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Development
2009 Mar 01;1365:729-38. doi: 10.1242/dev.025569.
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The non-methylated DNA-binding function of Kaiso is not required in early Xenopus laevis development.
Ruzov A
,
Savitskaya E
,
Hackett JA
,
Reddington JP
,
Prokhortchouk A
,
Madej MJ
,
Chekanov N
,
Li M
,
Dunican DS
,
Prokhortchouk E
,
Pennings S
,
Meehan RR
.
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Mammalian forms of the transcription repressor, Kaiso, can reportedly bind methylated DNA and non-methylated CTGCNA motifs. Here we compare the DNA-binding properties of Kaiso from frog, fish and chicken and demonstrate that only the methyl-CpG-binding function of Kaiso is evolutionarily conserved. We present several independent experimental lines of evidence that the phenotypic abnormalities associated with xKaiso-depleted Xenopus laevis embryos are independent of the putative CTGCNA-dependent DNA-binding function of xKaiso. Our analysis suggests that xKaiso does not play a role in the regulation of either xWnt11 or Siamois, key signalling molecules in the Wnt pathway during X. laevis gastrulation. The major phenotypic defects associated with xKaiso depletion are premature transcription activation before the mid-blastula transition and concomitant activation of a p53-dependent cell-death pathway.
Fig. 7. The Wnt signalling pathway is not activated in xKMO morphants. (A) The xKMO morphants exhibit a delay in closing the blastopore (short arrows) compared with wild-type embryos. This phenotype appears in 85-90% of the embryos and is identical to that presented by McCrea and colleagues (Kim et al., 2004; Park et al., 2005). By stage 15 (neurulation), the xKMO morphants (downward arrow) cannot form a neural fold; they are apoptotic and are shedding cells through the open blastopore. Control embryos are shown with a proper neural fold (long arrows). (B,C) Neither Siamois nor xWnt11 are ectopically activated in xKMO morphants when assayed by semi-quantitative RT-PCR according to Park et al. (Park et al., 2005) or real-time PCR relative to a histone H4 control at stage 10 (Siamois) or 12 (xWnt11). Caspase7 and Caspase9 expression is activated in 2.5-3.5 times compared to control in the same sets of KMO embryos at stages 10 and 12, respectively. (D) Whole-mount RNA in situ analysis demonstrates that xWnt11 is not prematurely activated in pre-MBT xKMO morphants in comparison to a control transcript, xID2, that is activated prematurely (Ruzov et al., 2004).
Fig. S3. Kaiso is a ubiquitously expressed methyl-dependent transcriptional repressor. Kaiso whole-mount RNA in situ hybridization on zebrafish (A), chicken (B) and Xenopus (C) embryos. The stages of development for Xenopus and hours after fertilisation for zebrafish embryos are indicated. An, animal pole; Veg, vegetal pole; Ant, anterial part of the embryo; Ecd, ectoderm; np, neural plate; nc, notochord; nt, neural tube; sc, spinal cord; tb, tailbud; mn, motoneurons; psm, perisomitic mesoderm; ba, branchial arches; sm, somites; YE, yolk extension layer; EEM, extraembryonic membranes; Vnt, ventral neural tube.
Fig. S7. No evidence for mis-expression of xWnt11 in xKaiso-depleted Xenopus laevis embryos. Whole-mount in situ hybridisation on control and xKMO Xenopus pre-MBT embryos using xWnt11 (A) or xID2 (B) probes. The stages of development are indicated. Animal and dorsal views are shown for every group of embryos. Note that only xID2 is ectopically expressed in pre-MBT xKMO morphants.
zbtb33 (zinc finger and BTB domain containing 33)gene expression in Xenopus laevis embryo via in situ hybridization, NF stage 8, lateral view, dorsal up.
zbtb33 (zinc finger and BTB domain containing 33 )gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 14, dorsolateral view, anterior up.
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