Integration of IGF, FGF, and anti-BMP signals via Smad1 phosphorylation in neural induction

Abstract

How do very diverse signaling pathways induce neural differentiation in Xenopus? Anti-BMP (Chordin), FGF8, and IGF2 signals are integrated in the embryo via the regulation of Smad1 phosphorylation. Neural induction results from the combined inhibition of BMP receptor serine/threonine kinases and activation of receptor tyrosine kinases that signal through MAPK and phosphorylate Smad1 in the linker region, further inhibiting Smad1 transcriptional activity. This hard-wired molecular mechanism at the level of the Smad1 transcription factor may help explain the opposing activities of IGF, FGF, and BMP signals not only in neural induction, but also in other aspects of vertebrate development.

Figure 1.

Comparison of neural induction by Chordin, IGF, and FGF8 in Xenopus embryos. (A-D) Sox2 expression: Microinjection of Chd (10 pg), IGF2 (800 pg), or FGF8 (50 pg) mRNA into a single animal blastomere at the 4-8-cell stage expanded the neural plate on the injected side. (E-H) N-tubulin expression: Chd (4 pg), IGF2 (100 pg), or FGF8 (5 pg) mRNA expanded the lateral-most sensory neurons (Rohon-Beard neurons) into lateral epidermis. (I-P) Embryos injected with protein into the blastocoele at blastula stage. Protein amounts per injection were 2.6 ng Chordin, 2 ng IGF2, and 1.4 ng FGF8, injected together with 40 ng BSA. (I-L) Unstained tail bud-stage embryos. (M-P) In situ hybridization for N-tubulin. (Q-S) Loss of N-tubulin-positive neurons after a single animal injection of 250 pg BMP7 mRNA, 16 ng IGFR-MO, or 500 pg DN-FGFR4a mRNA at the 4-cell stage. (T) Summary of signals involved in neural induction. (U) Animal cap explants from embryos injected with Chd mRNA (4 pg/blastomere) alone or in combination with control-MO (16 ng), IGFR-MO (16 ng), DN-FGFR4a mRNA (500 pg), or BMP7 mRNA (250 pg). Frequency of embryos with the indicated phenotypes was: B, 34/35; C, 27/32; D, 16/19; F, 14/22; G, 37/51; H, 117/120; Q, 26/30; R, 41/47; S, 34/38; (U) 10 explants per lane (tail bud stage), three independent experiments.

Figure 2.

Endogenous embryonic MAPK signals inhibit Smad1 activity in the Xenopus embryo. Wild type (WT), linker mutant (LM), double mutant (DM), or carboxy-terminal mutant (CM) Smad1 mRNAs were injected into each blastomere at the 4-cell stage (250 pg per injection). (A) Western blot showing similar levels of expression of Smad1 proteins at gastrula stage. (B) Diagram of Smad1 mutant proteins. (C-P) Uninjected and injected tail bud-stage embryos, unlabeled or after in situ hybridization with the ventral mesoderm marker Sizzled or the neuronal marker N-tubulin. Note that LM-Smad1 (I,J,K) is very active in inhibiting CNS differentiation and expanding ventral tissue. A minimum of 30 embryos were used per injected sample, 10 independent experiments.

Figure 3.

The MAPK phosphorylation mutant LM-Smad1 inhibits endogenous CNS formation and neural induction by FGF8 or IGF2 signals. (A-H) Neurula-stage embryos in dorsal view, uninjected (control) or injected four times into the animal pole with WT-Smad1, LM-Smad1, or DM-Smad1 mRNA. (I-L) FGF8-injected embryos in anterior view. (M-P) IGF2-injected embryos in lateral view. Embryos were injected at the 8-cell stage into a single animal blastomere together with nuclear lacZ mRNA as lineage tracer and stained for Sox2. Stippled lines mark the lacZ-positive injection sites. Note that LM-Smad1, but not WT-Smad1, blocks neural induction by FGF8 or IGF2 mRNA. (Q) RT-PCR analysis of stage 26 ectodermal explants from embryos injected at the 8-cell stage with the indicated mRNAs. WE, whole embryo; Co, uninjected animal cap explants used as controls. The amounts of injected mRNAs per blastomere were 250 pg Smad1, 4 pg Chd, 100 pg FGF8, 800 pg IGF2, and 200 pg nuclear lacZ. Endogenous neural marker expression was inhibited in: A, 0/72; B, 0/46; C, 58/58; D, 0/35; E, 0/44; F, 37/78; G, 67/68; H, 9/23 embryos. Ectopic Sox2 expression was inhibited in: J, 0/23; K, 0/34; L, 24/32; N, 0/69; O, 0/53; P, 91/109 embryos. In (Q) one whole embryo or 10 animal caps were used per lane, two independent experiments.

Figure 4.

FGF8 and IGF2 induce linker phosphorylation of Smad1 via MAPK in vivo. Proteins in cell lysates were analyzed by Western blot with antibodies against Flag (for Smad1) and Erk1/2. (A) FGF8 shifts the electrophoretic mobility of WT-Smad1 (lane 4), but not of LM-Smad1 (lane 6). NIH3T3 cells were transfected with Flag-tagged Smad1 constructs, serum-starved for 6 h, and recombinant mouse FGF8b protein (100 ng/mL) added for 10 min. (B) Pre-incubation of NIH3T3 cells with the MEK1 inhibitor U0126 (20 μM) 1 h before the addition of FGF8 suppresses the phosphorylation of Smad1 and Erk1/2 (lane 4). (C) in vivo ³²P incorporation into the linker MAPK sites of Smad1 in Xenopus. Animal caps from uninjected embryos, injected with CM-Smad1 or DM-Smad1 were explanted at late blastula stage and incubated with 2 mCi/mL ³²P orthophosphate for 45 min. (D) Xenopus embryos injected first with 400 pg Smad1 mRNA into each animal blastomere at the 8-cell stage, then with 40 nl FGF8 protein (1.4 ng) into the blastocoele at stage 8 and lysed 30 min later. FGF8 induces phosphorylation of Erk2 and WT-Smad1, but not of LM-Smad1 (lanes 3,5). Erk1 is not expressed in the early Xenopus embryo (Chesnel et al. 1997). (E) Xenopus oocytes at stages V-VI were injected with 4 ng Smad1 mRNA and 12 h later exposed to 18 ng/mL recombinant human IGF2 protein for 14 h. IGF2 induced phosphorylation of WT-Smad1 and Erk2. (F) Endogenous signals phosphorylate Smad1 linker region in Xenopus embryos. Equal numbers of Smad1 mRNA-injected embryos were lysed at mid-blastula (stage 8), early gastrula (stage 10.5), and late gastrula (stage 12.5), and protein extracts were analyzed by Western blot with antibodies against Erk, phosphorylated Erk (pErk), and Flag (for Smad1). Note the mobility shift of WT-Smad1 (lanes 2,3) but not LM-Smad1 (lanes 5,6) in samples with high levels of phosphorylated Erk.

Figure 5.

Model of the integration of multiple signaling pathways at the level of Smad1 phosphorylation in neural induction. MH1 and MH2 are evolutionarily conserved Mad-homology domains. Diagram modified from Kretzschmar et al. (1997).