Cellphysiological Analysis on Inductive Differentiation using Isolated Early Embryonic Cell-Roles of Inducer Receptor, Gap Junction, Retinoic Acid

使用分离的早期胚胎细胞进行诱导分化的细胞生理学分析-诱导受体、间隙连接、视黄酸的作用

• 批准号: 04454131
• 负责人: TAKAHASHI Kunitaro
• 金额: $ 4.86万
• 依托单位: University of Tokyo
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (B)
• 财政年份: 1992
• 资助国家: 日本
• 起止时间: 1992 至 1993
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-04454131/
• 关键词: Holocynthia embryo; 2-cell system; Neural induction; Tyrosine kinase; bFGF receptor; Gap junction; K252a; TuNaI gene; ホヤ2細胞系; 誘導性神経分化; bFGF; 受容体型チロシンキナーゼ

1) On anterior animal blastomeres isolated from cleavage-arrested Halocynthia 8-cell embryos, the neural inducing effect of bFGF in the concentration range from 10 to 100 ng/ml was confirmed with expression of Na channels as the marker. The neural inducing activity was identical with those by a protease, subtilisin, and by cell-contact in terms of developmental-time dependency and channel expression, indicating existence of a common neural inducer receptor. Cloning of receptor-tyrosine-kinase homologs from the cDNA libraries of Halocynthia unfertilized eggs and 100-cell embryos was carried out by PCR and 12 clones were obtained. Among those clones pTK6, pTK7, pTK9 and pTK11 were highly homologous to those of mammalian FGF receptors.2) Gap junctions between the 2 cell system separated from the cleavage-arrested Halocynthia 8-cell embryo increased or disappeared dependently upon either autonomous epidermal or induced neural differentiation respectively. The disappearance of gap junction … More after neural induction was inhibited and delayd by kinase inhibitor, K252a. Concomitantly the expression of Na and K channels was also delayd. However, in the case of the neurally induced and afterward separated single blastomere no delay of channel expression was observed. It was concluded that K252a specifically inhibited the gap-junctional disappearance and thereby delayd the channel expression. It is suggested that the disappearance of gap junction is caused by the phosphorylation as a result of neural induction while it can control the timing of the functional expression of neural differentiation.3) Na channel gene TuNa I was cloned and demonstrated to be transcribed specifically in neurons within the neural tissue of Halocynthia tadpole larvae by whole-mount in-situ hybridization. The transcription was also observed in the cleavage-arrested embryos. Further the cell-contact dependent transcription was confirmed by using the 2 cell neural induction system separated from the cleavage-arrested Halocynthia 8-cell embryo. Less

1)在从卵裂停滞的Halocynthia 8-细胞胚胎中分离的前动物卵裂球上，以Na通道的表达作为标记，证实了浓度范围为10至100 ng/ml的bFGF的神经诱导作用。的神经诱导活性是相同的蛋白酶，枯草杆菌蛋白酶，并通过细胞接触方面的发展时间依赖性和通道的表达，表明存在一个共同的神经诱导受体。利用PCR技术从盐爪蟾未受精卵和100细胞胚cDNA文库中克隆了受体酪氨酸激酶同源基因，共获得12个克隆。其中pTK 6、pTK 7、pTK 9和pTK 11与哺乳动物FGF受体高度同源。（2）从分裂停滞的盐爪草8-细胞胚胎分离的2-细胞系统之间的缝隙连接分别依赖于自主表皮分化和诱导神经分化而增加或消失。缝隙连接消失 ...更多信息 用激酶抑制剂K252 a抑制和延迟神经诱导。Na、K通道的表达也随之延迟。然而，在神经诱导和随后分离的单个卵裂球的情况下，没有观察到通道表达的延迟。结论：K252 a特异性抑制缝隙连接消失，从而延迟通道表达。结果表明，间隙连接的消失是由神经诱导的磷酸化引起的，但它可以控制神经分化功能表达的时间。3）克隆了Na通道基因TuNa I，并通过整体原位杂交证实其在盐蛙蝌蚪神经组织中特异性转录。在卵裂停滞的胚胎中也观察到了这种转录。此外，细胞接触依赖性转录通过使用从分裂停滞的盐辛西娅8细胞胚胎分离的2细胞神经诱导系统来证实。少

项目成果

Okamura,Y.: "Neural induction suppresses early expression of the inward-rectifier K^+ channel in the ascidian blastomere." J.Physiol.(London). 463. 245-268 (1993)

Okamura,Y.：“神经诱导抑制海鞘卵裂球中内向整流 K^ 通道的早期表达。”

Inazawa,T.: "Effect of bFGF and protein tyrosine kinase inhibitors on the differentiation of ascidian ectodermal blastomeres." Jap.J.Physiol.42. S120 (1992)

Inazawa,T.：“bFGF 和蛋白酪氨酸激酶抑制剂对海鞘外胚层卵裂球分化的影响。”

Okamura,Y.: "The ascidian embryo as a prototype of vertebrate neurogenesis." BioEssays. 15. 1-8. (1993)

Okamura,Y.：“海鞘胚胎作为脊椎动物神经发生的原型。”

Okamura, Y.Shigemoto, T.Chong, J.A.Mandel, G: "Transcription of an ascidian sodium channel gene." Jap.J.Physiol.42. S120 (1992)

Okamura，Y.Shigemoto，T.Chong，J.A.Mandel，G：“海鞘钠通道基因的转录。”

Okamura, Y.Okagaki, R.Ono, F.Chong, A.Mandel, G.: "Primary structure and neuronal gene expression of an ascidian sodium channel." Jap.J.Physiol.43, suppl.2. s165 (1993)

Okamura、Y.Okagaki、R.Ono、F.Chong、A.Mandel、G.：“海鞘钠通道的初级结构和神经元基因表达。”