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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.

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