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First published online 28 November 2007
doi: 10.1242/dev.004713

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Calcium fluxes in dorsal forerunner cells antagonize β-catenin and alter left-right patterning

¹ Department of Biological Sciences, University of Iowa, Iowa City, IA 52242, USA.
² Department of Anatomy and Cell Biology, University of Iowa, Iowa City, IA 52242, USA.

View larger version (41K): [in this window] [in a new window]   Fig. 1. Ca2+ manipulation impacts organ laterality. (A-C) Lateral view, dorsal to the right of 70-80% epiboly stage embryos with arrows indicating the DFC region in (A) bright field image, (B) an individual frame of Fura-2 (380) fluorescence and (C) the corresponding ratiometric image (340/380). The ratio image is converted to pseudo-color with yellow indicating high free intracellular Ca2+. (D) Summary plot of thapsigargin exposure vs heart jogging defects. (E) Summary of heart jogging defects in wt, control and after 10 and 20 minutes of thapsigargin treatment at 60% epiboly. Lateral view of wt (F) and thapsigargin-treated (G) embryos. (H) Summary of organ laterality defects. Thap, thapsigargin.

View larger version (61K): [in this window] [in a new window]   Fig. 2. Thapsigargin treatment disrupts organ laterality and molecular asymmetry. (A-C) Dorsal view, 30 hpf, showing nkx2.5 expression in the heart tube, denoting a left jog in wt (A), and no jog (B) or right jog (C) in thapsigargin-treated embryos. (D-F) Dorsal view, 48 hpf, white arrows indicate foxA3 gut expression in wt (D) and symmetrical (E) or reverse loop (F) in thapsigargin-treated embryos. (G-I) Dorsal view, 19-23 somites, arrows indicate left-sided spaw expression in (G) wt, and (H) bilateral or (I) right-sided expression in thapsigargin-treated. Dorsal view, 19-23 somites, arrows indicate LPM, dashed arrows indicate brain lefty1 expression on the left side in (J) wt, and (K) bilateral in thapsigargin-treated, arrowheads denote lefty1 expression in the midline. Lateral view of ntl expression in (L) wt and (M) thapsigargin-treated, and shh expression in (N) wt and (O) thapsigargin-treated embryos. (P) Summary of asymmetrical marker expression.

View larger version (79K): [in this window] [in a new window]   Fig. 3. The DFCs are specified and migrate to the tailbud in thapsigargin-treated embryos. sox17 expression at 80% epiboly in (A) wt and (B) thapsigargin-treated embryos. Enriched Ntl protein at 80% epiboly in the DFCs in (C) wt and (D) thapsigargin-treated embryos; arrowheads note the DFC region. Expression of sox17 at 4-5 somites in (E) wt and (F) XeC-treated embryos; asterisks indicate dispersed cells, arrows indicate DFC/KV region. Tg (Dusp6:d2EGFP) expression at 80% epiboly in (G) untreated and (H) thapsigargin-treated embryos; arrows indicate DFCs. EGFP expression at tailbud to 1-somite in (I) untreated and (J) thapsigargin-treated embryos and at 5 somites in (K) untreated and (L) thapsigargin-treated embryos. XeC, Xestospongin C; n, notochord.

View larger version (77K): [in this window] [in a new window]   Fig. 4. DFC regional Ca2+ is required for KV morphogenesis. (A) Summary of KV defects. (B-D) Bright field image of wt embryo with normal KV (B) and thapsigargin-treated with reduced (C) and absent (D) KV: dorsal view, 10 somites. Arrows indicate vesicle location; n, notochord. (E-G) charon expression (arrows) around KV in wt (E) and thapsigargin-treated embryos with reduced (F) and absent (G) signal: lateral view, 10 somites. Insets show dorsal view of charon expression. Thap, thapsigargin. Scale bars: 50 µm.

View larger version (79K): [in this window] [in a new window]   Fig. 5. Ca2+ inhibition activates β-catenin and β-catenin is sufficient to alter laterality. Confocal images of β-catenin immunolocalization at 70-80% epiboly in (A) wt and (B) thapsigargin-treated embryos. HRP staining of β-catenin protein oriented on the DFC region in (C) wt, (D) thapsigargin-treated and (E) DFC-targeted Axin-1 MO-injected embryos; arrowheads indicate β-catenin-positive nuclei. (F) Comparison of total nuclear β-catenin in wt and thapsigargin-treated embryos.* indicates P-value of 2.5x10-5. (G) TopFlash (top) vs. FopFlash (fop) luciferase reporter constructs analyzed for relative luminescence in wt or thapsigargin-treated embryos, normalized to control (Renilla) luciferase. Data are means ± s.e. (H,I) Combined lefty1 and lefty2 expression (arrows) in the left LPM and brain in wt (H) and bilateral (I) in DFC-targeted β-catenin RNA-injected embryos: dorsal-anterior view, 19-23 somites.

View larger version (79K): [in this window] [in a new window]   Fig. 6. Xenopus DFC-like cells and Ca2+ requirement for laterality. Xenopus embryos incubated with Syto-11 at stage 11.5 and sagittally bisected at (A) stage 14 and (B) stage 17 showing Syto-11-labeled cells enriched in the future tail region. (C) Stage 17 archenteron roof explant, showing Syto-11 staining of the node (arrow) and surrounding LPM. (D) Explant in C immunostained for acetylated tubulin (acTub), which is enriched in the node region (arrow). (E) Ventral view of stage 46 control-treated embryo, showing situs solitus, or normal lateral asymmetry. The heart is outlined, showing the left-sided position of the ventricle and right-sided outflow tract. The gut coils counterclockwise, as shown by the arrow. (F) Ventral view of a thapsigargin-treated embryo at stage 46, showing an example of situs inversus, or reversed laterality. The heart is outlined, showing the right-sided position of the ventricle and left-sided outflow tract. The gut coils clockwise, as shown by the arrow. (G) Left-sided xnr1 expression at stage 22-24 in a DMSO-treated embryo. (H) Bilateral xnr1 expression in a thapsigargin-treated embryo. (I,J) Lateral view of β-catenin protein localization in control-treated (I) and thapsigargin-treated (J) stage 13 embryos. Note increased number and intensity of nuclear staining. (K,L) Lateral views of control (K) and thapsigargin-treated (L) embryos, showing normal axial development. (M) Summary of Xenopus laterality data. Thap, thapsigargin.