Zambrowicz B P, Zimmermann J W, Harendza C J, Simpson E M, Page D C, Brinster R L, Palmiter R D
Howard Hughes Medical Institute, University of Washington SL-15, Seattle 98195.
Development. 1994 Jun;120(6):1549-59. doi: 10.1242/dev.120.6.1549.
The Zfy-1 and Zfy-2 genes, which arose by gene duplication, map to the mouse Y chromosome and encode nearly identical zinc-finger proteins. Zfy-1 is expressed in the genital ridge and adult testis and likely encodes a transcription activator. Although potential roles in sex determination and spermatogenesis have been hotly debated, the biological functions of Zfy-1 remain unknown. To study the gene's regulation, transgenes with 21-28 kb of Zfy-1 5' flanking DNA placed upstream of lacZ were constructed in plasmids or created by homologous recombination of coinjected DNA molecules. The resulting transgenic mice expressed beta-galactosidase in the genital ridge of both males and females starting between embryonic day 10 and 11 (E10-E11), peaking at E12-E13 and then declining to low levels by E15, a pattern that matches Zfy-1 mRNA as detected by RT-PCR. This lacZ expression in genital ridge was confined to somatic cells as demonstrated by its absence from the alkaline phosphatase-positive germ cells. It had been reported previously that Zfy-1 mRNA was absent from the embryonic gonad of homozygous W(e) embryos, which virtually lack germ cells. By contrast, we observed normal expression of the Zfy-1/lacZ transgene when introduced into the W(e) background, suggesting that germ cells are not necessary for expression. In the adult, the Zfy-1/lacZ transgene is expressed abundantly in developing germ cells. Extragonadal (kidney, meninges, arteries, choroid plexus) expression of the transgene was also observed in embryos. A smaller transgene with only 4.3 kb of Zfy-1 5' flanking DNA was expressed only in germ cells of adult mice. These results suggest that an enhancer for germ cell expression in the adult lies near the Zfy-1 promoter and that an enhancer for expression in the somatic cells of the embryonic gonad is located further 5'.
Zfy-1和Zfy-2基因是通过基因复制产生的,定位于小鼠Y染色体上,编码几乎相同的锌指蛋白。Zfy-1在生殖嵴和成年睾丸中表达,可能编码一种转录激活因子。尽管其在性别决定和精子发生中的潜在作用一直存在激烈争论,但Zfy-1的生物学功能仍然未知。为了研究该基因的调控,构建了将21 - 28 kb的Zfy-1 5'侧翼DNA置于lacZ上游的转基因质粒,或通过共注射DNA分子的同源重组来构建。所产生的转基因小鼠在胚胎第10至11天(E10 - E11)开始在雄性和雌性的生殖嵴中表达β-半乳糖苷酶,在E12 - E13达到峰值,然后在E15降至低水平,这种模式与通过RT-PCR检测到的Zfy-1 mRNA相匹配。如碱性磷酸酶阳性生殖细胞中不存在该表达所示,生殖嵴中的这种lacZ表达局限于体细胞。先前有报道称,纯合W(e)胚胎的胚胎性腺中不存在Zfy-1 mRNA,这些胚胎实际上缺乏生殖细胞。相比之下,当将Zfy-1/lacZ转基因引入W(e)背景时,我们观察到其正常表达,这表明生殖细胞对于表达不是必需的。在成年小鼠中,Zfy-1/lacZ转基因在发育中的生殖细胞中大量表达。在胚胎中还观察到该转基因在性腺外(肾脏、脑膜、动脉、脉络丛)的表达。一个仅含4.3 kb Zfy-1 5'侧翼DNA的较小转基因仅在成年小鼠的生殖细胞中表达。这些结果表明,成年生殖细胞表达的增强子位于Zfy-1启动子附近,而胚胎性腺体细胞表达的增强子位于更上游5'处。