O-fucosylation of DLL3 is required for its function during somitogenesis.

Delta-like 3 (DLL3) is a member of the DSL family of Notch ligands in amniotes. In contrast to DLL1 and DLL4, the other Delta-like proteins in the mouse, DLL3 does not bind in trans to Notch and does not activate the receptor, but shows cis-interaction and cis-inhibitory properties on Notch signaling in vitro. Loss of the DSL protein DLL3 in the mouse results in severe somite patterning defects, which are virtually indistinguishable from the defects in mice that lack lunatic fringe (LFNG), a glycosyltransferase involved in modifying Notch signaling. Like LFNG, DLL3 is located within the trans-Golgi, however, its biochemical function is still unclear. Here, we show that i) both proteins interact, ii) epidermal growth factor like repeats 2 and 5 of DLL3 are O-fucosylated at consensus sites for POFUT1, and iii) further modified by FNG proteins in vitro. Embryos double homozygous for null mutations in Dll3 and Lfng are phenotypically indistinguishable from the single mutants supporting a potential common function. Mutation of the O-fucosylation sites in DLL3 does not disrupt the interaction of DLL3 with LFNG or full length Notch1or DLL1, and O-fucosylation-deficient DLL3 can still inhibit Notch in cis in vitro. However, in contrast to wild type DLL3, O-fucosylation-deficient DLL3 cannot compensate for the loss of endogenous DLL3 during somitogenesis in the embryo. Together our results suggest that the cis-inhibitory activity of DLL3 observed in cultured cells might not fully reflect its assumed essential physiological property, suggest that DLL3 and LFNG act together, and strongly supports that modification of DLL3 by O-linked fucose is essential for its function during somitogenesis

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