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

我们已经使用染色质免疫沉淀来筛选Ncx的靶基因（Tlx 2，Enx，Hox 11 L1）。该筛选导致鉴定了称为Nczf的新型KRAB锌指蛋白。Nczf在肠神经元中与Ncx共表达。Nczf基因的启动子区包含4个Ncx结合基序的共有序列。用不同量的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。在小鼠发育过程中分析Nczf mRNA表达。RT-PCR检测到该mRNA在胚胎发生的7.5天（E7.5），并在E9.5最大。通过原位杂交进一步检测胚胎和新生儿各种组织中的表达。在E7.5和E9.5之间在整个身体中检测到。E10.5时在神经上皮中检测到高水平的表达，随后从E12.5开始在中枢神经系统的脑室区中检测到高水平的表达。在P7时，发育中小脑的外颗粒层也检测到强信号。Nczf mRNA也表达于颅神经节和背根神经节的有丝分裂后神经元，以及精子发生中第一次减数分裂的粗线期精母细胞。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细胞分化和减数分裂细胞分裂中起着至关重要的作用。Nczf KO小鼠是胚胎致死的，并在E8.5-9.5左右死亡。细胞数量减少，并观察到许多凋亡细胞。器官发生正常，但胚胎不能像E8.5中正常发生的那样转动身体。我们现在正在使用这些KO小鼠研究Nczf的功能，并使用Cre-LoaP系统创建条件性KO小鼠。

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.