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Biochem Biophys Res Commun
2004 Dec 24;3254:1360-6. doi: 10.1016/j.bbrc.2004.10.177.
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A downstream enhancer is essential for Xenopus FoxD5 transcription.
Schön C
,
Köster M
,
Knöchel W
.
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We have isolated and sequenced the pseudo-allelic versions of FoxD5 genes in Xenopus laevis, xlFoxD5a and xlFoxD5b, and the xtFoxD5 gene of Xenopus tropicalis. These genes show a highly conserved structure, they are composed of only one exon, and they exhibit a high degree of sequence conservation within their flanking sequences. The X. tropicalis gene is expressed like its X. laevis orthologues in progenitor cells of the neural floor plate. Serial deletions of the 5'- and 3'-flanking region in combination with reporter gene assays demonstrate that a distal and a proximal upstream element as well as a downstream-located enhancer do mainly contribute to transcriptional activity. The downstream enhancer cooperates with the proximal upstream element and also contributes to the spatial expression. Transgenic animals express enhanced green fluorescent protein in a spatial pattern like xlFoxD5b, when the 5'- and 3'-flanking regions of the xlFoxD5b gene are used to direct transgene expression.
Fig. 1. Xenopus laevis and X. tropicalis FoxD5 genes. (A) Schematic
presentation of recombinant phages, O1, F12.2, and F1 containing the
xlFoxD5a, xlFoxD5b, and xtFoxD5 genes. The localization of the
genes (black boxes) and EcoRI restriction sites (vertical bars) is
indicated. (B) Plot matrix comparison of O1 (xlFoxD5a) versus F12.2
(xlFoxD5b); number of matches: 20, mismatches: 3. (C) Plot matrix
comparison of F1 (xtFoxD5) versus F12.2 (xlFoxD5b); number of
matches: 20, mismatches: 3.
Fig. 2. Xenopus tropicalis FoxD5 (xtFoxD5). (A) Winged helix domain amino acid alignment of xtFoxD5 versus xlFoxD5a and xlFoxD5b.
Deviating positions are indicated in red. (B–G) Whole mount in situ hybridization of X. tropicalis embryos for xtFoxD5. (B) Gastrula, (C) early
neurula, (D) organogenesis, (E) tadpole, (F) magnification of the head (tadpole), and (G) magnification of the tail (tadpole). Orientation of embryos
shown in (D–G): anterior to the left, posterior to the right side.
Fig. 3. Promoter analysis of the 50-flanking region of the xlFoxD5b
gene. (A) Deletions of the xlFoxD5b upstream region direct luciferase
reporter gene expression. DNA was injected into dorsal blastomeres of
4-cell stage embryos. Luciferase activities were measured when
embryos had reached stage 12. (B–C) Plot matrix comparison of the
upstream regions of xlFoxD5b versus xlFoxD5a (B) and xlFoxD5b
versus xtFoxD5 (C); number of matches: 13, mismatches: 0. (D)
Nucleotide alignments of the proximal and distal upstream regions of
xlFoxD5a, xlFoxD5b, and xtFoxD5 genes. Identical nucleotide positions
are indicated by dots, deletions are indicated by dashes.
Fig. 4. A downstream enhancer cooperates with the proximal upstream region. (A) Schematic drawing of luciferase reporter constructs fused to
DNA fragments derived from the xlFoxD5b flanking regions. Enhancer efficiency is indicated by (+) or ( ). (B) Relative luciferase activities of
reporter constructs containing the 1465/+113 promoter fragment fused to intragenic/30-fragments obtained by several restriction enzymes (see A).
(C) Relative luciferase activities of reporter constructs containing 50-deletions fused to the 30-located BamHI/XhoI fragment. (D) Relative luciferase
activities of reporter constructs containing the 151/+113 fragment and the indicated 30-located elements (see A). DNA was injected into dorsal
blastomeres of 4-cell stage embryos. Luciferase activities were measured when embryos had reached stage 12.
Fig. 5. Spatial expression of a luciferase reporter gene directed by the
xlFoxD5b 1465/+113 promoter alone (A,B) or in cooperation with
the 30-localized BamHI/XhoI fragment (C,D). DNA constructs were
injected into all blastomeres of 4-cell stage X. laevis embryos. Embryos
were fixed at different developmental stages and luciferase transcripts
were visualized by whole mount in situ hybridization. (A,C) collection
of embryos at gastrula, early, and late neurula stages. (B,D) Embryos
at gastrula stage.
Fig. 6. Transgenic embryos expressing GFP directed by FoxD5 gene
50- and 30-flanking regions. Transgenic animals are illuminated by a
UV-binocular-microscope (Olympus) using the GFP module. Embryos
shown are at developmental (A) stage 10, (B) stage 13, (C) stage 15,
and (D) stage 17.
