|
Display additional annotations [+]
|
| |
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). |
|
Display additional annotations [+]
|
| |
Fig. 2. Ectopic expression of Tbx2 inhibits pronephric nephron formation. (A-J) Xenopus embryos injected with Tbx2 (200 pg), Tbx2-GR (200 pg), VP16-Tbx2δC (100 pg) or EnR-Tbx2δC (100 pg) mRNA were treated (B,D,F) or otherwise (A,C,E,G-J) with dexamethasone (DEX) from stage 22 to 31 or 34 and then subjected to in situ hybridization for the pronephric markers Pax2, WT1 and Gremlin. The left and right images in each panel indicate the injected and uninjected control sides of the embryo, respectively. Of note, VP16-Tbx2δC disorganizes the pronephric tubules (G) and expands the glomus domain (I). (K) Tbx2 constructs used in the injection experiments. T-BOX, T-box from Tbx2; R, repressor domain of Tbx2; GR, human glucocorticoid receptor ligand-binding domain; VP16, transactivation domain of VP16; EnR, transrepression domain of Engrailed. |
|
Display additional annotations [+]
|
| |
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. |
|
Display additional annotations [+]
|
| |
Fig. 5. Tbx2 downregulates the expression of Gremlin and Hey1 to control pronephric morphogenesis. (A) Diagram of the in vitro kidney induction assay. RA, retinoic acid. (B-N) Four-cell stage Xenopus embryos were injected in the animal pole region with Tbx2 mRNA (100 pg) or Tbx2 MO (10 ng) and then the animal explants were dissected at stage 9.5 and processed for in vitro kidney induction. Subsequently, the animal cap tissues were observed for morphology (B-E), sectioned for in situ hybridization with a Pax2 antisense probe (F-I) and immunohistochemistry with the tubule-specific antibody 3G8 (J-M) or subjected to RT-PCR analysis (N). 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. Arrowheads (G,K,I,M) indicate Pax2 expression or 3G8 staining in the induced animal caps. (O-R) Embryos injected with Tbx2 (200 pg), Tbx2-GR (200 pg), Tbx2 MO (10 ng) or Tbx2δC-GR (200 pg) were subject to in situ hybridization with a Hey1 antisense probe. Arrowheads indicate Hey1 expression in the pronephros. Embryos in P,R were treated with DEX from stage 22 to 32. (S-U) Impaired expression of Pax2 caused by Tbx2 was restored by co-injection of Gremlin or Hey1. Embryos injected with Tbx2 mRNA (200 pg) with or without Hey1 (100 pg) or Gremlin (10 pg) were subject to in situ hybridization for Pax2 at stage 35. n, total number of embryos analyzed; the percentage of embryos showing the phenotype illustrated is indicated. Left and right parts of each panel show the injected and uninjected control sides of embryos, respectively. |
|
Display additional annotations [+]
|
| |
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. |
