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In Xenopus embryonic development, the MEK5-ERK5 pathway, one of the MAPK pathways, lies downstream of SoxD and upstream of Xngnr1 in a signaling pathway regulating neural differentiation. It remains unclear, however, how the MEK5-ERK5 pathway is regulated in Xenopus neural development. As SoxD is a transcription factor, we hypothesized that some growth factor should be induced by SoxD and activate the MEK5-ERK5 pathway. As the expression level of fibroblast growth factor 13 (FGF13) is increased by SoxD, we analyzed the function of FGF13 in neural development. Knockdown of FGF13 with antisense morpholino-oligonucleotides (MOs) results in the reduced head structure and inhibition of neural differentiation. FGF13 MOs inhibit the SoxD-induced expression of Xngnr1 and the Xngnr1-induced expression of NeuroD, suggesting that FGF13 is necessary both upstream and downstream of Xngnr1 in neural differentiation. In addition, FGF13 MOs inhibit the activation of the MEK5-ERK5 pathway by dominant-negative bone morphogenetic protein receptor, a mimicker of neural inducers, indicating that FGF13 is involved in the activation of the MEK5-ERK5 pathway. Together, these results identify a role of FGF13 in Xenopus neural differentiation.
FIGURE 3.
Expression pattern of xFGF13.A, total RNA was isolated from embryos at the indicated stages, and expression of xFGF13 was analyzed by real time PCR. B, expression of each isoform of xFGF13 was analyzed by semiquantitative RT-PCR. XeODC was used as an RNA loading control. Xbra was also examined. C, whole-mount in situ hybridization against xFGF13 at the indicated stages.
FIGURE 4.
FGF13 is required for head development.A, indicated sets of morpholino-oligonucleotides (30 ng) and mRNA (0.8 ng) were injected, and the protein level was examined by immunoblotting with anti-Myc antibody. We used xERK2 as a loading control. B, control MO (30 ng), FGF13 MO1 (15 ng) plus MO2 (15 ng), or FGF13 MO1 mis5 (15 ng) plus MO2 mis5 (15 ng) were injected into the dorsal animal hemisphere on the right side at the four-cell stage. C, embryos injected with MOs were classified into four groups according to the eye size: class 1, normal or only slightly smaller; class 2, smaller; class 3, much smaller; class 4, none. About 80% of embryos injected with FGF13 MO1 plus MO2 (n = 108) showed definite defects in eyes, whereas only 20% of embryos injected with control MO (n = 101) and 6% of embryos injected with FGF13 MO1 mis5 plus MO2 mis5 (n = 118) showed defects. D, xFGF13-S (rMO) mRNA (3.2 ng) was co-injected with FGF13 MO1 plus MO2 for the rescue experiment. 58% of embryos co-injected with xFGF13-S (rMO) mRNA showed defects (n = 102), whereas 79% of embryos injected with FGF13 MO1 plus MO2 showed defects (n = 170) (p < 0.001 by Mann-Whitney test).
FIGURE 5.
FGF13 is required for neural differentiation.A, control MO (60 ng) or FGF13 MO1 (30 ng) plus MO2 (30 ng) (FGF13 MO) were injected into the right blastomeres of four-cell stage embryos. The expression pattern of SoxD, Sox2, N-tubulin, and MyoD were analyzed by whole-mount in situ hybridization. Nuclear localization signal sequence-fused β-galactosidase mRNA was co-injected and used as a tracer (red staining). Dorsal views of embryos are shown with the anterior side at the top. B, dominant-negative BMP receptor (dnBMPR) mRNA (0.4 ng) was injected with control MO (60 ng) or FGF13 MO1 (30 ng) plus MO2 (30 ng), and animal caps were cultured to stage 14. Expression levels of marker genes were analyzed by real time RT-PCR.
FIGURE 6.
xFGF13 lies downstream of SoxD and upstream of Xngnr1.A, SoxD mRNA (0.4 ng) was injected with control MO (80 ng), FGF13 MO1 (20 ng) plus MO2 (20 ng), FGF13 MO1 (40 ng) plus MO2 (40 ng), or ERK5 MO (20 ng). Animal caps were cultured to stage 21 and analyzed by real time RT-PCR. B, Myc-xERK5 (Xenopus ERK5) and dnBMPR mRNA were injected with control MO (60 ng) or FGF13 MO1 (30 ng) plus MO2 (30 ng), and animal caps were cultured to stage 14. The ERK5 activity was measured by kinase assays. The normalized values of the kinase activity are shown at the bottom.
FIGURE 7.
xFGF13 also functions downstream of Xngnr1.A, Xngnr1 mRNA (40 pg) was injected with control MO (60 ng) or FGF13 MO1 (30 ng) plus MO2 (30 ng). Animal caps were cultured to stage 20 and analyzed by real-time RT-PCR. B, Xngnr1 mRNA (40 pg) was injected with control MO (60 ng) or FGF13 MO1 (30 ng) plus MO2 (30 ng) into the right blastomeres of four-cell stage embryos. Expression of N-tubulin was analyzed by whole-mount in situ hybridization. C, Xngnr1 mRNA (40 pg) was injected, and animal caps were cultured to the indicated stages. An expression level of xFGF13 mRNA was analyzed by real time RT-PCR.
fgf13 (fibroblast growth factor 13 ) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 21, dorsal view, anteriorleft.
fgf13 (fibroblast growth factor 13 ) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 23, lateral view, anteriorleft, dorsal up.
fgf13 (fibroblast growth factor 13 ) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 23, transverse section, mid trunk section, dorsal up ( left image) and dorsal view, anteriorleft (right image).
