XB-ART-51112PLoS One. January 1, 2015; 10 (8): e0135504.
Xenopus Pkdcc1 and Pkdcc2 Are Two New Tyrosine Kinases Involved in the Regulation of JNK Dependent Wnt/PCP Signaling Pathway.
Protein Kinase Domain Containing, Cytoplasmic (PKDCC) is a protein kinase which has been implicated in longitudinal bone growth through regulation of chondrocytes formation. Nevertheless, the mechanism by which this occurs remains unknown. Here, we identified two new members of the PKDCC family, Pkdcc1 and Pkdcc2 from Xenopus laevis. Interestingly, our knockdown experiments revealed that these two proteins are both involved on blastopore and neural tube closure during gastrula and neurula stages, respectively. In vertebrates, tissue polarity and cell movement observed during gastrulation and neural tube closure are controlled by Wnt/Planar Cell Polarity (PCP) molecular pathway. Our results showed that Pkdcc1 and Pkdcc2 promote the recruitment of Dvl to the plasma membrane. But surprisingly, they revealed different roles in the induction of a luciferase reporter under the control of Atf2 promoter. While Pkdcc1 induces Atf2 expression, Pkdcc2 does not, and furthermore inhibits its normal induction by Wnt11 and Wnt5a. Altogether our data show, for the first time, that members of the PKDCC family are involved in the regulation of JNK dependent Wnt/PCP signaling pathway.
PubMed ID: 26270962
PMC ID: PMC4536202
Article link: PLoS One.
Genes referenced: chrd.1 ctnnb1 dvl2 fzd7 mcf2 myc nodal3.1 otx2 pkdcc.1 pkdcc.2 sia1 snai2 sox2 tbxt tnni3 wnt11 wnt5a wnt8a
Morpholinos referenced: pkdcc.1 MO1 pkdcc.2 MO1
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|ig 1. PKDCC protein family encode for a Serine/threonine/tyrosine protein kinase catalytic domain. Comparison of the predicted amino acid sequence of mouse (M. musculus) PKDCC with its X. laevis orthologs, Pkdcc1 and Pkdcc2. pkdcc1 encodes a 449 a.a. protein with a predicted molecular mass of 51.0 kDa and pkdcc2 encodes for a protein with 489 a.a. and 55.9 kDa of predicted molecular mass. Bioinformatic analysis showed that both proteins contain the Serine/Threonine/Tyrosine protein kinase catalytic domain (between a.a. 89 and 336 for Pkdcc1 and between a.a 134 and 381 for Pkdcc2; Grey box, STYKc domain). Identical amino acids among all are shown in red while identical amino acids in only two sequences are shown in blue. The absence of residues at the corresponding region is indicated by dashes.|
|Fig 2. pkdcc1 and pkdcc2 expression patterns during X. laevis embryonic development. (A-H) Expression pattern of pkdcc1 during early development. (I) Temporal expression pattern of Xenopus pkdcc1 and pkdcc2 analysed by quantitative PCR. GAPDH was used as reference gene. (J-O’) Expression pattern of pkdcc2 during Xenopus development. By early gastrula stages, pkdcc1 transcripts can be detected in the (A) dorsal blastopore lip and (B) ADE. (C) At stage 12, pkdcc1 is present in the involuting mesoderm. (D) During neurula stages pkdcc1 is expressed in neural folds and eye fields. (E-F’) pkdcc1 expression at tailbud stages is restricted to the lateral plate mesoderm, foregut, eye and isthmus. (G) Double whole mount in situ hybridization for pkdcc1 (blue) and otx2 (red) shows that pkdcc1 expression in the brain is restricted to the mid-hindbrain boundary. (H) Double whole mount in situ hybridization for pkdcc1 (blue) and cardiac troponin (red) showed that pkdcc1 expression is absent on the heart. (J) Zygotic pkdcc2 is detectable at early gastrula stages in the anterior dorsal endoderm. (K) At late-gastrula stages pkdcc2 mRNA is expressed in the involuting mesoderm. (L) During early neurula stages, pkdcc2 transcripts can be detected in the neural folds (dorsal view, anterior down). (M) At stage 17, pkdcc2 is co-expressed with pkdcc1 in the prospective eye field and neural folds. (N-O’) In tailbud stages (27 and 31; lateral view, anterior to the right and dorsal up), pkdcc2 is expressed in the neural tube roof, otic vesicle, notochord, eye, lateral plate mesoderm and head mesenchyme. D, dorsal; V, ventral; A, anterior; P, posterior.|
|Fig 3. Expression of both pkdcc1 and pkdcc2 is induced downstream of Wnt canonical signaling. (A-H) Axis perturbation assay. Whole-mount in situ hybridization of gastrula stages untreated embryos (A, E) or treated with UV (B, F) or LiCl (C, D, G, H) and hybridized with (A-D) pkdcc1 or (E-H) pkdcc2 probe. Embryos are shown in a vegetal view with the dorsal side at the top and ventral side at the bottom. Hemisections are displayed with the dorsal side to the right. (I) qPCR for pkdcc1 and pkdcc2 expression on wild type embryos and uninjected and β-catenin or wnt8 injected animal caps. (J) qPCR analysis of xnr3 and siamois (sia) expression on wild type embryos and uninjected and pkdcc1 or pkdcc2 injected animal caps. D, dorsal; V, ventral. (p<0.05, in the Student’s t-test).|
|Fig 4. In vivo requirement of Pkdcc1 and Pkdcc2 during early development. (A-I”) Four cell stages were injected either (A, D, G) dorsally or (B-C, E-F”, H-I”) unilaterally with (D-F”) pkdcc1Mo, (G-I”) pkdcc2Mo or (A-C) coMo and analysed at (A, D, G) stage 13, (B, E, H) 15 or (C, F-F”, I-I”) 18/19. Injection of pkdcc1Mo and pkdcc2Mo caused (D, G) gastrulation and (E-F, H-I) neural tube closure defects that were not observed in (A-C) coMo injected embryos. (F’-F”, I’-I”) Magnification of the stage 18/19 pkdcc1Mo and pkdcc2Mo unilaterally injected embryos. Yellow and red arrows show the delay of neural fold formation. A, anterior; P, posterior; D, dorsal; V, ventral; inj, injected side; co, uninjected side.|
|Fig 5. Absence of Pkdcc1 and Pkdcc2 does not change cell fate of mesoderm and neural tissues. Whole mount in situ hybridization for Xbra (A-C) and chd (D-F) of embryos injected with CoMo (A, D), pkdcc1Mo (B, E) or pkdcc2Mo (C, F) in the dorsal blastomeres. Whole mount in situ hybridization for myoD (G-I), slug (J-L), epk/chd (M-O), otx2 (P-R) and sox2 (S-U) of embryos injected with CoMo (G, J, M, P, S), pkdcc1Mo (H, K, N, Q, T) or pkdcc2Mo (I, L, O, R, U) in the right side. (A-F) Vegetal view of gastrula stage embryos with dorsal to the top. (G-U) Anterior view of embryos from late gastrula (G-I) to neurula stages (J-U) with dorsal to the top. inj, injected side; co, uninjected side.|
|Fig 6. Effect of Pkdccs downregulation on convergent extension movements. (A) the percentage of elongated animal caps. -, no elongation; +/- partial elongation; +, strong elongation. (B) Uninjected animal caps without activin treatment. Uninjected (C) or injected animal caps with (D) coMo, (E) pkdcc1Mo or (F) pkdcc2Mo treated with activin. (G) qPCR analysis of mesodermal markers chd, myoD and xbra expression on wild type embryos at st 14 and uninjected or coMo, pkdcc1Mo or pkdcc2Mo injected animal caps in the presence of activin.|
|Fig 7. Both Pkdcc1 and Pkdcc2 promote the recruitment of Dvl to the plasma membrane. (A-O) Embryos were injected with the indicated mRNAs, the ectodermal explants were extracted and Dvl.GFP localization was observed by confocal microscopy. Dvl tagged with GFP (green) is shown on the left panel, the membrane bound RFP (mbRFP, red) is shown on the middle panel and the merge pictures are shown on the right panel. (A-C) Dvl.GFP is localized in the cytoplasm of animal cap cells injected with 300 pg of dvl.GFP mRNA. (D-F) When 300 pg of dvl.GFP mRNA are co-injected with 150 pg of fz7 RNA, Dvl.GFP is recruited to the membrane. (G-I) Co-injection of 500 pg pkdcc1 RNA and 300 pg of dvl.GFP mRNA leads to membrane recruitment of Dvl.GFP as well as (J-L) the co-injection of 500 pg of pkdcc2 mRNA and 300 pg of dvl.GFP mRNA. (M-O) the same membrane localization of Dvl.GFP is observed when 300pg of dvl.GFP mRNA are co-injected with 500 pg of each pkdcc1 and pkdcc2 mRNAs.|
|Fig 8. Pkdcc1 and Pkdcc2 promote the recruitment of Dvl to the plasma membrane through DEP domain. (A) Schematic representation of Dvl domains involved in both Wnt canonical and non-canonical signaling pathways, and DEP.Dvl construct containing the DEP domain of Dvl fused to GFP reporter. (B-P) Embryos were injected with the indicated RNAs, the ectodermal explants were extracted and DEP.Dvl localization was observed by confocal microscopy. DEP domain tagged with GFP (green) is shown on the left panel, the membrane bound RFP (mbRFP, red) is shown on the middle panel and the merged pictures are shown in the right panel. (B-D) DEP.Dvl is localized in the cytoplasm of cells when is overexpressed alone but is recruited to the plasma membrane when co-overexpressed with fz7 (E-G). Co-injection of DEP.dvl and pkdcc1 (H-J), pkdcc2 (K-M) or both simultaneously (N-P) leads to the membrane recruitment of DEP.Dvl.|
|Fig 9. Pkdcc1 is an inducer of PCP signaling while Pkdcc2 is a repressor. (A, B) HEK293T cells were transfected with the indicated constructs in addition to an ATF2 luciferase reporter construct and a β-galactosidase expression vector. Luciferase activity was measured 48h after transfection and normalized with β-galactosidase activity. Each experiment was carried out in triplicates and error bars represent the standard deviation. (C) X. laevis embryos were injected radially at two cell stage with the indicated constructs in addition to an ATF2 luciferase reporter construct and a β-galactosidase expression vector. Luciferase activity was measured at gastrula stages (st11) and normalized with β-galactosidase activity. Error bars represent the standard deviation of the mean.|
|Fig 10. Rescue of pkdcc1Mo and pkdcc2Mo phenotype by JNK and dnJNK, respectively. (A- G) Four cell stage embryos were injected dorsally with coMo (A), pkdcc1Mo (B-D) or pkdcc2Mo (E-G) and incubated until blastopore closure. The pkdcc1Mo phenotype (delay in blastopore closure) was rescued by injection with JNK mRNA (C) but not with dominant negative JNK (dnJNK) mRNA(D). Contrary, pkdcc2Mo phenotype was rescued by the injection with the dnJNK mRNAbut not with JNK mRNA. (H-N) Four cell stage embryos were unilaterally injected with coMo (H), pkdcc1Mo (I-K) or pkdcc2Mo (L-N) and incubated until neural tube closure. Once again, the phenotype observed in the absence of Pkdcc1 (delay in neural tube closure) was rescued by the overexpression of JNK mRNA (J) but not of dnJNK mRNA (K). Contrary, pkdcc2Mo phenotype was rescued by co-injection of dnJNK mRNA (M) but not of JNK mRNA (N).|
|S1 Fig. Hemi-sections of Pkdcc1 and Pkdcc2 knockdown embryos during gastrula and neurula stages. (A-C) Hemi-section of X.laevis embryos injected dorsally with CoMo (A), pkdcc1Mo (B) or pkdcc2Mo (C) at gastrula stage. Auto-fluorescence of the embryo was observed by confocal microscopy. Dorsal to the left and animal to the top (A’-C’). Schematic representation of embryos A-C, respectively. (D, E) Hemisection of X.laevis embryos injected unilaterally with pkdcc1Mo (D) or pkdcc2Mo (E). Auto-fluorescence of the embryo was observed by confocal microscopy. Dorsal to the top. (D’, E’) Schematic representation of embryos D, E, respectively. bc, bottle cells; idm, involuting dorsal mesoderm; dbl, dorsal blastopore lip; sm, presomitic mesoderm; nc, neural crest; e, endoderm.|
|S2 Fig. Rescue of pkdcc1Mo and pkdcc2Mo phenotype by pkdcc1(mut) and pkdcc2(mut) mRNAs, respectively. (A-E) Four cell stage embryos were injected dorsally with coMo (A), pkdcc1Mo (B) or pkdcc2Mo (D) and incubated until blastopore closure. The pkdcc1Mo phenotype was rescued by co-injection with 1ng of pkdcc1(mut) mRNA (C) and pkdcc2Mo phenotype was rescued by the co-injection with the 1ng of pkdcc2(mut) mRNA. (F-J) Four cell stage embryos were unilaterally injected with pkdcc1Mo (G), pkdcc2Mo (I) or coMo (F) and incubated until neural tube closure. Once again, the phenotype obtained by the absence of Pkdcc1 was rescued by the overexpression of pkdcc1(mut) mRNA (H) and pkdcc2Mo phenotype was rescued by co-injection of pkdcc2(mut) mRNA (J). n is the number of injected embryos and the percentage stands for the embryos with the observed defect.|
|S3 Fig. Co-localization of Rab8, Pkdcc1 and Pkdcc2 in the Golgi apparatus. Transfection of HEK293T cells with (A-C) Rab8.GFP (50 ng) and Pkdcc1.HA (1 μg) or (D-F) with Rab8.GFP (50 ng) and Pkdcc2.myc (1 μg). Immunofluorescence against HA (B) and myc (E) was performed. Overlay of Rab8 and Pkdcc1 (C) or Pkdcc2 (F) are represented in the right side of the panel.|
|pkdcc.1 (protein kinase domain containing, cytoplasmic homolog, gene 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 26, lateral view, anterior right, dorsal up.|
|pkdcc.1 (protein kinase domain containing, cytoplasmic homolog, gene 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 35 & 36, lateral view, anterior right, dorsal up.|
|pkdcc.2 (protein kinase domain containing, cytoplasmic) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 26, lateral view, anterior right, dorsal up.|
|pkdcc.2 (protein kinase domain containing, cytoplasmic) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 35 & 36, lateral view, anterior right, dorsal up.|