Summary Characterization of the pufferfish Otx 2 cis-regulators reveals evolutionarily conserved genetic mechanisms for vertebrate head specification

The establishment of the body plan of the vertebrate embryo requires the expression of regulatory genes in precisely controlled spatial domains, such that their products can function appropriately in the patterning process. Elucidation of the underlying mechanisms involved in this regulatory cascade initially depends on the identification of cis-acting regions that mediate spatial and temporal regulation. Homeobox genes are thought to participate in such developmental processes, thereby providing an ideal system with which to examine the genetic regulatory mechanism. Notably, Hox genes play a crucial role in the body plan of the trunk region, caudal to the hindbrain (Krumlauf, 1994). An intriguing co-linearity exists between the order of vertebrate Hox genes on the chromosome, and their temporal and spatial expression (Krumlauf, 1994). Much is known regarding the genetic regulatory mechanism of Hox expression (Nonchev et al., 1997; Duboule, 1998). However, in the head region, the regulatory mechanism of homeobox gene expression remains uncertain (Logan et al., 1993; Kammandel et al., 1999; Zerucha et al., 2000). 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 Otx2is likely to be crucial for proper head specification. However, regulatory mechanisms of Otx2expression 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 Otx2possesses introns threefold smaller in size than those of the mouse Otx2 gene. Otx2mRNA was transcribed after MBT, and expressed in the rostral head region throughout the segmentation and pharyngula periods of wild-type Fuguembryos. To elucidate regulatory mechanisms of Otx2expression, 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 Otx2promoter acquires multiple, spatiotemporally specific cis-regulators in order to precisely control highly coordinated processes in head development.