Nitric Oxide Reverses the Position of the Heart during Embryonic Development.

Nitric oxide (NO) produced by endothelial nitric oxide synthase (eNOS) plays crucial roles in cardiac homeostasis. Adult cardiomyocyte specific overexpression of eNOS confers protection against myocardial-reperfusion injury. However, the global effects of NO overexpression in developing cardiovascular system is still unclear. We hypothesized that nitric oxide overexpression affects the early migration of cardiac progenitor cells, vasculogenesis and function in a chick embryo. Vehicle or nitric oxide donor DEAN (500 mM) were loaded exogenously through a small window on the broad side of freshly laid egg and embryonic development tracked by live video-microscopy. At Hamburg Hamilton (HH) stage 8, the cardiac progenitor cells (CPC) were isolated and cell migration analysed by Boyden Chamber. The vascular bed structure and heart beats were compared between vehicle and DEAN treated embryos. Finally, expression of developmental markers such as BMP4, Shh, Pitx2, Noggin were measured using reverse transcriptase PCR and in-situ hybridization. The results unexpectedly showed that exogenous addition of pharmacological NO between HH stage 7⁻8 resulted in embryos with situs inversus in 28 out of 100 embryos tested. Embryos treated with NO inhibitor cPTIO did not have situs inversus, however 10 embryos treated with L-arginine showed a situs inversus phenotype. N-acetyl cysteine addition in the presence of NO failed to rescue situs inversus phenotype. The heart beat is normal (120 beats/min) although the vascular bed pattern is altered. Migration of CPCs in DEAN treated embryos is reduced by 60% compared to vehicle. BMP4 protein expression increases on the left side of the embryo compared to vehicle control. The data suggests that the NO levels in the yolk are important in turning of the heart during embryonic development. High levels of NO may lead to situs inversus condition in avian embryo by impairing cardiac progenitor cell migration through the NO-BMP4-cGMP axis.

Assessment of teratogenic effects of lidocaine in rat embryos cultured in vitro.

BACKGROUND: Lidocaine has been reported to cause neural tube closure defects in vitro in mice at clinically relevant concentrations. However, no studies have been conducted to further investigate this potentially hazardous effect of lidocaine. This study was aimed to reassess teratogenic effects of lidocaine in vitro in rats. METHODS: Sprague-Dawley rat embryos were explanted at 8:00 AM on gestational day 9 and were cultured in medium containing various concentrations of lidocaine. (Embryos in the control group were cultured without lidocaine). After 50 h of culture, they were evaluated for growth size and morphology, including the neural tube closure. RESULTS: In the presence of 250 microM of lidocaine, embryos showed a increased incidence of situs inversus compared with control group but were otherwise normal. At 375 microM, embryos showed slight growth retardation but no significant morphologic abnormalities. At 500 microM, all viable embryos showed severe morphologic abnormalities. However, morphologic abnormalities were so-called nonspecific types and neural tube closure defects were not observed. CONCLUSIONS: Results from the current study indicate that lidocaine causes teratogenic effects in vitro in rats only at concentrations much higher than clinically relevant concentrations. Furthermore, lidocaine did not cause neural tube closure defects at any concentrations evaluated.

Specification of left-right asymmetry in mammals: embryo culture studies of stage of determination and relationships with morphogenesis and growth.

The internal mammalian body plan is laterally asymmetric with a consistent handedness such that some organs are placed on one side (stomach on the left, for example) and paired organs are not symmetric (for example, there are more lung lobes on the right). Some chemical teratogens can affect the development of asymmetry, and some can cause asymmetric defects in overtly symmetric structures, but the mechanisms are unknown. We have used chemical treatment of rat embryos in culture to examine the stage at which the left-right axis is determined and show that all effective treatments can affect left-right axis development up to the early headfold stage, but not from late headfold onwards. This suggests that the left-right axis is determined by the late headfold stage, even though the embryo is overtly symmetric at this stage. It appears to be much easier to induce an abnormal left-right axis from late allantoic bud and early headfold stages than the early allantoic bud stage, but we have not established the earliest stage at which a response can be induced. Complete situs inversus was the most common chemically induced abnormality, although heart looping and body turning could be inverted separately, suggesting that the two phenomena are linked but not wholly interdependent. The treatments appeared to cause a loss of handedness, rather than inducing inversion, since the incidence of an abnormal left-right axis never exceeded 50%. All treatments except methoxamine, an alpha1 adrenergic agonist, induced an abnormal left-right axis in association with other morphologic defects and growth retardation. However, there was no relationship between the severity or incidence of dysmorphology, nor growth retardation, and left-right abnormality, suggesting that although the process that specifies lateral asymmetry is labile, it is independent of general growth and morphogenesis.

Adrenergic neurotransmitters and calcium ionophore-induced situs inversus viscerum in Xenopus laevis embryos.

Xenopus laevis embryos at the blastula-early tail bud stage were exposed to norepinephrine or octopamine dissolved in culture saline until they reached the larval stage. The left-right asymmetry of the heart and gut was then examined. We found that these adrenergic neurotransmitters induced situs inversus in the heart and/or gut in up to 35% of tested neurula embryos. Norepinephrine-induced situs inversus was blocked by the alpha-1 adrenergic antagonist prazosin. Furthermore, A23187, a calcium ionophore, also increased the incidence of situs inversus up to 54% when late-neurula embryos were exposed to the solution. A23187 treatment initiated before neural groove formation was less effective. The incidence of situs inversus induced by these reagents decreased towards the control level (2.2%, 25 untreated embryos out of 1127 embryos in total) in embryos past the stage of neural tube closure. In the present experiments we obtained 22 gut-only situs inversus embryos having an inverted gut and a normal heart. In contrast, such embryos were not observed among the 1127 untreated embryos. An adrenergic signal mediated by an increase in intracellular free calcium may be involved in the asymmetrical visceral morphogenesis of Xenopus embryos.

Staurosporine does not prevent adrenergic-induced situs inversus, but causes a unique syndrome of defects in rat embryos grown in culture.

Staurosporine, an alkaloid isolated from Streptomyces species, is commonly used as a protein kinase C (PKC) inhibitor in animal investigations. In the present study, we used this compound to determine whether alpha 1-adrenergic stimulation-induced situs inversus in rats is mediated by PKC. Embryos were explanted at 8 A.M. on day 9 of gestation. Those with a neural groove but with no visible neural folds (Stage 11a) were selected and were cultured in medium containing various concentrations of staurosporine with or without 50 microM of phenylephrine, an alpha 1-adrenergic agonist. At 10 A.M. on day 11 of gestation, embryos were examined for situs inversus and other abnormalities. Staurosporine, tested at 0.0001, 0.001, 0.01, 0.1, 0.375, and 0.5 microM (lethal concentration), did not block phenylephrine-induced situs inversus at any concentration. However, staurosporine alone produced situs inversus at concentrations above 0.1 microM. At 0.5 and 1.0 microM, staurosporine also caused cyst-like lesions projecting dorsally from the mesencephalon that we named "mesencephalic vesicles" and the formation of secondary somites. To confirm and further examine these unique effects of staurosporine both grossly and histologically, we conducted additional experiments using staurosporine from another source. Embryos were explanted between 6 A.M. and 9 P.M. on day 9 of gestation and were placed in one of the following groups according to their stage of development: 10b, 11a, 11b, 11c, 12/s1-2, 12/s3-4, and 12/s5-6. Embryos were then cultured with various concentrations of staurosporine. Those cultured from Stage 11a exhibited similar lesions to those seen in the initial experiment but at somewhat higher concentrations of staurosporine. Embryos cultured from Stage 10b showed a similar pattern of lesions as seen at Stage 11a, except that higher concentrations of staurosporine were required to cause mesencephalic vesicles and secondary somites formation. Embryos cultured from Stage 11b showed similar effects to those cultured from younger stages except that maximum incidences of situs inversus were much lower. Those cultured from Stage 11c showed similar dose-response to those cultured from Stage 11b except that the incidence of secondary somites formation was much higher. In addition, in approximately 40% (n = 25) of embryos treated with greater than 1.0 microM of staurosporine, the growing end of the allantois did not reach the chorion and remained unattached in the exocoelomic cavity.(ABSTRACT TRUNCATED AT 400 WORDS)

Methionine prevents nitrous oxide-induced teratogenicity in rat embryos grown in culture.

BACKGROUND: Nitrous oxide (N2O)-induced teratogenicity in rats is commonly believed to be due to decreased tetrahydrofolate, which results in decreased DNA synthesis. The role of decreased methionine has been largely ignored as have the sympathomimetic effects of N2O. METHODS: A rat whole-embryo culture system was used to determine whether N2O-induced teratogenicity can be prevented with supplemental methionine or folinic acid and whether N2O-induced situs inversus is mediated by alpha 1-adrenergic stimulation. Embryos were explanted on day 9 of gestation, and those at stage 10b (late primitive streak stage) were cultured with or without N2O and the various chemicals, methionine (25 micrograms.ml-1), folinic acid (5 micrograms.ml-1), phenylephrine (range 0.5-50 microM) and prazosin (10 microM). Embryos in the N2O groups were exposed to a concentration of 75% for the first 24 h of culture. After 50 h of culture, embryos were examined for abnormalities including situs inversus. RESULTS: Treatment with N2O alone resulted in increased incidences of malformations and growth retardation. Methionine, but not folinic acid or prazosin, almost completely prevented N2O-induced malformations and growth retardation. N2O itself did not cause situs inversus but increased the incidence of phenylephrine-induced situs inversus. This additive effect was blocked by prazosin. CONCLUSIONS: Our results indicate that decreased methionine rather than decreased tetrahydrofolate plays the major role in N2O-induced teratogenicity in rats. They also indicate that N2O stimulates the alpha 1-adrenergic pathway in the embryo and thereby increases the incidence of phenylephrine-induced situs inversus.

Situs inversus in the developing mouse: proteins affected by the iv mutation (genocopy) and the teratogen retinoic acid (phenocopy).

To decipher genes that are important in the determination of laterality, we compared two-dimensional protein gels from wild-type C57BL/6J mice and C57BL/6J mice that carried the iv mutation, which confers random determination of visceral situs. To span the time period(s) during which laterality determination occurs, we compared computer-analyzed two-dimensional protein gels from wild-type mouse embryos and iv/iv mouse embryos at 7.5, 8.0, and 8.5 days post-coitum. One polypeptide that was expressed only on day 8.0 of development and only in wild-type embryos represents a particular candidate for determination of laterality. Day 8.5 postcoitum represents the earliest time in murine development that laterality is manifest. Two-dimensional gels were compared from 8.5 day embryos that were C57BL/6J wild-type, C57BL/6J iv/iv, or C57BL/6J wild-type and exposed to the teratogen retinoic acid late on day 7. Reproducible alterations of protein synthesis were observed in both the iv genocopy and retinoic acid phenocopy, yielding abnormal laterality determination. The intersection of these peptide changes identifies a protein likely to play a role in the determination of laterality.

Evidence for an adrenergic mechanism in the control of body asymmetry.

The effect of phenylephrine, an alpha-1 adrenergic agonist, on development of body asymmetry was studied using a rat whole embryo culture system. Embryos were explanted at the presomite stage, cultured in 100% rat serum containing various concentrations of phenylephrine, and examined at the 20-25 somite stage for sidedness of asymmetric body structures, namely, bulboventricular loop, allantoic placenta, and tail. Phenylephrine treatment resulted in a dose-dependent increase of situs inversus with a maximum incidence of 52%. Coadministration of prazosin, an alpha-1 adrenergic antagonist, almost completely prevented this effect. Our results suggest that receptor-mediated stimulation of the alpha-1 adrenergic pathway is involved in the control of normal body asymmetry in developing rat embryos.

[Aminoacetonitrile and fetogenesis of the rat. II. Malformations of organ systems (author's transl)]. / Aminoacetonitril und Fetogenese der Ratte. II. Fehlbildungen innerer Organe.

We investigated the action of the lathyrogenic compound aminoacetonitrile (300 mg/kg body weight) on fetogenesis of the Wistar rat. In a 1st section we dealt with influence on the outer body shape and common reproductive parameters (WENDLER and coworkers 1980). This 2nd section describes toxic effects on internal organs and malformations on organ systems detected by free-hand razor blade technique. Especially, we observed cardial ectopia, rupture of the ascendant aorta, situs inversus, hydrocephalia, hydronephroses, and dystopic kidneys. Enlargement of heart atria, and main thoracic veins, dilatations of salivary gland ducts and bronchial tree are considered to be nonspecific toxic effects of aminoacetonitrile.

[The influencing of blastogenesis and embryogenesis in rats through aminoacetonitrile]. / uber die Beeinflussung von Blastogenese und Embryogenese der Ratte durch Aminoacetonitril

The study was designed to determine the influence of the lathyrogenic substance aminoacetonitrile on blastogenesis and embryogenesis of the Wistar rat. 91 female Wistar rats, weighing from 190 through 380 g, received a single injection of 300 mg aminoacetonitrile (AAN)/kg body weight. Substance was given intraperitoneally on days 5, 7, 9, 11 or 13 of pregnancy. The detection of sperms in the vaginal smears was counted as day 1 post coitum. Animals were sacrificed on day 21 of pregnancy. The following parameters served as a base of interpretation: fetal body weight, numbers of implantations, resorptions, dead and living fetuses. Malformations were detected by outer inspection for gross anomalies, by the razor blade technique for internal malformations, and by skeletal preparations. All results were evaluated by statistical means. AAN influences pregnancy and fetal development. The rates of fetal resorptions are enhanced after application of AAN beyond the 7th day p. c. The maximum of fetal death is reached on day 11 p. c. There is no influence on the mean implantation number. With the exception of the 5th day of development AAN reduces the number of living fetuses. After application of AAN on day 13 p.c. the mean body weight decreases significantly. All over the investigated range malformations can be observed. Abnormalities of internal organs are frequent: Hydrocephalus, hydronephrosis, situs inversus. Malformations of the skeletal system are only observable on days 5, 7 and 9: sternal fissure, supernumerary ribs, destruction of the lumbar spine. AAN does not induce gross anomalies of the fetus.