Sequential gene expression during pronephric tubule formation in vitro in Xenopus ectoderm

The kidney has been used as a model organ to analyze organogenesis. In in vitro experiments using Xenopus blastula ectoderm, the development of pronephric tubules (the prototype of the kidney) may be induced by treatment with activin A and retinoic acid (RA). The present study examined whether pronephric tubules induced in ectodermal explants exhibited similar characteristics to those of normal embryos at the molecular level. The experimental conditions required for high frequency induction (100%) of pronephric tubule formation from presumptive ectoderm without the development of muscle and notochord were determined. The developmental expression of the pronephros marker genes Xlim-1 and Xlcaax-1 was examined in induced pronephric tubules. After treatment with 10 ng/mL activin A and 10⁻⁴ mol/L RA, only pronephric tubules were induced at a high frequency. Induced pronephric tubules showed the same timing and patterns of expression for the marker genes Xlim-1 and Xlcaax-1 as normal embryos. These results suggest that the in vitro development of pronephric tubules induced in the presumptive ectoderm by activin A and RA parallels normal development at the molecular level.     

Effects of vitamin D metabolites on axolotl limb regeneration

Vitamin D is essential for normal metabolism of phosphorus and calcium, and differentiation of skeletal elements. 1,25 dihydroxyvitamin-D₃, the biologically active metabolite, acts as an induction/proliferation switch in various cell types and promotes chondrogenesis of chick limb bud mesenchymal cells. The function of vitamin D is mediated through its nuclear receptor, the vitamin D receptor (VDR). The proliferative actions of 1,25(OH)₂-D₃ on limb bud mesenchymal cells are similar to the ones produced by retinoids, such as all- trans retinoic acid (RA) or 9- cis retinoic acid (9- cis ). The retinoids have been shown to be compounds of extreme importance in the field of limb development and regeneration. In order to examine possible roles of vitamin D metabolites on limb regeneration, the effects of 1,25(OH)₂-D₃, 24,25(OH)₂-D₃ and KH1060 (a more potent metabolite) alone or in conjunction with all- trans RA or 9- cis RA on the regenerating axolotl limb. Vitamin D affects limb morphogenesis by generating abnormalities in skeletal elements. Synergism of vitamin D with retinoic acid in affecting pattern formation is suggested by the results.     

In vitro induction of the pronephric duct in Xenopus explants

The earliest form of embryonic kidney, the pronephros, consists of three components: glomus, tubule and duct. Treatment of the undifferentiated animal pole ectoderm of Xenopus laevis with activin A and retinoic acid (RA) induces formation of the pronephric tubule and glomus. In this study, the rate of induction of the pronephric duct, the third component of the pronephros, was investigated in animal caps treated with activin A and RA. Immunohistochemistry using pronephric duct-specific antibody 4A6 revealed that a high proportion of the treated explants contained 4A6-positive tubular structures. Electron microscopy showed that the tubules in the explants were similar to the pronephric ducts of normal larvae, and they also expressed Gremlin and c-ret, molecular markers for pronephric ducts. These results suggest that the treatment of Xenopus ectoderm with activin A and RA induces a high rate of differentiation of pronephric ducts, in addition to the differentiation of the pronephric tubule and glomus, and that this in vitro system can serve as a simple and effective model for analysis of the mechanism of pronephros differentiation.     

Specific Developmental Defects in Molluscs after Treatment with Retinoic Acid during Gastrulation

All-trans retinoic acid is known as a teratogen in vertebrate development. To study whether molluscan morphogenesis is sensitive to retinoic acid, the development of Lymnaea stagnalis, Physa fontinalis and Bithynia tentaculata was examined after treatment with retinoic acid. Low concentrations retinoic acid (10^–7M) specifically affected eye formation in each of these species. In Lymnaea , it was shown that 10^–6M retinoic acid resulted in a wider spectrum of deficiencies, including eye defects, arrested development and shell deformations. Pulse treatments revealed that embryos were most sensitive during gastrulation. Soon after gastrulation the embryos had lost their sensitivity to retinoic acid, which indicates that the observed defects do not result from a general non-specific toxic effect on cells. Even though molluscan development differs in numerous respects from vertebrate development, the present results suggest that molluscs share common features with vertebrates in morphogenetic processes that operate in early development.     

Effects of retinoic acids on the dendritic morphology of cultured hippocampal neurons

Vitamin A-derived retinoic acids (RAs) are known to exert a variety of biological actions, including modulatory effects on cell differentiation and apoptosis. A recent study has demonstrated that 13- cis -RA and all- trans -RA suppressed neurogenesis in the dentate gyrus of the hippocampus in adult mice. The present experiments were performed to see whether 13- cis -RA and all- trans -RA could alter the dendritic morphology of cultured hippocampal neurons via RA receptors: retinoic acid receptor (RAR) and retinoid X receptor (RXR). High doses of 13- cis -RA and all- trans -RA exerted a negative effect on the cultured hippocampal neurons, while a low dose of 13- cis -RA but not all- trans -RA caused a positive effect. The negative changes induced by 13- cis -RA and all- trans -RA were antagonized by RXR antagonists and RAR antagonists, respectively. The positive changes induced by a low dose of 13- cis -RA were blocked by both RXR antagonists and RAR antagonists. These results suggest that RAs at high concentrations cause a negative effect on the dendritic morphology of cultured hippocampal neurons through RA receptors, while RAs at low concentrations exert a positive influence on cultured hippocampal neurons.     

Cloning and expression pattern of a Xenopus pronephros-specific gene, XSMP-30

The first step in kidney development is the formation of the pronephros which is derived from mesoderm. Xenopus is an appropriate model to study this process since the pronephros can be efficiently induced in animal cap explants by treatment with activin and retinoic acid (RA). Using this in vitro system, we isolated a Xenopus homologue of SMP-30 (Senescence marker protein-30), which is a Ca(2+)-binding protein that is highly conserved in vertebrates. This gene, termed XSMP-30, was found to be selectively expressed in pronephric tubules from the late tadpole stage, by whole mount in situ hybridization. Furthermore XSMP-30 was expressed in animal caps treated with both activin and RA, a condition in which the pronephros is formed in vitro. These data indicate that XSMP-30 is a specific marker for the pronephros.     

An increase in intracellular Ca2+ is involved in pronephric tubule differentiation in the amphibian Xenopus laevis

The pronephros is the first kidney to develop and is the functional embryonic kidney in lower vertebrates. It has previously been shown that pronephric tubules can be induced to form ex vivo in ectodermal tissue by treatment with activin A and retinoic acid. In this study, we investigated the role of Ca(2+) signaling in the formation of the pronephric tubules both in intact Xenopus embryos and ex vivo. In the ex vivo system, retinoic acid but not activin A stimulated the generation of Ca(2+) transients during tubule formation. Furthermore, tubule differentiation could be induced by agents that increase the concentration of intracellular Ca(2+) in activin A-treated ectoderm. In addition, tubule formation was inhibited by loading the ectodermal tissue with the Ca(2+) chelator, BAPTA-AM prior to activin A/retinoic acid treatment. In intact embryos, Ca(2+) transients were also recorded during tubule formation, and photo-activation of the caged Ca(2+) chelator, diazo-2, localized to the pronephric domain, produced embryos with a shortened and widened tubule phenotype. In addition, the location of the Ca(2+) transients observed, correlated with the expression pattern of the specific pronephric tubule gene, XSMP-30. These data indicate that Ca(2+) might be a necessary signal in the process of tubulogenesis both ex vivo and in intact embryos.     

The specification and growth factor inducibility of the pronephric glomus in Xenopus laevis

We report a study on the specification of the glomus, the filtration device of the amphibian pronephric kidney, using an explant culturing strategy in Xenopus laevis. Explants of presumptive pronephric mesoderm were dissected from embryos of mid-gastrula to swimming tadpole stages. These explants were cultured within ectodermal wraps and analysed by RT-PCR for the presence of the Wilm's Tumour-1 gene, xWT1, a marker specific for the glomus at the stages analysed, together with other mesodermal markers. We show that the glomus is specified at stage 12.5, the same stage at which pronephric tubules are specified. We have previously shown that pronephric duct is specified somewhat later, at stage 14. Furthermore, we have analysed the growth factor inducibility of the glomus in the presence or absence of retinoic acid (RA) by RT-PCR. We define for the first time the conditions under which these growth factors induce glomus tissue in animal cap tissue. Activin together with high concentrations of RA can induce glomus tissue from animal cap ectoderm. Unlike the pronephric tubules, the glomus can also be induced by FGF and RA.     

Biological activity of 6-(2,3,4-trihydroxy-3-methylbutylamino)purine, an oxidation product of zeatin

6-(2,3,4-Trihydroxy-3-methylbutylamino)purine was identified as an oxidation product of cis -zeatin and is biologically as active as the parent compound. A comparison of trans -zeatin, cis -zeatin and (±)-dihydrozeatin indicated that trans -zeatin and (±)-dihydrozeatin were more active in the soybean callus bioassay than cis -zeatin. Both the trans - and cis -isomers of zeatin did, however, give an optimum response at 10^-5 M. Dihydrozeatin was more active at concentrations of 10^-6 and 10^-5 M than trans -zeatin. The significance of the formation of 6-(2,3,4-trihydroxy-3-methylbutylamino)purine with respect to stereochemistry and the oxidation of cytokinins with an unsaturated isopentenyl side chain is discussed.     

Interaction of Wnt and activin in dorsal mesoderm induction in Xenopus.

Both the activin and Wnt families of peptide growth factors are capable of inducing dorsal mesoderm in Xenopus embryos. Presumptive ventral ectoderm cells isolated from embryos injected with Xwnt8 mRNA were cultured in the presence of activin A to study the possible interactions between these two classes of signaling proteins. We find that overexpression of Xwnt8 RNA alters the response of ventral ectoderm to activin such that ventral explants differentiate dorsoanterior structures including notochord and eyes. This response is similar to the response of dorsal ectoderm to activin alone. When embryos are irradiated with uv light to inhibit dorsal axis formation, ectodermal explants differentiate notochord when they are induced by a combination of both signaling factors, but not when cells receive only one inducing signal (activin or Xwnt8). This result is further supported by the observation that goosecoid (gsc) mRNA, an early marker for dorsal mesoderm, is expressed in these explants only when they are injected with Xwnt8 mRNA followed by exposure to activin. Early morphogenetic movements of the induced cells and activation of muscle-specific actin and Brachyury (Xbra) genes also reveal a cooperation of activin A and Xwnt8 in mesoderm induction.     

A role for Xlim-1 in pronephros development in Xenopus laevis

Xlim-1, a LIM class homeobox gene expressed in Xenopus laevis, is one of the earliest known marker genes of pronephros development and is expressed in pronephros rudiment. In this study, we examined the role of Xlim-1 in pronephros development. Temporal expression of Xlim-1 in explants was analyzed in a series of induction assays using RT-PCR analysis. Xlim-1 was expressed 9 to 15 h after activin/retinoic acid treatment, corresponding to pronephros differentiation in explants. We further examined the role of Xlim-1 using a series of microinjection experiments. Presumptive pronephric anlagen of embryos were injected with various Xlim-1 mutants, and effects of these Xlim-1 mutants on pronephrogenesis in embryos and in explants were analyzed by RT-PCR and immunohistochemistry. Dominant-negative Xlim-1 inhibited differentiation of pronephros in activin/retinoic acid-treated animal caps. In embryos injected with a dominant-negative form of Xlim-1, development of pronephric tubules was inhibited at the late tail-bud stage. Our results suggest that Xlim-1 may not initiate differentiation of the pronephros, but that it is necessary for growth and elongation in the development of pronephric tubules.     

Retinoic Acid Increases Hydroxyindole-O-Methyltransferase Activity and mRNA in Human Y-79 Retinoblastoma Cells

Abstract: Hydroxyindole- O -methyltransferase (HIOMT) plays an important role as the final enzyme in the synthesis of melatonin. Here we present the first evidence that retinoic acid (RA) stereoisomers are potent regulators of HIOMT in the human retinoblastoma-derived Y-79 cell line. Treatment with all- trans -, 13- cis -, and 9- cis -RA induced a gradual 10-fold increase in HIOMT activity and mRNA, without changing the levels of mRNA encoding glyceraldehyde-3-phosphate dehydrogenase, actin, S-antigen, and interphotoreceptor retinoid-binding protein. These findings point to the possibility that RA may play a physiological role in the regulation of human HIOMT.     

Effect of abscisic acid on CO2 exchange in Lemna gibba

The effects of abscisic acid (ABA) on photosynthesis, dark respiration, and photorespiration were studied in Lemna gibba L. plants. The initial concentration of ABA in the nutrient solution was 10⁻⁷M and in a few experiments, 10⁻⁶M. The cultures were grown in the same solution for time periods ranging from one hour to 12 days. Net photosynthesis, measured as CO₂ uptake by infrared gas analyser technique, was inhibited after four hours of ABA treatment and reached a minimum after four to seven days depending on the time of the year. After 12 days a substantial recovery of photosynthesis was observed. Dark respiration was significantly stimulated after two to seven days of ABA treatment but then returned to the control level. The transient effects of ABA on photosynthesis and dark respiration corresponded to the previously measured time course of [¹⁴C]-ABA uptake by Lemna . Photorespiration measured as oxygen inhibition of photosynthesis was not affected by ABA.     

Mesoderm and Neural Inductions on Newt Ectoderm by Activin A

Mesoderm-inducing activity of human recombinant activin A was examined on presumptive ectoderm of the Japanese newt, Cynops pyrrhogaster , by using the animal cap assay, Activin A induced neural tissues and mesodermal tissues such as brain, neural tube, notochord, muscle, mesenchyme, coelomic epithelium and blood-like cells after 14 days cultivation. These tissues were induced by activin A at concentrations ranging from 0.5– 100 ng/ml. Dose-dependent inducing activity of activity A on newt ectoderm was slightly different from that on other animals, including Xenopus . Wide range of concentration of activin A (0.5– 100 ng/ml) could induce the neural tube, notochord, mesenchyme and coelomic epithelium on the newt ectoderm. Though the percentage of induced explants (two out of 23 explants, 8.7%) was low, the pulsating heart was induced. This paper showed first that activin could induce the mesodermal and neural tissues in newt presumptive ectoderm. Since activin homologues were present In Xenopus and chick embryos, it is likely that activin may be one of the natural inducers in a wide range of species.     

Effect of plant growth hormones and polyamines on ornithine decarboxylase activity during the germination of barley seeds

Gibberellic acid (GA₃) and β-indolylacetic acid (IAA), two of the well known growth hormones, induce four fold the activity of ornithine decarboxylase (ODC) during the germination of barley seeds ( Hordeum vulgare L. var. Beca). The optimal concentration for induction of ODC was 10^–5 M for GA₃ and 10^–3 M for IAA. When 10^–3 M of a polyamine, putrescine or spermidine, is added to the growth medium, ODC activity is significantly inhibited. This inhibition is due to the induction of a protein inhibitor of ODC (antizyme), whose apparent molecular weight is 16 000 ± 2 000 daltons. Addition of GA₃ to cultures which have been grown for 50 or 98 h in the presence of polyamines, abolishes the observed inhibition of ODC activity, while in the reverse experiment, addition of polyamines at 50 or 98 h does not affect the ODC activity induced by GA₃. Cadaverine, a physiological plant diamine, enhances ODC activity; whereas 1,8-diaminooctane (the alkyl analogue of spermidine) does not have any effect.     

STIMULATORY EFFECTS OF SUBSTANCE P ON NEURITE EXTENSION AND CYCLIC AMP LEVELS IN CULTURED NEUROBLASTOMA CELLS

Abstract— Synthetic substance P initially increased cyclic AMP levels and subsequently induced neurite extension in cultured neuroblastoma N 18 cells. The magnitude of these effects depended on the concentration of fetal calf serum (FCS) in the culture medium, being more evident in the presence of a lower (0.1%) concentration of FCS.
In Eagle's medium containing 0.1% FCS, low concentrations of substance P (10⁻⁷-10⁻⁵ M) increased cyclic AMP levels and stimulated neurite extension.
In Eagle's medium containing 5%FCS, both substance P at concentrations of 10⁻⁵-10⁻³M and dibutyryl cyclic AMP at concentrations of 10⁻⁴-10⁻²M increased cyclic AMP levels and stimulated neurite extension. The activities of acetylcholinesterase, (Na⁺+ K⁺)-, HCO₃⁻ and Mg²⁺ -stimulated-ATPase were also increased. Cell growth was inhibited.
Substance P at concentrations of 10-⁷-10⁻⁵M also stimulated the adenylate cyclase activity of a particulate fraction of N 18 in a concentration-dependent manner.     

Effect of chelating agents on bud induction and accumulation of iron and copper by the moss Bartramidula bartramioides

The chelating agents, EDDHA, its iron salt, EDTA, and salicylic acid enhance bud formation in Bartramidula bartramioides (Griff.) Wijk & Marg. Salicylic acid elicits optimal response at 10^–4 M , whereas the other substances do so at 10^–7 M . Increased concentration of ferric citrate and cupric sulphate also stimulate bud induction. The accumulation of Fe³⁺ and Cu²⁺ is facilitated by chelators. The endogenous iron content is maximum at 10^–7 M EDDHA or EDTA, which is also the concentration optimal for bud induction.     

Isomerization of the proline in the M2–M3 linker is not required for activation of the human 5-HT3A receptor

Each subunit of the cation-selective members of the Cys-loop family of ligand-gated ion channels contains a conserved proline residue in the extracellular loop between the second and third transmembrane domains. In the mouse homomeric 5-hydroxytryptamine type 3A (5-HT₃A) receptor, the effects of substitution of this proline by unnatural amino acids led to the suggestion that trans - cis isomerization of the protein backbone at this position is integral to agonist-induced channel opening [ Nature (2005) vol. 438 , pp. 248–252]. We explored the generality of this conclusion using natural amino acid mutagenesis of the homologous human 5-HT₃A receptor. The conserved proline (P303) was substituted by either a histidine or tryprophan and the mutant receptors were expressed in Xenopus oocytes. These mutations did not significantly affect the magnitude of agonist-mediated currents, compromise channel gating by 5-HT or inhibition of 5-HT-induced currents by either picrotoxin or d -tubocurarine. The mutations did, however, result in altered dependence on extracellular Ca²⁺ concentration and a 10-fold increase in the rate of receptor desensitization. These results demonstrate an important role for P303 in 5-HT₃A receptor function but indicate that trans - cis isomerization at this proline is unlikely to be a general mechanism underlying the gating process.