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Developmental Biotechnological Analysis of Craniofacial Malformation in Mutant Rat and Mouse.

突变大鼠和小鼠颅面畸形的发育生物技术分析。

基本信息

批准号：
06454520
负责人：
ETO Kazuhiro
金额：
$ 4.54万
依托单位：
Tokyo Medical and Dental University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1994
资助国家：
日本
起止时间：
1994 至 1996
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-06454520/
关键词：
Uchida Rat (rSey)Transgenic Mouse Mammalian Whole Embryo Culture Pax-6 gene HIV gene Neural crest cells Craniofacial Malformation A Long-Term Culture 顎顔面奇形ミュータントラット(rSey)哺乳類全胚培養法脳神経顎顔面形態異常

项目摘要

1 Analysis of Uchida Rat (rSay)Pax-6 gene is a member of the Pax family of vertebrate developmental genes that hae the conserved "paired box motif", which is originally defined in Drosophila segmentation genes. Mutations that reduce Pax-6 dosage cause dominantly inherited eye malformations in the mouse, rat, and human. Uchida rat (rSey) is a mutant rat spontaneously found in the colony of Sprague-Dowley rats, and has a mutation in an exon of Pax-6 gene, resulting in generation of truncated transceipts.(1) We found a new type of Uchida rat (rSay^2) that has a mutation in an exon which is down stream of paired box.(2) From Dil labeling study, neural crest cells originated from the anterior midbrain, which migrate toward the frontonasal mass via the eye rudiment in normal embryos, do not reach the frontonasal region but stay at the area dorsal the optic vesicle in rSey/rSey embryos. Pax-6 gene is not expressed in the crest cells and orthotopic injection of normal midbrain crest cells into … More homozygous host embryos have confirmed that migration defect in rSey homozygotes is caused by abnormal environment to which midbrain crest cells encounter.(3) We revealed, from recombination experiments using the presumptive lens ectoderm and optic cup obtained from homozygous and/or wild type embryos, taht the filure of lens differentiation in the mutant is due to the ectoderm ; the ectoderm taken from the homozygous mutant cannot respond to the signal from the optic cup, while the homozygous optic cup can induce lens in the wild type ectoderm.(4) In the ethmoid region, the nasal septum and the derivative of the medial nasal prominence were present, while the rest of the nasal capsule, as well as the nasal and lachrymal bones, were totally absent except for a pir of cartilaginous rods in place of the nasal capsule. This suggest that the primary cranial defect is restricted to the lateral nasal prominence (LNP) derivatives.(5) Pax-6 gene expression patterns in the central nervous systems are spatially and temporally complex. In accordance, various structural devects were observed in the brain of the Uchida rat. These included lack of the olfactory bulb, lateral olfactory tract, and commissura rostralis, 'as well as deformity of the cerebral and cerebellar cortex, and of the thalamus.(6) We found impaired development of the peripheral nervous system in the Uchida rat. First, the homozygous ophthalmic nerve (N.V1) shows abnormal feature of innervation ; it is not well fasciculated and strays into ectopic regions. It is suggested that this abnormality is indirectly caused by the failure in formation of LNP,the structure being innervated by N.V1 in normal development. The other branches of the trigeminal ganglion did not seem to be affected in the homozygote. More interestingly, two cranial motor nerves, N.abducens (N.VI) and N.hypogossal (N.XII) nerves, whose nuclei lie at the postotic levels of the hidbrain, are absent in homozygous embryos, though spinal nerves (C1 and C2) appear to innervate the tongue muscles. Histological observation of homozygotes shows that Islet-1+/MAP1+cells are observed in the otic hindbrain but that they do not sprout axons out of the neural tube. Thus, Pax-6 is most likely to be involved in development of some motor neurons.2 alysis of HIV Transgenic Mouse.In order to elucidate the genetic effects on the growth and development of craniofacial regios, HIV I transgneic mice (TG mice) which exhibited the phenotype of anterior crossbites were investigated by various methods. Northern hybridization showed that the introduced gene was expressed from 14.5 days post coitum to adult. In morphological measurements using digital converter "GRADICON,significant differences betwee TG mice and/wild type mice (WT mice) were found at 5 weeks of age (5W) for most measurement items. Especially at 7W,the results indicated remarkable reductions in maxilla and catch up growth in the mandlble. On the other hand, the serrum alkaline phosphatase activity of TG mice (3W,5W,and 7W) was significantly higher than that of WT mice, and also the low level of inorganic phosphate was detected at 3W for TG mice. These results suggested that TG mice had a disorder of bone metabolism. Other TG mouse lines carrying the same DNA construct showed normal figures in the craniofacial regions, suggesting the possibility that the phenotype of anterior crossbites and the disorder of bone metabolism were not caused by functions of HIV-1 genome but by insertional mutation in craniofacial development. Less

内田大鼠（rSay）Pax-6基因是脊椎动物发育基因Pax家族的一名成员，该基因具有保守的“配对盒基序”，最初是在果蝇分割基因中定义的。在小鼠、大鼠和人类中，减少Pax-6剂量的突变主要导致遗传性眼睛畸形。内田大鼠（rSey）是一种在Sprague-Dowley大鼠群体中自发发现的突变大鼠，其Pax-6基因外显子发生突变，导致产生截短的光导细胞。(1)我们发现了一种新的内田大鼠（rSay^2），它在配对盒的下游有一个外显子突变。(2)从Dil标记研究来看，神经嵴细胞起源于前中脑，在正常胚胎中通过眼基底向额鼻肿块迁移，但在rSey/rSey胚胎中没有到达额鼻区，而是停留在视神经泡背侧区域。Pax-6基因在顶骨细胞中不表达，将正常的中脑顶骨细胞原位注射到宿主胚胎中，证实了rSey纯合子的迁移缺陷是由中脑顶骨细胞所处的异常环境引起的。(3)通过对纯合子和/或野生型胚胎的晶状体外胚层和视杯进行重组实验，我们发现突变体晶状体分化的失败是由于外胚层；纯合子突变体的外胚层对光学杯的信号没有反应，而纯合子的光学杯能在野生型外胚层诱导出晶状体。(4)在筛区，鼻中隔和中间鼻突的衍生物存在，而鼻囊的其余部分，以及鼻骨和泪骨，除了一组软骨棒代替鼻囊外，完全不存在。这表明原发性颅骨缺损局限于外侧鼻突（LNP）衍生物。(5) Pax-6基因在中枢神经系统中的表达模式具有时空复杂性。据此，内田大鼠脑内观察到各种结构缺陷。这些症状包括缺少嗅球、侧嗅束和鼻交，以及大脑、小脑皮层和丘脑的畸形。(6)我们发现内田大鼠周围神经系统发育受损。一是纯合子眼神经（N.V1）神经支配异常；它不是很好地束状，并误入异位区域。提示这种异常是由LNP的形成失败间接引起的，LNP是在正常发育中由N.V1支配的结构。三叉神经节的其他分支在纯合子中似乎没有受到影响。更有趣的是，尽管脊神经（C1和C2）似乎支配舌肌，但在纯合子胚胎中却没有两种颅运动神经，即外展神经（N.VI）和下颌骨神经（N.XII），它们的细胞核位于后脑水平。纯合子的组织学观察表明，胰岛1+/MAP1+细胞存在于后脑，但它们不从神经管中长出轴突。因此，Pax-6很可能参与了一些运动神经元的发育。2 . HIV转基因小鼠分析。为了阐明基因对颅面区生长发育的影响，采用多种方法对具有前交叉咬表型的HIV转基因小鼠（TG）进行了研究。Northern杂交表明，该基因从交媾后14.5天开始表达到成虫。在数字转换器GRADICON的形态学测量中，TG小鼠和野生型小鼠（WT小鼠）在5周龄（5W）时的大多数测量项目存在显著差异。特别是在7W时，结果显示上颌骨明显减少，肩胛迎头增长。另一方面，TG小鼠（3W、5W和7W）血清碱性磷酸酶活性显著高于WT小鼠，并且在3W时检测到低水平的无机磷酸盐。这些结果提示TG小鼠存在骨代谢紊乱。其他携带相同DNA结构的TG小鼠系在颅面区域显示正常，这表明前交叉咬的表型和骨代谢紊乱可能不是由HIV-1基因组功能引起的，而是由颅面发育中的插入突变引起的。少