XB-ART-5039Genes Dev July 1, 2003; 17 (13): 1663-76.
PKC delta is essential for Dishevelled function in a noncanonical Wnt pathway that regulates Xenopus convergent extension movements.
Protein kinase C (PKC) has been implicated in the Wnt signaling pathway; however, its molecular role is poorly understood. We identified novel genes encoding delta-type PKC in the Xenopus EST databases. Loss of PKC delta function revealed that it was essential for convergent extension during gastrulation. We then examined the relationship between PKC delta and the Wnt pathway. PKC delta was translocated to the plasma membrane in response to Frizzled signaling. In addition, loss of PKC delta function inhibited the translocation of Dishevelled and the activation of c-Jun N-terminal kinase (JNK) by Frizzled. Furthermore, PKC delta formed a complex with Dishevelled, and the activation of PKC delta by phorbol ester was sufficient for Dishevelled translocation and JNK activation. Thus, PKC delta plays an essential role in the Wnt/JNK pathway by regulating the localization and activity of Dishevelled.
PubMed ID: 12842914
PMC ID: PMC196137
Article link: Genes Dev
Species referenced: Xenopus laevis
Genes referenced: chrd.1 dvl1 dvl2 fn1 fzd7 gsc jun lgals4.2 map2k7 mapk8 myc myf5 nodal3.1 nodal3.2 otx2 prkcd sia1 tbxt wnt11 wnt8a
Antibodies: FLAG Ab2 Notochord Ab2 Somite Ab3
Morpholinos: prkcd MO1 prkcd MO2
Article Images: [+] show captions
|Figure 1. PKCδ is expressed during Xenopus embryogenesis. (A) Sequence alignment of Xenopus PKCδ1 and human PKCδ. The DAG-binding C1 domain is underlined. The pseudosubstrate region is indicated by dots. (B) Sequence comparison between Xenopus PKCδ and some human PKC family members. Xenopus PKCδ1 is the most similar to human PKCδ in both the regulatory and the catalytic domains. (C) RT–PCR analysis of PKCδ expression during Xenopus development. Primers whose sequences were common between PKCδ1 and PKCδ2 were used. Stages are according to Nieukoop and Faber (1994). (D) In situ hybridization probing with PKCδ1 and PKCδ2 showing their ubiquitous expression.|
|Figure 2. Overexpression of PKCδ lacking the catalytic domain inhibits gastrulation movements. (A) PKCδ1 lacking the catalytic domain (PKCδδC) inhibits gastrulation movements. RNA (100 pg) encoding PKCδδC was injected into the two dorsal blastomeres of four-cell embryos. PKCδδC severely inhibited gastrulation movements, and this effect was rescued by the coinjection of 1 ng of RNA encoding full-length PKCδ1. Embryos in the top panels are at the early neurula stage. (B) In situ hybridization of early gastrula embryos probed with a pan-mesodermal marker, Xbra, and dorsal mesodermal markers chordin (chd) and goosecoid (gsc). (Left) Uninjected. (Middle) PKCδδC RNA (200 pg) was injected into all four blastomeres of four-cell embryos. (Right) In order to trace the cell lineage, mRNA encoding β-galactosidase (β-gal) with a nuclear localization signal was coinjected with PKCδδC into two dorsal blastomeres of four-cell embryos. Cells expressing β-gal were stained in red. (C) Immunostaining of the notochord and somites in PKCδδC-injected embryos. (D) The PKCδδC mutant blocked the elongation of animal cap explants by activin. Activin RNA (0.5 pg) was injected with or without 100 pg of PKCδδC RNA. (E) The induction of mesodermal markers by activin in animal caps was not affected by PKCδδC. ui, uninjected; Em, whole embryo; gsc, goosecoid; chd, chordin.|
|Figure 3. PKCδ antisense morpholino also blocked gastrulation movements. (A) Morpholino oligonucleotides (MOs) for PKCδ1 and PKCδ2 inhibited the translation of mRNA that had the corresponding sequences. RNA encoding GFP-tagged PKCδ and unrelated GFP were coinjected with or without each MO. PKCδ1 (left panel) and PKCδ2 MO (right panel) blocked the production of each GFP-tagged PKCδ, but unrelated GFP was not affected. (B) Control MO or PKCδ MO (20 ng each) was injected into four-cell embryos (panels a,b, respectively). The PKCδ MO caused a gastrulation-defective phenotype that was indistinguishable from that of PKCδδC-injected embryos. This phenotype was rescued by 1 ng of full-length PKCδ1 RNA (panel c). (C) In situ hybridization of early gastrula embryos probed with chordin (chd), goosecoid (gsc), and Xbra. The left and middle panels show 20 ng of control or PKCδ MO was injected into all four blastomeres of four-cell embryos. (Right) To trace the cell lineage, mRNA encoding β-gal with a nuclear localization signal was coinjected with PKCδ MO into two dorsal blastomeres of four-cell embryos. (D) PKCδ MO inhibited the elongation of dorsal marginal zone explants. Twenty nanograms of MO were injected into the two dorsal blastomeres of four-cell embryos.|
|Figure 4. PKCδ is localized to the plasma membrane with Xdsh by Xfz7 signaling. (A) Flag-tagged PKCδ RNA (200 pg) and myc-tagged Xdsh RNA (100 pg) were coinjected with or without 500 pg of Xfz7 RNA in the animal cap explants of Xenopus embryos. Their localization was observed by laser-scanning confocal microscopy. (B) Coimmunoprecipitation of PKCδ and Xdsh. Flag-tagged PKCδ and myc-tagged Xdsh were expressed as indicated in HEK293T cells. PKCδ and Xdsh coimmunoprecipitated, indicating that they form a complex. (C) The indicated genes were expressed in HEK293T cells. PMA was added to the medium at a final concentration of 1 μM 2 h before the cell lysate preparation. The addition of PMA did not change the amount of coimmunoprecipitated Xdsh and PKCδ.|
|Figure 5. PKCδ is required for the activation of Xdsh by Xfz7 signaling. (A) Twenty nanograms of PKCδ MO were coinjected with 100 pg of myc-tagged Xdsh and 500 pg of Xfz7 mRNAs, and the localization of Xdsh in animal cap explants was observed. The coinjection of PKCδ MO blocked the membrane localization of Xdsh by Xfz7. (B) Myc-tagged Xdsh and Xfz7 mRNAs were coinjected with PKCδ MO or PKCδδC mRNA. Animal cap explants were isolated at stage 10, and their extracts were fractionated by SDS-PAGE. Myc-tagged Xdsh protein was detected by Western blotting using an anti-myc antibody. Two bands were detected in all four lanes for Xdsh-injected samples. (C) PKCδ is required for JNK activation by Xfz7. GAL4 DNA-binding domain (DBD)-tagged c-Jun mRNA was injected, and the phosphorylation levels of c-Jun were detected by Western blotting using anti-phosphorylated c-Jun (P-c-Jun) and anti-DBD antibodies (c-Jun). (D) Animal cap explants expressing myc-tagged Xdsh and Flag-tagged PKCδ were treated with PMA, and the localization of Xdsh and PKCδ was observed. Xdsh was translocated to the plasma membrane by PMA. (E) PMA can activate JNK in animal cap explants. Isolated explants were treated with or without 1 μM PMA for 1 h. The JNK activity was detected by an anti-phospho-c-Jun antibody.|
|Figure 6. PKCδ loss-of-function is rescued by overexpression of active MKK7. (A) Twenty nanograms of PKCδ MO were coinjected with 1 ng of PKCδ1, and 200 pg of constitutively active (CA) MKK7. The closure of the blastopore of injected embryos was compared at stage 14, when the blastopore in the control embryos was completely closed. PKCδ MO blocked gastrulation movement. PKCδ mRNA rescued the phenotype completely, and CA MKK7 mRNA partially rescued it. (B) At the tadpole stage, embryos coinjected with CA MKK7 showed a partially rescued phenotype. (C) To test whether PKCδ is essential for the canonical Wnt pathway, 100 pg of PKCδδC or 20 ng of PKCδ MO was coinjected with 10 pg of Xwnt8 into Xenopus embryos. PKCδδC and PKCδ MO did not inhibit the induction of siamois or Xnr3, indicating that PKCδ does not affect the canonical Wnt signaling pathway.|
|Figure 7. PKCδ is required for convergent extension movements. (A) PKCδ MO, Rhodamine dextran, and mRNA for Venus fused with a membrane localization signal (mb-Venus) were coinjected into one of the two dorsal blastomeres at the four-cell stage. As a control, mb-Venus mRNA alone was injected into the other dorsal blastomere of the same embryo. (B) At the gastrula stage, dorsal marginal zone (DMZ) explants were cut and cultured on a cover glass coated with fibronectin, and the convergent extension movements were observed by laser-scanning confocal microscopy. (C) The indicated cDNAs were fused to Venus and expressed in the dorsal mesodermal cells. DMZ explants were cultured and observed as described above. Bar, 50 μm.|
|prkcd (protein kinase C, delta ) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 11, animal and vegetal views.|
|prkcd (protein kinase C, delta ) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 15, dorsal view, anterior left.|
|prkcd (protein kinase C, delta ) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 22, lateral view, anterior left, dorsal up.|
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