Molecular cloning and expression of dead end homologue in chicken primordial germ cells
Molecular cloning and expression of dead end homologue in chicken primordial germ cells
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DOI：
10.1007/s00441-007-0435-1
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发表时间：
2007-10-01
影响因子：
3.6
通讯作者：
Hattori, Masa-Aki
中科院分区：
文献类型：
--
作者：
Aramaki, Shinya;Sato, Fuminori;Hattori, Masa-Aki
鸡原始生殖细胞（PGCs）在胚胎早期通过生发新月区和血流向未来性腺区动态迁移。迄今为止，鸡PGCs主要通过组织化学和免疫组织化学方法或形态特征进行鉴定。然而，由于缺乏特异性的PGC分子标记，它们的起源、迁移和分化尚不完全清楚。在这里，我们分离了鸡死端同源物（CDH），并克隆了全长cDNA，具有329个氨基酸的开放阅读框。CDH中54 ~ 133个氨基酸的rna结合基序与人、鼠、爪蟾的末端蛋白高度同源。以CDH cRNA为探针，采用原位杂交（ISH）技术研究了PGCs在全胚上的时空分布。对X期至20期的鸡胚进行ISH处理。然后将杂交样品切片以分析PGCs的易位。从X期到4期可以计数到cdh阳性细胞，囊胚至少有30个细胞，生发月牙约有260个细胞。因此，CDH mRNA通过ISH和逆转录/聚合酶链反应在鸡胚、生发月牙和未来性腺区PGCs中特异性表达。我们得出结论，分离的CDH在鸡胚发育过程中特异性表达。
Chicken primordial germ cells (PGCs) dynamically migrate towards the prospective gonadal area through the germinal crescent region and bloodstream at early embryonic stages. To date, chicken PGCs have been mainly identified by histochemical and immunohistochemical methods or by their morphological characteristics. However, their origin, migration and differentiation are not fully understood because of the lack of specific PGC molecular markers. Here, we have isolated the chicken dead end homologue (CDH) in order to clone its full-length cDNA with an open reading frame of 329 amino acids. The RNA-binding motif present in CDH at amino acids 54-133 was highly homologous to those in the dead end proteins of human, mouse and Xenopus. The temporal and spatial distribution of PGCs was also investigated by in situ hybridization (ISH) on whole-mount embryos with CDH cRNA as a probe. Chicken embryos from stage X to stage 20 were subjected to ISH. The hybridized samples were then sectioned to analyse the translocation of PGCs. CDH-positive cells could be counted from stage X to stage 4, with minimally 30 cells at the blastderm and approximately 260 cells at the germinal crescent. Thus, specific expression of CDH mRNA has been established in chicken PGCs located at the blastderm, germinal crescent and prospective gonadal area by ISH and reverse transcription/polymerase chain reaction. We conclude that isolated CDH is specifically expressed in chicken PGCs during embryogenesis.