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BMP signaling positively regulates Nodal expression during left right specification in the chick embryo

M. Elisa Piedra and Mana A. Ros*

Departamento de Anatomía y Biología Celular, Facultad de Medicina, Universidad de Cantabria, 39011 Santander, Spain



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  		Fig. 1. BMPs positively regulates Nodal expression. (A) Normal pattern of Nodal expression in a stage 8 embryo. (B) Schematic drawing delineating the experiment of bead placement (red circle) into the stage 7-8 lateral plate mesoderm (LPM). Only the placement on the right is depicted, but for each experiment left and right placements were made. (C) A BMP2-soaked bead induces ectopic expression of Nodal in the right LPM. (D) Detail of ectopic right-side expression of Nodal in another embryo with the same treatment. (E,F) Transverse sections of the embryos shown in C,D, respectively, at the level indicated. (G) A BMP2-soaked bead in the left LPM expands the normal domain of Nodal expression. (H) BMP2 maintains Nodal expression, as indicated by the arrow, when the normal domain of expression has moved posteriorly. Treatment with BMP4 (I) and BMP7 (J) had the same effect as treatment with BMP2. (K,L) Repression of Nodal expression after the application of a BMP2-soaked bead (1 mg/ml, K; 0.1 mg/ml, L) at earlier stages (4-6 HH). (M,N) Shh expression is downregulated (arrows) by BMP2 (1 mg/ml, M; 0.1 mg/ml, N) application at earlier stages (4-6 HH). The red asterisk marks the position of the bead in the sections. The bead of the embryo in I was dislodged during hybridization.

 


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  		Fig. 2. BMPs positively regulates Pitx2 expression. (A) Ectopic Pitx2 expression in the right LPM after BMP treatment. (B) A BMP-soaked bead applied to the left LPM (arrows) enhances Pitx2 expression. (C) A transverse section through the embryo shown in A at the level of ectopic Pitx2 expression. The red asterisk marks the bead (arrow) (D) A section through the same embryo at a level of normal Pitx2 expression. Note that the ectopic activation of Pitx2 by BMP also affects the neural tube. (E) A BMP-treated embryo 19 hours after the treatment, showing a right-looped heart and maintenance of ectopic right sided Pitx2 expression. . All the pictures are dorsal views.

 


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  		Fig. 3. Noggin interferes with Nodal and Pitx2 expression. (A) A Noggin-bead that has almost completely abolished Nodal expression in the LPM. (B) Another example in which Noggin had a partial effect on Nodal expression. (C) When the Noggin bead is applied at stage 7-8 HH, the effect is minimal. (D,E) Blocking of BMP signaling by Noggin in the right LPM, either by using recombinant protein (D) or an aggregate of Noggin-expressing cells (E), has no effect on Nodal expression. The insert in E shows the high level of Noggin expression by the cellular aggregate. (F) Applying Noggin to the left LPM before activation of Pitx2 expression prevents its normal expression. (G,H) Noggin applied after Pitx2 activation of expression represses Pitx2 expression around the bead (G) or in a more ample domain (H), as marked by the arrows. (I) A PBS-loaded bead has no effect on Pitx2 expression. (J) Noggin does not modify Pitx2 expression when applied on the right.

 


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  		Fig. 4. Car and Snail expression are positively regulated by BMP signaling. (A) A stage 7 embryo hybridized for Car. (B) The normal domain of expression of Car is expanded after ectopic BMP signaling to the left LPM. (C) Exogenous BMP signaling in the right LPM results in ectopic Car expression. (D) A Noggin-bead that has clearly inhibited the normal expression of Car, compare with the stage-matched control in A. (E) Application of Noggin at later stages only minimally downregulated Car expression (arrows). (F) Normal expression of Car in a stage-matched for comparison. (G) A control stage 8 embryo hybridized for Snail. (H) Exogenous BMP signaling in the left LPM results in ectopic activation of Snail. (I) BMP application on the right does not modify or upregulate the pattern of Snail expression. (J) Inhibition of Snail expression by Noggin, indicated by the arrows. (K) A pellet of Noggin-expressing cells applied to the right that completely abolished Snail expression.

 


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  		Fig. 5. BMP positively regulates Cfc expression. (A) A control stage 8 embryo hybridized for Cfc. Exogenous BMP signaling applied to the right (B) or to the left (C) enhances Cfc expression. Note that the ectopic expression is stronger in the somites. (D) Noggin application (here shown on the left) inhibits the normal expression of Cfc. (E) A transverse section through the embryo shown in B at the indicated level (arrows indicate the medial boundary of each side of Cfc expression). (F) A transverse section through the embryo shown in D at the indicated level. The red asterisk marks the position of the bead in the sections.

 


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  		Fig. 6. Exogenous BMP alters normal gene expression in the midline. (A) A PBS-bead in the left LPM does not perturb normal Lefty1 expression. BMP application, either to the left (B) or right (C) of the LPM represses Lefty1 (delimited by the red arrows). (D,E) Transverse sections through the embryo shown in B at the indicated levels. (F) A PBS-soaked bead in the left LPM does not perturb normal Bra expression. Treatment with BMP either to the left (G) or right (H) dramatically decreases Bra expression in the region facing the bead (delimited by the red arrows). (I,J) Transverse sections through the embryo shown in H at the levels indicated. (K) A PBS-soaked bead in the left LPM does not perturb normal Shh expression. (L,M) Loss of Shh expression in the midline (area between the red arrows) after BMP treatment either to the left (L) or to the right (M). The beads of the embryos in (G,L) were dislodged during hybridization. Red asterisks indicate the bead positions.

 


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  		Fig. 7. Barrier and removal experiments. (A) Schematic drawings illustrate the use of a barrier immediately to the right (top) or left (bottom) of the neural tube in a stage 7 embryo. (B) Picture of an embryo immediately after the operation. (C) Normal Nodal expression after this experimental manipulation. (D) Ectopic Nodal expression induced by a BMP-soaked bead on the right after the placement of the barrier. (E) A double hybridization for Nodal (blue) and Lefty1 (yellow) shows ectopic Nodal expression on the right induced by the BMP-bead but Lefty1 appears unaffected. (F) Schematic drawings illustrate the removal of the left LPM. (G) Stage 6 embryo immediately after the removal of the left LPM. (H) Nodal expression in an embryo 5 hours after the operation. (I) An embryo subjected to removal of the left LPM at stage 6 and implanted with a BMP-soaked bead on the right hybridized for Nodal and Lefty1.

 


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  		Fig. 8. Role of BMP signals on Nodal expression in the LPM. Asymmetric Shh in the left side of Hensen’s node induces the medial perinodal domain of Nodal expression. BMP endogenous signaling in the LPM induces Cfc expression making the cells competent to receive Nodal signaling. Nodal is presumed to diffuse from its medial domain of expression up to the area of competent cells where it activates its own expression. For details and references, see text.

 

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