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Xwig1, a novel putative endoplasmic reticulum protein expressed during epithelial morphogenesis and in response to embryonic wounding.
Klingbeil P
,
Frazzetto G
,
Bouwmeester T
.
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In a subtractive differential screening, we identified a novel gene with interesting characteristics, termed Xenopus wounding induced gene 1 (Xwig1). Xwig1 encodes a novel protein of 912 amino acids containing 13 putative transmembrane segments and an evolutionarily conserved carboxy-terminal domain. Protein localization studies revealed that Xwig1 is anchored in cytoplasmic structures, presumably the endoplasmic reticulum. Expression is largely confined to epithelial cells in regions that undergo morphogenetic processes, such as blastopore closure, hindgut closure, dorsal closure and optic vesicle invagination. Interestingly, Xwig1 transcription is activated in response to embryonic epidermal wounding. The wounding-induced transcription occurs downstream of the transient phosphorylation of extracellular signal-regulated protein kinases and is in part mediated by Elk-1, but independent of dissection-induced FGF signalling. Thus, Xwig1 provides a molecular link between epithelial morphogenesis and wound healing.
Fig. 1. Predicted amino acid sequence of Xwig1 and homology with putative proteins encoded by
different vertebrate ESTs. (A) Amino acid sequence of Xwig1. The black arrow indicates the presumptive
signal peptide cleavage site and underlined in red are the numbered hydrophobic segments. (B)
Schematic representation of Xwig1. Indicated are the putative transmembrane regions (blue) and the
evolutionarily conserved carboxy-terminal domain (red). (C) Amino acid sequence alignment between the
carboxy-terminus of Xwig1 (amino acids 646-814), zebrafish EST AW184075, human EST AV652546,
chick EST AJ394934 and Xenopus ESTs AW766117 and BE491372. Underlined in red are conserved
amino acids between all proteins. The genebank accession number for Xwig1 is AF310008.
Fig. 2. (A) Subcellular localization of Xwig1.GFP fusion protein in animal cap
explants. Confocal images showing Xwig1.GFP distribution (green channel), cortical
actin visualized by rhodamin phalloidin (red channel), nuclei visualized by DAPI
staining (blue channel) and the overlay. Individual channels were scanned and each
image is the merge of 4 optical sections. Xwig1.GFP is detected in cytoplasmic
structures, presumably the endoplasmic reticulum. (B) RT-PCR analysis of Xwig1
expression during embryonic development (stages according to Nieuwkoop and
Faber, 1967). Histone H4 serves as an internal loading control.
Fig. 3. Xwig1 is expressed in epithelial
cells of the Spemann-Mangold organizer.
Whole-mount in situ hybridization analysis
was used to determine the spatio-temporal
expression of Xwig1 during early (AC),
mid (D-F), late (G) gastrulation and neurulation
(H-L). A and D are wild-type
pigmented embryos to show the blastopore
rim. During gastrulation, expression is confined
to epithelial cells of the blastopore rim
(red arrows in C,F). At the end of neurulation
expression is observed in a dorsal axial row
of cells (red arrow in L). All whole embryos
are vegetal views, except H-K which are
dorsal views with anteriorleft.
Fig. 4. Late embryonic expression
pattern of Xwig1. Expression is confined
to the ciliary margin and epithelial
cells on the dorsal side of the head at
stage 25 (A-C) and stage 32 (D-F).
Additional expression is detected in
the proctodeum of the stage 32 embryo
(G,H). Abbreviations: cm, ciliary
margin and pr, proctodeum.
Fig. 5. Xwig1 transcription is activated in response to embryonic epithelial
wounding. (A) Wounding-induced Xwig1 expression in gastrula embryos. (B,C)
Transverse sections of embryos displayed in (A). (D) Time course (0’-60’) of Xwig1
activation compared to the transient phosphorylation of ERK-1 and –2, visualized
by anti-dpERK staining, in response to lateral epidermal wounding of stage 27
embryos. Red arrows indicate the embryonic wounds. Xwig1 transcription is
activated downstream of ERK activation.
Fig. 6. Wounding-induced activation
of Xwig1 is partially dependent on
Elk-1, but independent of FGF signalling.
(A) Time course of Xwig1,
Xfos and Xegr-1 activation in response
to ectodermal wounding in dissected
gastrula animal cap explants. (B) Xwig1,
Xfos and Xegr-1 activation in explanted
control caps (0 and 1 hr) and caps
injected with dominant-negative Elk-1
(1 hr+dnElk-1) and dnFGFR (1
hr+dnFGFR). EF-1a serves as loading
control.
loc398207 (putative transmembrane protein TA-2) gene expression in a Xenopus laevis embryo, NF stage 12, as assayed by in situ hybridization. vegetal view, dorsal up.
loc398207 (putative transmembrane protein TA-2) gene expression in a Xenopus laevis embryo, NF stage 32, as assayed by in situ hybridization. lateral view, dorsal up, anteriorleft.
