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In vertebrate early development, the neural tissue is specified along the antero-posterior (A-P) axis by the activity of graded patterning signals such as Wnt, Nodal and FGF. Attenuation of these signals has been shown to play critical roles in the determination of anterior neural region, but it remains poorly understood how FGF action is counteracted in the neural plate. Here, we show that BMP signal acts as an antagonist of FGF signaling for AP neural patterning in Xenopus embryo. During the neurula stages, BMP signal was up-regulated in the anterior neural plate, displaying a graded pattern along the AP axis. Inhibition of the late BMP signaling after mid-gastrulation abrogated the expression of anterior neural markers. We found that BMP signaling interfered with FGFs-induced ERK phosphorylation and neural caudalization. This inhibitory action of BMP signal involved repression of the expression of Flrt3, a positive regulator of FGF signaling. Furthermore, the gain- and loss-of-function of Flrt3 inhibited and expanded the expression of forebrain marker genes, respectively. Together, these results demonstrate that BMP signal can down-regulate FGF pathway via inhibition of Flrt3 expression for anterior neural formation, revealing stage-specific roles of BMP signaling and its novel crosstalk with FGF pathway in neural development.
BMP signal forms a graded pattern along the antero-posterior axis of a neurulae. (A) Western blotting analysis showing the activities of FGF and BMP signaling in the dorsal neural ectoderm, which is subdivided into four parts (I, II, III and IV), along the A-P axis of a neurula stage Xenopus embryo. An embryo in the cartoon is viewed laterally with anterior to the left. (B) Whole embryos were subjected to immunohistochemistry against phospho-Smad1 at stage 11 and 13. Embryos are viewed as indicated. A, anterior; P, posterior; D, dorsal; V, ventral.
BMP signals down-regulate FGF signaling. (A, C–I) Four-cell stage embryos were injected in the animal pole region with the indicated combination of BMP4 (200 pg), BMP4-HA (100, 250 pg), BMP2 (200 pg), noggin (10 pg), FGF8 (1 ng), eFGF (1 ng), IGF1 (1 ng) and Wnt8 (200 pg) mRNA, and then the animal cap explants were dissected at stage 8 and cultured until stage 12 for Western blotting (A, C, D, F–I) or until stage 15 (E) for RT-PCR analyses. WE, whole embryo; -RT, a control in the absence of reverse transcriptase; Con. AC, uninjected control animal caps. ODC and ERK serve as loading controls. (F) After excision, the animal caps were cultured to stage 12 in the presence or absence of cycloheximide (CHX; 10 μg/ml). (B) HEK 293T cells were transfected with BMP2 (0.5 μg) or BMP4 (0.5 μg) DNA and harvested for Western blotting analysis 24 h later.
BMP activation is necessary for maintenance of anterior neural fate. (A–D) Four-cell stage embryos were injected in the animal pole region as indicated with chordin (200 pg), noggin (10 pg), FGF8 (B, 100–500–1000 pg; C, 1000 pg), Smad1-GR (200 pg), ΔSmad7tevGR (250 pg) and TEVGR (10 pg) mRNA, and then the animal cap explants were dissected at stage 8 and cultured until stage 12 for Western blotting (D) or stage 15 for RT-PCR (A–C). (A, C, D) Animal caps were treated with SU5402 (20 μM) from stage 8 to 15 (A) or dexamethasone (DEX; 10 μM) from stage 8 or 10.5 to 15 (C) or from stage 8 to 12 (D) as indicated. (E) Smad7tevGR (250 pg) and TEVGR (10 pg) mRNAs were injected into the dorso-animal region of one blastomere at the 4-cell stage, and the injected whole embryos were cultured in the presence of DEX for the indicated periods and harvested for whole-mount in situ hybridization against Otx2, Bf1, En2 and Krox20. An arrow and arrowhead indicate the expansion of Bf1 and En2 expression, respectively. Asterisks denote Krox20 expression. Embryos are shown in anterior view with dorsal to the top. Inj., injected side.
BMP signal inhibits Flrt3 expression to down-regulate FGF pathway. (A–E) Four-cell stage embryos were injected in the animal pole region as indicated with noggin (10 pg), tBR (100 pg), FGF8 (1 ng), BMP4 (200 pg), Flrt3 (200 pg), Flrt3 MO (12.5 ng) and Co MO (12.5 ng), and then the animal cap explants were excised at stage 8 and cultured to stage 12 for RT-PCR (A, B) or Western blotting (C–E) analyses. (F–I) Four-cell stage embryos were injected dorso-animally with Flrt3 (200 pg) or Flrt3 MO (10 ng), cultured until stage 15 and then subjected to in situ hybridization against Otx2, Bf1, En2 and Krox20. Anterior view with dorsal to the top.