ANTISENSE OLIGONUCLEOTIDE DOWN-REGULATION OF E-CADHERIN IN THE YOLK-SAC AND CRANIAL NEURAL-TUBE MALFORMATIONS
ANTISENSE OLIGONUCLEOTIDE DOWN-REGULATION OF E-CADHERIN IN THE YOLK-SAC AND CRANIAL NEURAL-TUBE MALFORMATIONS
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DOI：
10.1095/biolreprod53.5.1229
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发表时间：
1995-11-01
影响因子：
3.6
通讯作者：
HALES, BF
中科院分区：
文献类型：
--
作者：
CHEN, BY;HALES, BF
钙黏蛋白是一类依赖钙的细胞黏附分子家族，在发育过程中受到空间和时间上的调控。上皮钙黏蛋白（E - 钙黏蛋白）在器官发生过程中存在于胚胎和卵黄囊的上皮细胞中。在发育的这个阶段破坏E - 钙黏蛋白表达的后果了解甚少。我们在此报道我们关于反义寡核苷酸对大鼠全胚胎培养系统中E - 钙黏蛋白影响的研究。四种18个碱基的单链硫代磷酸寡脱氧核苷酸（AS - 寡核苷酸），与小鼠E - 钙黏蛋白cDNA序列的不同区域互补，溶解在盐水中并注射到具有5 - 7个体节的大鼠胚胎的羊膜腔中；寡核苷酸1的正义链（S - 寡核苷酸）、一个18个碱基的随机序列寡核苷酸（C - 寡核苷酸）以及磷酸盐缓冲液（PBS）用作对照。胚胎培养长达45小时；检查胚胎形态和E - 钙黏蛋白的相对浓度。当每个胚胎注射25到50皮摩尔的寡核苷酸时，所有六种寡核苷酸（AS - 寡核苷酸和对照寡核苷酸）都诱导畸形。所有寡核苷酸诱导的畸形包括颅面发育不全、心包增大、脊髓扭曲、菱脑肿胀和前肢发育不良。注射AS - 寡核苷酸1（其序列从翻译起始密码子（ATG）下游第10个碱基开始），导致畸形胚胎，其中颅神经管畸形的发生率很高。通过蛋白质印迹分析检查了AS - 寡核苷酸1对E - 钙黏蛋白和神经（N） - 钙黏蛋白相对丰度的影响。在暴露于AS - 寡核苷酸1的畸形胚胎中，注射后24小时内E - 钙黏蛋白和N - 钙黏蛋白的相对丰度没有改变；注射后45小时胚胎中E - 钙黏蛋白和N - 钙黏蛋白浓度降低。相反，在注射AS - 寡核苷酸1后1 - 2小时，卵黄囊中E - 钙黏蛋白的相对丰度降低，并在4小时恢复到对照水平。S - 寡核苷酸1没有引起E - 钙黏蛋白或N - 钙黏蛋白相对丰度的任何变化。因此，在暴露于反义（AS - 寡核苷酸1）的胚胎卵黄囊中存在E - 钙黏蛋白表达的组织特异性和暂时性“敲低”；卵黄囊E - 钙黏蛋白的下调似乎导致胚胎中神经管缺陷的诱导。在培养中使整个胚胎暴露于反义寡核苷酸提供了一个模型系统，在该系统中可以阐明发育上重要的分子的作用及其对胚胎发生的空间和时间贡献。
The cadherins are a family of calcium-dependent cell adhesion molecules that are regulated both spatially and temporally during development. Epithelial cadherin (E-cadherin) is present in epithelial cells in both the embryo and yolk sac during organogenesis. The consequences of disrupting the expression of E-cadherin at this stage of development are poorly understood. We report here our studies on the effects of antisense oligonucleotides on E-cadherin in the rat whole embryo culture system. Four 18-base single strand phosphorothioate oligodeoxynucleotides (AS-oligos), complementary to Various regions of the mouse E-cadherin cDNA sequence, were dissolved in saline and injected into the amniotic cavities of 5-7 somite rat embryos; a sense (S-oligo) to oligo-1, an 18-base random sequence oligo (C-oligo), and PBS were used as controls. Embryos were cultured for up to 45 h; embryo morphology and the relative concentrations of E-cadherin protein were examined. All six oligonucleotides (AS-oligos and control oligos) induced malformations when amounts ranging from 25 to 50 pmol of oligonucleotide were injected per embryo. The malformations induced by all the oligos included craniofacial hypoplasia, an enlarged pericardium, twisted spinal cord, swelling of the rhombencephalon, and underdeveloped forelimb. injection of AS-oligo-1, a sequence starting at the tenth base downstream from the translation initiation codon (ATG), resulted in malformed embryos with a high incidence of cranial neural tube malformations. The effects of AS-oligo-1 on the relative abundance of E- and neural (N)-cadherin proteins were examined by Western blot analysis. In the AS-oligo-1-exposed malformed embryos, the relative abundance of E-and N-cadherin proteins was not altered up to 24 h after injection; E- and N-cadherin concentrations in the embryo were decreased at 45 h postinjection. In contrast, the relative abundance of the E-cadherin protein in the yolk sac was reduced at 1-2 h after injection of AS oligo-1 and returned to control levels by 4 h. S-oligo-1 did not induce any change in the relative abundance of E- or N-cadherins. Thus, there was a tissue-specific and temporary ''knockdown'' of E-cadherin expression in the yolk sac of embryos exposed to antisense (AS-oligo-1); the down-regulation of yolk sac E-cadherin appears to lead to the induction of neural tube defects in the embryo. The exposure of whole embryos in culture to antisense oligonucleotides provides a model system in which the roles of developmentally important molecules and their spatial and temporal contributions to embryogenesis can be elucidated.