Teratogenic Effects Of Retinoic Acid Research Articles

Retinoic Acid Teratogenicity in Mouse Embryo

The present study was designed to explore furthermore the relationship between abnormalities in mice whichwere used as a model to study the (NTDs) in human beings and retinoic acid. The study was conducted onthe Swiss white mice, with 40 females and 10 males. Females that have the vaginal plug are isolated inseparate plastic cages to study the teratogenic effect of retinoic acid in the concentration of 15, 25, 35 or45 mg/kg live body weight which were administered intraperitonially. Also, folic acid was used with dose35mg/kg to test the preventive effect against neural tube defects. Retinoic acid doses of 15 mg/kg, 25 mg/kg and 35 mg/kg caused different abnormalities in percent of 20% including (Small size with unclear face,NTDs), 32% including (NTDs, prolonged embryos and deformation eye and ears), 68% including (NTDs,prolonged embryos and deformation eye and ears and small size) respectively. While the dose of 45 mg/kgcaused 100% reabsorption.

The protective effects of curcumin on cytotoxic and teratogenic activity of retinoic acid in mouse embryonic liver.

Retinoic acid (RA) is a synthetic vitamin derivative. It exerts toxic and teratogenic effects on the development of embryonic organs in dose- and time-dependent manners in mice. Curcumin is a compound obtained from rhizomes of turmeric (Curcuma longa) and has protective effects on teratogenic agents. The current study examined the effects of curcumin on embryos treated with RA. A total of 24 female NMRI mice (8-week-old pregnant mice) were investigated in the current study. All of them were treated for 10 days during days 15 to 50 of pregnancy. In the first group, the animals were fed with normal diets (control); in the second group, with 60 mg/kg all- trans RA; in the third group, with 10 mg/kg curcumin; and in the fourth group, with RA and curcumin in their diets. The animals were killed by cervical dislocation at the 18th day of pregnancy and embryos were separated from the uteruses. The embryo weight and crown rump (CR) length were measured, and the SPSS software was used to analyze data. There was a significant increase in the lengths of CR and weights of embryos after using curcumin, but RA had no effect on the length of CR and weight of embryos at a dose of 60 mg/kg. Morphometric assay of liver tissue was performed, and data analysis indicated that there were significant differences between groups in terms of morphometric parameters of liver tissue. Therefore, RA increased the cell number and sinusoid diameter and decreased the cell areas in the embryonic liver tissue. However, curcumin decreased these side effects of RA on the embryonic liver tissue. The results indicated that curcumin could decrease the toxic and teratogenic effects of RA in mouse embryos.

Retinoic acid retards fetal and hindlimb skeletal development asymmetrically in a retinoic acid-induced clubfoot model

Retinoic acid (RA) has been shown to induce congenital clubfoot in animal models, but it is unknown whether the effect of RA on the formation of clubfoot in vivo results from generalized growth retardation or from the specific effects of hindlimb skeletal development. Our experimental research was based on a clubfoot model treated by maternal administration of RA (120, 130 or 140 mg/kg body weight) as an intragastric dose on embryonic day 10 (E10), and a control group was administered with an equivalent dose of solvent. Prenatal RA exposure reduced fetal body weight, length and skeletal ossification of the hindlimb compared with the control fetuses in a dose-dependent manner. The normal development curves indicated that the RA-exposed fetuses showed delayed increase in body weight and skeletal ossification development. However, there was no uniform effect on the skeletons of the hindlimb, not least retardation in ossification and induction malformation on the talus and calcaneus. Our results demonstrated that prenatal RA exposure had retardation effects on the developing hindlimb skeleton that was independent of those on the overall fetal growth. The normal skeletal ossification showed that the talus and calcaneus were poorly ossified and they were delayed by almost one day in the RA 120 mg/kg group. Therefore, during the susceptible stages, different regions of the limb bud responded differently to the teratogenic effects of RA.

Bmpr1bb is a novel gene involved in retinoic acid induced patterning of the zebrafish hindbrain

Retinoic acid signalling plays a critical role during zebrafish development. The teratogenic effects of retinoic acid have been demonstrated by embryonic deformation resulting from insufficient or excessive levels of this vitamin A derivative. During embryogenesis, bone morphogenetic proteins are closely linked to the physiological interpretation of RA gradients, particularly in the hindbrain. We describe an uncharacterized gene, Bmpr1bb, as being significantly downregulated in response to retinoic acid treatment. In situ expression demonstrates that Bmpr1bb is expressed ubiquitously at 10hpf, and is slowly downregulated until 48hpf where the expression is concentrated in the hindbrain. We propose that Bmpr1bb is a downstream target of RA signalling, strongly downregulated during embryogenesis and specified to a specific region of the hindbrain.

Pharmacogenetic model of retinoic acid-induced dyslipidemia and insulin resistance

Therapeutic administration of retinoids is often accompanied with undesirable side effects, including an increase in lipid levels in up to 45% of treated patients. We tested the hypothesis of whether spontaneously hypertensive rat (SHR) and congenic SHR.PD-(D8Rat42-D8Arb23)/Cub (SHR-Lx) strains, differing only in a 14-gene region of chromosome 8 and previously shown to display differential sensitivity to the teratogenic effects of retinoic acid, could serve as a pharmacogenetic model set of the metabolic side effects of retinoid therapy. Male, 15-week old rats (n = 12/strain) of SHR and SHR-Lx strains were fed a high-sucrose diet for 2 weeks and subsequently treated either with all-trans retinoic acid (15 mg/kg) or only with a vehicle for 16 days (n = 6/strain/treatment), while still on the high-sucrose diet. We assessed the morphometric and metabolic profiles of all groups, including glucose tolerance tests, levels of insulin, adiponectin, free fatty acids, concentrations of triglycerides and cholesterol in 20 lipoprotein fractions under conditions of both high-sucrose diet and high-sucrose diet plus all-trans retinoic acid administration. SHR-Lx displayed substantially greater sensitivity to a number of all-trans retinoic acid-induced metabolic dysregulations compared with SHR, resulting in impairment of glucose tolerance, increased visceral adiposity, and substantially greater increase of circulating triglyceride concentrations, accompanied by a shift towards their less favorable distribution into the lipoprotein fractions. These observations closely mimic the common side effects of retinoid therapy in humans, rendering SHR-Lx an experimental pharmacogenetic model of atRA-induced dyslipidemia.

Embriopatía por isotretinoína: Un daño evitable

Retinoic acid is a widely used drug in the treatment of cystic acne. It has teratogenic effects that depend on the gestational period in which it is used. We report a seven months old female whose mother was exposed to retinoic acid in both pre-gestational and gestational periods. She had a retardation of psychomotor development and a brain MRI showed frontal atrophy and a malformation of the posterior fossa. We discuss the mechanisms of the teratogenic effects of retinoic acid.

Teratogenic effect of retinoic acid in swiss mice

To identify the types of malformations resulting from the administration of retinoic acid (RA) to Swiss mice on different days of pregnancy. Twenty-four pregnant Swiss mice were divided into 4 groups of 6 animals each. The experimental groups received a single intraperitoneal injection of RA (70 mg/kg) on gestational days 7, 8 and 9 (D7, D8 and D9), while control animals (C) received only saline solution. Were obtained: exencephaly (C:0; D7:16.1%; D8:25.4%; D9:0), myelomeningocele (C:0; D7:25.8%, D8:30.9%, D9:0), spina bifida occulta (C:0, D7:29%, D8:41.8%, D90), gastroschisis (C:0, D7:6.4% D8:5.4%, D9:0), omphalocele (C:0, D7:6.4%, D8:14.5%, D9:0), lower limb alterations (C:0, D7:74.1%, D8:80%, D9:0), imperforated anus (C:0, D7:100%, D8:100%, D9:100%), and tail agenesis/alteration (C: D7:100%, D8:100%, D9:100%). The experimental model using Swiss mice proved to be efficient in the induction of the different types of defects, with the eighth gestational day being the one that most favored the induction of neural tube defect, omphalocele, gastroschisis, lower limb defects, imperforated anus and tail agenesis/alteration. On this basis, this is a useful model for future investigation of neural development and of the formation of the appendicular skeleton.

Teratogenic effects of retinoic acid on neurulation in mice embryos

Retinoic acids (RA) are natural chemicals that exert a hormone-like activity and a variety of biological effects on early development of mouse. In this study, the probable teratogenic effects of RA on CNS have been investigated in pregnant mice ( n = 20) divided into four groups: (1) untreated controls, (2) controls which received a single dose of DMSO, (3) a group that received 40 mg/kg, and (4) a group that received 60 mg/kg of all-trans RA in DMSO, respectively on the eighth day of gestation. Embryos whose dams had received 40 and 60 mg/kg doses of RA, showed malformations and decreased size. At 40 mg/kg dosage level, 50% of the embryos had closed neural tubes while at 60 mg/kg dosage level the neural tube failed to close. The neuroblast mantle layers were disorganized in the 40 mg/kg and even more in the 60 mg/kg exposed group compared to the controls. In mitosis, the density of chromatin was increased in the 60 mg/kg dose group. Compared to controls the 40 and 60 mg/kg dose groups of RA treated dams decreases in the luminal longitudinal and internal measures were observed. Also the thickness of ventricular, mantle and marginal layers was smaller. Wide intercellular spaces due to the degenerated cells at high doses of RA as well as an accumulation of intercellular fluid were observed. Therefore, the wedge shape of neuroepithelium was abolished, preventing the elevation of the neural wall.

Teratogenic effects of retinoic acid are modulated in mice lacking expression of epidermal growth factor and transforming growth factor-?

Epidermal growth factor (EGF) and transforming growth factor-alpha (TGFalpha) regulate cell proliferation and differentiation in the embryo. The induction of cleft palate (CP) by all trans-retinoic acid (RA) was associated with altered expression of TGFalpha, EGF receptor, and binding of EGF. This study uses knockout (KO) mice to examine the roles of EGF and TGFalpha in teratogenic responses of embryos exposed to RA. Pregnant wild-type (WT) mice of mixed genetic background, EGF KO, C57BL/6J, and TGFalpha KO mice were given a single oral dose of RA (100 mg/kg, 10 ml/kg) or corn oil on GD 10 at 12 PM, GD 11 at 12 PM or 4 PM, or GD 12 at 8 AM or 12 PM (plug day = GD 0). GD 18 fetuses were examined for external, visceral, and skeletal effects. After exposure to RA on GD 12, the incidence of CP in EGF KO was significantly reduced relative to WT. In TGFalpha KO fetuses, RA exposure on GD 10 increased the incidence of CP versus C57BL/6J. The incidence of skeletal defects in the limbs, vertebrae, sternebrae, and ribs were also affected by lack of expression of EGF or TGFalpha with region-specific amelioration or exacerbation of the effects of RA. In TGFalpha KO fetuses, incidences of forelimb long bone and digit defects increased relative to C57BL/6J. In EGF KO fetuses, relative to WT, the incidence of hindlimb oligodactyly was increased. In EGF KO, but not WT, RA produced short, bent radius, humerus, and ulna. Both TGFalpha and EGF KO mice had increased incidences of dilation of the renal pelvis and this was reduced by RA. RA exposure produced skeletal and visceral defects in all genotypes; however, EGF or TGFalpha KO influenced the incidence and severity of defects. This study supports a role for EGF and TGFalpha in the response to RA.

Ethanol increases retinoic acid production in cerebellar astrocytes and in cerebellum

Several characteristics of fetal alcohol syndrome (FAS) are similar to the teratogenic effects of retinoic acid (RA) exposure. It has been suggested that FAS may result from ethanol-induced alteration in endogenous RA synthesis, leading to abnormal embryonic concentrations of this morphogen. We examined whether ethanol may interfere with RA synthesis in the postnatal cerebellum, as a region of the developing CNS particularly vulnerable to both ethanol and RA teratogenesis. It was found that astrocytes are the predominant source of postnatal RA synthesis in the cerebellum. They express both retinaldehyde dehydrogenase 1 and 2. In vitro cytosolic preparations of astrocytes, as well as live cell preparations, have an increased capacity to synthesize RA in the presence of ethanol. A mechanism by which ethanol could stimulate RA synthesis is via the ethanol-activated short-chain retinol dehydrogenases, which we show to be present in the postnatal cerebellum. To determine whether ethanol stimulated RA synthesis in vivo, a sensitive and highly specific HPLC/MS n technique was used to measure cerebellar RA after administration of ethanol to postnatal day 4 rat pups. Cerebellar RA levels climbed significantly after such treatment. These results suggest that the cerebellar pathology exerted by ethanol may occur, at least in part, through increased production of RA.

Hyperglycaemia potentiates the teratogenicity of retinoic acid in diabetic pregnancy in mice.

We recently showed in mice that maternal diabetes increases embryonic susceptibility to caudal regression induced by vitamin A metabolite retinoic acid. Here we tested whether in the maternal diabetic milieu hyperglycaemia is the critical factor responsible for mediating this increased susceptibility. Non-diabetic pregnant mice were made hyperglycaemic by subcutaneous injections of glucose at regular intervals. Conversely, diabetic pregnant mice were treated with phlorizin to induce renal glucosuria and thus reduce blood glucose concentrations. Pregnant mice were treated with retinoic acid and the extent of caudal regression in mouse embryos, measured in terms of the ratio of tail length to crown-rump length was assessed. Embryos were also examined for Wnt-3a expression and cell death. Embryos of mice treated with glucose had a greater extent of caudal regression induced by retinoic acid than saline-treated controls, with enhanced down-regulation of Wnt-3a expression and exacerbated cell death specifically at the caudal end of the embryo. Embryos of diabetic mice treated with phlorizin had a similar extent of caudal regression to embryos of non-diabetic mice after treatment with retinoic acid. Hyperglycaemia increases embryonic susceptibility to caudal regression induced by retinoic acid, with the underlying cellular and molecular changes closely mimicking those that occur in maternal diabetes. Reduction of blood glucose concentrations in diabetic mice completely abolishes this increased susceptibility to retinoic acid. These results suggest that in maternal diabetes hyperglycaemia is the critical factor responsible for potentiating the teratogenic effect of retinoic acid.

Nutritional components affecting skeletal development in fish larvae

Marine fish larvae undergo major functional and morphological changes during the developmental stages and several factors can interfere with the normal development of larvae and affect fry quality. Skeletal malformations, such as spinal malformation—scoliosis, lordosis, coiled vertebral column-, missing or additional fin rays, bending opercle or jaw malformations, are frequently observed in hatchery-reared larvae. This paper reviews the effects of some nutritional components on skeletal development in larvae of a number of fish species. In the dietary lipid fraction, for instance, it was proven that the phospholipid concentration affected the spinal malformation rate in sea bass fed a compound diet from mouth opening onwards. Phosphatidylinositol, in particular, seems to prevent skeletal deformities. Highly unsaturated fatty acids, and particularly DHA enrichment in live prey, induce a decrease of opercular deformities in milkfish. It is known that highly unsaturated fatty acids have profound effects on gene expression, leading to changes in metabolism, growth and cell differentiation, and these effects are worth investigating in developing fish. The nature of the dietary protein fraction also affects the quality of fish larvae development. It appears that dietary incorporation of 20 amino acid peptides or di- and tripeptides leads to a reduction of spinal malformations in sea bass. Among vitamins, the teratogenic effect of retinoic acid is now well documented in vertebrates. High dietary retinoic acid levels result in higher incidence of bone deformities, such as vertebral curvature, central fusion and compression of vertebra in Japanese flounder larvae. The teratogenic effect of retinoic acid observed in embryonic and postembryonic stages was explained by a depression of shh expression. As for vitamin C, opercular abnormalities in milkfish larvae, associated with distortion of gill filament cartilages, were reduced by 50% when feeding larvae with ascorbic acid enriched rotifers and Artemia, compared to control fish.

Retinoic acid‐induced thymic abnormalities in the mouse are associated with altered pharyngeal morphology, thymocyte maturation defects, and altered expression of Hoxa3 and Pax1

Exogenous retinoic acid is teratogenic in animals and man, causing a spectrum of abnormalities termed retinoic acid embryopathy. Using a mouse model of retinoic acid embryopathy, our results show that exposure to all-trans retinoic acid (RA) on gestational day (gd) 9 results in thymic ectopia, hypoplasia, and thymocyte maturational defects. Immunohistochemical and flow cytometric analyses showed aberrant expression of stromal and thymocyte markers, and abnormalities in thymocyte development. RNA in situ hybridization for the transcription factors Hoxa3 and Pax1 was used to investigate the basis of this defect. Hoxa3 and Pax1 have been shown to be required for normal thymus development, and are normally expressed in the cells of the third pharyngeal pouch and third and fourth pharyngeal arches, involved in thymus organogenesis. RA-exposed embryos showed an increased level of Hoxa3 expression in the neural tube and caudal pharyngeal arches as soon as 6 hr after exposure. The Pax1 expression pattern, in conjunction with analysis of the external pharyngeal morphology, showed that the development and structure of the third pharyngeal pouch and cleft were disrupted, resulting in a reduced third pharyngeal arch and/or fusion of the third and fourth arches. Changes in the expression of cellular retinoic acid binding protein (CRABP) and in the morphology of the cranial ganglia were consistent with altered neural crest cell migration from the caudal hindbrain after RA exposure. Together, our findings suggest that the teratogenic effects of RA on thymus development include changes in both the cranial neural crest and pharyngeal endoderm that contribute to thymus development. Further, the observed defects in thymus development may be mediated by RA-induced alterations in the expression of Hoxa3. Teratology 58:263–275, 1998. © 1998 Wiley-Liss, Inc.

Retinoic acid-induced thymic abnormalities in the mouse are associated with altered pharyngeal morphology, thymocyte maturation defects, and altered expression ofHoxa3 andPax1

Exogenous retinoic acid is teratogenic in animals and man, causing a spectrum of abnormalities termed retinoic acid embryopathy. Using a mouse model of retinoic acid embryopathy, our results show that exposure to all-trans retinoic acid (RA) on gestational day (gd) 9 results in thymic ectopia, hypoplasia, and thymocyte maturational defects. Immunohistochemical and flow cytometric analyses showed aberrant expression of stromal and thymocyte markers, and abnormalities in thymocyte development. RNA in situ hybridization for the transcription factors Hoxa3 and Pax1 was used to investigate the basis of this defect. Hoxa3 and Pax1 have been shown to be required for normal thymus development, and are normally expressed in the cells of the third pharyngeal pouch and third and fourth pharyngeal arches, involved in thymus organogenesis RA-exposed embryos showed an increased level of Hoxa3 expression in the neural tube and caudal pharyngeal arches as soon as 6 hr after exposure. The Pax1 expression pattern, in conjunction with analysis of the external pharyngeal morphology, showed that the development and structure of the third pharyngeal pouch and cleft were disrupted, resulting in a reduced third pharyngeal arch and/or fusion of the third and fourth arches. Changes in the expression of cellular retinoic acid binding protein (CRABP) and in the morphology of the cranial ganglia were consistent with altered neural crest cell migration from the caudal hindbrain after RA exposure. Together, our findings suggest that the teratogenic effects of RA on thymus development include changes in both the cranial neural crest and pharyngeal endoderm that contribute to thymus development. Further, the observed defects in thymus development may be mediated by RA-induced alterations in the expression of Hoxa3.

Correlation between Loss of Middle Ear Bones and AlteredgoosecoidGene Expression in the Branchial Region Following Retinoic Acid Treatment of Mouse Embryosin Vivo

The homeobox genegoosecoidmarks the Spemann organizer in vertebrate gastrula embryos, and is expressed in the craniofacial region, body wall and limbs during organogenesis. Mouse mutants ofgoosecoiddisplayed a variety of phenotypes related to the expression pattern at mid-embryogenesis. These defects included loss of the tympanic ring and malformation of the malleus, phenotypes which were reminiscent of the teratogenic effects of retinoic acid (RA). Here we investigated the correlation ofgoosecoidgene expression and RA-teratogenicity following treatment of mouse embryosin vivoat embryonic day (E) 8 + 5 h. We found thatgoosecoidwas specifically affected at E10.5 in branchial arches I and II. Expression was either reduced to background levels or restricted to the branchial cleft region. This change ingoosecoidgene expression correlated with a loss of middle ear ossicles and a partial or complete deletion of the tympanic ring, suggesting a role forgoosecoidin executing the RA teratogenic effects.

Retinoids and Hox genes

The vertebrate embryonic body plan is constructed through the interaction of many developmentally regulated genes that supply cells with the essential positional and functional information they require to migrate to their appropriate destination and generate the proper structures. Some molecular cues involved in patterning the central nervous system, particularly in the hindbrain, are interpreted by the Hox homeobox genes. Retinoids can affect the expression of Hox genes in cells lines and embryonic tissues; the hindbrain and branchial region of the head are particularly sensitive to the teratogenic effects of retinoic acid. The presence of endogenous retinoic acid, together with the distribution of retinoid binding proteins and nuclear receptors in the developing embryo, strongly suggest that retinoic acid is a natural morphogen in vertebrate development. The molecular basis for the interaction between retinoic acid and the Hox genes has been aided in part by approaches using deletion analysis in transgenic mice carrying lacZ reporter constructs. Such studies have identified functional retinoic acid response elements within flanking sequences of some of the most 3' Hox genes, suggesting a direct interaction between the genes and retinoic acid. Furthermore, as demonstrated using transgenic mice carrying Hoxb-1/lacZ constructs, multiple retinoic acid response elements may cooperate with positive and negative regulatory enhancers to specify pattern formation in the vertebrate embryo. These types of studies strongly support the normal roles of retinoids in patterning vertebrate embryogenesis through the Hox genes.

Specific teratogenic effects of different retinoic acid isomers and analogs in the developing anterior central nervous system of zebrafish.

Vertebrate embryos are sensitive to retinoic acid, either in deficiency or in excess. Although all-trans retinoic acid (RA) and 9-cis RA are known to have distinct but overlapping activities in higher organisms, only the all-trans isomer has been investigated in detail as a teratogen in zebrafish. We have identified profound and specific effects of 9-cis RA when administered to zebrafish embryos, and have confirmed the results of prior studies on the teratogenic effects of exogenous all-trans RA. Moreover, we have identified a 1-hr period during gastrulation in which embryos are particularly sensitive to the teratogenic effects of RA. In the course of these investigations, we have also studied the effects of two synthetic retinoids-a 9-cis RA analog, SR11217, and an all-trans RA analog, TTAB. An application of all-trans RA to the early zebrafish gastrula leads to defects that are limited to the caudal midbrain and rostral hindbrain. Our experiments show that an application of exogenous 9-cis RA for a period as short as 1 hr and at a concentration as low as 0.1 mu M can block differentiation of the rostral CNS. We have observed abnormal phenotypes using DIC optics, and have demonstrated further abnormalities using whole-mount immunocytochemical staining with antibodies to HNK-1 and acetylated alpha-tubulin. Major axon tract formation in the anterior CNS is unambiguously disrupted by the administration of 9-cis RA but not all-trans RA. Furthermore, exogenous 9-cis RA produces a qualitative alteration in the multiple-site expression pattern of the hlx-1 gene within the rostral CNS, while treatment with all-trans RA leads only to a weakened expression signal. The administration of TTAB and SR11217 result in distinctive inhibitions of hlx-1 expression. Unlike all-trans RA, which causes premature par-2 expression in the posterior midbrains of a majority of embryos, 9-cis RA leads to a complete deletion of this domain throughout development. These results suggest that 9-cis RA is a more active teratogen than all-trans RA in rostral CNS structures of the zebrafish embryo.

Mice lacking all isoforms of retinoic acid receptor β develop normally and are susceptible to the teratogenic effects of retinoic acid

Retinoic acids (RA) are vitamin A derivatives essential for normal embryonic development and viability of vertebrates. The RA signal is mediated by two distinct classes of receptors, RA receptors (RARs) and retinoid X receptors (RXRs). The RAR family is composed of three genes: RARα, β, and γ. The expression of RARβ gene is spatially and temporally restricted in certain structures in the developing embryo, suggesting that RARβ could play specific roles during morphogenesis. Four isoforms of the RARβ gene (β1–β4) are generated by differential usage of promoters and alternative splicing. It has recently been demonstrated that the RARβ2 isoform is dispensable for normal development. To ascertain the function of all RARβ isoforms in vivo, we have generated a mutation that disrupts all isoforms of the RARβ gene in the mouse by gene targeting in embryonic stem cells. Mice homozygous for the mutation are viable and fertile with no externally apparent abnormalities. During development, 1 11 RARβ mutant embryos showed fusion of the ninth and tenth cranial ganglia on both sides of the hindbrain. However, no obvious alterations in the spatial pattern of expression of Hoxb-1, Hoxb-4 and Hoxb-5 were observed in day 9.5 p.c. embryos. The RARβ null mutation did not alter the pattern or extent of the limb and craniofacial malformations induced by RA excess, suggesting that RARβ may not be mandatory to mediate the observed teratological effects of RA in these structures. These experiments demonstrate that RARβ isoforms are not absolutely required for embryonic development and provide additional support to the concept of functional redundancy among members of the RAR family.

Teratogenic effects of retinoic acid and related substances on the early development of the zebrafish ( Brachydanio rerio) as assessed by a novel scoring system

The early development of the zebrafish Brachydanio rerio is shown to be suitable as a test system for prescreening drugs suspected to be hazardous to humans. The teratogenic effects of all-trans-retinoic acid (tretinoin) and eight chemically related substances, such as 3,4-didehydro-retinol (vitamin A 2), 4-oxo-retinoic acid and several cis isomers, are analysed. Zebrafish development offers several advantages as a test system: large amounts of eggs can be collected throughout the year. Because the embryo is translucent, organ development can be monitored in vivo. After 1 day, eyes, heart and blood circulation is visible already, and the development is synchronous up to day 3 after fertilization. The effects of the retinoids can be scored easily, and the substances can be ranked and be compared by their effectiveness. Further, a new scoring method is presented which also allows comparison of the relative strength of the teratogenic effects of the substances on the various organs enabling investigation of the structure-activity relationship. Concentrations of 10 −19−10 −6M all-trans-retinoic acid generate malformations such as oedema, brain deformities (anophthalmy, microcephaly and acephaly), duplication of the otic placodes and otoliths, and a shortened and bent tail. The related substances display similar effects but they differ in their effectiveness. With respect to the chemical structure, the order of potency is found to be: acid > aldehyde > alcohol; all-trans ⩾ 9- cis > 13- cis; tretinoin = 3,4-didehydro > 4-oxo. The same order of potency is reported for mammals. Because of this similarity early zebrafish development appears to be a proper model system for predicting teratogenic effects of drugs in mammals, especially of chemicals and their metabolites, which are able to cross the placental barrier and reach the mammalian embryo in utero.

Distribution of cellular retinoic acid‐binding proteins I and II in the chick embryo and their relationship to teratogenesis

The distribution of cellular retinoic acid-binding proteins I and II (CRABP I and II) during the first 6 days of chick development has been investigated using immunoblotting. Since retinoic acid (RA) is teratogenic to some parts of the embryo, stimulatory to other parts, and has no effect on others it may be that the distribution of cytoplasmic proteins such as CRABP I and II plays some role in this differential activity. Neither protein is expressed in the day 2 embryo, but from day 3 onwards both proteins are expressed and CRABP I is in considerable excess over CRABP II. Within the day 4 embryo there is some significant variation in the distribution according to tissue type. Neural tissues, neural crest derivatives, and limb buds most strongly express CRABP I whilst other tissues contain only moderate levels, and heart and epidermis do not express CRABP I at all. CRABP II has a widespread distribution, although at a lower level than CRABP I, with the exception of somites and ectoderm which do not express it at all. In the limb buds, there is a significant variation in CRABP I levels across the anteroposterior axis which suggests that these two CRABPs may have different functions during development. The relationship of these distributions in the embryo to the role of endogenous RA and the teratogenic effects of RA is discussed.