Kinoshita N et al. (2003), PKC delta is essential for Dishevelled function...

XB-ART-5039

Genes Dev 2003 Jul 01;1713:1663-76. doi: 10.1101/gad.1101303.

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PKC delta is essential for Dishevelled function in a noncanonical Wnt pathway that regulates Xenopus convergent extension movements.

Kinoshita N, Iioka H, Miyakoshi A, Ueno N.

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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.

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Species referenced: Xenopus
Genes referenced: chrd dvl1 dvl2 fn1 fzd7 gsc jun lgals4.2 map2k7 mapk8 myc myf5 nodal3.2 otx2 prkcd sia1 tbxt wnt11 wnt8a
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	Figure 1. PKCÎ´ is expressed during Xenopus embryogenesis. (A) Sequence alignment of Xenopus PKCÎ´1 and human PKCÎ´. The DAG-binding C1 domain is underlined. The pseudosubstrate region is indicated by dots. (B) Sequence comparison between Xenopus PKCÎ´ and some human PKC family members. Xenopus PKCÎ´1 is the most similar to human PKCÎ´ in both the regulatory and the catalytic domains. (C) RTâ€“PCR analysis of PKCÎ´ expression during Xenopus development. Primers whose sequences were common between PKCÎ´1 and PKCÎ´2 were used. Stages are according to Nieukoop and Faber (1994). (D) In situ hybridization probing with PKCÎ´1 and PKCÎ´2 showing their ubiquitous expression.
	Figure 2. Overexpression of PKCÎ´ lacking the catalytic domain inhibits gastrulation movements. (A) PKCÎ´1 lacking the catalytic domain (PKCÎ´Î´C) inhibits gastrulation movements. RNA (100 pg) encoding PKCÎ´Î´C was injected into the two dorsal blastomeres of four-cell embryos. PKCÎ´Î´C severely inhibited gastrulation movements, and this effect was rescued by the coinjection of 1 ng of RNA encoding full-length PKCÎ´1. 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) PKCÎ´Î´C RNA (200 pg) was injected into all four blastomeres of four-cell embryos. (Right) In order to trace the cell lineage, mRNA encoding Î²-galactosidase (Î²-gal) with a nuclear localization signal was coinjected with PKCÎ´Î´C into two dorsal blastomeres of four-cell embryos. Cells expressing Î²-gal were stained in red. (C) Immunostaining of the notochord and somites in PKCÎ´Î´C-injected embryos. (D) The PKCÎ´Î´C mutant blocked the elongation of animal cap explants by activin. Activin RNA (0.5 pg) was injected with or without 100 pg of PKCÎ´Î´C RNA. (E) The induction of mesodermal markers by activin in animal caps was not affected by PKCÎ´Î´C. ui, uninjected; Em, whole embryo; gsc, goosecoid; chd, chordin.
	Figure 3. PKCÎ´ antisense morpholino also blocked gastrulation movements. (A) Morpholino oligonucleotides (MOs) for PKCÎ´1 and PKCÎ´2 inhibited the translation of mRNA that had the corresponding sequences. RNA encoding GFP-tagged PKCÎ´ and unrelated GFP were coinjected with or without each MO. PKCÎ´1 (left panel) and PKCÎ´2 MO (right panel) blocked the production of each GFP-tagged PKCÎ´, but unrelated GFP was not affected. (B) Control MO or PKCÎ´ MO (20 ng each) was injected into four-cell embryos (panels a,b, respectively). The PKCÎ´ MO caused a gastrulation-defective phenotype that was indistinguishable from that of PKCÎ´Î´C-injected embryos. This phenotype was rescued by 1 ng of full-length PKCÎ´1 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 PKCÎ´ MO was injected into all four blastomeres of four-cell embryos. (Right) To trace the cell lineage, mRNA encoding Î²-gal with a nuclear localization signal was coinjected with PKCÎ´ MO into two dorsal blastomeres of four-cell embryos. (D) PKCÎ´ 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. PKCÎ´ is localized to the plasma membrane with Xdsh by Xfz7 signaling. (A) Flag-tagged PKCÎ´ 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 PKCÎ´ and Xdsh. Flag-tagged PKCÎ´ and myc-tagged Xdsh were expressed as indicated in HEK293T cells. PKCÎ´ 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 Î¼M 2 h before the cell lysate preparation. The addition of PMA did not change the amount of coimmunoprecipitated Xdsh and PKCÎ´.
	Figure 5. PKCÎ´ is required for the activation of Xdsh by Xfz7 signaling. (A) Twenty nanograms of PKCÎ´ 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 PKCÎ´ MO blocked the membrane localization of Xdsh by Xfz7. (B) Myc-tagged Xdsh and Xfz7 mRNAs were coinjected with PKCÎ´ MO or PKCÎ´Î´C 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) PKCÎ´ 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 PKCÎ´ were treated with PMA, and the localization of Xdsh and PKCÎ´ 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 Î¼M PMA for 1 h. The JNK activity was detected by an anti-phospho-c-Jun antibody.
	Figure 6. PKCÎ´ loss-of-function is rescued by overexpression of active MKK7. (A) Twenty nanograms of PKCÎ´ MO were coinjected with 1 ng of PKCÎ´1, 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. PKCÎ´ MO blocked gastrulation movement. PKCÎ´ 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 PKCÎ´ is essential for the canonical Wnt pathway, 100 pg of PKCÎ´Î´C or 20 ng of PKCÎ´ MO was coinjected with 10 pg of Xwnt8 into Xenopus embryos. PKCÎ´Î´C and PKCÎ´ MO did not inhibit the induction of siamois or Xnr3, indicating that PKCÎ´ does not affect the canonical Wnt signaling pathway.
	Figure 7. PKCÎ´ is required for convergent extension movements. (A) PKCÎ´ 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 Î¼m.
	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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Adler, Planar signaling and morphogenesis in Drosophila. 2002, Pubmed