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Effects of gravity on early embryogenesis [correction of embryogeneis] in Caenorhabditis elegans.

The embryonic development of the nematode Caenorhabditis elegans was examined under different gravitational conditions. The embryos after fertilization normally hatched under hypergravity (200 G) or 3D-clinorotation, whereas the number of eggs laid from an adult hermaphrodite decreased and their hatching rate was reduced under the hypergravity condition. The first cleavage plane in the 1-cell embryo was slid to some extent by re-orientation of liquid culture vessel, but the pattern and timing of cleavages were not affected.

Animals↗

Age related induction of platelet-derived growth factor A-chain mRNA in normal human fibroblasts.

We have previously found that stimulation of normal neonatal fibroblasts with PDGF or EGF leads to a transient induction of PDGF A-chain mRNA and the synthesis of PDGF-AA proteins. This finding may imply the existence of an autocrine feedback mechanism to amplify the mitogenic signal under certain conditions. We have now studied the PDGF-BB mediated PDGF A-chain induction in a set of fibroblasts from young and old donors to clarify if the levels of induction are correlated to the donor age and the replicative capacity of the cells. The PDGF A-chain induction was found to be reduced in cells from old donors compared with cells from embryonic and neonatal donors. The different cell strains were also characterized further with respect to PDGF receptor expression and PDGF binding properties. PDGF beta-receptors were found to be enhanced in old donor cell strains, whereas the PDGF alpha-receptors showed more variability in expression between the strains. The PDGF A-chain mRNA induction was also decreased or absent in late passage human fibroblasts (senescent cells) when compared with early passage cells. These data suggest that the PDGF A-chain mRNA induction is regulated by an age related mechanism in human fibroblasts.

Aged↗

Effects of protein phosphatase inhibition by okadaic acid on the differentiation of F9 embryonal carcinoma cells.

Okadaic acid, a protein phosphatase inhibitor, is a strong tumor promoter which apparently activates protein phosphorylation. To examine the role of protein phosphatases in stem cell growth and differentiation, embryonal carcinoma F9 cells were treated with okadaic acid. In the presence of this agent, the cells showed rapid morphological changes and arrest of proliferation at the M phase of the cell cycle, accompanied by a marked increase in the mRNA expression of various differentiation markers. Okadaic acid induced rapid increase in the mRNA levels of both c-jun and junB and results indicate that the inhibition of phosphatase by okadaic acid induces apparent activation of protein phosphorylation and may cause the expression of differentiation marker genes in F9 cells via the activation of AP-1.

Animals↗

Hrs, a FYVE finger protein localized to early endosomes, is implicated in vesicular traffic and required for ventral folding morphogenesis.

Hrs is an early endosomal protein homologous to Vps27p, a yeast protein required for vesicular trafficking. Hrs has a FYVE double zinc finger domain, which specifically binds phosphatidylinositol(3)-phosphate and is conserved in several proteins involved in vesicular traffic. To understand the physiological role of Hrs, we generated mice carrying a null mutation of the gene. Hrs homozygous mutant embryos developed with their ventral region outside of the yolk sac, had two independent bilateral heart tubes (cardia bifida), lacked a foregut, and died around embryonic day 11 (E11). These phenotypes arise from a defect in ventral folding morphogenesis that occurs normally around E8.0. Significant apoptosis was detected in the ventral region of mutant embryos within the definitive endoderm, suggesting an important role of this germ layer in ventral folding morphogenesis. Abnormally enlarged early endosomes were detected in the mutants in several tissues including definitive endoderm, suggesting that a deficiency in vesicular transport via early endosomes underlies the mutant phenotype. The vesicular localization of Hrs was disrupted in cells treated with wortmannin, implicating Hrs in the phosphatidylinositol 3-kinase pathway of membrane trafficking.

Androstadienes↗

Paternal effects in Drosophila: implications for mechanisms of early development.

The study of paternal effects on development provides a means to identify sperm-supplied products required for fertilization and the initiation of embryogenesis. This review describes paternal effects on animal development and discusses their implications for the role of the sperm in egg activation, centrosome activity, and biparental inheritance in different animal species. Paternal effects observed in Caenorhabditis elegans and in mammals are briefly reviewed. Emphasis is placed on paternal effects in Drosophila melanogaster. Genetic and cytologic evidence for paternal imprinting on chromosome behavior and gene expression in Drosophila are summarized. These effects are compared to chromosome imprinting that leads to paternal chromosome loss in sciarid and coccid insects and mammalian gametic imprinting that results in differential expression of paternal and maternal loci. The phenotypes caused by several early-acting maternal effect mutations identify specific maternal factors that affect the behavior of paternal components during fertilization and the early embryonic mitotic divisions. In addition, maternal effect defects suggest that two types of regulatory mechanisms coordinate parental components and synchronize their progression through mitosis. Some activities are coordinated by independent responses of parental components to shared regulatory factors, while others require communication between paternal and maternal components. Analyses of the paternal effects mutations sneaky, K81, paternal loss, and Horka have identified paternal products that play a role in mediating the initial response of the sperm to the egg cytoplasm, participation of the male pronucleus in the first mitosis, and stable inheritance of the paternal chromosomes in the early embryo.

Animals↗

Early ontogeny of the secondary proliferative population of the embryonic murine cerebral wall.

The present report is an analysis of the proliferative behavior of the secondary proliferative population (SPP) of the dorsomedial region of the embryonic mouse cerebral wall. It is based upon experiments undertaken on embryonic days 14-16 (E14-E16) and exploits methods in which proliferative cells are labeled in S phase with either or both bromodeoxyuridine and tritiated thymidine. The SPP, which arises from the PVE by E13, is principally the progenitor population to the neuroglial population of the mature neocortex and subjacent cerebral wall. By the end of E14 the SPP comes to be distributed diffusely from the outer margin of the ventricular zone throughout subventricular zone and intermediate zone. The length of the cell cycle of the SPP is constant at approximately 15 hr throughout this interval; thus, this population undergoes 1.6 cell cycles/24 hr or 3.2 cycles in the course of the 48 hr period, E14-E16. Over this 48 hr period, the SPP increases from 11% to 35% of the total proliferative population of the dorsomedial cerebral wall. The absolute size of the SPP increases nearly sixfold. With these values taken together it may be estimated that approximately 87% of postmitotic cells of the SPP reenter S phase after each cell division in this interval which means that only approximately 13% of the proliferative population exits the cycle. These findings illustrate the massive expansion of the SPP antecedent to the explosive diffusion of glial cells through the neocortex and subjacent cerebral wall as neuronal migration comes to completion and neocortical growth and differentiation accelerate.

Animals↗

Recombinant human leukemia inhibitory factor enhances the development of preimplantation mouse embryo in vitro.

OBJECTIVE: To assess the effect of recombinant human leukemia inhibitory factor (rhLIF) on mouse embryos in vitro. DESIGN: Controlled prospective study. SETTING: Academic research environment. ANIMAL(S): Female CB6F1 mice between 6 and 8 weeks old. INTERVENTION(S): Mice were divided randomly into three groups, which included a control group in an in vivo study (group I) and two groups in an in vitro study (groups II and III). Mice were killed at 116-120 hours (group I) and 44-48 hours (groups II and III) after hCG injection. Two-cell embryos (groups II and III) and blastocysts (group I) were obtained. Embryos in group II were cocultured with human tubal fluid (HTF) + 0.5% human serum albumin and in group III with HTF + rhLIF (1,000 U/mL) under paraffin oil. MAIN OUTCOME MEASURE(S): The embryonic numbers in different stages were recorded and compared. RESULT(S): Similar early embryo development to the four- to eight-cell and morula stages was noted between groups II and III (87.3% versus 91.0% and 74.6% versus 87.1%, respectively). However, further embryo development to the blastocyst, expanded blastocyst, and hatching blastocyst in group II (48.1%, 31.7%, and 18.5%, respectively) was lower than that in group III (83.6%, 53.7%, and 37.8%). CONCLUSION(S): RhLIF does not provide obvious stimulation in the early mouse embryo. However, rhLIF has positive effects on preimplantation blastocyst growth, differentiation, and hatching.

Animals↗

Spatial and temporal distribution of Indian hedgehog mRNA in the embryonic mouse mandible.

Hedgehog genes are involved in pattern formation during embryonic development. A recent report showed that Sonic hedgehog is expressed in the mouse mandible in the presumptive incisor region. In the present study, Indian hedgehog (Ihh) transcripts were present from gestational day 9 to 14 in the mouse mandible (reverse transcription/polymerase chain reaction analysis). Ihh mRNA was present in the dental lamina in both incisor and molar regions and in the developing whiskers (in-situ hybridization). Ihh may be involved in the site-specific proliferation of mandibular epithelium during the formation of the dental lamina. This is consistent with the observation that endogenous synthesis of retinoic acid is necessary for the initiation of odontogenesis and that retinoic acid induces hedgehog expression.

Animals↗

Histone H1(0) mRNA and protein accumulate early during retinoic acid induced differentiation of synchronized embryonal carcinoma cells.

The very lysine-rich replacement histone variant H1(0) is found to be present in different murine (C1003, PC13, P19) and human (Tera-2) embryonal carcinoma cell lines. The proportion of H1(0) increases upon induction of differentiation of the different cell lines by various treatments. In undifferentiated PC13 EC cells H1(0) mRNA is present at a low level. During retinoic acid induced differentiation of mitotically synchronized PC13 EC cells, accumulation of H1(0) mRNA starts in the first cell cycle. The H1(0) protein level starts to increase in the second synchronous cycle preceding changes in the cycle parameters that become apparent in the third cycle. The results provide further support for an important role of H1(0) in the control of cellular differentiation in early mammalian development.

Animals↗

Comparative effects of essential and nonessential metals on preimplantation mouse embryo development in vitro.

It is well recognized that deficiencies of essential trace elements during early development can result in structural abnormalities and/or embryonic death. Recently, there has been increasing interest in the concept that small excesses of essential metals can also have negative effects on the developing embryo. We hypothesized that, with respect to toxicity, metals with similar physico-chemical properties would act by similar mechanisms to influence the preimplantation embryo. In the current study we investigated the influence of four essential (Cu, Mn, Fe, Zn), and eight nonessential (Cr, Hg, Pb, V, Al, Ag, Cd, As) metals on mouse preimplantation embryonic development. Two cell stage mouse embryos were cultured for 72 h in media containing varying metal concentrations (0.05 - 200 microM). Embryo cell differentiation and proliferation were respectively assessed by scoring for blastocyst formation and final embryo cell number. Both nonessential and essential metals were embryotoxic at relatively low concentrations. However, in contrast to our expectations, at similar molar concentrations, redox active essential metals were less toxic than non-redox active nonessential metals. These data suggest that direct metal binding to critical membrane sites and/or intracellular ligands, including protein and nucleic acids, may trigger abnormal development and death prior to metal-associated oxidative damage.

Animals↗

Genetic regulation of preimplantation mouse embryo survival.

The preimplantation period of mammalian development is characterized by cleavage of a one-cell embryo to a blastocyst stage embryo. During preimplantation development, 15%-50% of the embryos die as a result of factors that are largely unknown. Two parameters of preimplantation development, a fast rate of development and a low degree of fragmentation, are indicative of good embryo quality. There is mounting evidence that genes control both rate of development and degree of fragmentation. We have discovered a gene, Ped (preimplantation embryo development), which controls the rate of preimplantation embryonic cleavage. The Ped gene is encoded by two similar genes, Q7 and Q9, in the Q region of the mouse major histocompatibility complex (MHC). The Ped gene product is an MHC class Ib protein, the Qa-2 antigen. The mechanisms by which the Ped gene controls rate of embryonic cleavage division are being explored. In order to understand genetic mechanisms underlying the second criterion of embryo quality, degree of fragmentation, we have begun to assess expression of the genes that could potentially regulate apoptosis in preimplantation embryos. We have shown that staurosporine can induce apoptosis in mouse blastocysts. By using RT-PCR, we have shown that genes encoding protein in the two major gene families that regulate apoptosis, the Bcl-2 and caspase gene families, are present in preimplantation embryos. We hypothesize that there is a homeostatic mechanism by which genes that regulate cell survival and those that regulate cell death determine the overall viability of preimplantation embryos.

Animals↗

Bmpr encodes a type I bone morphogenetic protein receptor that is essential for gastrulation during mouse embryogenesis.

Bone morphogenetic proteins (BMPs) are secreted proteins that interact with cell-surface receptors and are believed to play a variety of important roles during vertebrate embryogenesis. Bmpr, also known as ALK-3 and Brk-1, encodes a type I transforming growth factor-beta (TGF-beta) family receptor for BMP-2 and BMP-4. Bmpr is expressed ubiquitously during early mouse embryogenesis and in most adult mouse tissues. To study the function of Bmpr during mammalian development, we generated Bmpr-mutant mice. After embryonic day 9.5 (E9.5), no homozygous mutants were recovered from heterozygote matings. Homozygous mutants with morphological defects were first detected at E7.0 and were smaller than normal. Morphological and molecular examination demonstrated that no mesoderm had formed in the mutant embryos. The growth characteristics of homozygous mutant blastocysts cultured in vitro were indistinguishable from those of controls; however, embryonic ectoderm (epiblast) cell proliferation was reduced in all homozygous mutants at E6.5 before morphological abnormalities had become prominent. Teratomas arising from E7.0 mutant embryos contained derivatives from all three germ layers but were smaller and gave rise to fewer mesodermal cell types, such as muscle and cartilage, than controls. These results suggest that signaling through this type I BMP-2/4 receptor is not necessary for preimplantation or for initial postimplantation development but may be essential for the inductive events that lead to the formation of mesoderm during gastrulation and later for the differentiation of a subset of mesodermal cell types.

Alleles↗

On functions of cholinesterases during embryonic development.

Expression of cholinesterase (ChE) activity during phases of embryonic development is a general phenomenon in embryonic tissues. To elucidate the role(s) of ChEs during embryonic development, one line of research followed the assumption of a primitive muscarinic system involved in morphogenesis (Hohmann et al., 1995). This means that ChE functioning during development fits into the classical cholinergic neurotransmitter system: acetylcholine (ACh), as a signal, binds to ACh receptors and then is degraded by acetylcholinesterase (AChE) as the terminating enzyme. However, this is just one of the possible mechanisms. The other line of research was driven by evidence for noncholinergic functions of ChE proteins (AChE and butyrylcholinesterase [BChE]). There is accumulating data that other sites on AChE could exert nonclassical roles related to cell differentiation, neurite outgrowth, and adhesion.

Acetylcholinesterase↗

Neurotransmitter receptors in the proliferative zones of the developing primate occipital lobe.

Film autoradiography was used to investigate the expression of several neurotransmitter receptor subtypes in the transient ventricular and subventricular proliferative zones of the developing occipital lobe in two groups of macaque monkey fetuses. The first group of fetuses were between 60 and 93 days after conception (E60-E93), when the ventricular and subventricular zones of the monkey occipital lobe produce neurons destined for the visual cortex. In the second group, fetuses were between E107 and E128, after generation of cortical neurons has ceased. In the E60-E93 group of fetuses, ventricular and subventricular zones displayed high densities of 5-HT1-serotonergic, D1-dopaminergic, alpha 1- and alpha 2-adrenergic and high affinity kainate receptors. The activation of these receptors has previously been shown to stimulate cell proliferation in other cell systems. The possible involvement of these receptors in regulation of neuronal production is also supported by their absence in the deep laminae of the embryonic cerebral wall after E107, after cortical neurogenesis has been completed. The only exception is a high density of alpha 2-adrenergic receptors maintained near the ventricular surface long after all cortical neurons have been generated. We also found that during neurogenesis, proliferative zones in E66-E90 fetuses displayed virtually no 5-HT2-serotonergic, D2-dopaminergic, beta-adrenergic, M1-muscarinic cholinergic, gamma aminobutyric acid (GABA)A, N-methyl-D-aspartate (NMDA), or alpha-amino-3-hydroxy-5-menthy-4-isoxazole proprionate (AMPA) sites; most of these receptor subtypes have been reported to mediate the suppression of cell proliferation. The present findings suggest that dividing and/or newly generated cortical neurons are capable of receiving specific signals from multiple neurotransmitters present in their environment.

Animals↗

Shp2-mediated molecular signaling in control of embryonic stem cell self-renewal and differentiation.

A key issue to be addressed in stem cell biology is the molecular signaling mechanism controlling embryonic stem (ES) cell pluripotency. Stem cell properties are dictated by specific transcription factors and epigenetic processes such as DNA methylation and chromatin remodeling. Several cytokines/growth factors have been identified as critical ES cell regulators. However, there is a gap in our knowledge of the intracellular signaling pathways linking extracellular signals to transcriptional regulation in ES cells. This short review discusses the physiological role of Shp2, a cytoplasmic tyrosine phosphatase, in the molecular switch governing ES cell self-renewal versus differentiation. Shp2 promotes ES cell differentiation, mainly through bi-directional modulation of Erk and Stat3 pathways. Deletion of Shp2 in mouse ES cells results in more efficient self-renewal. This observation provides the impetus to develop Shp2 inhibitors for maintenance and amplification of ES cells in culture.

Animals↗

Immunolocalization of the heterotrimeric kinesin-related protein KRP(85/95) in the mitotic apparatus of sea urchin embryos.

We have used monoclonal antibodies to perform confocal light microscopic immunolocalization of KRP(85/95), a heterotrimeric plus-end-directed microtubule motor protein, in dividing cells of sea urchin embryos. Embryos were stained during the first division cycle, and dissociated blastomeres were stained at the 32- to 64-cell stages. Double labeling of the dividing cells with anti-tubulin and anti-KRP(85/95) showed a clear concentration of the motor protein in the mitotic apparatus; KRP(85/95) appeared to associate with pericentriolar regions during prophase, with kinetochore-to-pole microtubules during metaphase, and, in a striking fashion, with the spindle interzone during anaphase. KRP(85/95) began to accumulate in the interzone immediately following chromosome separation and the area of concentration expanded with the lengthening of the interzonal region during anaphase. During telophase KRP(85/95) appeared to disperse with the establishment of the cleavage furrow and did not concentrate in the midbody. KRP(85/95) staining in the mitotic apparatus was punctate and detergent-sensitive, suggesting an association with membranous vesicles, but unlike kinesin, KRP(85/95) did not appear to codistribute with calsequestrin-containing endoplasmic reticulum. Finally, KRP(85/95) appears to be present in dividing blastomeres up to at least the blastula stage, but, unlike kinesin, it is not expressed in terminally differentiated, nonmitotic coelomocytes of the adult animal. These results suggest that the expression and targeting of KRP(85/95) and kinesin differ and that KRP(85/95) may play a role in vesicle transport during embryonic cell division.

Animals↗

Disrupted retinal development in the embryonic belly spot and tail mutant mouse.

The Belly spot and tail (Bst) semidominant mutation, mapped to mouse Chromosome 16, leads to developmental defects of the eye, skeleton, and coat pigmentation. In the eye, the mutant phenotype is characterized by the presence of retinal colobomas, a paucity of retinal ganglion cells, and axon misrouting. The severity of defects in the Bst/+ retina is variable among individuals and is often asymmetric. In order to determine the role of the Bst locus during retinal morphogenesis, we searched for the earliest observable defects in the developing eye. We examined the retinas of Bst/+ and +/+ littermates from embryonic day 9.5 (E9.5) through E13.5 and measured retinal size, cell density, cell death, mitotic index, and cell birth index. We have found that development of the Bst/+ retina is notably dilatory by as early as E10.5. The affected retinas are smaller than their wildtype counterparts, and optic fissure fusion is delayed. In the mutant, there is a marked lag in the exit of retinal cells from the mitotic cycle, even though there are no observable differences in the rate of cellular proliferation or cell death between the two groups. We hypothesize that Bst regulates retinal cell differentiation and that variability of structural defects in the mutant, such as those affecting optic fissure fusion, is a reflection of the extent of developmental delay brought about by the Bst mutation.

Animals↗

Changes in epidermal growth factor receptor expression and competence to generate glia regulate timing and choice of differentiation in the retina.

Previous studies demonstrated that the level of epidermal growth factor receptors (EGF-Rs) expressed by progenitor cells in the newborn (P0) rat retina was limiting for the generation of Muller glial cells but not for proliferation. To determine whether EGF-R signaling biases cells to generate a specific cell type or regulates more general processes during progenitor cell development, we have introduced extra copies of the EGF-R into progenitor cells at earlier stages (E15 and E18), when different cell types are produced. We show that progenitor cells in early embryonic retina (E15) normally express lower levels of EGF-Rs than progenitor cells in later retina (E18 and P0). Whereas lower levels of stimulation of endogenous and virally transduced EGF-Rs enhanced proliferation, higher levels reduced proliferation, resulting in premature differentiation. At E15, very few EGF-R-Infected progenitor cells differentiated prematurely into Muller glial cells, unlike E18 and P0 cells, even when they were exposed to an older retinal environment. Higher levels of EGF-R-mediated signaling alone therefore do not specify a glial fate, indicating that competence to generate glia is temporally regulated by additional mechanisms. The differences in EGF-R expression observed among retinal progenitor cells at distinct developmental stages may instead help to define signaling thresholds which delay or accelerate their differentiation.

3T3 Cells↗