Keith E. Latham - US grants

Mouse embryos that lack either a maternal (androgenones) or paternal (gynogenones) genetic contribution fail to develop due to functional differences between the two genomes established through genetic imprinting during gametogenesis. It has been suggested that the genetic imprints are translated into differences in gene expression through modification of imprinted genes by factors in the egg. These "modifier" factors are proposed to influence the methylation status or chromatin structure of imprinted genes. The nature and function of such modifiers have yet to be determined. Recent experiments indicate that androgenetic embryos offer a useful system for studying modifiers. The developmental capacity of androgenones varies with the maternal genotype (i.e. egg composition) due to strain-dependent modification of the male pronuclei. This modification is most likely mediated by one or more strain-specific modifiers which influence the expression of genes that are required for blastocoel formation. In this proposal,l will determine whether a single or multiple modifiers is/are responsible for this modification and, if a single modifier, determine its chromosomal location by recombinant inbred mapping. Immunofluorescence microscopy will be used to test the hypothesis that developmental failure in androgenones results from a defect in the establishment or differentiation of the putative trophectoderm lineage. These experiments will provide critical background information to be used in the future to pursue the identification and characterization of the genes encoding these modifiers and the genes that are regulated by them. This will further our understanding of imprinting, which contributes to specific disorders in man.

DESCRIPTION (Adapted from the Investigator's Abstract): The first major task of the mammalian embryo is to transform the information provided from independent parental genomes, into a single totipotent genome able to direct the formation of a new individual. Transcriptional initiation is required to sustain embryonic metabolism before depletion of maternal mRNA and protein. Nuclear reprogramming and early genome activation (EGA) must be coordinated to avoid inappropriate gene expression while providing appropriate gene expression. If both requirements are not met, the embryo may develop abnormally or die. Due to the lack of transcription at the start of development, both nuclear reprogramming and EGA must be mediated by post-transcriptional mechanisms. It is hypothesized that this is accomplished by cytoplasmic factors through two forms of regulation. One is the post-translational modification, e.g., phosphorylation, of maternally-inherited transcription factors. A second is the translational control of maternal mRNA expression. These hypotheses will be tested: 1) nuclear transplantation experiments to determine if cytoplasmic factors of early embryos can repress or activate transcription; 2) examination of the recruitment of mRNA encoding widely used transcription factors into polysomes as an index of translational control; and 3) identification of mRNA encoding additional transcription factors that are recruited into polysomes during EGA. These studies should provide fundamental insights into the role of maternal mRNA in EGA and early development.

Latham
9807542
The genetic material that is transmitted from gamete to zygote and from cell to cell during development contains a combination of genetic information in the form of DNA base sequence but also in the form of epigenetic information that affects gene expression. One such epigenetic phenomenon is parental genomic imprinting which renders maternal and paternal chromosomes functionally non-equivalent. Both the mechanism and function of genomic imprinting remain poorly characterized. This project utilizes the strain-dependent difference in paternal genome modification between the C57BL/6 and DBA/2 mouse strains to search for the factors responsible for the difference in egg phenotype. An analysis of egg phenotypes of the 25 C57BL/6 x DBA/2 recombinant inbred mouse strains (2000 nuclear transplantations) revealed the chromosomal locations of two modifier loci, to within 8.7 and 5 centimorgans (cM) on chromosomes 1 and 2. More complete gene mapping studies will be done by 1) fine mapping of the modifier locus on chromosome 2 which lies within an interval containing many polymorphic markers and for which a significant number of recombinant chromosomes exist and 2) to narrow the candidate region on chromosome 1 by identifying additional polymorphic markers that can be used for further fine mapping at this locus.

Because the biochemical and functional properties of imprinting modifiers are unknown, genetic systems provide invaluable opportunities for identifying the factors that are involved in the process of mammalian genomic imprinting. These studies should also improve our understanding of how egg cytoplasm controls nuclear reprogramming during normal development. So little is known about the fundamental process of genomic imprinting that this study could have an impact beyond just locating relevant genes. All cells carry the same DNA in most muticellular organisms but how the genes are regulated ultimately defines the function of the cell. Some human diseases are being recognized as lacking the usual parental genomic imprint and ultimately, these studies have the potential to contribute to our understanding of those genetic defects in humans.

The preimplantation period of mammalian development is notable for the many crucial events that must occur in order to permit the developmental program to be executed correctly. Many such events have lasting effects on the pattern of gene expression and success of development in the embryo and even on gene function in the adult animal. Because of the importance of these early events to successful embryogenesis, an understanding of the molecular changes that occur during normal preimplantation development is of fundamental importance to the field of mammalian embryology. While preimplantation embryogenesis has been studied in detail in such species as mouse and rabbit, very little is known about preimplantation embryogenesis in primates. A detailed knowledge of the preimplantation period for primate embryos and molecular tools for its study are becoming increasingly in demand, particularly in the area of human reproductive medicine. Improvements in the rates of success of, e.g., in vitro fertilization and in vitro oocyte maturation may be sought through improvements in methods for in vitro culture of oocytes and embryos. The identification of specific gene products that are indicative of good oocyte and embryo health will be useful in the selection of embryos for implantation. New methods for preimplantation genetic diagnosis can also become available if a gene associated with a given disease is known to be expressed during the preimplantation period. Ethical and legal constraints preclude experimental analyses of human embryos. Detailed studies focusing on a suitable non-human primate embryo model have been prevented by limited access and great costs of obtaining these embryos. To increase research capacity for non-human preimplantation embryology, we propose to develop a Non-Human Primate Embryo Gene Expression Resource (PREGER) that can be used by any investigator to study primate preimplantation embryogenesis. Two approaches will be taken to produce large amounts of new and valuable molecular data from small numbers of embryos, along with valuable tools for further investigations. The availability of these resources and new data will permit previously unapproachable scientific questions about the primate embryo to be addressed.

The feasibility of producing viable cloned mammalian offspring by transplantation of adult somatic cell nuclei into oocytes provides striking evidence of the exquisite ability mammalian oocytes to reprogram nuclear function, and of the comparative plasticity of the programmed state of a variety of adult somatic cell nuclei. The advent of these cloning technologies also provides valuable new research opportunities for investigating the molecular mechanisms through which differentiative programming can be reversed in adult somatic cell nuclei, and the molecular mechanisms by which mammalian oocytes reprogram nuclear function, which is an essential part of the process of converting sperm and egg genomes into an embryonic genome. Of special interest for understanding nuclear reprogramming is to what degree the oocyte is able to erase or modify epigenetic information that is present in adult somatic cell nuclei. We hypothesize that some forms of epigenetic inheritance, such as genomic imprinting, must be maintained in order for a somatic cell nucleus to support normal development. Other forms of epigenetic inheritance, such as those associated allelic exclusion or positional identity, most likely need to be erased. The likely fate of still other forms of epigenetic modification, such as X chromosome inactivation is unclear. We also hypothesize that failure of the oocyte to elicit the necessary epigenetic changes, or the loss of essential epigenetic information in the somatic cell, will result in reduced viability of cloned embryos. To test these hypotheses, we will determine the degree to which the DNA methylation that is associated with genomic imprinting, positional identity, and allelic exclusion is altered during cloning, and whether these changes in DNA methylation are correlated with embryo viability. We also hypothesize that, because genetic differences in the ooplasm how pronuclei are modified, these same differences will affect how somatic cell nuclei are modified, and hence the viability of the clonal embryos. This will be tested by comparing the developmental capacities of cloned embryos prepared with oocytes of specific genotypes that are known to modify pronuclei differently. These studies are relevant to problems of human infertility and a variety of diseases involving inappropriate epigenetic regulation of gene function.

DESCRIPTION (provided by applicant): The interactions between oocyte and sperm or between ooplasm and the paternal genome that support early development remain poorly understood. We have discovered an interaction between oocyte and sperm that affects cellular integrity in the 2-cell mouse embryo. Cellular fragmentation and apoptosis during the preimplantation period of development have been observed in a variety of species, including human, mouse, and bovine, but this process is poorly understood. Blastomere fragmentation and apoptosis diminish fertility and may negatively affect the success rates of in vitro methods for treating infertility in humans. We have shown that the genotypes of both the mother and the father affect the incidence of fragmentation. Moreover, a significant parental origin effect is observed for reciprocal F1 hybrid mothers. This may reflect a strain difference in mitochondria or the involvement of at least one imprinted gene in controlling early blastomere fragmentation. Our data also reveal that the magnitude of the effect exerted by both maternal and paternal genotype are each dependent upon the genotype of the other. These observations establish a novel experimental system with which to examine the fundamental biological mechanisms that operate in embryos to maintain cellular integrity. Our findings also have important implications for human reproductive medicine, because they indicate that parameters of gamete fitness, particularly oocyte quality, or genetic factors affecting early development, cannot necessarily be evaluated independently of the genotype of the opposite parent. We propose to (a) determine whether maternal and paternal genotypes affect fragmentation through cytoplasmic constituents of the gametes or through allelic differences in genes expressed in the early embryo, (b) determine whether blastomere fragmentation at the 2-cell stage reflects activation of apoptotic processes or is the result of other aberrant cellular events, and (c) determine the long-term effects of partial blastomere fragmentation on embryo developmental competence. The proposed studies, when completed, should provide essential and valuable new information about the basic biology of the early embryo, oocyte-sperm interactions and their consequences, the roles of gamete-derived factors in controlling early embryo viability, and useful information for improving assisted reproduction techniques or for devising novel approaches to contraception. They should also provide the necessary cellular information with which to design biochemical, or genetic approaches to the identification of molecules involved in fragmentation. Last, because studies such as those proposed cannot be conducted in the human, the proposed studies will provide unprecedented data regarding the relationship between fragmentation and viability.

[unreadable] DESCRIPTION (provided by applicant): Cloning by somatic cell nuclear transfer offers exciting new possibilities for basic research in embryology, development of stem cell therapies, and important agricultural applications. Cloning remains highly inefficient, and recent studies indicate that nuclear reprogramming may be defective in cloned embryos. However, no detailed study of reprogramming has been undertaken, and little information has been obtained about the phenotypic effects of incomplete reprogramming in the early embryo. We have found that early cloned mouse embryos differ substantially from normal fertilized embryos with respect to culture medium preference and DNA methyltransferase expression, exhibit enhanced glucose uptake relative to control embryos, and are deficient in post-transcriptional gene regulation. Some of these differences can be seen even before the first cleavage division, indicating an immediate effect of the donor nucleus on cloned embryo properties. We hypothesize that continued expression of the somatic cell genome after transfer, combined with the translation of maternal mRNAs present in the oocyte, generates a gene expression repertoire in the cloned embryo intermediate between that of a somatic cell and that of an embryo. This altered gene expression pattern may cause cloned embryos to differ markedly from normal embryos with regard to basic physiological and metabolic parameters. This may lead to defects in such basic functions as internal pH regulation, osmoregulation, homeostasis, and ATP production. We also hypothesize that as a result of this aberrant metabolic or physiological state, typical mouse embryo culture environments are grossly sub-optimal for the cloned embryo, and as a result the cloned embryo exists in a state of poor health that is not conducive to efficient nuclear reprogramming, leading to the low efficiency of overall success. Indeed, we find strikingly different culture requirements manifested by cloned embryos. Last, we hypothesize that different somatic cell types may differ in compatibility with the cloning process due to differences in initial donor cell state. To test these hypotheses, we will pursue three complementary and essential Aims. First, we will determine to what degree the transferred somatic cell nucleus continues to express its pre-programmed repertoire of genes. Second, we will determine to what degree these nuclei can direct embryo-specific gene expression patterns, including appropriate posttranscriptional recruitment of matemal mRNA. Third, we will determine to what degree specific metabolic and physiological aspects of cell function are altered in cloned embryos. Fulfillment of these Aims will provide novel information about basic regulatory and homeostatic mechanisms of normal embryos, and will also a basis for further studies examining the molecular basis of reprogramming, and for improving cloning success. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): [unreadable] The effects of ooplasmic factors on genome function and development remain poorly understood, despite an extensive body of literature indicating the existence of such effects. Understanding these effects is essential for a thorough understanding of the developmental mechanisms that initiate each new life, and that direct normal growth and development. Moreover, understanding these effects has significant clinical relevance in the area of assisted reproduction technology (ART), as the treatment of human infertility continues to advance through the implementation of novel microsurgical procedures being implemented in the absence of any controlled scientific data addressing their safety or efficacy. Abundant data from mouse studies reveal the potential for epigenetic effects of 'foreign' cytoplasm on the embryonic genome, leading to effects on embryo viability or developmental abnormalities, some of which are transmitted to the next generation. This proposal addresses the biology of epigenetic programming during oocyte development and maturation, the epigenetic effects of ooplasm on the embryonic genome, the potential for aberrant epigenetic modifications arising as a result of ARTs methods for application at other stages. We will exploit a model system that is based on the existence of genetically diverse egg modifiers, which differentially modify, epigenetically, the maternal and paternal genomes. The proposed studies will provide new basic data about ooplasm-nuclear interactions as well as data relevant to the clinical application of microsurgical procedures. [unreadable] [unreadable]

embryo /fetus; gene expression; developmental genetics; Primates; animal colony;

This application seeks continued support for the Primate Embryo Gene Expression Resource (PREGER). PREGER was established to address constraints of limited availability and enormous costs of obtaining non- human primate (NHP) oocytes and embryos for research purposes, and as a model to improve human assisted reproduction technologies. Constraints of oocyte and embryo availability have greatly limited the scope and pace of NHP embryology, and the number of scientists attempting NHP embryology studies, preventing an increase in molecular studies in parallel with the growth of other model systems. PREGER is a comprehensive resource that was developed employing a well-established and well-documented RT-PCR method for quantitative amplification of entire mRNA populations from as little as a single oocyte or embryo. The >200 cDNA libraries, encompassing a diverse array of stages and procedures, can be amplified and used to prepare dot blots for gene expression analysis. The amplified libraries can also be employed for gene discovery approaches. Because the libraries can be repeatedly amplified by PCR, the samples constitute a sustainable resource. This means that the initial investment that has been made to produce our sample collection yields an immense pay-off by permitting rapid, quantitative analysis of gene expression without the need to obtain additional oocytes or embryos. The savings in both time and cost are therefore vast. The three objectives for the continued development of PREGER are: (1) Maintain the resource and continue to make PREGER samples, dot blots, molecular reagents and methods, the gene expression database, and the web-based utilities available to the community as the premier molecular resource for NHP embryology, (2) Vastly expand the database portion of our resource using DNA arrays to advance our knowledge of NHP embryos and provide unique data to support the further development of safe and efficient ART methods, (this continues our function of converting small-and precious-initial investments of oocytes/embryos into permanent molecular and data resources that can be widely shared and distributed in an easy-to-use form), and (3) Provide training opportunities and support to young scientists, to enable them to enter more readily the field of NHP reproductive biology, and help them to address the increasingly urgent need for appropriate NHP modeling of human embryos. Meeting these objectives will permit the further development and application of the PREGER resource to facilitate the growth and expansion of NHP embryology research, increase our understanding of human and NHP embryos, and provide the critical foundation of information needed to develop improved clinical ARTs.

DESCRIPTION (provided by applicant): The Primate Embryo Gene Expression Resource (PREGER) is an integrated resource combining an extensive collection of amplified cDNA: libraries from rhesus monkey oocytes and embryos, available services, molecular materials for distribution and a website that offers online gene expression databases (including an expanding set of array data sets), important links to other web based resources, protocols, and other tools for embryologists. The PREGER sample collection represents diverse hormonal stimulation, oocyte maturation, and embryo culture procedures mimicking clinical procedures. The sample collection also includes rhesus monkey and human embryonic stem cells lines. This large, biologically diverse resource enables interested investigators to undertake a wide array of molecular studies, including gene expression analysis and gene discovery studies using array based and subtraction hybridization approaches. Most recently, the resource has expanded to offer a two-week training course in mammalian molecular preimplantation biology as a tool for developing human resources for the field. This proposal requests a supplement to extend Aim 3 of our project, by specifically recruiting young professionals with DVM or MD training into the course through a competitive scholarship program. Additionally, we will promote enrollment of graduate level or higher Ph D scientists by providing partial coverage for course related expenses, thereby minimizing required course fees. To enhance the ability of the course to fulfill its mission of attracting qualified young investigators to the field and training them, we are requesting, first, funds to provide partial support to 10 students each year. These students will either posses or be pursuing DVM or MD degrees, and will be selected through a competitive screening process. By targeting this population of students, we should foster the development of young scientists with the ideal training to engage in non human primate studies or clinical human studies to address the scientific needs of the field. Second, we are requesting support for the course to cover costs associated with speaker travel, molecular reagents consumed by the students, and other charges associated with student activities, so that we can offer students the most comprehensive training experience possible. Third, we seek funds support a small research mini-symposium to coincide with the last day of the course, which will provide an outstanding opportunity for the trainees to build new professional contacts in the field. PUBLIC HEALTH RELEVANCE (provided by applicant): This supplement seeks support for training young professionals in the area of molecular embryology of preimplantation mammalian embryos, with emphasis on developing non human primate models of human reproduction. This will advance the study of human infertility and its treatment.

[unreadable] DESCRIPTION (provided by applicant): Correct segregation of chromosomes during meiosis and mitosis is essential for embryonic development and perpetuation of the species. Nuclear transfer (cloning) provides a powerful means for studying spindle formation and function during both processes. We find that removal of the spindle-chromosome complex (SCC) from the oocyte during the first step in cloning by adult somatic cell nuclear transfer (SCNT) depletes the oocyte of a number of proteins, but these proteins become replenished within a few hours. We also find that spindles that form in clones made by SCNT are deficient in calmodulin, whereas the spindles that form in clones made with embryonic nuclei (ECNT) acquire calmodulin appropriately. This correlates with better chromosome congression, reduced incidence of tetraploidy and enhanced development in ECNT clones. Because calmodulin remains abundant in the ooplasm, these observations indicate that the association of calmodulin, and possibly other proteins, with the spindle is controlled by factors associated with the nuclei, and that these factors differ between embryonic and somatic nuclei. It is our hypothesis that the factors that recruit the appropriate proteins to the SCC constitute a novel form of non-genetic, nuclear inheritance present in the oocyte and expressed in cells of the early embryo, but not in somatic cells. These factors are required for correct SCC formation specifically within the context of the ooplasm, and are thus dispensable in somatic cells. This inheritance is removed during cloning, and thus lacking in SCNT embryos, but is present in ECNT embryos. The deficiency in SCNT embryos likely contributes to the poor success of cloning. Identifying the factors that direct SCC formation SCC in the oocyte and early embryo will provide an important new advancement in our basic understanding of meiosis and mitosis in early embryos, and should provide an important key to improving cloning for applied and therapeutic purposes. We will pursue these objectives using a combination of state-of-the-art proteomics approaches and manipulation of gene expression in oocytes and cloned embryos: We will (1) Identify the protein(s) that recruit calmodulin to the meiotic SCC, (2) Undertake a proteomics analysis focused on SCC proteins to identify proteins that are depleted from oocytes and lacking specifically in the SCNT SCC, and (3) Restore or augment expression of these proteins in SCNT embryos and donor cells, and test for enhanced viability of SCNT embryos. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): This proposal is submitted in response to the RFA, RFA-OD-09-003, NIH Challenge Grants in Health and Science Research. The Broad Challenge Area is "Stem Cells", and the specific Challenge is 14-HD-102: Identifying Reprogramming Factors for Oocytes. The advent of new stem cell approaches to cure disease and repair tissue damage is one of the most exciting developments in recent science. Some of the most exciting stem cell technology rests with the ability to reprogram nuclei. The oocyte is uniquely able to reprogram somatic cell nuclei to an embryonic, totipotent state, albeit with a low percentage of success in supporting term development. This power may be harnessed to derive stem cells. A key goal in pursuit of these exciting possibilities is to discover the specific oocyte factors that drive nuclear reprogramming, so that the reprogramming capacity of the oocyte can be manipulated to improve cloning, and so that those same factors can be exploited to advance stem cell technologies. However, there may be hundreds of factors in the oocyte that affect chromatin structure and gene transcription, but only a few of these may be relevant to reprogramming. Thus, simply cataloguing potential reprogramming factors that are expressed in an oocyte is of limited value. A clear relationship of expressed genes to reprogramming capacity of a cell is needed. Genetic systems that can correlate variations in a trait with a combination of gene mapping data and array expression differences offer unparalleled opportunity for circumventing such restrictions. The gene mapping data can greatly facilitate the interpretation of array data, and correlating array data with different variants in phenotype is likewise highly informative. We have available a genetic system that is ideal for this purpose. We have shown that clones made with C57BL/6 (B6) eggs progress beyond the 2-cell stage much more efficiently than those made with D2 eggs;F1 hybrid eggs support a higher still rate of development indicative of a hybrid vigor effect. Ability to direct cloned embryo development beyond the 2-cell stage is a clear indicator of oocyte reprogramming potential. Thus, D2 oocytes are inferior at reprogramming somatic cell nuclei to support early embryogenesis compared to B6 and F1 hybrids. We will employ B6xD2 recombinant inbred strains to determine the number and chromosomal locations of reprogramming factor genes that account for this difference. We will combine those data with array expression data for known transcription factors and chromatin regulators to identify candidates, and then perform functional studies to confirm which genes determine reprogramming capacity of the oocyte. This combined genetic and molecular approach, built on a foundation of phenotype difference will thus result in identification of novel reprogramming factors. PUBLIC HEALTH RELEVANCE: There is great interest in identifying factors in the egg that are responsible for nuclear programming during cloning, because such knowledge may lead to enhanced methods for generating stem cells, and for cloning animals for a range of basic and applied purposes. The difficulty is how to determine which of the myriad of expressed transcription factors and chromatin regulators in the egg are responsible for reprogramming capacity in mice, and we have obtained already a wealth of array expression data for the relevant mouse strains. We will combine these data here to identify gene mapping, gene expression, and functional studies those specific genes that determine oocyte reprogramming capacity, and hence serve as key oocyte reprogramming factors.

DESCRIPTION (provided by applicant): Oocytes and preimplantation stage embryos are exquisitely sensitive to their environments, and even minor alterations can lead to significant effects on adult health (e.g,. adult hypertension following maternal low-protein diet during the preimplantation period). Learning how minor, transient changes in the oocyte/early embryo environment can have such long-term, persistent, and serious effects is vital for improving human health. This proposal is founded on three central hypotheses: (1) in order for transient treatments of oocytes/early embryos to exert long-term effects on adult phenotype, heritable, stable, epigenetic changes must arise that modify gene expression, development, and physiology~ (2) Because these changes arise a result of oocyte/early embryo exposure, and persist, they should exist in all cells and tissues of the adult body, and will likely affect a brod range of characteristics. (3) Because placental function is key to post-natal phenotype, epigenetic changes also arise in the placenta to affect its function, which in turn affects post-natal health. The objectives of this proposal are to determine when epigenetic changes occur, their stability, their affected genes, and their affected processes. Microsurgical oocyte manipulation and manipulation of embryo culture medium together provide a unique system to do this. We observed in mice that inter-strain germinal vesicle transfer (iGVT) results in a pronounced growth deficiency in a large fraction of female progeny. Additionally, altering the zygotic REDOX state (ZRS) by changing the pyruvate and lactate content in the culture medium for 10 h of culture can lead to transient or persistent post-natal growth effects. Together, these results establish iGVT and ZRS manipulation as ideal approaches that can be combined for studying the origins and nature of epigenetic changes that underlie abnormal fetal, post-natal and adult phenotypes that arise from early effects on oocytes and zygotes, and testing whether such effects can be prevented. Our Aims are to determine the mechanistic connections between oocyte (iGVT) and embryo (altered ZRS) perturbations in modifying post- natal growth, to identify the nature, timing, and stability of epigenetic changes and the array of affected genes and to determine possible overlap with epigenetic effects observed for human assisted reproduction and other variables affecting progeny growth.

? DESCRIPTION (provided by applicant): Because research into both animal and human reproductive biology has such widespread implications for human health we propose to establish a new Reproductive and Developmental Sciences Training Program (RDSTP) that will train young scientists to pursue research in High Priority Program Topic Areas identified by the NICHD/Fertility and Infertility (FI) Branch and the Gynecologic Health and Disease Branch. To provide training related to these NICHD target areas, the trainees in the proposed program will select from among four Areas of Specialization: Gonad and Gamete biology, Early Development, Reproductive Tract Biology and Gynecological Pathologies, and Environmental Factors impacting Reproduction, with multiple faculty trainers offering outstanding research opportunities and training environments in each of their specific areas of expertise. The Reproductive and Developmental Sciences program at Michigan State University has expanded extensively in the past six years with substantial institutional commitment, and is continuing to expand which provides a dynamic and rich environment for training young scientists. The RDSTP will pursue dual objectives of providing competence in emerging, cutting-edge, NICHD-recognized areas of high research interest, and providing essential skills for long-term career and professional development, with qualifications to participate in the broader roles of scientists in our community. The latter will include developing verbal and written communication skills, management and mentoring skills, career advancement skills, teaching skills, and other expertise that will be essential for future success in both academics as well as alternate career opportunities. The training program is designed to ensure that our trainees are fully equipped to meet the challenges required to succeed in any professional environment be it academics or alternate career paths in which their scientific training can be fully utilized.

In mammals, the ability to regulate transcription is absent at the start of life. This fundamental ability is acquired during early cleavage stages through the formation of a transcriptionally repressive chromatin state (TRCS), wherein transcriptional enhancers first become necessary. Establishing the TRCS soon after fertilization is vital for two reasons: 1) it is important to suppress activation of endogenous transposable elements, which if activated can be mutagenic, and 2) it is essential to correctly execute the correct transcriptional program for embryo viability. This includes activating and repressing thousands of genes during four successive waves of embryonic genome activation (EGA1 to 4). Failure to establish the TRCS and to regulate EGA waves correctly kills embryos. We discovered that Structural maintenance of chromosomes flexible hinge domain containing protein one (SMCHD1) is a maternal effect gene that potentially orchestrates all of these events. SMCHD1 1) promotes EGA1 termination, 2) is implicated in repressing genes that are up-regulated during EGA2 to 4, and 3) supports inner cell mass (ICM) formation and embryo viability revealing long-term impacts of early SMCHD1 actions. Our overall model is that oocyte-expressed SMCHD1 terminates EGA1 and helps to establish the TRCS to allow correct gene regulation during EGA2. Embryo-expressed SMCHD1 then maintains and extends gene repression and enables optimum control of EGA3 & EGA4 necessary for embryo viability. The study of SMCHD1 mechanisms of action thus provides an important new entry for discovering fundamental mechanisms regulating embryonic genome function and viability. We created a novel floxed Smchd1 allele, which can be used to achieve oocyte-specific ablation of SMCHD1 function, and thus create embryos that lack maternal, embryonic or both sources of SMCHD1, as needed to dissect SMCHD1 earl functions. This proposal will provide essential preliminary data on the phenotype of these knockout animals to allow more in-depth mechanistic studies to be pursued. In Aim 1 we will determine the effects of oocyte-specific knockout on oogenesis and early embryo viability. In Aim 2 we will assess SMCHD1?s role in controlling genes that are activated during the first two waves of gene expression during the 2-cell stage. Overall, this project seeks to solve long-standing fundamental mysteries of how mammalian embryos become competent for life.