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First published online 4 August 2004
doi: 10.1242/dev.01269

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Chick Pcl2 regulates the left-right asymmetry by repressing Shh expression in Hensen's node

¹ Division of Developmental Biology, Department of Cell and Molecular Biology and Center for Bioenvironmental Research, Tulane University, New Orleans, LA 70118, USA
² College of Bioengineering, Fujian Normal University, Fuzhou, Fujian Province, 350007, P. R. China

View larger version (9K): [in a new window]   Fig. 1. The conserved domains and position on the phylogenetic tree of chick Pcl2 protein. (A) Chick Pcl2 encodes a putative 595 amino acid residue protein that contains one Tudor domain (AA:45-100) and two PHD finger domains (AA:105-147; AA:204-238). (B) Phylogenetic tree generated from known Pcl proteins from Drosophila, Xenopus, chick, mouse and human using Clustal method with PAM250 residue weight table.

View larger version (107K): [in a new window]   Fig. 2. Expression and regulation of chick Pcl2 in Hensen's node. (A-D) Whole-mount in situ hybridization analyses reveal chick Pcl2 expression in early chick embryos at stages 4 (A), 5 (B), 6 (C) and 8 (D). Asymmetric chick Pcl2 expression in the node appears from stage 5, and is restricted in the ectoderm (insert in C). (E-H) Ectopic chick Pcl2 expression is induced by Activin-A (E) and BMP4 (G) around the node, while Follistatin represses chick Pcl2 expression (F). BSA control bead does not alter chick Pcl2 expression (H). All embryos are dorsal views. Arrows indicate the node; asterisks indicate bead or bead position.

View larger version (71K): [in a new window]   Fig. 3. Overexpression chick Pcl2 represses Shh expression in the node and alters Shh downstream genes in the LPM. (A,B) A stage 7 chick embryo shows Gfp expression in the node and its derivatives after pMES-Gfp expression vector was targeted to Hensen's node at stage 4. (C,E,G,I) Control embryos transfected with pMES-Gfp vectors show unaltered expression of Shh (C) in the node and notochord, and of Nodal (E) and Pitx2 (G) in the left LPM, and of Lefty1 (I) in the prospective floor plate. (D,F,H,J) Embryos overexpressing chick Pcl2 show downregulation of Shh (D) in the node and notochord, of Nodal (F) and Pitx2 (H) in the left LPM, and of Lefty1 (J) in the prospective floor plate. (K,L) SnR is ectopically activated in the left PLM of embryo overexpressing chick Pcl2 (L), but not in the control embryo (K). Arrows in A-D indicate the node; arrows in E-H,K,L indicate the left LPM, and arrows in I,J indicate the midline. Images are all dorsal views.

View larger version (86K): [in a new window]   Fig. 4. Ectopic expression of chick Pcl2 suppresses Shh expression in the ZPA of the chick and mouse limb buds and in the chick feather buds. (A) Chick Pcl2 transcripts are detected in the medial mesenchyme of a stage 18 chick wing bud. The anteroposterior orientation is indicated. (B) A stage 22 chick embryo shows complete repression of Shh expression in the ZPA (red arrow) of the right wing infected with RCAS-Pcl2 and normal Shh expression in the control wing (black arrow). (C,D) Chick Pcl2 expression is detected in the mesenchyme of a feather bud from a stage 31 chick embryo (C), while Shh expression is restricted to the epithelium of a feather bud from the same stage (D), exhibiting a complementary expression pattern to that of chick Pcl2. (E) Shh expression is inhibited in feather buds (arrows) infected with RCAS-chick Pcl2. There is normal Shh expression (arrowheads) in adjacent uninfected feather buds. (F,G) Chick Pcl2 transgene expression (arrow) is detected in a hindlimb of an E10.5 transgenic embryo (G), but not in a wild-type limb (F). (H,I) Transgenic expression of chick Pcl2 in the mesenchyme of mouse developing hindlimb suppresses Shh expression in the ZPA (arrow in I), when compared with a control hindlimb from E10.5 nontransgenic littermate (H). Scale bars: 100 µm in A; and 50 µm in C,D.

View larger version (140K): [in a new window]   Fig. 5. Knock-down of chick Pcl2 randomizes cardiac looping direction and alters gene expression. (A-C) Ventral view of stage 11 embryo treated with control oligonucleotide shows normal rightwards heart looping (A), while embryo treated with antisense oligonucleotide specific to chick Pcl2, which abolished chick Pcl2 expression as detected by whole-mount in situ hybridization (C), exhibits reversed cardiac looping direction (B). (D,E) Fgf8 expression is unaltered (arrows) in embryos treated with control oligonucleotide (E), but is repressed in the right side of the node (arrows) of embryos treated with antisense oligonucleotide to chick Pcl2 (D). (F-M) Embryos treated with control oligonucleotide show normal expression of Shh (F) in the node, of Caronte (H), Nodal (J) and Pitx2 (L) in the left LPM. By contrast, embryos treated with antisense oligonucleotide show ectopic expression of Shh in the right side (arrow) of the node (G), and ectopic expression of Caronte (I), Nodal (K) and Pitx2 (M) in the left LPM (arrows). Embryos in C-M are shown dorsal views.

View larger version (34K): [in a new window]   Fig. 6. Mapping of repression domain of chick Pcl2 and inhibition of the mouse Shh promoter activities by chick Pcl2. (A) P19 cells were transfected with the reporter plasmid, pG5tkCAT, and the constructs indicated, and CAT (chloramphenicol acetyltransferase) activities were assayed. The N-domain of chick Pcl2, which covers the PHD fingers, represses about a third of the promoter activities, while the PHD domain suppresses the promoter activities to a half. C-terminal domain of chick Pcl2 exhibits similar effect to the controls on the promoter activities. (B) The 3 kb mouse Shh upstream region drives the expression of the CAT reporter gene in P19 cells (lane 1). The promoter activities are suppressed by co-expression of chick Pcl2 (lane 2) but not the PHD fingers (lane 3). Lanes 4-6 show the TK promoter activities that are not suppressed by co-expression of either chick Pcl2 (lane 5) or the PHD domain (lane 6), when compared with controls (lane 4). Data shown here represent at least three independent experiments.

View larger version (22K): [in a new window]   Fig. 7. Interaction of the PHD domain of chick Pcl2 with EZH2. 293T cells were transfected with expression vectors expressing the FLAG-tagged chick Pcl2, the FLAG-tagged PHD fingers (AA103-238) and the Myc-tagged mouse EZH2. Cell extracts were immunoprecipitated and blotted with antibodies against FLAG or Myc, reciprocally. Lane 1, negative control; lane 2, positive control for Myc pull-down and blot (arrow indicates the blotted band); lanes 3 and 4, positive control for FLAG pull-down and blot; lane 5, pull-down and blot of chick Pcl2-FLAG and EZH2-Myc reciprocally; lane 6, pull-down and blot of PHD-FLAG and EZH2-Myc reciprocally. Data shown are representatives of at least three independent experiments.

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