Fate of embryonal carcinoma cells injected into postimplantation mouse embryos

Embryonal carcinoma (EC) cells, stem cells of teratocarcinoma, represent an excellent model to study the developmental mechanisms that, inappropriately reactivated, can drive tumorigenesis. EC cells are very aggressive, and grow rapidly when injected into adult syngeneic mice. However, when injected into blastocysts, they revert to normality, giving rise to chimeric animals. In order to study the ability of postimplantation embryonic environment to "normalize" tumorigenic cells, and to study their homing, we transplanted F9, Nulli-SCC1, and P19 EC cells into 8 to 15-day allogenic CD1 mouse embryos, into allogenic CD1 newborns, and into syngeneic adult mice, and evaluated tumor formation, spreading, and homing. We found that, although at all embryonic stages successful transplantation occurred, the chances of developing tumors after birth increased with the time of injection of EC cells into the embryo. In addition, using enhanced green fluorescent protein-expressing F9 cells, we demonstrated that the cells not giving rise to tumors remained latent and could be tracked down in tissues during adulthood. Our data indicate that the embryonic environment retains a certain ability to "normalize" tumor cells also during post-implantation development. This could occur through yet unknown epigenetic signals triggering EC cells' differentiation.     

Subtractive differential display: a modified differential display technique for isolating differentially expressed genes

Differential display (DD) is a novel PCR-based technique, very commonly used to study differentially expressed genes at the mRNA level. In this paper we report a modified version of this technique that we have used to study the differences between the mRNA population from brain tissue of adult and old rats. We have modified the technique to enhance reproducibility and reduce false positives and redundancy. It is fast and does not require any expensive or uncommon reagent. We choose to call it as subtractive differential display as it is a differential display performed over subtracted mRNA population. We have used this protocol successfully to clone a number of age-related differentially expressed sequences from rat brain that need to be sequenced to establish the gene identity.     

Correlation of receptors for growth factors on mouse embryonal carcinoma cells with growth in serum-free, hormone-supplemented medium

High affinity receptors for insulin, transferrin, epidermal growth factor (EGF) and a multiplication-stimulating activity (MSA) have been identified and partially characterized on a mouse embryonal carcinoma cell line, OTT-6050, using various ¹²⁵I-ligands. With the exception of MSA receptors which bound both MSA and insulin, the receptors for EGF, insulin and transferrin exhibited specificity of binding for their respective ligands. There is a correlation between the saturation of these receptors and the concentration of growth factors necessary for optimal growth of OTT-6050 cells in serum-free medium supplemented with insulin (or MSA), transferrin, EGF, fibroblast growth factor (FGF) and Pedersen fetuin on culture surfaces treated with polylysine or various types of collagen. Cells cultured in this medium exhibit growth rates equivalent to that observed with cells maintained in medium containing 5% fetal calf serum (FCS). These results suggest that relatively undifferentiated mouse embryonal carcinoma cells or endoderm cells possess receptors for various growth factors and that their presence on these cells is correlated with the ability of these cells to mitogenically respond to these growth factors.     

安徽省不同人群及各型肝炎患者庚型肝炎病毒感染检测分析

应用酶联免疫试验（ＥＩＡ）和逆转录套式聚合酶链反应（ＲＴ－ｎＰＣＲ）对１００ 例一般人群、３８５例献血员、５４ 例血液透析患者、７２ 例乙型肝炎、４１ 例丙型肝炎２７ 例非甲－戊型肝炎患者进行检测。结果抗－ＨＧＶ 阳性率分别为２．００％ 、７．５３％ 、２７．７８％ 、１８．０６％ 、１９．５１％ 和１４．８１％ ;抗－ＨＧＶ 阳性者中ＨＧＶＲＮＡ 阳性率分别为１００．００％ 、６２．０７％ 、６６．６７％ 、６９．２３％ 、７５．００％ 和 １００．００％ ,提示本地区不同人群存在ＨＧＶ 感染。献血员、血透患者、乙型肝炎、丙型肝炎、非甲－戊型肝炎患者的ＨＧＶ 感染率显著高于一般人群,提示献血员,血透患者及ＨＢＶ、ＨＣＶ 感染者是ＨＧＶ 感染的高危人群。ＨＧＶ 常与ＨＢＶ 或ＨＣＶ 重叠／联合感染,也可单独感染。抗－ＨＧＶ 阳性者中ＨＧＶ ＲＮＡ 阳性率为８３．８２％ ,提示抗－ＨＧＶＥＩＡ 可用于ＨＧＶ 感染的检测。ＡＬＴ 正常和异常献血员中抗－ＨＧＶ 阳性率无显著性差异。     

Differential expression of mouse Disabled 2 gene in retinoic acid-treated F9 embryonal carcinoma cells and early mouse embryos.

Using a differential display PCR, we identified a differentially expressed cDNA fragment which was detectable in retinoic acid (RA) treated F9 embryonal carcinoma (EC) cells but not in untreated F9 cells. A homology search of the Gene Bank indicated that the cDNA fragment is part of the mouse homolog of the Drosophila Disabled (mDab2) gene. Aggregate cultures of F9 EC cells grown in the presence of the RA differentiated into nonmalignant cells resembling the visceral endoderm of the mouse embryo. Upon induction of endodermal differentiation with 10(-7) M RA, the gene expression of mDab2 was increased gradually during the first 96 h. Neither undifferentiated F9 cells, nor the undifferentiated aggregate cells without RA expressed mDab2. Whole-mount in situ hybridization and quantitative RT-PCR also showed that the temporal expression pattern of the mDab2 gene coincides with the initiation pattern of RA synthesis that occurs during mouse embryogenesis. Also, two alternative splicing messages of mDab2 were detected in a tissue specific manner. All the data indicate that mDab2 may play an important role in RA-induced signal transduction during mouse development.     

Teratogen-induced activation of caspase-6 and caspase-7 in early postimplantation mouse embryos

BACKGROUND: Previous work has shown that teratogens such as hyperthermia (HS), 4-hydroperoxycyclophosphamide (4CP), and staurosporine (ST) induce cell death in day 9 mouse embryos by activating the mitochondrial apoptotic pathway. Key to the activation of this pathway is the activation of a caspase cascade involving the cleavage-induced activation of an initiator procaspase, caspase-9, and the downstream effector procaspase, caspase-3. For example, procaspase-3, an inactive proenzyme of 32 kDa is cleaved by activated caspase-9 to generate a large subunit of approximately 17 kDa and a small subunit of approximately 10 kDa. In turn, caspase-3 is known to target a variety of cellular proteins for proteolytic cleavage as part of the process by which dying cells are eliminated. Previous work has also shown that neuroepithelial cells are sensitive to teratogen-induced activation of this pathway and subsequent cell death whereas cells of the heart are resistant. Although caspase-3 is a key effector caspase activated by teratogens, two other effector caspases, caspase-6 and caspase-7, are known; however, their role in teratogen-induced cell death is unknown. METHODS: Because cleavage-induced generation of specific subunits is the most specific assay for activation of caspases, we have used antibodies that recognize the procaspase and one of its active subunits and a Western blot approach to assess the activation of caspase-6 and caspase-7 in day 9 mouse embryos (or heads, hearts and trunks isolated from whole embryos) exposed to HS, 4CP, and ST. To probe the relationship between teratogen-induced activation of caspase-9/caspase-3 and the activation of caspase-6/caspase-7, we used a mitochondrial-free embryo lysate with or without the addition of cytochrome c, recombinant active caspase-3, or recombinant active caspase-9. RESULTS: Western blot analyses show that these three teratogens, HS, 4CP, and ST, induce the activation of procaspase-6 (appearance of the 13 kDa subunit, p13) and caspase-7 (appearance of the 19 kDa subunit, p19) in day 9 mouse embryos. In vitro studies showed that both caspase-6 and caspase-7 could be activated by the addition of cytochrome c to a lysate prepared from untreated embryos. In addition, caspase-6 could be activated by the addition of either recombinant caspase-3 or caspase-9 to a lysate prepared from untreated embryos. In contrast, caspase-7 could be activated by addition of recombinant caspase-3 but only minimally by recombinant caspase-9. Like caspase-9/caspase-3, caspase-6 and caspase-7 were not activated in hearts isolated from embryos exposed to these three teratogens. CONCLUSIONS: HS, 4CP and ST induce the cleavage-dependent activation of caspase-6 and caspase-7 in day 9 mouse embryos. Results using DEVD-CHO, a caspase-3 inhibitor, suggest that teratogen-induced activation of caspase-6 is mediated by caspase-3. In addition, our data suggest that caspase-7 is activated primarily by caspase-3; however, we cannot rule out the possibility that this caspase is also activated by caspase-9. Finally, we also show that teratogen-induced activation of caspase-6 and caspase-7 are blocked in the heart, a tissue resistant to teratogen-induced cell death.     

Involvement of insulin in early development of mouse one-cell stage embryos

Recent studies have suggested that growth factors and hormones play important roles in cell prolif-eration and differentiation during early embryonic development. In the present study, we examined the expression and localization of insulin in the mouse oocytes and one-cell stage embryos by quantitative ELISA, RT-PCR, Western blot and immunofluorescence. In the mouse oocytes and one-cell stage em-bryos, expression of insulin was uniformly distributed in the cytoplasm. We also examined the expres-sion, activity and localization of mTOR (mammalian target of rapamycin) and p70S6K. The expression of mTOR and p70S6K was not significantly different at the cell cycle of mouse one-cell stage embryos. mTOR and S6K were distributed evenly in the cytoplasm at G1, G2 and M phase phase, but at S phase, the distribution of mTOR and S6K was around the pronucleus. At different phases, the activity of mTOR fluctuated. We also used the PI3K specific inhibitor-Wortmannin to investigate the cleavage rate of eggs. The result showed that the rate obviously decreased. When the mTOR specific inhibitor Rapa-mycin was used, the first mitotic division of the mouse one-cell stage embryo was delayed. These re-sults suggested that insulin was expressed both in mouse oocytes and one-cell stage embryos, and may play functional roles in regulation of mouse early embryogenesis by activating the signal pathway of PI3K/PKB/mTOR/S6K.     

Expression of endogenous and microinjected hsp 30 genes in early Xenopus laevis embryos

In the present study, we have examined the regulation of expression of a newly isolated member of the hsp 30 gene family, hsp 30C. Using RT-PCR, we found that this gene was first heat-inducible at the tailbud stage of development. We also examined the expression of two microinjected modified hsp 30C gene constructs in Xenopus embryos. One of the constructs had 404 bp of hsp 30C 5′-flanking region, whereas the other had 3.6 kb. Both gene constructs had 1 kb of 3′-flanking region. RT-PCR assays were employed to detect the expression of these microinjected genes. The presence of extensive 5′- and 3′-flanking regions of the hsp 30C gene did not confer proper developmental regulation, since heat-inducible expression of both of the microinjected constructs was detectable at the midblastula stage. The premature expression of the microinjected hsp 30 gene was not a result of high plasmid copy number or the presence of plasmid DNA sequences. These results suggest that the microinjected genes contain all the cis-acting DNA sequences required for correct heat-inducible regulation but do not contain the elements required for the proper regulation of hsp 30 gene expression during development. It is possible that regulatory elements controlling the developmental expression of the hsp30 genes may reside upstream or downstream of the entire cluster. © 1993Wiley-Liss, Inc.     

Comparative functional analysis of the cow and mouse myostatin genes reveals novel regulatory elements in their upstream promoter regions

Myostatin is a paracrine/autocrine factor that inhibits muscle growth, and mutations that affect myostatin activity or expression produce dramatic increases in muscle mass in several species. However, at present it is less clear whether differences in myostatin expression or activity exist between species with differing body sizes. Here we demonstrate that mouse muscle expresses far greater levels of myostatin mRNA than cow. In addition, activity of a 1200 bp mouse myostatin promoter construct was significantly greater than that of a 1200 bp cow myostatin promoter construct in C₂C₁₂ myotubes. In contrast, activity of reporter constructs flanked by one or both untranslated regions (UTRs) was not significantly different between the two species. Sequence analysis identified a number of promoter regions which differed between larger species (cow, pig, goat, sheep, human) and smaller (mouse, rat), including a TATA-box sequence, a CACCC box, two AT-rich regions (AT1 and AT2), and a palindromic sequence (PAL). We therefore used mutagenesis to alter the mouse sequence for each of these elements to that of the cow. Mutagenesis of the TATA, CACC, and AT1 sequences of the mouse to those of the cow significantly decreased activity of the mouse myostatin promoter compared to the wild type mouse promoter, while mutation of the AT2 and PAL sequences tended to increase promoter activity. Finally, the cow myostatin promoter was less responsive to FoxO signaling than the mouse myostatin promoter. Together these data support the hypothesis that differences in promoter activity between mouse and cow may contribute to differences in expression of the myostatin gene between these species.     

Temporally regulated expression of insulin and insulin-like growth factors and their receptors in early mammalian development

Recent studies of early development in a number of invertebrate and vertebrate species have suggested that growth factors and their receptors may play important roles in differentiation as well as cell proliferation. In the mouse embryo, the expression of the receptors for insulin and insulin-like growth factors I and II (IGF-I and -II) are temporally regulated. The ontogeny of receptor and ligand expression within the insulin and IGF gene family suggests that the very earliest stages of mammalian embryogenesis may be subject to regulation by autocrine and paracrine factors from maternal and embryonic sources.     

Radioactive labeling of proteins in cultured postimplantation mouse embryos II. Dose and time dependency

Summary The conditions for optimum incorporation of radioactive amino acids into proteins of cultured postimplantation mouse embryos were investigated under the aspect of using these proteins for two-dimensional electrophoretic separations and fluorography. The aim was to obtain highly radioactively labeled proteins under conditions as physiological as possible. Mouse embryos of Days 8, 10, and 11 of gestation were cultured in Tyrode’s solution. Incubation time and concentration of [³H (or¹⁴C)]amino acids in the culture medium were varied over a broad range. Embryos were prepared with placenta and yolk sac or without any embryonic envelopes. After culturing, the physiologic-morphologic state of the embryos was registered on the basis of several criteria. The radioactivity taken up by the total protein of each embryo was determined and calculated in disintegrations per minute per milligram protein per embryo. To approach our aim, embryos of different developmental stages had to be cultured under different conditions. A good compromise for Day-8, Day-10, and Day-11 embryos was: embryos prepared with yolk sac (opened) and placenta, 150 μCi radioactive amino acids added per milliliter medium, incubation for 4 to 5 h. For maximum labeling of proteins it is advisable to culture Day-10 embryos without embryonic envelopes under particular conditions. This work was supported by grants from the Deutsche Forschungsgemeinschaft awarded to the project K1 237/3-2 (Systematic analysis of cell proteins).     

Expression of fibroblast growth factor receptors in peri-implantation mouse embryos

FGF receptor (FGFR) function is essential during peri-implantation mouse development. To understand which receptors are functioning, we tested for the expression of all four FGF receptors in peri-implantation blastocysts. By RT-PCR, FGFR-3 and FGFR-4 were detected at high levels, FGFR-2 at lower levels, and FGFR-1 was detected at background levels compared to control tissues. Because FGFR-3 and FGFR-4 were detected at the highest levels, we studied these in detail. Between 3.5 days after fertilization (E3.5) and E6.0, FGFR-4 mRNA was detected ubiquitously in the peri-implantation embryo, restricted to the inner cell mass (ICM) and its derivatives and primitive endoderm by E6.0, and was not detected at E6.5. FGFR-3 mRNA was detected ubiquitously in the peri-implantation embryo with a tendency towards extraembryonic cells. We tested blastocyst outgrowths, a model for implantation, for FGFR-3 and FGFR-4 protein. FGFR-3 protein was detected in all cells early during the outgrowth. Later, FGFR-3 was detected in the extraembryonic endoderm and trophoblast giant cells (TGC), but not in the ICM. FGFR-4 protein was detected in all cells of the implanting embryo, but was restricted to the ICM/primitive endoderm in later stage outgrowths. The distribution of the receptor proteins in the blastocyst outgrowths is similar to the distribution of the mRNA detected by in situ hybridization of sections of embryos. The data suggest roles for FGFR-3 and FGFR-4 in peri-implantation development. Mol. Reprod. Dev. 51:254–264, 1998. © 1998 Wiley-Liss, Inc.