DEV018986 Supplementary Material

Files in this Data Supplement:

• Supplemental Table S1 -

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• Supplemental Table S2 -

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• Supplemental Figure S1 -

  Fig. S1. Distribution of phosphorylated Smad1/5/8, Bmp4, Nog, Chrd and Nodal in LPM. (A-E) Five-somite-stage (5s) wild-type embryos sectioned at the same level. (A) Immunohistochemistry for phosphorylated Smad1/5/8 (p-Smad1). (B) lacZ staining of Bmp4^lacZ/+ embryo. (C) lacZ staining of Nog^lacZ/+ embryo. (D) In situ hybridization for Chrd. (E) In situ hybridization for Nodal. Left (b) and right (a) LPM (boxed areas) are shown at high magnification to right. Dashed red lines indicate LPM region.

• Supplemental Figure S2 -

  Fig. S2. Expression of L-R axis-related genes. Whole-mount in situ hybridization for (A,B) Pitx2, (C,D) Lefty1 and Lefty2 and (E,F) Cryptic in (A,C,E) wild type (WT) or (B,D,F) Chrd;Nog double mutant (DKO) at (A,B) 8s or (C-F) 4-5s. The number of embryos analyzed is indicated at bottom right.

• Supplemental Figure S3 -

  Fig. S3. Reduced Notch signaling in Chrd;Nog double mutant. (A,B) Immunohistochemistry for phosphorylated Smad1 (p-Smad1) in (A) wild type and (B) Chrd;Nog double mutant (DKO). (C-H) Whole-mount in situ hybridization for Lfng in (C,E,G) wild type and (D,F,H) Chrd;Nog DKO. (C,D) 5s stage. Dashed red circle encircles node. Number of embryos analyzed is shown at bottom right. (I-L) In situ hybridization for (I,J) Nodal and (K,L) Lfng in (I,K) BSA-treated control and (J,L) BMP-treated cultured explants. Red arrows indicate Nodal or Lfng expression in each explant. (M) Quantitative RT-PCR analysis for Nodal, Lfng, Dll1, Notch1 and Rbpjk. Nodal, Lfng and Dll1 expression levels were reduced by BMP treatment, whereas levels of Notch1 and Rbpjk were not. *, significant difference (mean value<0.05).

• Supplemental Figure S4 -

  Fig. S4. Procedure for manipulating BMP signaling in lateral plate mesoderm.

• Supplemental Figure S5 -

  Fig. S5. Nodal expression in LPM of Nog-null mutant. Whole-mount in situ hybridization for Nodal. (A) Wild-type embryo (n=10). (B) Nog-null embryo (n=5). Ventral views. Arrowhead indicates Nodal expression in left LPM.

• Supplemental Figure S6 -

  Fig. S6. Nog expression in left LPM correlates with spatiotemporal Nodal expression pattern. (A-D) Whole-mount in situ hybridization for Nodal in wild-type embryos at (A) 2s (n=3), (B) 3s (n=5), (C) 4s (n=8) and (D) 5s (n=10). Left lateral views. Arrowhead indicates starting Nodal expression point in left LPM at 3s. Red arrow indicates expansion direction of Nodal expression in left LPM. (E-J) Whole-mount lacZ staining of Nog^lacZ/+ at (E) 2s (n=4), (F) 3s (n=5) and (G) 4s (n=6). The boxed regions in E-G are shown at high magnification in H-J, respectively. (K-M′) High-magnification pictures of left LPM from E-G at (K) 2s, (L) 3s and (M) 4s. Dashed line indicates the adjacent part to node. Arrowhead indicates Nog expression in left LPM. (K′-M′) Diagrams of spatiotemporal Nog expression pattern in left LPM from 2s to 4s.

• Supplemental Figure S7 -

  Fig. S7. Effects of perturbing Nodal activity on expression of Chrd and Nog in LPM. (A,B) Experimental strategy for quantitative RT-PCR analysis. (A) Steps of the experiment. (B) Method of microdissection of cultured embryos. LPM pieces were isolated along the dashed red lines. (C,D) Comparison of fold expression of Nodal (C) and of Chrd and Nog (D) in left LPM. DMSO, DMSO-treated control; SB, SB431542-treated LPM. Each analysis was normalized to β-actin expression. Fold expression was calculated by normalizing right side expression to 1.00. *, significant difference (mean <0.05).