April 1, 2012;
TAK1 promotes BMP4/Smad1 signaling via inhibition of erk MAPK: a new link in the FGF/BMP regulatory network.
FGFs and BMPs act in concert to regulate a wide range of processes in vertebrate development. In most cases, FGFs and BMPs have opposing effects, and specific developmental outcomes arise out of a balance between the two growth factors. We and others have previously demonstrated that signaling pathways activated by FGFs and BMPs interact via inhibitory crosstalk. Here we demonstrate a role for the BMP effector TGF-β Activated Kinase 1 (TAK1
) in the maintenance of Smad1
activity in Xenopus embryos, via the inhibition of erk MAPK
. Up- or downregulation of TAK1
levels produces an inverse alteration in the amount of activated erk MAPK
. The inhibition of erk MAPK
is mediated by p38
and a corresponding decrease in phosphorylation of MEK. TAK1
morphant embryos show a decrease in the nuclear accumulation of Smad1
. Conversely, reduction of erk MAPK
activity via overexpression of MAP Kinase
Phosphatase1 (MKP1) leads to an increase in nuclear Smad1
. Both TAK1
treated with FGF show a decrease in the expression of several Smad1
-inducible genes. Neural-specific gene expression is inhibited in isolated ectoderm
, suggesting that TAK1
is sufficient to inhibit neural specification. Introduction of TAK1
morpholino oligonucleotide expands the expression of organizer
genes, disrupts formation of the boundary between organizer
and non-organizer mesoderm
, and increases the spatial range of MAPK
activation in response to localized FGF. Our results indicate that inhibitory interactions between FGF and BMP4
effector pathways increase the robustness of BMP signaling via a feed-forward mechanism.
[+] show captions
Fig. 1. Increase in ERK MAPK phosphorylation following TAK1 knockdown. Embryos were injected with 20 ng of either the TAK1 MO (TAK MO) or the 5-base mispair (mis MO). Tissues were isolated when embryos reached early gastrula (10–10.25). (A) Activated (diphospho; dp-MAPK) and total ERK MAPK (MAPK) from isolated ectoderm. (B) Activated (diphospho; dp-MAPK), and total ERK MAPK (MAPK) from isolated mesoderm. This mesoderm consists of the lower (i.e., vegetalward) marginal zone minus the superficial layer, around the entire circumference of the embryo. (20 tissue isolates/sample; N=3 independent experiments).
Fig. 2. TAK1 regulates p38 phosphorylation. (A) Embryos were injected with one of the following: 20 ng TAK1 MO; 20 ng Mispair MO; 250 pg β-galactosidase mRNA; 250 pg TAK1 mRNA. Embryos were lysed at st. 10.25 for immunoblotting to detect TAK1, phospho-p38, and tubulin (loading control). Relative accumulation of TAK1 (B) and phospho-p38 (C) normalized to tubulin. Bars represent mean±S.E.M. (N=3 independent experiments). Unpaired t-tests were used to assess statistical significance; asterisks indicate that p-values: *, p<0.05; **, p<0.007; ***, p<0.001.
Fig. 3. TAK1 inhibits MEK activity via p38. Embryos were injected with 20 ng of either TAK1 MO (TAK1) or the mispair MO (mis); additional embryos were injected with 250 ng of mRNA encoding either TAK1 or β-galactosidase (β-gal). Embryos were lysed at st. 10.25; immunoblots were probed with antibodies directed against MEK1/2 and phospho-MEK1/2 (A). Quantification of phospho-MEK levels normalized to that of β-galactosidase embryos is shown in (B). Bars represent mean±S.E.M. **, p<0.001. (N=4 independent experiments). (C) Embryos were injected with mRNA encoding either TAK1 or β-galactosidase and incubated with or without the p38 inhibitor SB203580. Embryos were lysed at st. 10.25 in preparation for immunoblotting; blots were probed to show levels of phospho-MEK (pMEK), total MEK (MEK), phospho-p38 (pp38), and tubulin. (N=3 independent experiments).
Fig. 4. Reduction in Nuclear Smad1 in TAK1 morphants. Embryos were injected with 20 ng of either the TAK1 MO or the mispair MO and lysed at st. 10.25. (A) Nuclear and cytoplasmic fractions were immunoblotted and probed with antibodies directed against Smad1, diphospho-ERK MAPK (dp-MAPK), α-tubulin, and lamin B2. (B) Nuclear–cytoplasmic distribution of Smad1 in TAK1 morphant and control embryos. (C=Cytoplasmic, N=Nuclear). ***, p<0.0001. (C) Nuclear–cytoplasmic distribution of diphospho-ERK MAPK. Bars represent mean±S.E.M. N=3 independent experiments.
Overexpression of MKP promotes nuclear accumulation of Smad1. Embryos were injected with 1 ng of mRNA encoding either MKP1 or the phosphatase-inactive ΔP-MKP (ΔP). Embryos were lysed at st. 10.25, separated into nuclear (N) and cytoplasmic (C) fractions, and immunoblotted. (A) Blots were sequentially probed with antibodies directed against Smad1, diphospho-erk-MAPK (dp-MAPK), α-tubulin, and lamin B2. The distribution of Smad1 between the nucleus and the cytoplasm is shown in (B); **, p<0.002. (C) Effects of MKP on accumulation of diphospho-ERK MAPK (dp-MAPK) in the cytoplasm and the nucleus. Bars represent mean±S.E.M. N=3 independent experiments.
Fig. 6. TAK1 overexpression does not increase nuclear accumulation of Smad1. (A) Immunoblots of embryos injected with 250 pg mRNA encoding TAK1 or β-galactosidase (β-gal). Embryos were lysed at st. 10.25 and fractionated prior to immunoblot analysis. (C=Cytoplasmic, N=Nuclear). (B) Distribution of Smad1 in nuclear and cytoplasmic fractions in embryos overexpressing TAK1 or β-gal. Bars represent mean±S.E.M. N=3 independent experiments.
Fig. 7. Effects of TAK1 knockdown on gene expression and responsiveness to FGF. (A, B) Embryos were injected with 20 ng of either TAK1 MO or the mispair MO; animal caps were isolated at st. 9 and cultured in VLCMR±400 ng/ml bFGF until intact controls reached st. 11. Tissues were then lysed in preparation for RNA isolation and Q-RT-PCR. Relative fold enrichment for each gene was calculated in comparison to ODC using the δδCt method. Bars represent mean±S.E.M. (N=3 independent experiments). (A) Expression of Smad1-inducible genes in control and TAK1 morphant ectoderm treated with FGF. (B) Effects of TAK1 knockdown and FGF on expression of NCAM. (C) Overexpression of TAK1 inhibits neural specification in response to noggin. Embryos were injected with 1 ng noggin RNA±1 ng TAK1 RNA, or with 1 ng LacZ RNA alone. Animal cap ectoderm (AC) was isolated at st. 8 and collected for RNA isolation and RT-PCR when sibling control embryos reached st. 13. Samples were also prepared from uninjected whole embryos (WE) and animal caps. RT-PCR for Xbra is included as a control for mesodermal contamination, and EF-1α is included to monitor relative RNA recovery. N=3 independent experiments.
Fig. 8. Effects of TAK1 knockdown on dorsal–ventral pattern. Embryos were injected in both dorsal blastomeres at the 4-cell stage with 20 ng of either the TAK1 MO (TAK MO) or the 5-base mispair (Mis MO); embryos were fixed at early gastrula stages (10+–10.25) and used for in situ hybridization. Dorsal-specific probes include not1 (A, B, A′, B′), otx2 (C, D), and shh (E, F). Ventrolateral probes include Xvent2 (G, H), and Xvent1 (I, J). Expression of each gene was evaluated in samples from 4 independent experiments, prepared in 2 rounds of in situ hybridization. Each set of images (for a given probe) was taken from the same round of in situ hybridization; thus, mispair and TAK1 MO embryos were processed identically, and in situs were developed for the same period of time. Images show expression patterns characteristic of at least 2/3 of embryos (TAK vs. mispair MO).
Fig. 9. Effects of TAK1 MO on the spatial extent of FGF/MAPK signaling. All animal caps (AC) were held under a coverslip for 30 min, fixed, and stained with the anti-diphospho-MAPK antibody. (A) AC without a bead. (B) AC with bead soaked in PBS. (C–E), AC exposed to beads soaked in FGF. Lines indicate extent of diphospho-MAPK immunoreactivity. The holes indicate bead sites. (C) AC injected with β-galactosidase mRNA. (D) AC injected with noggin mRNA. (E) AC injected with TAK1 MO. For each preparation, N=≥6 explants in each of ≥3 independent experiments for each type of sample.