Alexei V. Evsikov

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.

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.

The Protein Ontology: a structured representation of protein forms and complexes

The Protein Ontology (PRO) provides a formal, logically-based classification of specific protein classes including structured representations of protein isoforms, variants and modified forms. Initially focused on proteins found in human, mouse and Escherichia coli, PRO now includes representations of protein complexes. The PRO Consortium works in concert with the developers of other biomedical ontologies and protein knowledge bases to provide the ability to formally organize and integrate representations of precise protein forms so as to enhance accessibility to results of protein research. PRO (http://pir.georgetown.edu/pro) is part of the Open Biomedical Ontology Foundry.

MouseCyc: a curated biochemical pathways database for the laboratory mouse.

Linking biochemical genetic data to the reference genome for the laboratory mouse is important for comparative physiology and for developing mouse models of human biology and disease. We describe here a new database of curated metabolic pathways for the laboratory mouse called MouseCyc http://mousecyc.jax.org. MouseCyc has been integrated with genetic and genomic data for the laboratory mouse available from the Mouse Genome Informatics database and with pathway data from other organisms, including human.

Gene expression during the oocyte-to-embryo transition in mammals

The seminal question in modern developmental biology is the origins of new life arising from the unification of sperm and egg. The roots of this question begin from 19th to 20th century embryologists studying fertilization and embryogenesis. Although the revolution of molecular biology has yielded significant insight into the complexity of this process, the overall orchestration of genes, molecules, and cells is still not fully formed. Early mammalian development, specifically the oocyte‐to‐embryo transition, is essentially under “maternal command” from factors deposited in the cytoplasm during oocyte growth, independent of de novo transcription from the nascent embryo. Many of the advances in understanding this developmental period occurred in tandem with application of new methods and techniques from molecular biology, from protein electrophoresis to sequencing and assemblies of whole genomes. From this bed of knowledge, it appears that precise control of mRNA translation is a key regulator coordinating the molecular and cellular events occurring during oocyte‐to‐embryo transition. Notably, oocyte transcriptomes share, yet retain some uniqueness, common genetic motifs among all chordates. The common genetic motifs typically define fundamental processes critical for cellular maintenance, whereas the unique genetic features may be a source of variation and a substrate for sexual selection, genetic drift, or gene flow. One purpose for this complex interplay among genes, proteins, and cells may allow for evolution to transform and act upon the underlying processes, at molecular, structural and organismal levels, to increase diversity, which is the ultimate goal of sexual reproduction. Mol. Reprod. Dev. 76: 805–818, 2009.

Voltage sensitive phosphoinositide phosphatases of Xenopus: their tissue distribution and voltage dependence.

Voltage‐sensitive phosphatases (VSPs) are unique proteins in which membrane potential controls enzyme activity. They are comprised of the voltage sensor domain of an ion channel coupled to a lipid phosphatase specific for phosphoinositides, and for ascidian and zebrafish VSPs, the phosphatase activity has been found to be activated by membrane depolarization. The physiological functions of these proteins are unknown, but their expression in testis and embryos suggests a role in fertilization or development. Here we investigate the expression pattern and voltage dependence of VSPs in two frog species, Xenopus laevis and Xenopus tropicalis, that are well suited for experimental studies of these possible functions. X. laevis has two VSP genes (Xl‐VSP1 and Xl‐VSP2), whereas X. tropicalis has only one gene (Xt‐VSP). The highest expression of these genes was observed in testis, ovary, liver, and kidney. Our results show that while Xl‐VSP2 activates only at positive membrane potentials outside of the physiological range, Xl‐VSP1 and Xt‐VSP phosphatase activity is regulated in the voltage range that regulates sperm–egg fusion at fertilization. J. Cell. Physiol. 226: 2740–2746, 2011.

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.

Novel Alu retrotransposon insertion leading to Alström syndrome.

Alström syndrome is a clinically complex disorder characterized by childhood retinal degeneration leading to blindness, sensorineural hearing loss, obesity, type 2 diabetes mellitus, cardiomyopathy, systemic fibrosis, and pulmonary, hepatic, and renal failure. Alström syndrome is caused by recessively inherited mutations in the ALMS1 gene, which codes for a putative ciliary protein. Alström syndrome is characterized by extensive allelic heterogeneity, however, founder effects have been observed in some populations. To date, more than 100 causative ALMS1 mutations have been identified, mostly frameshift and non-sense alterations resulting in termination signals in ALMS1. Here, we report a complex Turkish kindred in which sequence analysis uncovered an insertion of a novel 333 basepair Alu Ya5 SINE retrotransposon in the ALMS1 coding sequence, a previously unrecognized mechanism underlying the mutations causing Alström syndrome. It is extraordinarily rare to encounter the insertion of an Alu retrotransposon in the coding sequence of a gene. The high frequency of the mutant ALMS1 allele in this isolated population suggests that this recent retrotransposition event spreads quickly, and may be used as a model to study the population dynamics of deleterious alleles in isolated communities.

The representation of protein complexes in the Protein Ontology (PRO)

BackgroundRepresenting species-specific proteins and protein complexes in ontologies that are both human- and machine-readable facilitates the retrieval, analysis, and interpretation of genome-scale data sets. Although existing protin-centric informatics resources provide the biomedical research community with well-curated compendia of protein sequence and structure, these resources lack formal ontological representations of the relationships among the proteins themselves. The Protein Ontology (PRO) Consortium is filling this informatics resource gap by developing ontological representations and relationships among proteins and their variants and modified forms. Because proteins are often functional only as members of stable protein complexes, the PRO Consortium, in collaboration with existing protein and pathway databases, has launched a new initiative to implement logical and consistent representation of protein complexes.DescriptionWe describe here how the PRO Consortium is meeting the challenge of representing species-specific protein complexes, how protein complex representation in PRO supports annotation of protein complexes and comparative biology, and how PRO is being integrated into existing community bioinformatics resources. The PRO resource is accessible at http://pir.georgetown.edu/pro/.ConclusionPRO is a unique database resource for species-specific protein complexes. PRO facilitates robust annotation of variations in composition and function contexts for protein complexes within and between species.

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.