Click here to close
Hello! We notice that you are using Internet Explorer, which is not supported by Xenbase and may cause the site to display incorrectly.
We suggest using a current version of Chrome,
FireFox, or Safari.
???displayArticle.abstract???
Transforming growth factor-beta1-stimulated clone 22 (TSC-22) encodes a leucine zipper-containing protein that is highly conserved. During mouse embryogenesis, TSC-22 is expressed at the site of epithelial-mesenchymal interaction. Here, we isolated Xenopus laevis TSC-22 (XTSC-22) and analyzed its function in early development. XTSC-22 mRNA was first detected in the ectoderm of late blastulae. Translational knockdown using XTSC-22 antisense morpholino oligonucleotides (XTSC-22-MO) caused a severe delay in blastopore closure in gastrulating embryos. This was not due to mesoderm induction or convergent-extension, as confirmed by whole-mount in situ hybridization and animal cap assay. Cell lineage tracing revealed that migration of ectoderm cells toward blastopore was disrupted in XTSC-22-depleted embryos, and these embryos had a marked increase in the number of dividing cells. In contrast, cell division was suppressed in XTSC-22 mRNA-injected embryos. Co-injection of XTSC-22-MO and mRNA encoding p27Xic1, which inhibits cell cycle promotion by binding cyclin/Cdk complexes, reversed aberrant cell division. This was accompanied by rescue of the delay in blastopore closure and cell migration. These results indicate that XTSC-22 is required for cell movement during gastrulation though cell cycle regulation.
Fig. 1. Sequence of Xenopus
TSC-22. (a) Structure of XTSC-22
protein. LZ, leucine zipper; RD1
and RD2, repression domain 1
and 2. (b) Comparison of TSC-22
proteins from Xenopus laevis
(accession no. AB191720), Xenopus
tropicalis EST (AL776759),
mouse (X62940) and human
(U35048). White letters on black
background indicate amino acid
residues that are conserved
across species. Note that the
leucine zipper and TSC-box is
100% identical to mammalian
TSC-22.
Fig. 2. Expression pattern of
TSC-22 (a) Temporal expression
pattern of XTSC-22 mRNA detected
by reverse transcriptionpolymerase
chain reaction (RTPCR).
Transcripts were first
detected at late blastula (stage 9)
and persisted until tadpole stage
(stage 35). Ornitine decarboxylase
(ODC) expression serves as
the quantitative control. RT-PCR
was carried out to avoid contamination
with genomic DNA. (b–f)
Spatial expression pattern of
XTSC-22 determined by wholemount
in situ hybridization. At
stage 10 (b,c), the ectoderm was
stained. (c) Sagittal section of the
stage-10 embryo in (b). The black
lines indicate the blastopore. At
neurula stage (d,e), transcripts
were observed in the neural
region including eyes and brain.
This localization was clear at
stage 35 (f). Bars, 0.3 mm (g)
Induction of XTSC-22 transcription
by growth factors: there was
no change in XTSC-22 expression
with any treatment. WE,
whole embryo.
Fig. 3. Overexpression of XTSC-
22 resulted in apoptosis (a,b)
Morphology of control embryos at
stage 11 (a) and XTSC-22 mRNAinjected
embryos (b). Expulsion of
cells at animal pole was observed
in XTSC-22 injected embryos.
(c,d) Terminal deoxyribonucleotidyl
transferase-mediated dUTP–
digoxigenin nick end-labeling
(TUNEL) staining of control
embryos (c) and XTSC-22 injected
embryos (d). TUNEL-positive cells
were seen in XTSC-22-injected
embryos (d). Bars, 0.5 mm.
Fig. 4. XTSC-22 depletion affects gastulation movements (a)
Morphology of XTSC-22-morpholino oligonucleotides (MO)-
injected embryos at stage 11. XTSC-22 depletion led to delayed
gastrulation. This phenotype was rescued by co-injection of
XTSC-22 mRNA. (b) Frequency of phenotypes seen in
experiment shown in (a). (c) Cell lineage tracing of XTSC-22-MOinjected
embryos at stage 12. Epiboly was perturbed in XTSC-
22-depleted embryos. Co-injection of RNA with XTSC-22-MO
partially rescued the perturbation. (d) Animal cap assay using
explants excised from uninjected embryos and XTSC-22-MOinjected
embryos. Explants treated with activin elongated
whether the explants were injected with the MO or not. (e)
Expression of mesodermal gene in control embryos and XTSC-
22-MO-injected embryos. No difference in expression of Xbra
and Chordin was observed. (f) Expression of mesodermal gene
in activin-treated animal caps from uninjected embryos and
XTSC-22-MO-injected embryos. Activin treatment induced Xbra
and Chordin in both uninjected and XTSC-22-MO injected
animal caps.
Fig. 5. XTSC-22 depletion affects gastrulation movements
through cell cycle promotion (a) PH3 staining of XTSC-22-MO
injected embryos. The number of PH3-positive cells increased
in XTSC-22-depleted embryos. This increase was reversed by
co-injection of XTSC-22 mRNA. The number of PH3-positive
cells decreased when XTSC-22 mRNA alone was injected. (b)
PH3 staining of embryos injected with XTSC-22-MO and
p27Xic1 mRNA. Single injection of p27Xic1 mRNA led to a
decrease in the number of PH3-positive cells. The increase in
number of dividing cells by XTSC-22 depletion was reversed by
co-injection of p27Xic1 mRNA. (c) Morphology of embryos
injected with XTSC-22-MO and p27Xic1 mRNA. p27Xic1 alone
did not affect gastrulation. Delay of blastopore closure seen in
XTSC-22-depleted embryos was rescued by co-injection of
p27Xic1 mRNA. (d) Frequency of phenotypes seen in
experiment shown in (c). (e) Cell lineage tracing of rescued
embryos. Disruption of epiboly movement in XTSC-22-depleted
embryos was rescued in embryos injected with XTSC-22-MO
and p27Xic1 mRNA.
tsc22d1 (TSC22 domain family, member 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 10, vegetal view, dorsal up.
tsc22d1 (TSC22 domain family, member 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 18, anterior view, dorsal up.
tsc22d1 (TSC22 domain family, member 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 35, lateral view, anteriorleft, dorsal up.
