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First published online 11 June 2008
doi: 10.1242/dev.018986

Development 135, 2425-2434 (2008)
Published by The Company of Biologists 2008
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BMP antagonism is required in both the node and lateral plate mesoderm for mammalian left-right axis establishment

Naoki Mine, Ryan M. Anderson* and John Klingensmith{dagger}

Department of Cell Biology, Duke University Medical Center, Durham, NC 27710, USA.,


Figure 1
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  		Fig. 1. L-R asymmetric Smad1/5/8 activation and BMP antagonist expression in mouse embryo lateral plate mesoderm. (A-C) Whole-mount immunohistochemistry for phosphorylated (p-) Smad1/5/8 in 4s (A) (n=7/10) and 5s (B) (n=8/8) embryos (anterior view). Anti p-Smad1/5/8 staining in lateral plate mesoderm (LPM) is stronger on the right side than on the left (arrowhead). (C) High magnification of the boxed region from A. Rectangles demark left and right LPM. (D,E) Whole-mount lacZ staining of Bmp4lacZ/+ embryo at 4s (n>10). Bmp4 expression is observed bilaterally at the edge of the LPM (arrowhead). Anterior (D) and posterior (E) views. The node is boxed. (F-H) Whole-mount lacZ staining of NoglacZ/+ embryo at 4s (F) (n=6/6) and 5s (G) (n=6/6). Nog expression in LPM is stronger on the left side than right (arrowhead). (F,G) Anterior view. (H) High magnification of the boxed region from F. (I-K) Whole-mount in situ hybridization (WMISH) for Chrd at 4s (I) (n=6/12) and 5s (J) (n=10/16). Chrd expression is stronger in left LPM than right (arrowhead). Ventral views. The dashed line indicates the extent of LPM on each side. (K) High magnification of the boxed region from I. (L) Quantitative RT-PCR results for Nog, Chrd, Bmp4 and Nodal in right versus left LPM at 4s. R, right; L, left. (M) Summary of BMP signaling activity and BMP antagonist expression at 4-5s. BMP signaling activity is represented in red (left), Nog and Chrd expression in green (right), with higher levels in a darker hue.

 

Figure 2
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  		Fig. 2. Chordin and noggin are both required for correct heart looping and expression of left-side determinants. (A-C) Arrows indicate laterality of heart looping from left ventricle to outflow tract. Dashed lines outline hearts. (A) E9.0 wild-type (WT) mouse embryo showing normal rightward heart looping. (B) E9.0 Chrd-/-;Nog-/- (DKO) embryo showing no directional looping of the heart. (C) E9.0 Chrd-/-;Nog-/- embryo showing reversed, leftward looping. (D) Summary of cardiac looping at E9.5 in various genotypes, each investigated in >20 embryos. (E-H) Embryos subjected to WMISH at 5s. WMISH for Nodal probe in wild type (E) and Chrd-/-;Nog-/- (F) and for Lefty2 in wild type (G) and Chrd-/-;Nog-/- (H). (I) Summary of expression patterns of the L-R asymmetric markers Nodal, Lefty2 and Pitx2 in Chrd-/-;Nog-/- embryos. The percentages and numbers of embryos in each class are shown.

 

Figure 3
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  		Fig. 3. Noggin and chordin promote Nodal expression around the node, the level of which correlates with Nodal expression in left LPM. (A-D) Ventral views of 4s mouse embryos hybridized with Nodal probe. The node area is boxed. The approximate level of Nodal expression around the node is judged as normal (++), weak (+) or undetectable (-). Arrowheads indicate Nodal expression in LPM. (A) Wild-type embryo. (B-D) Chrd-/-;Nog-/- (DKO) embryos showing variable levels of perinodal Nodal expression. (E) Summary of perinodal Nodal expression in non-mutant control (wild type, Chrd-/-;Nog+/+ and Chrd-/-;Nog+/-) and Chrd-/-;Nog-/- embryos. Numbers above bars indicate embryos of a given result among the total assayed. (F) Summary of the relationship between Nodal expression around the node and in LPM of Chrd-/-;Nog-/- embryos. (G,H) Wild-type (G) and Chrd-/-;Nog-/- (H) nodes at 1s stained for acetylated tubulin, marking cilia. Dashed lines indicate node boundaries. Confocal sections along planes G-a, -b and H-a, -b are shown alongside. Red arrowheads, example cilia. (I) Node cilia density at 1s, comparing wild-type (n=3) and Chrd-/-;Nog-/- (n=3) embryos. *, significant difference (mean <0.05).

 

Figure 4
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  		Fig. 4. Endogenous asymmetric BMP antagonism by noggin and chordin regulates Nodal expression in LPM. (A,B) Wild-type (A) and Chrd-/-;Nog-/- (B) mouse embryos subjected to immunohistochemistry for p-Smad1/5/8 (p-Smad1). Arrowhead indicates p-Smad1 in LPM. n>3 embryos per genotype assayed at 5s. (C-L) Cultured embryos assayed for Nodal and GFP expression by two-color WMISH. Red staining shows GFP expression, purple staining Nodal expression. (C) Wild-type embryo showing the normal level (++) of perinodal Nodal expression (boxed). (D) Chrd-/-;Nog-/- embryo injected with GFP vector alone into the left LPM, showing GFP expression (red arrows) but no Nodal expression in LPM. Weak Nodal expression (+) occurs in the node (boxed). (E) Chrd-/-;Nog-/- embryo injected with both GFP and Nog vectors into left LPM. Nodal expression in the left LPM (arrowhead) occurs in the vicinity of GFP (red arrows). (F) Summary of rescued Nodal expression in left LPM. (a) Graph of Nodal expression in left LPM of manipulated Chrd-/-;Nog-/- embryos. Numbers above bars indicate embryos of a given result among total embryos assayed. (b) Subdivision of results as a function of Nodal expression around the node [normal (++), weak (+) or absent (-)], comparing Chrd-/-;Nog-/- embryos injected with GFP vector alone (left column) versus embryos injected with both GFP and Nog vectors (right column). Orange indicates presence of Nodal expression in left LPM, gray indicates its absence (value in each bar is percentage of embryos with absent or present left LPM Nodal expression). (G) Wild-type embryo injected with GFP vector into left LPM. Arrowheads indicate Nodal expression in LPM, red arrows indicate GFP. (H) Wild-type embryo injected with GFP and Bmp4 vectors into left LPM. (I) Wild-type embryo injected with GFP vector into right LPM. (J) Wild-type embryo injected with GFP and Nog vectors into right LPM. (K) Summary of Nodal expression in embryos injected in left LPM with GFP alone or Bmp4 plus GFP. (L) Summary of Nodal expression in embryos injected in right LPM with GFP alone or Nog plus GFP.

 

Figure 5
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  		Fig. 5. Genetic reduction of chordin, noggin and Nodal causes molecular laterality defects that are rescued by decreasing Bmp4 dosage. (A-K) Ventral views of 5s mouse embryos hybridized with Nodal probe. The node region is boxed. Arrowheads indicate Nodal expression in LPM. (A) Wild type, (B) Nodal+/-, (C) Chrd-/-, (D,E) Chrd-/-;Nodal+/-, (F) Chrd-/-;Nog+/-, (G,H) Chrd-/-;Nog+/-;Nodal+/-, (I) Chrd-/-;Nog+/-;Bmp4+/- and (J,K) Chrd-/-;Nog+/-;Nodal+/-;Bmp4+/-. At the bottom of each panel is a summary of the Nodal expression in the left LPM and the level of perinodal Nodal expression [normal (++) or weak (+)]. Orange indicates the presence of Nodal expression in left LPM, gray its absence.

 

Figure 6
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  		Fig. 6. Nodal induces elevated noggin expression in left LPM. (A-D) Ventral views of lacZ staining in 5s Nog-/- (A,C) and Chrd-/-;Nog-/- (DKO) (B,D) mouse embryos. C and D are high magnification views of the boxed regions from A and B, respectively, showing the left and right LPM regions. (E-H) Cultured embryos at 5s hybridized with Lefty2 (E,F) or Nog (G,H) probe. Embryos were cultured with DMSO (E,G) or with the Nodal signaling inhibitor SB-431542 in DMSO (F,H). White lines surround left LPM in G and H. (I,J) Cultured 5s embryos hybridized with Lefty2 (purple) and GFP (red) probes. (I) Control embryo with GFP vector introduced into right LPM. (J) Embryo with GFP and Nodal vectors introduced into right LPM. Red arrows indicate expression of GFP in LPM, arrowheads that of Lefty2. (K,L) Cultured 5s embryos hybridized with Nog (purple) and GFP (red) probes. (K) Control embryo with GFP vector introduced into right LPM. (L) Embryo with GFP and Nodal vectors introduced into right LPM. Gray line surrounds right LPM. Red arrows indicate expression of GFP in LPM, arrowheads that of Nog.

 

Figure 7
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  		Fig. 7. Three-step model for BMP antagonism by noggin and chordin in promoting Nodal expression during left-right axis establishment in mouse. (A) Step 1, from presomitic to 1s. Nog and Chrd promote Nodal expression around the node. Leftward flow is created by cilia rotation. (B) Step 2, 2-3s. Nodal expression is higher on the left than right side of the node, but both left and right LPM receive some level of Nodal-dependent signals. Bilateral BMP signals in LPM repress Nodal signaling and its positive-feedback loop. Stronger Nodal signals to left LPM induce increased BMP antagonist expression. (C) Step 3, 4-5s. Increased Nog and Chrd expression in left LPM elevates left-side-specific BMP antagonism, inhibiting the repressive action of BMP on Nodal expression. As a result, a robust Nodal positive-feedback loop is established in left LPM.

 

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© The Company of Biologists Ltd 2008