Epigenetic Control of Early Mouse Development.

Although the genes sequentially transcribed in the mammalian embryo prior to implantation have been identified, understanding of the molecular processes ensuring this transcription is still in development. The genomes of the sperm and egg are hypermethylated, hence transcriptionally silent. Their union, in the prepared environment of the egg, initiates their epigenetic genomic reprogramming into a totipotent zygote, in which the genome gradually becomes transcriptionally activated. During gametogenesis, sex-specific processes result in sperm and eggs with disparate epigenomes, both of which require drastic reprogramming to establish the totipotent genome of the zygote and the pluripotent inner cell mass of the blastocyst. Herein, we describe the factors, DNA and histone modifications, activation and repression of retrotransposons, and cytoplasmic localizations, known to influence the activation of the mammalian genome at the initiation of new life.

Reprogramming and differentiation in mammals: motifs and mechanisms.

The natural reprogramming of the mammalian egg and sperm genomes is an efficient process that takes place in less than 24 hours and gives rise to a totipotent zygote. Transfer of somatic nuclei to mammalian oocytes also leads to their reprogramming and formation of totipotent embryos, albeit very inefficiently and requiring an activation step. Reprogramming of differentiated cells to induced pluripotent stem (iPS) cells takes place during a period of time substantially longer than reprogramming of the egg and sperm nuclei and is significantly less efficient. The stochastic expression of endogenous proteins during this process would imply that controlled expression of specific proteins is crucial for reprogramming to take place. The fact that OCT4, NANOG, and SOX2 form the core components of the pluripotency circuitry would imply that control at the transcriptional level is important for reprogramming to iPS cells. In contradistinction, the much more efficient reprogramming of the mammalian egg and sperm genomes implies that other levels of control are necessary, such as chromatin remodeling, translational regulation, and efficient degradation of no longer needed proteins and RNAs.

Genome plasticity in the mouse oocyte and early embryo.

In dissecting the molecules and molecular mechanisms that control mammalian oocyte-to-embryo transition, we found abundant transcripts representing developmentally regulated ERVs (endogenous retroviruses) in mouse oocyte and two-cell stage embryo cDNA libraries. These retrotransposons can act as alternative promoters and first exons for diverse genes, synchronizing their expression. Heritable genetic change due to replication of these retrotransposons probably occurs specifically in oocytes and early embryos. ERVs are usually epigenetically silenced, through DNA methylation and chromatin-based mechanisms. Their activation and silencing indicates a change in the epigenetic state of the genome. The thousands of endogenous retro-elements in the mouse genome provides potential scope for large-scale co-ordinated epigenetic fluctuations and leads to the hypothesis that differential transposable element expression triggers sequential reprogramming of the embryonic genome during the oocyte-to-embryo transition.

Systems biology of the 2-cell mouse embryo.

The transcriptome of the 2-cell mouse embryo was analyzed to provide insight into the molecular networks at play during nuclear reprogramming and embryonic genome activation. Analysis of ESTs from a 2-cell cDNA library identified nearly 4,000 genes, over half of which have not been previously studied. Transcripts of mobile elements, especially those of LTR retrotransposons, are abundantly represented in 2-cell embryos, suggesting their possible role in introducing genomic variation, and epigenetic restructuring of the embryonic genome. Analysis of Gene Ontology of the 2-cell-stage expressed genes outlines the major biological processes that guide the oocyte-to-embryo transition. These results provide a foundation for understanding molecular control at the onset of mammalian development.

Expression of genes involved in mammalian meiosis during the transition from egg to embryo.

The ooplasm of higher eukaryotes provides substances necessary for completing the last stages of meiosis and initiating the first mitotic division. These processes are firmly attuned to other events in the egg and newly formed embryo, such as switching from the use of maternal transcripts to the onset of zygotic transcription. In mammals little is known about the molecular mechanisms guiding this transition, largely due to the lack of information about genes expressed in the egg and early embryos. Studies of yeast mitosis have contributed much of what is known about the vertebrate cell cycle, and recent reports indicate that homologs of yeast DNA repair genes also function during mammalian gametogenesis. To examine whether this conservation can be expanded to include genes operative in oocyte meiosis, we performed a computer-based search for homologs of yeast genes that are induced during sporulation in C. elegans, Drosophila, and mammals. Results from this study suggest that yeast and higher eukaryotes share genes that coordinate the overall process of meiosis. However intriguing differences exist, reflecting the distinctive mechanisms governing the progression of meiosis in each organism. ESTs representing more than half of the mammalian homologs are present in mouse cDNA libraries that contains genes controlling the meiosis/mitosis transition. About 50% of these genes contain potential cis-elements for cytoplasmic polyadenylation in their 3'-UTR, suggesting the importance of controlled translation in the egg and zygote.

Expression and immune recognition of SV40 Tag in transgenic mice that develop metastatic osteosarcomas.

Mature adult mice of the C57BL/6-TgN(Amy1TAg)501Knw transgenic mouse lineage, 501, containing a liver alpha-amylase promoted-SV40 Tag hybrid gene, routinely develop SV40 Tag-induced metastatic osteosarcomas. This form of alpha-amylase was known to be expressed in the liver, salivary glands, pancreas, and fat. Cells in the normal rib adjacent to the periosteum also express alpha-amylase suggesting that transgene expression is correctly targeted to generate osteosarcomas. 501 mice express SV40 Tag in the salivary glands but do not develop abnormalities in these organs by the time of their death from SV40-induced osteosarcomas. Mice of the C57BL/6 strain make a strong and effective anti-tumor immune response to SV40 Tag immunization. However, immunization of 501 mice with SV40 Tag early in life does not alter or prevent SV40 Tag-induced osteosarcomagenesis. 501 mice mount a significantly less effective cytotoxic T-lymphocyte response following SV40 Tag immunization while 501 osteosarcoma-derived cells are fully susceptible to SV40 Tag-specific T-cell lysis. This suggests that partial tolerance, not loss of antigen presentation by tumor cells, characterizes this mouse model of endogenous bone tumor development. To determine whether the immune recognition of endogenous SV40 Tag could influence tumorigenesis, the metastatic potential and time of death from tumor was investigated in CD4-null mutant 501 mice and beta-2 microglobulin-null mutant 501 mice. The size and number of metastases in these strains and longevity of these strains varied. We suggest that components of both the innate and adaptive immune response control tumor appearance and progression.

Timely translation during the mouse oocyte-to-embryo transition.

In the mouse, completion of oocyte maturation and the initiation of preimplantation development occur during transcriptional silence and depend on the presence and translation of stored mRNAs transcribed in the growing oocyte. The Spin gene has three transcripts, each with an identical open reading frame and a different 3' untranslated region (UTR). (Beta)-galactosidase-tagged reporter transcripts containing each of the different Spin 3'UTRs were injected into oocytes and zygotes and (beta)-galactosidase activity was monitored. Results from these experiments suggest that differential polyadenylation and translation occurs at two critical points in the oocyte-to-embryo transition - upon oocyte maturation and fertilization - and is dependent on sequences in the 3'UTR. The stability and mobility shifts of ten other maternal transcripts were monitored by reprobing a northern blot of oocytes and embryos collected at 12 hour intervals after fertilization. Some are more stable than others and the upward mobility shift associated with polyadenylation correlates with the presence of cytoplasmic polyadenylation elements (CPEs) within about 120 nucleotides of the nuclear polyadenylation signal. A survey of the 3' UTRs of expressed sequence tag clusters from a mouse 2-cell stage cDNA library indicates that about one third contain CPEs. We suggest that differential transcript stability and a translational control program can supply the diversity of protein products necessary for oocyte maturation and the initiation of development.

Sequential loss of cytotoxic T lymphocyte responses to simian virus 40 large T antigen epitopes in T antigen transgenic mice developing osteosarcomas.

The role of CTL tolerance in tumor immunity to SV40 large T antigen (T ag)-induced tumors was studied using T ag transgenic mice of the line 501 (H2b). 501 mice express SV40 T ag under the influence of the alpha-amylase promoter, which leads to the development of osteogenic osteosarcomas late in life and eventual death between 12 and 17 months of age. We determined the ability of 501 mice to respond to the four H2b-restricted T ag CTL epitopes, which include epitope I (T ag 206-215), epitope II/III (T ag 223-231), the immunorecessive epitope V (T ag 489-497), restricted by H2-Db, and epitope IV (T ag 404-411), restricted by H2-Kb. We demonstrate that 501 mice are partially tolerant to the H2b-restricted T ag epitopes. Immunization of 4-month-old 501 mice with T ag-transformed syngeneic cell lines or a recombinant vaccinia virus expressing full-length T ag elicited CTL responses against the H2-Kb-restricted T ag epitope IV only. In contrast, immunization of 4-month-old 501 mice with recombinant vaccinia viruses expressing individual T ag epitopes as minigenes elicited CTLs against epitopes I, IV, and V, but not against epitope II/III. Complete tolerance to epitopes I, IV, and V developed in 501 mice, but the age when tolerance was detected varied for each epitope. Tolerance to epitope I occurred by 6 months of age and was accelerated in the absence of CD4+ T cells. Tolerance to the immunorecessive epitope V was observed in 12-month-old 501 mice but was independent of the presence of osteosarcomas. In contrast, CTLs specific for epitope IV were detected in mice from 3 to 14 months of age but not in mice that had developed osteosarcomas. Analysis of epitope IV-specific CD8+ cells derived from 3-month-old 501 mice with H2-Kb/epitope IV tetramers revealed decreased numbers of epitope IV-specific CD8+ cells in 501 mice relative to C57BL/6 mice, with a further decrease in older 501 mice. Tumor progression resulted in loss of H2-Kb/epitope IV tetramer staining CD8+ cells. Thus, progression to tolerance to individual T ag CTL epitopes in 501 mice is epitope dependent.

The mouse cornichon gene family.

As part of a large scale mouse Expressed Sequence Tag (EST) project to identify molecules involved in the initiation of mammalian development, a homolog of the Drosophila cornichon gene was detected as a mouse maternal transcript present in the two-cell embryo. Cornichon is a multigene family in the mouse: the new gene, Cnih, maps to mouse chromosome 10, another cornichon homolog, Cnil, maps to chromosome 14 and two additional cornichon-related loci, possibly pseudogenes, localize to chromosomes 3 and 10, respectively. Cnih encodes an open reading frame (ORF) of 144 amino acids that is 93% homologous (68% identical) to the Drosophila protein, whereas the ORF of Cnil contains two extra polypeptide regions not found in these other proteins. Transcripts of Cnih are highly abundant in the full grown oocyte and the ovulated unfertilized egg, while Cnil message is only detectable after activation of the embryonic genome at the eight-cell stage. In situ hybridization shows specific localization of Cnih transcripts to ovarian oocytes. The lack of cytoplasmic polyadenylation of the maternally inherited Cnih transcript suggests that Cnih mRNA is translated in the full grown oocyte before, but not after, ovulation. In Drosophila, cornichon is involved in the establishment of both anterior-posterior and dorso-ventral polarity via the epidermal growth factor (EGF)-receptor signaling pathway. Finding Cnih in the mammalian oocyte opens a new perspective on the investigation of EGF-signaling in the oocyte.

Lactation-induced WAP-SV40 Tag transgene expression in C57BL/6J mice leads to mammary carcinoma.

Two transgenic lineages were generated by directing the expression of SV40 T antigen to the mammary gland of inbred C57BL/6J mice using the whey acidic protein (WAP) promoter. In one lineage, WAPTag 1, multiparous female mice developed mammary adenocarcinoma with an average latency period of 13 months. The histopathological phenotype was heterogeneous, tumours occurred in a stochastic fashion, normal tissue was located next to neoplastic tissue, the mammary tumours usually developed and were remarkably similar to that observed in human cases. In addition, male and virgin females developed a poorly differentiated SV40 T antigen-positive soft tissue sarcoma, also at 13 months of age. In the other lineage, WAPTag 3, some parous females developed mammary tumours, but most mice succumbed to osteosarcomas arising from the os petrosum at 5.5 to 6 months of age and on necropsy, renal adenocarcinomas were also found. Appearance of these unexpected tumour types demonstrates the non-specific expression of SV40 Tag under the control of the WAP promoter. The expression of SV40 Tag in mammary glands at different stages of development was also examined, and only actively lactating glands were positive. This suggests that the abundant cyclic synthesis of SV40 Tag associated with pregnancy is required for mammary tumorigenesis in these lineages.

Cloning and mapping of the UNC5C gene to human chromosome 4q21-q23.

The vertebrate Unc5 genes, like their Caenorhabditis elegans counterpart, define a family of putative netrin receptors. One member of this family, Unc5h3, has been shown to have an important role during cell migration in the developing murine cerebellum. Mice homozygous for mutations in Unc5h3 are ataxic and have cerebellar hypoplasia and laminar structure defects. In addition, these mice have ectopic granule and Purkinje cells in the midbrain and brainstem. We have identified the human homologue of this gene, UNC5C, and shown it to have a restricted expression pattern in adult human tissues. By radiation hybrid analysis, we have determined that UNC5C localizes to chromosome 4q21-q23 between markers D4S1557 and D4S836 and is closely linked to the Parkinson disease gene.

Differential expression of the zinc finger gene TCF17 in testicular tumors.

TCF17, the human homologue of the rat zinc finger gene Kid1, is highly expressed in neurons derived from the retinoic acid-treated human embryonal carcinoma (EC) cell line, NTERA-2. This differentiation-related up-regulation of TCF17 prompted us to investigate its expression during human spermatogenesis and in human testicular germ cell tumors considered to be precursors of EC. Expression of TCF17 increases as spermatogonia differentiate into spermatocytes, indicating that this gene is developmentally regulated during spermatogenesis. TCF17 mRNA levels are high in carcinoma in situ and in seminoma, a tumor derived from carcinoma in situ but still of low-grade malignancy. However, TCF17 expression is decreased in highly malignant EC. The differential regulation of TCF17 during neoplastic germ cell differentiation may be of predictive value in germ cell tumor diagnosis.

Differential expression of the zinc finger gene Zfp105 during spermatogenesis.

We report the isolation of Zfp105, the mouse homolog of the human ZNF35 zinc finger gene. Zfp105 and ZNF35 are highly conserved at the protein and nucleotide level, and Zfp105 maps to a region of mouse Chromosome (Chr) 9 that is homologous to the human region containing ZNF35. Zpf105 is highly expressed in the testis, especially in pachytene spermatocytes and round spermatids. The possible role of this gene product in maintaining an ordered germ cell differentiation process is discussed.

SPIN, a substrate in the MAP kinase pathway in mouse oocytes.

The newly cloned gene Spin encodes a 30-kDa protein, a well-defined abundant molecule found in mouse oocytes and early embryos. This protein SPIN undergoes metaphase-specific phosphorylation and binds to the spindle. To understand the role of SPIN in oocyte meiosis, oocytes were treated with drugs that affect the cell cycle by activating or inactivating specific kinases. The posttranslational modification of SPIN in the treated oocytes was then investigated by one- and two-dimensional gel electrophoresis. Modification of SPIN is inhibited by treatment with 6-dimethylaminopurine (DMAP), suggesting that SPIN is phosphorylated by a serine-threonine kinase. Furthermore, SPIN from cycloheximide-treated oocytes that lack detectable MAP kinase activity is only partially phosphorylated, indicating that SPIN may be phosphorylated by the MOS/MAP kinase pathway. To confirm this observation, SPIN was analyzed in Mos-null mutant mice lacking MAP kinase activity. Normal posttranslational modification of SPIN did not occur in Mos-null mutant oocytes. In addition, there is reduced association of SPIN with the metaphase I spindle in Mos-null mutant oocytes, as determined by immunohistochemical analysis. These findings suggest that SPIN is a substrate in the MOS/ MAP kinase pathway and further that this phosphorylation of SPIN may be essential for its interaction with the spindle.

Embryonic phenotype of Unc5h3 mutant mice suggests chemorepulsion during the formation of the rostral cerebellar boundary.

Mutation of the Unc5h3 (formally known as rcm) gene has important consequences on neuronal migration during cerebellar development. Unc5h3 transcripts are expressed early (embryonic day 8.5) in the hindbrain region and later in the cerebellar primordia. In Unc5h3 mutant embryos, both the development and initial migration of Purkinje cell progenitors occur as in wild-type controls. The rhombic lip, from which granule cell precursors arise, also appears to form normally in mutants. However, at E13.5, an abnormal subpopulation of granule cell and Purkinje cell precursors becomes detectable in rostral areas of the Unc5h3 mutant brain stem. These ectopic cerebellar cells increase in number and continue moving in a rostral direction throughout the remainder of embryogenesis and early stages of postnatal development invading the lateral regions of the pontine area and eventually the inferior colliculus. Cell proliferation markers demonstrate the mitotic nature of these subpial ectopic granule neurons indicating the displacement of the rostral external germinal layer in mutant animals. Our data suggest that establishment of the rostral cerebellar boundary may rely on chemorepulsive signaling events that require UNC5H3 expressed by cerebellar neurons and extracellular ligands that are functionally related to the UNC5H3-binding, guidance molecule netrin1. Although the phenotype resulting from the Unc5h3 mutation is apparently limited to the formation of the cerebellum, additional sites of Unc5h3 expression are also found during development suggesting the compensatory function of other genes.

Maid: a maternally transcribed novel gene encoding a potential negative regulator of bHLH proteins in the mouse egg and zygote.

We isolated an abundant novel cDNA SSEC-8 from a subtraction cDNA library enriched for maternal transcripts that are still present in the mouse 2 cell stage embryo. This gene is evolutionarily conserved and maps to the distal region of mouse chromosome 2. The deduced polypeptide sequence of the encoded protein contains a conserved helix-loop-helix (HLH) motif without a basic DNA binding domain, suggesting that it functions as a negative regulator of basic (b) HLH transcription factors. Gel mobility shift assays show that in vitro translated protein prevents the E12/MyoD bHLH dimer from binding to DNA. Also, transient overexpression of this protein in C2C12 cells reduced the transcription of a CAT-reporter regulated by an E12/MyoD driven enhancer. The 3'-UTR contains consensus sequences of cytoplasmic polyadenylation elements (CPE's), and the length of its poly (A) tail changes during oocyte maturation, indicating that its expression is controlled by timely activation of translation. This new gene, Maid, models the translational and transcriptional regulation of gene expression during the transition from gamete to embryo.