Regulation of neural crest cell proliferation, differentiation and death in normal development and diseases

正常发育和疾病中神经嵴细胞增殖、分化和死亡的调节

基本信息

批准号：
18590284
负责人：
HATANO Masahiko
金额：
$ 2.57万
依托单位：
Chiba University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2006
资助国家：
日本
起止时间：
2006 至 2007
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-18590284/
关键词：
Neural Crest cells Transcription factor Knockout mice Ncx Nczf Cell proliferation 腸管神経細胞標的遺伝子 zinc finger蛋白

项目摘要

We have used chromatin immunoprecipitation to screen for target genes for Ncx (Tlx2, Enx, Hox11 L1). This screen led to the identification of novel KRAB zinc finger protein termed Nczf. Nczf is co-expressed with Ncx in enteric neurons. Promoter region of Nczf gene contains 4 consensus sequences for Ncx binding motifs. The luciferase reporter gene analysis with various amount of Ncx expression vector showed dose dependent increase of the Nczf promoter activity. The Nczf mRNA expression was analyzed in mouse development. The mRNA was detected in 7.5 days of embryogenesis (E7.5) and was maximal at E9.5 by RT-PCR. The expression was further examined in various tissues from embryos and newborns by in situ hybridization. It was detected throughout the body between E7.5 and E9.5. High level of expression was detected in neuroepithelium at E10.5, and subsequently in the ventricular zone of central nervous system from E12.5. Strong signal was also detected in external granular layer of the developing cerebellum at P7. Nczf mRNA was also expressed in postmitotic neurons of the cranial ganglia and dorsal root ganglia, and pachytene stage spermatocytes at first meiotic cell division in spermatogenesis. These results suggest that Nczf plays a crucial role in rapid cell proliferation at early organogenesis as well as neuronal cell differentiation and meiotic cell division in spermatogenesis.In order to elucidate the function of Nczf, we generated Nczf deficient (KO) mice by homologous recombination. Nczf KO mice were embryonic lethal and died around E8.5-9.5. Cell numbers are reduced and many apoptotic cells were observed. Organogenesis took place normally but embryos could not turn their body as normally occurred in E8.5. We are now investigating the function of Nczf using these KO mice and also creating conditional KO mice using Cre-LoaP system.

我们已经使用染色质免疫沉淀来筛选NCX的靶基因（TLX2，ENX，HOX11 L1）。该屏幕导致鉴定新型的Krab锌指蛋白称为NCZF。 NCZF与肠神经元中的NCX共表达。 NCZF基因的启动子区域包含4个用于NCX结合基序的共有序列。具有不同量的NCX表达载体的荧光素酶报告基因分析显示NCZF启动子活性的剂量依赖性增加。在小鼠发育中分析了NCZF mRNA的表达。在7.5天的胚胎发生（E7.5）中检测到mRNA，并通过RT-PCR在E9.5时最大。通过原位杂交从胚胎和新生儿的各种组织中进一步检查了该表达。在E7.5和E9.5之间检测到它。在E10.5的神经上皮中检测到高度表达，随后在E12.5的中枢神经系统心室中检测到。在P7发育中的小脑的外部颗粒层中也检测到了强信号。 NCZF mRNA也在颅神经节和背根神经节的有丝分裂后神经元中表达，在精子发生中首次减数分裂细胞分裂的pachytene阶段精子细胞。这些结果表明，NCZF在早期器官发生的快速细胞增殖以及精子发生中的神经元细胞分化和减数分裂细胞分裂中起着至关重要的作用。为了阐明NCZF的功能，我们通过同源性重组产生了NCZF缺乏症（KO）小鼠。 NCZF KO小鼠是胚胎致死的，死于E8.5-9.5。细胞数减少，观察到许多凋亡细胞。器官发生通常发生，但胚胎无法转动其身体，因为通常在E8.5中发生。我们现在正在使用这些KO小鼠研究NCZF的功能，并使用CRE-LOAP系统创建有条件的KO小鼠。