Excess Retinoic Acid Research Articles

Retinoic Acid Signaling Regulates Sonic Hedgehog and Bone Morphogenetic Protein Signalings During Genital Tubercle Development

Retinoic acid (RA) plays pivotal roles in organogenesis, and both excessive and reduced amounts of RA cause developmental abnormalities. Reproductive organs are susceptible to teratogen toxigenicity, and the genital tubercle (GT) is one such representative organ. The physiological function of endogenous RA signaling and the mechanisms of RA-induced teratogenicity are poorly understood during the GT development. The objective of this study is to understand the developmental and teratogenic roles of RA during GT development by analyzing genetically modified mouse models. We found dynamic patterns of gene expression for the RA-synthesizing enzyme, Raldh2, and for the RA-catabolizing enzyme, Cyp26b1, during GT development. Rarb, an indicator gene for RA signaling, starts its expression in the prospective corpus cavernosum penis and in the urethral plate epithelium (UE), which plays central roles during GT development. Excessive RA signaling in Cyp26b1(-/-) mutants leads to abnormal extents of cell proliferation and differentiation during GT development, and also upregulates expression of growth factor signalings. They include Sonic hedgehog (Shh) signaling and Bone morphogenetic protein (Bmp) signaling, which are expressed in the UE and its bilateral mesenchyme. RA signaling positively regulatesShh and Bmp4 expression during GT development as testified also by the experiment of RA administration and analyses of loss-of-function of RA signaling mutants. Thus, RA signaling is involved in the developmental cascade necessary for UE formation and GT development.

Tail regression induced by elevated retinoic acid signaling in amphioxus larvae occurs by tissue remodeling, not cell death

The vitamin A derived morphogen retinoic acid (RA) is known to function in the regulation of tissue proliferation and differentiation. Here, we show that exogenous RA applied to late larvae of the invertebrate chordate amphioxus can reverse some differentiated states. Although treatment with the RA antagonist BMS009 has no obvious effect on late larvae of amphioxus, administration of excess RA alters the morphology of the posterior end of the body. The anus closes over, and gut contents accumulate in the hindgut. In addition, the larval tail fin regresses, although little apoptosis takes place. This fin normally consists of columnar epidermal cells, each characterized by a ciliary rootlet running all the way from an apical centriole to the base of the cell and likely contributing substantial cytoskeletal support. After a few days of RA treatment, the rootlet becomes disrupted, and the cell shape changes from columnar to cuboidal. Transmission electron microscopy (TEM) shows fragments of the rootlet in the basal cytoplasm of the cuboidal cell. A major component of the ciliary rootlet in amphioxus is the protein Rootletin, which is encoded by a single AmphiRootletin gene. This gene is highly expressed in the tail epithelial cells of control larvae, but becomes downregulated after about a day of RA treatment, and the breakup of the ciliary rootlet soon follows. The effect of excess RA on these epidermal cells of the larval tail in amphioxus is unlike posterior regression in developing zebrafish, where elevated RA signaling alters connective tissues of mesodermal origin. In contrast, however, the RA-induced closure of the amphioxus anus has parallels in the RA-induced caudal regression syndrome of mammals.

Uncoupling of retinoic acid signaling from tailbud development before termination of body axis extension

During the early stages of body axis extension, retinoic acid (RA) synthesized in somites by Raldh2 represses caudal fibroblast growth factor (FGF) signaling to limit the tailbud progenitor zone. Excessive RA down-regulates Fgf8 and triggers premature termination of body axis extension, suggesting that endogenous RA may function in normal termination of body axis extension. Here, we demonstrate that Raldh2-/- mouse embryos undergo normal down-regulation of tailbud Fgf8 expression and termination of body axis extension in the absence of RA. Interestingly, Raldh2 expression in wild-type tail somites and tailbud from E10.5 onwards does not result in RA activity monitored by retinoic acid response element (RARE)-lacZ. Treatment of wild-type tailbuds with physiological levels of RA or retinaldehyde induces RARE-lacZ activity, validating the sensitivity of RARE-lacZ and demonstrating that deficient RA synthesis in wild-type tail somites and tailbud is due to a lack of retinaldehyde synthesis. These studies demonstrate an early uncoupling of RA signaling from mouse tailbud development and show that termination of body axis extension occurs in the absence of RA signaling.

Signals controlling olfactory neurogenesis

To fulfil a chemosensory role, olfactory neurons extend dendrites externally. This makes them prone to damage; however, function is maintained thoughout life by a series of progenitors present in the olfactory epithelium. Key amongst these is the horizontal basal cell, a quiescent cell that upon stimulation proliferates slowly to give rise to transit amplifying olfactory precursors. In the embryo, the horizontal basal cell is the founding progenitor cell of the olfactory epithelium. Thus the mechanism of olfactory founder cell formation and how quiescence is regulated are not known. We identify a role for retinoic acid, a metabolite of vitamin A, in the control of olfactory neurogenesis. Normally retinoic acid is provided from the neural crest and the olfactory epithelium itself. However, excess retinoic acid promotes the formation of HBCs, and prevents progression into committed olfactory precursors. In contrast, RA depletion in chicks or mice results in dysregulation of quiescence and precocious neurogenesis. Furthermore, due to progenitor depletion over time, neurogenesis is not sustained.

Retinoid signaling in inner ear development: A “Goldilocks” phenomenon

Retinoic acid (RA) is a biologically active derivative of vitamin A that is indispensable for inner ear development. The normal function of RA is achieved only at optimal homeostatic concentrations, with an excess or deficiency in RA leading to inner ear dysmorphogenesis. We present an overview of the role of RA in the developing mammalian inner ear, discussing both how and when RA may act to critically control a program of inner ear development. Molecular mechanisms of otic teratogenicity involving two members of the fibroblast growth factor family, FGF3 and FGF10, and their downstream targets, Dlx5 and Dlx6, are examined under conditions of both RA excess and deficiency. We term the effect of too little or too much RA on FGF/Dlx signaling a Goldilocks phenomenon. We demonstrate that in each case (RA excess, RA deficiency), RA can directly affect FGF3/FGF10 signaling within the otic epithelium, leading to downregulated expression of these essential signaling molecules, which in turn, leads to diminution in Dlx5/Dlx6 expression. Non-cell autonomous affects of the otic epithelium subsequently occur, altering transforming growth factor-beta (TGFβ) expression in the neighboring periotic mesenchyme and serving as a putative explanation for RA-mediated otic capsule defects. We conclude that RA coordinates inner ear morphogenesis by controlling an FGF/Dlx signaling cascade, whose perturbation by deviations in local retinoid concentrations can lead to inner ear dysmorphogenesis.

A Role for All-Trans-Retinoic Acid in the Early Steps of Lymphatic Vasculature Development

The molecular mechanisms that regulate the earliest steps of lymphatic vascular system development are unknown. To identify regulators of lymphatic competence and commitment, we used an in vitro vascular assay with mouse embryonic stem cell-derived embryoid bodies (EBs). We found that incubation with retinoic acid (RA) and, more potently, with RA in combination with cAMP, induced the expression of the lymphatic competence marker LYVE-1 in the vascular structures of the EBs. This effect was dependent on RA receptor (RAR)-α and protein kinase A signaling. RA-cAMP incubation also promoted the development of CD31+/LYVE-1+/Prox1+ cell clusters. In situ studies revealed that RAR-α is expressed by endothelial cells of the cardinal vein in ED 9.5–11.5 mouse embryos. Timed exposure of mouse and Xenopus embryos to excess of RA upregulated LYVE-1 and VEGFR-3 on embryonic veins and increased formation of Prox1-positive lymphatic progenitors. These findings indicate that RA signaling mediates the earliest steps of lymphatic vasculature development.

The role of CYP26 enzymes in defining appropriate retinoic acid exposure during embryogenesis

Retinoic acid (RA) is a pleiotropic derivative of vitamin A, or retinol, which is responsible for all of the bioactivity associated with this vitamin. The teratogenic influences of vitamin A deficiency and excess RA in rodents were first observed more than 50 years ago. Efforts over the last 15-20 years have refined these observations by defining the molecular mechanisms that control RA availability and signaling during murine embryonic development. This review will discuss our current understanding of the role of RA in teratogenesis, with specific emphasis on the essential function of the RA catabolic CYP26 enzymes in preventing teratogenic consequences caused by uncontrolled distribution of RA. Particular focus will be paid to the RA-sensitive tissues of the caudal and cranial regions, the limb, and the testis, and how genetic mutation of factors controlling RA distribution have revealed important roles for RA during embryogenesis.

Disruption of Retinoic Acid Receptor Signaling by Environmental Pollutants

Retinoic acid (RA) receptors (RARs) are nuclear receptors that play a critical role in regulating cellular proliferation, development and differentiation in vertebrates in response to endogenous RAs, i.e., all-trans RA and 9-cis RA. On the other hand, it has been well-known that both a deficiency and an excess of RA and related retinoids can cause a variety of teratogenic effects on developing embryos of vertebrates, which has been proven to be an RAR-mediated process. Therefore, the occurrence of xenobiotic environmental pollutants that interfere with RARs and disrupt the RAR signaling (i.e., show RAR agonistic or antagonistic effects) may pose a threat to the health of wild animals and humans. This review mainly focuses on RAR agonists. We summarize the RAR agonistic activity of natural and xenobiotic compounds determined using in vitro bioassay systems and present recent field research showing the occurrence of RAR agonist contamination in the aquatic environment in North America, China and Japan. Environmental pollution by RAR agonists is a new endocrine disruption issue discovered very recently, and relevant knowledge is very limited. Further research will be required to obtain accurate information to assess the possible risks of RAR agonists in the environment.

Retinoic acid is both necessary for and inhibits myogenic commitment and differentiation in the chick limb

Retinoic acid (RA) plays an essential role in the development of many embryonic tissues, including the developing tetrapod limb bud. At early stages of limb development, RA levels are highest proximally and regulate the migration of myoblasts into the limb. As the premyogenic progenitor cells migrate into the limb and accumulate in premuscle masses, they express Pax3 and Meox2. Myogenic differentiation is initiated by expression of Myf5 and MyoD, and both Pax3 and Meox2 are required for normal Myf5 expression. We show by loss of function using the inhibitor citral, that RA signalling within the limb bud is required to maintain Pax3 and Meox2 in the progenitor and Myf5 and MyoD in the differentiating myoblasts. Treatment with excess RA showed a differential effect: Meox2 and Pax3 showed localised down-regulation of expression in the limb. In contrast, there was a dramatic down-regulation of expression of MyoD, Myf5 and Meox1. The down-regulation of myogenic gene expression in response to inhibition of RA signalling, and differential response to application of excess RA, in the absence of changes to cell proliferation and apoptosis, indicate that myogenic specification and differentiation in the developing limb possess a complex sensitivity to RA concentrations.

Retinoic acid signaling targets Hox genes during the amphioxus gastrula stage: Insights into early anterior–posterior patterning of the chordate body plan

Previous studies of vertebrate development have shown that retinoic acid (RA) signaling at the gastrula stage strongly influences anterior-posterior (A-P) patterning of the neurula and later stages. However, much less is known about the more immediate effects of RA signaling on gene transcription and developmental patterning at the gastrula stage. To investigate the targets of RA signaling during the gastrula stage, we used the basal chordate amphioxus, in which gastrulation involves very minimal tissue movements. First, we determined the effect of altered RA signaling on expression of 42 genes (encoding transcription factors and components of major signaling cascades) known to be expressed in restricted domains along the A-P axis during the gastrula and early neurula stage. Of these 42 genes, the expression domains during gastrulation of only four (Hox1, Hox3, HNF3-1 and Wnt3) were spatially altered by exposure of the embryos to excess RA or to the RA antagonist BMS009. Moreover, blocking protein synthesis with puromycin before adding RA or BMS009 showed that only three of these genes (Hox1, Hox3 and HNF3-1) are direct RA targets at the gastrula stage. From these results we conclude that in the amphioxus gastrula RA signaling primarily acts via regulation of Hox transcription to establish positional identities along the A-P axis and that Hox1, Hox3, HNF3-1 and Wnt3 constitute a basal module of RA action during chordate gastrulation.

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Retinoic acid homeostasis: retinoic acid regulates liver retinol esterification as well as its own catabolic oxidation in liver.

Retinol is stored in liver in the form of its esters. The retinol-esterifying enzyme lecithin:retinol acyltransferase (LRAT) catalyzes the conversion of retinol into its storage form. Expression of the LRAT mRNA is induced by retinoic acid (RA), or by dietary vitamin A, and is downregulated upon vitamin A depletion. RA also induces the expression in liver of the mRNA for CYP26, the enzyme that disposes of excess RA by oxidizing RA to 4-oxo-RA. CYP26 is downregulated upon vitamin A depletion.

Increased Hox activity mimics the teratogenic effects of excess retinoic acid signaling

Excess retinoic acid (RA) signaling can be teratogenic and result in cardiac birth defects, but the cellular and molecular origins of these defects are not well understood. Excessive RA signaling can completely eliminate heart formation in the zebrafish embryo. However, atrial and ventricular cells are differentially sensitive to more modest increases in RA signaling. Increased Hox activity, downstream of RA signaling, causes phenotypes similar to those resulting from excess RA. These results suggest that Hox activity mediates the differential effects of ectopic RA on atrial and ventricular cardiomyocytes and may underlie the teratogenic effects of RA on the heart.

The involvement of cytochrome p450 (CYP) 26 in the retinoic acid metabolism of human epidermal keratinocytes

All-trans retinoic acid (RA) levels are controlled by enzymes of the vitamin A metabolism (RDH16, RalDH2, and LRAT) and RA catabolism (CYP26 and CYP2S1). Here, the mRNA expression of these enzymes was investigated in human keratinocytes at different Ca 2+concentrations and after exposure to RA and CYP26 inhibitors. Cellular differentiation (high Ca 2+) increased the expression of LRAT, RDH16 and RalDH2, and decreased CYP26B1. RA (1 μM) induced CYP26A1, CYP26B1, CYP2S1, CRABPII and LRAT mRNA. The CYP26 inhibitor talarozole altered CYP26A1 and LRAT mRNA expression in a similar way as RA, increased the cellular accumulation of [ 3H]RA, and induced a punctate CRABPII staining, also observed after siRNA knock-down of CYP26B1 (but not after RA exposure). Furthermore, CYP26B1 siRNA increased the accumulation of [ 3H]RA and the CRABPII mRNA, suggesting an augmented retinoid signalling. Thus CYP26B1 appears essential for RA catabolism under physiological conditions, whereas CYP26A1 might play a greater role during RA excess.

Cloning of endothelin-1 (ET-1) from European sea bass ( Dicentrarchus labrax) and its gene expression analysis in larvae with retinoic acid-induced malformations

It is known in vertebrates that endothelin-1 (ET-1) plays a key role in morphogenesis whose expression level greatly influences the development of craniofacial malformations. In the present study, the complete cDNA fragment encoding a precursor of endothelin-1, the preproendothelin-1 (PPET1), was cloned by RACE-PCR from European sea bass . The cDNA encoded a 199 amino acid polypeptide that was composed of the “mature” and “big” ET-1. Relative ET-1 expression levels were investigated in European sea bass larvae fed microparticulate diets containing the standard amount (0.08 g retinol/kg diet: group N) or an excess of retinoic acid which induces skeletal malformations (0.5 g retinol/kg diet: group RA). Real-time reverse transcription polymerase chain reaction analysis revealed that PPET1 mRNA levels were sensitively reduced in the RA group during early development (days 10 and 15 post hatching). Regulation of ET-1 gene expression in larvae fed the teratogenic level of vitamin A confirmed the involvement of ET-1 in the molecular mechanism involved in craniofacial deformities. These results suggest that the expression level of ET-1 may be used as a precocious molecular marker to predict malformations during European sea bass development.

Retinoid signaling can repress blastula Wnt signaling and impair dorsal development in Xenopus embryo

Vitamin A derivatives (retinoids) are actively involved during vertebrate embryogenesis. However, exogenous retinoids have also long been known as potent teratogens. The defects caused by retinoid treatment are complex. Here, we provided evidence that RAR-mediated retinoid signaling can repress Xenopus blastula Wnt signaling and impair dorsal development. Exogenous retinoic acid (RA) could antagonize the dorsalizing effects of lithium chloride-mediated Wnt activation in blastula embryos. The Wnt-responsive reporter gene transgenesis and luciferase assay showed that excess RA can repress the Wnt signaling in blastula embryos. In addition, the downstream target genes of the Wnt signaling that direct embryonic dorsal development, were also down-regulated in the RA-treated embryos. Mechanically, RA did not interfere with the stability of β-catenin, but promoted its nuclear accumulation. The inverse agonist of retinoic acid receptors (RAR) rescued the Wnt signaling repression by RA and relieved the RA-induced nuclear accumulation of β-catenin. Our results explain one of the reasons for the complicated teratogenic effects of retinoids and shed light on the endogenous way of interactions between two developmentally important signaling pathways.

Retinoic acid down‐regulates Tbx1 expression and induces abnormal differentiation of tongue muscles in fetal mice

Excess retinoic acid (RA) during pregnancy can cause various developmental anomalies in both humans and rodents. We investigated the mechanisms underlying the aberrant differentiation of tongue muscles in fetal mice exposed to exogenous RA in utero. RA-degrading enzymes (Cyp26a1 and Cyp26b1) were expressed at early stages of normal tongue development, but exogenous RA perturbed their expression in the fetal tongue. RA is normally distributed in the developing tongue muscles but its localization was disrupted by exogenous RA. After RA treatment, myogenic determination factors were reduced and the differentiation was significantly suppressed in tongue muscles. Tbx1, a candidate gene of DiGeorge syndrome, was down-regulated in the fetal tongue in response to excess RA. Moreover, Tbx1 as well as myogenic determination factors were not observed in tongue muscle primordia of Cyp26b1-/- fetuses. Our study suggests that RA signaling may play an essential role in tongue muscle differentiation via the regulation of Tbx1.

Retinoic acid‐induced inner ear teratogenesis caused by defective Fgf3/Fgf10‐dependent Dlx5 signaling

Retinoic acid (RA) is essential for inner ear development. However, exposure to excess RA at a critical period leads to inner ear defects. These defects are associated with disruption in epithelial-mesenchymal interactions. This study investigates the role of Dlx5 in the epithelial-mesenchymal interactions that guide otic capsule chondrogenesis, as well as the effect of excess in utero RA exposure on Dlx5 expression in the developing mouse inner ear. Control of Dlx5 by Fgf3 and Fgf10 under excess RA conditions is investigated by examining the developmental window during which Fgf3 and Fgf10 are altered by in utero RA exposure and by testing the ability of Fgf3 and Fgf10 to mitigate the reduction in chondrogenesis and Dlx5 expression mediated by RA in high-density cultures of periotic mesenchyme containing otic epithelium, a model of epithelial-mesenchymal interactions in which chondrogenic differentiation of periotic mesenchyme ensues in response to induction by otic epithelium. Dlx5 deletion alters expression of TGFbeta(1), important for otic capsule chondrogenesis, in the developing inner ear and compromises the ability of cultured periotic mesenchyme containing otic epithelium, harvested from Dlx5 null embryos, to differentiate into cartilage when compared with control cultures. Downregulation in Dlx5 ensues as a consequence of in utero RA exposure in association with inner ear dysmorphogenesis. This change in Dlx5 is noted at embryonic day 10.5 (E10.5), but not at E9.5, suggesting that Dlx5 is not a direct RA target. Before Dlx5 downregulation, Fgf3 and Fgf10 expression is modified in the inner ear by excess RA, with the ability of exogenous Fgf3 and Fgf10 to rescue chondrogenesis and Dlx5 expression in RA-treated cultures of periotic mesenchyme containing otic epithelium supporting these fibroblast growth factors (FGFs) as intermediary genes by which RA mediates its effects. Disruption in an Fgf3, -10/Dlx5 signaling cascade is operant in molecular mechanisms of inner ear teratogenesis by excess RA.

Abstract 2335: Transposition Of The Great Arteries And Double Outlet Right Ventricle Have Related Early Developmental Origins: Fundamental Insights From In Vivo Imaging

Congenital heart defects (CHD) are the most common birth defect. Proper retinoic acid (RA) signaling is required for normal development, and RA excess produces conotruncal CHD such as transposition of the great arteries (TGA) and double outlet right ventricle (DORV). We hypothesized that origins and evolution of TGA and DORV follow specific developmental patterns, definable in vivo by noninvasive ultrasound biomicroscopy (UBM)-Doppler imaging. Timed-pregnant ICR mice were injected with 70 mg/kg IP RA at 8.5 days post-conception (E8.5). Individual embryos were tracked with 40 MHz UBM-Doppler longitudinally on 5 consecutive days (E11.5–15.5: N=34 injected, N=8 uninjected controls). CHD included TGA (24%) and DORV (21%, all with right aorta, left pulmonary artery), as well as ventricular septal defect (VSD, defined at E15.5) and double-inlet left ventricle. External gross examination and histology confirmed ~90% concordance of UBM diagnosis of CHD, here (at E15.5) and in separate experiments (E11.5–15.5, N=111). All E11.5 embryos showed normal cardiac looping, with a normal unseptated heart and outflow tract (OFT) - the truncus arteriosus -arising from the right ventricle/bulbus cordis. Of embryos destined for TGA, 7/8 showed partial septation of the distal truncus into parallel OFT at E12.5; 4 showed DORV at E12.5 before evolving into TGA by E13.5. Of embryos destined for DORV, 6/7 showed partial septation of the distal truncus into parallel OFT at E12.5 as well. Thus, abnormal septation commences in the more distal truncus arteriosus as early as E12.5. In contrast, embryos destined for normal OFT development showed partial OFT septation in spiral (non-parallel) fashion, with complete/near-complete OFT septation and morphology at E13.5. All TGA/DORV embryos displayed normal cardiac function. CONCLUSIONS. TGA and DORV are developmentally related, differing only in complete (TGA) vs. incomplete (DORV) leftward truncal shift. Given normal heart, proximal OFT, and cardiac function, flow streams do not explain TGA/DORV morphogenesis. Corresponding to 4–6 weeks’ human gestation, these first in vivo data support longstanding hypotheses of TGA/DORV embryogenesis, and implicate RA signaling in the pathogenesis of abnormal truncal septation and shift.

Apoptotic Extinction of Germ Cells in Testes of Cyp26b1 Knockout Mice

Cyp26b1 encodes a retinoic acid (RA) metabolizing cytochrome P450 enzyme that is expressed in embryonic tissues undergoing morphogenesis, including the testes. We have generated transgenic mice lacking Cyp26b1 and have observed increased RA levels in embryonic testes. Cyp26b1(-/-) germ cells prematurely enter meiosis at embryonic d 13.5 and appear to arrest at pachytene stage. Furthermore, after embryonic d 13.5, a rapid increase in apoptosis is observed in male germ cells derived from Cyp26b1(-/-) embryos; germ cells are essentially absent in mutant male neonates. In contrast, testicular somatic cells appear to develop normally in the absence of Cyp26b1. Moreover, ovarian germ and somatic cells appear unaffected by the lack of CYP26B1. We also show that the synthetic retinoid Am580, which is resistant to CYP26 metabolism, induces meiosis of male germ cells in cultured gonads, suggesting that abnormal development of germ cells in the Cyp26b1(-/-) testes results from excess RA rather than the absence of CYP26B1-generated metabolites of RA. These results provide evidence that CYP26B1 maintains low levels of RA in the developing testes that blocks entry into meiosis and acts as a survival factor to prevent apoptosis of male germ cells.