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

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.     

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

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.     

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

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.     

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

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.     

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.     

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.
In the present study, we demonstrated that 5442 bp of early retrotransposon was inserted into intron 3 of Gli3 gene in the Pdn mouse (Gli3^Pdn). This transposon had almost the same sequence as MMY17106 (EMBL). It had 317-bp long terminal repeat at both ends followed by the identical 6-bp target duplication sequence, GAGACT. Forward and reverse PCR primers were constructed in intron 3 near the insertion point, and a forward primer in the transposon was also constructed. These primers allowed us to discriminate +/+, Pdn /+ and Pdn/Pdn embryos by the PCR products. Morphological determination of the genotypes in the Pdn mouse embryos is impossible before day 12 of gestation. Quick discrimination method of genotypes developed in the present study allows us to investigate the early dysmorphogenetic mechanisms in the brain and limbs in the Pdn/Pdn embryos. Then, the dysmorphogenetic mechanisms in the Pdn/Pdn may be extrapolated to those in GCPS.     

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

ABSTRACT  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, Xt^J/Xt^J 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 Xt^J/Xt^J mice. The Xt^J/Xt^J 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 Xt^J/Xt^J mice.     

The Role of Apoptosis in the Manifestation of Polydactyly and Arhinencephaly in Genetic Mutant Mouse Pdn/Pdn*

Abstract Pdn/Pdn mouse exhibits preaxial polydactyly and arhinencephaly, including various brain abnormalities such as the absence of corpus callosum and hydrocephaly. We have investigated the mechanism of the manifestation of polydactyly. The abolishment of the deep preaxial mesodermal programmed cell death, foyer primaire préxial (fpp), has been considered to be the starting point of the manifestation of preaxial polydactyly. With this hypothesis, we electrocauterized the fpp region of Pdn/Pdn embryos, because Pdn/Pdn lacks fpp. After surgery, Pdn/Pdn embryos were developed using whole embryo culture system or exo utero method. They exhibited 5 digits in their limbs which received surgery in fpp region. Meanwhile, much more apoptotic degenerations were observed in the brains of Pdn/Pdn newborns and embryos than normals. Anti-single-stranded DNA antibody was used to detect the localization of apoptotic cell death in the brains of Pdn/Pdn mice. TRPM-2 gene has been considered to be an indicator of apoptosis. The brains of Pdn/Pdn newborns and embryos showed higher TRPM-2 gene expression in Northern blot analysis. From these results, we speculated that abnormal apoptosis in the periventricular zone induced the expansion of the ventricle followed by hydrocephaly.     

Shh与Wnt5a基因在Cornelia De Lange综合征中的表达及意义

目的 通过研究Shh、Wnt5a基因在NIPBL^+/-胎鼠肢芽中的表达情况,探讨两基因与CdLS综合征的相关性。方法 实验组取NIPBL^+/-胎鼠肢芽（n=36）和对照组取NIPBL^+/+胎鼠肢芽（n=36）,分别于妊娠（E）10 d、E11 d、E12 d （n=12）取胎鼠肢芽组织,应用real-time PCR、Western blot技术分别检测Shh、Wnt5a基因转录及蛋白表达水平。结果 E10 d、E11 d、E12 d时,Shh、Wnt5a基因及其蛋白在两组胎鼠肢芽中均有表达,其表达水平在实验组与对照组比较均降低（P < 0.01）。实验组和对照组胎鼠肢芽内Shh、Wnt5a基因及其蛋白表达水平在E10 d时均较少,E11 d时升高,随后E12 d时表达水平减少,且低于E10 d时的水平（P < 0.01）。结论 Shh及Wnt5a基因与其相关蛋白水平表达存在一致性;CdLS综合征的发病可能与NIPBL基因低表达,从而抑制Shh及Wnt5a基因及其蛋白的表达相关。     

邻苯二甲酸二丁酯致雄性仔鼠肛门直肠畸形与相关基因表达的研究

目的 邻苯二甲酸二丁酯(di-n-butyl phthalate,DBP)孕晚期染毒诱导子代雄性大鼠发生肛门直肠畸形(anorectal malformations,ARMs),探讨形态学变化及相关基因的表达.方法 孕鼠20只,随机分为两组,在怀孕天数第12～18天,每日分别给予DBP及大豆油850mg/kg灌胃.出生后(PND)1d统计雄性仔鼠肛门闭锁发生率,测量雄性仔鼠体重和肛门生殖器距离(AGD),观察肛门闭锁雄性仔鼠末端直肠及肛周病理学改变.Real-time PCR法检测雄性仔鼠末端直肠组织中Shh、Gli2、Gli3、Bmp4、Wnt5a、Hoxa13、Hoxd13、Fgf10、Fgfr2基因mRNA的表达水平.结果 与对照组相比,DBP染毒组母鼠孕期体重增量减少,孕期延长,仔鼠产量降低(P＜0.05),PND 1d肛门闭锁雄性仔鼠体重及AGD较对照组明显降低(P＜0.05).染毒组雄性仔鼠肛门闭锁发生率大约为39.5％.肛门闭锁雄性仔鼠PND1末端直肠及肛周组织病理显示典型的ARMs.与对照组相比,肛门闭锁雄性仔鼠PND1末端直肠中Shh、Gli2、Gli3、Bmp4、Wnt5a、Hoxa13、Hoxd13、Fgf10、Fgfr2的mRNA表达水平较对照组也明显降低(P＜0.05).结论 DBP孕晚期染毒能明显诱导雄性仔鼠ARMs发生,并对其生长发育具有不良影响,其末端直肠组织中与肛门直肠分化发育相关的基因Shh、Gli2、Gli3、Bmp4、Wnt5a、Hoxa13、Hoxd13、Fgf10、Fgfr2的mRNA的表达水平降低可能促进ARMs的发生.     

肛门直肠畸形胎鼠直肠末端Gli2、BMP4基因的表达

目的探讨乙烯硫脲（ETU）致畸胎鼠直肠末端Gli2、BMP4基因在胎鼠直肠肛门及其畸形发生过程中的表达和作用。方法取妊娠SD大鼠30只,按受孕时间配对分成2组,实验组（n=15）于孕10d灌胃注入10g／LETU （125mg／kg）,对照组n=15）予等量蒸馏水。两组分别于孕13d、14d、15d、16d和17d剖宫取胎鼠直肠末端1cm提取RNA,采用RT—PCR和RealtimePCR法检测Gli2、BMP4基因在直肠肛门发育不同时期的表达情况。结果采用RT-PCR法检测Gli2、BMP4基冈在直肠肛门发育不同时期的表达情况．发现在胎鼠第13～17天,实验组直肠末端Gli2、BMP4mRNA的表达水平均明显低于正常胎鼠直肠末端（P〈0．05）。而Realtime PCR检测Gli2、BMP4mRNA表达时,发现实验组Gli2的表达在胚胎第13天、第14天、第15天分别为（0．73＋0．07、0．60＋0．09、0．81＋0．06）,勺对照组相比显著降低（P〈0．05）,而在孕第16天、第17天实验组Gli2表达比对照组虽有下降,但无统计学意义;实验组BMP4的表达在孕第3～17天均较对照组下降,差异有统计学意义。结论直肠肛门的正常发育需要Shh信号通路的正常表达,Shh信号通路中转录因子Gli2和靶基囚BMP4的异常表达在肛门直肠畸形发生过程中起重要作用,ETU可能影响Shh信号转导通路和肛门直肠畸形发生。     

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 Bouin's solution and were subsequently decalcified in 0.5 mol/L of EDTA solution, paraffin sectioned, and stained with hematoxylin and eosin.
Ink spread into the 3rd and right lateral ventricles and flowed to the 4th ventricle and Magendie's foramen rapidly in Pdn/Pdn mice. This rapid spread was due to the dilatation of the interventricular foramen and that the lateral ventricle was directly connected with enlarged 3rd ventricle in Pdn/Pdn. In spite of the rapid spread of ink in the cerebrospinal fluid pathway, ink was not observed in the subarachnoid space around the superior sagittal sinus at 3.5 or 10 hours in Pdn/Pdn mice.
The superior sagittal sinus was narrower in Pdn/Pdn than in +/+, and the arachnoid villi were not observed in Pdn/Pdn. From these observations, we suggested that absorption of cerebrospinal fluid from the arachnoid villi in the superior sagittal sinus stagnated and that stagnation of the fluid in the ventricles was the cause of hydrocephalus in Pdn/Pdn mice.     

Cross-regulatory interactions between Fgf8 and Shh in the avian frontonasal prominence

The frontonasal prominence of the developing avian embryo contains an organizing center, defined by juxtaposition of the Sonic hedgehog (Shh) and Fibroblast growth factor 8 (Fgf8) expression domains. This molecular interface presages any detectable growth of the frontonasal prominence, and experiments involving transplantation of this boundary epithelium have demonstrated it is a source of dorsal-ventral and rostral-caudal patterning information for the neural crest-derived mesenchyme of the upper beak. We explored the ontogeny of this organizing center by mapping the expression domains of both genes and their receptors and downstream targets. We tested the extent to which Shh and Fgf8 regulate each other's expression in this frontonasal organizer by either blocking or ectopically activating these pathways. Our experiments revealed mutual antagonism between the two molecules, which aids in establishing and maintaining a molecular boundary that subsequently influences patterning and growth of the middle and upper face.     

Prevention of Genetic Polydactyly in Polydactyly Nagoya (Pdn) Mice in Vitro by Surgical Treatment of Foot Plate during Embryogenesis

Abstract Pdn/Pdn fetuses show preaxial Polydactyly of duplicated or triplicated metacarpal/metatarsal type in the fore- and hindlimbs. Pdn /+ fetuses show one extra digit of distal phalangeal type preaxially in the hindlimb and deformity of distal phalanx of the 1st digit in the forelimb. Normal patterns of physiological cell death in the preaxial apical ectodermal ridge (AER) and the deep preaxial mesoderm (fpp) were disrupted in Pdn/Pdn embryos. It was supposed that delayed involution of preaxial AER might have caused the abolishment of fpp, which in turn could have induced polydactyly in Pdn/Pdn .
In order to induce cell death in the fpp region artificially, tissue destruction of the fpp region of right fore- and hindfoot plates of Pdn/Pdn , Pdn /+ and +/+ embryos was performed by an electric knife on day 11.5 of gestation, and the embryos were cultured in the rotator culture system for 20 hours. The non-treated left foot plates served as the controls.
In Pdn/Pdn embryos, the non-treated left foot plates showed abnormal protuberance and/or the extra digital rays preaxially. But the treated right foot plates did not exhibit these abnormal characteristics. Instead they revealed 5 digital rays of mesodermal condensation in the histological sections.
These results indicated that the restriction of the artificial tissue destruction in the fpp region could prevent the manifestation of preaxial extra digits in Pdn mice.     

Effects of thalidomide on Fgf8, Bmp4 and Hoxa11 expression in the limb bud in Kbl:JW rabbit embryos

Thalidomide (TM) induces limb defects in humans and some animal species including rabbits. Although the mechanism of TM‐induced limb defects has been investigated for a long period, the limb development‐related genes expressions have not been vigorously characterized in rabbits. In this study, we investigated the Fgf8, Bmp4 and Hoxa11 expressions in TM‐treated JW rabbit embryos on gestation days (GDs) 10, 11 and 12 by whole mount in situ hybridization. On GDs 10 and 11, growth retardation of the embryo was induced by TM treatment. The Fgf8 expression lengths on GDs 10 and 11 in the forelimb bud were significantly or tended to be decreased in the TM‐treated embryos, which was correlated to the growth retardation and was not considered to be directly relevant to the teratogenic effect of TM in the forelimb. The TM‐induced characteristic changes in the expression pattern of Hoxa11 and Bmp4 on GDs 10 and/or 11 were not noted. On GD 12, TM‐induced growth retardation was not noted and the Fgf8 and Bmp4 expressions were not changed. On the contrary, Hoxa11 expression was narrowed at the anterior region, which was located on the radial side, and was not changed at the middle and posterior regions in the forelimb bud and in all regions in the hindlimb bud. Because the radius malformations were induced by TM treatment, we concluded the decrease in the Hoxa11 expression was related to the TM‐induced limb defects and can be a good marker for early prediction of the teratogenic effect of TM.     

叶酸拮抗剂甲氨喋呤导致斑马鱼心脏发育异常及BMP2b HAS2表达下调

目的：该研究利用叶酸拮抗剂甲氨喋呤（MTX）构建叶酸生物学活性受抑制的斑马鱼模型后，观察叶酸生物学活性受抑后对斑马鱼心脏发育的干扰作用以及对斑马鱼心脏发育相关基因BMP2b及HAS2表达的影响。 方法：用不同浓度的MTX处理不同发育时段的斑马鱼胚胎，于48 hpf（hours post fertilization）观察胚胎心脏发育情况并计数各组心脏发育异常个体的百分比及心率，评定MTX 对斑马鱼心脏发育的影响程度。用1.5×10-3 M的MTX处理6~10 hpf 发育时段的斑马鱼胚胎作为MTX处理组。于24 hpf及48 hpf 在显微镜下观察MTX处理组斑马鱼胚胎心脏发育情况。借助胚胎整体原位杂交和Real-time PCR 的方法检测BMP2b和HAS2在正常对照组及MTX处理组胚胎的表达水平。结果：胚胎早期发育阶段6~12hpf是斑马鱼胚胎对MTX的敏感时期。显微镜下观察结果显示MTX处理组斑马鱼心脏发育延迟，并有心脏形态发育明显异常。胚胎整体原位杂交结果显示MTX处理组斑马鱼心脏发育相关基因BMP2b及HAS2在心脏的表达于36 hpf及48 hpf下调。Real-time PCR 结果显示MTX处理组斑马鱼BMP2b的相对表达量在12，24，36及48 hpf减少，HAS2的相对表达量在24，36及48 hpf减少。结论：叶酸生物学活性受抑对早期胚胎的心脏发育影响较大，可导致斑马鱼心脏发育延迟及心脏形态异常，并下调斑马鱼心脏发育相关基因BMP2b及HAS2的表达，这可能是叶酸生物学活性受抑后导致心脏发育异常的机制之一。［中国当代儿科杂志，2007，9（2）：159-163］     

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), Xt^H/Xt^H (Extra toes) and Xt^J/Xt^J (Extra toes Jackson) are the mouse homolog of GCPS, and Gli3^tmlUrtt/Gli3^tmlUrt 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 , Xt^H/Xt^H , Xt^J/Xt^J and Gli3^tmlUrt/Gli3^tmlUrt , 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.     

Bmp4 is an essential growth factor for the initiation of genital tubercle (GT) outgrowth

The external genitalia are appendage organs outgrowing from the posterior body trunk. Murine genital tubercle (GT), anlage of external genitalia, initiates its outgrowth from embryonic day (E) 10.5 as a bud structure. Several growth factors such as fibroblast growth factor (FGF), Wnt and Sonic hedgehog (Shh) are essential for the GT outgrowth. However, the mechanisms of initiation of GT outgrowth are poorly understood. We previously identified bone morphogenetic protein (Bmp) signaling as a negative regulator for GT outgrowth. We show here novel aspects of Bmp4 functions for GT outgrowth. We identified the Bmp4 was already expressed in cloaca region at E9.5, before GT outgrowth. To analyze the function of Bmp4 at early stage for the initiation of GT outgrowth, we utilized the Hoxa3-Cre driver and Bmp4 ^flox/flox mouse lines. Hoxa3 ^Cre/+; Bmp4 ^flox/flox mutant mice showed the hypoplasia of GT with reduced expression of outgrowth promoting genes such as Wnt5a, Hoxd13 and p63, whereas Shh expression was not affected. Formation of distal urethral epithelium (DUE) marked by the Fgf8 expression is essential for controlling mesenchymal genes expression in GT and subsequent its outgrowth. Furthermore, Fgf8 expression was dramatically reduced in such mutant mice indicating the defective DUE formation. Hence, current results indicate that Bmp4 is an essential growth factor for the initiation of GT outgrowth independent of Shh signaling. Thus, Bmp4 positively regulates for the formation of DUE. The current study provides new insights into the function of Bmp signaling at early stage for the initiation of GT outgrowth.