Etsuko Ueta

Gender-dependent differences in the incidence of ochratoxin A-induced neural tube defects in the Pdn/Pdn mouse.

Genetic polydactyly/arhinencephaly mouse embryo, Pdn/Pdn, exhibits suppression of Gli3 gene expression. Ochratoxin A (OTA) is a teratogen that causes neural tube defects (NTD) in mice. We investigated gender‐dependent differences in the incidence of NTD induced by OTA in the Pdn/Pdn mouse. After administering 2 mg/kg OTA to Pdn/+ female mice, mated with Pdn/+ males, on day 7.5 of gestation, we examined the genotypes, sex and NTD of fetuses on day 18. Non‐treated Pdn/Pdn had a 15.8% risk of NTD, and all NTD fetuses were female. When Pdn/Pdn embryos were exposed to OTA, the incidence of NTD increased to 16 (51.6%) of 31 Pdn/Pdn fetuses, and 10 (71.4%) of 14 male Pdn/Pdn fetuses exhibited NTD. From these results, it was speculated that NTD in OTA‐treated male Pdn/Pdn were due to the synergistic effect between depressed Gli3 and altered sex‐correlated gene expression from OTA treatment. After treatment with OTA, the embryos were recovered on day 9 and gene expressions, which were correlated with Gli3, telencephalic morphogenesis, formation of gonadal anlage, and gender‐dependent differentiation were investigated. From real‐time polymerase chain reaction analysis results, it was suggested that the manifestation of NTD in the male OTA‐treated Pdn/Pdn might be due to the complicated altered gene expressions among Gli3, Wnt7b, Wnt8b, Fez1, Barx1, Lim1, Dmrt1, Igf1, Fog2, Dax1 and Sox9, and in particular, upregulation and gender‐dependent difference in Barx1 and gender‐dependent difference in Sox9 gene expressions might be noteworthy findings.

Birth defects caused by mutations in human GLI3 and mouse Gli3 genes

GLI3 is the gene responsible for Greig cephalopolysyndactyly syndrome (GCPS), Pallister–Hall syndrome (PHS) and Postaxial polydactyly type‐A (PAP‐A). Genetic polydactyly mice such as Pdn/Pdn (Polydactyly Nagoya), XtH/XtH (Extra toes) and XtJ/XtJ (Extra toes Jackson) are the mouse homolog of GCPS, and Gli3tmlUrtt/Gli3tmlUrt is produced as the mouse homolog of PHS. In the present review, relationships between mutation points of GLI3 and Gli3, and resulting phenotypes in humans and mice are described. It has been confirmed that mutation in the upstream or within the zinc finger domain of the GLI3 gene induces GCPS; that in the post‐zinc finger region including the protease cleavage site induces PHS; and that in the downstream of the GLI3 gene induces PAP‐A. A mimicking phenomenon was observed in the mouse homolog. Therefore, human GLI3 and mouse Gli3 genes have a common structure, and it is suggested here that mutations in the same functional regions produce similar phenotypes in human and mice. The most important issue might be that GCPS and PHS exhibit an autosomal dominant trait, but mouse homologs, such as Pdn/Pdn, XtH/XtH, XtJ/XtJ and Gli3tmlUrt/Gli3tmlUrt, are autosomal recessive traits in the manifestation of similar phenotypes to human diseases. It is discussed here how the reduced amounts of the GLI3 protein, or truncated mutant GLI3 protein, disrupt development of the limbs, head and face.

Integration of a transposon into the Gli3 gene in the Pdn mouse

ABSTRACT  The phenotype of the genetic polydactyly/arhinencephaly mouse (Pdn/Pdn) is similar to Greig cephalopolysyndactyly syndrome (GCPS), whose responsible gene is GLI3. Suppression of Gli3 gene expression has been observed in the Pdn/Pdn and integration of retrotransposon in Gli3 gene in the Pdn mouse has been reported. Thus, the responsible gene for Pdn/Pdn is thought to be Gli3, but the site of mutation within the gene has not been demarcated.

Hydrocephalus manifestation in the genetic polydactyly/arhinencephaly mouse (Pdn/Pdn)

ABSTRACT  The genetic polydactyly/ariiinencephaly mouse, Pdn/Pdn, exhibits severe polydactyly both in the fore‐and hindlimbs, hydrocephalus, and agenesis of the olfactory bulbs, corpus callosum, and anterior commissure. The mechanism of hydrocephalus manifestation in Pdn/Pdn was investigated in the present study. Ink was injected into the left lateral ventricle in the Pdn/Pdn and +/+ newborn mice. After incubation at 32°C for different time intervals, the heads were fixed in Bouins solution and were subsequently decalcified in 0.5 mol/L of EDTA solution, paraffin sectioned, and stained with hematoxylin and eosin.

Sonic hedgehog expression in Gli3 depressed mouse embryo, Pdn/Pdn

ABSTRACT  The phenotype of the genetic polydactyly/arhinencephaly mouse (Pdn/Pdn) is similar to Greig cephalopolysyndactyly syndrome (GCPS), which is induced by mutation of GLI3. Suppression of Gli3 gene expression has been observed in Pdn/Pdn. Thus, the gene responsible for Pdn/Pdn has been considered to be Gli3. Recently, the mutation point was demarcated, that is, a transposon was inserted into intron 3 of the Gli3 gene in the Pdn mouse. Forward and reverse primers were constructed in intron 3 near the insertion point. A forward primer in the long terminal repeat region of the transposon was also constructed. Now we can discriminate +/+, Pdn/+, Pdn/Pdn embryos from the PCR products. After genotyping of the Pdn embryos, Gli3 and other correlated gene expressions, such as sonic hedgehog (Shh), Bmp‐2, Bmp‐4, ptc‐1, were analyzed by real‐time PCR method. Gli3 gene expression in Pdn/Pdn was suppressed to 20–30% of +/+, and that in Pdn/+ was about 60% of +/+ through all the embryonic and neonatal periods examined. As Shh has been considered to be an antagonist of Gli3, Shh expression was analyzed, and a difference among genotypes was observed only on day 9 of gestation. We could not detect any alterations among genotypes in other gene expressions examined. Gli3 and Shh gene expression were also analyzed on day 9 by whole‐mount in situ hybridization in the +/+ and Pdn/Pdn embryos. Neuroectoderm was positive by Gli3 probe in +/+ but not in Pdn/Pdn. Notochord, floor plate and prechordal mesoderm were positive by Shh probe both in +/+ and Pdn/Pdn embryos, but ectopic and/or over‐expression of Shh were not observed in Pdn/Pdn embryos.

Prevention of ochratoxin a-induced neural tube defects by folic acid in the genetic polydactyly/arhinencephaly mouse, Pdn/Pdn

ABSTRACT  The gene responsible for the polydactyly/arhinencephaly (Pdn/Pdn) mouse, which exhibits polysyndactyly and arhinencephaly and has a 13.2% risk of neural tube defects (NTD), has been identified as Gli3. Ochratoxin A (OTA) is a teratogen causing NTD in mice. When Pdn/Pdn embryos were exposed to 2 mg/kg of OTA on day 7.5, the incidence of NTD in Pdn/Pdn fetuses increased to 51.6%. Pre‐treatment with folinic acid (FA), metabolically the most active form of folic acid, before OTA‐treatment decreased the incidence of NTD to 20.8%. We investigated the effect of OTA and FA on gene expression in day 9 embryos using whole‐mount in situ hybridization and real‐time PCR. Over‐expression of Fgf8 was observed at the anterior neural ridge (ANR) in the non‐treated Pdn/Pdn. Over‐expression at the ANR expanded in the OTA‐treated Pdn/Pdn, and it was ameliorated by pretreatment with FA. Emx2 signal was observed in the dorsal forebrain in the non‐treated +/+, but disappeared in the OTA‐treated +/+, and was recovered by FA. The Emx2 signal was pale and the expression amount was depressed in the non‐treated and OTA‐treated Pdn/Pdn embryos. It was suggested that down‐regulation of Gli3 induced the over‐expression of Fgf8 at the ANR, that OTA treatment accelerated the over‐expression, and that pretreatment with FA ameliorated the OTA‐induced over‐expression of Fgf8 in the Pdn/Pdn. It was also suggested that down‐regulation of Gli3 induced the down‐regulation of Emx2 in the Pdn/Pdn. It was further speculated that the over‐expression of Fgf8 at the ANR and down‐regulation of Emx2 in the dorsal forebrain may contribute to NTD induction.

Response of anti-oxidant enzymes mRNA in the neonatal rat liver exposed to 1,2,3,4-tetrachlorodibenzo-p-dioxin via lactation.

Background : The aim of this study was to assess the response to dioxin‐induced oxidative stress in neonates via lactation in the model we have described previously.

Genetic susceptibility in the neural tube defects induced by ochratoxin A in the genetic arhinencephaly mouse, Pdn/Pdn

ABSTRACT  It is well known that ochratoxin A (OTA) induces neural tube defects (NTDs) in mice. In the present study, OTA was administered to the genetic polydactyly/arhinencephaly mouse (Pdn/Pdn) to investigate the synergistic effect between gene and environmental toxin. OTA treatment on day 7.5 of gestation increased NTDs in the Pdn/Pdn mouse. The responsible gene for Pdn/Pdn is Gli3. So, it was speculated that specific susceptibility for OTA in the Pdn/Pdn mouse embryo may be due to the severe depression of Gli3 gene expression. As correlated genes, Gli3, Shh and Fgf8 gene expressions were examined in the Pdn mouse embryo on day 9 of gestation after administration of OTA on day 7.5. No alteration of Shh expression was observed in the non‐treated Pdn/Pdn, and OTA‐treated +/+ and Pdn/Pdn. Fgf8 signal was observed at the anterior neural ridge (ANR) in the non‐treated +/+, and that was elongated in the non‐treated Pdn/Pdn, and further elongated and more intensive in the OTA‐treated Pdn/Pdn. It was suggested that Fgf8 gene expression was affected by the depression of Gli3, and alteration of Fgf8 gene expression was accelerated by the toxicity of OTA in the Pdn/Pdn.

Exencephaly induction by valproic acid in the genetic polydactyly/arhinencephaly mouse, Pdn/Pdn

ABSTRACT  Non‐treated homozygous polydactyly/arhinencephaly (Pdn/Pdn) mouse fetuses exhibited exencephaly in 16.7% of cases. Treatment of Pdn/Pdn mice with 350 mg/kg of valproic acid (VPA) on days 8.5 and 9.5 of gestation increased the rate of exencephaly to 66.7%. The responsible gene for the Pdn mouse phenotype has been determined to be Gli3, and the suppression of Gli3 gene expression has been documented in Pdn/Pdn embryos. We investigated how the sonic hedgehog (Shh) and Fgf8 genes, the correlated genes of Gli3, are expressed in the VPA‐treated exencephalic Pdn/Pdn embryos on day 10 of gestation, using whole mount in situ hybridization (WISH) and real‐time PCR methods. We could not detect any alterations in Shh expression by real‐time PCR, or WISH in the non‐treated Pdn/Pdn and VPA‐treated exencephalic Pdn/Pdn embryos. Altered Fgf8 expression patterns were observed in the commissural plate and dorsal isthmal neuroepithelium in the non‐treated Pdn/Pdn embryos. We speculated that the altered expression of Fgf8 might be the result of down‐regulation of Gli3 in Pdn/Pdn embryos. Fgf8 gene expression in the commissural plate and dorsal isthmal neuroepithelium exhibits wide or altered signal patterns in the VPA‐treated exencephalic Pdn/Pdn embryo. From these findings, it was suggested that down‐regulation of Gli3 gene expression induced the altered expression of Fgf8 in the Pdn/Pdn embryos, and that VPA treatment accelerated the alterations of Fgf8 gene expression in the Pdn/Pdn embryos. It was further speculated that altered expression of Fgf8 in the commissural plate may be the fundamental cause of exencephaly, and that the synergistic effect between gene and drug shown in this experiment may explain the differences of sensitivity in the side‐effects of the drug.

Altered signaling pathway in the dysmorphogenesis of telencephalon in the Gli3 depressed mouse embryo, Pdn/Pdn

ABSTRACT  The responsible gene of genetic polydactyly/arhinencephaly mouse (Pdn/Pdn) is Gli3. Pdn/Pdn exhibits absence of the olfactory bulb, suggesting telencephalic dysmorphogenesis. It has been cleared that a transposon was inserted into intron 3 of the Gli3 gene in the Pdn mouse. Adequate PCR primers in the intron 3 and transposon allowed us to discriminate +/+, Pdn/+ and Pdn/Pdn embryos. After genotyping of the Pdn embryos using genomic DNA from the yolk sac membrane, gene expressions in the embryo proper were analyzed by DNA microarray, real‐time PCR and whole‐mount in situ hybridization (WISH) methods. DNA microarray detected 368 depressed and 425 over‐expressed genes in the Pdn/Pdn mouse embryos on day 9 of gestation. In these genes, six signaling pathway and 20 transcription factor genes were included. From these genes, we further investigated Gli3, Emx2, Wnt8b and Wnt7b gene expressions using real‐time PCR and WISH, and depression of these gene expression amounts and altered expression patterns were confirmed. Although alterations of Shh and Fgf8 gene expressions were not detected in the DNA microarray, as these genes have been closed up in the telencephalic morphogenesis, we investigated these gene expressions by real‐time PCR and WISH. Shh gene expression amount and pattern were not changed. Alteration of Fgf8 gene expression amount was not detected also in the real‐time PCR, but altered expression pattern was detected in the Pdn/Pdn embryos by WISH. From the present data, we suggested that Emx2, Wnt8b, Wnt7b and Fgf8 are the important Gli3 signaling pathway in the morphogenesis of telencephalon.