XB-ART-496Int J Dev Biol January 1, 2006; 50 (5): 473-9.
Bowline, a novel protein localized to the presomitic mesoderm, interacts with Groucho/TLE in Xenopus.
Cells in the prospective somite of Xenopus laevis embryos rotate in an orchestrated manner to form a segregated somite. The prospective somite boundaries are prepatterned by gene expressions in the unsegmented presomitic mesoderm (PSM). However, the roles of polarized gene expression in this boundary formation are not well elucidated. Here we identified a novel gene, bowline, which localizes to the anterior halves of S-II, III in the PSM of X. laevis. Bowline associated with corepressor XGrg-4, a Xenopus homolog of Groucho/TLE protein. A WRPW tetrapeptide motif in Bowline was prerequisite for coprecipitation with XGrg-4 and for downregulation of X-Delta-2 by bowline RNA injection. This study indicates that Bowline is a novel protein interacting with Groucho/TLE and may play a role in somitogenesis in X. laevis.
PubMed ID: 16586348
Article link: Int J Dev Biol
Genes referenced: dlc dll1 dll4 gal.2 hes4 mespa myc notch1 pcdh8 ripply2.1 ripply2.2 ripply3 tbx2 tle4
Antibodies: GFP Ab6 Myc Ab2
Article Images: [+] show captions
|Fig. 1. Structural features of Bowline. The complete peptide sequence of Bowline deduced from its nucleotide sequence is aligned with human �similar to DSCR6� (s-DSCR6; NP001009994), chick hypothetical protein (XP419859) and X. laevis Ledgerline (BAB90857). Asterisks under the sequences indicate the position of conserved amino acids. A yellow box indicates the position of the conserved WRPW sequence and a red box indicates the BDLC-region. Sequences were aligned using GENETYX software. The Genbank accession number for the bowline nucleotide sequence is AB105905.|
|Fig. 2. Localization and transcriptional regulation of bowline transcripts. (A-C) Xenopus embryos from different stages were stained for expression of bowline mRNA by whole-mount in situ hybridization at (A) stage 13, (B) stage 18 and (C) stage 30. Embryos are oriented in dorsal view with anterior to the top except for (C), for which the anterior is to the left. (D-I) Longitudinal section through stage 20 embryos stained for expression of XDelta- 2 (D), bowline (E,G,I) and Thy1 (F,H). Embryos are oriented with anterior to the left. Arrowheads on the lower side of the embryos indicate the anterior end of X-Delta-2-and Thy1-expressing regions. The probes used are shown in the upper right corner. In (H and I), the width of the expression region for Thy1 and bowline are indicated as red and black lines, respectively. (J) Diagram showing the position of bowline expression relative to that of other genes known to be expressed segmentally in X. laevis embryos (Jen et al., 1997, Kim et al., 2000, Sparrow et al., 1998). (KM) Embryos were unilaterally injected with β-gal RNA (K), XotchδE RNA (L), or XSu(H)DBM RNA (M) and analyzed for the expression of bowline (K,L,M) by whole-mount in situ hybridization at stage 20. RNA encoding the lineage tracer β-gal was co-injected to identify the injected side (red staining). Dorsal views with anterior towards the top are shown. Injected sides are indicated as �inj.� Somitomeres were demarcated as S0, S-I, S-II, S-III based on the annotation of Pourqui� and Tam (Pourqui� and Tam, 2001). Bars, 100 μm.|
|BowlineδWRPWFig. 3. The WRPW motif is required for Bowline interaction with XGrg-4. (A) Extracts prepared from embryos injected with bowline- EGFP alone (lane 1), Myc-XGrg4 and bowline-EGFP (lane 2), or Myc- XGrg4 and bowlineδWRPW-EGFP (lane 3) were subjected to immunoprecipitation (IP) with an anti-myc antibody followed by Western blotting (WB) with either anti-GFP or anti-myc antibody. (B) Extract prepared from embryos injected with Myc-XGrg4 alone (lane 1), Myc-XGrg-4 and bowline- EGFP (lane 2), or Myc-XGrg-4 and bowlineδWRPW-EGFP (lane 3) were subjected to IP with an anti-GFP antibody, followed by WB with the anti-myc antibody. Lane 4 was an uninjected negative control. (A,B) 5% of embryo extracts were loaded as input to indicate the expression (A,B; lanes 5-8). (C,D) Subcellular localization of Bowline-EGFP protein. EGFP RNA (1 ng) (C) or bowline-EGFP RNA (1 ng) (D) was injected into two animal blastomeres of two-cell stage embryos. Animal caps were excised at stage 9-10 and examined for GFP fluorescence. Bars, 50 μm. (EG) Embryos injected unilaterally at the 4-cell stage with β-gal RNA (E), bowline RNA (F) or bowlineδWRPW (G) and analyzed for the expression of X-Delta-2 (E,F,G) by whole-mount in situ hybridization at stage 20. RNA encoding the lineage tracer β-gal was co-injected to identify the injected side (red staining). Dorsal views with anterior towards the top are shown. Injected sides are indicated as �inj.�|
|ripply2.2 (ripply2 homolog, gene 2) gene expression in Xenopus laevis embryo, NF stage 13, as assayed by in situ hybridization. Dorsal view: anterior up.|
|ripply2.2 (ripply2 homolog, gene 2) gene expression in Xenopus laevis embryo, NF stage 30, as assayed by in situ hybridization. lateral view: anterior left, dorsal up.|