Overview
Our laboratory is among the first to intensively investigate the molecules and processes associated with the oocyte-to-embryo transition in mammals. The genetic material from two completely differentiated cells, sperm and egg, are reprogrammed in the egg with the result that the first two cells of the early embryo are capable of differentiating into all of the cells of the body. The egg environment is the natural reprogramming environment and is the one scientists are trying to mimic to reprogram any differentiated cell into one with totipotent potential.
We are also using a mouse model of human breast cancer to determine how tumor cells arise from somatic stem cells of more restricted developmental potential.
Research details
Transitions: Oocytes to embryos and stem cells to cancer
How are nuclei from differentiated cells (eggs and sperm or transferred somatic cells) reprogrammed to totipotency in the oocyte? What are the first steps in the stem to malignant precursor cell transition? We are addressing these questions by investigating the molecular processes that occur during the reprogramming of the gametic nuclei into the totipotent cells of the preimplantation embryo and at the initiation of mammary tumorigenesis.
Oocyte to embryo transition: Molecular changes and evolution
Dr. Alexei Evsikov published his analysis of 19,000 expressed sequence tags of the Mus musculus (mouse) fully-grown oocyte cDNA library, reporting expression of 5,400 genes and transposable elements. His comparative analysis noted the regulatory cascades underlying oogenesis in chordates as well as the rapid evolution of specific groups of genes in mammals. Overall, this research illustrates how genes acquire and lose reproductive function during evolution creating a molecular mechanism for reproductive isolation and speciation.
There is no transcription during the oocyte to embryo transition. Accordingly, the molecular processes underlying the progression from differentiated gamete to the totipotent embryonic blastomeres at the 2-cell stage include: signal transduction; selective destruction of proteins and RNAs; and controlled RNA stability and translation. Dr. Evsikov's detection of a previously undescribed oocyte-specific eukaryotic translation initiation factor, 4E (Eif4eoo) in mammals suggests a novel system for translational regulation in the transition between maternal and embryonic control of gene expression. In an ongoing collaboration with Dr. Joel Graber, motifs in the 3?-untranslated regions of these maternal mRNAs were found to be associated with transcript stability and translational control. Dr. de Vries and Dr. Graber are now investigating the role of these motifs and others in translational regulation of individual mRNAs.
Signal transduction during the oocyte-to-embryo transition
Dr. Mimi de Vries devised a Cre/loxP-based strategy to conditionally delete maternal genes as the oocyte begins its growth phase, thereby eliminating a specific mRNA/protein from the oocyte and from the embryo until new transcription from the paternal genome is initiated. This enabled her to query the role of two proteins, E-cadherin and β-catenin, during the oocyte- to-embryo transition.
E-cadherin is involved in cell adhesion while β-catenin binds to a number of other proteins, which in turn specify the activity of β-catenin as an adhesion molecule, a transcriptional co-activator, and a recruiter of chromatin modifying proteins to DNA. Surprisingly, embryo development is not negatively affected by elimination of either of these maternal proteins; the zona pellucida surrounding the developing embryo ensures the blastomeres are kept in close proximity until the protein encoded by the paternal allele is translated (by the 8 cell stage) mediating interblastomere adhesion.
The entire β-catenrin gene is not targeted by any of the known β-catenin floxed alleles; a shorter transcript and truncated protein is produced. The missing N-terminal portion of the β-catenin protein interacts with E-cadherin and the Wnt-pathway. The truncated protein, representing the C-terminal portion of the molecule, contains motifs that interact with proteins involved in nucleosome and protein modification and this truncated protein translocates to the nucleus. Even though the Wnt/β-catenin pathway is not active during the oocyte-to-embryo transition, mRNAs and proteins of the alternative Wnt-signaling pathways are present. At present, Dr. de Vries is investigating these pathways and delineating the role of β-catenin, and other chromatin remodeling proteins, during oogenesis and the oocyte-to-embryo transition.
Endogenous retroviruses and genomic reprogramming
In dissecting the molecules and molecular mechanisms that control the mammalian oocyte-to-embryo transition, Drs. Evsikov and Peaston previously reported developmentally regulated expression of specific endogenous retrovirus families. Endogenous retroviruses are usually epigenetically silenced, through DNA methylation and chromatin-based mechanisms, so their activation and silencing indicates a changing epigenetic state, which potentially affects all members of an endogenous retrovirus family simultaneously. Many endogenous retrovirus families exist as multiple, sometimes thousands, of copies in the mouse genome. Thus, the scope for coordinated epigenetic fluctuations is potentially large and leads to the hypothesis that differential transposable element expression triggers sequential reprogramming of the embryonic genome during the oocyte-to-embryo transition and in preimplantation embryos.
One way to address this hypothesis is to systematically investigate the expression and epigenetic status of multiple discrete loci of specific endogenous retroviruses. Genetic and biochemical evidence has shown that transcriptional activation of endogenous retroviruses is regulated by DNA cytosine methylation of CpG dinucleotides in their long terminal repeat promoter sequences. However, expression of some families of retrotransposons does not correlate with known global changes in methylation during these early stages of development. Moreover, sequence analysis shows that long terminal repeats of these retrotransposons are deficient in CpG dinucleotides. Dr. Anne Peaston is continuing to investigate whether CpG methylation in these long terminal repeats or flanking genomic DNA is related to their transcriptional regulation. She is using bisulfite sequencing to analyze long terminal repeat CpG methylation of selected retrotransposons in defined genomic loci in oocytes and in embryos of different stages of preimplantation development. In a complementary investigation, Dr. Keith Hutchison used a bioinformatics approach to identify specific putative active and inactive retrotransposon loci. Dr. Peaston is currently using an RT-PCR and sequencing approach to test whether transcription of the specific retrotransposons is restricted to the loci predicted by Dr. Hutchison. These studies will provide clues to the process of genomic reprogramming in the zygote and cleavage-stage embryo.
Mammary tumorigenesis
Preclinical mouse models facilitate the study of early stages of human tumorigenesis by elucidating the molecules and mechanisms involved in tumor progression. The latent, adult-onset mammary cancer transgenic mouse model C57BL/6J-Tg(WapTAg)1Knw (Waptag1) develops characteristic stages of tumorigenesis, progressing in a highly predictable manner. In multiparous mice, atypical cells appear in otherwise histologically normal mammary tissue at 6-8 months of age, ductal carcinoma in situ arises at 6-9 months of age, papillary adenocarcinoma is observed between 9 and 12 months of age, and solid invasive tumors are found by 10-14 months of age.
Karen Fancher has performed microarray analyses on mammary samples taken throughout the lifespan of Waptag1 as well as age-matched C57BL/6J normal controls, using R/MAANOVA. Genes involved in antigen presentation and defense response were up-regulated in all stages of Waptag1 mammary glands compared with normal controls. Pair-wise comparison of stages of Waptag1 tumorigenesis revealed: up-regulation of mitochondrial genes and ribosomal protein genes in atypia compared with C57BL/6J controls; over-expression of genes involved in DNA replication, histone variants, and multiple classes of retrotransposable elements in DCIS compared with atypia; massive up-regulation of cell cycle, cell division, and immune response genes in papillary adenocarcinoma compared with DCIS; up-regulation of heat shock protein genes and genes involved in glucose metabolism in solid/invasive tumors compared with papillary adenocarcinomas. Laser capture microdissection, linear amplification and microarray analysis of mammary cells from each of the specific stages are under way, as is comparison of these results with human ductal carcinoma in situ. The long-term goal of this project is to find new markers of early stages of mammary tumorigenesis so that prevention strategies can be developed.
Strong evidence from many sources indicates that cancers arise from tumor stem cells, which are difficult to eradicate and are the principle reason for metastasis, relapse and death in many different tumor types of malignancy. Moreover, epigenetic dysregulation is thought to be a common underlying process in the first stages of tumorigenesis, and may be fundamental to the diversion of normal tissue stem cells to a tumorigenic stem cell phenotype. Indeed, the up-regulation of retrotransposable elements early in Waptag1 tumorigenesis suggests that epigenetic alterations may already be present in tumorigenic stem cells prior to morphologic abnormalities in this model. Dr. Peaston is continuing a project attempting to mark tumor stem cells in the Waptag1 mice by preparing a triple transgenic mouse in which a parity-induced (i.e., induced by pregnancy and lactation) mammary stem cell population targeted for tumor development will be labeled with green fluorescent protein. The cells thus marked will be separated from normal mammary stem cell populations and identified by surface immunophenotype, opening the way to investigate molecular mechanisms involved in the normal stem to tumorigenic stem cell transition.
Lab staff
Principal Investigator: Barbara B. Knowles, Ph.D.Adjunct Staff Scientists: Davor Solter, M.D., Ph.D., Keith Hutchison, Ph.D.
Collaborator: Joel Graber, Ph.D.
Research Scientists: Wilhelmine de Vries, Ph.D., Anne Peaston, Ph.D.
Associate Research Scientist: Alexei Evsikov, Ph.D.
Graduate Student: Karen Fancher, B.S.
Research Assistants: Lan Ying Shi, M.S., Ben Harwood, B.S. (Dr. de Vries)
Publication listings
Books, Book Chapters, and Reviews:
Solter D, deVries WN, Peaston A, Evsikov A, Knowles BB. 2002. Fertilization and
activation of the embryonic genome. In: Mouse development: Morphogenesis and
Organogenesis, Tamm P, Rossant J, [eds], Academic Press, New York, N.Y.
