Characterization of the pufferfishOtx2cis-regulators reveals evolutionarily conserved genetic mechanisms for vertebrate head specification

Abstract

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-lacZreporter 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.

In vivo characterization of a vertebrate ultraconserved enhancer

“…Comparative genomics is continuing to help localize conserved noncoding sequences with gene regulatory activity, and distant evolutionary comparisons between mammals and teleosts have proven especially efficient [1,13,15,27]. In this study we characterized a gene enhancer (Dc2) identified through human-Fugu comparative genomics, and refined the sequences necessary and sufficient for its function, as well as assessed the impact of insertional mutations on its gene regulatory activity.…”

“…These characteristics led to the original proposal to sequence the Fugu genome in order to assist with the annotation of human genes based on comparative genomics [10]. Furthermore, the availability of Fugu genomic sequence also revealed that human-Fugu conserved noncoding sequences can be used to delineate gene regulatory sequences [1,[11][12][13][14][15].…”

Genome sequence, comparative analysis and haplotype structure of the domestic dog

“…Although the function of conserved non-coding elements is unknown, on the basis of recent studies 59,[63][64][65][66] it seems likely that many regulate gene expression. If so, the above results suggest that ,50% of all mammalian HCNEs may be devoted to regulating ,1% of all genes.…”

Genomic regulatory blocks underlie extensive microsynteny conservation in insects

“…The clusters most often coincide with genes encoding developmental and differentiation-related transcription factors. Many HCNEs have been characterized as long-range enhancers, first in studies of individual genes (Gottgens et al 2000;Sumiyama and Ruddle 2003;Kimura-Yoshida et al 2004;Milewski et al 2004), followed by systematic studies in zebrafish, Xenopus, and mouse (de la Calle-Mustienes et al 2005;Shin et al 2005;Woolfe et al 2005;Pennacchio et al 2006). Genome-wide analyses of HCNE sequences have detected several overrepresented motifs that are believed to be associated with context-specific enhancer activity (Bailey et al 2006;Pennacchio et al 2007).…”

“…The emerging model is that an array of HCNEs defines a region of regulatory inputs of its target gene(s), and that the full complement of those inputs results in the actual expression pattern of the gene (Kimura-Yoshida et al 2004;de la CalleMustienes et al 2005;Woolfe et al 2005;Pennacchio et al 2006). It is plausible to speculate that the genes with the most complex spatiotemporal expression should have more complex regulatory inputs.…”