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BMP2 is a positive regulator of Nodal signaling during left-right axis formation in the chicken embryo

Cell and Molecular Biology, Technical University Braunschweig, Germany

View larger version (144K): [in a new window]   Fig. 1. Expression of Nodal and Pitx2 in the left lateral plate mesoderm is dependent on BMP. Ventral views of stained embryos at HH stage 7 (A-C), or HH stage 8 (D-P). (H) A transverse section through the embryos shown in G. The plane of sectioning is indicated. Normal control embryos in A-E were hybridized with a probe for Nodal. The number of somites is given in the bottom right-hand corner in A-E. All other embryos were placed in New culture and implanted with BMP2-expressing cells (F,G,L,M), control cells (I,K,N,P), or with Noggin-expressing cells (J,O). Cell aggregates were placed in the lateral plate mesoderm at HH stages 4 (F), 5 (G,I-L,N-P) or 6 (M). The embryos were cultured until four somites had formed and subjected to whole-mount in situ hybridization with probes for Nodal (A-K) and Pitx2 (L-P). Green arrowheads indicate the tongue-like expression domain of Nodal that connect the paraxial and LPM expression domains in D,E. Red arrowheads indicate ectopic or loss-of expression as a result of the manipulation. Asterisk marks the implanted cell aggregate.

View larger version (146K): [in a new window]   Fig. 2. Expression Nkx3.2, ActrIIa and Snr is dependent on BMP signaling. Ventral views of stained embryos at HH stage 11 (A,B), HH stage 7 (E,F) or HH stage 8 (G-J). (C,D) Transverse section through the embryos shown in A,B. The plane of sectioning is indicated. Embryos were placed in New culture and implanted with BMP2-expressing cells (A,E,G,I) or with Noggin-expressing cells (B,F,H,J) in the left (A,B,E,F,I,J), or right (G,H) LPM at HH stage 5. The embryos were subjected to whole-mount in situ hybridization with probes for Nkx3.2 (A,B), ActrII2a (E,F), or Snr (G-J). Red arrows indicate ectopic or loss-of expression as a result of the manipulation. Asterisk marks the implanted cell aggregate.

View larger version (86K): [in a new window]   Fig. 3. Analysis of expression domains of determinants of left-right asymmetry. Expression domains of Nodal, CFC, BMP2 and Car were analyzed by double color whole-mount in situ hybridization of HH stage 7 chick embryos. Expression of (A) Nodal (blue) and Cfc (red), (B) Nodal (blue) and Bmp2 (red), (C) Nodal (blue) and Car (red), and (D) BMP2 (blue) and Car (red) are shown. (E-H) Transverse sections through the embryos shown in A-D as indicated.

View larger version (120K): [in a new window]   Fig. 4. Car and BMP2 synergize to upregulate Pitx2 in mesoderm. Ventral views of chicken embryos implanted with cell aggregates expressing BMP2 (A,C-E), Nodal (F,G), Car (H-K,M), and BMP2 and Car (N). Cell implants were placed in left and right LPM (A-D), left LPM (E,K,M) or right LPM (F-J,N). Embryos were cultured in New culture and subsequently subjected to whole-mount in situ hybridization with probes for Nodal (C,F,H,I,) Pitx2 (D,G,J,K,N), Cfc (A,M) or human BMP2 (E). (B,L,O) Transverse sections through the embryos shown in (A,K,N). Insets in H,I show cell implants expressing Car. Red arrowheads indicate ectopic expression as a result of the manipulation. Asterisk marks the implanted cell aggregates.

View larger version (117K): [in a new window]   Fig. 5. BMP signaling interferes with a functional midline barrier. Ventral views of stained embryos at HH stage 7 (A) or HH stage 8 (B,E-H). (C,D) Transverse sections through the embryos shown (A,B). Plane of sectioning is indicated in the individual panels. Chicken embryos are shown that were implanted with BMP2-expressing cells (A,B,E,G) or control cells (F,H) adjacent to the midline on the left side and stained for Nodal (A), Nodal and Lefty (B), Lefty (E,F), or Shh (G,H). Red arrowheads indicate ectopic expression (A,B) or loss-of expression (E-H). Asterisk marks the implanted cell aggregates.

View larger version (14K): [in a new window]   Fig. 6. The proposed interactions among different signaling molecules involved in the lateralization of the left LPM in the chick. Unilateral Shh induces left-sided expression domains of Nodal and Car adjacent to Hensen’s node (Levin et al., 1995; Rodriguez-Esteban et al., 2001). Car probably allows the spread of Nodal to the lateral plate. BMP2, which is present in mesendoderm at stage 4 and in pharyngeal endoderm beginning at HH stage 5 (Andrée et al., 1998) maintains the expression of Cfc and ActrIIa. Both genes provide competence to respond to the incoming Nodal signal. Nodal upregulates Pitx2 and Nkx3.2 (Liu et al., 2001; Logan et al., 1998; Schneider et al., 1999). Expression of Pitx2 at least in mice is maintained by the homeobox gene Nkx2.5 (Shiratori et al., 2001). Expression of Nkx2.5 in the mesoderm is dependent on BMP2 signals and thus, BMP2 ochestrates competence for long-range Nodal signals and maintenance of Pitx2 expression. Expression of Lefty in the midline is dependent on CFC (Schlange et al., 2001) and Shh (Tsukui et al., 1999). Ectopic BMP interferes with Lefty expression probably by downregulating Shh expression. The main function of Lefty is probably to prevent Nodal signals from spreading to the right side. On the right side, FGF8 controls the expression of Snr (Boettger et al., 1999). The proposed function of Snr is to prevent expression of Nodal and Pitx2 on the right side (Patel et al., 1999).