|
Display additional annotations [+]
|
| |
Fig. 3. Loss of Tbx2 function expands the territory of the pronephric duct and glomus but not the tubule. (A-L) Xenopus embryos injected with Tbx2 MO (10 ng), Tbx2δC-GR (200 pg) or control MO (Co MO, 10 ng) were subject to in situ hybridization for the glomus-specific markers WT1 and Nephrin and the duct-specific marker Gremlin at stage 35 or the tubule-specific marker SMP30 at stage 31. To activate injected Tbx2δC-GR mRNA, embryos were treated with DEX from stage 22 to 35 (B,E) or to 31 (H). (A-I) Stained embryos are shown in the upper part of each panel and transverse sections at the levels indicated by the dashed lines are shown below. Left and right parts of each panel show the injected and uninjected control sides, respectively. cl, coelom; g, glomus; pd, pronephric duct; pt, pronephric tubule. Arrows in A,B indicate WT1 and Nephrin expression in the somatic layer of the intermediate mesoderm, respectively. Arrows and bracket in D,E indicate migrating Gremlin-expressing cells and the diameter of pronephric duct, respectively. (J-L) Control MO has no effect on the expression of Nephrin, Gremlin or SMP30. |
|
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. 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. |
|
Display additional annotations [+]
|
| |
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. |
