Limb reduction defects in humans associated with prenatal isotretinoin exposure.

Retinoic acid has long been used to induce limb reductions defects in experimental animal studies. No limb malformations, however, have been reported among malformed retinoic acid-exposed human fetuses from case reports or epidemiologic studies. We report a child and a fetus with limb reduction malformations following maternal use of isotretinoin (13-cis-retinoic acid) during the first trimester of pregnancy. The child had a unilaterally absent clavicle and nearly absent scapula, with a short humerus and short, synostotic forearm bones. He also had ventriculomegaly and developmental delay, minor dysmorphic facial features, and a short sternum with a sterno-umbilical raphe. The fetus had a unilaterally absent thumb with normal proximal bony structures. Other findings included hydrocephalus, craniofacial anomalies, thymic agenesis, supracristal ventricular septal defect, single umbilical artery, anal and vaginal atresia, and urethral agenesis with dysplastic, multicystic kidneys. Although the limb malformations were quite dissimilar, a number of anomalies that are frequently found among isotretinoin-exposed fetuses/infants were present in both cases. This increases the probability that retinoic acid caused these limb defects, but a causal association cannot be conclusively drawn on the basis of these two retrospective case reports.     

Studies on the mechanism of retinoid-induced pattern duplications in the early chick limb bud: temporal and spatial aspects

All-trans-retinoic acid causes striking digit pattern changes when it is continuously released from a bead implanted in the anterior margin of an early chick wing bud. In addition to the normal set of digits (234), extra digits form in a mirror-symmetrical arrangement, creating digit patterns such as a 432234. These retinoic acid-induced pattern duplications closely mimic those found after grafts of polarizing region cells to the same positions with regard to dose-response, timing, and positional effects. To elucidate the mechanism by which retinoic acid induces these pattern duplications, we have studied the temporal and spatial distribution of all-trans-retinoic acid and its potent analogue TTNPB in these limb buds. We find that the induction process is biphasic: there is an 8-h lag phase followed by a 6-h duplication phase, during which additional digits are irreversibly specified in the sequence digit 2, digit 3, digit 4. On average, formation of each digit seems to require between 1 and 2 h. The tissue concentrations, metabolic pattern, and spatial distribution of all- trans-retinoic acid and TTNPB in the limb rapidly reach a steady state, in which the continuous release of the retinoid is balanced by loss from metabolism and blood circulation. Pulse-chase experiments reveal that the half-time of clearance from the bud is 20 min for all-trans- retinoic acid and 80 min for TTNPB. Manipulations that change the experimentally induced steep concentration gradient of TTNPB suggest that a graded distribution of retinoid concentrations across the limb is required during the duplication phase to induce changes in the digit pattern. The extensive similarities between results obtained with retinoids and with polarizing region grafts raise the possibility that retinoic acid serves as a natural "morphogen" in the limb.     

Retinoic-acid-induced pattern completion in regenerating double anterior limbs of urodeles

The effects of retinoic acid on the regeneration of double anterior lower arms in the adult newt, Notophthalmus viridescens, were investigated. Normally, double anterior lower arms regenerate a hypomorphic, symmetrical pattern of structures, which are distally complete; and double anterior upper arms regenerate a hypomorphic, symmetrical but distally incomplete pattern of structures. In limbs with a normal anteroposterior axis, the major effect of retinoic acid is to alter the proximodistal (PD) positional value of cells local at the amputation level to a much more proximal value, thereby creating duplications in the regenerate of structures proximal to the amputation plane (Thoms and Stocum, '84). Therefore, we predicted that double anterior lower arms treated with retinoic acid would regenerate like double anterior upper arms. However, in a substantial number of cases, each half of these double anterior lower arms regenerated a limb that was complete in the anteroposterior (AP) axis, with asymmetry corresponding to the half of origin. In addition, these regenerates were serially duplicated in the PD axis. These results indicate that retinoic acid can posteriorize the positional value of midline cells, leading to restoration of normal AP pattern, when the set of posterior-half positional values is removed from the cross section of the limb.     

Teratogenic effects of retinoic acid in pigtail monkeys (Macaca nemestrina). I. General features.

Daily oral administration of 10 mg/kg retinoic acid to pregnant Macaca nemestrina monkeys on days 20 to 44 resulted in a high frequency of craniofacial and musculoskeletal malformations. Craniofacial defects including cleft palate and anomalies of the prinna were common as were ectrodactyly, kyphosis, and muscular-joint contractures. Transposition of the great vessels of the heart occurred in one animal and polycystic kidney and associated urogenital anomalies in another. Shorter treatment periods with similar or higher dosages were not teratogenic and were less fetocidal. Although only relatively long treatment courses were teratogenic, the defects that resulted were morphologically similar to those induced with retinoic acid or other vitamin A compounds in other animal orders.     

The respecification of limb pattern by new synthetic retinoids and their interaction with cellular retinoic acid-binding protein

We describe here experiments to examine the role of cellular retinoic-acid-binding protein (CRABP) during the induction of limb duplication in the chick limb bud and regenerating axolotl limb by retinoids. A newly synthesised class of retinoic acid analogues have been used because among them, some have been specifically designed with the property of binding to the retinoic acid receptors, but not to CRABP. We can thus test whether binding to CRABP is an obligatory step during limb respecification. The binding of four of these compounds to chick limb bud and axolotl CRABP was tested in sucrose density gradient assays and then their potencies at inducing limb duplications tested. Two of the four compounds do not bind to limb CRABP and yet are able to induce limb duplications, suggesting that an interaction with CRABP is not an obligatory step in the process. However, the two compounds which do bind to CRABP are more potent than the two which do not, suggesting that an interaction with CRABP may, nevertheless, increase the potency of a retinoid.     

Explaining certain human limb anomalies and the limb-hematopoiesis community of syndromes using a model of determination

Disturbances of determination, the process of limitation of developmental potential, can cause structural as well as histologic anomalies. A polar coordinate model (PCM) developed from studies in animals, in which determination depends upon positional information arranged along polar coordinates, can be used to explain certain classes of human limb anomalies. Under the model, interference with this system primarily affects distal patterning. If the radial area is distal to the zone of polarizing activity in embryological development, as it appears to be, the PCM explains the teratologic equivalence of preaxial duplications and deficiencies in certain circumstances and the prevalence of ulnar dimelias in forearm duplications. Also, failures of hematopoiesis can be considered late problems with determination and may be markers for abnormalities of a determinative process that also has earlier developmental consequences. Abnormalities of retinoic acid morphogen receptors would be one possible mechanism. This would provide a rationale for the known association of postnatal hematologic problems with developmental anomalies preferentially affecting the radial area. Syndromes with limb and hematopoietic problems may well be a community of determinative disorders.     

Craniosynostosis and multiple skeletal anomalies in humans and zebrafish result from a defect in the localized degradation of retinoic acid

Excess exogenous retinoic acid (RA) has been well documented to have teratogenic effects in the limb and craniofacial skeleton. Malformations that have been observed in this context include craniosynostosis, a common developmental defect of the skull that occurs in 1 in 2500 individuals and results from premature fusion of the cranial sutures. Despite these observations, a physiological role for RA during suture formation has not been demonstrated. Here, we present evidence that genetically based alterations in RA signaling interfere with human development. We have identified human null and hypomorphic mutations in the gene encoding the RA-degrading enzyme CYP26B1 that lead to skeletal and craniofacial anomalies, including fusions of long bones, calvarial bone hypoplasia, and craniosynostosis. Analyses of murine embryos exposed to a chemical inhibitor of Cyp26 enzymes and zebrafish lines with mutations in cyp26b1 suggest that the endochondral bone fusions are due to unrestricted chondrogenesis at the presumptive sites of joint formation within cartilaginous templates, whereas craniosynostosis is induced by a defect in osteoblastic differentiation. Ultrastructural analysis, in situ expression studies, and in vitro quantitative RT-PCR experiments of cellular markers of osseous differentiation indicate that the most likely cause for these phenomena is aberrant osteoblast-osteocyte transitioning. This work reveals a physiological role for RA in partitioning skeletal elements and in the maintenance of cranial suture patency.     

Retinoids reprogramme pre-bud mesenchyme to give changes in limb pattern

Retinoic acid was locally applied to presumptive limb regions of chick embryos to find out the earliest time at which the limb pattern can be reprogrammed. When beads soaked in retinoic acid were placed in the appropriate positions in embryos at stage 10 or older, duplicated or reduced leg patterns resulted. To pin point the time at which the cells in the limb rudiment respond to the retinoid, beads were removed at various times and the lengths of exposure required to reprogramme limb development found. The early limb rudiments require longer exposures to give duplications than late rudiments. The effective treatment periods last at least until stage 17 when the limb bud and apical ectodermal ridge develop. In contrast, the length of exposure to reduce the limb is constant at early stages. Retinoids first start acting to produce duplicated structures between stages 10 and 13. Therefore, retinoids appear to begin to reprogramme the cells as soon as they are determined to give rise to a limb.     

The cellular retinoic-acid-binding protein is expressed in tissues associated with retinoic-acid-induced malformations

Retinoic acid (RA) is thought to play a role in embryonic pattern formation in vertebrates. A naturally occurring gradient of endogenous RA has been demonstrated in the developing chick limb bud, while local application of RA leads to the formation of additional digits. In mammals, a well-defined spectrum of birth defects has been reported as a result of fetal exposure to excess RA. In analogy to the chick limb bud, it may be speculated that these malformations are the result of disturbance of morphogenetic RA concentration gradients. A candidate gene involved in the regulation of endogenous RA concentrations is the gene encoding cellular RA binding protein (CRABP). We have isolated a partial cDNA clone corresponding to the chicken homolog of CRABP, and performed in situ hybridization experiments on sections of embryos at various stages of development. CRABP expression was detected in the CNS, the craniofacial mesenchyme, ganglia of the peripheral nervous system, the limb bud, and the visceral arch area. Our results indicate that the spatiotemporally specified expression pattern displayed by the CRABP gene exhibits a striking correspondence to the tissues that are affected by exposure of avian or mammalian embryos to RA. We hypothesize that CRABP plays an important role in normal embryogenesis and that embryonic tissues showing high CRABP expression are susceptible to the adverse effects of excess RA.     

Distribution of retinoids in the chick limb bud: analysis with monoclonal antibody

Retinoic acid induces anteroposterior duplicate formation in developing chick limb bud, and it may be a natural morphogen involved in limb pattern formation. Retinoic acid is produced from retinol locally in the limb bud via retinal, and thus, to elucidate the distribution of these retinoids in the limb bud seems to be important for the understanding of the morphogen formation. We produced a monoclonal antibody against the retinoids with BSA-RA (bovine serum albumin-retinoic acid) conjugate for antigen, and investigated the distribution of retinoids in the chick limb bud. The antibody predominantly bound to retinoic acid, but weakly to retinol and retinal. Retinoids appeared in the limb bud at stage 18 and were distributed through stages 20-24, when the pattern formation in distal mesoderm was in progress. Initially they were found evenly in the whole mesoderm, but disappeared gradually from core mesoderm and remained only in the region of peripheral mesoderm at stage 24. At stage 26, retinoids were detected only in ectoderm. These results support the idea that the retinoids actually play roles in limb pattern formation and suggest that the retinoids in the peripheral mesoderm are important for pattern formation. Further, the role of retinoids in epidermis development at later limb bud stages is also suggested.     

Retinoic acid inhibits growth in agarose of early chick embryonic cells and may be involved in regulation of axis formation

The mechanisms involved in the generation of axial structures in the chick are well documented, yet, little is known about the actual factors that generate such a complex pattern. The recent demonstrations that all-trans-retinoic acid (RA) acts as a morphogen during limb development (Thaller and Eichele, 1987) lead us to examine whether during axis formation in the developing chick, RA could be one of the factors involved. We now show that retinoic acid can block a very unusual property of normal early chick embryonic cells, mainly their capacity to grow in semisolid medium. We also present experiments that suggest that RA may play a direct role during axis formation in the developing chick.     

Retinoic acid and mammalian craniofacial morphogenesis

Retinoic acid is a morphogenetic signalling molecule in vertebrate embryos, one being known to perform a specific function in organizing the body pattern along the anteroposterior axis. This molecule has especially attracted research attention because retinoic acid treatment will also induce abnormal morphogenesis, particularly in the craniofacial structures. The present review discusses recent molecular insights revealing how the retinoic acid signal is transduced within a cell, specifically focusing on the involvement of cranial neural crest cells in retinoic acid-induced abnormal morphogenesis in the mammalian head     

The role of gap junctions in patterning of the chick limb bud

The role of gap junctional communication during patterning of the chick limb has been investigated. Affinity-purified antibodies raised against rat liver gap junctional proteins were used to block communication between limb mesenchyme cells. Co-injection of the antibodies and Lucifer yellow into mesenchyme cultures demonstrated that communication was inhibited almost immediately. When antibodies were loaded into mesenchyme tissue by DMSO permeabilization, [3H]nucleotide transfer was prevented for at least 16 h. Polarizing region tissue from the posterior limb bud margin causes digit duplications when grafted to the anterior margin. Quail polarizing region cells were loaded with gap junction antibody and grafted into chick wing buds. The antibody had no effect on growth or survival of the grafted cells. As very few polarizing region cells are required to initiate duplications, the number of polarizing region cells in the grafts was reduced by diluting 1:9 with anterior mesenchyme tissue. When either polarizing region or anterior mesenchyme tissue in the graft was loaded separately with antibody, there was little effect on respecification of the digit pattern. However, loading both tissues in the graft caused a significant decrease in duplications. This indicates that a major role of gap junctions in limb patterning may be to enable polarizing region cells to communicate directly with adjacent anterior mesenchyme. A role for gap junctional communication between anterior mesenchyme cells cannot be excluded. The results are discussed in relation to the role of retinoic acid as a putative morphogen.     

The cellular retinoic acid binding proteins

The two cellular retinoic acid binding proteins, CRABP I and CRABP II, belong to a family of small cytosolic lipid binding proteins and are highly conserved during evolution. Both proteins are expressed during embryogenesis, particularly in the developing nervous system, craniofacial region and limb bud. CRABP I is also expressed in several adult tissues, however, in contrast, CRABP II expression appears to be limited to the skin. It is likely that these proteins serve as regulators in the transport and metabolism of retinoic acid in the developing embryo and throughout adult life. It has been proposed that CRABP I sequesters retinoic acid in the cytoplasm and prevents nuclear uptake of retinoic acid. A role in catabolism of retinoic acid has also been proposed. Recent gene targeting experiments have shown that neither of the two CRABPs are essential for normal embryonic development or adult life. Examination of CRABP I expression at subcellular resolution reveals a differential cytoplasmic and/or nuclear localization of the protein. A regulated nuclear uptake of CRABP I implies a role for this protein in the intracellular transport of retinoic acid. A protein mediated mechanism which controls the nuclear uptake of retinoic acid may play an important role in the transactivation of the nuclear retinoic acid receptors.     

Characterization of retinoid metabolism in the developing chick limb bud

Retinoids (vitamin A derivatives) have been shown to have striking effects on developing and regenerating vertebrate limbs. In the developing chick limb, retinoic acid is a candidate morphogen that may coordinate the pattern of cellular differentiation along the anteroposterior limb axis. We describe a series of investigations of the metabolic pathway of retinoids in the chick limb bud system. To study retinoid metabolism in the bud, all-trans-[3H]retinol, all-trans-[3H]retinal and all-trans-[3H]retinoic acid were released into the posterior region of the limb anlage, the area that contains the zone of polarizing activity, a tissue possibly involved in limb pattern formation. We found that the locally applied [3H]retinol is primarily converted to [3H]retinal, [3H]retinoic acid and a yet unidentified metabolite. When [3H]retinal is locally applied, it is either oxidized to [3H]retinoic acid or reduced to [3H]retinol. In contrast, local delivery of retinoic acid to the bud yields neither retinal nor retinol nor the unknown metabolite. This flow of metabolites agrees with the biochemical pathway of retinoids that has previously been elucidated in a number of other animal systems. To find out whether metabolism takes place directly in the treated limb bud, we have compared the amount of [3H]retinoid present in each of the four limb anlagen following local treatment of the right wing bud. The data suggest that retinoid metabolism takes place mostly in the treated limb bud. This local metabolism could provide a simple mechanism to generate in a controlled fashion the biologically active all-trans-retinoic acid from its abundant biosynthetic precursor retinol. In addition, local metabolism supports the hypothesis that retinoids are local chemical mediators involved in pattern formation.     

Selective stimulation of in vitro limb-bud chondrogenesis by retinoic acid

Embryonic exposure to pharmacologic doses of vitamin A analogs (retinoids) is a well-known cause of limb-skeletal deletions, limb truncation and other skeletal malformations. The exclusively inhibitory effect of retinoic acid (RA) on chondrogenesis in standard serum-containing cultures of limb-bud mesenchymal cells is equally well known and has provided a means to explore the cellular basis for RA-mediated skeletal teratogenesis. Recent studies showing that lower RA concentrations can cause skeletal duplication when applied directly to the anterior border of a developing limb, suggest that RA may have a role in normal limb development as a diffusible morphogen capable of regulating skeletal pattern. While RA treatment causes both, skeletal deletions and duplications are clearly different (if not opposing) effects, the latter of which is difficult to reconcile with RA's heretofore exclusively inhibitory effect on in vitro chondrogenesis. In the present study. RA's effects on chondrogenesis and myogenesis were examined in serum-free cultures of chick limb-bud mesenchymal cells and compared with its effects on similar cultures grown in serum-containing medium. When added to serum-free medium, concentrations of RA known to cause skeletal duplication in vivo dramatically enhanced in vitro chondrogenesis (to over 200% of control values) as judged by both Alcian-blue staining and [35S]sulfate incorporation, while having little effect on myogenesis. Higher concentrations inhibited both chondrogenesis and myogenesis. The results indicate that at physiological concentrations. RA can selectively modulate chondrogenic expression and suggest that at higher concentrations, RA's inhibitory effects are less specific.(ABSTRACT TRUNCATED AT 250 WORDS)     

Craniofacial abnormalities induced by ectopic expression of the homeobox gene Hox-1.1 in transgenic mice

Hox-1.1 is a murine homeobox-containing gene expressed in a time- and cell-specific manner during embryogenesis. We have generated transgenic mice that ectopically express Hox-1.1 from the chicken beta-actin promoter. In these mice Hox-1.1 expression was changed to an almost ubiquitous pattern. Ectopic expression of Hox-1.1 leads to death of the transgenic animals shortly after birth and is associated with multiple craniofacial anomalies, such as cleft palate, open eyes at birth, and nonfused pinnae. This phenotype is similar to the effects seen after systemic administration of retinoic acid during gestation. This suggests that retinoic acid embryopathy and the specific developmental defects caused by ectopic expression of a potential developmental control gene share a common pathogenic mechanism.