A genome-wide study of gene activity reveals developmental signaling pathways in the preimplantation mouse embryo

The preimplantation development of the mammalian embryo encompasses a series of critical events: the transition from oocyte to embryo, the first cell divisions, the establishment of cellular contacts, the first lineage differentiation-all the first subtle steps toward a future body plan. Here, we use microarrays to explore gene activity during preimplantation development. We reveal robust and dynamic patterns of stage-specific gene activity that fall into two major phases, one up to the 2-cell stage (oocyte-to-embryo transition) and one after the 4-cell stage (cellular differentiation). The mouse oocyte and early embryo express components of multiple signaling pathways including those downstream of Wnt, BMP, and Notch, indicating that conserved regulators of cell fate and pattern formation are likely to function at the earliest embryonic stages. Overall, these data provide a detailed temporal profile of gene expression that reveals the richness of signaling processes in early mammalian development.     

Signaling pathways and preimplantation development of mammalian embryos

The mammalian preimplantation embryo is a critical and unique stage in embryonic development. This stage includes a series of crucial events: the transition from oocyte to embryo, the first cell divisions, and the establishment of cellular contacts. These events are regulated by multiple signal-transduction pathways. In this article we describe patterns of stage-specific expression in several signal-transduction pathways and try to give a profile of the signaling transduction network in preimplantation development of mammalian embryo.     

Mapping the gene expression universe

Methods to globally survey gene expression provide valuable insights into gene function during development. In particular, comprehensive in situ hybridization studies have demonstrated that gene expression patterns are extraordinarily diverse and new imaging techniques have been introduced to capture these patterns with higher resolution at the tissue, cellular, and subcellular levels. The analysis of massive image databases can be greatly facilitated by computer vision techniques once annotated image sets reach the crucial mass sufficient to train the computer in pattern recognition. Ultimately, genome-wide atlases of gene expression during development will record gene activity in living animals with at least cellular resolution and in the context of morphogenetic events. These emerging datasets will lead to great advances in the field of comparative genomics and revolutionize our ability to decipher and model developmental processes for a variety of organisms.     

New member of the Snf1/AMPK kinase family,Melk, is expressed in the mouse egg and preimplantation embryo

The initial phase of mammalian preimplantation development is directed by stored maternal mRNAs and their encoded proteins, yet most of the molecules controlling this process have not been described. We have used differential display analysis of cDNA libraries prepared from unfertilized eggs and preimplantation embryos to isolate three maternal cDNAs that represent novel genes exhibiting different patterns of expression during this developmental period. One of these, Melk, encodes a protein with a kinase catalytic domain and a leucine zipper motif, a new member of the Snf1/AMPK family of kinases. This gene product may play a role in the signal transduction events in the egg and early embryo. Mol. Reprod. Dev. 47:148–156, 1997. © 1997 Wiley-Liss, Inc.     

Comparative aspects of early lineage specification events in mammalian embryos – insights from reverse genetics studies

Within the mammalian class, formation of the blastocyst is morphologically highly conserved among different species. The molecular and cellular events during preimplantation embryo development have been studied extensively in the mouse as model organism, because multiple genetically defined strains and a plethora of reverse genetics tools are available to dissect specific gene functions and regulatory networks. However, major differences in preimplantation developmental kinetics, implantation, and placentation exist among mammalians, and recent studies in species other than mouse showed, that even regulatory mechanisms of the first lineage differentiation events and maintenance of pluripotency are not always conserved. Here, we focus on the first and the second lineage segregation in mouse and bovine embryos, when the first differentiated cell types emerge. We outline their common features and differences in the regulation of these essential events during embryonic development with a glance at further species. In addition, we show how new reverse genetics strategies aid the study of regulatory circuits in embryos of domestic species, enhancing our overall understanding of mammalian preimplantation development.     

Orphan nuclear receptor betaFTZ-F1 is required for muscle-driven morphogenetic events at the prepupal-pupal transition in Drosophila melanogaster

In Drosophila melanogaster, fluctuations in 20-hydroxyecdysone (ecdysone) titer coordinate gene expression, cell death, and morphogenesis during metamorphosis. Our previous studies have supported the hypothesis that betaFTZ-F1 (an orphan nuclear receptor) provides specific genes with the competence to be induced by ecdysone at the appropriate time, thus directing key developmental events at the prepupal-pupal transition. We are examining the role of betaFTZ-F1 in morphogenesis. We have made a detailed study of morphogenetic events during metamorphosis in control and betaFTZ-F1 mutant animals. We show that leg development in betaFTZ-F1 mutants proceeds normally until the prepupal-pupal transition, when final leg elongation is delayed by several hours and significantly reduced in the mutants. We also show that betaFTZ-F1 mutants fail to fully extend their wings and to shorten their bodies at the prepupal-pupal transition. We find that betaFTZ-F1 mutants are unable to properly perform the muscle contractions that drive these processes. Several defects can be rescued by subjecting the mutants to a drop in pressure during the normal time of the prepupal-pupal transition. Our findings indicate that betaFTZ-F1 directs the muscle contraction events that drive the major morphogenetic processes during the prepupal-pupal transition in Drosophila.     

Phospholipid transfer protein (PLTP) mRNA expression is stimulated by developing embryos in the oviduct

In mammal, fertilization and early preimplantation embryo development occurs in the oviduct. Evidence is accumulating that the oviductal epithelia secrete various biomolecules to the lumen during the secretory phase of the estrus cycle to enhance embryo development. This secretory activity of the oviduct is under the regulation of steroid hormones. Observations also suggested that the gametes and embryos modulate the physiology and gene-expressing pattern of the oviduct. However, the underlying molecular changes remain elusive. We hypothesize that the developing embryos interact with the surrounding environment and affect the gene expression patterns of the oviduct, thereby modulating the oviductal secretory activity conducive to the preimplantation embryo development. To test this hypothesis, suppression subtractive hybridization (SSH) was used to compare the gene expressions in mouse oviduct containing transferred in vitro cultured preimplantation embryos with that of oviduct containing oocytes during the preimplantation period. We reported here the identification and characterization of phospholipids transfer protein (PLTP), which is highly expressed in the embryo-containing oviduct and localized at the oviductal epithelium by in situ hybridization. PLTP contains signal peptide putative for secretory function. More importantly, PLTP mRNA increases in the oviductal epithelia of pregnant, but not pseudo-pregnant mice when assayed by real-time PCR. Taken together, our data suggested that PLTP may play important role(s) during in vivo preimplantation embryo development. This molecule would be a target to delineate the mechanisms and the roles of oviductal secretory proteins on early embryonic development.     

Somatic cell-like features of cloned mouse embryos prepared with cultured myoblast nuclei

Cloning by somatic cell nuclear transfer requires silencing of the donor cell gene expression program and the initiation of the embryonic gene expression program (nuclear reprogramming). Failure to silence the donor cell program could lead to altered embryonic phenotypes. Cloned mouse embryos produced using myoblast nuclei fail to thrive in standard embryo culture media but flourish in somatic cell culture media favored by the donor myoblasts themselves, forming blastocysts at a significant rate, with robust morphologies, high total cell number, and a normal allocation of cells to the inner cell mass in most embryos. Myoblast cloned embryos continue expressing the GLUT4 glucose transporter, which is typically expressed in muscle but not in preimplantation stage embryos. Myoblast clones also exhibit precocious enrichment of GLUT1 at the cell surface. Both myoblast and cumulus cell cloned embryos exhibit enhanced rates of glucose uptake. These observations indicate that silencing of the donor cell genome during cloning either is incomplete or occurs progressively over the course of preimplantation development. As a result, cloned embryos initially exhibit many somatic cell-like characteristics. Tetraploid constructs, which possess a transplanted somatic cell genome plus the oocyte-derived chromosomes, exhibit a more embryonic-like pattern of gene expression and culture preference. We conclude that preimplantation stage cloned embryos have profoundly altered characteristics that are donor cell type specific and that exposure of cloned embryos to standard embryo culture conditions may lead to disruptions in basic homeostasis and inhibition of a range of essential processes including further nuclear reprogramming, contributing to cloned embryo demise.     

The stage-specific expression of TEC-1, -2, -3, and -4 antigens on bovine preimplantation embryos

The preimplantation developmental period is associated with constant changes within the embryo, and some of these changes are apparent on the embryo cell surface. For example, during transition from maternal to embryonic genome control and the compaction and differentiation of embryonic cells, the cell surface undergoes morphologic alterations that reflect changes in gene control. In order to gain insight into the events occurring during embryonic development and cellular differentiation, monoclonal antibodies specific for cell surface antigens (TEC antigens) of embryonic cells have been generated previously and shown to recognise either the carbohydrate moiety of embryoglycan or a developmentally regulated protein epitope. The TEC antigens have been identified on mouse preimplantation embryos, and their expression is specific to particular developmental stages. To determine whether these antigens are conserved in higher mammals, we examined the expression of four TEC antigens (TEC-1 to TEC-4) on in vitro–derived bovine and murine embryos during the preimplantation stage of development. It was found that bovine oocytes and embryos derived from in vitro maturation (IVM) and in vitro fertilisation (IVF) showed stage-specific expression of each of the TEC antigens investigated, with the pattern of expression overlapping but not identical to that seen in the mouse. Immunoprecipitation together with Western blot analysis showed that the TEC monoclonal antibodies recognised a single glycoprotein band with an apparent molecular weight of 70 kDa. Confocal microscopy of immunofluorescence staining of the bovine cells showed this protein to be located on the cell surface. The apparent negative expression of these TEC antigens by immunohistochemistry and immunoprecipitation at particular stages of development appears to be due to the epitopes being inaccessible to the TEC antibodies, since Western blotting revealed the TEC antigens to be present at all stages of development examined. Antibodies identifying stage-specific antigens will provide useful markers to characterise early embryonic cells, monitor normal embryonic development in vitro, and identify cell surface structures having a function in cell-cell interactions during embryogenesis and differentiation. Mol. Reprod. Dev. 49:19–28, 1998. © 1998 Wiley-Liss, Inc.     

Identification and analysis of stage-specific expression of lysosome-associated protein transmembrane 4alpha gene during development of preimplantation rabbit nuclear transfer embryo

The stage-specific expression of Lysosome-associated protein transmembrane 4alpha (LAPTM4alpha) in preimplantation rabbit nuclear transfer (NT) embryo was identified with the DDRT-PCR and reverse Northern Blot. The full length (1,364 bp) cDNA of LAPTM4alpha was screened out from cDNA library constructed with rabbit ovary and in situ hybridization (ISH) was used to trace the distribution of the LAPTM4alpha mRNA in intra-ovary, especially the follicle which proved that the LAPTM4alpha gene expression is involved in the follicles development, maturation, ovulation, luteinization, and preimplantation development in the rabbit (Oryctolagus cuniculus domestica). To our knowledge, this is the first characterization of LAPTM4alpha gene expression and mRNA distribution in the rabbit ovary and first evidence for this gene involving in follicle development and rabbit preimplantation development.