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First published online 28 August 2008
doi: 10.1242/dev.018861

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A ryanodine receptor-dependent Ca_i²⁺ asymmetry at Hensen's node mediates avian lateral identity

¹ Department of Nutritional Sciences, University of Wisconsin-Madison, Madison, WI 53706, USA.
² Department of Food Science and Human Nutrition, Michigan State University, East Lansing, MI 48824, USA.

View larger version (76K): [in this window] [in a new window]   Fig. 1. High magnification imaging of Cai2+ in early embryos. Ratiometric images of Fura-2-loaded HH4 or HH5 embryos. (A-C) Bright-field images at HH4 (A), HH4+ (B) and HH5- (C). (D-F) Fura-2 ratiometric imaging of embryos depicted in A-C. (Bottom row) Line-scan quantitation of the Fura-2 signal in embryos depicted in D-F plotted against right-left axis, at the position indicated by the white line in D-F. A-F are ventral views with anterior at the top; right (R) and left (L) are as indicated in A. (A,D) At HH4, a modest and symmetric Cai2+ enrichment appears at the anterior margin of the node. (B,E) At HH4+, Cai2+ enrichment expands posteriorly. (C,F) At HH5-, the prechordal plate splits the Fura-2 signal into distinct left and right Cai2+ fields; the left Cai2+ is more posterior and is enriched relative to the right. The blue spot in F is an artifact. *, Hensen's node; pc, prechordal plate; ps, primitive streak. Scale bar in A,D: 100 µm.

View larger version (76K): [in this window] [in a new window]   Fig. 2. Regulation of LR Cai2+ levels along the node. Fura-2-loaded embryos were treated with the indicated pharmacological agent or left untreated. The upper panels show a representative Fura-2 image; lower images are the line-scan quantitation of fluorescent intensity versus right-left axis for that embryo, at the position indicated by the yellow line in the upper panel. Images are ventral views with anterior at the top; right (R) and left (L) are as indicated in A; asterisks indicate Hensen's node. (A,B) Untreated embryos at HH5 (A) and HH6 (B). (C) HH6 embryo treated with the intracellular Cai2+ chelator Bapta-AM. (D) HH6 embryo treated with EGTA. (E) HH6 embryo treated with the IP3 antagonist xestospongin C. (F) HH6 embryo treated with the phospholipase C antagonist U73122. (G) HH6 embryo treated with the RyR antagonist 9,21-dehydroryanodine. (H) HH6 embryo treated with the calmodulin antagonist calmidizolium. Some treatments had inconsistent, minor effects on morphology but did not affect the Fura-2 measurements.

View larger version (23K): [in this window] [in a new window]   Fig. 3. Heart laterality in embryos treated with Cai2+ antagonists. The indicated agents were implanted to the left or right of Hensen's node at HH3++/4; heart laterality was scored 20 hours later. The percentage of embryos with a right-looped (R, situs solitus) or left-looped (L, situs inversus) heart is shown; the number of embryos per treatment is indicated in parentheses. Treatments were: DMSO carrier solvent, Bapta-AM (30 mM), ionomycin (1 mM), U73122 (10 mM), EGTA (5 mM), ryanodine (2.5 mM), ryanodine (2.5 mM) plus EGTA (5 mM), and calmidizolium (5 µM). Asterisks indicate situs inversus frequencies that significantly differ from controls by 2 analysis (P<0.05).

View larger version (72K): [in this window] [in a new window]   Fig. 4. Situs inversus induced by antagonists of Cai2+ asymmetry. Shown are ventral views of embryos treated at HH3++/HH5 with calcium antagonists and analyzed 20 hours later. (A) DMSO control at 15 somites; (B) 30 mM Bapta-AM applied to the left, 14 somites; (C) 1 mM ionomycin applied to the right, 14 somites; (D) 1 µM calmidizolium applied to left, 13 somites; (E) 2.5 mM ryanodine plus 5 mM EGTA applied to left, 12 somites. Heart shape is outlined for clarity.

View larger version (60K): [in this window] [in a new window]   Fig. 5. Nodal expression after treatment with antagonists of Cai2+ asymmetry. Embryos received the indicated agent on the left or the right at HH3++/HH4 and Nodal expression was evaluated at HH8 or HH9- by using in situ hybridization. Images are dorsal views with anterior to the top, left (L) and right (R) are as indicated in A; arrows indicate expression. (A,B) Normal, left-sided Nodal expression in a DMSO-treated embryo having 3 somites (A) or 5 somites (B). (C) Bilateral Nodal expression in a 3-somite embryo that received left-sided Bapta-AM. (D) Normal Nodal expression in 4-somite embryo receiving right-sided Bapta-AM. (E) Loss of lateral Nodal expression in a 3-somite embryo receiving right-sided ionomycin (arrow); a faint signal is seen at the left node (arrowhead). (F) Six-somite embryo receiving left-sided ryanodine plus EGTA; Nodal expression is absent apart from a small amount to the left of the tailbud (arrowhead); compare with the 5-somite control embryo in B. (G) Loss of Nodal expression in a 4-somite embryo that received left-sided calmidizolium; note Nodal expression in the tailbud region (arrowhead).

View larger version (141K): [in this window] [in a new window]   Fig. 6. RyR expression in early chick. (A-F) Antibodies directed against all three RyR isoforms. Symmetric signal is detected around Hensen's node at HH4 (arrows; B,D). RyR signal expands into the primitive streak at HH5 (left arrow, E) and neural plate by HH6 (arrows, F). Signal was absent from embryos reacted with irrelevant antibody (A,C). Images are dorsal views with anterior at the top.

View larger version (46K): [in this window] [in a new window]   Fig. 7. Effect of serotonin agonists and antagonists upon LR Cai2+ levels. Fura-2-loaded HH6 embryos were treated with serotoneric agents and imaged ventrally. The upper panel shows a representative Fura-2 image; the lower panel is the line-scan quantitation of fluorescent intensity versus left-right axis for that embryo, at the position indicated by the yellow line in the upper panel. (A) Untreated embryo. (B) The serotonin reuptake inhibitor fluoxetine reduced LR Cai2+. (C) The 5-HT3 receptor agonist 2-methyl-5-HT reduced LR Cai2+. (D) The 5-HT3 receptor antagonist ondansetron elevated right-side Cai2+. (E) The 5-HT4 receptor agonist ML10302 reduced LR Cai2+. (F) The 5-HT4 receptor antagonist GR125487 elevated right-sided Cai2+. Asterisk indicates Hensen's node; right (R) and left (L) are as indicated in A.

View larger version (14K): [in this window] [in a new window]   Fig. 8. Heart laterality after treatment with serotonin agonists or antagonists. The indicated agents were implanted to the left or right of Hensen's node at HH4-HH6; heart laterality was scored 20 hours later. The percentage of embryos with right-looped (R, situs solitus) or left-looped (L, situs inversus) hearts is shown; the number of embryos per group is indicated in parentheses. Treatments were: DMSO, serotonin reuptake inhibitor fluoxetine (100 µM), 5-HT3 receptor agonist 2-CH3-5HT (5 mM), 5-HT3 receptor antagonist ondansetron (5 mM), 5-HT4 receptor agonist ML10302 (5 mM) and the 5-HT4 receptor antagonist GR125487 (5 mM). Asterisk indicates situs inversus frequencies that significantly differ from DMSO by 2 analysis (P<0.05).

View larger version (38K): [in this window] [in a new window]   Fig. 9. Cai2+ levels and heart laterality after treatment with proton ATPase antagonists. (A-C) Fura-2-loaded HH6 embryos were treated with the indicated agents and imaged ventrally. The upper panel shows a representative Fura-2 image; the lower panel is the line-scan quantitation of fluorescent intensity versus left-right axis for that embryo, at the position indicated by the yellow line in the upper panel. Hensen's node is indicated by an asterisk. (A) Untreated embryo. (B) The H+V-ATPase antagonist concanamycin reduces LR Cai2+. (C) The H+K+-ATPase antagonist lansoprazole elevates right Cai2+. (D) Embryos were treated with the indicated proton ATPase antagonists at HH4-HH6 and heart laterality was scored 20 hours later. The percentage of embryos with a right-looped (R, situs solitus) or left-looped (L, situs inversus) heart is shown; the number of embryos per treatment is indicated in parentheses. Treatments were: DMSO, concanamycin (100 µM) and lansoprazole (7 mM). Asterisk indicates situs inversus frequencies that significantly differ from controls by 2 analysis (P<0.05).

View larger version (72K): [in this window] [in a new window]   Fig. 10. LR Cai2+ levels in VAS or VAD quail embryos. Fura-2-loaded HH6 embryos were imaged ventrally, anterior to the top. (A,B) The upper panel shows a representative Fura-2 image; the lower panel presents the line-scan quantitation of fluorescent intensity versus the right-left axis for that embryo, at the position indicated by the yellow line in the upper panel. Asterisk indicates Hensen's node. (A) VAS, vitamin A sufficient. (B) VAD, vitamin A deficient.

View larger version (21K): [in this window] [in a new window]   Fig. 11. Model of Cai2+ action in laterality. (A) At HH3++/HH4-elevated Cai2+ (green) appears along the anterior Hensen's node (HN), overlapping with RyR expression; its appearance requires RyR and proton ATPases. A transient depolarization is also observed to the left of Hensen's node (HN) and the primitive streak (PS). Gap junctions (blue dots) may relay and stabilize these early signals. (B) Left-side Cai2+ enrichment occurs at HH4+/HH5-, and the head process (HP) bifurcates the Cai2+ field. Shh (dark blue) becomes left-side restricted in the node. Midline expression of serotonin, 5-HT3, 5-HT4 and H+K+ATPase reduces Cai2+ and makes a barrier (light blue) for autonomous LR regulation of Cai2+. (C) By HH6, left Cai2+ is substantially enriched. Factors including RA, RyR, extracellular calcium and the H+V-ATPase sustain left Cai2+ elevation and Nodal induction, whereas serotonin effectors and the H+K+-ATPase suppress right Cai2+. Extracellular calcium activates Notch and supports CICR RyR. See text for details.

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