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The role of an endogenous PKA inhibitor, PKI, in organizing left-right axis formation

Minoru Kawakami^* and Nobuki Nakanishi

The Burnham Institute, Center for Neuroscience and Aging, La Jolla, CA 92037, USA
Harvard Medical School, Department of Neurobiology, Boston, MA 02115, USA
^* Present address: The Queen’s Medical Center, Honolulu, HI 96813, USA

View larger version (70K): [in a new window]   Fig. 1. Expression of PKI in chick embryos. Whole-mount in situ hybridization with a PKI probe. (A,C,E,G) Dorsal views of chick embryos from stages 4 to 8. (B,D,F,H) Close-ups of Hensen’s node. (I,J) Stage 10+ and stage 16 embryos. At stage 4 (A,B), expression is uniform on both sides of the node and primitive streak. At stage 6 (C,D), expression becomes stronger in the right side of the node. The right-handed expression at the node continues through stage 7+ (E,F). Black and white arrowheads indicate the areas where PKI signal is asymmetric. PKI seems symmetrically expressed in the head process, head fold and emerging somites at these stages. At stage 8 (G,H), expression at the node becomes symmetric. At stage 10+ (I) and 16 (J), PKI is expressed at the notochord, somites, heart (h) and branchial arch (b).

View larger version (104K): [in a new window]   Fig. 2. Treatments with PKI antisense oligonucleotides interfere with the proper formation of L-R axis. S, sense; AS, antisense. (A,B) Examples of heart situs (indicated by red arrowheads) in embryos treated with S (A) and AS (B) oligonucleotides. Ventral views are shown. AS treatment caused reversed heart looping in some embryos. (C,D) Examples of Shh expression in embryos treated with S (C) and AS (D) oligonucleotides. Dorsal views are shown. AS treatment did not change the asymmetric expression of Shh. (E,F,I,J) Examples of Nodal expression in embryos treated with S (E,I) and AS (F,J) oligonucleotides. Dorsal views (E,F) and coronal sections (I,J) are shown. AS treatment induced bilateral expression of Nodal. (G,H,K,L) Examples of Pitx2 expression in embryos treated with S (G,K) and AS (H,L) oligonucleotides. Ventral views (G,H) and coronal sections (K,L) are shown. AS treatment induced bilateral expression of Pitx2. (M,N) Examples of PKI expression in embryos treated with S (M) and AS (N) oligonucleotides. Dorsal views are shown. AS treatment suppressed the expression of PKI, while S treatment did not affect PKI mRNA expression. Black arrowheads point to areas of high expression, and white arrowheads point to areas of low expression.

View larger version (93K): [in a new window]   Fig. 3. Treatment with PKI antisense oligonucleotides suppressed right sided expression of SnR. Examples of SnR expression in embryos treated at stage 4 with sense (A) and antisense (B) oligonucleotides. Dorsal views at stage 9 are shown. Note that SnR signal is symmetrical within somites. Suppression of SnR signal by AS treatment is seen most prominently in the posterior area of lateral mesoderm. Black arrowheads point to areas of high expression and white arrowheads point to areas of low expression.

View larger version (26K): [in a new window]   Fig. 4. Sequence specificity of antisense oligonucleotides. Percentages of embryos exhibiting bilateral expression of Nodal following treatments with oligonucleotides. Values below treatments represent the number of embryos with bilateral Nodal expression/ the number of embryos treated with oligonucleotides. The nucleotide sequences of oligonucleotides are shown in inset. Mismatches with chick PKI are underlined. AS, antisense; S, sense.

View larger version (12K): [in a new window]   Fig. 5. Effects of PKA activators on Nodal expression and heart looping. (A) Percentages of embryos exhibiting bilateral Nodal expression after drug treatments. (B) Percentages of embryos exhibiting heart reversal after drug treatments.

View larger version (39K): [in a new window]   Fig. 6. PKI is regulated by ectopic activin, but not by Shh. Whole-mount in situ hybridization with PKI probe after beads implantation adjacent to the node. Beads were placed in embryos at stage 4 at the location indicated by red arrowheads, and in situ hybridization was performed at stages 6-7. In one set of experiments, embryos received a buffer-soaked bead (A) or an activin-soaked bead (B) at the left side of the node. An activin-soaked bead induced the expression of PKI mRNA on the left side of the node. In the second set of experiments, embryos received beads soaked in anti-Shh antibody (C), and a bead soaked in Shh protein (D). Black arrowheads point to areas of high expression, and white arrows point to areas of low expression.

View larger version (16K): [in a new window]   Fig. 7. The role of PKI in signal transduction cascades determining L-R asymmetry. See text for explanation.

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