Expression of genes for two C-type lectins during budding of the ascidian Polyandrocarpa misakiensis

Two closely related cDNA fragments, named pTC14-1 and pTC14-2, encoding C-type lectins were cloned from the budding ascidian Polyandrocarpa misakiensis by means of the polymerase chain reaction. The amino acid sequence deduced from pTC14-1 was identical to that of a 14-kDa calcium-dependent galactose-binding lectin, TC-14, that had been purified from this species. Between the two clones, nucleotide sequence similarity was 90%, whilst that of the deduced amino acid sequences was 82%. The cDNA inserts of these clones hybridized weakly with each other. Antisense RNA probes prepared from these clones gave intense hybridization signals on Northern blots of the W strain, but very weak signals on those of the other strains. Therefore, both clones were suggested to originate from the W strain, but from two separate genes, since the base substitution was scattered throughout the entire translated region. The amount of TC14-1 mRNA increased during bud development, and peaked at 36 h after separation of the bud from the parental body wall. At this stage, extracellular matrix containing TC-14 lectin developed in the mesenchymal space around the morphogenetic region of the bud. There was much less TC14-2, than TC14-1 mRNA at every stage of bud development. TC14-1 and TC14-2 mRNAs were detected on Northern blots of RNAs from adults and growing buds, suggesting that these genes can be used as the earliest markers of budding in this species.     

Retinoic acid disrupts anterior ectodermal and endodermal development in ascidian larvae and postlarvae

 In vertebrates, excess all-trans retinoic acid (RA) applied during axis formation leads to the apparent truncation of anterior structures. In this study we sought to determine the type of defects caused by ectopic RA on the development of the ascidian Herdmania curvata. We demonstrate that H. curvata embryos cultured in the presence of RA develop into larvae whose trunks are shortened and superficially resemble those of early metamorphosing postlarvae. Despite RA-treated larvae lacking papillar structures they respond normally to natural cues that induce metamorphosis, indicating that chemosensory functionality previously mapped to the most anterior region of normal larvae is unaffected by RA. Excess RA applied during postlarval development leads to a graded loss of the juvenile pharynx, apparently by respecifying anterior endoderm to a more posterior fate. This structure is considered homologous to the gill slits of amphioxus, which are also lost upon RA treatment. This suggests that RA may have had a role in the development of the pharynx of the ancestral chordate and that this function has been maintained in ascidians and cephalochordates and lost in vertebrates. Received: 27 February 1998 / Accepted: 20 April 1998     

Retinoid X receptor-mediated transdifferentiation cascade in budding tunicates

In the budding tunicate, Polyandrocarpa misakiensis, retinoic acid (RA) applied to buds promotes transdifferentiation of somatic cells to form the secondary body axis. This study investigated the gene cascade regulating such RA-triggered transdifferentiation in tunicates. Genes encoding retinoic acid receptor (RAR) and retinoid X receptor (RXR) were induced during transdifferentiation, and they responded to all-trans RA or 13-cis RA in vivo, whereas 9-cis RA had the least effects, demonstrating differences in the ligand preference between budding tunicates and vertebrates. In contrast to RAR mRNA, RXR mRNA could induce transdifferentiation-related genes such as RXR itself, ERK, and MYC in an RA-dependent manner and also induced β-catenin (β-CTN) RA-independently when it was introduced in vitro into tunicate cell lines that do not express endogenous RAR or RXR. Small interfering RNA (siRNA) of RXR dramatically attenuated not only RXR but also ERK and β-CTN gene activities. An ERK inhibitor severely blocked wound healing and dedifferentiation. β-CTN siRNA suppressed morphogenesis and redifferentiation, similar to RXR siRNA. These results indicate that in P. misakiensis, the main function of RA is to trigger positive feedback regulation of RXR rather than to activate RAR for unlocking downstream pathways for transdifferentiation. Our results may reflect an ancient mode of RA signaling in chordates.     

Effects of retinoic acid on the endoderm in Xenopus embryos

 When Xenopus embryos from mid-tailbud to early tadpole stages were exposed to retinoic acid (RA), the gut developed with an uncoiled, straight intestinal tube, morphogenesis of the liver and stomach was affected and intestinal epithelial cells developed without a brush border and alkaline phosphatase activity. However, the temporal and spatial expression pattern of XlHbox 8, the only homeobox gene expressed in the endoderm was unaffected. In lateral plate mesodermal cells the expression of α-smooth muscle (SM) actin was delayed. A similar syndrome has been reported in a study of embryos lacking functional FGF receptors in which it was proposed that the uncoiled intestinal tube and the delayed differentiation of the intestinal muscle cells are causally related. Our results support this proposition and further suggest that mesenchymal-epithelial interactions concerned with regional specification of the endoderm may be impaired resulting in other defects in the gut. Received: 3 October 1997 / Accepted: 3 February 1998     

Response of human rhabdomyosarcoma cell lines to retinoic acid: relationship with induction of differentiation and retinoic acid sensitivity

The ability of retinoids to induce growth inhibition associated with differentiation of diverse cell types makes them potent anti-cancer agents. We examined the effect of retinoic acid (RA) in cell lines derived from rhabdomyosarcoma (RMS), a malignant soft-tissue tumor committed to the myogenic lineage, but arrested prior to terminal differentiation. We showed that several RMS derived cell lines, including RD human rhabdomyosarcoma cells, are resistant to the growth-inhibitory and differentiation effects of RA. We established that this RA-resistance correlates with reduced expression and activity of RA-receptors in RD cells. We stably expressed either RARalpha, RARbeta, RARgamma, or RXRalpha expression vector into RD cells and found that only RARbeta or RARgamma induced a significant RA growth arrest without promoting differentiation indicating that changes in the amounts of RARs and RXRs are not sufficient to determine the RA myogenic response of rhabdomyosarcoma cells. Activation of RD cell differentiation by ectopic MRF4 expression enhanced RA-receptor activity and led to RA induction of differentiation. These studies demonstrate that RA-resistance of RD cells is linked to their lack of differentiation and suggest that the differentiation-promoting activity of RA requires factors other than RAR-RXR heterodimers.     

Expression and role of retinoic acid receptor alpha in lens regeneration

The role of retinoids in eye development has been well studied. Retinoids and their receptors regulate gene expression and morphogenesis of the eye. In this study, a highly specific antagonist of retinoic acid receptor (RAR)-alpha was used in an attempt to study its function in lens regeneration. It was found that this antagonist inhibited lens regeneration and lens fiber differentiation. It was also shown that RAR-alpha is expressed in the lens during the process of regeneration. These results indicate that different RAR might have unique as well as redundant effects and patterns of expression in the regenerating lens.     

A robust characterization of retinoic acid response elements based on a comparison of sites in three species

The availability of high-throughput genomic sequencing has allowed us to construct a more robust characterization of retinoic acid response elements than was possible in the past. We located human, mouse, and rat homologs for each of 51 well-documented, conserved retinoic acid response elements. Mathematical and statistical analyses of these 153 sites, 78 of which are new, shows that 92% of response elements have direct-repeat symmetry, but that only 76% exhibit canonical spacing attributes. While the familiar ‘(a/g)g(g/t)tca’ hexamer motif is upheld, the more relaxed sequence, ‘(a/g)g(g/t)(g/t)(g/c)a’, represents a 10% consensus. Sites are as likely to be on the coding strand as on the non-coding strand, and 86% of them are in upstream locations. From a statistical point of view, DR1 elements are fundamentally different from DR2 and DR5 elements, but this is only evident in the 5′ hexamer. While there is considerable variation in core positions, and while no nucleotide can be considered forbidden at any position, variation among species at a fixed locus appears surprisingly constrained once a functional site has been attained.     

Transient expression of a novel serine protease in the ectoderm of the ascidian Herdmania momus during development

 We have studied gene expression during ascidian embryonic development using the technique of differential display and isolated partial cDNA sequences of 12 genes. Developmental regulation of these genes has been confirmed by northern hybridization analysis. Further cDNA cloning and sequence analysis of an mRNA that is present during gastrulation, neurulation and tailbud formation reveals that it encodes a novel serine protease containing a single kringle motif and catalytic domain. The spatial expression of this gene, designated Hmserp1, is restricted to precursor cells of the epidermis. The structure and expression of Hmserp1 is discussed in relation to possible functions during development. Received: 8 October 1996 / Accepted: 17 December 1996     

Identification of a decarboxylation product of retinoic acid

The oxidative decarboxylation of retinoic acid was investigated utilizing a model system concisting of all-trans-retinoic acid, H₂O₂ and horseradish peroxidase. The decarboxylation products were purified by high-performance liquid chromatography on bonded, octadecylsilane columns. Based on mass spectral, nuclear magnetic resonance, ultraviolet and Fourier transform infrared analyses, the major decarboxylation product was identified as a 4-oxo-C₁₉ aldehyde with a hyrdoxyl group on the side chain at C₉, specifically 8-(2,6,6,-trimethyl-3-oxocyclohex-1-enyl)2,6-dimethyl-6-hydroxyoctatrienal.     

Effect of marginal vitamin A deficiency during pregnancy on retinoic acid receptors and N-methyl-D-aspartate receptor expression in the offspring of rats

This study examined whether pregnancy-related marginal vitamin A deficiency (MVAD) influences postnatal development of retinoic acid receptors (RARs) and N-methyl-d-aspartate (NMDA) receptor subunit 1 (NR1) in hippocampus of rat pups. Sixteen female rats were randomized equally into control and MVAD groups. Dams and pups were fed with either a normal control diet or one deficient in vitamin A. Eight female pups in each group were killed at 1 day, 2 weeks, 4 weeks and 8 weeks after birth, respectively. Serum retinol levels were monitored. The messenger RNA (mRNA) and protein expressions and subcellular localization of RARα, RARβ and NR1 in postnatal hippocampus were detected. At 1 day, 2 weeks and 8 weeks after birth, serum retinol levels in the MVAD group were significantly lower than those in the control group. Results of Morris water maze test at 7 weeks of age showed that spatial learning and memory in the MVAD group were affected. Vitamin A deficiency resulted in decreased mRNA levels of RARα, RARβ and NR1 (P<.05). The protein level of RARα and NR1 in the MVAD group was lower than that of the control group (P<.05). There was no significant difference in RARβ between the groups (P>.05). A mass of RARα and NR1 colocalized in hippocampal cell cytoplasm on postnatal day 1. Our data suggested that vitamin A deficiency in pregnancy may affect the postnatal expression of RARα and NR1, affecting learning and memory function in the hippocampus and synaptic plasticity of the calcium signaling pathway.     

Inhibition of invasiveness and expression of epidermal growth factor receptor in human colorectal carcinoma cells induced by retinoic acid

INTRODUCTIONAsaderivativeofvitaminA,RAcaninhibittheproliferati0nofmanymalignantcel1sallde1icitdifferentiationinsometumorce1lsI1-5].RecentstudieshaveshownthatRAmodu1atessynthesisofover4Oproteinsthroughitsnucleicrecept0r[6].Forinstance,RAinducesthesynthesis0ffibronectin(FN)incertaintumorcellsandactivatesthegenecodingfOrB1sllbunitoflaminin(LN)causingitsexpressi0n[7,8].FNandLN'areknownt0bethemostimportantcomponentsofnon-c0llageng1ycoproteinsintheextracellu1armatrixandareclose1yrelatedt…