Desmin and titin expression in early postimplantation mouse embryos

The expression of the intermediate filament (IF) constituents desmin, vimentin and keratin, as well as the striated-muscle-specific marker titin, was studied in mouse embryos of 8.0 to 9.5 days post coitum (d.p.c.), using the indirect immunofluorescence technique in combination with polyclonal and monoclonal antibodies. During the development of the embryo, desmin was first detected at 8.25 d.p.c. in the ectoderm, where it was transiently coexpressed with keratin and vimentin. At later stages, the ectoderm contained only keratin and to a certain extent also vimentin IF. At 8.5 d.p.c., desmin was found exclusively in the heart rudiment, and remained present with increasing intensity in the myocardial cells during later cardiogenesis. Striation of desmin in the heart muscle cells was observed in 9.5 d.p.c. embryos. At these stages (8.5-9.5 d.p.c.), triple expression of the IF proteins desmin, vimentin and keratin was evident in these cells. From 9.0 d.p.c. onwards, desmin could be detected in the myotomes as well. Immunoblotting studies of 9.5 d.p.c. mouse embryos confirmed the immunohistochemical data. Titin was found in the early heart anlage at stage 8.25 d.p.c., when no desmin expression was observed in this tissue. At this stage the titin appeared in a punctate pattern, similar to that observed in cardiac myofibrils of early chicken embryos (Tokuyasu and Maher, 1987; J. Cell Biol. 105, 2781-2793). In 8.5 d.p.c. mouse embryos, this punctate titin staining pattern was still observed, while, at this stage, a filamentous staining reaction could be seen with the desmin antibodies. During further development, cross-striation was detected within myocardial cells using the polyclonal titin antibody from 9.0 d.p.c. onwards, i.e. before such striation could be detected with the desmin antibodies. From these data, we conclude that titin synthesis may anticipate desmin expression in the developing mouse myocard, although the level of expression of the former protein remains low until 9.0 d.p.c.     

Synthesis of steroids in postimplantation mouse embryos cultured in vitro

Steroid and total lipid synthesis have been assessed in postimplantation stage mouse embryos cultured in vitro from the blastocyst to early somite stage. A large increase in acetate incorporation into these compounds is observed during this period. Cholesterol (60–70%), lanosterol (1–15%), and a fraction containing pregnenolone (0–5%) are the major components of the embryo-associated steroid fraction. When embryos are labeled with [³H]pregnenolone, ³H-labeled progesterone, pregnanedione, and a compound identified as acylpregnenolone are produced and secreted into the medium. Production of progesterone and pregnanedione, but not acylpregnenolone, is severely inhibited by the drug cyanoketone (1 μM). Another drug, SU-10603 (10 μM), severely inhibits pregnanedione production, with only a partial repression of progesterone synthesis, and no effect on acylpregnenolone synthesis. Neither drug affects embryonic development. When embryonic tissues were carefully separated and analyzed for their ability to metabolize [³H]pregnenolone it was observed that all tissues (embryo/yolk sac, yolk sac, and trophoblast) can produce progesterone and acylpregnenolone from pregnenolone. Only embryo/yolk sac and yolk sac, but not trophoblast tissue, can produce pregnanedione. The significance of these observations in relation to metabolic communication between the embryo and its mother is discussed.     

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).     

酒精诱导小鼠海马齿状回神经细胞的增殖:神经酰胺的可能作用

本文旨在探讨神经酰胺(ceramide,Cer)在酒精诱导神经细胞增殖及新生神经元形成过程中的作用及机制.因为Cer主要的代谢途径是经神经鞘磷脂合成酶(sphingomyelin synthase,SMS)作用转化成神经鞘磷脂(sphingomyelin,SM),所以我们用SMS2基因敲除(sphingomyelin ...     

RNA inhibition of BMP-4 gene expression in postimplantation mouse embryos

Short, hairpin RNA (shRNA) directed against bone morphogenetic protein 4 (Bmp-4) was delivered to early postimplantation staged mouse embryos via tail vein injection of pregnant dams. As early as 24 h postinjection, embryos expressed a DsRed marker and later exhibited defects of neural fold elevation and closure and of cardiac morphogenesis. Immunohistochemical analysis of sectioned embryos indicated that Bmp-4 protein was depleted and gene expression analysis indicated there was a reduction in Bmp-4 mRNA and an upregulation of the Bmp-4 antagonists, noggin and chordin, in embryos exposed to the shRNA, but not in control embryos. There was no change in the expression of Gata4, brachyury, or claudin6 in RNAi exposed embryos, indicating that RNA silencing was specific to Bmp-4 rather than producing widespread gene inhibition. Delivery of shRNA to embryos has the potential to specifically knockdown the expression of developmentally essential genes and to rescue gene mutations, significantly decreasing the time required to analyze the function(s) of individual genes in 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.     

Differentiation and grafting of haemopoietic stem cells from early postimplantation mouse embryos

Haemopoietic stem cells evidently arise in early post-implantation mouse embryos at day 6 of gestation, a day earlier than previously thought (Moore & Metcalf, 1970). Disaggregated embryonic cells were injected into mice given a lethal dose of X-irradiation. The presence of donor haemoglobin (Whitney, 1978) and donor lymphocytic glucose phosphate isomerase (GPI) (Siciliano & Shaw, 1976) to detect donor erythrocytes and lymphocytes, respectively, were monitored by starch gel electrophoresis. The presence of donor cells was also assessed by using donor embryos carrying the T6 marker chromosomes. Decidual cells dissected free of embryos did not colonize any recipients. Disaggregated cells from early mouse embryos first colonized the liver and then repopulated the haemopoietic systems of recipients, producing adult donor haemoglobin within 2-3 days and donor GPI within 3-5 days. 80% of grafted X-irradiated recipients survived and donor markers were found in each of them. All nongrafted controls died within 14 days of X-irradiation and none of them showed donor markers. Disaggregated embryonic cells could be grafted across major histocompatibility barriers unlike adult bone marrow. Haemopoietic stem cells could not be identified in disaggregated cells from embryos aged less than 6 days gestation.     

Transfer of a mutant dihydrofolate reductase gene into pre- and postimplantation mouse embryos by a replication-competent retrovirus vector.

In order to explore the potential of retrovirus vectors for efficiently transferring foreign genes into mouse embryos, a replication-competent recombinant Moloney murine leukemia virus (Mo-MLV) vector carrying a mutant dihydrofolate reductase (DHFR) cDNA insert in the U3 region of the viral long terminal repeat was used to infect pre- and postimplantation embryos. When preimplantation mouse embryos were infected with the vector, as expected, the provirus integrated into the embryos and the germ line with the same efficiency as that observed with wild-type Mo-MLV, leading to inactivation of the recombinant virus. In contrast, when postimplantation mouse embryos were microinjected with virus-producing cells, between 90 to 100% of the surviving animals proved to be infected with the virus. The recombinant virus spread as efficiently as wild-type Mo-MLV in the infected embryos, resulting in up to three to five proviral copies per genome in heart, thymus, and brain tissues. Substantial expression of mutant DHFR*-coding viral message was found in all somatic tissues analyzed, the amounts correlating with the proviral copy number in the respective organ. These results suggest that replication-competent vectors are useful for efficient transfer and expression of foreign genes into tissues or whole animals when virus spread is needed.     

Teratogen-induced cell death in postimplantation mouse embryos: differential tissue sensitivity and hallmarks of apoptosis

Teratogen-induced cell death is a common event in the pathogenesis associated with tissues destined to be malformed. Although the importance of this cell death is recognized, little information is available concerning the biochemistry of teratogen-induced cell death. We show that three teratogens, hyperthermia, cyclophosphamide and sodium arsenite induce an increase in cell death in day 9.0 mouse embryos with concurrent induction of DNA fragmentation, activation of caspase-3 and the cleavage of poly (ADP-ribose) polymerase (PARP). Teratogen-induced cell death is also selective, i. e., some cells within a tissue die while others survive. In addition, cells within some tissues die when exposed to teratogens while cells in other tissues are relatively resistant to teratogen-induced cell death. An example of the latter selectivity is seen in the cells of the developing heart, which are resistant to the cytotoxic potential of many teratogens. We show that the absence of cell death in the heart is accompanied by the complete lack of DNA fragmentation, activtion of caspase-3 and the cleavage of PARP.     

Enhancement of radiation effects by mercury in early postimplantation mouse embryos in vitro

Summary Two-cell mouse embryos were X-irradiated (1 Gy) and immediately thereafter exposed to mercuric chloride (3 µM) up to the blastocyst stage. When combined treatment started shortly (about 1 to 2 h) before mitosis to the four-cell stage, blastocyst formation, hatching of blastocysts, trophoblast outgrowth and ICM formation were impaired stronger than expected from the addition of the single effects. The enhancement of risk was maximal for hatching of blastocysts and no further increase was observed for trophoblast outgrowth and ICM formation. When exposure of embryos to X-rays and mercury began about 5 to 6 h before mitosis to the four-cell stage, only additive effects were obtained for the endpoints mentioned above.     

The sex-chromosome constitution and early postimplantation development of diandric triploid mouse embryos

Diandric triploid mouse embryos were produced by standard micromanipulatory techniques, using eggs isolated from female mice with a normal chromosome constitution that had been mated to homozygous Rb(1.3)1Bnr males (which carry a large metacentric "marker" chromosome, viz., a Robertsonian translocation involving chromosomes 1 and 3). The tripronucleate embryos were transferred to the oviducts of pseudopregnant mice, which were subsequently autopsied at about midday on the 10th day of gestation. Although a relatively small number of the isolated conceptuses consisted of morphologically abnormal egg-cylinder-like structures or empty gestational sacs, most were at clearly distinguishable embryonic stages, from the primitive streak stage to embryos with about 20 pairs of somites present. These embryos all appeared to be morphologically normal but were substantially smaller than normal (diploid) fertilized embryos analyzed at similar stages of development. A total of 63 diandric triploid conceptuses were recovered and analyzed cytogenetically. They were G-banded to determine their sex-chromosome constitution and confirm their diandric triploid status. No obvious difference was observed in the developmental potential of the 58,XXX class of diandric triploids, compared to that of the 58,XXY class. The ratio of 58,XXX to 58,XXY embryos was close to the expected ratio of 1:2, assuming that unfertilized eggs have an equal chance of becoming fertilized by an X- or a Y-bearing spermatozoon and that the additional (i.e., "donor") male pronucleus also has an equal chance of having either an X or a Y sex chromosome present. However, the development of the 58,XYY class appeared to be restricted, even at the stage of gestation analyzed, in that no embryos with this genetic constitution were observed that had progressed beyond the early somite stage. The present findings are discussed in relation to the cytogenetic findings in human triploid conceptuses, the majority of which are spontaneously aborted during the first half of pregnancy. In man, the 69,XYY class (equivalent to the 58,XYY class in our study) is only rarely encountered, and it has been assumed that these triploid embryos are probably lost at a very early stage of gestation.     

Changes in temporal and spatial patterns of Gi protein expression in postimplantation mouse embryos

We previously demonstrated the presence of GTP-binding proteins, G proteins, in the preimplantation mouse embryo (Jones and Schultz, 1990. Dev. Biol. 139, 250-262). These studies have been extended to the Day 6.5, 7.5, and 8.5 gestation embryo by employing PT-catalyzed ADP-ribosylation and immunoblotting techniques. We report here that the amount of embryonic alpha i increases from Day 6.5 to Day 7.5 of gestation, and remains at about the same level at Day 8.5. In contrast, the extent of PT-catalyzed ADP-ribosylation of Gi alpha protein(s) decreases between Days 6.5 and 7.5--this decrease is global and not restricted to a particular germ layer of the Day 7.5 embryo--and then dramatically increases by Day 8.5 of gestation. In the Day 8.5 gestation embryo, the extent of PT-catalyzed ADP-ribosylation of Gi alpha proteins increases along the anterior-posterior axis, whereas the amount of immunoreactive alpha i subunit decreases along this axis. By using a combination of PT-catalyzed ADP-ribosylation and immunoprecipitation with antisera specific for alpha i1, alpha i2, or alpha i3, we report that all three alpha i subtypes are present in the Day 8.5 gestation mouse embryo. Results of these experiments suggest that an activation of Gi proteins occurs between Days 6.5 and 7.5 of gestation in the postimplantation embryo, a time during which the embryo is gastrulating, and that a decreasing gradient of activation exists along the anterior to posterior axis in the Day 8.5 gestation embryo. Last, we report that oocytes, eggs, and preimplantation embryos possess all three subtypes of alpha i.     

Partial characterization of skeletal myoblast mitogens in mouse crushed muscle extract.

We have utilized a model system to investigate myotrophic factors released by normal adult mouse muscles following a crush injury. We found that saline extracts from gently crushed mouse muscles (CME) contain potent mitogenic activities which act on primary newborn mouse myoblast cultures, as well as on mouse C2 cells, a mouse myoblast cell line. We compared the activity of CME on mouse myoblasts with that of basic fibroblast growth factor (bFGF) and insulin-like growth factor I (IGF-I), two growth factors known to be mitogenic for primary myoblasts (Allen, Dodson, and Lutein: Exp. Cell. Res., 152:154-160, 1984; DiMario and Strohman: Differentiation, 39:42-49, 1988; Allen and Boxhorn: J. Cell. Physiol., 138:311-315, 1989; Dodson, Allen, and Hossner: Endocrinology, 117:2357-2363, 1985; Florini and Magri: Am. J. Physiol., 256:C701-C711, 1989). We found that CME could act in an additive fashion to saturating doses of bFGF to increase proliferation in myoblast cultures. Additionally, CME acted additively to the combination of saturating amounts of bFGF and IGF-I on both C2 and primary myoblast cultures. We also examined additivity of CME with the combination of saturating doses of bFGF, IGF-I, transferrin (Tf), platelet-derived growth factor (PDGF), epidermal growth factor (EGF), adrenocorticotrophin (ACTH), and macrophage colony-stimulating factor (M-CSF). Our data indicate that CME contains Tf, as well as one or more uncharacterized mitogens for myoblasts which are distinct from Tf, the IGFs, bFGF, EGF, PDGF, M-CSF, and ACTH. These uncharacterized mitogens may act independently of known growth factors to stimulate myoblast proliferation, or may act through modulation of known growth factor activities.     

Radioimmunochemical characterization of ACTH-like peptides in the antropyloric mucosa.

Extracts of feline and human antropyloric mucosa contain two ACTH immunoreactive components. The main molecular component is radioimmunochemically and gel chromatographically indistinguishable from pituitary ACTH (1–39). Upon chromatography at two different pH, the main component and ACTH (1–39) displays identical changes in elution behaviour. In addition, antropyloric extracts contain a minor ACTH immunoreactive component, eluting in the void volume on Sephadex G-50 columns. The nature of this component is still undecided, but circumstantial evidence suggest that it may represent a biosynthetic precursor to the main ACTH-like component.     

FOXO1 delays skeletal muscle regeneration and suppresses myoblast proliferation

Unloading stress, such as bed rest, inhibits the regenerative potential of skeletal muscles; however, the underlying mechanisms remain largely unknown. FOXO1 expression, which induces the upregulated expression of the cell cycle inhibitors p57 and Gadd45α, is known to be increased in the skeletal muscle under unloading conditions. However, there is no report addressing FOXO1-induced inhibition of myoblast proliferation. Therefore, we induced muscle injury by cardiotoxin in transgenic mice overexpressing FOXO1 in the skeletal muscle (FOXO1-Tg mice) and observed regeneration delay in skeletal muscle mass and cross-sectional area in FOXO1-Tg mice. Increased p57 and Gadd45α mRNA levels, and decreased proliferation capacity were observed in C2C12 myoblasts expressing a tamoxifen-inducible active form of FOXO1. These results suggest that decreased proliferation capacity of myoblasts by FOXO1 disrupts skeletal muscle regeneration under FOXO1-increased conditions, such as unloading.