes blastocyst abnormalities and programming of postnatal hypertension.
Interspecies chimera between primate embryonic stem cells and mouse embryos: monkey ESCs engraft into mouse embryos, but not post-implantation fetuses.
Declaration: The author reports no financial or commercial conflicts of interest.
Footnotes
Agnieszka Jedrusik gained a First in developmental biology from University of Warsaw in Professor Marek Maleszewski’s group, investigating the nature of sperm-activating factor during fertilization of the mouse oocyte. In 2006 she moved to University of Cambridge, to Professor Magdalena Zernicka-Goetz’s group at the Wellcome Trust/Cancer Research UK Gurdon Institute, where she completed her PhD. Here, she is investigating the molecular and cellular mechanisms behind the cell fate decisions in the mammalian embryos.
Agnieszka Jedrusik gained a First in developmental biology from University of Warsaw in Professor Marek Maleszewski’s group, investigating the nature of sperm-activating factor during fertilization of the mouse oocyte. In 2006 she moved to University of Cambridge, to Professor Magdalena Zernicka-Goetz’s group at the Wellcome Trust/Cancer Research UK Gurdon Institute, where she completed her PhD. Here, she is investigating the molecular and cellular mechanisms behind the cell fate decisions in the mammalian embryos.
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