DLL3 physically interacts with LFNG and is modified by POFUT and LFNG at the predicted O-fucosylation consensus motifs in EGF2 and EGF5.

<p>(A) Detection of DLL3 and LFNG interaction by coimmunoprecipitations of Flag-tagged DLL3 and HA-tagged LFNG expressed in CHO cells (red arrowheads point to co-precipitated proteins). (B) Schematic overview of expression constructs used for the analysis of DLL3 modifications. DLL3 carries a consensus motif for O-fucosylation (C²XXGG(S/T)C³) in EGF2 (S at position 286) and EGF5 (T at position 403). T83* and T206* are amino acids in the N-terminal and DSL region predicted to be modified by GalNAc O-glycosylation (NetOGlyc3.1 Server: <a href="http://www.cbs.dtu.dk/services//NetOGlyc" target="_blank">www.cbs.dtu.dk/services//NetOGlyc</a>, [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123776#pone.0123776.ref047" target="_blank">47</a>]. Sig, Signal sequence; DSL, DSL domain; 1–6, EGF-like repeats 1 to 6; TM, transmembrane region; F, Flag tag, S286A, Serin mutation into Alanin; T403V, Threonin mutation into Valin. (C) Immunoprecipitations of wild type DLL3, DLL3-S286A, DLL3-T403V and DLL3-S286A,T403V proteins from lysates of metabolically labeled CHO cells stably expressing the respective protein. No tritiated fucose was incorporated into the DLL3-S286A,T403V protein (red arrowhead, lower row), indicating that these are the only O-fucosylation sites in DLL3. As a positive control for the metabolic labeling procedure cells were labeled with S³⁵-Methionine (middle row). Western blot analysis (upper row) with anti-Flag antibody shows different expression levels of used clones, consistent with different signal intensities after labeling with methionine. (D) Schematic representation of the strategy to analyze O-fucose elongation by LFNG using click-iT chemistry. LFNG catalyses elongation of O-linked fucose (black square) bound to Serin or Threonin in EGF-like repeats with N-Acetylglucosamine (grey circle) followed by Galactose (grey triangle) and Sialic Acid (white diamond) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123776#pone.0123776.ref045" target="_blank">45</a>]. With the "click" reaction azide modified Sialic acid is chemoselectively ligated to alkyne-tagged Biotin (white circle), which can be detected with Streptavidin or anti-Biotin Antibodies. (E and F) Immunoblots of Flag-tagged DLL3 variants shown in (B) immunoprecipitated from lysates of metabolically Ac₄ManNAz (sialic acid precursor) labeled CHO cells using anti-Flag antibodies. Incorporation of sialic acid (see D) was visualized with peroxidase-conjugated Streptavidin (E) or with anti-Biotin Antibody (F). Presence of immunoprecipitated DLL3 variant proteins was verified using anti-DLL3 antibodies (E) or on input Lysate with anti-Flag antibodies (F). DLL3 and the O-fucosylation mutant DLL3-S286A, T403V showed incorporation of sialic acid (E, red arrowheads), indicating the presence of additional O-Glycosylation sites. Sialic acid was incorporated into wild type DLL3 lacking the N-terminus and DSL domain (F, upper row, red arrowhead) but not into the truncated variant when S286 and T403 were mutated (DLL3ΔN,DSL-S286A, T403V, see B) indicating further modification of O-fucose residues at S283 and T403.</p

Somite patterning and vertebral column defects in single and double <i>Dll3</i> and <i>Lfng</i> mutants.

<p>Whole-mount in situ hybridizations of E9.5 embryos with an <i>Uncx4.1</i> specific probe (a-d) and lateral (e-h) and dorsal (e‘-h‘) views of skeletal preparations of wild type (a, e,e‘), homozygous <i>Dll3</i>^<i>pu</i> (b, f, f‘), homozygous <i>Lfng</i>^<i>lacZ</i> (c, g, g‘) and double homozygous <i>Dll3</i>^<i>pu</i>; <i>Lfng</i>^<i>lacZ</i> E18.5 embryos (d, h, h‘). Absence of both proteins does not enhance somite A-P patterning or vertebral column defects present in single mutants.</p

The DLL3-S286A,T403V mutant does not complement the loss of endogenous DLL3 in somitogenesis.

<p>(A) Schematic representation of constructs introduced into HPRT deficient homozygous <i>Dll3</i> mutant ES cells. MSD2 is a dimer of the mesoderm-specific promoter element (MSD) from the <i>Dll1</i> locus [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123776#pone.0123776.ref059" target="_blank">59</a>]. Locations of primers and restriction sites used for genotyping of embryos derived from tetraploid complementation are indicated below and above schemes b and c, respectively. (B) The MSD2 promoter element drives transgene expression in the PSM similar to endogenous <i>Dll3</i> expression. E8.5 MSD2::LacZ chimeric embryos were stained for β-galactosidase activity for one hour (a) and over night (b), or were analyzed by whole-mount in situ hybridization with a <i>lacZ</i> specific probe (c). (d) Expression pattern of endogenous <i>Dll3</i> transcripts in an E8.5 wt embryo for comparison. (C) Whole mount in situ hybridizations of E9.5 wt (a), homozygous <i>Dll3</i>^<i>pu</i> (b), and <i>Dll1</i>^{<i>Dll3ki</i>}, <i>Dll3</i>^<i>pu/pu</i> [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123776#pone.0123776.ref038" target="_blank">38</a>] (c) embryos. In <i>Dll1</i>^{<i>Dll3ki</i>}, <i>Dll3</i>^<i>pu/pu</i> embryos, which lack endogenous DLL3 but express DLL3 from the <i>Dll1</i> locus, expression of the anterior-posterior (A-P) somite patterning marker <i>Uncx4.1</i> is restored except for minor irregularities (arrowhead in c). (d-h) Completely ES cell-derived embryos hybridized with <i>Uncx4.1</i>. Embryos generated from ES cells homozygous mutant for <i>Dll3</i> (<i>Dll3</i>^<i>pu/pu</i>) and carrying the HPRT deletion (ΔHPRT) display the same A-P somite patterning defects as homozygous <i>Dll3</i>^<i>pu</i> embryos (compare d with b). Expression of wild type DLL3 in ES cell-derived embryos almost completely rescues the <i>Dll3</i>^<i>pu</i> A-P somite patterning defect except for minor irregularities (arrowheads in e, g), whereas ES cell-derived embryos expressing mutant DLL3 display a <i>pudgy</i>-like somite phenotype (f, h) indicating that DLL3-S286A, T403V is not functional during somitogenesis. (D) Genotyping of tetraploid embryos shown in (C) using PCR and restriction digests as indicated in (A). The wild type <i>Dll3</i> PCR product is cut by <i>AleI</i> but not <i>NarI</i> (left panel), whereas the <i>Dll3</i>-S286A, T403V PCR product is cut by <i>NarI</i> (due to the presence of S286A) but not by <i>AleI</i> (due to the presence of T403V; middle and right panel; see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123776#sec009" target="_blank">Material and Methods</a> for further details). Letters in parentheses refer to embryos shown in (C), arrowheads indicate cleavage products of the expected sizes. (E) Western blot analysis of lysates of wild type embryos (lane 1) or embryos obtained with ES cell clone B5 (lane 2) and lysates of CHO cells overexpressing Flag-tagged DLL3-S286A, T403V (lane 3) or wild type DLL3 (lane 4) using anti-Flag antibodies confirmed expression of DLL3-S286A, T403V-Flag in completely ES cell-derived embryos (red arrowhead). The equivalent of 4 trunks of E9 embryos was loaded in lanes 1 and 2. Asterisk between lanes 1 and 2 indicates a background band detected in embryo lysates.</p

O-fucosylation is not essential for DLL3 interaction with LFNG, full length Notch1 and DLL1, and Notch1 <i>cis</i>-inhibition.

<p>(A) Flag-tagged wild type DLL3 or DLL3-S286A, T403V, and HA-tagged LFNG, Myc-tagged Notch1 and MycHis-tagged DLL1, respectively, were immunoprecipitated from lysates of CHO cells expressing the indicated combinations of these proteins demonstrating that the O-fucosylation defective DLL3 S286A, T403V protein interacts with LFNG, full length Notch1 and DLL1 (red arrowheads). The protein running above full length Notch (marked by asterisks) represents a background band recognized by the anti-Myc antibody in immunoprecipitated material. (B) Notch transactivation assay. HeLa cells, transfected with the (4xRBP-Jκ)-luciferase reporter, Renilla luciferase and expression vectors for DLL3, DLL3-S286A,T403V or empty vector respectively, were cocultivated either with wild type CHO cells or CHO cells stably expressing DLL1. Luciferase activity from HeLa cocultured with CHO cells (set to 1) and CHO cells stably expressing DLL1 were measured and normalized to the expression levels of the appropriate constructs. Error bars represent SD. * = p<0.05; **0 p<0.01.</p

Wild type DLL3 but not DLL3-S286A,T403V affects the subcellular localization of DLL1.

<p>Confocal images of CHO cells stably expressing Flag-tagged DLL1 (A), transfected either with HA-tagged wild type or fucosylation mutant DLL3 (B, C). (A) Costaining of DLL1-Flag (using MAb 1F9) with the cell surface marker Sodium potassium ATPase (a-c) detects DLL1 on the cell surface (arrowheads in a and c), costaining with GM130 (d-f) in the trans-Golgi (arrow heads in d and f). In contrast, staining of DLL1-Flag using anti-DLL1 antibody H-265 detects predominantly Golgi localized DLL1 (white arrowheads in g and i) and reacts only weakly with DLL1 on the cell surface (g and i, grey arrowhead). DLL1 staining with anti-Flag detects DLL1-Flag similar to staining with 1F9 (compare j with a or d) overlapping with H-265 staining in the Golgi (arrowhead in l). (B) Coexpression of wild type DLL3 in DLL1 CHO cells (a-f) leads to an intracellular accumulation of DLL1 protein (white arrowheads in a, c and d, f), whereas in cells coexpressing the fucosylation mutant of DLL3 (g-l) localization of DLL1 is similar to untransfected cells and readily detected at the cell surface (white arrowheads in g, i and j, l). (C) Coexpression of wild type DLL3 with DLL1 protein leads to colocalization of DLL3 and DLL1 in vesicular structures or punctae outside the Golgi (white arrowheads in a, c, e, f, h, j; 18 out of 21 cells). The asterisk in a-e marks a neighboring DLL1 expressing cell without DLL3 expression showing intracellular DLL1 protein confined to the trans-Golgi (grey arrowhead in a and c). Coexpression of the O-fucosylation-defective DLL3 mutant (k-t) had no obvious effect on the localization of intracellular DLL1 protein (k, m, o, p, r, t; 10 our of 14 cells) or resulted only in weak detection of DLL overlapping with DLL3 outside the Golgi (u-y). The asterisk in k-o marks a neighboring DLL3 non-expressing cell.</p

Somite patterning and vertebral column defects in single and double <i>Dll3</i> and <i>Lfng</i> mutants.

<p>Whole-mount in situ hybridizations of E9.5 embryos with an <i>Uncx4.1</i> specific probe (a-d) and lateral (e-h) and dorsal (e‘-h‘) views of skeletal preparations of wild type (a, e,e‘), homozygous <i>Dll3</i>^<i>pu</i> (b, f, f‘), homozygous <i>Lfng</i>^<i>lacZ</i> (c, g, g‘) and double homozygous <i>Dll3</i>^<i>pu</i>; <i>Lfng</i>^<i>lacZ</i> E18.5 embryos (d, h, h‘). Absence of both proteins does not enhance somite A-P patterning or vertebral column defects present in single mutants.</p