Fig. 4. Alteration of PKA activity induces changes in heart looping that are
preceded by changes in both Nodal and Pitx2 expression.
(A-E) A bead (red in A) soaked in the PKA activator Forskolin applied to the
right side of the node of HH stage 4 chick embryos, results in ectopic
induction of Nodal (D, compare with B) and Pitx2 (E, compare
with C) in the right LPM, indicated by red arrows (black arrows indicate
normal domains). (G) Treated embryos show reversals of heart looping (compare
with normal heart in F). (H,I) Application of a bead (green in A) soaked in
the PKA inhibitor H89 to the left side of the node of HH stage 4 chick
embryos, represses Nodal (H) and Pitx2 (I), indicated by red
arrows. (J) H89-treated embryos also develop reversals of heart looping
(compare with normal heart in F). Although we applied beads to specific
locations, as indicated, these chemicals most likely reach both sides of the
embryo. (K,L) The gene encoding PKI, an inhibitor of PKA activity, is
expressed symmetrically in Hensen's node (arrows in K) and the adjacent
portion of the primitive streak at HH stage 4. At HH stages 5-6, PKI
expression becomes much stronger on the right side of the node (arrow in L).
At these stages, PKI transcripts appear to be excluded from cells expressing
Nodal mRNA. (M,N) At HH stage 4, Nodal transcripts are
present throughout most of the primitive streak, but they are clearly absent
from the streak adjacent to the node and from the node itself (indicated by
the arrow in M), both regions where PKI is transcribed at the same stage
(compare with K). Later on, at HH stage 6, Nodal transcripts are
detected on the left side of the node (arrow in N), but they are absent from
the right side of the node, where PKI is strongly expressed at the same stage
(compare with L). We have overexpressed chick PKI in the early embryo using a
retroviral vector (data not shown), but so far we have failed to detect any
alteration of LR development. This may be due to inefficient production of PKI
protein by the retroviral construct, a phenomenon we have previously observed
when trying to retrovirally express small proteins (the chick PKI protein
comprises only 76 amino acid residues).
