Kimura-Yoshida Chiharu, Kitajima Kuniko, Oda-Ishii Izumi, Tian E, Suzuki Misao, Yamamoto Masayuki, Suzuki Tohru, Kobayashi Makoto, Aizawa Shinichi, Matsuo Isao
Head Organizer Project, Vertebrate Body Plan Group, RIKEN Center for Developmental Biology, 2-2-3 Minatojima Minami Cho, Chuou-Ku, Kobe, Hyougo 650-0047, Japan.
Development. 2004 Jan;131(1):57-71. doi: 10.1242/dev.00877. Epub 2003 Nov 26.
The Otx2 gene, containing a highly conserved paired-type homeobox, plays a pivotal role in the development of the rostral head throughout vertebrates. Precise regulation of the temporal and spatial expression of Otx2 is likely to be crucial for proper head specification. However, regulatory mechanisms of Otx2 expression remain largely unknown. In this study, the Otx2 genome of the puffer fish Fugu rubripes, which has been proposed as a model vertebrate owing to its highly compact genome, was cloned. Consistently, Fugu Otx2 possesses introns threefold smaller in size than those of the mouse Otx2 gene. Otx2 mRNA was transcribed after MBT, and expressed in the rostral head region throughout the segmentation and pharyngula periods of wild-type Fugu embryos. To elucidate regulatory mechanisms of Otx2 expression, the expression of Otx2-lacZ reporter genes nearly covering the Fugu Otx2 locus, from -30.5 to +38.5 kb, was analyzed, by generating transgenic mice. Subsequently, seven independent cis-regulators were identified over an expanse of 60 kb; these regulators are involved in the mediation of spatiotemporally distinct subdomains of Otx2 expression. Additionally, these expression domains appear to coincide with local signaling centers and developing sense organs. Interestingly, most domains do not overlap with one another, which implies that cis-regulators for redundant expression may be abolished exclusively in the pufferfish so as to reduce its genome size. Moreover, these cis-regions were also able to direct expression in zebrafish embryos equivalent to that observed in transgenic mice. Further comparative sequence analysis of mouse and pufferfish intergenic regions revealed eight highly conserved elements within these cis-regulators. Therefore, we propose that, in vertebrate evolution, the Otx2 promoter acquires multiple, spatiotemporally specific cis-regulators in order to precisely control highly coordinated processes in head development.
Otx2基因含有一个高度保守的配对型同源异型框，在整个脊椎动物的头部前端发育中起着关键作用。Otx2在时间和空间上表达的精确调控对于正确的头部特化可能至关重要。然而，Otx2表达的调控机制在很大程度上仍然未知。在本研究中，河豚红鳍东方鲀的Otx2基因组被克隆，因其基因组高度紧凑，河豚已被提议作为模式脊椎动物。一致的是，河豚Otx2的内含子大小比小鼠Otx2基因的内含子小三倍。Otx2 mRNA在中囊胚转换后转录，并在野生型河豚胚胎的整个分节期和咽胚期在头部前端区域表达。为了阐明Otx2表达的调控机制，通过生成转基因小鼠，分析了几乎覆盖河豚Otx2基因座（从-30.5 kb到+38.5 kb）的Otx2-lacZ报告基因的表达。随后，在60 kb的范围内鉴定出七个独立的顺式调节因子；这些调节因子参与介导Otx2表达在时空上不同的亚结构域。此外，这些表达结构域似乎与局部信号中心和发育中的感觉器官一致。有趣的是，大多数结构域彼此不重叠，这意味着冗余表达的顺式调节因子可能仅在河豚中被消除，以减小其基因组大小。此外，这些顺式区域也能够在斑马鱼胚胎中指导表达，与在转基因小鼠中观察到的表达相当。对小鼠和河豚基因间区域的进一步比较序列分析揭示了这些顺式调节因子内的八个高度保守元件。因此，我们提出，在脊椎动物进化过程中，Otx2启动子获得了多个时空特异性的顺式调节因子，以便精确控制头部发育中高度协调的过程。
