Tanaka M, Chen Z, Bartunkova S, Yamasaki N, Izumo S
Cardiovascular Division, Beth Israel Deaconess Medical Center, and Department of Medicine, Harvard Medical School, Boston, MA 02215, USA.
Development. 1999 Mar;126(6):1269-80. doi: 10.1242/dev.126.6.1269.
Csx/Nkx2.5 is a vertebrate homeobox gene with a sequence homology to the Drosophila tinman, which is required for the dorsal mesoderm specification. Recently, heterozygous mutations of this gene were found to cause human congenital heart disease (Schott, J.-J., Benson, D. W., Basson, C. T., Pease, W., Silberbach, G. M., Moak, J. P., Maron, B. J., Seidman, C. E. and Seidman, J. G. (1998) Science 281, 108-111). To investigate the functions of Csx/Nkx2.5 in cardiac and extracardiac development in the vertebrate, we have generated and analyzed mutant mice completely null for Csx/Nkx2.5. Homozygous null embryos showed arrest of cardiac development after looping and poor development of blood vessels. Moreover, there were severe defects in vascular formation and hematopoiesis in the mutant yolk sac. Interestingly, TUNEL staining and PCNA staining showed neither enhanced apoptosis nor reduced cell proliferation in the mutant myocardium. In situ hybridization studies demonstrated that, among 20 candidate genes examined, expression of ANF, BNP, MLC2V, N-myc, MEF2C, HAND1 and Msx2 was disturbed in the mutant heart. Moreover, in the heart of adult chimeric mice generated from Csx/Nkx2.5 null ES cells, there were almost no ES cell-derived cardiac myocytes, while there were substantial contributions of Csx /Nkx2.5-deficient cells in other organs. Whole-mount &bgr;-gal staining of chimeric embryos showed that more than 20% contribution of Csx/Nkx2. 5-deficient cells in the heart arrested cardiac development. These results indicate that (1) the complete null mutation of Csx/Nkx2.5 did not abolish initial heart looping, (2) there was no enhanced apoptosis or defective cell cycle entry in Csx/Nkx2.5 null cardiac myocytes, (3) Csx/Nkx2.5 regulates expression of several essential transcription factors in the developing heart, (4) Csx/Nkx2.5 is required for later differentiation of cardiac myocytes, (5) Csx/Nkx2. 5 null cells exert dominant interfering effects on cardiac development, and (6) there were severe defects in yolk sac angiogenesis and hematopoiesis in the Csx/Nkx2.5 null embryos.
Csx/Nkx2.5是一种脊椎动物同源异型盒基因，其序列与果蝇tinman基因具有同源性，tinman基因是背侧中胚层特化所必需的。最近，发现该基因的杂合突变会导致人类先天性心脏病（肖特，J.-J.，本森，D.W.，巴森，C.T.，皮斯，W.，西尔伯巴赫，G.M.，莫克，J.P.，马龙，B.J.，塞德曼，C.E.和塞德曼，J.G.（1998年）《科学》281卷，第108 - 111页）。为了研究Csx/Nkx2.5在脊椎动物心脏和心脏外发育中的功能，我们构建并分析了Csx/Nkx2.5完全缺失的突变小鼠。纯合缺失胚胎在心脏环化后心脏发育停滞，血管发育不良。此外，突变体卵黄囊中血管形成和造血存在严重缺陷。有趣的是，TUNEL染色和PCNA染色显示突变体心肌中既没有凋亡增加也没有细胞增殖减少。原位杂交研究表明，在所检测的20个候选基因中，突变体心脏中ANF、BNP、MLC2V、N - myc、MEF2C、HAND1和Msx2的表达受到干扰。此外，在由Csx/Nkx2.5缺失的胚胎干细胞产生的成年嵌合小鼠心脏中，几乎没有胚胎干细胞来源的心肌细胞，而Csx/Nkx2.5缺陷细胞在其他器官中有大量贡献。嵌合胚胎的整体β - 半乳糖苷酶染色显示，心脏中Csx/Nkx2.5缺陷细胞的贡献超过20%会使心脏发育停滞。这些结果表明：（1）Csx/Nkx2.5的完全缺失突变并未消除心脏的初始环化；（2）Csx/Nkx2.5缺失的心肌细胞中没有凋亡增加或细胞周期进入缺陷；（3）Csx/Nkx2.5调节发育中心脏中几种重要转录因子的表达；（4）Csx/Nkx2.5是心肌细胞后期分化所必需的；（5）Csx/Nkx2.5缺失的细胞对心脏发育产生显性干扰作用；（6）Csx/Nkx2.5缺失的胚胎中卵黄囊血管生成和造血存在严重缺陷。
