Endogenous retinoids in the early avian embryo

Vitamin A is required to rescue the vitamin A-deficient quail embryo from early death, but the endogenous presence of bioactive retinoids has not been documented in these embryos. The analysis of 2000 pooled stage 5-8 normal quail embryos described here provides for the first time direct evidence for the presence of endogenous all-trans-retinoic acid (5 nM) and 3,4-didehydroretinoic acid (4 nM), signaling molecules known to be potent ligands for nuclear retinoic acid receptors. The demonstration of all-trans-retinal (80 nM), all-trans-retinol (100 nM), 3,4-didehydroretinol (200 nM), and retinyl esters (100 nM) suggests the capability of the early avian embryo to generate in situ the vitamin A-bioactive molecules required for development. Analysis of 2100 pooled stage 5-8 quail embryos from vitamin A-deficient eggs revealed no vitamin A-like molecules, supporting the evidence that links vitamin A deficiency in these embryos to abnormalities and early death.     

The retinoid ligand 4-oxo-retinoic acid is a highly active modulator of positional specification

Retinoids (vitamin A and its metabolites) are suspected of regulating diverse aspects of growth, differentiation, and patterning during embryogenesis, but many questions remain about the identities and functions of the endogenous active retinoids involved. The pleiotropic effects of retinoids may be explained by the existence of complex signal transduction pathways involving diverse nuclear receptors of the retinoic acid receptor (RAR) and retinoid X receptor (RXR) families, and at least two types of cellular retinoic acid binding proteins (CRABP-I and -II). The different RARs, RXRs, and CRABPs have different expression patterns during vertebrate embryogenesis, suggesting that they each have particular functions. Another level at which fine tuning of retinoid action could occur is the metabolism of vitamin A to active metabolites, which may include all-trans-retinoic acid, all-trans-3,4-didehydroretinoic acid, 9-cis-retinoic acid, and 14-hydroxy-4,14-retroretinol. Formation of the metabolite all-trans-4-oxo-retinoic acid from retinoic acid was considered to be an inactivation pathway during growth and differentiation. We report here that, in contrast, 4-oxo-retinoic acid is a highly active metabolite which can modulate positional specification in early embryos. We also show that this retinoid binds avidly to and activates RAR beta, and that it is available in early embryos. The different activities of 4-oxo-retinoic acid and retinoic acid in modulating positional specification on the one hand, and growth and differentiation on the other, interest us in the possibility that specific retinoid ligands regulate different physiological processes in vivo.     

Expression of regeneration-associated cytoskeletal proteins reveals differences and similarities between regenerating organs

The unique events which allow regeneration of an entire organ to occur are formation of a specialized wound epidermis and accumulation of progenitor cells (blastemal cells) at the amputated surface to form a blastema. In order to identify some of the molecular events underlying the early stages of the regenerative process which are either common to different systems or specific to one of them, we have investigated whether molecules which are induced in limb blastemas are also expressed in skin repair and during regeneration of other complex body structures (lower jaws, upper jaws, and tails). In addition, we have addressed the issue of the identity of progenitor cells during jaw development and regeneration by analyzing the expression of limb blastemal markers in the developing head and face. We have focused on cytoskeletal components, and particularly on the epidermal keratin NvKII, the simple epithelial keratins 8 and 18 and 22/18, because they are among the few molecules which have been shown to be associated with regeneration in the limb and may play significant roles in various developmental processes. Some important findings emerge from this study: 1) Expression of the epidermal keratin NvKII, unlike that of its mammalian homologue K6, is not simply induced in response to wounding, but is associated with regeneration of specific organs. In fact, NvKII is expressed in regenerating limbs and tails, but not in upper or in lower jaw regenerates, demonstrating the existence of molecular differences in the composition of the wound epidermis in these systems. This, together with the fact that NvKII mRNA is regulated by retinoic acid, which differentially affects patterning of limbs and jaws, argues for distinct inductive abilities of the wound epidermis in different organs. 2) In contrast to the differential expression of the epidermal keratin NvKII, the regeneration-associated cytoskeletal molecules identified in limb blastemal cells are expressed in a similar fashion in jaw and tail blastemas. Therefore, it appears that similar cellular events lead to the establishment of an actively proliferating population of progenitor cells from the stump of different organs. Finally, the mesenchyme of the facial rudiments, unlike that of developing limb buds, expresses simple epithelial keratins. Thus, it appears that mesenchymal progenitor cells of developing and regenerating jaws are alike in regard to their intermediate filament content, and this may be related to nerve-dependent growth control of progenitor cells in different developing and regenerating systems.     

Pattern of retinoid-induced teratogenic effects: possible relationship with relative selectivity for nuclear retinoid receptors RAR alpha, RAR beta, and RAR gamma

Retinoic acid, an oxidative metabolite of vitamin A, is involved in the control of many biological processes including embryonic development. Excess as well as deficiency of retinoids were found to be teratogenic. The effects of retinoids in normal as well as abnormal development may be mediated by two members of retinoid receptors, the RAR's and RXR's, which exhibit a specific temporal and spatial expression during development. The significance of the retinoid receptors was investigated here by studying the teratogenic effects of retinoid ligands with relative selectivity for binding and transactivation of the retinoic acid receptors RAR alpha, RAR beta and RAR gamma. Pregnant NMRI mice were administered 5 or 15 mg/kg of CD 336 (Am 580) (alpha-ligand), CD 2019 (beta-ligand), CD 437 (gamma-ligand) or 37.5 mg/kg all-trans-retinoic acid in 25% Cremophor EL on day 8.25 or day 11 of gestation by gastric intubation. External, visceral and skeletal malformations were observed on day 18 of gestation. The order of teratogenic potency was: alpha-ligand > beta-ligand > gamma-ligand. In addition, these retinoids also produced a different spectrum of defects. The alpha-ligand induced the most varied defects including severe ear, mandible, and limb malformations. The beta-ligand induced defects of the urinary system and liver in greater frequency than expected from its relative potency. The gamma-ligand preferentially induced ossification deficiencies and defects of the sternebrae and vertebral body. Our results show that these three retinoids, which were previously demonstrated to exhibit retinoid-like activities in several systems, exert differing teratogenic activities, in regard to both potency and regioselectivity: we hypothesize that the relative selectivity for binding and transactivation of the three retinoic acid receptors could possibly be related to the differences of teratogenic effects observed in this study. The low potency of the gamma-ligand may lead the way to interesting new retinoids with improved therapeutic ratio.     

A comparative two-dimensional gel protein database of the intact and regenerating newt limbs

In this paper we describe a two-dimensional gel database of the regenerating newt limb. Protein synthesis was compared in the intact limb, in the 1-week regenerating limb, representing the dedifferentiation stage, and in the 2-week regenerating limb, representing the formation of the blastema. This comparative database provided data on differential expression of about 800 proteins during the process of limb regeneration. In addition, a map has been generated for these proteins for future guidance in characterizing further new, unknown proteins. The overall expression patterns of the proteins indicated that the dedifferentiation stage was marked by down-regulation of most proteins, while the blastema formation was marked by the appearance of many new proteins. The potential use of such a database in isolating factors involved during limb regeneration is discussed.     

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.     

Retinoid-regulated gene expression in neural development

The discovery and development of information surrounding the retinoic acid receptors (RAR and RXR) has ushered in a new era in understanding the molecular mechanism of action of vitamin A in embryonic development and cellular differentiation. The mechanisms involved in the regulation of gene expression by the retinoids is at least partially known and involves binding of the RAR and RXR to retinoic acid response elements. Additional factors, including coregulatory proteins, associated regulatory elements, and cell-specific factors, may also be involved in determining the specificity of retinoid-regulation of gene expression during development. During embryogenesis, retinoids are required for the development of the posterior hindbrain and its associated structures, as well as for the survival and differentiation of certain classes of neurons and neural crest cell derivatives. At least some of the effects of retinoid on hindbrain development are related to the regulation of Hox gene expression. Additional retinoid-regulated genes have been implicated in nervous system development, and the manner in which they lead to phenotypic changes during embryogenesis remains to be determined.     

Arg278, but not Lys229 or Lys236, plays an important role in the binding of retinoic acid by retinoic acid receptor gamma

The diverse biological actions of retinoic acid (RA) are mediated by retinoic acid receptors (RARalpha, beta and gamma) and retinoid X receptors (RXR alpha, beta, and gamma). Although the ligand-binding domains of RARs share the same novel folding pattern, many RAR subtype-specific retinoids have been synthesized indicating that the ligand-binding pocket of each RAR subtype has unique features. Previously we have demonstrated the importance for RA binding and RA-dependent transactivation of Arg276 of RARalpha alone and in RARbeta Arg269 in conjunction with Lys220. In this study, we have examined the role of the homologous amino acid residues (Lys229 and Arg278) in RARgamma for these activities. Like RARalpha but dissimilar to RARbeta, Arg278 in RARgamma alone was found to play an important role in RA binding and RA-dependent transactivation. Since Lys236 in RARgamma was suggested from the crystal structure of holo-RARgamma to interact with RA, we also examined its role and that of its homologs in RARalpha and RARbeta. Despite the suggestion from the crystal structure, neither Lys236 nor its homologs in RARalpha and RARbeta play a role in the binding of RA or RA-dependent transactivation. It is likely that Lys236 in RARgamma and its homologs in RARalpha and RARbeta are solvent exposed rather than pointing into the RA-binding pocket.     

Epidemiology of violence and theft in the workplace

Using sequence information derived from the Drosophila melanogaster (Dm) ecdysteroid receptor (EcR)- and retinoid X receptor (RXR)-encoding gene homologs, we have isolated cDNA clones corresponding to the DNA-binding domains (DBD) for these two nuclear receptors from the fiddler crab, Uca pugilator (Up). Both genes appear to be represented in 1-2 copies in the Up genome, and unlike Dm, contain an intron within the DBD-encoding region. Sequence comparisons to the Dm EcR and RXR homologs indicate 76 and 82% nucleotide identity, respectively, corresponding to 6 and 4 single-amino acid substitutions which primarily cluster in the region of the molecule involved in dimerization. RT-PCR analysis indicates that both the EcR and RXR homologs are expressed during the initial stages of limb regeneration, temporally concomitant with early blastema formation and the secretion of a flexible sac cuticle at the site of limb loss.     

Differential effects of synthetic nuclear retinoid receptor-selective retinoids on the growth of human non-small cell lung carcinoma cells

Retinoids are promising agents for cancer chemoprevention and therapy. Nuclear retinoic acid receptors (RARs; RARalpha, -beta, and -gamma) and retinoid X receptors (RXRs; RXRalpha, -beta, and -gamma) are thought to mediate most of retinoids' effects on cell growth and differentiation. Because the majority of human non-small cell lung carcinoma (NSCLC) cell lines are resistant to all-trans-retinoic acid, we searched for more potent retinoids. Therefore, we examined the effects of 37 natural and synthetic retinoids that exhibit specific binding to and transactivation of individual RARs or RXRs on the proliferation of eight human NSCLC cell lines. All of these cells expressed mRNAs of the three RXRs; however, they expressed varying levels of RARalpha and RARgamma, and only three of the eight cell lines expressed RARbeta mRNA. Cellular retinoic acid-binding proteins (CRABPs) I and II were detected in one and three of the eight cell lines, respectively. Only 8 of the 37 retinoids exhibited growth-inhibitory activity (IC50, < 10 microM) against at least two of the eight NSCLC cell lines. The active retinoids included one (TD550) of five RARalpha-selective, one (Ch55) of three RARbeta-selective, three (CD437, CD2325, and SR11364) of six RARgamma-selective, and one (CD271) of four RARbeta/gamma-selective retinoids. The potency of these retinoids was low (IC50, > 1 microM), except for CD437, which was very potent (IC50, 0.1-0.5 microM). The six RXR-selective retinoids were mostly inactive even at 10 microM. However, combinations of RAR-selective and RXR-selective retinoids exhibited additive effects. There appeared to be no simple correlation among the histological type of the NSCLC (adeno- or squamous), the levels of nuclear receptors or CRABPs, and the response of the cells to the growth-inhibitory effects of retinoids. Nevertheless, in contrast with former studies with natural retinoids, these results suggest that several synthetic retinoids do exhibit inhibitory activity against NSCLC cells, and some of them may be useful clinically.     

Susceptibility to in vitro lipid peroxidation of low density lipoproteins and erythrocyte membranes from liver cirrhotic patients

All retinoic acid receptors (RAR alpha, beta, gamma) have two isoforms, whose function is unknown. We now show that at least for RAR gamma, the isoforms are differentially distributed in the embryo. RAR gamma 1 and RAR gamma 2 are detected in the head region, whereas RAR gamma 2 is the sole isoform expressed in the tail region. Specifically, it is expressed in the chordoneural hinge, a region of the tailbud that has organizing properties. Treatment with high doses of retinoic acid (RA) reduces expression in this region. The results are discussed in terms of the known teratogenic effects of RA in the tail region.     

Retinoic acid signaling is required during early chick limb development

In the chick limb bud, the zone of polarizing activity controls limb patterning along the anteroposterior and proximodistal axes. Since retinoic acid can induce ectopic polarizing activity, we examined whether this molecule plays a role in the establishment of the endogenous zone of polarizing activity. Grafts of wing bud mesenchyme treated with physiologic doses of retinoic acid had weak polarizing activity but inclusion of a retinoic acid-exposed apical ectodermal ridge or of prospective wing bud ectoderm evoked strong polarizing activity. Likewise, polarizing activity of prospective wing mesenchyme was markedly enhanced by co-grafting either a retinoic acid-exposed apical ectodermal ridge or ectoderm from the wing region. This equivalence of ectoderm-mesenchyme interactions required for the establishment of polarizing activity in retinoic acid-treated wing buds and in prospective wing tissue, suggests a role of retinoic acid in the establishment of the zone of polarizing activity. We found that prospective wing bud tissue is a high-point of retinoic acid synthesis. Furthermore, retinoid receptor-specific antagonists blocked limb morphogenesis and down-regulated a polarizing signal, sonic hedgehog. Limb agenesis was reversed when antagonist-exposed wing buds were treated with retinoic acid. Our results demonstrate a role of retinoic acid in the establishment of the endogenous zone of polarizing activity.