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First published online August 24, 2007
doi: 10.1242/10.1242/dev.02881

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Porcupine-mediated lipid-modification regulates the activity and distribution of Wnt proteins in the chick neural tube

Lisa M. Galli¹, Tiffany L. Barnes^1,*, Stephanie S. Secrest^1,*, Tatsuhiko Kadowaki² and Laura W. Burrus^1,

¹ Department of Biology, San Francisco State University, 1600 Holloway Avenue, San Francisco, CA 94132, USA.
² Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa, Nagoya, 464-8601, Japan.

View larger version (24K): [in this window] [in a new window]   Fig. 1. Antibodies generated against chick Wnt1 and Wnt3a can be utilized for western blots and immunohistochemistry. (A) Western blot analysis of 293T cells transfected with control, Wnt1 and Wnt3a shows that antibodies specifically immunoreact with their cognate Wnt protein. A cluster of Wnt proteins migrates at approximately 40 kDa. The Wnt3a blot was also probed with anti-tubulin antibodies as a loading control. (B) Immunohistochemical analysis of COS cells transfected with Wnt1 or Wnt3a. Wnt1/Wnt3a were visualized by the addition of Cy3-labeled secondary antibody (green); nuclei were visualized with DAPI (blue).

View larger version (70K): [in this window] [in a new window]   Fig. 2. Localization of Wnt proteins in the chick neural tube. Wnt1 and GFP (A-C) or Wnt3a and GFP (D-F) were expressed in the neural tube following electroporation. Diagram to left depicts GFP (green) expression in an electroporated neural tube; the boxed region is shown in A-F. GFP-positive embryos were harvested 24 hours post-electroporation (HH stage 18/19), fixed and cryosectioned prior to immunostaining with anti-Wnt1/Wnt3a antibodies. Goat anti-mouse-Cy3 secondary antibodies were used to visualize Wnt proteins (red). All images were collected by confocal microscopy. White arrows point to perinuclear staining; arrowheads indicate immunopositive punctae. No punctae were observed when primary antibody was omitted (data not shown). (G-O) A wild-type chick embryo (HH stage 18) was cryosectioned and stained with Wnt1 primary antibody and a Cy3-labeled secondary antibody (red). Diagram to left shows endogenous Wnt1 expression (red) in the neural tube; the boxed region is shown in G-O. G and H are adjacent sections, as are I-L. In H, primary antibody was omitted. In I, excess GST-Wnt1 blocking peptide was added. In J-L, we compare the localization of Wnt1 protein (J) with that of Wnt1 transcript (K). In M-O, we compare the localization of Wnt1 (red) with NCAM (green) in a single optical confocal layer. In M and O, white arrows point to staining on or just interior to the plasma membrane, whereas the white arrowheads indicate staining that is inside the cell. nt, neural tube; no, notochord.

View larger version (37K): [in this window] [in a new window]   Fig. 3. The conserved histidine residue in the predicted porcupine active site is required for the lipid-modification of Wnt3a. 293T cells were transfected with chick (c) Wnt3a in the presence of either GFP (control), mouse (m) PorcD, mPorcD(H341D) or mPorcD(H341Q). Cells were lysed 24 hours post-transfection and extracted with TX-114 before analysis by SDS-PAGE and western blot. The blots were probed with Wnt3a and tubulin (control) antibodies. Monomeric Wnt3a proteins migrate at 40 kDa, whereas tubulin migrates at 50 kDa. In the presence of GFP alone, Wnt3a is in both aqueous (A) and detergent (T) phases. Whereas the presence of ectopic porcupine promotes the partitioning of Wnt3a into the hydrophobic phase, ectopic mPorcD(H341D) and mPorcD(H341Q) failed to show a similar effect.

View larger version (21K): [in this window] [in a new window]   Fig. 4. Porcupine promotes the lipid-modification of both wild-type Wnt1/Wnt3a and mutant Wnt1/Wnt3a, which possess C-to-S point mutations in the cysteine that is known to be palmitoylated. 293T cells were transfected with Wnt1, Wnt3a, Wnt1(C93S) or Wnt3a(C77S) in the presence of GFP (control) or mPorcD. Cells were lysed 24 hours post-transfection and extracted with TX-114 before analysis by SDS-PAGE and western blot. The blots were probed with Wnt1 (left), Wnt3a (right) and tubulin antibodies. Monomeric Wnt1/Wnt3a proteins migrate at 40 kDa, whereas tubulin migrates at 50 kDa. In the absence of ectopic porcupine, Wnt1 and Wnt3a are found in both aqueous (A) and detergent (T) phases. The presence of ectopic porcupine promotes the partitioning of Wnt1, Wnt3a, Wnt1(C93S) and Wnt3a(C77S) into the hydrophobic phase.

View larger version (37K): [in this window] [in a new window]   Fig. 5. Porcupine/palmitoylation regulates the activity of Wnt1 and Wnt3a in 293T cells. 293T cells were transfected with Top/FopFlash, Renilla luciferase and the indicated pHIPPY (siRNA) and pcDNA expression constructs (Kaykas and Moon, 2004). DNA concentrations were held constant within each series of experiments. The effects of mPorcD and mPorcD mutants on Wnt3a activity are shown in light blue. The effects of co-transfection of hPORC RNAi constructs with Wnt3a are shown in medium blue. The activity of Wnt3a(C77S) and Wnt1(C93S) mutants is shown in dark blue. Finally, TopFlash activity in cells co-transfected with Wnt3a(C77S) and human PORC RNAi constructs is shown in purple. `FOP' indicates samples in which FopFlash was transfected in place of TopFlash. Error bars indicate standard error. Each experiment was repeated a minimum of three times with five independent replicates each time.

View larger version (31K): [in this window] [in a new window]   Fig. 6. Porcupine/palmitoylation regulates Wnt-induced proliferation in the chick neural tube. Embryos were electroporated in the neural tube as described in Materials and methods. GFP-positive embryos were harvested for analysis 24 hours post-electroporation. Embryos were fixed, cryosectioned and immunostained with anti-phosphohistone H3 (red), which marks cells in late G2/M phase. (A) A section from a representative embryo electroporated with mPorcD. The right side of the neural tube has been electroporated (experimental), whereas the contralateral side serves as a control. (B) The ratio of experimental/control proliferation indices for embryos electroporated with Wnt1, Wnt3a, mPorcD, mPorcD(H341Q), Wnt1(C93S) and Wnt3a(C77S). Error bars show the standard error. N, number of embryos analyzed for each data point; n, number of sections analyzed. *, Significantly different from the control (P<0.05). nt, neural tube; no, notochord.

View larger version (25K): [in this window] [in a new window]   Fig. 7. Ectopic expression of porcupine in the chick neural tube steepens the Wnt1/Wnt3a proliferation gradient. mPorcD was transfected into the neural tube by electroporation. GFP-positive embryos were harvested for analysis 48 hours post-electroporation. Embryos were fixed, cryosectioned and immunostained with anti-phosphohistone H3 (red), which marks cells in late G2/M phase. The neural tube was divided into quintiles for quantitation of proliferation, as illustrated in schematic to left. The red dots represent mitotic cells in each quintile. The proliferation index for the control and experimental sides of the neural tube was determined and graphed for each quintile. A total of 43 sections derived from three different embryos were analyzed. Error bars indicate standard error.

View larger version (72K): [in this window] [in a new window]   Fig. 8. Ectopic expression of porcupine in the chick neural tube shortens the BAT-gal reporter gradient. The BAT-gal reporter construct was co-electroporated into chick neural tubes along with either pCIG (control), Wnt1, Wnt3a, mPorcD or mPorcD(H341Q) constructs. GFP-positive embryos were harvested for analysis 24 hours post-electroporation. Embryos were fixed, cryosectioned and immunostained with antibodies against ß-galactosidase. Sections were imaged with confocal microscopy. Merged images of the GFP and ß-galactosidase expression are shown above, separate channels below. The vertical green line demarcates the distance of GFP expression from the dorsal-most extent of the neural tube; a red line demarcates the distance of BAT-gal expression from the dorsal neural tube. A graphic representation of the ratio of the BAT-gal distance divided by the GFP distance is shown. N, number of embryos analyzed; n, number of sections analyzed. Error bars indicate standard error.