Distinct myogenic programs of embryonic and fetal mouse muscle cells: expression of the perinatal myosin heavy chain isoform in vitro.

Early embryonic and late fetal mouse myogenic cells showed distinct patterns of perinatal myosin heavy chain (MHC) isoform expression upon differentiation in vitro. In cultures of somite or limb muscle cells isolated from Day 9 to Day 12 embryos, differentiated cells that expressed perinatal MHC were rare and perinatal MHC was not detectable by immunoblotting. In cultures of limb muscle cells isolated from Day 13 to Day 18 fetuses, in contrast, the perinatal MHC isoform was easily detected and was expressed in a substantial percentage of myocytes and myotubes. Analyses of clonally derived muscle colonies and cytosine arabinoside-treated fetal muscle cell cultures suggested that different fetal muscle cell nuclei initiated perinatal MHC expression at different times. In both embryonic and fetal cell cultures, the embryonic MHC isoform was expressed by all differentiated cells examined. A small number of myotubes in fetal muscle cell cultures showed a mosaic distribution of MHC isoform accumulation in which the perinatal MHC isoform accumulated in a restricted region of the myotube near particular nuclei, whereas the embryonic MHC isoform accumulated throughout the myotube. Thus, the myogenic program of fetal, but not embryonic, mouse myogenic cells includes expression of the perinatal MHC isoform upon differentiation in culture.     

Development of bovine and porcine embryonic teratomas in athymic mice

Inner cell masses (ICM) and embryonic discs from bovine and porcine blastocysts of various ages were transplanted under the kidney capsule of athymic (nude) mice to evaluate growth of teratocarcinomas containing both differentiated tissues and undifferentiated stem cells. Inner cell masses were isolated immunosurgically from Day 8, Day 9 and Day 10 porcine blastocysts and from Day 8, Day 10 and Day 12 bovine blastocysts. Embryonic discs were mechanically dissected from Day 11 and Day 12 porcine embryos and from Day 14 bovine embryos. Day 6 egg cylinders were dissected from embryos and from hybrid embryos of a cross between BALB/C and an outbred strain of mouse. Two to four ICM, embryonic discs or egg cylinders were transplanted under the kidney capsule of each athymic host. After 8 weeks, graft hosts were killed and their tumors removed, fixed and prepared for histological and immunohistochemical examination. Embryonic teratomas developed at high frequency from murine egg cylinders and from Day 11 and Day 12 porcine and Day 14 bovine embryos. Tumors were observed only infrequently from younger bovine and porcine blastocysts. Murine embryonic tumors were composed of numerous differentiated cell types of ectodermal, mesodermal and endodermal origins, but representation of the three embryonic germ layers was somewhat more restricted in bovine and porcine embryonic tumors. No undifferentiated stem cells were detected in tumors of any of the three species. These results demonstrate that teratomas will develop from bovine and porcine embryos when grafted to an immunocompromised host, but the presence of undifferentiated teratocarcinoma stem cells from these species has yet to be achieved.     

Immunocytochemical detection of neuronal nitric oxide synthase (nNOS)-IR in embryonic rat stomach between days 13 and 21 of gestation.

In this study, the localization and appearance of neuronal nitric oxide synthase-immunoreactive (nNOS-IR) nerve cells and their relationships with the developing gastric layers were studied by immunocytochemistry techniques and light microscopy in embryonic rat stomach. The stomachs of Wistar rat embryos aged 13-21 days were used. The first nerve cells containing nNOS-IR were seen on embryonic Day 14. The occurrence of mesenchymal cell condensation near nNOS-IR neuroblasts on embryonic Day 15 may reflect an active nerve element-specific mesenchymal cell induction causing the morphogenesis of muscle cells. Similarly, the appearance of glandular structures after nNOS-IR neuroblasts, on embryonic Day 18, suggests that the epithelial differentiation may depend on inputs coming from nNOS-IR neuroblasts, as well as other factors. Observation of nNOS-IR nerve fibers on embryonic Day 21 demonstrates that at this stage they contribute to nonadrenergic noncholinergic relaxation. In conclusion, depending on this study's results, it can be said that cells and tissues might be affected by NO secreted by nNOS-IR nerve cells during the development and differentiation of embryonic rat stomach.     

Ontogeny of the carotid body and glomus cells distributed in the wall of the common carotid artery and its branches in the chicken

Summary Developmental patterns of immunoreactivity for serotonin and neuropeptide Y were investigated immunohistochemically in the carotid body and glomus cells in the wall of the common carotid artery and around its branches of chickens at various developmental ages. The development of peptidergic nerve fibers was also studied. Serotonin immunoreactivity began to appear in the glomus cells of the carotid body and around arteries at 10 days of incubation and became very intense from 12 days onwards. Neuropeptide Y immunoreactivity also appeared in these cells at 10 days, became intense at 14 days, and was sustained until 20 days. After hatching, neuropeptide Y immunoreactivity in the carotid body rapidly decreased with age and almost cisappeared at posnatal day 10. However, it persisted for life in the glomus cells distributed in the wall of the common carotid artery. Substance P- and calcitonin gene-related peptide (CGRP)-immunoreactive fibers first penetrated into the carotid body parenchyma at 12 days of incubation. These peptidergic nerve fibers in the carotid body and glomus cell groups in and around arteries gradually increased with age, and approached the adult state at 18 days of incubation. Only a few galanin-and vasoactive intestinal peptide (VIP)-immunoreactive fibers were observed in the late embryonic carotid bodies. They rapidly developed after hatching and reached adult numbers at postnatal day 10. During late embryonic and neonatal development, considerable numbers of met-enkephalin-immunoreactive fibers were detected in the connective tissue encircling the carotid body.     

Ontogeny of Fc and complement receptors in mouse embryonic tissues.

Mouse embryonic tissues were tested for their ability to form rosettes with EA and EAC. It was found that as early as the day after implantation (day 6) a small percentage of cells was able to bind both. After the 8th day of gestation the development of rosette-forming cells paralleled that of the immune system. However, the detection of Fc and C'3 receptors on cells at least 2 days prior to the earliest known demonstration of lymphoid stem cells suggests that they may be expressed on cells other than those of the immune and phagocytic series.     

The determination of the developmental stages in the hamster using erythroid cell development.

A technique was developed for the determination of developmental stages by measuring the electronically determined volume of fetal blood cells. The size shifts of the nucleated and nonnucleated cell populations as well as the release of liver reticulocytes that occurs between the twelfth and thirteenth days provided hematological correlates of gestational development.     

Glutathione in the red blood cells of embryonic mice

A micro-method for the determination of red blood cell glutathione levels has been developed. It has been shown that, in order to obtain true values of glutathione concentrations within red blood cells, it is necessary to correct for loss of labelled glutathione and also to take account of the time taken to complete the analytical procedure. Glutathione is the major small molecular weight thiol present in embryonic red blood cells. The glutathione to haemoglobin ratio is maintained at 0.6 from 13 days gestation to adulthood in the mouse. Decay of glutathione in both adult and embryonic red blood cells can be avoided by incubation of the red blood cells in glucose containing buffers.     

Keratan sulfate proteoglycan during embryonic development of the chicken cornea

Antibodies to corneal keratan sulfate proteoglycan (KSPG) were used to characterize the pattern of KSPG accumulation during differentiation of neural crest cells in the stroma of embryonic chick cornea. Immunohistochemistry with monoclonal antibody I22 to keratan sulfate found this KSPG antigen localized inside stromal cells at stage 29 (Day 6), ca. 12 hr after migration into the primary stroma. A 2- to 3-day lag then occurred before appearance of extracellular keratan sulfate, first seen on Day 9 (Stage 35) in the posterior stroma. Keratan sulfate antigen accumulated in a posterior to anterior direction during subsequent development. Uniform staining of the stroma for keratan sulfate did not occur until after Day 16. Among several tissues, only corneal stroma contained an extracellular matrix which stained for keratan sulfate, though intracellular staining of some cartilage cells was observed. Accumulation of KSPG antigens in developing cornea was measured in unfractionated guanidine extracts with a quantitative ELISA using three different antibodies against KSPG. Increases were first detected after Day 9 using monoclonal I22, and somewhat later with the other two antibodies. Assays with all three antibodies detected a sustained, exponential increase of KSPG throughout the 5 days prior to hatching. Keratan sulfate continued to accumulate after hatching, but an antibody with specificity to KSPG core protein, detected no relative increase in antigen after hatching. This suggests a modulation of KSPG primary structure late in development and after hatching. Overt differentiation of individual neural crest cells thus appears to begin ca. 12 hr after their arrival in the primary stroma; a lag of 2-3 days precedes active secretion of KSPG.     

Embryonic growth and mobilization of energy and material in oviposited eggs of the red-necked keelback snake, Rhabdophis tigrinus lateralis

We used the red-necked keelback (Rhabdophis tigrinus lateralis) as a model animal to study embryonic growth and mobilization of energy and material in oviposited snake eggs. Females (N=12) laid eggs between late May and early June. Eggs were incubated at 30 (+/-0.3) degrees C. One egg from each clutch was dissected at five-day intervals starting at oviposition. Incubation length averaged 27.9 days. Three phases of embryonic growth or yolk depletion could be detected in this study. The first phase, between oviposition and Day 10, was one of minimal transfer of energy and material from yolk to embryo. The second phase, between Day 10 and Day 22-23, was characterized by increasingly rapid embryonic growth and yolk depletion. The third phase, between Day 22-23 and hatching, was characterized by a gradual reduction in embryonic growth and yolk depletion. Approximately 73.6% of dry mass, 50.0% of non-polar lipids and 57.8% of energy were transferred from egg to embryo during incubation. Embryos withdrew mineral from the eggshell mainly during the last quarter of incubation. Our data show that oviposition does not coincide with the onset of rapid embryonic growth in oviparous species of squamate reptiles that are positioned midway within the oviparity-viviparity continuum, and that the greater conversion efficiencies of energy and material from egg to hatchling in snakes can be mainly attributed to their lower energetic costs of embryonic development and greater residual yolk sizes.     

A timetable of embryonic development, and ovarian and uterine changes during pregnancy, in the stripe-faced dunnart, Sminthopsis macroura (Marsupialia: Dasyuridae)

Aged stages (63) were available for establishment of a timetable of embryonic development of the stripe-faced dunnart. On Day 0 oocytes reaching maturity were found in the ovary. Within +/- 24 h of time 0 (time of minimum morning weight) polymorphonuclear leucocytes appeared and spermatozoa were last detected in the urine of 70% of females. Embryos were collected at intervals during pregnancy by hemihysterectomy and the embryos in the contralateral uterus either were examined at a later stage of pregnancy or allowed to develop to term. Cleavage to the unilaminar blastocyst stage with around 32 cells took 3 days with a cleavage arrest of 24 h at the 4-cell stage. Expansion of the unilaminar blastocyst occurred over the next 3 days. Primitive endoderm cells appeared on Day 6, fully bilaminar blastocysts by the end of Day 7 and trilaminar blastocysts on Day 8. Shell loss and implantation of 13-15-somite stage embryos occurred on Day 8 and organogenesis over the next 2-3 days. The gestation period was 9.5-12.0 days with most births occurring between 10.5 and 11.0 days. Major steps in embryonic development were correlated with stages in the development of the corpora lutea, which were maximal in size, and possibly in secretory activity, when the embryos were at the bilaminar blastocyst stage. Regression commenced when the embryos were at the primitive streak stage. At the time the corpora lutea were maximal the uterine epithelium reached its greatest height and the endometrium was thick and folded. Later in pregnancy villous-like projections of the epithelium formed, and the luminal epithelial cells became rounded. Two cell populations, a tier of 8 smaller cells above the yolk mass and a tier of 8 larger cells around the sides of the yolk mass appeared at the 16-cell stage. From the 16-cell stage to the blastocyst stage, with 150-200 cells, two cell populations distinguished by size, cell cycle time, cytoplasmic appearance and position relative to the yolk mass were present. The two populations were indistinguishable in blastocysts with greater than 200 and less than 2000 cells. They reappeared in blastocysts with greater than 2000 cells, as the darker cells of the embryoblast, and as the paler cells of the trophoblast. The darker cells lay in the yolky hemisphere and the paler cells in the non-yolky hemisphere.     

The effect of heterologous antisera on embryonic development. XIII. Lack of effect of antisera to alpha fetoprotein.

This investigation was carried out to determine whether heterologous antisera to alpha fetoprotein (AFP) are embryotoxic to developing rat embryos. Homogeneous rat AFP was isolated and antisera directed against this glycoprotein were produced in rabbits, horse and goat. The effect of the antisera on embryonic development was examined by injecting the antisera intraperitoneally into pregnant rats on the ninth, eleventh and thirteenth days of gestation. The results demonstrated that there was no evidence of increased incidence of fetal abnormalities in 472 surviving fetuses of 42 injected rats. There was no evidence of increase embryonic death or retardation of intrauterine growth following administration of the antisera on the ninth, eleventh and thirteenth days of gestation. The localization of the injected antisera was examined by the indirect immunofluorescent method. The results showed that the heterologous AFP antibodies localized specifically in the visceral yolk sac placenta. No antibody localization was observed in the embryo proper or the chorioallantoic placenta. It is speculated that the localization of AFP antibodies in the visceral yolk sac does not interfere with the embryotrophic function of the visceral yolk sac placenta.     

Embryonic growth and mobilization of energy and material during incubation in the checkered keelback snake, Xenochrophis piscator

We collected 20 checkered keelback snakes (Xenochrophis piscator) to study embryonic growth and mobilization of energy and material during incubation. Females laid eggs between late May and late June. The eggs were incubated at 27 degrees C (+/-0.3). One egg from each clutch was dissected at five-day intervals starting at oviposition. The mean incubation length at 27 degrees C was 48.9 days. We identified three phases of embryonic growth or yolk depletion in X. piscator. Phase 1, between oviposition and Day 20, was one of minimal transfer of energy and material from yolk to embryo. Phase 2, between Day 20 and Day 39-40, was characterized by increasingly rapid embryonic growth or yolk depletion. Phase 3, between Day 39-40 and hatching, was characterized by reduced embryonic growth or yolk depletion. Approximately 71% of dry mass, 53% of non-polar lipids and 66% of energy were transferred from the egg contents to the hatchling during incubation. Our data confirm that oviposition is not timed to coincide with the onset of rapid embryonic growth in oviparous squamate reptiles. The greater conversion efficiencies of energy and material from egg to hatchling in snakes can be attributed to their lower energetic costs of embryonic development and greater residual yolk sizes.     

Assessment of embryonic growth in chicken eggs by means of visible transmission spectroscopy

During this work, it was investigated whether spectral measurements can be used to monitor embryonic growth. An experiment was conducted in which both the transmission spectra and embryonic weight were determined on 240 eggs (Cobb, 37 weeks) between Day 5 and Day 10 of incubation. The spectral data were linked to embryonic weight by means of a partial least squares analysis. Different preprocessing procedures were compared during this work, that is, smoothing, multiplicative scatter correction (MSC), and first‐ and second‐order derivative. Compared to the remainder of the preprocessing procedures, MSC leads to a considerable improvement of the prediction capability of the embryonic weight. The ratio of performance to deviation obtained for the MSC spectra equaled 4.5 indicating that a very accurate prediction of embryonic weight is feasible based on the VIS/NIR transmission measurements. Important regions for the prediction are situated around 685–740 nm. It is suggested that the spectral changes in these spectral regions result from the displacement of carotenoids from the yolk into the blood circuitry. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Chick embryonic plasma proteins and binding capacity for corticosterone

Plasma was collected from White Leghorn embryos on alternate days from Day 4 of incubation to Day 22 (day after hatch). The plasma-binding capacity for corticosteroid was essentially zero before Day 10, but rose rapidly between Days 10 and 12. Binding capacity remained high until Day 16 and then declined before hatch. The increase after Day 10 was concurrent with the appearance of α-globulin whose rate of migration on acrylamide gel electrophoresis was similar to that of purified chicken corticosteroid binding globulin. Corticosterone, which was present in the plasma of the 4-day embryo, rose to its highest level on Day 20. The relatively high corticosterone concentration, with low plasma-binding capacity, suggests that in the chick embryo levels of free or active corticosterone are highest before Day 10 and just prior to hatching.     

A pituitary-salivary mixed gland induced by tissue recombination of embryonic pituitary epithelium and embryonic submandibular gland mesenchyme in mice

Renal subcapsular syngrafts of Day 9 to 11 mouse embryonic pituitary epithelium with Day 14 mouse embryonic submandibular gland mesenchyme produced mixed organs that include residual cleft structure surrounded by anterior pituitary cells some which are stained by anti-ACTH antiserum and submandibular gland-like structure with differentiated acinar cells which are stained by anti-alpha-amylase antiserum. However, when Day 8.5 or 12 embryonic pituitary epithelium was recombined with submandibular gland mesenchyme and syngrafted, development of submandibular gland-like or anterior pituitary tissues resulted, respectively. Thus, during organogenesis of the mouse anterior pituitary, there exists a developmental stage (Day 8.5-11 in utero), when prospective pituitary epithelium can respond to heterotypic submandibular gland mesenchyme with the development of a submandibular gland-like tissue.     

Chemical and immunological characterization of proteoglycans of embryonic chick calvaria

We have previously demonstrated in quail embryos grafted on chick yolk sacs the existence of intraembryonic stem cells responsible for definitive hemopoiesis. In order to determine the origin of these cells, we now examine the diffuse hemopoietic processes within the avian embryo's mesoderm. At 4–5 days of incubation in the two species, basophilic cells were found throughout the dorsal mesentery. At 6–8 days these cells became very numerous and built up dense foci at the level of branching of the anterior and posterior cardinal veins. These cells often infiltrated the wall of lymph spaces and channels and were also present in the lumen of blood vessels. Such locations support the interpretation that these basophilic cells represent early stages of hemopoietic differentiation. At 8–10 days, erythropoiesis or granulopoiesis was seen in the foci, which then regressed rapidly. The foci maximal development coincided with the period of colonization of the intraembryonic organ rudiments. In “yolk sac chimeras,” the foci were always constituted by quail cells, indicating their intraembryonic origin. The primordial origin of the intramesodermal cells remains to be determined. A likely source might be the ventral wall of the aorta which appeared to shed cells into the lumen and into the mesentery in the 3-day embryo.     

Engineered early embryonic cardiac tissue retains proliferative and contractile properties of developing embryonic myocardium

Embryonic myocardium has a high rate of cell proliferation and regulates cellular proliferation, contractile function, and myocardial architecture in response to changes in external mechanical loads. However, the small and complex three-dimensional (3D) structure of the embryonic myocardium limits our ability to directly investigate detailed relationships between mechanical load, contractile function, and cardiomyocyte proliferation. We developed a novel 3D engineered early embryonic cardiac tissue (EEECT) from early embryonic ventricular cells to test the hypothesis that EEECT retains the proliferative and contractile properties of embryonic myocardium. We combined freshly isolated White Leghorn chicken embryonic ventricular cells at Hamburger-Hamilton (HH) stage 31 (day 7 of a 46-stage, 21-day incubation period), collagen type I, and matrix factors to construct cylindrical-shaped EEECTs. We studied tissue architecture, cell proliferation patterns, and contractile function. We then generated engineered fetal cardiac tissue (EFCT) from HH stage 40 (day 14) fetal ventricular cells for direct comparison with EEECT. Tissue architecture was similar in EEECT and EFCT. EEECT maintained high cell proliferation patterns by culture day 12, whereas EFCT decreased cell proliferation rate by culture day 9 (P < 0.05). EEECT increased active contractile force from culture day 7 to day 12. The culture day 12 EEECT contractile response to the beta-adrenergic stimulation was less than culture day 9 EFCT (P < 0.05). Cyclic mechanical stretch stimulation induced myocardial hyperplasia in EEECT. Results indicate that EEECT retains the proliferative and contractile properties of developing embryonic myocardium and shows potential as a robust in vitro model of developing embryonic myocardium.     

Development of isolated sheep inner cell masses/embryonic discs in vitro

Ovine inner cell masses (ICMs)/embryonic discs cultured in vitro, in conditions copying those in which mouse embryonic stem cells (ESCs) arise from mouse blastocysts, give rise to ectodermal colonies. Day 10–11 ICMs/epiblasts produce ectodermal colonies sufficiently often (55–60%) for it to be considered worthwhile trying to generate presumed ESCs from them. Younger ICMs can only be taken into account if culture conditions can be improved so that ICM/ectodermal cells are more numerous. Older embryonic discs (12–13 day) are inconvenient because of the problem of endoderm overgrowing ectoderm. Secondary cultures of ectodermal colonies form epithelial or mesenchymal cells, which can be passaged at least seven times (50 days).This study was financed by the State Council for Scientific Research (grant no 5.5701.91.02 to J.A.M) and by funds from Edison Biotechnology Institute, Ohio University     

Role of progesterone in mobility, fixation, orientation, and survival of the equine embryonic vesicle

Luteal progesterone was removed by an injection of prostaglandin F(2alpha) or bilateral ovariectomy on Day 12 of pregnancy in pony mares. The embryonic vesicle remained mobile in the uterus until loss occurred on Days 13, 13, 15, or 19 in four prostaglandin-treated mares and Days 15, 17, 19, or 26 in four ovariectomized mares. Exogenous progesterone given daily, starting on Day 12, maintained pregnancy until Day 40 in five of five prostaglandin-treated and three of four ovariectomized mares. During two-hour mobility trials on Day 14, embryonic vesicles in mares without luteal or exogenous progesterone (n = 9) moved to a different uterine segment less frequently (mean number of location changes per two-hour trial: 7.2 +/-1.0 vs 10.4 +/-1.1, P < 0.05) and were observed more often in the uterine body (14.9 +/-2.9 vs 8.9 +/-1.3, P < 0.10) compared to vesicles in mares with a progesterone influence (n = 15). Of mares that still had a vesicle present on Day 18, fixation occurred by Day 17 in all (12 12 ) mares under the influence of luteal or exogenous progesterone but failed to occur in the three mares that were not under progesterone influence. Progesterone replacement was started on Day 16 in three mares that received prostaglandin F(2alpha) on Day 12 and still had a vesicle on Day 16. The vesicle was maintained and continued to develop in all three mares, indicating that the vesicles were viable four days after PGF(2alpha) treatment. However, fixation tended to be delayed (P < 0.15) and orientation of the embryo proper was altered (P < 0.005) compared to mares that were continuously under the influence of progesterone. The results demonstrated the importance of luteal progesterone to mobility, fixation, orientation, and survival of the embryonic vesicle.     

The control of the differentiation of female embryonic germ cells in the bird

The left gonad from female chick embryos at 4–12 days of incubation was cultured in vitro as pieces of intact gonad, pieces of isolated cortex, and groups of pure germ cells. All cultures were maintained for a time equal to 17 days in ovo. At the end of the culture period, a cytological and quantitative study was made on the germ cells.The results show that some germ cells in pieces of intact 6-day gonad and pieces of 6-day cortex complete their normal developmental sequence and enter zygotene. This shows that the factors that control the differentiation of the germ cells reside in the cortex of the gonad and their expression does not depend upon the pituitary and the medullary estrogens after 6 days of incubation.Germ cells that are cultured as isolated cells do not attach to the tissue culture substrate, do not divide mitotically, and do not enter zygotene. Evidence is presented that suggests 12-day germ cells do enter zygotene when cultured with pieces of 12-day cortex. These data suggest the differentiation of the female germ cells is regulated by the somatic cells of the cortex.