December 1, 2018;
Role of dipeptidyl peptidase-4 as a potentiator of activin/nodal signaling pathway.
(dipeptidyl peptidase-4), a highly conserved transmembrane glycoprotein with an exo-peptidase activity, has been shown to contribute to glucose metabolism, immune regulation, signal transduction, and cell differentiation. Here, we show that DPP4
is involved in control of activin/nodal
signaling in Xenopus early development. In support of this, gain of function of DPP4
phosphorylation as well as expression of target genes induced by activin or nodal
signal. In addition, Dpp4
showed synergistic effect on induction of ectopic dorsal body axis, when co-injected at suboptimal doses in early embryos. Conversely, saxagliptin, a DPP4
inhibitor repressed activin induction of Smad2
phosphorylation. Notably, overexpression of Dpp4
disrupted specification of dorsal body axis of embryo
, leading to malformed phenotypes such as spina bifida and a shortened and dorsally bent axis. Together, these results suggest that DPP4
functions as a potentiator of activin/nodal
signaling pathway. [BMB Reports 2018; 51(12): 636-641].
nodal signaling pathway
[+] show captions
Fig. 1. Dpp4 enhances activin/nodal signaling but not BMP4 signaling. (A–D) Four-cell stage embryos were injected in the animal pole region as indicated with increasing doses of Dpp4 (10, 1000 pg) alone or with Xnr1 (50 pg) or BMP4 (100 pg) mRNAs, and then animal cap explants were excised at stage 8.5 from injected or uninjected embryos, cultured to stage 10.5 for RT-PCR analysis (C and D) or 11 for Western blotting (A and B) in the presence or absence of activin protein (5 ng/ml) as shown. Smad1, Smad2 and ODC serve as loading controls. Co AC, uninjected control animal caps. (−), no injection of Dpp4. WE, stage 10.5 whole embryo. —RT, control in the absence of reverse transcriptase. (E) One blastomere of four-cell stage embryos was injected in the ventral marginal region with Dpp4 (50 pg) and/or Xnr1 (5 pg) mRNAs as indicated, and injected embryos were cultured until uninjected sibling embryos reached tadpole stages. Arrows denote the induced secondary dorsal axis. Embryos are shown in lateral views with anterior to the left.
Fig. 2. Saxagliptin, a DPP4 inhibitor represses Smad2 phosphorylation induced by activin. HEK293T cells were pre-treated for 1 hour with DPP4 inhibitors as indicated and subsequently incubated for 3 hours with or without activin (20 ng/ml), and harvested for Western blotting. Smad2 is a loading control.
Fig. 3. Spatio-temporal expression pattern of Dpp4 in Xenopus early embryogenesis. (A) Temporal expression pattern of Dpp4 analyzed by RT-PCR. St., a developmental stage. ODC serves as a loading control. —RT, stage 27 control whole embryos in the absence of reverse transcriptase. (B and C) Whole mount in situ hybridization (B) and RT-PCR (C) showing spatial expression pattern of Dpp4 transcripts at stage 10.25. Embryos in (B) are shown in lateral (upper panel) or dorso-vegetal (lower panel) view with animal to the top. For (C), respective regional explants were dissected from stage 10.25 whole embryos. D, dorsal; V, ventral; VP, vegetal pole; MZ, marginal zone; AP, animal pole; DMZ, dorsal marginal zone; VMZ, ventral marginal zone. Chordin is a dorsal mesodermal marker, Vg1 is a member of TGF-β family expressed vegetally, and EF1-α is a loading control. —RT, stage 10.25 control whole embryos in the absence of reverse transcriptase.
Fig. 4. Embryonic morphological phenotypes caused by overexpression of Dpp4. (A) Four-cell stage embryos were injected in the dorsal or ventral marginal regions with Dpp4 (500 pg) mRNA and cultured to tadpole stages. Embryos are shown in lateral views with anterior to the left. Uninjected, uninjected control embryos. (B) Four-cell stage embryos were injected in the dorsal marginal region with LacZ (50 pg) alone or with Dpp4 (1 ng) mRNA, cultured to stage 10.25, and fixed for LacZ staining (red) and subsequent in situ hybridization against Goosecoid (Gsc). Arrows indicate expression of Gsc in the dorsal lip. Embryos are shown in dorso-vegetal views with anterior to the top. Control, an embryo injected with LacZ alone.
dpp4 (dipeptidyl-peptidase 4) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 10.25, horizontal view, dorsal left.