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Fig. 1. Pronephric expression of Xenopus Tbx2 analyzed by in situ hybridization. (A,B) Expression of Tbx2 around the pronephric anlage (pa) at stage 21 (A) and 23 (B). Arrowhead indicates weakly expressed Tbx2 around the pronephric anlage. (C) Transverse section of the embryo in B at the level indicated by the dashed line. (D) Tbx2 expression surrounding the developing pronephric tubule and duct at stage 31/32. (E) Magnified view of the boxed area in D. Arrowheads indicate the increased expression of Tbx2 in the dorsal region of the pronephric tubule anlage. (F,G) Transverse sections of the embryo in D at the levels indicated by the dashed lines. The arrow (F) indicates the absence of expression of Tbx2 in the medial layer of the intermediate mesoderm (IM). (H) Tbx2 expression enclosing the pronephric tubule (pt) and duct (pd) at stage 35/36. (I) Enlarged view of the boxed region in H. Arrowheads point to the expression of Tbx2 around the tips of nephrostomes. (J,K) Transverse sections of the embryo in H at the levels indicated by the dashed lines. The pronephric glomus (g) is outlined in red in J. The arrow indicates Tbx2 expression in the non-glomus mesenchyme in the medial layer of IM. (L) Pax2 expression in the pronephric anlage at stage 21. (M-O) Pax2 expression in transverse sections of the pronephric anlage, tubule and duct at the indicated stages. (P) Strong expression of Pax2 in the tips of nephrostomes (arrowheads). |
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Fig. S4. Ectopic expression of WT1 expands the glomus domain and inhibits proximal tubule formation. (A-C) Embryos injected with WT1 RNA (200 pg) were subject to in situ hybridization for Pax2, Nephrin and Tbx2 at stage 32. Note that WT1 specifically impairs Pax2 expression in the pronephric tubules (arrowhead), whereas it increases Nephrin expression. Tbx2 expression is still detectable around the expanded pronephric mesoderm in the WT1-injected side of the embryo. Control, uninjected side. |
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Fig. 6. BMP signaling inhibits the expression of Hey1 and Gremlin via Tbx2. (A-J) Xenopus embryos injected with the indicated combinations of Smad1-GR (250 pg), Delta-Smad7tevGR (250 pg)/TEV2GR (10 pg) and Tbx2-GR (200 pg) mRNA were subjected to in situ hybridization for Tbx2, Pax2, Hey1 or Gremlin. Control shows the uninjected side of the embryo. DEX treatment was from stage 22 to 35. Arrowheads (G,H) indicate Hey1 expression in the pronephros. (K-N) Quantification of rescue experiments shown in A-J. n, total number of embryos analyzed. |
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Fig. 7. Gremlin and Hey1 downregulate Tbx2 by antagonizing BMP signaling. (A) Animal cap tissues from Xenopus embryos injected with Hey1 (100 pg) or Gremlin (10 pg) mRNA were subjected to RT-PCR analysis. (B-E) Embryos injected with Hey1 (100 pg) or Gremlin (10 pg) mRNA were subject to in situ hybridization for Tbx2, Gremlin or Hey1. Control shows the uninjected side of the embryo. Arrowheads (E) indicate Hey1 expression in the pronephros. (F) Animal caps from embryos injected with a combination of Bmp4 (200 pg), Gremlin (200 pg) and Tbx2 MO (10 ng) were treated with RA and activin for in vitro kidney induction and then subjected to RT-PCR analysis. W, whole embryo; AC, uninjected animal caps without RA and activin treatment; (–), uninjected animal caps with RA and activin treatment; –RT, negative control without reverse transcriptase; ODC, ornithine decarboxylase loading control. |
