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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. |
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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. |
