The Xenopus LIM homeodomain protein Xlim-1 is specifically expressed in the Spemann organizer region and assumed to play a role in the establishment of the body axis as a transcriptional activator. To further elucidate the mechanism underlying the regulation of its transcriptional activity, we focused on the region C-terminal to the homeodomain of Xlim-1 (CT239-403) and divided it into five regions, CCR1-5 (C-terminal conserved regions), based on similarity between Xlim-1 and its paralog, Xlim-5. The role of Xlim-1 CT239-403 in the Spemann organizer was analyzed by assaying the axis-forming ability of a series of CCR-mutated constructs in Xenopus embryos. We show that high doses of Xlim-1 constructs deleted of CCR1 or CCR2 initiate secondary axis formation in the absence of its coactivator Ldb1 (LIM-domain-binding protein 1), suggesting that CCR1 and CCR2 are involved in negative regulation of Xlim-1. In contrast, while Xlim-1 is capable of initiating secondary axis formation at low doses in the presence of Ldb1, deletion of CCR2 (aa 275-295) or substitution of five conserved tyrosines in CCR2 with alanines (CCR2-5YA) abolished the activity. In addition, UAS-GAL4 one-hybrid reporter assays in Xenopus showed that CCR2, but not CCR2-5YA, with its flanking regions (aa 261-315) functions as a transactivation domain when fused to the GAL4 DNA-binding domain. Finally, we show that none of the known transcriptional coactivators tested (CBP, SRC-1, and TIF2) interacts with the Xlim-1 transactivation domain (aa 261-315). Thus, Xlim-1 not only contains a unique tyrosine-rich activation domain but also contains a negative regulatory domain in CT239-403, suggesting a complex regulatory mechanism underlying the transcriptional activity of Xlim-1 in the organizer.
A cDNA library derived from the anterior neuroectoderm (ANE) of Xenopus lategastrula embryos was systematically screened to isolate novel developmental regulatory genes involved in early brain development. We isolated 1,706 5´ expressed sequence tags (ESTs), which were subdivided into 1,383 clusters and categorized into 19 classes based on predicted functions according to their similarities to other known genes. Of these, 757 clusters that were considered possible novel regulatory genes or unknown genes were subjected to expression pattern analysis using whole-mount in situ hybridization. Genes from 69 clusters (9%) were expressed in the ANE region. Based on their expression patterns and predicted amino acid sequences, 25 genes were selected for further analysis as novel Xenopus genes expressed broadly or region-specifically in the ANE. Eighteen genes were expressed in postulated patterning centers in the neuroectoderm, including the anterior (four genes) and lateral (nine genes) neural ridges, the midbrain-hindbrain boundary region (one gene) and the midline region of the neural plate (two genes), whereas 13 genes were expressed in the eye anlagen. Therefore, early regionalization of the neuroectoderm appears to occur mainly in those neural patterning centers and the eye anlagen. We determined the entire coding regions of p54nrb, Semaphorin 6D and a novel gene designated scribble-related protein 1 (SCRP1). Interestingly, Semaphorin 6D is expressed in the mesoderm with a dorsoventral gradient, as well as in the ectoderm at the gastrula stage, implying a new role for this protein in development other than in axon guidance.
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