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Fig. 5. Inhibition of BMP signaling is required for the expression ofpitxgenes, while BMP/Smad1 signaling is required for cement gland development. (A) Pitx1-mediated inhibition of the BMP target gene sizzled is rescued by co-expression of either Smad1 or CA-BMPRIa. Pitx1-mediated induction of two pitx2 transcript variants, pitx2b and pitx2c, as well as endogenous pitx1, are down-regulated following enhancement of BMP signaling. 50 pg pitx1 RNA was either injected alone or co-injected with 1.6 ng smad1 or CA-BMPRIa RNA at early cleavage stages. RT-PCR analysis was performed on animal caps harvested at mid-neurula stages after dissection at late blastula stages. (B) 90% of neurula stage control embryos (n = 20) display strong pitx1 staining. Water was injected into the animal pole of 4-cell stage Xenopus embryos. (C) 88% of embryos (n = 16) exhibit weak and spatially restricted pitx1 staining following injection of 500 pg CA-BMPRIa RNA at early cleavage stages. Embryo views are anterior, ventral to bottom. (D) Injection of noggin RNA inhibits Pitx1-mediated induction of cement gland differentiation genes. 400 pg pitx1-GR RNA and/or 20 pg noggin RNA were injected into Xenopus embryos. RT-PCR analysis was performed on animal caps dissected at late blastula stages and cultured with or without dexamethasone until mid-neurula stages. Dexamethasone (10 µM) was added to animal caps at stage 12. (E) Pitx1 binds to Smad1. 2 ng pitx1-myc RNA was injected at early cleavage stages. Pull-down of native Smad1 from injected embryos leads to co-immunoprecipitation of exogenous Pitx1. Normal rabbit IgG antibodies was used in parallel studies as a negative control.
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