Ichiro Naruse

Extensive brain hemorrhage and embryonic lethality in a mouse null mutant of CREB-binding protein.

CREB-binding protein (CBP) is a transcriptional co-activator which is required by many transcription factors. Rubinstein-Taybi syndrome (RTS), which is an autosomal dominant syndrome characterized by abnormal pattern formation, is associated with mutations in the human CBP gene. Various abnormalities occur at high frequency in the skeletal system of heterozygous Cbp-deficient mice, but some features of RTS such as cardiac anomalies do not, suggesting that some symptoms of RTS are caused by a dominant-negative mechanism. Here we report the characterization of homozygous Cbp-deficient mice. Homozygous mutants died around E10.5-E12.5, apparently as a result of massive hemorrhage caused by defective blood vessel formation in the central nervous system, and exhibited apparent developmental retardation as well as delays in both primitive and definitive hematopoiesis. Cbp-deficient embryos exhibited defective neural tube closure which was similar to those observed in twist-deficient embryos. However, a decrease in the level of twist expression was not observed in Cbp-deficient embryos. Anomalous heart formation, a feature of RTS patients and mice mutated in the CBP-related molecule, p300, was not observed in Cbp-deficient embryos. Since both Cbp and p300 are ubiquitously expressed in embryonic tissues including the developing heart, these results suggest that cardiac anomalies observed in RTS patients may be caused by a dominant negative effect of mutant CBP.

Suckling Dysfunction Caused by Defects in the Olfactory System in Genetic Arhinencephaly Mice

Mouse newborns find their mother’s nipples and suckle milk by themselves. It has been argued which sense organ they use when locating their mother’s nipples to suckle milk. Olfactory or tactile sensory systems are primary candidates. In the present study, we investigated the trigeminal-whisker sensory and olfactory systems in genetic arhinencephaly mouse embryos (Pdn/Pdn). Pdn/Pdn newborns do not suckle milk and die within 1 day after birth. Dysfunction of nipple-searching behavior was clear in Pdn/Pdn newborns. Pdn/Pdn newborns had a complete developmental failure in the olfactory nerve projection to the central nervous system and no olfactory bulb architecture. The trigeminal-whisker system was intact in this strain. From the results of these experiments, it was suggested that the olfactory system is essential for nipple-searching behavior and suckling milk and that the trigeminal-whisker system is not able to substitute for the lack of olfactory input in mouse newborns.

Caudal dysgenesis in staged human embryos: Carnegie stages 16–23

The severity of expression of malformations of the median axis in the caudal region of human embryos is highly variable and ranges from caudal dysgenesis and sirenomelia to simple sacral hypoplasia. Several forms of sacral dysgenesis may be discovered later in life. This shows that caudal malformations of relatively lesser severity should occur at a greater frequency than actually reported. In the present study we looked at the morphology and histology of some human embryos with caudal dysgenesis. Several developmental alterations of the median axis were observed. These included significant reduction in the craniofacial mesenchyme characterized by hypoplasia of the pharyngeal arches, palatal shelves, and agenesis or hypoplasia of the auricular hillocks at the rostral end, absence of the caudal trunk from midsacral to all coccygeal segments, vertebral fusion or agenesis, defective development of the primary and secondary neural tubes, rectal and urinary tract dysgenesis, and deficiency, malrotation, and deficiency of the limbs at the caudal end. Hindlimb malformations included bilateral agenesis (one case), meromelia, and various forms of abnormal rotation, but no instances of sirenomelia were present. Radial dysgenesis has been reported to be associated with caudal dyplasia in the literature, however, we observed agenesis of the ulna in one and of the fibula in another embryo. There was an impressive association between limb malformations and body wall defects. The histological studies demonstrated caudal vascular deficiency and hemorrhagic lesions in the limbs of the dysplastic embryos. The data suggest that these polytopic field defects arise very early in development possibly as result of disturbances to fundamental developmental events that share common molecular and cellular mechanisms.

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 2u2003mg/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

ABSTRACTu2002 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)

ABSTRACTu2002 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.

Phenotypic differences in the brains and limbs of mutant mice caused by differences of Gli3 gene expression levels

ABSTRACTu2002 The genetic polydactyly/arhinencephaly mouse, Pdn/Pdn, exhibits severe polydactyly both in the fore‐and hindlimbs, agenesis of the olfactory bulbs, corpus callosum, anterior commissure, and hydrocephalus. A candidate gene for the Pdn mouse has been speculated to be Gli3, because Pdn has been considered to be an allele of Xt whose responsible gene has been clarified to be Gli3. Recently, it has been cleared that retro‐transposons are inserted into nitron 3, upstream of zinc finger domain, of the Gli3 gene in the Pdn mouse, resulting to the severe suppression of Gli3 gene expression in Pdn/Pdn embryos. Meanwhile, XtJ/XtJ mice exhibit more severe polydactyly than that of Pdn/Pdn. Arhinencephaly and microholoprosencephaly including agenesis of the olfactory bulbs, corpus callosum, anterior commissure, hippocampal commissure, habenular commissure, and posterior commissure, and moreover, the cerebral cortical plates and hippocampus are not formed in the XtJ/XtJ mice. The XtJ/XtJ mouse has a large deletion in Gli3 structural gene and shows null expression. From these corroborations, we speculated that the differences in the Gli3 gene expression levels resulted in the phenotypic differences between the Pdn/Pdn and XtJ/XtJ mice.

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

ABSTRACTu2003 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 2u2003mg/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.

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

ABSTRACTu2003 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.