Sugimoto K et al. (2005), XBtg2 is required for notochord differentiation...

XB-ART-1334

Dev Growth Differ 2005 Sep 01;477:435-43. doi: 10.1111/j.1440-169X.2005.00819.x.

Show Gene links Show Anatomy links

XBtg2 is required for notochord differentiation during early Xenopus development.

Sugimoto K , Hayata T , Asashima M .

???displayArticle.abstract???
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 notochord tissue, 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.

???displayArticle.pubmedLink??? 16179070
???displayArticle.link??? Dev Growth Differ

Species referenced: Xenopus laevis
Genes referenced: btg2 chrd myc not

???attribute.lit??? ???displayArticles.show???

	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 62–63% similar to the mammalian Btg2 sequence and 52% similar to zebrafish Btg-b. The two conserved domains of XBtg2 are 85–90% 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 pCS2–9m 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 pCS2–9m 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 (A–C; 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.