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Int J Dev Biol
2007 Jan 01;511:27-36. doi: 10.1387/ijdb.062211mi.
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XSu(H)2 is an essential factor for gene expression and morphogenesis of the Xenopus gastrulaembryo.
Ito M
,
Katada T
,
Miyatani S
,
Kinoshita T
.
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The CSL (CBF-1, Suppressor of Hairless, Lag-1) transcriptional factor is an important mediator of Notch signal transduction. It plays a key role in cell fate determination by cell-cell interaction. CSL functions as a transcriptional repressor before the activation of Notch signaling. However, once Notch signaling is activated, CSL is converted into a transcriptional activator. It remains unclear if CSL has any function during early development before neurogenesis, while transcriptional products exist from the maternal stage. Here, we analyzed the function of Xenopus Suppressor of Hairless (XSu(H)) using morpholino antisense oligonucleotides (MO), which interfere with the translation of transcripts. In Xenopus embryos, maternal transcripts of both XSu(H)1 and XSu(H)2 were ubiquitously observed until the blastula stage and thereafter only XSu(H)1 was zygotically transcribed. Knockdown experiments with MO demonstrated that XSu(H)2 depletion caused a decrease in the expression of the Xbrachyury, MyoD and JNK1 genes. Morphological and histological examinations indicated that XSu(H)2 depletion caused abnormal gastrulation, which resulted in severe defects of the notochord and somitic mesoderm. The effect of XSu(H)2-MO was completely rescued by co-injection of XSu(H)2 mRNAs, but not by XSu(H)1 mRNAs. XESR-1, a Notch signaling target gene, inhibited Xbrachyury expression. However, expression of the XESR-1 gene was not induced by depletion of XSu(H)2. Co-injection of the dominant-negative form of XESR-1 could not rescue the suppression of Xbrachyury expression in the XSu(H)2-depleted embryo. These results suggest that XSu(H)2 is involved in mesoderm formation and the cell movement of gastrula embryos in a different manner from the XESR-1-mediated Notch signaling pathway.
Fig. 1. The temporal expression pattern of XSu(H)1 and XSu(H)2. (A)
Developmental profile of XSu(H)1 and XSu(H)2 expression. Both XSu(H)1 and XSu(H)2 transcripts were detected ubiquitously from unfertilized eggs (E) to stage 35. Enriched maternal transcripts of XSu(H)2 were recognized from unfertilized eggs to the gastrula stage. Histone H4 was used as a loading control. -RT, PCR without reverse transcriptase. (B) Quantification of XSu(H)1 and XSu(H)2 expression at each stage. The vertical line indicates the relative value of XSu(H)2/Histone H4 ratio calculated with sample E as 1. Experiments were carried out in triplicate. (C) Distribution of XSu(H)1 and XSu(H)2 transcripts. Blastula stage (stage 9) embryos were dissected into animal cap (An), marginal zone (MZ) and vegetal cap (Vg) and gene expression was detected by RT-PCR.
Fig. 2. Effect of XSu(H) morpholinos on early embryonic development. (A) Specificity of morpholino oligonucleotides (MO). Five ng of myc-XSu(H)1 or myc-XSu(H)2 mRNA was injected into the animal pole of each blastomere of 2-cell stage with or without 25 ng of XSu(H)1-MO or XSu(H)2-MO. XSu(H)1 or XSu(H)2 protein was detected by anti-c-myc antibody at stage 10.5. 43 kDa actin bands were used as loading controls (Coomassie stained). (B) Phenotype of the MO-injected embryo. Em- bryos were injected with control-MO (B1, B6), XSu(H)1-MO (B2, B7), XSu(H)2-MO (B3, B8), mRNAs of XSu(H)1 (B4, B9) or XSu(H)2 (B5, B10) into one dorsal blastomere at the 4-cell stage and morphological changes were analyzed at stage 13 (B1-5) or stage 20 (B6-10). To identify the injection side of the embryo, 1 ng of GFP mRNA as the tracer was co- injected with each mRNA or MO. Upper panels (B1-5) show the vegetal view and lower panels (B6-10) show the dorsal view. XSu(H)2-depleted embryos showed delayed gastrulation (arrow in B3) and defective neurogenesis (arrow in B8), while XSu(H)1-depleted embryos developed normally (B2, B7).
Fig. 3. Histological examination of XSu(H)2-depleted embryos. (A)
Morphological change of XSu(H)2-depleted embryos. Dwarf embryos were induced by XSu(H)2-MO but not by XSu(H)1-MO. (B) Cross section of XSu(H)2-depleted embryos. Histological analysis shows that embryos injected with XSu(H)2-MO had tissue defects in the somite (so) and notochord (no) without lacking a neural tube (n). Ten ng of each MO was injected into the marginal zone of one blastomere at the 2-cell stage. The injected embryos were fixed at stage 35-36 for histological examination. Scale bar indicates 100 μm. (C) Gene expression in XSu(H)2-depleted embryos. Twenty five ng of XSu(H)1-MO or XSu(H)2-MO was injected into the marginal zone of both blastomeres at the 2-cell stage. The injected embryos were sacrificed at stage 10.5 for quantitative RT-PCR. XSu(H)2-MO reduced Xbrachyury, MyoD, Xvent1, chordin and JNK1 expressions, but had no effect on goosecoid expression in gastrula stage embryos. XSu(H)1-MO, XSu(H)1 XSu(H)2, XSu(H)1 + XSu(H)2 showed no effect on the gene expression of mesodermal markers.
Fig. 4. XSu(H)2 can rescue the defective phenotype caused by XSu(H)2-MO. (A) Embryos were injected with control-MO (25 ng) (A1), XSu(H)2-MO (25 ng) (A2), XSu(H)2-MO (25 ng) + mRNAs of ∆5TR XSu(H)1 (2 ng or 5 ng) (A3) or XSu(H)2-MO (25 ng) + mRNAs of ∆5TR XSu(H)2 (2 ng or 5 ng) (A4) into one dorsal blastomere at the 4-cell stage and morphological phenotype was examined at stage 20. Upper panel shows the dorsal view and lower panel indicates the injected side shown by GFP fluorescence on the same view. (B) Embryos were injected into the marginal zone of both blastomeres at the 2-cell stage and used for the assay of quantitative RT-PCR at stage 11. The defective neurogenesis and the reduction of Xbrachyury expression caused by XSu(H)2-MO were rescued by ∆5TR XSu(H)2 (A4, B), but not by ∆5TR XSu(H)1 (A3, B).
Fig. 5. Xbrachyury expression suppressed by XSu(H)2-MO cannot be rescued by activation of Notch signaling. (A) Embryos were injected with XSu(H)2ANK (2 ng), XSu(H)2DBM (2 ng), XSu(H)2-MO (50 ng), control-MO (50 ng) + NICD (2 ng) or XSu(H)2-MO (50 ng) + NICD (2 ng). Animal cap explants were isolated from the injected embryos at stage 8 and were cultured until stage 11 for RT-PCR. XSu(H)2ANK as an activation construct of Notch signaling increased the gene expression of XESR-1, while XSu(H)2DBM as a dominant-negative form of Notch signaling inhibited it. Even under depleted XSu(H)2 protein, NICD could activate the expression of XESR-1. (B) Embryos were injected with control-MO (B1), XSu(H)2- MO (B2), XSu(H)2-MO + mRNAs of ∆5TR XSu(H)2 (B3), mRNAs of NICD (B4) or XSu(H)2-MO + mRNAs of NICD (B5) into the marginal zone of one blastomere at the 2-cell stage. The injected embryos were cultured until stage 10.5 and the expression of Xbrachyury was examined using whole- mount in situ hybridization. All embryos were injected with 1 ng of β-galactosidase mRNA as a tracer of the injection side. The injected side was colored blue by staining the activity of β-galactosidase. The expression of Xbrachyury was colored brown. Upper and lower panels show vegetal and lateral views, respectively. Suppression of Xbrachyury gene expression by XSu(H)2-MO occurred widely (B2) and could be rescued by co-injection of ∆5TR XSu(H)2 (B3); however, activation of Notch signaling by NICD could not rescue the XSu(H)2-MO-induced reduction of Xbrachyury expression (B5). NICD alone did not suppress Xbrachyury expression (B4). (C) Synthesized RNAs of 50 pg Xnr2 were injected into the animal pole of 2-cell stage embryos with 50 ng various MO or 2 ng mRNAs. Animal caps were dissected from the injected embryos at stage 8 and were harvested at stage 11 for RT-PCR analysis. XSu(H)2-MO reduced Xbrachyury expression induced by Xnr2. ∆5TR XSu(H)2, but not NICD could rescue the XSu(H)2-MO- induced suppression of Xbrachyury gene expression.
Fig. 6. Xbrachyury expression by XSu(H)2 is independent of regulation by XESR-1. Synthesized RNAs of 50 pg Xnr2 were injected into the animal pole of 2-cell stage embryos together with XESR-1 (1 ng)), XESR- 1 (1 ng) + DN XESR-1 (4 ng), XSu(H)2-MO (50 ng), XSu(H)2-MO (50 ng) + ∆5TR XSu(H)2 (4 ng), XSu(H)2-MO (50 ng) + DN XESR-1 (4 ng). Animal caps were dissected from the injected embryos at stage 8 and were cultured until stage 11 and then the gene expression of Xbrachyury was examined using quantitative RT-PCR. Overexpression of XESR-1 reduced Xbrachyury induced by Xnr2. This inhibition could be rescued by co- injection with DN XESR-1. The reduction of Xbrachyury expression caused by XSu(H)2-MO could be rescued by co-injection of ∆5TR XSu(H)2, but not dominant-negative XESR-1, DN XESR-1.
