Fig. 1. Regulation of chick Cer and Nodal expression by Nodal
and Shh signaling pathways. (A-H) Beads soaked in
phosphate-buffered saline (PBS, control; A,C,E,G), Nodal protein (B,D,F) or
Shh protein (H) were implanted on the right side at Hamburger and Hamilton
stage 7 (HH7; A-F) or HH4 (G,H). Chick embryos were fixed after 1 hour (A,B),
2 hours (C,D), 4 hours (E,F) or 5 hours (G,H) and processed for single-label
[chick Cer (cCer); A,B] or double-label (cCer and
Nodal; C-H) whole-mount in situ hybridization (cCer, purple;
Nodal, orange). Nodal protein induced the right-side expression of
cCer transcripts in less than 1 hour (B; arrow), and Nodal
transcripts in approximately 2 hours (D; arrows). After 4 hours, both
cCer and Nodal expression levels were higher on the right
than on the left side (F; arrows). On the other hand, Shh protein took
approximately 5 hours to induce the transcription of both cCer and
Nodal (H; arrow). (I-L) cCer transcripts detected by
whole-mount in situ hybridization.
(I',J',K',L') Merge of bright-field
and RFP red fluorescence images. (I-J') Effect of the Nodal antagonist
Xenopus CerS (XCerS) on cCer expression. (J,J') Chick
embryos were electroporated with pCAGGS-XCerS on the left side of the
node at HH4 (i.e. in the cells that express Nodal).
pCAGGS-RFP was electroporated alone (control; I,I') or with
pCAGGS-XCerS (J,J'), and used to label the populations of electroporated
cells. In contrast to the control electroporation (I; arrowhead), the
inhibition of Nodal by XCerS suppressed the left-sided expression of
cCer (J; arrowhead). (K-L') Effect of the Nodal antagonist
XCerS on Shh-induced cCer expression. HH4 chick embryos were
electroporated with pCAGGS-RFP alone (control; K,K') or
co-electroporated with pCAGGS-RFP and pCAGGS-XCerS
(L,L'), and grafted on the right side with beads soaked in Shh protein.
Ectopic induction of cCer expression by Shh (K; arrow) was suppressed
by the Nodal inhibitor XCerS (L; arrow). All embryos are viewed from the
ventral side. cCer, chick Cer; RFP, red fluorescent protein;
XCerS, Xenopus CerS.