Howard L et al. (2007), Regulation of the Xenopus Xsox17alpha(1) promot...

XB-ART-36372

Dev Biol 2007 Oct 15;3102:402-15. doi: 10.1016/j.ydbio.2007.07.028.

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Regulation of the Xenopus Xsox17alpha(1) promoter by co-operating VegT and Sox17 sites.

Howard L , Rex M , Clements D , Woodland HR .

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The gene encoding the Sox F-group transcription factor Xsox17alpha(1) is specifically expressed throughout the entire region of the Xenopus blastula fated to become endoderm, and is important in controlling endodermal development. Xsox17alpha(1) is a direct target of the maternal endodermal determinant VegT and of Sox17 itself. We have analysed the promoter of the Xenopus laevis Xsox17alpha(1) gene by transgenesis, and have identified two important control elements which reside about 9 kb upstream at the start of transcription. These elements individually drive transgenic endodermal expression in the blastula and gastrula. One contains functional, cooperating VegT and Sox-binding consensus sites. The Sox sites in this region are occupied in vivo. The other responds to TGF-beta signals like Activin or Nodals that act through Smad2/3. We propose that these two regions co-operate in regulating the early endodermal expression of the Xsox17alpha(1) gene.

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Species referenced: Xenopus laevis
Genes referenced: actl6a smad2 sox17a sox17b.1 sox17b.2 tbx2 tgfb1 vegt

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	Fig. 1. Initial transgenics made with large fragments of the Xsox17α1 promoter. Top: Map of the endogenous gene, with its single intron, and below are maps of two GFP constructs with large fragments of the Xsox17α1 promoter (red), and also a small promoter fragment with just 260 bp of upstream sequence (black). (A, B) Fluorescent expression of the MR21 construct in stage 10.5 gastrulae. (C) In situ hybridisation to GFP mRNA from MR19 in an embryo similar to that in panels A, and (D) an optical section of this embryo after clearing. (E, F) In situ to the endogenous Xsox17 transcripts for comparison. The blastopore lip is marked by an arrowhead in panels A–F. At stage 12, the endogenous gene is expressed as in panel G, and the MR21 GFP is shown in panel H. A minimal promoter, with only 260 bp of 5′ sequence is not expressed in the vegetal region (I), but it is expressed elsewhere. (J) MR21 is expressed throughout the endoderm at tailbud stages, just like the endogenous gene, as shown in an in situ hybridisation (K). The arrowheads mark the blastopore.
	Fig. 2. Transgenic expression of deletion constructs. Top: Diagrams of a series of 5′ and internal deletion fragments fused to GFP, as in Fig. 1. The endodermal element is marked and fragments giving vegetal expression in the early gastrula are colored red. Below is GFP expression in various transgenics, with the corresponding expression panels indicated on the left of the constructs. (A, D, G, J, K) are stage 10.5; (B, E, H) are stage 12; (C, F, I, L) are tailbud stages. (A–C) Construct − 12δ10-5 is negative in early endoderm. (D–F) Positive construct − 10; the embryo in (D) is a hemi-transgenic, providing a good control for the background fluorescence; the insert panel is the animal pole at the same exposure. (G–H) Positive construct − 10.5δ7.7-5. (I) Construct − 5.7 is positive in the later foregut, as well as in the axis, like other 5′ constructs; (J) − 9.5δ8.4-1.7 is positive in the involuting and non-involuting endoderm, whereas − 7.5 is not (K), but it gives low-level expression in the posterior endoderm of the tailbud (L). In panel J, the main tissues of the early gastrula are marked: end, endoderm; ect, ectoderm; ee, the extra-blastoporal endoderm, which involutes over the blastopore.
	Fig. 3. (A) Dissection of the E-element. At the top are the sub-regions of the E-element that were tested in GFP transgenics, attached to a cytoskeletal-type muscle actin basal promoter. All constructs except C3 overlapped to avoid disrupting a possible control element. Images of the transgenics are shown below, labelled by construct, the basal promoter alone being labelled BAct (Latinkic et al., 2002). Expression of the entire E-element is shown in Fig. 2J. (B, C) Mutational analysis of the B1 element. In panel B are maps of selected transcription factor binding sites in B1, and also C3. The sequences of B1 and C3 are shown in Supplementary Fig. 3. In panel C is a transgenic analysis of mutants of B1 in which the transcription factor binding sites were disrupted. The green panels show GFP fluorescence and the others show in situ hybridisation to GFP mRNA. The third B1 panel shows a cleared embryo, with GFP expression in the deep involuting endoderm and also the extra-blastoporal, epithelial, involuting endoderm, enlarged in the fourth panel. The arrow indicates the blastopore lip. (D) Electrophoretic mobility shift analysis of B1 T-box motif. EMSA assays were conducted with the variant VegT-binding sequence in B1, with a mutant of it and with the consensus sequence in the Derrière gene. In each case, the reactions were performed with or without competitor (±).

References [+] :

Afouda, GATA4, 5 and 6 mediate TGFbeta maintenance of endodermal gene expression in Xenopus embryos. 2005, Pubmed, Xenbase