Fukuda M et al. (2010), Zygotic VegT is required for Xenopus paraxial m...

XB-ART-40819

Int J Dev Biol 2010 Jan 01;541:81-92. doi: 10.1387/ijdb.082837mf.

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Zygotic VegT is required for Xenopus paraxial mesoderm formation and is regulated by Nodal signaling and Eomesodermin.

Fukuda M , Takahashi S , Haramoto Y , Onuma Y , Kim YJ , Yeo CY , Ishiura S , Asashima M .

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The T-box gene VegT plays a crucial role during mesendoderm specification of the amphibian embryo. While the function of maternal VegT (mVegT) has been extensively investigated, little is known about the function and transcriptional regulation of zygotic VegT (zVegT). In the present study, we used comparative genomics and a knockdown experiment to demonstrate that zVegT is the orthologous gene of zebrafish Spadetail/Tbx16 and chick Tbx6L/Tbx6, and has an essential role in paraxial mesodermal formation. zVegT knockdown embryos show several defects in the patterning of trunk mesoderm, such as abnormal segmentation of somites, a reduction in muscle, and the formation of an abnormal mass of cells at the tail tip. We also identified the cis-regulatory elements of zVegT that are necessary and sufficient for mesoderm-specific expression. These cis-regulatory elements are located in two separate upstream regions of zVegT, corresponding to the first intron of mVegT. The results of in vitro binding and functional assays indicate that Forkhead box H1 (FoxH1) and Eomesodermin (Eomes) are the trans-acting factors required for zVegT expression. Our results highlight the essential role of zVegT in organization of paraxial mesoderm, and reveal that zVegT is regulated by a coherent feedforward loop of Nodal signaling via Eomes.

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Species referenced: Xenopus laevis
Genes referenced: eomes foxh1.2 gal.2 nodal odc1 tbx2 tbx5 tbx6 tbxt vegt
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	Fig. 1. Zygotic VegT is the Xenopus ortholog of the VegT/Spadetail gene subfamily. (A) Phylogenetic tree of the T-box gene family associated with vertebrate mesoderm development. The phylogenetic tree was calculated by MacVector 7.2.3 software. Human Tbx6 splicing variant1 (GenBank accession no. NM_004608), human Tbx6 splicing variant2 (GenBank accession no. NM_080758), human Brachyury (T) (GenBank accession no. NM_003181), human Eomesodermin (GenBank accession no. NM_003181), mouse Tbx6 (GenBank accession no. NM_011538), mouse T (GenBank accession no. NM_009309), mouse Eomesodermin (GenBank accession no. NM_010136), chick TbxL/Tbx6 (GenBank accession no. AB193180), chick Brachyury (GenBank accession no. U25176), X. tropicalis maternal VegT (GenBank accession no. AB451530), X. tropicalis zygotic VegT (GenBank accession no. AB451530), X. tropicalis Tbx6 (GenBank accession no. NM_001007994), X. tropicalis Xbra (GenBank accession no. BC081350), X. tropicalis Eomesodermin (GenBank accession no. NM_001128652), zebrafish Spadetail/Tbx16 (GenBank accession no. AF077225), zebrafish Tbx6 (GenBank accession no. U80951), zebrafish No tail (GenBank accession no. AB088068), zebrafish Eomesodermin a (GenBank accession no. NM_131679), and zebrafish Eomesodermin b (GenBank accession no. NM_001083575) were tested. Zebrafish Tbx5 (GenBank accession no. NM_130915) was used as the outgroup. (B) Genomic structure of the X. tropicalis VegT locus. First exon of mVegT, first exon of zVegT, and common exons of both variants are indicated as orange, green, and red boxes, respectively. (C) Comparison of the exon structure of genes in the VegT/Spadetail subfamily between six vertebrate genomes. The numbers shown on the left side of the box indicate the base pair size of each exon. (D) Conservation of synteny. By using Metazome, the flanking upstream and downstream genes of the putative X. tropicalis VegT orthologs were compared between six vertebrates. This set of genes is comprised of common orthologous genes among five vertebrate species including X. tropicalis, chick, fugu, medaka, and stickleback. Red arrows indicate the locus of the VegT ortholog. The gene and the transcriptional orientation are indicated by the color and direction of the arrow, respectively. (E-G) X. tropicalis zVegT expression patterns at stage 10, 11, and 12. The antisense probe for WISH recognized both mVegT and zVegT. Panels at the left and right show the lateral and vegetal views with the dorsal side up, respectively.
	Fig. 2. Zygotic VegT knockdown embryos show severe defects in the structure of the posterior mesoderm. (A,C,E) Control MO-injected embryos. (B,D,F) zVegT MO-injected embryos. 12 ng of MOs were injected into the marginal zone of both blastomeres at the two-cell stage in X. tropicalis embryos, harvested at stage 34 (C-F) or 40 (A,B). (C,D) Immunochemistry with the muscle-specific antibody, MF-20. (E,F) Immunochemistry with the notochord-specific antibody, Tor70. Panels at the right indicate magnification of the trunk region.
	Fig. 4. Nodal signaling, but not FGF signaling, is necessary for expression of zygotic VegT. (A,B) CerS inhibits zVegT expression in X. laevis embryos. WISH of the zVegT gene was performed on CerS- injected or uninjected embryos. 300 pg of CerS mRNA was injected into the marginal zone of one blastomere at the two-cell stage, and embryos were harvested at stage 11. Panels at the left and right for the CerS- injected embryo show the vegetal view with the dorsal side up and lateral view, respectively. (C) Expression of the zVegT gene is inhibited by coinjection of CerS mRNA in animal caps. CerS (600 pg) was coinjected with zVegT, Eomes, Tbx6, or Xbra (1 ng) into the animal pole of both blastomeres at the two-cell stage. Animal cap cells were explanted at stage 9 and then cultured until stage 11 for RT-PCR. Injected mRNAs were not detected by these experiments. (D-K) FGF signaling is not required for zVegT gene expression. The overexpression of XFD inhibited the expression of Xbra (H,I) and Tbx6 (J,K), but not the expression of zygotic VegT (D,E) and Eomes (F,G) in X. laevis embryos. Embryos were injected with 1 ng of XFD mRNA into the dorsal marginal zone of two blastomeres at the four-cell stage, and were fixed at stage 11 for WISH. Vegetal views are shown with dorsal side up. (L) Expression of zVegT and FGFs is induced dose-depen- dently by Activin in animal cap cells. The animal caps dissected at stage 9 were treated with 1, 10, or 100 ng/ml Activin, and harvested at stage 11 for RT-PCR analysis. (M) Expression of zVegT is induced by Activin independent of FGF signaling. The animal caps dissected at stage 9 were treated with or without Activin (10 ng/ml) and SU5402 (80 μM), and were harvested at stage 11 for RT-PCR analysis. The induction of expression of zVegT and Eomes by Activin was not affected by inhibition of FGF signaling. The expression of Xbra and Tbx6 was downregu- lated by SU5402 in animal cap cells.
	Fig. 6. Comparative gene expression patterns of the T-box genes in X. laevis embryos. (A-Q) Spatial expression of zVegT (A-E), Eomes (F- J), Xbra (K-N), and Tbx6 (O-Q) was detected by WISH from stage 9 to stage 11. Arrowheads indicate the initial zygotic expression of individual genes. The VegT antisense probe recognized both mVegT and zVegT. The top and bottom panels show the vegetal view with dorsal side up and dorsolateral view, respectively. (R) RT-PCR reveals temporal expression of T-box genes.
	Fig. 7. Eomesodermin is essential for expression of zygotic VegT. (A) Diagram of the exon-intron structure of Eomes and design of two Eomes splice-inhibiting antisense MOs. The X. tropicalis Eomes gene has 6 exons. The splicing sites targeted by the Eomes MO1 and Eomes MO2 (purple) were the boundary of intron1-exon2 and exon2-intron2, respectively. The light blue arrows indicate the PCR primers to confirm Eomes splicing. (B) Eomes MOs effectively inhibited the proper splicing of Eomes, and suppress expression of zVegT. For these experiments, 12 ng of Eomes MO1 or Eomes MO2 was injected into the marginal zone of both blastomeres at the two-cell stage in X. tropicalis embryos and then harvested at stage 10+. Eomes MO1 or Eomes MO2 effectively inhibited the splicing of Eomes pre-mRNA and the expression of zVegT transcripts. Sequence analysis confirmed that Eomes MO1 and Eomes MO2 caused marked premature termination of Eomes transcripts (arrowheads). Arrows show the band corresponding to normal Eomes transcripts. (C-H) VegT expression is eliminated in the Eomes MOs-injected region. Eomes MOs or control MO (6 ng) and β-gal mRNA (100 pg) were coinjected into the marginal zone of one blastomere at the two-cell stage, and were fixed at stage 10 for WISH. Red gal staining indicates the injected side. Lateral views are shown.
	Supplementary Fig. S3. X. laevis transgenic embryos expressed the EGFP reporter gene in the marginal region. Reporter EGFP expression was detected by WISH (Figs. 3A and 5 A,B). Panels at the left and right indicate lateral and vegetal views with dorsal side up, respectively. All embryos show equivalent expression patterns for endogenous zVegT.
	Supplementary Fig. S1. Temporal and spatial gene expression patterns of VegT during oogenesis and embryogenesis of X. tropicalis. (A) Temporal expression was analyzed by semi-quantitative RT-PCR. Primer pairs specific for X. tropicalis ODC were described previously (Haramoto et al., 2004). The primers designed for X. tropicalis mVegT are as follows: 5 ATGAGAAACTGCTGTCAGGAACAC-3and 5TGAAACCTGGGCTTGTAGCG- 3 (B-R) Spatial expression was analyzed by WISH. Roman figure shows stage of oogenesis.