Uchiyama H et al. (2001), Cloning and characterization of the T-box gene ...

XB-ART-7997

Dev Growth Differ 2001 Dec 01;436:657-69. doi: 10.1046/j.1440-169x.2001.00606.x.

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Cloning and characterization of the T-box gene Tbx6 in Xenopus laevis.

Uchiyama H , Kobayashi T , Yamashita A , Ohno S , Yabe S .

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Tbx6 is a member of the T-box gene family. Studies of knockout mice indicate that Tbx6 is involved in somite differentiation. In the present study, we cloned Tbx6 from another vertebrate species, namely Xenopus laevis, and studied its roles in development. The expression of Tbx6 in Xenopus started from the early gastrula stage, reached a peak during the late gastrula to neurula stages and then declined. Initial expression of Tbx6 was observed in the paraxial mesoderm during the gastrula stage. The Tbx6-expressing region spread anteriorly and ventrally in the neurula stage. In the tailbud stage, the area of expression shrank caudally and was finally restricted to the tip of the tailbud. Overexpression of Tbx6 mRNA in dorsal blastomeres caused atrophy of the neural tube and inhibited differentiation of the notochord. Animal cap explants overexpressing Tbx6 or Tbx6VP16 mRNA, but not Tbx6EnR mRNA, differentiated mainly into ventral mesodermal tissues. This suggests that Tbx6 is a transcriptional activator. Higher doses of Tbx6 or Tbx6VP16 mRNA caused hardly any muscular differentiation. However, coinjection of Tbx6 mRNA with noggin mRNA elicited marked muscle differentiation. These results suggest that Tbx6 is implicated in ventral mesoderm specification but is involved in muscle differentiation when acting together with the dorsalizing factor noggin.

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Species referenced: Xenopus laevis
Genes referenced: actc1 actl6a bmp4 eomes hba4 nog odc1 pdx1 tbx2 tbx6 tbxt vegt wnt8a

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	Fig. 1. (A) Structure of Tbx6 cDNA. The white box is a coding sequence; the hatched box is the T-box. Solid lines are 5 and 3 untranslated regions. The initial DNA fragment obtained by degenerate polymerase chain reaction and the BamHI fragment obtained from the 5 RACE product are shown by bold lines. (b) Deduced amino acid sequence of Xenopus laevis Tbx6. The T-box is enclosed with a solid line. The putative nuclear localization signal (NLS) and SPXX sequences are underlined. E311 is shown in bold. After this glutamic acid, DNA was exchanged with VP16 or EnR in making fusion constructs (see also Fig. 5A). (C) Dendrogram of the T-box amino acid sequences of Tbx6-related proteins calculated by GeneWorks 2.5.1. Zebrafish tbx16 (Ruvinsky et al. 1998) is shown as spadetail. Chick Tbx6L (Knezebic et al. 1997) falls into the VegT/Apod group rather than the Tbx6 group.
	Fig. 2. (A) Northern blot analysis of Tbx6. Two solid lines on the left indicate rRNA. An arrow on the right shows the Tbx6 band. (B) Embryos at stages indicated were analyzed by reverse transcriptionâpolymerase chain reaction using specific primers for Tbx6. The expression of ornithine decarboxylase (ODC) is also shown as a control.
	Fig. 3. Whole-mount in situ hybridization of Tbx6. (A) Stage 12. (B) Stage 13. (C) Stage 16. (D) Stage 32. (E) Stage 40. (F) Stage 14 frontal section at the trunk level. (G) Stage 32 section near the proctodeum. (Hâ€“J) Sagittal sections of stage 13 (H), stage 15 (I) and stage 17 (J) embryos. Arrows indicate segmental borders. (Aâ€“E) Anterior is to the left. Dorsal view. (F,G) Dorsal is to the top. (Hâ€“J) Anterior is at the left, dorsal is at the top. Ar, archenteron; Bc, blastocoele; Bp, blastopore; Np, neural plate; No, notochord; Lp, lateral plate mesoderm; Pc, proctodeum; PS, presomitic mesoderm.
	Fig. 4. Overexpression of Tbx6 in embryos. (A) Stage 31 embryo injected dorsally at the four-cell stage with 400 pg Tbx6 mRNA. Anterior is to the left. (B) Stage 31 embryo injected ventrally at the four-cell stage with 400 pg Tbx6 mRNA. Arrowheads indicate incomplete anal closure. (C) Enlarged view of the anal region of (B). (D) Enlarged view of the anal region of a normal stage 31 embryo. (E) Transverse section of (A) at the trunk level. Note that the neural tube is disorganized and the notochord has few vacuoles compared with (F). In contrast, the somite is enlarged. Nt, neural tube; No, notochord; S, somite. (F) Transverse section of a normal stage 31 embryo.
	Fig. 5. Appearance and histology of the animal cap explant. (A) The Tbx6 gene was fused to EnR and VP16. (BâE) Animal cap explants at stage 13. (B) No injection control. (C) Tbx6 mRNA-injected cap. (D) Noggin mRNA-injected cap. (E) Tbx6 mRNA- and noggin mRNAcoinjected cap. (FâK) Sections of the animal cap explants at stage 30. (F) No injection control. (G) Tbx6 mRNA-injected cap. (H) Tbx6VP16 mRNA-injected cap. (I) Tbx6EnR mRNA-injected cap. (J) Noggin mRNA-injected cap. (K) Tbx6 mRNA- and noggin mRNAcoinjected cap. Bl, blood-like cells; Cg, cement gland; Mt, mesothelium; Mu, muscle; N, neural-like cells. (BâE) and (FâK) are at the same magnifications, respectively.
	Fig. 6. Reverse transcriptionâ polymerase chain reaction (RTPCR) analysis of animal cap explants. (A) Animal caps were mesodermalized by treatments with proteins (activin and basic fibroblast growth factor), microinjected mRNAs (Xbra, Apod, Tbx6 and BMP-4) or microinjected plasmid (CSKA-Xwnt8). Total RNA was extracted at stage 13 and analyzed with RTPCR using specific primers for Tbx6, Apod, Xbra, Eomes, Xwnt8 and ornithine decarboxylase (ODC). The Xbra band in Xbra mRNA-injected animal caps, Apod band in Apod mRNA-injected animal caps and the Tbx6 band in Tbx6 mRNA-injected animal caps indicate auto-inductions, respectively, because the Xbra R primer, Apod R primer and Tbx6 F and R primers are located in a portion of the 3 untranslated region not included in the template for Xbra, Apod or Tbx6 mRNA synthesis. âRT is a negative control from RNA extracted from Tbx6 mRNA-injected animal caps. (B) Animal caps overexpressing Tbx6, noggin or Tbx6 + noggin mRNA were harvested at stage 30 and analyzed with RT-PCR for T4 globin, cardiac actin and XlHbox8. âRT is a negative control from RNA extracted from Tbx6 mRNA-injected caps.