Wilhelmine N. de Vries

Expression of Cre recombinase in mouse oocytes: a means to study maternal effect genes.

., 1998). Thesematernally derived transcripts are responsible for thecompletion of meiosis, initiation of mitosis, activation ofthe embryonic genome, and completion of the transfor-mation of the highly differentiated oocyte into a totipo-tent embryonic cell. To study the effect of specific ma-ternal transcripts on these processes, we have sought toestablish a Cre-

Trim28 Is Required for Epigenetic Stability During Mouse Oocyte to Embryo Transition

Trimprinting the Genome Reprogramming the parental genomes during the oocyte-to-embryo transition requires highly controlled epigenetic mechanisms. Although resetting the genome to a ground state is essential, conservation of inheritable marks is equally important. Now, Messerschmidt et al. (p. 1499) demonstrate that maternal deletion of the epigenetic modifier Trim28 in mice results in a strongly variable, yet ultimately embryonic, lethal phenotype. Aberrant loss of DNA methylation at imprinting control regions and thus partial loss of epigenetic memory was responsible for the phenotype. The stochastic time and mode of embryonic death reflect the exquisitely balanced interplay of maternal and zygotic factors in the early mammalian embryo. In early mouse embryos, the loss of a single maternal gene results in lethal phenotypic and epigenetic variability. Phenotypic variability in genetic disease is usually attributed to genetic background variation or environmental influence. Here, we show that deletion of a single gene, Trim28 (Kap1 or Tif1β), from the maternal germ line alone, on an otherwise identical genetic background, results in severe phenotypic and epigenetic variability that leads to embryonic lethality. We identify early and minute epigenetic variations in blastomeres of the preimplantation embryo of these animals, suggesting that the embryonic lethality may result from the misregulation of genomic imprinting in mice lacking maternal Trim28. Our results reveal the long-range effects of a maternal gene deletion on epigenetic memory and illustrate the delicate equilibrium of maternal and zygotic factors during nuclear reprogramming.

Maternal β-catenin and E-cadherin in mouse development

The oocyte to embryo transition in metazoans depends on maternal proteins and transcripts to ensure the successful initiation of development, and the correct and timely activation of the embryonic genome. We conditionally eliminated the maternal gene encoding the cell adhesion molecule E-cadherin and partially eliminated the β-catenin gene from the mouse oocyte. Oocytes lacking E-cadherin, or expressing a truncated allele of β-catenin without the N-terminal part of the protein, give rise to embryos whose blastomeres do not adhere. Blastomere adhesion is restored after translation of protein from the wild-type paternal alleles: at the morula stage in embryos lacking maternal E-cadherin, and at the late four-cell stage in embryos expressing truncated β-catenin. This suggests that adhesion per se is not essential in the early cleavage stage embryos, that embryos develop normally if compaction does not occur until the morula stage, and that the zona pellucida suffices to maintain blastomere proximity. Although maternal E-cadherin is not essential for the completion of the oocyte-to-embryo transition, absence of wild-type β-catenin in oocytes does statistically compromise developmental success rates. This developmental deficit is alleviated by the simultaneous absence of maternal E-cadherin, suggesting that E-cadherin regulates nuclear β-catenin availability during embryonic genome activation.

Stabilization of β-catenin in the mouse zygote leads to premature epithelial-mesenchymal transition in the epiblast

Many components of the Wnt/β-catenin signaling pathway are expressed during mouse pre-implantation embryo development, suggesting that this pathway may control cell proliferation and differentiation at this time. We find no evidence for a functional activity of this pathway in cleavage-stage embryos using the Wnt-reporter line, BAT-gal. To further probe the activity of this pathway, we activated β-catenin signaling by mating a zona pellucida3-cre (Zp3-cre) transgenic mouse line with a mouse line containing an exon3-floxedβ -catenin allele. The result is expression of a stabilized form ofβ -catenin, resistant to degradation by the GSK3β-mediated proteasome pathway, expressed in the developing oocyte and in each cell of the resulting embryos. Nuclear localization and signaling function of β-catenin were not observed in cleavage-stage embryos derived from these oocytes. These results indicate that in pre-implantation embryos, molecular mechanisms independent of the GSK3β-mediated ubiquitination and proteasome degradation pathway inhibit the nuclear function of β-catenin. Although the mutant blastocysts initially developed normally, they then exhibited a specific phenotype in the embryonic ectoderm layer of early post-implantation embryos. We show a nuclear function of β-catenin in the mutant epiblast that leads to activation of Wnt/β-catenin target genes. As a consequence, cells of the embryonic ectoderm change their fate, resulting in a premature epithelial-mesenchymal transition.

Gene replacement reveals a specific role for E-cadherin in the formation of a functional trophectoderm

During mammalian embryogenesis the trophectoderm represents the first epithelial structure formed. The cell adhesion molecule E-cadherin is ultimately necessary for the transition from compacted morula to the formation of the blastocyst to ensure correct establishment of adhesion junctions in the trophectoderm. Here, we analyzed to what extent E-cadherin confers unique adhesion and signaling properties in trophectoderm formation in vivo. Using a gene replacement approach, we introduced N-cadherin cDNA into the E-cadherin genomic locus. We show that the expression of N-cadherin driven from the E-cadherin locus reflects the expression pattern of endogenous E-cadherin. Heterozygous mice co-expressing E- and N-cadherin are vital and show normal embryonic development. Interestingly, N-cadherin homozygous mutant embryos phenocopy E-cadherin-null mutant embryos. Upon removal of the maternal E-cadherin, we demonstrate that N-cadherin is able to provide sufficient cellular adhesion to mediate morula compaction, but is insufficient for the subsequent formation of a fully polarized functional trophectoderm. When ES cells were isolated from N-cadherin homozygous mutant embryos and teratomas were produced, these ES cells differentiated into a large variety of tissue-like structures. Importantly, different epithelial-like structures expressing N-cadherin were formed, including respiratory epithelia, squamous epithelia with signs of keratinization and secretory epithelia with goblet cells. Thus, N-cadherin can maintain epithelia in differentiating ES cells, but not during the formation of the trophectoderm. Our results point to a specific and unique function for E-cadherin during mouse preimplantation development.

Members of the WNT signaling pathways are widely expressed in mouse ovaries, oocytes, and cleavage stage embryos

The mammalian oocyte‐to‐embryo transition, characterized by a period of transcriptional silence, is dependent on maternal RNAs and proteins produced during the growth phase of the oocyte. Signaling pathways control timely transcription and translation of RNA, as well as post‐translational modification of proteins. The WNT/β‐catenin pathway is clearly not active during preimplantation embryo development. However, alternative Wnt signaling pathways may play a role during early embryo development. This study describes the extensive expression, at the transcript and protein level, of receptors, ligands, and intracellular molecules known to play a role in WNT signaling, as well as those known to negatively regulate the canonical WNT/β‐catenin pathway in developing oocytes and preimplantation embryos. This expression of a wide array of molecules involved in WNT signaling suggests that the alternative WNT pathways may be active during oogenesis and the oocyte‐to‐embryo transition. Developmental Dynamics 237:1099–1111, 2008.

Mutations in a P-Type ATPase Gene Cause Axonal Degeneration

Neuronal loss and axonal degeneration are important pathological features of many neurodegenerative diseases. The molecular mechanisms underlying the majority of axonal degeneration conditions remain unknown. To better understand axonal degeneration, we studied a mouse mutant wabbler-lethal (wl). Wabbler-lethal (wl) mutant mice develop progressive ataxia with pronounced neurodegeneration in the central and peripheral nervous system. Previous studies have led to a debate as to whether myelinopathy or axonopathy is the primary cause of neurodegeneration observed in wl mice. Here we provide clear evidence that wabbler-lethal mutants develop an axonopathy, and that this axonopathy is modulated by Wlds and Bax mutations. In addition, we have identified the gene harboring the disease-causing mutations as Atp8a2. We studied three wl alleles and found that all result from mutations in the Atp8a2 gene. Our analysis shows that ATP8A2 possesses phosphatidylserine translocase activity and is involved in localization of phosphatidylserine to the inner leaflet of the plasma membrane. Atp8a2 is widely expressed in the brain, spinal cord, and retina. We assessed two of the mutant alleles of Atp8a2 and found they are both nonfunctional for the phosphatidylserine translocase activity. Thus, our data demonstrate for the first time that mutation of a mammalian phosphatidylserine translocase causes axon degeneration and neurodegenerative disease.

Chronic consumption of a western diet induces robust glial activation in aging mice and in a mouse model of Alzheimer’s disease

Studies have assessed individual components of a western diet, but no study has assessed the long-term, cumulative effects of a western diet on aging and Alzheimer’s disease (AD). Therefore, we have formulated the first western-style diet that mimics the fat, carbohydrate, protein, vitamin and mineral levels of western diets. This diet was fed to aging C57BL/6J (B6) mice to identify phenotypes that may increase susceptibility to AD, and to APP/PS1 mice, a mouse model of AD, to determine the effects of the diet in AD. Astrocytosis and microglia/monocyte activation were dramatically increased in response to diet and was further increased in APP/PS1 mice fed the western diet. This increase in glial responses was associated with increased plaque burden in the hippocampus. Interestingly, given recent studies highlighting the importance of TREM2 in microglia/monocytes in AD susceptibility and progression, B6 and APP/PS1 mice fed the western diet showed significant increases TREM2+ microglia/monocytes. Therefore, an increase in TREM2+ microglia/monocytes may underlie the increased risk from a western diet to age-related neurodegenerative diseases such as Alzheimer’s disease. This study lays the foundation to fully investigate the impact of a western diet on glial responses in aging and Alzheimer’s disease.

1 – Fertilization and Activation of the Embryonic Genome

This chapter discusses the fertilization and activation of the embryonic genome. The full-grown oocyte arrested in prophase of the first meiotic division contains all of the molecules that are utilized to bridge the period of transcriptional silence that begins with the completion of oocyte growth. Under hormonal stimulation the full-grown oocyte begins maturation, completing the first meiosis and the first half of the second meiotic division before arresting in metaphase of the second meiotic division. During this period the extensive stores of maternal messages are selectively utilized, which can result in the synthesis of a new and perhaps a different set of proteins. Simultaneously, preexisting maternal proteins can undergo post-translational modification and degradation. These programmed events result in an oocyte that is ready for fertilization. Fertilization initiates a cascade of events, also dependent on protein modifications and on the timely synthesis of new proteins from maternal mRNA stores that leads to completion of the second meiotic division, remodeling of egg and sperm chromatin, DNA synthesis, entry into the first mitosis, and activation of the embryonic genome.

Crim1 regulates integrin signaling in murine lens development

The developing lens is a powerful system for investigating the molecular basis of inductive tissue interactions and for studying cataract, the leading cause of blindness. The formation of tightly controlled cell-cell adhesions and cell-matrix junctions between lens epithelial (LE) cells, between lens fiber (LF) cells, and between these two cell populations enables the vertebrate lens to adopt a highly ordered structure and acquire optical transparency. Adhesion molecules are thought to maintain this ordered structure, but little is known about their identity or interactions. Cysteine-rich motor neuron 1 (Crim1), a type I transmembrane protein, is strongly expressed in the developing lens and its mutation causes ocular disease in both mice and humans. How Crim1 regulates lens morphogenesis is not understood. We identified a novel ENU-induced hypomorphic allele of Crim1, Crim1glcr11, which in the homozygous state causes cataract and microphthalmia. Using this and two other mutant alleles, Crim1null and Crim1cko, we show that the lens defects in Crim1 mouse mutants originate from defective LE cell polarity, proliferation and cell adhesion. Crim1 adhesive function is likely to be required for interactions both between LE cells and between LE and LF cells. We show that Crim1 acts in LE cells, where it colocalizes with and regulates the levels of active β1 integrin and of phosphorylated FAK and ERK. The RGD and transmembrane motifs of Crim1 are required for regulating FAK phosphorylation. These results identify an important function for Crim1 in the regulation of integrin- and FAK-mediated LE cell adhesion during lens development. Summary: Crim1, a type I transmembrane protein, acts in lens epithelial cells where it colocalizes with and regulates the levels of active β1 integrin to control cell adhesion during mouse lens morphogenesis.