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The notochord is essential for normal vertebrate development, serving as both a structural support for the embryo and a signaling source for the patterning of adjacent tissues. Previous studies on the notochord have mostly focused on its formation and function in early organogenesis but gene regulation in the differentiation of notochord cells itself remains poorly defined. In the course of screening for genes expressed in developing notochord, we have isolated Xenopus homolog of Btg2 (XBtg2). The mammalian Btg2 genes, Btg2/PC3/TIS21, have been reported to have multiple functions in the regulation of cell proliferation and differentiation but their roles in early development are still unclear. Here we characterized XBtg2 in early Xenopus laevis embryogenesis with focus on notochord development. Translational inhibition of XBtg2 resulted in a shortened and bent axis phenotype and the abnormal structures in the notochordtissue, which did not undergo vacuolation. The XBtg2-depleted notochord cells expressed early notochord markers such as chordin and Xnot at the early tailbud stage, but failed to express differentiation markers of notochord such as Tor70 and 5-D-4 antigens in the later stages. These results suggest that XBtg2 is required for the differentiation of notochord cells such as the process of vacuolar formation after determination of notochord cell fate.
Fig. 1. Alignment and comparison
of Btg2 from Xenopus and
other vertebrates. (A) Amino acid
sequence alignments of XBtg2
and human, mouse, rat and
zebrafish Btg2. The blue and red
boxes indicate the two highly
conserved domains, i.e. BTG Box
A and Box B. (B) Amino acid
sequence similarity between
XBtg2 and other vertebrate Btg2.
XBtg2 is 62â63% similar to the
mammalian Btg2 sequence and
52% similar to zebrafish Btg-b.
The two conserved domains of
XBtg2 are 85â90% similar to those
of other vertebrate Btg2.
Fig. 2. Spatio-temporal expression patterns of XBtg2.
(A) Expression of XBtg2 at various stages as detected by RTPCR.
XBtg2 transcripts were present maternally and increased
markedly after the neurula stage (stage 13). (B–I) Whole-mount
in situ hybridization analysis of XBtg2 expression. Lateral (B, E–
F) and dorsal (C, D) view of whole embryos at indicated stages.
(H,I) Transverse sections at the sites indicated by the black
lines in (G). XBtg2 was slightly expressed in the animal
hemisphere of the embryo at stage 10.5 (B). The black
arrowhead indicates dorsal blastopore lip. From stage 12.5,
XBtg2 was expressed strongly in the presomitic mesoderm (C).
At stage 15, XBtg2 expression was detected in the anterior
neural ectoderm (D). At tailbud stages, XBtg2 was expressed in
somite, neural tube, notochord, eyes, midbrain and pronephric
anlagen (E–I). The expression patterns in dorsal structures
showed a dynamic change along the anterior-posterior axis (H,
I); no, notochord; s, presomitic mesoderm; ne, neuraltube. (J) In
situ hybridization analysis of XBtg2 expression on sectioned
embryos. XBtg2 was expressed in the trunk-tail part of
notochord, which vanished subsequent to vacuolation.
Fig. 3. XBtg2 is required for
notochord development. (A) The
specificity of XBtg2 MO. Embryos
injected with 400 pg of XBtg2-
Myc or 9m XBtg2-Myc mRNA
either with or without 40 ng of
XBtg2 MO or control MO into both
cells at the 2-cell stage were
harvested and the lysates were
analyzed by western blot analysis.
XBtg2 MO but not control MO
inhibited the translation of XBtg2-
Myc, whereas XBtg2 MO did not
inhibit the translation of 9m
XBtg2-Myc. (B) XBtg2 depletion
causes abnormal structures in
notochord tissue. XBtg2 MO (7 ng),
control MO (7 ng), and a mixture
of XBtg2 MO (7 ng) and pCS2â9m
XBtg2 (7 pg) were co-injected
with 50 pg of a lineage tracer,
lacZ mRNA, into a single B1
blastomere of 32-cell stage
embryos. Embryos were analyzed
at stage 35. β-galactosidase
activity was visualized in red.
The sections were processed for
hematoxylin and eosin (HE) staining.
Embryos injected with control MO
showed no defect, while those
injected with XBtg2 MO exhibited
the shortened and bent axis and
the abnormal morphology of the
notochord cells. The cells were
not very swollen and appeared
disordered. Also, the sheath
surrounding notochord tissue
was disrupted. The XBtg2 MOinherent
cells in the notochord
(stained red) formed into clumps (white arrowheads). These altered phenotypes were partially rescued by co-injection of linearized
pCS2â9m XBtg2. (C) The effect of XBtg2 depletion on notochord cells before and after vacuolation. XBtg2 MO- and lacZ mRNAinjected
embryos were sectioned transversely at indicated stages and processed for HE staining. An abnormal cell mass was formed
at an early stage and hardly vacuolated onward. Scale bars indicate 100 μm.
Fig. 4. XBtg2 depletion affects differentiation of notochord cells.
7 ng of XBtg2 MO and 50 pg of lacZ mRNA were injected into
the a single B1 blastomere of 32-cell stage embryos. Embryos
were sectioned transversely at stage 23 or stage 30 and processed
for in situ hybridization (chordin, Xnot and collagen type II) and
antibody staining (Tor70 and 5-D-4). β-galactosidase activity is
visualized in red. At stage 23, chordin, Xnot, collagen type II
were all expressed in red-gal-stained cells (AâC; arrows). At
stage 30, only collagen type II was expressed in the red-stained
cells (F; arrows); the other early notochord makers chordin and
Xnot (D, E; arrowheads) and the mature notochord markers,
Tor70 and 5-D-4 (G, H; arrowheads) were not expressed.
