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. 2003 May 15;17(10):1213-8.
doi: 10.1101/gad.1084403. Epub 2003 May 2.

Notch activity induces Nodal expression and mediates the establishment of left-right asymmetry in vertebrate embryos

Angel Raya  1 , Yasuhiko Kawakami, Concepcion Rodriguez-Esteban, Dirk Buscher, Christopher M Koth, Tohru Itoh, Masanobu Morita, R Marina Raya, Ilir Dubova, Joaquin Grego Bessa, Jose Luis de la Pompa, Juan Carlos Izpisua Belmonte
Affiliations

Notch activity induces Nodal expression and mediates the establishment of left-right asymmetry in vertebrate embryos

Angel Raya et al. Genes Dev. .

Abstract

Left-sided expression of Nodal in the lateral plate mesoderm is a conserved feature necessary for the establishment of normal left-right asymmetry during vertebrate embryogenesis. By using gain- and loss-of-function experiments in zebrafish and mouse, we show that the activity of the Notch pathway is necessary and sufficient for Nodal expression around the node, and for proper left-right determination. We identify Notch-responsive elements in the Nodal promoter, and unveil a direct relationship between Notch activity and Nodal expression around the node. Our findings provide evidence for a mechanism involving Notch activity that translates an initial symmetry-breaking event into asymmetric gene expression.

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Figures

Figure 1
Figure 1
Increased Notch activity alters left–right development. (A–H) NotchIC (A,B), hes5 (C,D), deltaC (E,F), and cyclops (G,H) transcripts in 12-hpf control (A,C,E,G), and NotchIC mRNA-injected (B,D,F,H) zebrafish embryos. Embryo views are posterior, dorsal to the top. (I,J) Transgenic zebrafrish embryos expressing a heart-specific mlc2a-EGFP reporter were injected with NotchIC mRNA (J). This treatment resulted in alterations in heart looping and positioning (arrow), when compared to control embryos (I). Embryo views are ventral, anterior to the top. (K–N) Whole-mount in situ hybridization for the Nodal-related gene cyclops (K,L) and pitx2 (M,N) revealed the appearance of ectopic transcripts for both genes in the right LPM of 24-hpf NotchIC-injected embryos (L,N, arrows). Embryo views are dorsal, anterior to the top. Left (L) and right (R) sides are indicated.
Figure 2
Figure 2
Notch activity is necessary for Nodal expression and proper left–right determination. (A–D) Frontal views of E9.5 wild-type (A), Dll1−/− (B,C), and RBPjk−/− (D) embryos. Normal rightward heart looping is evident in wild-type embryos (A), while Dll1−/− and RBPjk−/− mutants display alterations in heart looping, ranging from incomplete looped heart (C), to leftward looping (B,D). OT, outflow tract; RV, right ventricle; LV, left ventricle; AT, atrium. (E–P) Whole-mount in situ hybridization for genes implicated in normal LR development revealed that Nodal transcripts were absent in the perinodal region of Dll1−/− and RBPjk−/− mice (E–G, arrow in E denotes presence in wild type) and also, later on, were absent in the left LPM (H–J). Pitx2 transcripts were also absent from the left LPM (K–M; arrowhead in H and K denotes presence in wild type). Similarly, the midline and LPM domains of expression of Lefty1 were absent in both Dll1−/− and RBPjk−/− mice (N–P). Embryo views are ventral, anterior to the top in E–G, and dorsal, anterior to the top in H–P. Please note that coloring reaction for in situ hybridizations was allowed to proceed longer than usual to verify the absence of signal in the mutants; hence, the increased background. Left (L) and right (R) sides are indicated.
Figure 3
Figure 3
Nodal is a direct target of the Notch pathway. (A) Sequences representing 15 kb of genomic DNA 5′ of the starting codon of human and mouse Nodal were aligned using the AVID algorithm and the output generated by the VISTA set (http://www.gsd.lbl.gov/vista/index.html). Exon 1 is labeled in magenta. Only one noncoding region (pale red) shows more than 75% homology between human and mouse DNA sequences. The sequence of a 355-bp BglII fragment of the mouse gene containing the evolutionarily conserved region is shown in B. Gaps introduced by the alignment algorithm are indicated by dashes. Some regions in the human sequence not aligning with mouse Nodal have been removed for clarity, and are indicated by spaces. Colored boxes mark the putative transcription factor binding sites identified using MatInspector with TRANSFAC 4.0 matrices (http://transfac.gbf.de/TRANSFAC). (C) A 32P-labeled probe representing the mouse Nodal sequence depicted in B was incubated in the absence (lanes 1,7) or in the presence (lanes 2–6,8–12) of purified recombinant Su(H), and the indicated competitors. Oligonucleotides (20-mer) representing the putative RBPjk binding sites 1 (ON-Site 1) and 2 (ON-Site 2), and their respective flanking regions, or equivalent oligonucleotides where the putative binding site was mutated (ON-Site 1-mut and ON-Site 2-mut), were used to compete the binding at the indicated molar excess (×). The arrow indicates the change in the probe's mobility caused by binding to Su(H). The arrowhead points to the free probe. (D–H) A genomic DNA fragment representing the sequences shown in B was used to drive the expression of lacZ in transgenic mouse embryos. β-Galactosidase staining of E7.5–E8.0 embryos (E) recapitulates the endogenous Nodal expression around the node (D). Transgenes in which the putative RBPjk binding site 1 (F) or 2 (G) are mutated to a PmeI recognition site drive weak ectopic expression of lacZ. (H) When both sites are mutated, no lacZ expression could be detected.
Figure 4
Figure 4
Notch activity during left–right determination does not depend on SHH activity. Implantation on the right side of HH3–HH4 chick embryos of SHH soaked beads (B), or of anti-SHH blocking antibody on the left side (C) did not have any effect on Dll1 expression around Hensen's node. Similarly, Lfng transcripts were not altered after implanting SHH soaked beads (E) or anti-SHH antibody (F). (G–J) Whole-mount in situ hybridization in E7.5–E8.0 Shh−/− mouse embryos did not reveal any alteration in the expression pattern of either Dll1 or Lfng. Left (L) and right (R) sides are indicated. Relative position of the node is denoted by a circle.
Figure 5
Figure 5
Notch activity regulates left–right asymmetry independently of nodal cilia function. Lfng is expressed in E7.5–E8.0 control mouse embryos as two sharp bilateral perinodal stripes (A), barely observable in Dll1−/− (B) or RBPjk−/− (C) embryos, indicating that Lfng expression depends of Notch activity during mouse gastrulation. Analysis of Dll1 expression in E7.5–E8.0 iv (E) and KIF3A−/− (F) embryos did not reveal any alteration in the expression pattern when compared to control littermates (D). Similarly, Lfng expression is not altered in iv (H) or KIF3A−/− (I) embryos, when compared to that of wild-type (G). Relative position of the node is denoted by a circle.
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References

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