Mitogen-activated protein kinase and neural specification in Xenopus

Abstract

We have investigated the activity and function of mitogen-activated protein kinase (MAPK) during neural specification in Xenopus. Ectodermal MAPK activity increased between late blastula and midgastrula stages. At midgastrula, MAPK activity in both newly induced neural ectoderm and ectoderm overexpressing the anterior neural inducer noggin was 5-fold higher than in uninduced ectoderm. Overexpression of MAPK phosphatase-1 (MKP-1) in ectoderm inhibited MAPK activity and prevented neurectoderm-specific gene expression when the ectoderm was recombined with dorsal mesoderm or treated with fibroblast growth factor (FGF). Neurectoderm-specific gene expression was observed, however, in ectoderm overexpressing both noggin and MKP-1. To evaluate the role of MAPK in posterior regionalization, ectodermal isolates were treated with increasing concentrations of FGF and assayed for MAPK activity and neurectoderm-specific gene expression. Although induction of posterior neural ectoderm by FGF was accompanied by an elevation of MAPK activity, relative MAPK activity associated with posterior neural fate was no higher than that of ectoderm specified to adopt an anterior neural fate. Thus, increasingly posterior neural fates are not correlated with quantitative increases in MAPK activity. Because MAPK has been shown to down-regulate Smad1, MAPK may disrupt bone morphogenetic protein 4 (BMP-4) signaling during neural specification. Our results suggest that MAPK plays an essential role in the establishment of neural fate in vivo.

Figure 1

Ectodermal MAPK activity before and during gastrulation. (A) Effects of PD098059 on FGF-induced MAPK activity. Animal caps were isolated at stage 8, aged in VLCMR until stage 11, and treated with 50 μM PD098059. Animal caps were pretreated with PD098059 in dimethyl sulfoxide or dimethyl sulfoxide alone for 30 min before addition of 15 ng/ml Xenopus bFGF + 0.5 mg/ml BSA. After 1 hr of bFGF treatment, the animal caps were lysed and assayed for phosphorylation of the Xnf7 substrate peptide. Pretreatment with PD098059 reduces the amount of substrate phosphorylated by lysates of FGF-treated ectoderm by approximately 90% (n = 4). (B) MAPK activity in ectoderm isolated from midblastula (stage 8) and early gastrula (stage 10) embryos and in neural ectoderm isolated at midgastrula (NP-11). Positive controls include thiophosphorylated MAPK (thio-phos MAPK) and midblastula animal cap ectoderm treated with 100 ng/ml bFGF (FGF). The lysate of FGF-treated ectoderm was also run in the absence of substrate (FGF-no sub). Autoradiograms in A and B show an immunoblot probed with anti-MAPK 42-kDa antibody (Upper) and an autoradiogram of the phosphorylated 2.8-kDa peptide (Lower). A ratio of the intensity of the phosphorylated substrate band to that of the MAPK protein provides a relative measure of MAPK specific activity. Relative specific activity for MAPK in ectoderm increases 6-fold between stage 8 and stage 10, with an additional increase during neural specification. (Mean ± SEM, n = 5).

Figure 2

MAPK activity increases during neural specification. (A) MAPK assays were performed on animal cap ectoderm isolated at stage 8 and cultured until midgastrulation (AC 11), and midgastrula neural-plate ectoderm (NP 11). MAPK activity is >5-fold higher in newly induced neural ectoderm than in uninduced ectoderm at the same stage. (B) Embryos were microinjected with 100 pg per embryo noggin or LacZ mRNA at the 2-cell stage. Animal caps were isolated at the midblastula stage and cultured until midgastrulation, when they were assayed for MAPK activity. MAPK activity is ≈5-fold higher in midgastrula ectoderm overexpressing noggin (nog) than in control ectoderm (LacZ) at this stage. The induction of anterior neural-gene expression in ectoderm overexpressing noggin was confirmed by RT-PCR. The assay for Xbra provides a way to detect mesoderm, whereas EF-1α serves as an indicator of RNA recovery and the presence of genomic DNA.

Figure 3

MAPK activity is required for neural induction by endogenous signals. (A) Ectoderm from embryos microinjected with 4 ng per embryo ΔMKP-1 or ΔP-MKP-1 mRNA was isolated at stage 10 and assayed for MAPK activity. Overexpression of MKP leads to a loss of MAPK activity. (B) Ectoderm from embryos overexpressing MKP-1 was isolated at stage 10 and recombined with involuted dorsal mesoderm from midgastrula embryos; recombinates were cultured until controls reached midneurulation (stage 15) and subjected to RT-PCR assays (n = 3).

Figure 4

MAPK activity is required for neural induction by bFGF. (A and B) Ectoderm from embryos microinjected with 4 ng of MKP-1 (WE) or ΔP-MKP-1 mRNA was isolated at stage 8 and treated with 150 ng/ml FGF at stage 10.5. At this stage, treatment with FGF does not elicit muscle actin expression. MAPK assays were performed after 1 hr (A), and gene expression was assayed by RT-PCR at stage 24 (B). (C and D) Ectoderm also was treated at stage 11 with 150 ng/ml FGF ± 5 μg/ml cycloheximide (CHX). Assays for gene expression (C) and MAPK activity (D) were performed after 1 hr. Ac juntreated animal cap ectoderm.

Figure 5

MKP-1 does not prevent neurectoderm-specific gene expression in noggin-overexpressing ectoderm. Animal caps were isolated at stage 8 from embryos coinjected with 100 pg of noggin plus 4 ng of MKP-1 or ΔP-MKP mRNA. Half of them were assayed for MAPK activity when controls reached stage 11, and half of them were used for RT-PCR assays when controls reached stage 13. (A) Overexpression of MKP-1 decreases MAPK activity in expressing noggin-injected animal cap ectoderm. (B) Expression of NCAM and otx2 is not affected by MKP-1 (n = 6).

Figure 6

MAPK and the induction of anteroposterior positional identity by bFGF. Animal caps were isolated at stage 8, held in VLCMR until intact control embryos reached stage 11, and treated with increasing concentrations of FGF. (A) Each sample was assayed for MAPK activity when control embryos reached stage 11. The maximal activation of MAPK observed over a range of 0.15 to 150 ng/ml was ≈4-fold. (B) The remainder were collected for RT-PCR assays when control embryos reached stage 24 (n = 6).