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Wnt signaling and PKA control Nodal expression and left-right determination in the chick embryo

Concepción Rodríguez-Esteban^*, Javier Capdevila^*, Yasuhiko Kawakami and Juan Carlos Izpisúa Belmonte

The Salk Institute for Biological Studies, Gene Expression Laboratory, 10010 North Torrey Pines Road, La Jolla, CA 92037, USA
^* Equal first authors
Author for correspondence (e-mail belmonte{at}salk.edu )

View larger version (109K): [in a new window]   Fig. 1. Wnt-8c signaling regulates heart looping in the chick embryo. (A) Wnt-8c expression in a HH stage 5 chick embryo. Transcripts are observed throughout the entire length of the primitive streak and symmetrically in the presumptive mesoderm cells lateral to the streak. (B) Detail of the same embryo, where Wnt-8c expression in the node is restricted to the right side (indicated by top arrow). (C) At HH stages 5+ to 6, Nodal becomes restricted laterally and anteriorly to the left side of Hensen's node. (D) A close-up of the Hensen's node area of the embryo in C, showing left-specific expression of Nodal mRNA in the node (arrow), a pattern complementary to that of Wnt-8c in the node (compare B with D). (E) At the same stage, ß-catenin transcripts appear to be symmetrically expressed (arrows). (F) Scheme showing the implantation of Wnt-8c cell pellets to the right side of Hensen's node of a HH stage 4 chick embryo. The syringe indicates the area to the left of the node where the Axin adenovirus is injected at the same stage. (G,H) 24-36 hours after implantation of Wnt-8c cells to the right side of the node, specimens were processed for scanning electron microscopy. Normal heart looping (G) is altered after implantation of Wnt-8c cells. Like right-sided ectopic expression of Nodal (Levin et al., 1995; Levin et al., 1997), right-sided Wnt-8c cell implants induce bilaterally symmetric hearts (not shown) or reversed heart looping (H). (I) Injection of an adenovirus expressing the Axin protein to the left of the node of a HH stage 4 chick embryo (see F) also results in reversed heart looping (I). White semicircular arrows in (G-I) indicate direction of heart looping. In this and the following figures, WT indicates wild-type or normal embryos, which are the controls mentioned in the Materials and Methods section.

View larger version (99K): [in a new window]   Fig. 2. Wnt-8c signaling regulates Nodal expression. (A) Scheme showing the implantation of Wnt-8c cell pellets to the right side of Hensen's node of a HH stage 4 chick embryo. (B) Shh expression on the left side (arrow) of the node in a HH stage 6 wild-type chick embryo. (C) Shh expression was not altered after implantation of Wnt-8c cell pellets to the right side of the node. (D,E) Wnt-8c cells to the right side of the node do not alter the asymmetric expression pattern of Fgf-8 on the node (arrows point to stronger expression on the right side). (F,G) Expression of Caronte (Car; arrows), is not altered after implantation of Wnt-8c cells to the right side of the node. (H-M) However, expression of Nodal (H,I), cSnR (J,K) and Pitx2 (L,M) is altered by grafting Wnt-8c cells to the right side of Hensen's node (red arrows in I,M indicate ectopic expression in the right LPM of Nodal and Pitx2, respectively, and red arrow in K indicates repression of cSnR expression in the right LPM, where it is normally strongly expressed, as shown in J). (N) Injection of an adenovirus expressing Axin to the left side of Hensen's node of a HH stage 4 chick embryo. Both Nodal (O) and Pitx2 (P) are strongly repressed in their normal domains (red arrows). Conversely, the left domain of cSnR, which is very weak outside the somites in normal embryos (see J), is strongly activated (red arrow in Q; compare with the normal strong expression of cSnR in the right side of the embryo, indicated by the black arrow).

View larger version (61K): [in a new window]   Fig. 3. Involvement of Shh, Wnt-8c and N-cadherin in the control of Nodal expression. (A) Blocking of Shh signaling with a bead (in red) soaked in anti-Shh antibody implanted on the left side of the node at HH stage 4. (B) This treatment abolishes normal Nodal expression in the left LPM (Pagán-Westphal et al., 1998; red arrow). (C,D) Nodal expression in the left LPM is maintained (black arrow in D) if Wnt-8c cells are implanted together with the bead soaked in the anti-Shh antibody (as shown in C). (E) Strategy to block N-cadherin activity with a bead (in green) soaked in anti-N-cadherin antibody (Garcia-Castro et al., 2000) implanted on the right side of the node at HH stage 4. (F) This results in ectopic activation of Nodal in the right side of the embryo (red arrow; compare with normal domain indicated by the black arrow).

View larger version (82K): [in a new window]   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).

View larger version (22K): [in a new window]   Fig. 5. A model for the role of Wnts and PKA in LR determination in the chick embryo. At the time at which Nodal becomes restricted to the left side of Hensen's node (HH stage 5+), Wnt/ß-catenin and PKA act as positive regulators of Nodal transcription through Shh-independent mechanisms. Activation (or maintenance) of Nodal on the right side of the node might be prevented by at least two mechanisms: first, the presence of N-cadherin on the right side, which could inhibit activation of Nodal transcription by ß-catenin; second, the presence of high levels of PKI, which could interfere with the activation of Nodal by PKA. Expression of N-cadherin and PKI is biased towards the right side of the node at this stage (indicated in brown); expression of Shh, Nodal and lefty-1 is left-specific (indicated in blue). At this stage, several Wnts that are known to signal through ß-catenin are expressed in or around the node, and thus in this model we consider a sum of Wnt activities mediated by ß-catenin. In the mouse, Wnt-8c is expressed in a pattern very similar to that of its chick counterpart, but its role in LR development has not been described yet. Ectopic expression of Wnt-8c in a transgenic line induces an ectopic embryonic axis and causes a truncation of the anterior neuroectoderm (Popperl et al., 1997), but effects of more localized expression of Wnt-8c had not been analyzed. Also, mice deficient in ß-catenin have been shown to display severe defects of the anterior-posterior axis (Huelsken et al., 2000), which prevents an analysis of possible defects in LR determination. This is a simplified model; only the factors mentioned in the text are indicated.