Akihiko Kashiwagi

Effect of atrazine on metamorphosis and sexual differentiation in Xenopus laevis

There is a growing international concern that commonly used environmental contaminants have the potential to disrupt the development and functioning of the reproductive system in amphibians. One such chemical of interests is the herbicide atrazine. Effects of atrazine on sex differentiation were studied using wild-type Xenopus laevis tadpoles and all-ZZ male cohorts of X. laevis tadpoles, produced by mating wild-type ZZ male to sex-reversed ZZ male (female phenotype). Stage 49 tadpoles were exposed to 0.1-100 ppb atrazine or 0.27 ppb (1 nM) 17beta-estradiol (E(2)) until all larvae completed metamorphosis (stage 66). Metamorphosis, gonadal morphology and histology, CYP19 (P450 aromatase) mRNA induction, and hepatic vitellogenin (VTG) induction were investigated. Effects of atrazine on VTG-induction were also assessed in vitro in primary-cultured X. laevis hepatocytes. Atrazine had no effect on metamorphosis of developing wild-type or all-male X. laevis larvae. Statistical increase in female ratios was observed in 10 and 100 ppb atrazine groups in comparison with control group. While no hermaphroditic froglet was observed in all atrazine groups. In ZZ males, sex reversal was induced by 0.27 ppb E(2), but not by atrazine at concentrations of 0.1 and 1.0 ppb. In addition, neither P450 aromatase mRNA in the gonad nor hepatic VTG were induced by atrazine. Furthermore, VTG was not induced by 1000 ppb atrazine in primary-cultured hepatocytes. Our results indicate that female ratios in developing X. laevis tadpoles were increased by 10 and 100 ppb atrazine under the present experimental conditions. While the other endpoints showed no effect in the range of 0.1-100 ppb atrazine. These results suggest that effect of atrazine on sexual differentiation was not caused by estrogenic action and has no induction ability of P450 aromatase gene in gonad.

High efficiency TALENs enable F0 functional analysis by targeted gene disruption in Xenopus laevis embryos

Summary Recently, gene editing with transcription activator-like effector nucleases (TALENs) has been used in the life sciences. TALENs can be easily customized to recognize a specific DNA sequence and efficiently introduce double-strand breaks at the targeted genomic locus. Subsequent non-homologous end-joining repair leads to targeted gene disruption by base insertion, deletion, or both. Here, to readily evaluate the efficacy of TALENs in Xenopus laevis embryos, we performed the targeted gene disruption of tyrosinase (tyr) and pax6 genes that are involved in pigmentation and eye formation, respectively. We constructed TALENs targeting tyr and pax6 and injected their mRNAs into fertilized eggs at the one-cell stage. Expectedly, introduction of tyr TALEN mRNA resulted in drastic loss of pigmentation with high efficiency. Similarly, for pax6, TALENs led to deformed eyes in the injected embryos. We confirmed mutations of the target alleles by restriction enzyme digestion and sequence analyses of genomic PCR products. Surprisingly, not only biallelic but also paralogous, gene disruption was observed. Our results demonstrate that targeted gene disruption by TALENs provides a method comparable to antisense morpholinos in analyzing gene function in Xenopus F0 embryos, but also applies beyond embryogenesis to any life stage.

Thyroxine enhancement and the role of reactive oxygen species in tadpole tail apoptosis

Our objective is to clarify the role of reactive oxygen species (ROS) in the atrophying tail of anuran tadpoles (tail apoptosis). Changes in catalase, superoxide dismutase (SOD) and caspase activity, genomic DNA, and nitric oxide (NO) generation were investigated biochemically using Rana japonica tadpole tails undergoing regression during thyroid hormone enhancement. DNA fragmentation and ladder formation with concomitant shortening of tadpole tail were induced by DL-thyroxine (T4) in culture medium. Catalase activity was also decreased by T4 treatment. T4 was also found to increase NO synthase (NOS) activity in cultured tadpole tail with concomitant increase in the concentration of NO2- plus NO3- (NOx) in the culture medium. Additional treatment with N-monomethyl-L-arginine (NMMA), a potent inhibitor of NOS, suppressed the enhancing effects of T4 on tail shortening and catalase activity reduction. It was also found that treatment with isosorbide dinitrate (ISDN), a NO generating drug, alone also had an enhancing effect on tail shortening and catalase activity reduction similar to that seen with T4. Both NO and an NO donor (ISDN) strongly suppressed catalase activity. Kinetic analysis revealed that catalase activity decreased and caspase-3-like activity increased during normal tadpole tail atrophy (apoptosis). These results suggested that T4 enhances NO generation, thereby strongly inhibiting catalase activity, resulting in an increase in hydrogen peroxide, and that the oxidative stress elicited by excess hydrogen peroxide might activate cysteine-dependent aspartate-directed protease-3 (caspase-3-like protease), which is thought to cause DNA fragmentation, leading to apoptosis.

COMPARISON OF CATALASE IN DIPLOID AND HAPLOID RANA RUGOSA USING HEAT AND CHEMICAL INACTIVATION TECHNIQUES

The present study examines differences in the hydrogen peroxide (H2O2) detoxifying enzyme, catalase, found in the tails and livers of diploid and haploid Rana rugosa. Investigative techniques include measurement of catalase activity and tests for temperature stability and chemical inhibition. Catalase from the tails of pre-climactic (stage XXIII) haploids was found to be over three times as H2O2 destructive as catalase from similar tails of diploids. Catalase from the livers of newly metamorphosed (stage XXV) froglets, on the other hand, displayed only one third the activity seen in diploid livers. The catalase in haploid tail and liver proved to be more heat resistant, retaining 40-60% of its original activity after 5 min of treatment at 55 degrees C, whereas diploid catalase was totally inactivated under the same conditions. Haploid and diploid catalase also responded differently to inhibition using urea and aminotriazole. These differences suggest that haploid catalase has diverged from normal diploid catalase through molecular modification, resulting in abnormal systems for H2O2 metabolism, which in turn are thought to be responsible for organ dysfunction and early death seen in haploid individuals.

Suppression of T3- and fatty acid-induced membrane permeability transition by l-carnitine

Cytochrome c (Cyt. c) is known to be released from the mitochondria into the cytosol by means of the membrane permeability transition (MPT) mechanism, thereby activating caspase cascade activity, and inducing cell apoptosis. Recently we reported that L-carnitine suppressed palmitoyl-CoA-induced MPT as well as apoptosis in some cell types (Biochem. Pharmacol, in press). In the present study T(3) was found to induce MPT and Cyt. c release, while cyclosporin A (CsA), bovine serum albumin (BSA) and L-carnitine were found to inhibit this action in a concentration-dependent manner. Similarly, long chain fatty acid (LCFA) also induced MPT and Cyt. c release, which was then inhibited by CsA, BSA and L-carnitine. From these results the authors postulate that T(3)-induced MPT is in part regulated by fatty acid metabolism through a dynamic balance between LCFAs and L-carnitine.

Free radical theory of apoptosis and metamorphosis

Abstract Reactive oxygen species (ROS) are the major factors that induce oxidative modification of DNA and gene mutation. ROS can elicit oxidative stress and affect a wide variety of physiological and pathological processes including embryonal development, maturation and aging.

Structural differences between XX and ZW sex lampbrush chromosomes in Rana rugosa females (Anura: Ranidae).

In this study we investigated the morphology and pairing behavior of sex lampbrush chromosomes of XX and ZW females ofRana rugosa from five localities in Japan. Whereas lampbrush chromosomes of XX females from Hiroshima and Isehara had subterminally located centromeres and showed remarkable similarity, those of XX females from Hamakita had the centromeres in the middle. Analysis of landmark configurations revealed that chromosome Xq of Hamakita females closely resembled a part of Xq of Hiroshima and Isehara females, whereas Xp of Hamakita females was inverted compared with the other part of Xq of Hiroshima and Isehara females. Z chromosomes from Kanazawa and Niigata closely resembled the Hiroshima X, whereas the W closely resembled the Hamakita X. XX pairings from Hiroshima, Iserara, and Hamakita were found to be joined by one to four chiasmata at various points all along the axis in both the short and long arms, whereas chromosomal pairs from Kanazawa and Niigata showed only one chiasma between Zp and the distal region of Wq. From these findings we conclude that (1) both the W and the Hamakita X must have evolved from the more primitive Hiroshima and Isehara X chromosomes by a series of pericentric inversions; and (2) females distributed in Hamakita possess two X chromosomes similar to the W, suggesting that either sex-determining or sexmodifying genes on the Hamakita X are clearly different from those on the Kanazawa and Niigata W chromosome.

Targeted mutagenesis of multiple and paralogous genes in Xenopus laevis using two pairs of transcription activator‐like effector nucleases

Transcription activator‐like effector nucleases (TALENs) have been extensively used in genome editing in various organisms. In some cases, however, it is difficult to efficiently disrupt both paralogous genes using a single pair of TALENs in Xenopus laevis because of its polyploidy. Here, we report targeted mutagenesis of multiple and paralogous genes using two pairs of TALENs in X. laevis. First, we show simultaneous targeted mutagenesis of three genes, tyrosinase paralogues (tyra and tyrb) and enhanced green fluorescent protein (egfp) by injection of two TALENs pairs in transgenic embryos carrying egfp. Consistent with the high frequency of both severe phenotypic traits, albinism and loss of GFP fluorescence, frameshift mutation rates of tyr paralogues and egfp reached 40–80%. Next, we show early introduction of TALEN‐mediated mutagenesis of these target loci during embryogenesis. Finally, we also demonstrate that two different pairs of TALENs can simultaneously introduce mutations to both paralogues encoding histone chaperone with high efficiency. Our results suggest that targeted mutagenesis of multiple genes using TALENs can be applied to analyze the functions of paralogous genes with redundancy in X. laevis.

Evidence for two successive pericentric inversions in sex lampbrush chromosomes of Rana rugosa (Anura: Ranidae)

Abstract.The objective of this study was to clarify the course of inversions by which a ZW sex chromosome dimorphism has become established in Rana rugosa. Fortunately, R. rugosa preserves three different forms of sex chromosomes in the several isolated populations. In both males and females, the homomorphic sex chromosomes from Hiroshima were closely similar to Z, while those from Isehara were slightly different from the Z. Females from Hirosaki demonstrated heteromorphic sex chromosomes. In this study, the configuration and pairing behavior of sex lampbrush chromosomes were examined in the female offspring produced from a cross between a female from Hiroshima and a male from Isehara, as well as the female offspring of a female from Hirosaki and the male from Isehara. For the sex lampbrush chromosomes from Hiroshima and Isehara, chiasmata were exclusively formed between the distal regions of the long arms of one sex chromosome and the terminal regions of the short arms of the other. As a result, landmarks arranged in reverse order were observed in the achiasmatic regions of these chromosomes. For the sex lampbrush chromosomes from Isehara and Hirosaki, on the other hand, chiasma formation was mainly confined to the lower half of the chromosomes corresponding to the long arms, and the landmarks in the achiasmatic regions of these chromosomes were disposed in the opposite direction to each other. These results seem to indicate that in the primitive sex chromosomes of the Hiroshima type two pericentric inversions occurred, leading to the differentiation of the W chromosomes. This is the first report to substantiate the process of sex chromosome differentiation experimentally.

Molecular features of thyroid hormone-regulated skin remodeling in Xenopus laevis during metamorphosis

Amphibian body skin provides an opportunity to investigate the molecular mechanism of thyroid hormone (TH)‐dependent organ remodeling during metamorphosis. Global gene expression changes in the TH‐dependent body skin remodeling were studied with microarray analysis. We identified 401 genes that were differentially expressed more than fourfold for 7 days after TH‐treatment. As expected, larval‐ and adult‐type keratin genes were significantly inactivated and activated, respectively. The expression changes of the Gene Ontology annotated genes demonstrated significant correlation with the morphological and physiological changes in body skin metamorphosis. The ‘transcription and proteolysis’ category genes were first upregulated 1 day after TH‐treatment. Subsequently, the ‘cell cycle’ category genes were activated at 3 days. The ‘defense response’ and ‘immune response’ category genes were the late TH‐response genes, which were downregulated and upregulated at 5 and 7 days, respectively. From these genes, adult‐type keratin‐c (xak‐c) gene was selected as a suitable gene to visually monitor the emergence of adult‐type epidermal cells during skin remodeling, because the gene is specifically expressed in adult epidermal basal cells. We generated enhanced green fluorescent protein (EGFP)‐transgenic Xenopus laevis driven by the promoter of xak‐c gene. The keratin promoter faithfully expressed the EGFP gene in adult‐type basal cells. Spatial and temporal EGFP‐fluorescence patterns of filial 1 (F1)‐offspring tadpoles visually demonstrated an event of sequential replacement of larval keratinocytes with the newly generated adult counterparts.