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Prior to the somite segmentation, the cells in the anterior presomitic mesoderm (PSM) express a set of genes that is required for defining the segmental border and polarity of the prospective somite. However, little is known how the expression of these genes is repressed upon segmentation. Here we report that Bowline, an associate protein of the transcriptional corepressor XGrg-4, repressed Tbx6 dependent transcription of Thylacine1 by mediating interaction of Tbx6 with XGrg-4 in Xenopus laevis. In bowline-deficient embryos, segmental border formation was disturbed, and expression of Thylacine1, X-Delta-2, and bowline expanded anteriorly. Tbx6-dependent transcription of Thylacine1 was suppressed by Bowline, together with XGrg-4. We also found that Bowline mediated the interaction of Tbx6 and XGrg-4. Based on our findings, we conclude that a part of the transcriptional repression at the anterior end of the PSM is caused by Bowline mediated transcriptional repression of Tbx6-dependent gene expression in X. laevis.
Fig. 1. Alterations in somite formation and expression of somitogenesisrelated
genes in Bowline-deficient embryos. (A) Western blotting analysis
of Bowline-myc protein. Two-cell stage embryos were injected in the
animal hemisphere with 200 pg of RNA encoding myc-tagged Bowline
(lanes: 1–3) or myc-tagged Bowline2 (lanes: 4–6), and 20 ng of bowline
MO (lane 2), bowline2 MO (lane 5), or 20 ng of CoMO (lanes: 3 and 6).
The 9E10 anti-myc antibody was used for immunoblotting. As a positive
control, a monoclonal antibody was used to detect a-tubulin. (B,C)
Embryos at 16-cell-stage were injected at equatorial region of 1 blastomere
(V2.2 or D2.2 position [27]) with combination of bowline MO (5 ng) and
bowline2 MO (5 ng) (BlnMO) or CoMO (10 ng) as indicated. RNA
encoding the lineage tracer b-gal was co-injected to identify the injected
side (red staining). Injected sides are indicated as ‘‘inj’’. (B) Eembryos were
fixed and stained with Red-gal and hematoxylin at stage 28–32. Embryos
are oriented with anterior to the left. Frontal sections. Bars, 100 nm. The
arrowheads indicate cells introduced with MOs. (C) Changes in expression
patterns of somitomeric markers. Embryos were analyzed for expression
of Thy1, X-Delta-2, bowline intron, X-hairy-2, or X-Delta-1 by wholemount
in situ hybridization at stage 20–21. Dorsal views are shown with
anterior towards the top. The arrowheads indicate positions where the
gene expression patterns are different from those of the uninjected side.
The arrows indicate ectopic expression of Thy1 between stripes. The
dotted lines indicate regions of Thy1 or bowline expression along the
anteroposterior axis (black line, uninjected sides; red line, injected sides).
(For interpretation of the references in color in this figure legend, the
reader is referred to the web version of this article.)
Fig. 2. Bowline and XGrg-4 synergistically repressed Tbx6-dependent
transcription. Relative luciferase activities driven by the transcriptional
regulatory region of Thy1 (pGL4.2Thy1) are indicated for COS7 cells
transfected with reporter gene plasmids and combinations of expression
vectors, as shown. Luciferase activity was normalized to the activity
obtained with the reporter plasmid transfected with b-gal.
Fig. 3. Bowline mediated the interaction between Tbx6 and XGrg-4. (A)
Extract prepared from embryos co-injected with RNAs coding for Tbx6-
HA (500 pg) and Bowline-myc (500 pg) (lane 1), with Tbx6-HA (500 pg)
and BowlineDBDLC-myc (500 pg) (lane 2), or with Tbx6-HA (500 ng) and
b-gal (500 ng) (lane 3) were subjected to immunoprecipitation (IP) with an
anti-myc antibody, followed by Western blotting (WB) with anti-HA
antibody. (B) Extract prepared from embryos injected with RNAs coding
for Tbx6-HA (250 pg), Bowline (500 pg) and XGrg-4-myc (250 pg) (lane
1), Tbx6-HA (250 pg), XGrg-4-myc (250 pg) and b-gal (500 pg) (lane 2), or
Tbx6-HA (250 ng) and b-gal (750 pg) (lane 3) were subjected to IP with an
anti-myc antibody, followed by WB with anti-HA antibody. To indicate
the expression, 0.9% of each embryo extract was loaded as input followed
by WB with anti-HA antibody and anti-myc antibody (A,B).
Fig. 4. Model for a role of Bowline in regulation of gene expression in somitogenesis. (A) Model for Bowline-mediated conversion of Tbx6; diagram of
hypothetical mechanism. In the absence of Bowline, Tbx6 activates transcription of genes including Thy1 in combination with other transcription factors;
in the presence of Bowline protein, the transcriptional corepressor XGrg-4 is recruited to Tbx6, thus suppressing transcription dependent on Tbx6. (B)
Hypothetical role of Tbx6-dependent transcription-repression via Bowline in somitogenesis. Accumulation of Bowline protein in the anterior-most PSM
represses expression of Tbx6-dependent genes and confines them to the anterior PSM.
