Interaction of epitope-tagged Vangl2 and Vangl1 proteins.

<p>(<b>A</b>) Schematic diagram of Vangl1 and full-length and truncated mutants of Vangl2 used for this study. (<b>B</b>) Transient co-expression of myc-Vangl1 with GFP-Vangl2, GFP-Vangl2ΔN, GFP-Vangl2ΔC, GFP-Vangl2ΔNΔC a control protein (GFP in T47D cells and immunoprecipitation with myc antibody shows co-immunoprecitation of Vangl2 with Vangl1). (<b>C</b>) Colocalization of ectopically expressed Myc-Vangl1 and GFP-Vangl2 in T47D cells and analysis by immunofluorescence and confocal analysis. All images were taken under a 40×objective. Scale bar corresponds to 10 µm and is labelled in white. (<b>D</b>) Vangl2 homodimerization is displayed by immunoprecipitation with Myc antibody shows co-immunoprecitation of Myc-Vangl2 with GFP-Vangl2. Protein complexes were separated using SDS-PAGE and western blot analysis.</p

List of peptides observed for Vangl1 identification.

<p>List of peptides observed for Vangl1 identification.</p

List of peptides observed for Vangl2 identification.

<p>List of peptides observed for Vangl2 identification.</p

Identification of an endogenous protein complex containing the Vangl2 and Vangl1 proteins.

<p>(<b>A</b>) Specificity of 2G4 mAb was verified in western blot using T47D cell extracts expressing GFP, GFP-Vangl1, Vangl2 or GFP. Proteins were separated on SDS-PAGE and analyzed with western blot with specific antibodies. Specificity of 2G4 mAb in immunoprecipitation experiments GFP-Vangl1, GFP-Vangl2 or GFP expressed in T47D cells. Western blotting was carried out using 2G4 mAb, anti-GFP and Vangl1/2 antibodies. (<b>B</b>) SKBR7 cells were treated with shLuc or shVangl2 and assessed for Vangl expression. Vangl2 was immunoprecipitated from cell lysates and detected with the same 2G4 antibody. (<b>C</b>) Proteins were subsequently separated using Bis-Tris gradient gels and silver stained. Bands specific to 2G4 mAb were excised followed by in-gel trypsin digestion, chromatographic separation and orbitrap analysis. An asterisk indicates the bands corresponding to Vangl2. (<b>D</b>) Endogenous Vangl2 and Vangl1 were identified with the presence of multiple peptides and associated with a high probability Mascot scores (at least 40 arbitrary units). Bold characters indicate the peptides identified by mass spectrometry as shown with alignment of the two sequences. Regions shaded in grey correspond to the amino-terminal regions of Vangl1 and Vangl2.</p

Sulf specific deficits during the postnatal development of the cerebellum are primarily mediated by altered signal transduction pathways.

<p>The outer half of the external granular layer (EGL) is the zone where precursors actively proliferate. Under the influence of GDNF, the cell survival of Sulf1 deficient precursor neurons is inhibited. A further proapoptotic effect was shown for NGF on Sulf2 deficient cells. Following the trail of a cerebellar precursor neuron, the cell enters the inner half of the EGL, stop dividing, and undergo neurite extension and tangential migration to reach their final destination in the internal granular layer (IGL) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139853#pone.0139853.ref001" target="_blank">1</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139853#pone.0139853.ref002" target="_blank">2</a>]. Neurite outgrowth deficits in Sulf2 knockout cells could be compensated by the addition of the growth factors FGF2 and GDNF, whereas no influence of growth factors such as FGF2, GDNF or NGF on neurite outgrowth was detectable for Sulf1 deficient neurons. The observed unaltered migration capacity (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139853#sec010" target="_blank">Discussion</a>) of Sulf deficient cerebellar granule cells is in line with inconspicuous cerebellar lamination in Sulf deficient mice.</p

Generation of a highly specific anti-Vangl2 monoclonal antibody.

<p>(<b>A</b>) Expression of GST, GST-NVangl1 and GST-NVangl2 fusion proteins verified by SDS-PAGE and Coomassie staining. (<b>B</b>) Selected hybridomas were screened using an ELISA against GST, GST-NVangl1 and GST-NVangl2. mAb: monoclonal antibody. (<b>C</b>) Specificity of 2G4 mAb for GST-NVangl2 shown by western blot experiments, using antibodies specific for GST, Vangl2 and Vangl1/2. (<b>D</b>) SPR analysis using 2G4 mAb (10 µg/ml) injected over immobilized GST, GST-NVangl1 or GST-NVangl2. Sensorgrams show total signal from GST fusion proteins normalised with the non-specific signal (GST).</p

Differential expression of Vangl2 in different murine tissues and mouse cochlea.

<div><p>(<b>A</b>) Expression profile of Vangl2 from murine tissues (brain, kidney and lung) detected with 2G4 antibody in western blot. (<b>B</b>) Vangl2 protein expression in mouse cochlear tissue deriving from homozygous <i>Lp</i> and heterozygous Vangl2^Lp/+ heterozygous mice compared to wild-type littermates, using 2G4 mAb antibody. Corresponding densitometry measurements representing relative Vangl2 protein expression normalized to GADPH protein levels. (<b>C</b>) Absence of staining with 2G4 mAb in Vangl2 mutant. Surface views of cochleae from WT (A-A’) and Looptail homozygote (B-B’) from E17.5 mice processed for immunocytochemistry with 2G4 mAb. At this stage, the cochlea comprises a single row of inner hair cells (#) and three rows of outer Hair Cells (OHC1 *, OHC2 **, OHC3 ***) surrounded by supporting cells. Phalloidin staining (actin, red) reveals hair cells borders. The image is taken at the level of the zonula adherens for the second and third row of OHC2 and OHC3, where Vangl2 accumulates. In the WT sample, we observe accumulation of Vangl2 at the junction between hair cells and supporting cells (A-A’, arrows). In contrast, in Lp/Lp cochlea, there is a complete absence of Vangl2 staining at the membrane of the cells (B,B’). Scale bar = 3 µm. (<b>D</b>) Co-localization of staining with 2G4 mAb and Vangl1 Ab.</p> <p>Surface view of a cochlea from a newborn mouse processed for immunocytochemistry with 2G4 mAb and Vangl1 Ab. The two antibodies reveal a co-localization of Vangl1 (A”) and Vangl2 (A’”) proteins at the junction between hair cells and supporting cells (arrows). Phalloidin is in blue, Vangl1 in red, and Vangl2 in green. Scale bar = 3 µm. Note: in the green channel, remains of the tectorial membrane covering normally the cochlear epithelium lead spots of non-specific green labeling. (<b>E</b>) Schematic diagram representing the two possible models of Vangl1/Vangl2 interaction.</p></div

Sulf1 and Sulf2 Differentially Modulate Heparan Sulfate Proteoglycan Sulfation during Postnatal Cerebellum Development: Evidence for Neuroprotective and Neurite Outgrowth Promoting Functions

<div><p>Introduction</p><p>Sulf1 and Sulf2 are cell surface sulfatases, which remove specific 6-O-sulfate groups from heparan sulfate (HS) proteoglycans, resulting in modulation of various HS-dependent signaling pathways. Both Sulf1 and Sulf2 knockout mice show impairments in brain development and neurite outgrowth deficits in neurons.</p><p>Methodology and Main Findings</p><p>To analyze the molecular mechanisms behind these impairments we focused on the postnatal cerebellum, whose development is mainly characterized by proliferation, migration, and neurite outgrowth processes of precursor neurons. Primary cerebellar granule cells isolated from Sulf1 or Sulf2 deficient newborns are characterized by a reduction in neurite length and cell survival. Furthermore, Sulf1 deficiency leads to a reduced migration capacity. The observed impairments in cell survival and neurite outgrowth could be correlated to Sulf-specific interference with signaling pathways, as shown for FGF2, GDNF and NGF. In contrast, signaling of Shh, which determines the laminar organization of the cerebellar cortex, was not influenced in either Sulf1 or Sulf2 knockouts. Biochemical analysis of cerebellar HS demonstrated, for the first time in vivo, Sulf-specific changes of 6-O-, 2-O- and N-sulfation in the knockouts. Changes of a particular HS epitope were found on the surface of Sulf2-deficient cerebellar neurons. This epitope showed a restricted localization to the inner half of the external granular layer of the postnatal cerebellum, where precursor cells undergo final maturation to form synaptic contacts.</p><p>Conclusion</p><p>Sulfs introduce dynamic changes in HS proteoglycan sulfation patterns of the postnatal cerebellum, thereby orchestrating fundamental mechanisms underlying brain development.</p></div

Disaccharide compositional analysis of HS from cerebella of postnatal day 6 Sulf deficient mice and wildtype littermates.

<p>HS from Sulf1 matched wildtype (black) and null (dark grey) as well as Sulf2 matched wildtype (light grey) and null (white) mice was purified using TRIzol, digested to disaccharides and labelled with BODIPY-FL hydrazide. Resulting disaccharides (with variant sulfation patterns depicted in panel A) were separated using strong anion exchange HPLC as described in Materials and Methods. Data are expressed in % of total disaccharide composition (<b>B</b>) and as fold change relative to wildtype (<b>C</b>) (mean +/- SD; n = 3, from individual cerebella). (<b>D</b>) Total NS, 2S and 6S containing disaccharides in postnatal cerebellum HS of Sulf deficient mice. Data are expressed in % of disaccharide composition. 1, ΔUA-GlcNAc; 2, ΔUA-GlcNAc(6S); 3, ΔUA-GlcNS; 4, ΔUA-GlcNS(6S); 5, ΔUA(2S)-GlcNS; 6, ΔUA(2S)- GlcNS(6S); 7, ΔUA-2S-GlcNAc; and 8, ΔUA-2S-GlcNAc(6S).</p

Sulf deficiency impairs the postnatal development of the cerebellum.

<p>| (<b>A</b>) The postnatal cerebellar development is mainly characterized by proliferation, migration and neurite outgrowth of granule precursor cells[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139853#pone.0139853.ref001" target="_blank">1</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139853#pone.0139853.ref002" target="_blank">2</a>]. The coordination and regulation of these processes involve growth factors such as Shh, FGF2, NGF and GDNF in the local environment of these precursor neurons (for details see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139853#sec004" target="_blank">Introduction</a>). (<b>B</b>) Poly-L-lysine dependent migration of Sulf1 deficient cerebellar granule cells is reduced. Cerebellar microexplant cultures from wildtype (wt), Sulf1 (S1) or Sulf2 (S2) deficient mice were plated onto glass cover slips coated with PLL (P, filled bars) or a combination of PLL and laminin (L, hatched bars). Explants were fixed, stained with DAPI and migration of cerebellar granule cells quantitated as described in Experimental Procedures. The asterisk indicates a statistically significant difference of Sulf deficient neurons as compared to wildtype cells (* p < 0.05). (<b>C, D</b>) Sulf1 and Sulf2 deficient cerebellar neurons show significant reduction of cell survival. Cerebellar neurons from wildtype (wt), Sulf1 (S1) and Sulf2 (S2) deficient mice were plated as single cell suspensions onto PLL coated cover slips (P, filled bars) or cover slips coated with a combination of PLL and laminin (L, hatched bars). For cells shown in D, 44 hours after plating 500 nM staurosporine was added to the medium to induce cell death. The cells were cultured for additional 4 h with (D) or without (C) staurosporine before cell death was determined by counting calcein versus propidium iodide positive cells of five independent experiments. Cell survival of wildtype cells cultured without staurosporine was set to 100%. Asterisks indicate a statistically significant difference of Sulf deficient neurons as compared to wildtype cells (* p < 0.05).</p