Early Stages Of Mouse Development Research Articles

Enforced cytokinesis without complete nuclear division in embryonic cells depleting the activity of DNA topoisomerase IIalpha.

There are two distinct DNA topoisomerase II (topo II) isoforms, designated topo IIalpha and topo IIbeta, in mammalian cells. The function of topo IIalpha in the development of mammalian cells has not been elucidated because of a lack of topo IIalpha mutants. We generated mice with a targeted disruption of the topo IIalpha gene. The development of topo IIalpha-/- embryos was terminated at the 4- or 8-cell stage. When wild-type embryos at the 2- or 4-cell stage were treated with ICRF-193, a catalytic inhibitor of topo II, nuclear division occurred followed by cytokinesis to form 4 or 8 cells, respectively, then development was terminated. Microscope analysis of 4,6-diamidino-2-phenylindole (DAPI)-stained nuclei of both topo IIalpha-/- and ICFR-193-treated embryonic cells revealed a droplet-like structure connecting the terminals of two adjacent nuclei forming a bridge-like structure. Phosphorylated histone H3, a marker for the M phases, disappeared from the nuclei of the topo IIalpha-depleted embryonic cells. Laser scanning cytometry of the topo IIalpha-depleted cells revealed the presence of 2N DNA cells. Our results indicate that topo IIalpha has an essential role in the early stages of mouse development and that depletion of topo IIalpha from the embryonic cells causes incomplete nuclear division followed by enforced cytokinesis.

The collagen type XVIII endostatin domain is co-localized with perlecan in basement membranes in vivo.

The C-terminal globular endostatin domain of collagen type XVIII is anti-angiogenic in a variety of experimental tumor models, and clinical trials to test it as an anti-tumor agent are already under way. In contrast, many of its cell biological properties are still unknown. We systematically localized the mRNA of collagen type XVIII with the help of in situ hybridization (ISH) and detected it in epithelial and mesenchymal cells of almost all organ systems throughout mouse development. Light and electron microscopic immunohistochemistry (IHC) revealed that the endostatin domain is a widespread component of almost all epithelial basement membranes in all major developing organs, and in all basement membranes of capillaries and blood vessels. Furthermore, quantitative immunogold double labeling demonstrated a co-localization of 50% of the detected endostatin domain together with perlecan in basement membranes in vivo. We conclude that the endostatin domain of collagen type XVIII plays a role, even in early stages of mouse development, other than regulating angiogenesis. In the adult, the endostatin domain could well be involved in connecting collagen type XVIII to the basement membrane scaffolds. At least in part, perlecan appears to be an adaptor molecule for the endostatin domain in basement membranes in vivo.

Embryonic lethality of mutant mice deficient in the p116 gene.

We report a lethal phenotype of mouse embryo with a disruption in the gene encoding p116, a subunit of the translation initiation factor, eIF3. The amino acid sequence of mouse p116, as deduced from the cDNA, shows high homology (97%) with human p116, and contains the conserved RNA binding sites, RNP1 and RNP2. The p116 mRNA is ubiquitously expressed in various organs, suggesting a house-keeping function of the p116 protein. To obtain genetic evidence for the essential role of the p116 protein in mouse cells, we constructed mice with a disruption in the p116 gene. Heterozygous p116(+/-) mice were intercrossed, and the genotypes of the offspring were determined. The results indicated no p116(-/-) pups among 84 neonates. Also, there were no p116(-/-) embryos 13.5 days postcoitum (d.p.c.). Among 77 embryos, there was only one p116(-/-) embryo at the blastocyst stage (3.5 d.p.c.). These results indicate that p116 plays an essential role in the early stages of mouse development.

Expression patterns of the Ets transcription factors from the PEA3 group during early stages of mouse development.

erm, er81 and pea3 are three related genes that define a novel Ets-related subfamily of transcription factors. The expression patterns of these genes has been previously characterized in the mouse from embryonic day (E) 9.5 to birth (Oncogene 15 (1997) 937). In this study, we report differential expression patterns of the PEA3 group genes during early mouse post-implantation development. erm and pea3 expression patterns were partly overlapping. erm was activated prior to pea3 in the distal tip of the embryonic epiblast but, at primitive streak-stages, both genes were coexpressed in the posterior region of the embryo in epiblast, primitive streak and adjacent mesoderm. Similar erm and pea3 expression patterns were seen later in posterior neural plate, presomitic and lateral mesoderm, mesonephros, and tail bud. Only erm, however, was expressed in specific brain regions corresponding to prospective midbrain and ventral forebrain. erm was also strongly expressed as early as E8 in the developing branchial region, especially at the level of branchial pouches, whereas pea3 transcripts appeared later in frontonasal and first arch mesenchyme. er81 transcripts were not detected prior to E9.0-9.5, suggesting that this gene may not be involved in early developmental events.

The polycomb-group gene Ezh2 is required for early mouse development.

Polycomb-group (Pc-G) genes are required for the stable repression of the homeotic selector genes and other developmentally regulated genes, presumably through the modulation of chromatin domains. Among the Drosophila Pc-G genes, Enhancer of zeste [E(z)] merits special consideration since it represents one of the Pc-G genes most conserved through evolution. In addition, the E(Z) protein family contains the SET domain, which has recently been linked with histone methyltransferase (HMTase) activity. Although E(Z)-related proteins have not (yet) been directly associated with HMTase activity, mammalian Ezh2 is a member of a histone deacetylase complex. To investigate its in vivo function, we generated mice deficient for Ezh2. The Ezh2 null mutation results in lethality at early stages of mouse development. Ezh2 mutant mice either cease developing after implantation or initiate but fail to complete gastrulation. Moreover, Ezh2-deficient blastocysts display an impaired potential for outgrowth, preventing the establishment of Ezh2-null embryonic stem cells. Interestingly, Ezh2 is up-regulated upon fertilization and remains highly expressed at the preimplantation stages of mouse development. Together, these data suggest an essential role for Ezh2 during early mouse development and genetically link Ezh2 with eed and YY1, the only other early-acting Pc-G genes.

Getting your head around Hex and Hesx1: forebrain formation in mouse.

An increasing amount of evidence suggests that in mouse there are two signalling centres required for the formation of a complete neural axis: the anterior visceral endoderm (AVE), and the node and its derivatives. Embryological and genetic studies suggest that the AVE has a head-inducing activity. In contrast, the node appears to act first as a head inducer in synergy with the AVE initiating anterior neural patterning at early stages of mouse development, and later, node derivatives are necessary for maintenance and embellishment of anterior neural character. Hex and Hesx1 are homeobox genes that are expressed in relevant tissues involved in anterior patterning. The analysis of the Hex and Hesx1 mutant mice has revealed that the lack of these genes has little or no effect on the early steps of anterior neural induction. However, both genes are required subsequently for the proper expansion of the forebrain region. We suggest that disturbance in the specification of an Fgf8 signalling centre in the anterior neural ridge may account for the anterior defects observed in these mutants.

Identification of Endogenous Retinoids, Enzymes, Binding Proteins, and Receptors during Early Postimplantation Development in Mouse: Important Role of Retinal Dehydrogenase Type 2 in Synthesis of All-trans-Retinoic Acid

Specific combinations of nuclear retinoid receptors acting as ligand-inducible transcription factors mediate the essential role of retinoids in embryonic development. Whereas some data exist on the expression of these receptors during early postimplantation development in mouse, little is known about the enzymes controlling the production of active ligands for the retinoid receptors. Furthermore, at early stages of mouse development virtually no data are available on the presence of endogenous retinoids. In the present study we have used a recently developed high-performance liquid chromatographic (HPLC) technique to identify endogenous retinoids in mouse embryos down to the egg cylinder stage. All-trans-retinoic acid, a ligand for the retinoic acid receptors, was detected in embryos dissected as early as 7.5 dpc (i.e., a combination of midstreak until late allantoic bud stage embryos). At these stages, we detected mRNA coding for all the retinoid receptors, retinoid binding proteins, and two enzymes able to convert retinol to retinal (retinol dehydrogenase 5 (RDH5) and alcohol dehydrogenase 4 (ADH4)). We also detected retinal dehydrogenase type 2 (RALDH2), an enzyme capable of oxidising the final step in the all-trans-retinoic acid synthesis. In egg cylinder stage mouse embryos no all-trans-retinoic acid was detected. However, at this stage its precursor all-trans-retinal was present. In accordance with these HPLC observations, RDH5 and ADH4 were expressed, but no transcripts coding for enzymes that oxidise retinal to retinoic acid. Therefore, our results suggest that RALDH2 is a key regulator in initiating retinoic acid synthesis sometime between the mid-primitive streak stage and the late allantoic bud stage in mouse embryos.

Regulation of left-right patterning in mice by growth/differentiation factor-1

The transforming growth factor-beta (TGF-beta) superfamily encompasses a large group of structurally related polypeptides that are capable of regulating cell growth and differentiation in a wide range of embryonic and adult tissues. Growth/differentiation factor-1 (Gdf-1, encoded by Gdf1) is a TGF-beta family member of unknown function that was originally isolated from an early mouse embryo cDNA library and is expressed specifically in the nervous systemin late-stage embryos and adult mice. Here we show that at early stages of mouse development, Gdfl is expressed initially throughout the embryo proper and then most prominently in the primitive node, ventral neural tube, and intermediate and lateral plate mesoderm. To examine its biological function, we generated a mouse line carrying a targeted mutation in Gdf1. Gdf1-/- mice exhibited a spectrum of defects related to left-right axis formation, including visceral situs inversus, right pulmonary isomerism and a range of cardiac anomalies. In most Gdf1-/- embryos, the expression of Ebaf (formerly lefty-1) in the left side of the floor plate and Leftb (formerly lefty-2), nodal and Pitx2 in the left lateral plate mesoderm was absent, suggesting that Gdf1 acts upstream of these genes either directly or indirectly to activate their expression. Our findings suggest that Gdf1 acts early in the pathway of gene activation that leads to the establishment of left-right asymmetry.

Focal expression of glial cell line-derived neurotrophic factor in developing mouse limb bud.

Glial cell line-derived neurotrophic factor (GDNF) is known to support the survival of motoneurons in vitro and in vivo, as well as subpopulations of sensory neurons in vitro. To clarify the mechanisms by which GDNF supports these neurons, we examined the patterns of GDNF mRNA expression in relation to motor and sensory axons during early stages of mouse development. Between embryonic days (E) 10 and 12, a time when motor and sensory axons are entering the periphery, GDNF mRNA is expressed at high levels in a restricted region in proximal limb buds where axons converge and enter the limb. At later ages (E14-16), GDNF mRNA was detected in non-neuronal cells along peripheral nerve, in dermis, and in some muscles. To characterize cells that express GDNF in the proximal limb, GDNF expression in the forelimb was compared to expression patterns of two markers of muscle, Pax 3 and myogenin, as well as with the pan neurotrophin receptor (p75) which is expressed by Schwann cell precursors. We show that expression of GDNF in the proximal limb bud at E11-12 does not correlate with markers of muscle or Schwann cell precursors, which supports the idea that GDNF is expressed by mesenchymal cells in this region. Our results suggest that GDNF expression in proximal limb buds may function as a transient survival factor, particularly for motor neurons, before they reach their final targets. GDNF expression in muscle and dermis at later stages suggests that GDNF may have additional functions as motor and sensory neurons mature.

Conserved regulatory element involved in the early onset ofHoxb6 gene expression

We have identified a 338 bp DNA fragment, the lateral plate mesoderm (LPM) enhancer, that is highly conserved between mouse and human. The LPM enhancer directs gene expression into the posterior lateral plate mesoderm and hindgut endoderm at early stages of development. By reporter gene analysis in transgenic mice, we demonstrate that both mouse and human DNA sequences possess similar enhancer activity. The expression patterns of the transgene and Hoxb6 during early stages of mouse development are identical, suggesting that the LPM enhancer is involved in the initial activation of Hoxb6 gene expression in posterior regions of mammalian embryos.

Genomic structure and chromosomal localization of the mouse LIM/homeobox gene Lhx3.

We have previously cloned the murine Lhx3 cDNA that encodes a predicted protein containing two tandemly repeated LIM domains and a homeodomain. Early expression of Lhx3 in oral ectoderm that is committed to contribute to the anterior and intermediate lobes of the pituitary and its perseverance in the adult gland strongly suggest an involvement of the gene in mediating and maintaining the differentiation program of this important endocrine system. Additional functions are suggested by the fact that Lhx3 is also expressed bilaterally along the spinal cord and the hindbrain at early stages of mouse development. Here we report the structural organization and chromosomal localization of the Lhx3 gene. The gene is composed of six exons and five introns. Two different exons, Ia and Ib, appear to be alternatively spliced to exon II. The first LIM domain is encoded by exon II and the second by exon III. The homeobox is shared by exons IV and V. We have mapped Lhx3 to the proximal region of mouse chromosome 2 in a region that shares homology with human chromosomes 9q and 10p.

Transcription enhancer factor-1 (TEF-1) DNA binding sites can specifically enhance gene expression at the beginning of mouse development.

In an effort to identify transcriptional elements that are recognized at different stages of early mouse development, polyomavirus (PyV) enhancer mutations were selected for their ability to support PyV transcription and replication in various mouse undifferentiated embryonal carcinoma (EC) and embryonic stem (ES) cell lines. Several of these enhancer mutations were then isolated, sequenced and tested for their ability to stimulate the PyV early gene promoter in plasmid DNA that was either transfected into EC, ES and fibroblast cell lines, or injected into the nuclei of mouse 1-cell and 2-cell embryos. EC, ES and fibroblast cell lines showed clear preferences for different enhancer configurations, and cleavage-stage embryos (2- to 8-cell stage) strongly preferred the same enhancer configuration favored by ES cells. This 'embryo responsive' (ER) enhancer configuration was characterized by a tandem duplication of the region containing a single point mutation that created a DNA binding site for Transcription Enhancer Factor-1 (TEF-1). ER enhancers stimulated the PyV promoter up to 350-fold in embryos, and were up to 74-fold more active than the wild-type PyV enhancer. Most of the activity from PyER enhancers could be duplicated in 2-cell embryos by synthesizing only the tandemly repeated sequence. Comparison of these synthetic enhancers with ER enhancers confirmed that TEF-1 DNA binding sites were highly preferred in ES cells and cleavage-stage embryos, and suggested that ER enhancer activity resulted primarily from cooperative interaction between either two closely spaced TEF-1 DNA binding sites or two TEF-1 DNA binding sites separated by a third, as yet unidentified, transcription factor binding site. These results provide a prototype of a mammalian embryo responsive enhancer, and suggest that TEF-1 plays an important role in activation of gene expression at the beginning of mammalian development.

Distinct patterns of expression of MHC class I and beta 2-microglobulin transcripts at early stages of mouse development.

The class I surface Ag recognized during tissue graft rejection are composed of a 40- to 45-kDa H chain and a 12-kDa L chain, beta 2-microglobulin (beta 2m). Regulation of MHC gene expression during early development is thought to play an essential role in maternal tolerance of the fetal allograft. Here we used in situ hybridization techniques to characterize the temporal and spatial pattern of expression of MHC class I mRNA in the developing mouse embryo. MHC class I transcripts were initially detected at day 9.5 postcoitus in the primary and secondary trophoblast giant cell populations. At this stage, none of the remaining fetal components of the developing placenta expresses MHC class I mRNA. By contrast, the outer zone of trophoblast giant cells is the only trophectoderm-derived tissue in the developing placenta that does not express detectable levels of beta 2m mRNA. These findings indicate that the onset of MHC class I and beta 2m gene expression in early postimplantation stage embryos is not coordinately regulated. In addition, we made use of pluripotent embryonic stem (ES) cells to study developmental regulation of MHC class I and beta 2m gene expression in vitro. ES cells can be induced to form endoderm-like cells by the technique of suspension culture. In contrast to retinoic acid-treated F9 cells, the endoderm derived from ES cells expressed high levels of beta 2m mRNA, but no detectable MHC class I transcripts. These findings are consistent with results of our in situ hybridization experiments showing high levels of expression of beta 2m mRNA, and extremely weak expression of MHC class I transcripts in derivatives of the visceral endoderm in vivo.

Oct-4: a germline-specific transcription factor mapping to the mouse t-complex.

Oct-4 is a maternally expressed octamer-binding protein encoded by the murine Oct-4 gene. It is present in unfertilized oocytes, but also in the inner cell mass and in primordial germ cells. Here we show that the ectopic expression of Oct-4 in HeLa cells is sufficient for transcriptional activation from the octamer motif, indicating that Oct-4 is a transcription factor. Therefore, Oct-4 is the first transcription factor described that is specific for the early stages of mouse development. The spatial and temporal expression patterns were further determined using in situ hybridization. With this technique Oct-4 expression is detected in the oocyte, in the blastocyst and before gastrulation in the embryonic ectoderm. After day 8 Oct-4 expression decreases and is restricted to primordial germ cells from about day 8.5 onwards. Therefore Oct-4 is a transcription factor that is specifically expressed in cells participating in the generation of the germline lineage. Linkage analysis using B X D recombinant inbred mouse strains demonstrates that Oct-4 maps to chromosome 17 in or near the major histocompatibility complex. Several mouse mutants in the distal region of the mouse t-complex affecting blastocyst and embryonic ectoderm formation also map to this region.

Genetic and Developmental Control of Multiple Forms of l-Glycerol 3-Phosphate Dehydrogenase

Abstract A molecular form of l-glycerol 3-phosphate dehydrogenase (EC 1.1.1.8) is found during early stages of mouse development that differs in several respects from the major form found in adult tissues. This early or embryonic form can be separated from the major adult form by ion exchange chromatography, it is more sensitive to thermal denaturation, and has lower Km values for dihydroxyacetone phosphate and lglycerol-3-P. The embryonic form is found in brain up to 9 days of age, in the carcass up to 16 days of gestation, and in mouse teratoma; however, it has not been detected in fetal liver as early as 13 days of gestation nor in fetal kidney. The embryonic form of l-glycerol-3-P dehydrogenase is probably coded for by a genetic locus distinct from the locus for the adult enzyme, for although there is a difference in the rate of thermal denaturation for the enzyme from adult tissues of BALB/cBy and C57BL/6J mice, no strain-dependent differences are observed with the embryonic enzyme.

Chapter 5 Cell Determination and Biochemical Differentiation of The Early Mammalian Embryo

The chapter discusses the difference between the approach of an embryologist and of a biochemist to the problem of cell differentiation within the mammalian embryo. It discusses the experiments that identify the time, at which the developmental fate of cells become fixed (the time of determination). The experiments attempt to analyze the determination to form trophoblast or not to form trophoblast in the preimplantation embryo. The mammalian egg develops into an embryo consisting of the trophoblast, the extraembryonic membranes, and the fetus. The decision to form trophoblast or not to form trophoblast appears to be the earliest determinative event inside the embryo, and this chapter is largely concerned with trophoblast determination. The appearance of biochemical differences between the cells of the embryo during the first 10 days of development is described in this chapter. The chapter also discusses the development of tissue-specific biochemical markers. The discussion is limited to the experiments concerned with the early development of the mouse unless a phenomenon is particularly clearly illustrated by another mammalian embryo. The early stages of mouse development are illustrated diagrammatically in this chapter.