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. 2003 May 15;17(10):1207-12.
doi: 10.1101/gad.1084703. Epub 2003 May 2.

Notch signaling regulates left-right asymmetry determination by inducing Nodal expression

Luke T Krebs  1 , Naomi Iwai, Shigenori Nonaka, Ian C Welsh, Yu Lan, Rulang Jiang, Yukio Saijoh, Timothy P O'Brien, Hiroshi Hamada, Thomas Gridley
Affiliations

Notch signaling regulates left-right asymmetry determination by inducing Nodal expression

Luke T Krebs et al. Genes Dev. .

Abstract

Generation of left-right asymmetry is an integral part of the establishment of the vertebrate body plan. Here we show that the Notch signaling pathway plays a primary role in the establishment of left-right asymmetry in mice by directly regulating expression of the Nodal gene. Embryos mutant for the Notch ligand Dll1 or doubly mutant for the Notch1 and Notch2 receptors exhibit multiple defects in left-right asymmetry. Analysis of the enhancer regulating node-specific Nodal expression revealed the presence of binding sites for the RBP-J protein, the primary transcriptional mediator of Notch signaling. Mutation of these sites destroyed the ability of this enhancer to direct node-specific gene expression in transgenic mice. Our results demonstrate that Dll1-mediated Notch signaling is essential for generation of left-right asymmetry, and that the Notch pathway acts upstream of Nodal expression during left-right asymmetry determination in mice.

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Figures

Figure 1
Figure 1
Heart looping defects in Notch pathway mutants. (A–F) Scanning electron micrographs of embryos at E9.5. The direction of heart looping is indicated by the arrow. The left (L) and right (R) sides of the embryo are indicated. (A) Wild-type embryo exhibiting normal right-to-left heart looping. (B) Dll1−/− mutant embryo exhibiting reversed heart looping. (C) Dll1−/− mutant embryo exhibiting ventral heart looping. (D) Embryo homozygous for the Notch2del1 hypomorphic allele (designated N2−/−) exhibiting normal right-to-left heart looping. (E) Notch1−/− Notch2−/− double-mutant embryo exhibiting mild ventral looping. (F) Severely affected Notch1−/− Notch2−/− double-mutant embryo exhibiting reversed looping and cardiac hypoplasia. (G–I) Expression of Notch pathway genes around the node (asterisk). (G) Dll1 expression. (H) Notch1 expression. (I) Notch2 expression. Both Notch1 and Notch2 are also expressed in the paraxial mesoderm (arrows). (J–L) Double-label whole-mount in situ hybridization with a digoxigenin-labeled riboprobe (blue) for Nodal and fluorescein-labeled riboprobes (orange) for Dll1 (J), Notch1 (K), or Notch2 (L).
Figure 2
Figure 2
Dll1−/− mutant embryos exhibit defects in the expression of left–right determinant genes. (A,B) Lefty1/2 expression. Embryos were hybridized with an antisense riboprobe that detects both the Lefty1 and Lefty2 genes. (A) In the wild-type embryo, Lefty1 is expressed along the midline in the prospective floorplate of the neural tube (arrowhead), and Lefty2 is expressed in the left LPM (arrow). (B) In the Dll1−/− embryo, no Lefty1 or Lefty2 expression is observed. Weak background staining is observed in the head of the mutant embryo. (C,D) Pitx2 expression. (C) In the wild-type embryo, Pitx2 is expressed in the head and in the left LPM (arrowhead). (D) In the Dll1−/− embryo shown, Pitx2 is expressed bilaterally in the LPM. Other Dll1−/− mutant embryos did not express Pitx2 in the LPM, or expressed it normally in the left LPM (see Supplementary Table 3). (E,F) Nodal expression at early somite stages. (E) In the wild-type embryo, Nodal is expressed in the node (arrowhead) and left LPM (arrow). (F) In the Dll1−/− embryo, Nodal is not expressed. (G,H) Nodal expression in headfold-stage embryos (E7.5). (G) In the wild-type embryo, Nodal is expressed in the perinodal region. (H) Nodal is not expressed in the Dll1−/− embryo. All images are ventral views, so that the left side of the embryo is toward the right of the panel (indicated in panel A).
Figure 3
Figure 3
Presence of monocilia on node cells of Dll1−/− mutant embryos. (A,B) Monocilia (some indicated with arrows) were detected by immunofluorescence with an antibody against acetylated tubulin. (C,D) Monocilia (arrows) were observed by scanning electron microscopy on both wild-type (C) and Dll1−/− mutant (D) node cells.
Figure 4
Figure 4
The Nodal node-specific enhancer (NDE) contains two RBP-J-binding sites essential for directing lacZ reporter gene expression. (A) The NDE enhancer resides in a 0.8-kb region upstream of Nodal coding sequences (−9.5 to −8.7 kb). Within the NDE, two motifs identical to the consensus binding sequence of RBP-J were present. The relative positions of the 5′ motif (site R1) and 3′ motif (site R2) are indicated. (B) The nucleotide sequences of sites R1 and R2 and of R1m and R2m containing four base pair substitutions (shown in red) in the RBP-J-binding consensus (shown in bold). (C) Electrophoretic mobility shift assay was performed with in vitro translated RBP-J protein (R; lanes 2,3,5–7,9–11) or control translates without RBP-J (c; lanes 1,4,8). Three labeled oligonucleotides were used: a consensus RBP-J sequence (RBP; lanes 1–3), site R1 from NDE (lanes 4–7), and site R2 from NDE (lanes 8–11). Binding reactions were performed in the absence (−) or presence of a 50-fold excess of unlabeled homologous oligonucleotides (lanes 3,6,10), or unlabeled mutated oligonucleotides R1m and R2m (lanes 7,11). (D) Summary of transient transgenic analysis of NDE constructs bearing single or double mutations in the two RBP-J-binding sites. The wild-type construct (NDE) contains the 0.8-kb NDE region linked to a hsp68–lacZ promoter (white circle) lacZ gene (gray bar) reporter cassette. The 5′ RBP-J-binding site, the 3′ RBP-J-binding site, and both RBP-J-binding sites were mutated in NDE-R1m, NDE-R2m, and NDE-R1m2m, respectively. For each construct, the level of NDE activity in the node, the number of embryos that showed NDE activity (column #1), and the number of embryos that carried the transgene but showed either only ectopic expression or no expression at all (column #2) are indicated. Four of the nine R1m2m embryos (indicated by an asterisk in column #2 of the table) showed weak, patchy expression in the center of the node, where the endogenous Nodal gene is not expressed. (E) Ventral views of X-gal-stained E8.2 embryos transgenic for the indicated constructs. The left (L) and right (R) sides of the axis are shown. Bar, 250 μm. (F) X-gal-stained iv/iv embryos transgenic for the NDE construct. One-somite (1s) and nine-somite (9s) iv/iv embryos exhibiting NDE activity are shown. The nine-somite embryo shown had reversed axial rotation.
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References

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