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TheRpx/Hesx1 homeobox gene is expressed during gastrulation in the anterior visceral and definitive endoderm and the cephalic neural plate. At later stages of development, its expression is restricted to Rathke's pouch, the primordium of the pituitary gland. This expression pattern suggests the presence of at least two distinct regulatory regions that control early and late Rpx transcription. Using transgenic mice, we have demonstrated that regulatory sequences in the 5' upstream region of Rpx are important for early expression in the anterior endoderm and neural plate and regulatory elements in the 3' region are required for late expression in Rathke's pouch. We have found that the genetically required LIM homeodomain-containing proteins Lim1/Lhx1 and Lhx3 are directly involved in the regulation of Rpx transcription. They bind two LIM protein-binding sites in the 5' upstream region of Rpx, which are required for Rpx promoter activity in both mice and Xenopus. Furthermore, we have found that a conserved enhancer in the 3' regulatory sequences of Rpx is not only required, but is also sufficient for the expression of Rpx transgenes in the developing Rathke's pouch.
Fig. 6. Structural and functional conservation of Rpx regulatory elements in mouse and Xenopus. (A) The sequence of the mouse Rpx promoter was compared with human Rpx and Xenopus Xanf upstream sequences. There are high degrees of similarity among them. The LIM-HD binding sites, dTAAT (red) and pTAAT (blue), are highly conserved among mouse, human and Xenopus. The identical bases are marked by black shading and the similar bases are marked by gray shading. (B) Xenopus embryos were injected with RpxW in the two animal–dorsal blastomeres of eight-celled embryos. RpxW contains both 5′ and 3′ regulatory regions and the first intron of Rpx. The β-galactosidase activity was detected in the anterior neural plate at stage 14 (anterior to the left). (C) At stage 32, the β-galactosidase activity was detected in anterior structures, such as forebrain, eye and cement gland. (D) Expression of lacZ in the pituitaryprimordium in longitudinal sections. (E) Xenopus embryos were injected with reporter DNA in the animal–dorsal or vegetal–ventral blastomeres of eight-celled embryos. LacZ staining was done at stage 14. RpxD contains 570 bp of upstream sequence and the first intron of Rpx. When RpxD was injected into animal dorsal blastomeres at the 8-cell stage, most of the injected embryos showed lacZ expression in the anterior neural plate (front view). (F) When RpxD was injected into vegetal–ventral cells, lacZ staining is not detectable (back view). (G, H) When CMV-lacZ was injected into animal–dorsal cells or vegetal–ventral cells, strong lacZ staining was detected in the anterior region (G, front view) or the posterior region (H, back view), respectively. (I) The RpxD-mut contains mutations in dTAAT and pTAAT sites (mutated sequences shown in Fig. 2A). When RpxD-mut was injected into animal dorsal cells, in most embryos, the lacZ staining is not detectable in anteriorneuroectoderm (front view).
