The effects of retinoic acid on heart formation in the early chick embryo.

The vitamin A derivative retinoic acid has previously been shown to have teratogenic effects on heart development in mammalian embryos. The craniomedial migration of the precardiac mesoderm during the early stages of heart formation is thought to depend on a gradient of extracellular fibronectin associated with the underlying endoderm. Here, the effects of retinoic acid on migration of the precardiac mesoderm have been investigated in the early chick embryo. When applied to the whole embryo in culture, the retinoid inhibits the craniomedial migration of the precardiac mesoderm resulting in a heart tube that is stunted cranially, while normal or enlarged caudally. Similarly, a local application of retinoic acid to the heart-forming area disrupts the formation of the cardiogenic crescent and the subsequent development of a single mid-line heart tube. This effect is analogous to removing a segment of endoderm and mesoderm across the heart-forming area and results in various degrees of cardia bifida. At higher concentrations of retinoic acid and earlier developmental stages, two completely separate hearts are produced, while at lower concentrations and later stages there are partial bifurcations. The controls, in which the identical operation is carried out except that dimethyl sulphoxide (DMSO) is used instead of the retinoid, are almost all normal. We propose that one of the teratogenic effects of retinoic acid on the heart is to disrupt the interaction between precardiac cells and the extracellular matrix thus inhibiting their directed migration on the endodermal substratum.     

Inhibition of heart formation by lithium is an indirect result of the disruption of tissue organization within the embryo

Lithium is a commonly used drug for the treatment of bipolar disorder. At high doses, lithium becomes teratogenic, which is a property that has allowed this agent to serve as a useful tool for dissecting molecular pathways that regulate embryogenesis. This study was designed to examine the impact of lithium on heart formation in the developing frog for insights into the molecular regulation of cardiac specification. Embryos were exposed to lithium at the beginning of gastrulation, which produced severe malformations of the anterior end of the embryo. Although previous reports characterized this deformity as a posteriorized phenotype, histological analysis revealed that the defects were more comprehensive, with disfigurement and disorganization of all interior tissues along the anterior-posterior axis. Emerging tissues were poorly segregated and cavity formation was decreased within the embryo. Lithium exposure also completely ablated formation of the heart and prevented myocardial cell differentiation. Despite the complete absence of cardiac tissue in lithium treated embryos, exposure to lithium did not prevent myocardial differentiation of precardiac dorsal marginal zone explants. Moreover, precardiac tissue freed from the embryo subsequent to lithium treatment at gastrulation gave rise to cardiac tissue, as demonstrated by upregulation of cardiac gene expression, display of sarcomeric proteins, and formation of a contractile phenotype. Together these data indicate that lithium's effect on the developing heart was not due to direct regulation of cardiac differentiation, but an indirect consequence of disrupted tissue organization within the embryo.     

Heat shock and thermotolerance during early rat embryo development

Effects of heat shock on the development of early pre-somite embryos have been studied using cultured rat embryos. The results illustrate the sensitivity of the developing head and brain to elevated temperatures prior to neural tube closure and the capacity of embryos to acquire thermotolerance. Embryos exposed briefly to an elevated temperature (43 degrees C for 7.5 min) developed severe craniofacial defects including microphthalmia, microcephaly, gross reduction of the forebrain region, and open neural tubes. In contrast, a nonteratogenic heat shock (42 degrees C for 10 min) caused embryos to acquire thermotolerance during a 15-min recovery period at 38.5 degrees C. Acquired thermotolerance was effective in protecting embryos from a subsequent more severe heat treatment which would have been teratogenic in an unprotected embryo. Recovering embryos mounted a heat shock response as evidenced by the induction of a 71 kilodalton heat shock protein. Activation of the heat shock response was not a teratogenic event in the developing embryo.     

Critical phases of the brain development

Various effects were shown to act alter the proceeding of histogenetic processes in the embryonic neural tissue. Studies in mice revealed that, depending on the embryogenesis stage when a lack of serotonin occurred, the lack of this monoamine entails either death of the embryo or a teratogenic effect, or underdevelopment of brain structures. In the rat model of a shorten hypoxia, a disorder of neural tissue histogenesis in early stages of development, was found, as well as a reduction of its sensitivity to lack of oxygen by the end of embryogenesis.     

N-cadherin localization in early heart development and polar expression of Na+,K(+)-ATPase, and integrin during pericardial coelom formation and epithelialization of the differentiating myocardium.

N-cadherin, a Ca(2+)-dependent cell adhesion molecule, has been localized previously to the mesoderm during chick gastrulation and to adherens junctions in beating avian hearts. However, a systematic study of the dynamic nature of N-cadherin localization in the critical early stages of heart development is lacking. The presented work defines the changes in the spatial and temporal expression of N-cadherin during early stages of chick heart development, principally between Hamburger and Hamilton stages 5-8, 18-29 hr of development. During gastrulation N-cadherin appears evenly distributed in the heart forming region. As development proceeds to form the pericardial coelom (stages 6, 7, and 8, i.e., between 22 and 26 hr of development) N-cadherin localization becomes restricted to the more central areas of the mesoderm. The localization also shows a periodicity that correlates closely with the distance between foci of cavities that eventually coalesce to form the coelom. This distribution suggests that N-cadherin may have a function in the sorting out of somatic and splanchnic mesoderm cells to form the coelom. This separation of the mesoderm in the embryo for the first time physically delineates the precardiac mesoderm population. Concomitant with cell sorting during coelom formation, the precardiac cells change shape and show a distinct polarity as conveyed by (1) the apical expression of N-cadherin on precardiac cell surfaces lining the pericardial coelom, (2) the primarily lateral expression of Na+,K(+)-ATPase, and (3) an enrichment of integrin (beta 1 subunit) on basal cell surfaces. The somatic mesoderm cells apparently down-regulate N-cadherin expression. N-cadherin is also absent from the precardiac cells close to the endoderm. The latter cells eventually form the endocardium, i.e., the endothelial lining of the heart. By contrast, in the tubular, beating heart N-cadherin is found throughout the myocardium. In summary, immunolocalization patterns of N-cadherin during early cardiogenesis suggest that this cell adhesion molecule has a major role in the dynamics of pericardial coelom formation. Subsequently, its continued expression during cell differentiation of the cardiomyocyte to form the myocardium, but not endocardium, suggests N-cadherin is an essential morphoregulatory molecule in heart organogenesis.     

Cellular retinoic acid-binding protein and the role of retinoic acid in the development of the chick embryo

The distribution of cellular retinoic acid-binding protein (CRABP) in four stages of chick development is described using an affinity-purified antibody against rat CRABP. CRABP is the protein to which retinoic acid (RA) binds when it enters cells and may reflect the requirement of those cells for RA. We found several discrete cell populations which showed high levels of immunoreactivity. Some were in the neural tube such as the commissural neurons and the dorsal roof plate. Some were of neural crest origin such as the dorsal root ganglia, sensory axons, sympathetic ganglia, and enteric ganglia. The remaining populations were certain connective tissue cells, limb bud cells, and the myotome. These results suggest that certain organ systems, particularly the nervous system, have a requirement for RA during development and they may further our understanding of the teratogenic effects of retinoids on the embryo.     

Analysis of high dysmorphogenic activity of Ro 13-6307, a tetramethylated tetralin analog of retinoic acid.

Certain synthetic retinoids differ widely from retinoic acid (RA) in teratogenic potency, being much more or much less effective than RA. It is assumed that the potency of a retinoid may depend on the nature of its interaction with cellular binding components (nuclear retinoic acid receptors or cytoplasmic binding proteins) and, as in the case of retinoids that are mammalian teratogens, on factors that determine its accessibility to the embryo. To investigate some of the factors that contribute to potency, we used a new synthetic retinoid Ro 13-6307 that differs in structure from RA in having an aromatic ring inserted in its side chain along with gem dimethyl modification of the natural cyclohexenyl ring. Pregnant ICR mice were given a single oral dose (0, 1, or 10 mg/kg) on day 11 of gestation, and the resultant teratogenic outcome was monitored on day 17. Direct effects on cell differentiation were obtained by exposing high density cultures of limb bud mesenchymal cells to a range of concentrations (0.3 ng/ml-3 micrograms/ml) of Ro 13-6307 and scoring for chondrogenic suppression. Concentrations reaching the embryo after maternal administration of Ro 13-6307 were measured by HPLC to quantify the analog for a period of 4 h after administration of the oral dose. We found that this retinoid was 40-fold as active as RA in both inducing teratogenesis and suppressing chondrogenesis, yet its concentration in the affected embryo was only a fraction of that achieved after an equivalent dose of RA was employed in a similar protocol.(ABSTRACT TRUNCATED AT 250 WORDS)     

Retinoic acid modifies development of the midbrain-hindbrain border and affects cranial ganglion formation in zebrafish embryos.

Considerable evidence now suggests that retinoic acid (RA) is an important modulator of patterning events in early neuronal development in vertebrates. In this paper, we describe the effects of exogenously applied RA on early neural development in the zebrafish embryo. Neural anatomy is assessed by immunocytochemical and histochemical analysis of the developing embryo in whole mounts at 24 h post-fertilization. RA was applied for one hour at concentrations ranging from 10(-9) to 10(-6) M to embryos at 50% epiboly, the midgastrula stage, and at 10(-7) M to embryos at early and late gastrula stages. The neuroanatomical analysis shows that 10(-7) M RA causes a defined lesion to the developing central nervous system which corresponds to a loss of a region of the brain in the caudal midbrain-rostral hindbrain area, the precursor of the cerebellum and associated neural structures. The region that fails to develop corresponds to the cranial expression domain of the engrailed protein as assessed by the monoclonal antibody 4D9 (Patel et al. 1989: Expression of engrailed proteins in arthropods, annelids and chordates. Cell 58, 955-968). Structures caudal to rhombomere 4 are unaffected by 10(-7) M RA, as are the cranial midbrain and forebrain: 10(-7) M RA also affects the development of cranial ganglia, principally the Vth, anterior lateral line and VIIIth ganglia, suggesting that RA affects normal development of the cranial neural crest. Effects of RA at stages immediately prior to and after gastrulation show some similar and some distinct features. Results are discussed in terms of the possible role of RA as an endogenous moderator of normal head development.     

Combinatorial signaling in the heart orchestrates cardiac induction, lineage specification and chamber formation

The complexity of mammalian cardiogenesis is compounded, as the heart must function in the embryo whilst it is still being formed. Great advances have been made recently as additional cardiac progenitor cell populations have been identified. The induction and maintenance of these progenitors, and their deployment to the developing heart relies on combinatorial molecular signalling, a feature also of cardiac chamber formation. Many forms of congenital heart disease in humans are likely to arise from defects in the early stages of heart development; therefore it is important to understand the molecular pathways that underlie some of the key events that shape the heart during the early stages of it development.     

达氏鳇不同发育期胚胎对低温的耐受研究

研究了达氏鳇12个发育期胚胎经过不同低温（2 ℃、3 ℃、5 ℃、7 ℃和8 ℃）处理12 h、24 h、2 d、3 d、6 d、10 d、15 d、20 d和30 d后的孵化率和仔鱼成活率.结果表明,卵黄栓期、隙状胚孔期、神经管闭合期胚胎在2～8 ℃水温下,处理24 h后孵化率为0;卵裂期、囊胚早期、原肠中期胚胎在2～8 ℃水温下,处理3 d后孵化率低于30%;囊胚晚期、原肠早期、眼基期、尾芽期、心跳期和尾达头部期胚胎在5～8 ℃水温下,处理3 d后孵化率、仔鱼成活率超过70%;随低温处理时间延长,胚胎和仔鱼的死亡率增加,处理时间与孵化率、仔鱼成活率呈负相关;囊胚晚期、原肠早期、眼基期胚胎在5 ℃水温下耐受力较强,处理10 d后孵化率、仔鱼成活率超过70%.本研究表明,达氏鳇胚胎发育过程中囊胚晚期、原肠早期和眼基期胚胎可以在某一低温下进行短期保存,其孵化率、仔鱼成活率与常温（16～17 ℃）下没有显著差异.这对于达氏鳇胚胎（受精卵）的长途运输有重要意义.     

Pharmacokinetic assessment of teratologically effective concentrations of an endogenous retinoic acid metabolite

Retinoic acid is a natural vitamin A derivative that undergoes oxidative metabolism in the body to yield several metabolites, which apparently represent the products of a detoxification pathway. To assess if such metabolic conversions diminished teratogenic potency, one of the major metabolites (4-oxo-all-trans-retinoic acid) was tested for its teratogenic activity in pregnant ICR mice and further investigated for its pharmacokinetic features to determine if it accumulated in the embryo in concentrations sufficient to elicit a teratogenic response. Administration of single oral doses (10, 25, 50, or 100 mg/kg) of the compound to ICR mice on day 11 of gestation (plug day = day 0) produced dose-dependent frequencies of serious fetal anomalies of the type usually associated with the use of retinoic acid and other retinoids. The metabolite was equivalent in teratogenic potency to retinoic acid, and, in the instance of cleft palate frequency, it was even more active. Concentrations of 4-oxo-all-trans-retinoic acid and its 13-cis isomer were measured in the maternal plasma and whole embryos at 30 min to 10 hr after administration of the lowest (10 mg/kg) and the highest (100 mg/kg) teratogenic dose of 4-oxo-all-trans-retinoic acid by means of high-performance liquid chromatography methodology. Distribution of the compound in the maternal system and transfer to the embryo occurred rapidly with either dose. Peak concentration in the maternal plasma and the embryo persisted for 3-4 hr after the higher dose but not with the lower dose; however, elimination kinetics for the two dose levels were similar.(ABSTRACT TRUNCATED AT 250 WORDS)     

Retinoids--their metabolites, action, and role in heart development

Retinoids constitute a group of active compounds known as vitamin A. Apart from an unquestionable function in adults, retinoids also play a profound role in many events during embryonic development for instancje in axial patterning and organogenesis. Retinoic acid is the most active biological form of vitamin A. Its signaling both in adults and during embryonic development occurs at different levels through interaction with specific proteins and nuclear receptors. Retinoic acid signaling in heart development occurs mostly via interaction with secondary heart field cells by restricting their spatial expansion and controlling proper addition of these cells to the cardiac tube. This signal requires precise level of local retinoic acid, excess or insufficiency of which causes various malformations of the embryo and embryonic heart. Although retinoid signaling in the developing heart is a highly significant developmental factor, it is not yet fully understood. The following review summarises recent developments regarding this subject.     

Retinoid teratogenicity in the macaque: verification of dosing regimen

To further define teratogenicity associated with 13-cis-retinoic acid (13-cis-RA) in the cynomolgus monkey, the drug was orally administered on three different treatment regimens. Experiment (Exp.) 1 (2.5 mg/kg/day, gestational day [GD] 12-27, n = 11) investigated the teratogenicity of a single daily dose of 13-cis-RA administered shortly after embryo implantation. Pharmacokinetic sampling was done to determine retinoid profiles on the first (GD12) and last (GD27) days of treatment. Exposure to 13-cis-RA during early organogenesis in Exp. 2 (2.5 mg/kg/day, GD20-27, and 2 x 2.5 mg/kg/day, GD28-30, n = 5) investigated the potential adverse effects of 13-cis-RA on the developing limb. The use of multiple doses of 13-cis-RA in Exp. 3 (2 x 2.5 mg/kg/day, GD26-27, n = 5) investigated the necessity of double dosing on the induction of retinoid embryopathy in the macaque. Malformations of retinoid target organs as well as embryolethality were most prevalent when single daily doses of 13-cis-RA were administered during pre- and early organogenesis in Exp. 1. Moreover, multiple doses on GD26-27 failed to induce any manifestation of abnormal development in Exp. 3. These results confirm that the lowest observed adverse effect level (LOAEL) in macaques is 2.5 rather than 5.0 times greater than that observed in human pregnancies. Exposure during forelimb development (GD20-30) in Exp. 2 was unsuccessful in inducing defects of this skeletal region, although defects in several retinoid target organs (i.e., cerebellum and internal ear) were present, indicating that a teratogenic threshold was achieved. Pharmacokinetic analysis of 13-cis-RA and its metabolites on GD12 and 27 in Exp. 1 showed considerable exposure to the administered drug and its 4-oxo-metabolite. In contrast, the exposure to all-trans-RA was negligible. The results support the use of a specific treatment schedule in early gestation in the macaque as the most appropriate model for characterizing the teratogenic potential of retinoids in humans.     

Effects of excess vitamin A on development of cranial neural crest-derived structures: a neonatal and embryologic study

BACKGROUND: Vitamin A and its metabolites have been shown to be teratogenic in animals and humans producing defects of neural crest derived structures that include abnormalities of the craniofacial skeleton, heart, and thymus. Our prior studies with retinoic acid have established that gestational day (gd) 9 is a sensitive embryonic age in the mouse for inducing craniofacial and thymic defects. METHODS: We exposed pregnant mice to variable doses of vitamin A (retinyl acetate) on gd 9 and embryos were evaluated for changes in developing pharyngeal arch and pouch morphology, neural crest cell migration and marker gene expression. Additionally, we investigated whether a single organ system was more sensitive to low doses of vitamin A and could potentially be used as an indicator of vitamin A exposure during early gestation. RESULTS: High (100 mg/kg) and moderate (50 and 25 mg/kg) doses of vitamin A resulted in significant craniofacial, cardiac outflow tract and thymic abnormalities. Low doses of vitamin A (10 mg/kg) produced craniofacial and thymic abnormalities that were mild and of low penetrance. Exposed embryos showed morphologic changes in the 2nd and 3rd pharyngeal arches and pouches, changes in neural crest migration, abnormalities in cranial ganglia, and altered expression of Hoxa3. CONCLUSIONS: These animal studies, along with recent epidemiologic reports on human teratogenicity with vitamin A, raise concerns about the potential for induction of defects (perhaps subtle) in offspring of women ingesting even moderate to low amounts of supplemental vitamin A during the early gestational period.     

Epidemiologic analysis of prenatal exposure to cough medicines containing dextromethorphan: no evidence of human teratogenicity

BACKGROUND: In a recent experimental study on chick embryos, Andaloro et al. (1998, Pediatr. Res. 43:1-7) observed a relationship between neural crest/neural tube defects (NTDs) and prenatal exposure to dextromethorphan. These authors made an extrapolation of their results to the human embryo, indicating that this drug could produce NTDs in humans. Rosenquist (1999, Teratology 60:58-60) based on the results of the epidemiologic study performed by Ferencz et al. (1997, Perspect. Pediatr. Cardiol. 5:50-162), concluded that dextromethorphan could cause congenital heart defects in humans. Because this drug is an over-the-counter drug, the suggestion of those authors has led to great controversy and public concern about the possible teratogenic effect of this drug on the human embryo. METHODS: We present the results of a case-control study by using logistic regression analyses on the effect of prenatal exposure to drugs containing dextromethorphan only, or in combination with other drugs, on human development. We mostly analyzed dextromethorphan. The study was designed in part as hypothesis confirmation for NTDs and congenital heart defects and, in part, as hypotheses generation by testing the association with many other congenital defects. RESULTS: The results do not show a relation between the occurrence of NTDs and heart defects or other defects with exposure to drugs containing dextromethorphan. CONCLUSIONS: The usual use of dextromethorphan in cough medications during pregnancy does not increase the risk for congenital defects.