Ahmed N et al. (2004), Early endodermal expression of the Xenopus Endo...

XB-ART-3507

Differentiation 2004 Apr 01;724:171-84. doi: 10.1111/j.1432-0436.2004.07204005.x.

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Early endodermal expression of the Xenopus Endodermin gene is driven by regulatory sequences containing essential Sox protein-binding elements.

Ahmed N , Howard L , Woodland HR .

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The Endodermin gene is expressed in the early endoderm and the Spemann organizer of Xenopus embryos. It has previously been shown to be a direct target of the early endodermal transcription factor Xsox17 (Clements et al., 2003, Mech Dev 120:337-348). Here we identify two adjacent control elements in the Endodermin promoter; these drive transcription of the gene in late-gastrula endoderm and contain consensus Sox-binding sites. We have analyzed one element in detail and show that it responds directly to Xsox17 and that the Sox sites are essential for endodermal expression in transgenic embryos. However, flanking regions on both sides are also essential, indicating that Xsox17 acts in concert with several DNA-binding partners.

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Species referenced: Xenopus
Genes referenced: a2m actc1 sox17a tbx2

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	Fig. 1 (A) Fluorescence reporting from the vegetal pole. (A1) shows an embryo expressing GFP from mRNA injected bilaterally into the vegetal pole at the two-cell stage. There is little fluorescence in the vegetal pole itself, apart from yellow autofluorescence. (A2) strong vegetal fluorescence after injecting fluorescein-lysine-dextran. (A3) shows fluorescence from GFP is easily detected in the vegetal pole of a GFP mRNA-injected oocyte. (B) Primer extension analysis of transcription initiation from the endogenous Endodermin gene. The Endodermin promoter is diagrammed, marking the two TATA boxes at 65 and 285 bp, as well as the position of exon 1 and the primer used, 74 bp downstream of the ATG (green arrow). Below is an autoradiograph of the primer extension products of Endodermin, as well as Xsox17a as a control. On the left are dideoxy sequencing reactions, using the primer extension primers. The primer positions on the extension reactions are marked with white arrows and the predominant product with black arrows. In the case of Endodermin there is no single long extension band. The primer used is 50-AGGATGAGGAGGAGGTGCAG ACT-30.

	Fig. 3 Two promoter fragments, E1 and E2, contain putative Soxbinding sites and can drive gastrula-stage expression in the endoderm. The position of the two Pst1/Spe1 fragments, E1 and E2, are shown at the top. Their expression in partial transgenics is shown below, using GFP fluorescence and in situ hybridization to GFP mRNA in different embryos. On the right are shown the sequences of E and E2, indicating Sox-binding sites (yellow boxes, contained the core sequence ACAAT), Smad sites (green), FAST-1 sites (blue), and GATA sites (red). The three Sox sites in E2, of which the central one is a complex of three overlapping sites, are labelled A–C.
	Fig. 4 Xsox17 can induce expression of the E2 reporter construct. The GFP in the E1 or E2/basal 169 bp promoter fusions used for transgenesis was replaced with firefly luciferase, and 20 pg of these constructs was injected bilaterally into the animal pole, with or without 200 pg of Xsox17b or Xsox17b::EnR mRNA, or into the vegetal pole, with or without Xsox17b::EnR. The basal 169/luciferase reporter is also shown. Whole embryos were harvested at early gastrula stages (10.5– 11) and groups of five embryos were assayed in triplicate for firefly luciferase activity. (A) Both E1 and E2 reporter constructs are induced vegetally, but the basal promoter is not. (B) In contrast, only E2 was induced by Xsox17 in animal caps, and it was repressed by Xsox17- b::EnR. (C) Xsox17b::EnR repressed E2 reporter expression by 80% in both the animal and vegetal halves. E1 reporter expression was also down-regulated by 40% and 50% in the animal or vegetal halves, respectively.
	Fig. 5 A minimal promoter fragment E2:112, derived from E2, drives early expression in the endoderm of early Xenopus gastrulae. Deletion of site A from E2 does not affect endoderm expression (1), nor is this Sox site sufficient for endoderm expression (2). A GATA site and flanking sequence is also insufficient for endoderm expression (3). A reporter construct containing sites B and C drives endoderm expression (4), but a derivative that lacked site B did not (5). A reporter construct containing sequence upstream of site C, but which did not include site C itself, was also insufficient for endoderm expression (6). Endoderm expression was restored by addition of site B and upstream flanking sequence (7). A shorter version of this construct (E2:112) can also drive endoderm expression (8). The constructs are: (1) 2698/2326; (2) 2733/2698; (3) 2519/2326; (4) 2698/2508; (5) 2609/2508; (6) 2636/2550; (7) 2698/ 2550; (8) 2698/2586.
	Fig. 6 (1) Site B is necessary but not sufficient for endoderm expression of E2:112. In each case, the constructs are shown on the left and GFP expression by fluorescence or in situ is shown on the right. (A, B) Endodermal expression of E2:112. (C–F) Reporter constructs containing site B, with either a 2 or 10 bp flanking sequence on either side, were not sufficient for endoderm expression. (G, H) Deletion of site B from E2:112 abolished reporter expression in the endoderm. (I–L) Endodermal expression was not restored in reporter constructs containing site B together with upstream or downstream sequences (I–L). (2) The reporter construct E2:112 is vegetally induced, but only if the B Sox site is present. Without this site, Xsox17b represses luciferase expression, but the B Sox site relieves this. The constructs are indicated.
	Fig. 7 (1) The E2 element is induced by Xsox17 in oocytes, showing a direct effect. A circular plasmid containing E112 fused to the actin basal promoter and driving luciferase was injected into the nuclei of oocytes, with or without Xsox17a mRNA and incubated for 18 hr. (2) Late expression of Endodermin promoter constructs in transgenics. (A) The Endodermin 196 promoter is expressed in most regions of the stage 32 embryo, including the pharynx, but not the rest of the endoderm. (B) The basal cardiac actin promoter shows a similar expression pattern at stage35. (C) The 3487/1 promoter region does not drive high-level endodermal expression, beyond that in the pharynx, already seen with the 196 bp region at stage 32. (D) The 7 kb/ 2632 region, which includes E1, drives general endodermal expression at stage 33. (E) A white light image of this embryo. (F) E2, fused to the basal 196 bp region, gives similar endodermal expression. (G) A white light image of this embryo. (H–K) Embryos expressing the 3487/2632 region (encompassing E1) fused to the basal cardiac actin promoter. (H) Three embryos at stage 33. (I, J) Ventral fluorescence and white light images of stage 42 embryos. (K) The same embryo from the right-hand side, with the pancreas indicated (pc).