Inefficient nuclear translocation and cleavage of DICD.

<p>(A) Detection of DICD stably (c, d) or transiently (e, f) expressed in CHO cells in the absence (c, e) or presence (d, f) of the proteasome inhibitor MG132. (B) Schematic representation of DICD-LexAVP16 fusion constructs. (C) Activation of lexA operator driven luciferase in CHO cells. (D) Western blot of cell lysates of CHO cells stably expressing DICD and fDICD.</p

Normal Notch target gene expression in embryos expressing DL1ICD variants.

<p>(A) Whole mount in situ hybridization of wild type and DL1ICD-expressing embryos showing normal Hey1 expression. (B) qRT-PCR analysis of Hes5 and Hey2 expression in wild type and DL1ICD-expressing embryos. Indicated are means and SEM of expression levels determined in individual wild type and transgenic embryos. ns: not significant (p>0.05).</p

Normal development of embryos expressing DL1ICD variants.

<p>(A) GFP expression in male, and hetero-and homozygous female transgenic embryos indicating Cre-mediated activation of transgene expression. (B) Western blot analysis of cell lysates from wild type and DL1ICD-expressing embryos. (C) Whole mount in situ hybridization of wild type (a, h) and DL1ICD-expressing (b-g, and i-n) embryos showing normal anterior-posterior somite patterning (a-g) and muscle differentiation (h-n). (D) Whole mount in situ hybridization of wild type (a) and DL1ICD-expressing (b-g) embryos showing normal neuronal differentiation. (E) qRT-PCR analysis of Nfem and Isl1 expression in wild type and DL1ICD-expressing embryos. Indicated are means and SEM of expression levels determined in individual wild type and transgenic embryos. ns: not significant (p>0.05).</p

Normal proliferation and neuronal differentiation of ES cells expressing DL1ICD variants.

<p>(A) Doubling times of targeted E14tg2a cells before and after Cre-mediated activation of DL1ICD expression. Doubling times were calculated from cell counts after non-linear regression using Prism software (GraphPad). Indicated are mean doubling times and upper and lower limit of 95% confidence intervals. (B) Western blot analysis of cell lysates of wild type and DL1ICD-expressing ES cells, CHO cells with or without transient expression of mouse p21, and HeLa nuclear extract. The arrow points to the position of p21, the asterisk marks a non-specific background band detected in ES cells. (C) Expression of the pan-neuronal marker Nefm in differentiated wild type and DL1ICD-expressing ES cells analyzed by qRT-PCR. Indicated are means and SEM of expression levels determined in differentiated wild type (n=16 pools of aggregates ) RA treated (n=14 pools of aggregates) and transgenic (DICD: n=13 pools of aggregates; fDICD: n=12 pools of aggregates; DΔECD: n=10 pools of aggregates) ES cells. ns: not significant (p>0.05). </p

A Novel Mammal-Specific Three Partite Enhancer Element Regulates Node and Notochord-Specific <em>Noto</em> Expression

<div><p>The vertebrate organizer and notochord have conserved, essential functions for embryonic development and patterning. The restricted expression of developmental regulators in these tissues is directed by specific cis-regulatory modules (CRMs) whose sequence conservation varies considerably. Some CRMs have been conserved throughout vertebrates and likely represent ancestral regulatory networks, while others have diverged beyond recognition but still function over a wide evolutionary range. Here we identify and characterize a mammalian-specific CRM required for node and notochord specific (NNC) expression of NOTO, a transcription factor essential for node morphogenesis, nodal cilia movement and establishment of laterality in mouse. A 523 bp enhancer region (NOCE) upstream the <em>Noto</em> promoter was necessary and sufficient for NNC expression from the endogenous <em>Noto</em> locus. Three subregions in NOCE together mediated full activity in vivo. Binding sites for known transcription factors in NOCE were functional in vitro but dispensable for NOCE activity in vivo. A FOXA2 site in combination with a novel motif was necessary for NOCE activity in vivo. Strikingly, syntenic regions in non-mammalian vertebrates showed no recognizable sequence similarities. In contrast to its activity in mouse NOCE did not drive NNC expression in transgenic fish. NOCE represents a novel, mammal-specific CRM required for the highly restricted <em>Noto</em> expression in the node and nascent notochord and thus regulates normal node development and function.</p> </div

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

HPRT targeting strategy.

<p>Schematic view of wild type and E14tg2a <i>Hprt</i> locus, targeting construct and targeted locus. The targeting construct introduces the reporter construct and the promoter and first exon of human <i>HPRT</i> to restore <i>Hprt</i> function. Wild type and targeted allele are HAT resistant (HAT^R) whereas the E14tg2a deletion results in HAT sensitivity (HAT^S).</p

NOCE is a three partite enhancer element.

<p>(A) Schematic view of transgenes containing parts of NOCE. (B) 1 hr β-galactosidase staining of different promoter-reporter transgenes in chimeric embryos. (C) Over night β-galactosidase staining of chimeric embryos carrying the transgenes depicted under (A). The stage is indicated at the top and the transgene at the left site. Arrow heads point at stained notochords.</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

An orphan binding site together with a FOXA2 binding site are essential for NOCE function.

<p>(A) Schematic view of location and sequences of transcription factor binding sites in NOCE and the alignment of OBS to CE <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047785#pone.0047785-Sawada1" target="_blank">[27]</a>. Red characters indicate exchanged nucleotides in mutated sites. (B) β-galactosidase stained chimeric embryos carrying a promoter-reporter transgene of NOCE with mutated OBS and/or FOXA2 binding sites. Transgenes are indicated at the left site and the stage at the top.</p