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Evidence for a role of protein kinase C in FGF signal transduction in the developing chick limb bud

Hui-Chen Lu1,2, Eric C. Swindell3,4, Walter D. Sierralta4, Gregor Eichele4,* and Christina Thaller3


1 Developmental Biology Program, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA
2 Division of Neuroscience, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA
3 V. and M. McLean Department of Biochemistry, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA
4 Max Planck Institute of Experimental Endocrinology, Feodor Lynen Strasse 7, 30625 Hannover, Germany



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  		Fig. 1. Expression profile of RACK1 during limb development and its induction by ectopic FGF. (A) At stage 13, RACK1 was expressed uniformly in the lateral plate. (B) By stage 17 the expression of RACK1 was augmented in the limb-forming region but was low in the interlimb flank. (C,D) By stage 17, RACK1 protein detected by immunostaining (brown deposit) was high in the wing bud (C) but was low in the interlimb region (D). (E) RACK1 was expressed in the limb buds of a stage 18 embryo. (F,G) RACK1 mRNA (F) and protein (G) in sections through a stage 22 wing bud. (H) Ectopic expression of RACK1 (white arrows) induced by FGF released from a bead (red arrow) was observed after 12 hours of treatment. (I,J) Two different embryos showing strong ectopic expression of RACK1 (black arrows) upon FGF4 treatment for 24 hours. (K) A plain heparin bead (red arrow) did not induce RACK1. AER, apical ectodermal ridge; dm, dermomyotome; ec, ectoderm; fl, forelimb; hl, hindlimb; if, interlimb flank; lp, lateral plate; m, mesenchyme.

 


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  		Fig. 2. Subcellular localization of RACK1 and the autophosphorylated forms of PKC{alpha}/PKCßII. (A,B) RACK1 (A) and PKC{alpha}/PKCßII (B) in wing bud mesenchymal cells of a stage 16/17 embryo. (C,D) PKC{alpha}/PKCßII in a mesenchymal cell from an ectopic nascent bud that developed from lateral plate treated with FGF4 (C) and in a cell on the contralateral side of the same embryo (D). In A-C, black arrows point at molecules at the nuclear pore; these areas are enlarged in the inserts. Arrowheads in C,D indicate clusters of gold particles. White arrows point to the nuclear envelope. C, cytoplasm; N, nucleus.

 


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  		Fig. 3. The effects of PKC inhibitors on PKC activation and limb development. (A) Western blot (top panel) and quantification (lower panel) of activated PKC{alpha}/PKCßII in primary limb bud cell cultures treated with PKC inhibitors. 1, medium only; 2, 100 nM Go 6976; 3, 1 µM Go 6976; 4, 13 µM chelerythrine chloride; 5, 130 µM chelerythrine chloride; 6, 30 µM sphingosine; 7, 300 µM sphingosine. In all cases, except for the low dose treatment with chelerythrine chloride, PKC inhibitors were effective in reducing PKC{alpha}/PKCßII autophosphorylation. Quantification of PKC{alpha}/PKCßII autophosphorylation was achieved by densitometry. Percent activity was normalized to PKCß detected by a pan PKCß antibody. Lane 1 (no inhibitor added) was defined as 100% activity. (B) Treatment of the presumptive wing bud with chelerythrine chloride produced a completely truncated wing. The scapula (s) was shortened and the clavicle (cl) had a small protrusion (arrow). (C) Contralateral wings were always normal showing humerus (h), radius (r), ulna (u) and digits 2, 3, and 4.

 


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  		Fig. 4. Expression of various limb patterning genes in chelerythrine chloride-treated wing buds. Embryos treated with chelerythrine chloride were analyzed for gene expression at the stage indicated. Green asterisks mark the slow release beads and yellow arrows point to the normal expression in untreated buds. (A,B) Shh transcripts were never detected in the treated bud. (C) Fgf4 expression in the AER of the treated wing bud (red arrow) has lost its posterior bias. (D) At stage 18, Fgf10 expression was not markedly different between the treated (red arrow) and the contralateral wing bud. (E) At stage 19, Fgf10 expression in the treated wing (red arrow) was downregulated. (F) By stage 24, Fgf10 mRNA was not detectable in the treated bud. In the embryo shown in G, the level of Fgf8 expression in the AER of the treated wing bud is reduced. By contrast in H, Fgf8 expression in the AER was not affected. (I) Msx-1 expression (red arrow). (J) The size of Rel/NF-{kappa}B expression domain (red arrow) was reduced in the treated wing bud.

 


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  		Fig. 5. Rescue of chelerythrine chloride-treated limb buds by RA, a ZPA or SHH (A) Ectopic Shh expression in chelerythrine chloride treated wing buds. A yellow arrow indicates normal posterior Shh expression in the left wing bud. A red arrow indicates ectopic Shh expression induced by a now buried RA bead implant indicated by a green asterisk. (B) An example of the typical skeletal pattern of 10-day-old RA-rescued wing. A complete hand plate is produced, but reversed in polarity, owing to the placement of the RA bead at the anterior side of the wing bud. (C) Placement of chelerythrine chloride beads (cc) and a SHH bead in the wing bud. (D) Skeletal pattern of 10-day-old ZPA-rescued wing. Polarity is reversed due to placement of ZPA at anterior of wing bud. (E,F) Skeletal patterns of 10-day-old SHH-rescued wings. In E, polarity is reversed, owing to placement of SHH bead at anterior side of the wing bud. 2, digit 2; 3, digit 3; 4, digit 4; d, digit; f, forearm element; h, humerus; r, radius; s, scapula; u, ulna.

 

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