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

Development 135, 3271-3280 (2008)
Published by The Company of Biologists 2008
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A ryanodine receptor-dependent Cai2+ asymmetry at Hensen's node mediates avian lateral identity

Ana Garic-Stankovic1, Marcos Hernandez1, George R. Flentke1, Maija H. Zile2 and Susan M. Smith1,*

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.

* Author for correspondence (e-mail: suesmith{at}nutrisci.wisc.edu)

Accepted 31 July 2008


   SUMMARY
TOP
 SUMMARY
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In mouse, the establishment of left-right (LR) asymmetry requires intracellular calcium (Cai2+) enrichment on the left of the node. The use of Cai2+ asymmetry by other vertebrates, and its origins and relationship to other laterality effectors are largely unknown. Additionally, the architecture of Hensen's node raises doubts as to whether Cai2+ asymmetry is a broadly conserved mechanism to achieve laterality. We report here that the avian embryo uses a left-side enriched Cai2+ asymmetry across Hensen's node to govern its lateral identity. Elevated Cai2+ was first detected along the anterior node at early HH4, and its emergence and left-side enrichment by HH5 required both ryanodine receptor (RyR) activity and extracellular calcium, implicating calcium-induced calcium release (CICR) as the novel source of the Cai2+. Targeted manipulation of node Cai2+ randomized heart laterality and affected nodal expression. Bifurcation of the Cai2+ field by the emerging prechordal plate may permit the independent regulation of LR Cai2+ levels. To the left of the node, RyR/CICR and H+V-ATPase activity sustained elevated Cai2+. On the right, Cai2+ levels were actively repressed through the activities of H+K+ ATPase and serotonin-dependent signaling, thus identifying a novel mechanism for the known effects of serotonin on laterality. Vitamin A-deficient quail have a high incidence of situs inversus hearts and had a reversed calcium asymmetry. Thus, Cai2+ asymmetry across the node represents a more broadly conserved mechanism for laterality among amniotes than had been previously believed.

Key words: Chick embryo, Hensen's node, Intracellular calcium, Left-right asymmetry, Proton ATPases, Ratiometric calcium imaging, Retinoic acid, Ryanodine receptors, Serotonin


   INTRODUCTION
TOP
 SUMMARY
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Establishment of the vertebrate left-right (LR) body plan initiates proper positioning of the internal organs. Early differences in small molecules and signals across the midline organizer are translated into an asymmetric genetic cascade that enforces LR identity. Effectors of these early events include cilia motility (Essner et al., 2005Go; McGrath et al., 2003Go; Tabin and Vogan, 2003Go), cell coupling (Levin and Mercola, 1999Go), ion pumps (Levin et al., 2002Go; Adams et al., 2006Go; Shu et al., 2007Go), inositol polyphosphates (Sarmah et al., 2005Go), serotonin (Fukumoto et al., 2005aGo; Fukumoto et al., 2005bGo) and retinoic acid (Chauzad et al., 1999; Tsukui et al., 1999Go; Zile et al., 2000Go). Although genetic effectors, including sonic hedgehog (Shh), Nodal, lefty and Pitx2, are largely conserved across vertebrates, there may be significant differences as to which upstream participants are used. Some differences might reflect evolutionary divergence and structural changes in the embryo. The precise relationships among these participants are also unclear.

One poorly understood early asymmetry signal is calcium. In mouse and zebrafish, intracellular calcium (Cai2+) is enriched along the left margin of the node and Kupffer's vesicle, respectively (McGrath et al., 2003Go; Sarmah et al., 2005Go). In mouse, a loss-of-function mutation in left-right dynein (iv/iv) or polycystin 2 collapses calcium asymmetry at the node and randomizes LR identity (McGrath et al., 2003Go), prompting suggestions that it is cilia-generated flow across the ventral node that generates the calcium asymmetry and subsequent left-sided gene expression. A role for calcium asymmetry in zebrafish is less clear, as Cai2+ fluxes are also linked to KV formation and subsequent laterality establishment (Sarmah et al., 2005Go; Schneider et al., 2008Go).

Whether and how Cai2+ asymmetry across the node might contribute to lateral identity in other vertebrates is unknown. Although the node architecture of the avian embryo might preclude nodal flow (Manner et al., 2001), its node monocilia are positioned toward the ventral endoderm (Essner et al., 2002Go) and it expresses several ciliary-related genes, including left-right dynein, kinesin 3B and polycystin 2 (Qiu et al., 2005Go). Thus, at least some elements of this laterality mechanism might be conserved in chick. Cai2+ levels have not been examined in chick, although, at neurulation, a left-sided external calcium pool affects laterality through the activation of notch signaling (Raya et al., 2004Go).

We report here that the avian embryo uses an asymmetric enrichment of Cai2+ at the node to mediate laterality. This Cai2+ enrichment emerges at early gastrulation and originates from the activity of ryanodine receptors and calcium-induced calcium release (CICR). Several previously identified laterality mediators, specifically serotonin, H+V-ATPase and H+K+ATPase, regulate LR identity by directly affecting node Cai2+ concentrations. Our data significantly advance the understanding of how the avian embryo achieves its LR identity, and show that, despite differences in spatial architecture, Cai2+ asymmetry across the node might be a conserved mechanism of laterality in amniotes.


   MATERIALS AND METHODS
TOP
 SUMMARY
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Embryos
Chick eggs (Hyline W98) were obtained from the UW Poultry Research Laboratory, Madison, WI. Normal and retinoid-deficient Japanese quail eggs were generated as described previously (Zile et al., 2000Go). Embryos were staged according to Hamburger and Hamilton (HH) (Hamburger and Hamilton, 1951Go).

Fura-2 ratiometric imaging
HH3+ to HH6 embryos were incubated (60 minutes, 37°C) in a slide chamber with Tyrode's buffer (TWC) containing 25 µM Fura-2-AM and 0.1% Pluronic F-127 (Molecular Probes), rinsed and then incubated (20 minutes) with one of the following pharmacological agents: Bapta-AM (1 mM), EGTA (1.0-2.5 mM), xestospongin C (1 µM), U73122 (10 µM), 9,21-didehydroryanodine (2 µM), ryanodine isomers (10 µM), dantrolene (2 µM), fluoxetine (10 µM), 2-methyl-5HT (25 µM), ondansetron (25 µM), ML10302 (25 µM), GR125487 (25 µM), concanamycin (100 nM) or lansoprazole (7 µM) (concentrations were determined experimentally). Embryos were rinsed and immediately imaged. Studies of CICR (ryanodine plus EGTA) used calcium-free TWC.


Figure 1
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  		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.

 
Ca2+ imaging used an inverted microscope (Nikon TE2000-U) with a Fluor-S x10 lens, Xenon lamp, and Sutter filter wheel with Fura-2 filters (Chroma Technology, Brattleboro, VT); HH4/HH5 analysis was carried out at a magnification of x20. Using MetaFluor imaging software (Universal Imaging, West Chester, PA), the left and right regions of interest were defined and images collected at 1 second intervals for 3 minutes using a CCD digital camera (Cool Snap-ES, Photometrics, Tucson, AZ). Fluorescent signal emission at 510 nm, following dual excitation at 340 and 380 nm, was calculated for each region and timepoint. Mean response per region and per embryo were calculated from 10 consecutive images. At the end of the assay, ionomycin challenge (0.1 mM) affirmed embryo viability and determined Rmax; non-responding embryos were discarded. Subsequent treatment with 2 M MnCl2 quenched Ca2+ -bound Fura-2 and defined the background fluorescence (Rmin).

Data were analyzed using two approaches. The line-scan function in MetaFluor quantified the mean Fura-2 signal within the left and right side at a fixed distance from the node; results were expressed as the mean Fura-2 signal of six to 12 embryos per treatment. To calculate the fold change in left versus right regions, we used the equation Cai2+=Kdx[(R-Rmin)/(Rmax-R)]x (Fmax380/Fmin380) (Grynkiewicz et al., 1985Go), where the constant terms Kd and Fmax380/Fmin380 were cancelled because a ratio was calculated. Rmin is the emission ratio after MnCl2 treatment, Rmax is the emission ratio after ionomycin treatment, and R represents the emission ratio of experimental interest. Rmin and Rmax were calculated individually for each region. Results were expressed as the mean left-right ratio for five to nine embryos per treatment.

Determination of cardiac laterality
A microbead soaked in the agent of interest was implanted to the left or right of Hensen's node of in ovo HH3++/HH4 embryos: Bapta-AM (30 mM), ionomycin (1 mM), U73122 (10 mM), mixed ryanodine isomers (2.5 mM), EGTA (5 mM), calmidizolium (5 µM), fluoxetine (100 µM), 2-CH3-5HT (5 mM), ondansetron (5 mM), ML10302 (5 mM), GR125487 (5 mM), concanamycin (100 µM), lansoprazole (7 mM) or DMSO only. Ryanodine and EGTA were applied as small agarose plugs. Beads were removed 4 hours later. At HH10/HH11, embryos with significant cranial or midline defects were discarded. The remainder were scored for cardiac laterality as left or right loop, by two treatment-blinded individuals. We analyzed eight to 25 embryos per treatment.

In situ hybridization and immunostaining
cDNA encoding chick Nodal (cNR-1) (Levin et al., 1995Go) was provided by C. Tabin. HH4 embryos were implanted to the left or right of Hensen's node with DMSO, Bapta-AM, ionomycin, ryanodine-EGTA or calmidizolium-soaked microbeads. At HH8, embryos were processed for whole-mount in situ hybridization as described previously (Smith et al., 1997Go). For whole embryo immunostaining, antibody directed against the carboxy-terminus of all three RyR isoforms (C-18, 1/500, Santa Cruz) was reacted with HH3+ to HH6 embryos as described (Smith et al., 1997Go). Signal was visualized using alkaline phosphatase-conjugated secondary antibody (Southern Biotech) and BM-Purple (Roche). Embryos were processed batch-wise to ensure consistent treatment. We analyzed eight to 16 embryos per treatment.

Statistical analysis
Binary data (e.g. heart laterality) were analyzed using {chi}2 analysis (SAS 9.1, SAS Institute, Cary, NC). Normally distributed data were subjected to an unpaired, two-tailed t-test employing the appropriate variance parameter using SigmaStat v.2.0 (Systat Software, Point Richmond, CA). Data not normally distributed were examined using the Mann-Whitney U-test. P<0.05 was set as the level of significance.


   RESULTS
TOP
 SUMMARY
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Cai2+ is enriched to the left of Hensen's node
We used Fura-2-based ratiometric imaging to evaluate Cai2+ concentrations at the chick Hensen's node. At HH3++/HH4, a modest and bilaterally symmetric Cai2+ elevation was first detected along the anterior margin of the node (Fig. 1A,D). A modest left-side enrichment was occasionally observed (Fig. 1D). At HH4+/HH5-, the calcium signal extended posteriorly and the emerging prechordal plate split this signal into bilateral fields, with levels being lower at the midline (Fig. 1B,E). By HH5-, left Cai2+ levels were consistently elevated when compared with the right side (Fig. 1C,F); anterior node levels were reduced. By HH6, the Cai2+ signal extended more posteriorly and it was significantly enriched along the left side (LR ratio 2.85±0.44; Table 1, Fig. 2B). The left Cai2+ field appeared to overlap with a left-sided extracellular calcium enrichment previously described (Raya et al., 2004Go). Treatment with the intracellular Cai2+ chelator Bapta-AM reduced Cai2+ levels throughout, and ablated the left-right differential (Table 1, Fig. 2C), affirming that this signal represented Cai2+.


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  		Table 1. Left-right Cai2+ levels in normal chick embryos and those treated with Cai2+ effectors

 
LR Cai2+ asymmetry contributes to lateral identity
We manipulated Cai2+ levels to the left or right side of Hensen's node and analyzed the effects on asymmetry. An early asymmetry indicator in chick is the right loop of the heart tube at HH10. DMSO treatment had little effect and most embryos had normal, right-looped hearts (Fig. 3, Fig. 4A). A small percentage had midline-positioned heart tubes that failed to loop or fully fuse; their incidence was not treatment dependent and they were excluded from the analysis. Left but not right Bapta-AM treatment at HH4 significantly increased the incidence of left-looped hearts (40% situs inversus; Fig. 3, Fig. 4B). To test whether Cai2+ enrichment alone was sufficient to influence laterality, we applied the potent Cai2+ ionophore ionomycin. Right but not left ionomycin treatment increased the frequency of left-looped hearts (31%; Fig. 3, Fig. 4C).


Figure 2
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  		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.

 
Nodal is an early laterality determination gene that is restricted to the left-side of Hensen's node and the lateral plate mesoderm during early somitogenesis (Levin et al., 1995Go). Although DMSO did not affect Nodal expression (left treatment, 10/10 normal; right treatment, 8/8 normal; Fig. 5A,B), left-sided Bapta-AM treatment at HH4 caused bilateral Nodal expression in the lateral plate mesoderm in 60% of embryos (n=10; Fig. 5C) and normal expression in the remainder. Right-sided Bapta-AM did not affect Nodal (8/8 normal; Fig. 5D). Conversely, right-sided ionomycin reduced Nodal expression (4/6 embryos, 67%; Fig. 5E), but left-sided treatment did not (1/7 lacked Nodal). Both calcium effectors also affected cranial morphology consistent with the known effects of calcium on neural fold elevation. Although we cannot rule out that such cranial changes may have disrupted midline formation and thus affected LR identity (Schneider et al., 2008Go), this seems unlikely because, while left and right treatment equally affected cranial morphology, only left Bapta and right ionomycin significantly affected Nodal, suggesting that the effects on Nodal and the neural folds were separable events. This suggested that Cai2+ acted upstream of Nodal to affect laterality.


Figure 3
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  		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 {chi}2 analysis (P<0.05).

 


Figure 4
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  		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.

 
Cai2+ originates from CICR/RyR activity
Both extracellular (Raya et al., 2004Go) and intracellular calcium (McGrath et al., 2003Go; Sarmah et al., 2005Go; Shu et al., 2007Go) affect LR laterality and could originate the Cai2+ enrichment. The left Cai2+ enrichment overlaps at later stages (HH6) with a left-side enriched extracellular calcium pool that has previously been demonstrated to govern chick laterality, heart looping and Nodal expression (Raya et al., 2004Go). EGTA treatment ablated the Cai2+ asymmetry and attenuated LR Cai2+ levels (Fig. 2D, Table 1), suggesting that extracellular calcium contributed to the Cai2+ enrichment. EGTA also reversed heart looping when applied to the left side (25%, P=0.006) but not the right (0%, Fig. 3).

Intracellular calcium could originate from either phosphoinositide or RyR activity. Phosphoinositides are implicated in zebrafish laterality through roles in Kupffer's vesicle formation (Schneider et al., 2008Go) and ciliary beating (Sarmah et al., 2005Go; Sarmah et al., 2007Go). However, neither xestospongin C, which inhibits IP3-mediated Cai2+ release, nor the phosphoinositidyl-phospholipase C antagonist U73122, affected absolute Cai2+ levels (Fig. 2E,F) or the LR Cai2+ ratio (Table 1), nor did U73122 affect asymmetry (Fig. 3). Thus the Cai2+ did not originate from IP3-mediated sources.

RyRs mediate calcium mobilization from sarco/endoplasmic stores following stimulation by smaller calcium quantities that often originate from extracellular sources, a process known as calcium-induced calcium release (CICR) (Zucchi and Roncha-Testoni, 1997Go). RyRs have not been previously implicated in laterality. Treatment with the RyR antagonist 9,21-dehydroryanodine ablated the LR Cai2+ asymmetry and significantly lowered Cai2+ levels (Fig. 2G, Table 1), as did a ryanodine isomer mixture and a distinct RyR antagonist, dantrolene (Table 1). Cai2+ levels were not as low as those obtained with Bapta-AM, probably because the CICR component of RyR activity was still present. Accordingly, the ryanodine-EGTA combination was more potent than was each separately in reducing Cai2+ (Table 1), implicating CICR as the RyR stimulus. Ryanodine/EGTA also abolished the Cai2+ enrichment in the HH3++/4 node (Table 2), suggesting that CICR/RyR originated this signal. Similarly, left-side ryanodine treatment had little effect on heart looping (12.5%), whereas the ryanodine/EGTA combination significantly reversed heart looping (36%; Fig. 3, Fig. 4E). Left-side ryanodine/EGTA treatment at HH3++/4 repressed Nodal expression in five out of nine embryos (Fig. 5F).


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  		Table 2. Fura-2 levels at the anterior node of HH3++/HH4 embryos treated with Cai2+ effectors

 


Figure 5
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  		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).

 


Figure 6
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  		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.

 
An important Cai2+ effector is the Cai2+ sensor protein calmodulin, which interacts with and activates numerous signaling proteins, including RyRs (Berridge et al., 2000Go; Zalk et al., 2007Go). Treatment with the calmodulin inhibitor calmidizolium significantly reduced Cai2+ levels at both HH3+/4 (Table 2) and HH6 (Table 1), actions that are consistent with the known ability of calmodulin to directly regulate RyR activity (Zalk et al., 2007Go). Left but not right calmidizolium treatment also caused a significant incidence of cardiac situs inversus (36%; Fig. 3, Fig. 4D). Left calmidizolium treatment abolished Nodal expression in 59% of embryos (10/17, Fig. 5G).

Avians, like mammals, have three RyR isoforms with differing expression and regulatory control. Their contributions to early development are unknown. Immunostaining against all RyR isoforms at HH4 revealed low but discernable levels along the anterior node margin (Fig. 6B,D), overlapping the Cai2+ Fura-2 signal. Expression expanded along the primitive streak at HH5 (Fig. 6E) and the neural plate at HH6 (Fig. 6F). Its expression was symmetric, indicating that its asymmetric activity was regulated post-translationally, a mechanism that commonly governs RyR activity (Zucchi and Ronca-Testoni, 1997Go). Thus, RyRs are present at the right time and location to mediate the Cai2+ enrichment.

Serotonin signaling represses right-sided Cai2+
The relationship between node Cai2+ asymmetry and other laterality effectors is unknown. The initial symmetry of the Cai2+ signal, followed by its left-side enrichment, suggested the existence of asymmetric regulators. The laterality effector serotonin affects both Nodal expression and heart looping in chick (Fukumoto et al., 2005aGo; Fukumoto et al., 2005bGo). Treatment at HH3++/4 with the serotonin re-uptake inhibitor fluoxetine, which prolongs serotonin signaling, repressed Cai2+ at the H3++/4 anterior node (Table 2). At HH6, fluoxetine ablated the LR Cai2+ differential (1.38±0.13; Table 3) by reducing left-side Cai2+ levels (Fig. 7B; Table 3), suggesting that serotonin might normally suppress right-side Cai2+. Consistent with this, left but not right fluoxetine treatment significantly randomized heart looping (36%; Fig. 8).


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  		Table 3. Left-right Cai2+ levels in chick embryos treated with serotonin agonists and antagonists, or proton ATPase antagonists

 


Figure 7
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  		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.

 
Serotonin affects laterality through its 5-HT3 and 5-HT4 receptors (Fukumoto et al., 2005bGo). 5-HT3 (2-methyl-5-HT) and 5-HT4 (ML10302) agonists rapidly reduced left-side Cai2+ levels and ablated Cai2+ asymmetry (Fig. 7C,E; Table 3), whereas their respective antagonists (ondansetron, GR125487) elevated right Cai2+ and sustained left Cai2+ levels (Fig. 7D,F; Table 3), indicating that serotonin acted to keep right Cai2+ low. Similarly, right but not left application of 5-HT3 and 5-HT4 antagonists significantly randomized heart-looping direction (ondansetron, 32%; GR125487, 30%), whereas their agonists reversed heart-loop direction only when applied to the left (2-methyl-5HT, 31%; ML10302, 32%; Fig. 8). These findings endorse the model of Fukumoto et al. (Fukumoto et al., 2005aGo; Fukumoto et al., 2005bGo) that serotonin operates on the right side of the chick to affect LR identity. The repression of right-sided Cai2+ by serotonin is a novel mechanism for this laterality effector.

Also implicated in avian laterality are the H+-V-ATPase (Adams et al., 2006Go) and H+K+-ATPase proton pumps (Levin et al., 2002Go; Raya et al., 2004Go); their inhibition randomizes heart laterality and alters asymmetric gene expression. The H+-V-ATPase antagonist concanamycin prevented the node Cai2+ enrichment at HH3++/4 (Table 2) and at HH6 (Table 3; Fig. 9B). Left but not right concanamycin treatment also increased the incidence of cardiac situs inversus (58%; Fig. 9D). Thus, H+V-ATPase may affect laterality through its ability to initiate and sustain node Cai2+ asymmetry.

H+K+-ATPase affects avian laterality by preventing a transient depolarization to the right of Hensen's node (Levin et al., 2002Go); its inhibition flattened the LR asymmetry of extracellular calcium (Raya et al., 2004Go). It affected Cai2+ in a complex manner. At HH4, the H+/K+-ATPase antagonist lansoprazole significantly reduced the node Cai2+ enrichment (Table 2). However, at HH6, its action opposed H+V-ATPase, and it abolished Cai2+ asymmetry (1.03±0.19; Table 3) by elevating right Cai2+ (Fig. 9C), an action consistent with suggestions that it acts upon the right-side of the embryo (Levin et al., 2002Go). Interestingly, right-side inhibition of H+K+-ATPase did not affect heart looping (Fig. 9D), a finding that is at odds with its ability to elevate right Cai2+. Previous laterality studies of H+K+-ATPase used bilateral inhibitor treatment (Levin et al., 2002Go; Raya et al., 2004Go). These complex actions of lansoprazole may indicate shifting roles for the ATPase in establishing asymmetry. Nonetheless, both H+V-ATPase and H+K+-ATPase contribute to laterality through their regulation of node Cai2+.


Figure 8
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  		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 {chi}2 analysis (P<0.05).

 
Retinoic acid, laterality and Cai2+
Vitamin A-deficient (VAD) quail embryos have a marked tendency (72%) to form left-sided hearts (Zile et al., 2000Go); the expression of laterality genes, such as Nodal and Pitx2, is also altered (Zile et al., 2000Go). Vitamin A-sufficient (VAS) HH6 quail had a modest LR Cai2+ enrichment compared with Gallus gallus chicks of equivalent stage (LR ratio 1.44±0.40, Fig. 10A), although the mean Fura-2 signal of the quails did not differ appreciably from that of the chick (right, 168.4±13.7; left, 187.0±14.9; L versus R, P=0.014). Interestingly, HH6 VAD quail had an inverted Cai2+ asymmetry, and, in a majority (5/7), Cai2+ levels were significantly higher on the right of the node versus the left (Fig. 10B): the mean LR Cai2+ ratio for those five embryos was 0.60±0.21. This was not due to increased right Cai2+ but to significantly reduced left Cai2+ levels (right, 164.1±9.9; left, 161.6±17.0; n=7; left Fura-2 of VAD versus VAS, P=0.016). Although unexpected, this inverted LR Cai2+ ratio in the majority of VAD quail embryos is consistent with their high incidence of reversed cardiac laterality (Zile et al., 2000Go).


   DISCUSSION
TOP
 SUMMARY
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Here, we show that, like mouse, the chick embryo uses node Cai2+ asymmetry to mediate laterality. Cai2+ is first enriched along the anterior margin of the node at HH3++/4, and overlaps with RyR expression. Its induction and left-side enrichment requires RyR and extracellular calcium, implicating CICR as a novel origin for this signal. H+K+-ATPase and H+V-ATPase activity are also contributory, although by HH6 their activities are distinct and opposing. 5-HT3 and 5-HT4 keep right-side Cai2+ levels low, representing a novel mechanism for the laterality effects of serotonin. Although the avian node structure may preclude nodal flow, its use of Cai2+ asymmetry supports the hypothesis that aspects of this laterality mechanism are conserved among amniotes.

Figure 11 summarizes our findings and details underlying this model are discussed below. At HH2 and HH3 (not shown), the elongating primitive streak already expresses much of the machinery that governs CICR/RyR activity, including serotonin and its receptors and transporters (Fukumoto et al., 2005aGo; Fukumoto et al., 2005bGo), H+V-ATPase (Adams et al., 2006Go) and H+K+-ATPase (Levin et al., 2002Go); the surrounding blastoderm expresses Cx43 (Levin and Mercola, 1999Go). At HH3+, the first asymmetric gene, cAct-RIIa, appears (Levin et al., 1995Go). At this same time, Cai2+ enrichment emerges along the anterior margin of Hensen's node commensurate with RyR expression; its ablation by EGTA/ryanodine and calmidizolium suggests that it originates from CIRC/RyR. Cai2+ emergence coincides with a transient depolarization of cells along the left side of the node (Levin et al., 2002Go), and could enhance CICR/RyR activity. Left-sided depolarization, the instigator of which is unknown, may thus be a crucial early step to create asymmetry. By HH4+/HH5, Cai2+ levels are asymmetric and enriched along the left side of the node. The previously symmetric Shh expression also becomes restricted to the left side of the node (Levin et al., 1995Go), and, importantly, the sodium-calcium transporter NCX1 is enriched along the right node (Linask et al., 2001Go). This right-side restricted NCX1, which uses Na+ influx to drive Cai2+ export, may steepen further the LR Cai2+ asymmetry. By neurulation (HH6), the Cai2+ asymmetry has increased and extends posteriorly. H+V-ATPase sustains the left-side Cai2+ elevation, whereas H+K+-ATPase keeps right Cai2+ levels low. Although the mechanism underlying Cai2+ repression by serotonin is unclear, the physiological properties of the right side of the node suggest that 5HT3 may function as a Na+K+ exchanger, perhaps in coordination with NCX1 and/or H+K+-ATPase, to maintain low Cai2+. Gap junctions in the blastoderm (Levin and Mercola, 1999Go) could serve to coordinate and stabilize the Cai2+ and ATPase signals across the anterior node. Bifurcation of the anterior Cai2+ field by the prechordal plate, along with the proposed barrier activity of the midline (Danos and Yost, 1996Go), could then allow LR Cai2+ levels to be regulated autonomously.


Figure 9
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  		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 {chi}2 analysis (P<0.05).

 


Figure 10
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  		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.

 
Cai2+ mediates LR asymmetry decisions in chick
Although Cai2+ asymmetry across the node is necessary for laterality in mouse (McGrath et al., 2003Go) and now chick, its origins and relationship to other laterality mediators has been unclear. The identification of CICR/RyR as the origin of Cai2+ enrichment and asymmetry at Hensen's node is a novel function for RyRs in the early embryo. The requirement for CICR/RyR offers a second mechanism for left-side enriched extracellular calcium to govern avian LR identity, in addition to its regulation of Notch signals (Raya et al., 2004Go); any interaction between Cai2+ and Notch remains to be determined. Because RyR proteins are expressed symmetrically along the anterior node and the primitive streak, their activity is likely to be regulated post-translationally. One important mediator is membrane potential and pH, through direct effects upon the protein and indirectly by controlling the activity of calcium exchangers and channels that mediate calcium influx. The complex abilities of the proton ATPases to regulate Cai2+ levels may reflect a possible involvement in Ca2+ release from CICR/RyR-gated stores and this is discussed in greater detail below.

In mouse, movement by monocilia within the ventral node is proposed to generate a fluid current that creates the Cai2+ asymmetry across the node; mutation of left-right dynein heavy chain (Lrd; Dnahc11 - Mouse Genome Informatics) leads to immotile cilia, the collapse of Cai2+ asymmetry and the randomization of lateral identity (McGrath et al., 2003Go). Although the avian node architecture is quite different from that of mouse and it may lack a `ventral node' (Manner, 2001Go), HH4-chick node possesses monocilia that project ventrally from the epiblast and towards the ventral endoderm (Essner et al., 2002Go). Unfortunately, it was not possible to determine which cell layer at the node was Cai2+ enriched. However, the existence of Cai2+ enrichment at the avian node shows that at least some aspects of the mechanisms used to establish LR laterality are conserved among amniotes. In this light, it may be worth noting that polycystin-2, a calcium channel linked to LR identity in mouse and zebrafish (Pennekamp et al., 2002Go; Bisgrove et al., 2005Go; Obara et al., 2006Go; Schottenfeld et al., 2007Go) can initiate CIRC/RyR-mediated calcium release (Nauli et al., 2003Go) in a protonation-dependent manner (Gonzalez-Perrett et al., 2002Go). The contribution of polycystins to avian lateral identity remains unexplored.

Serotonin and proton ion channels mediate Cai2+ asymmetry
How serotonin and the proton ion channels affect lateral identity is unclear. Our data suggest that serotonin operates specifically on the right side of the node to reduce Cai2+ and maintain asymmetry; 5-HT3 and 5-HT4 inhibition rapidly elevated right-side Cai2+ and randomized heart looping. Because serotonin and its 5-HT3 and 5-HT4 receptors are bilaterally expressed at these stages (Fukumoto et al., 2005bGo), there must exist signals that preclude left-sided serotonin action. These could include the serotonin transporters, two of which facilitate serotonin activity and have right-biased expression (Fukumoto et al., 2005aGo), and monoamine oxidase, which is expressed along the right node margin (Fukumoto et al., 2005bGo) and which could create a degradative barrier to prevent LR serotonin communication. Thus, several components of serotonin signaling might be good candidates to reduce right-side calcium levels and steepen the Cai2+ asymmetry.


Figure 11
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  		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.

 
How could serotonin rapidly decrease Cai2+? The 5-HT4 is a G protein-coupled receptor that signals through G{alpha}s and protein kinase A, and thus could regulate diverse elements of calcium homeostasis, including RyR itself (Zucchi and Roncha-Testoni, 1997Go). The 5-HT3 is a relatively non-selective, ligand-gated cation channel that acts either as a Na+K+ exchanger or to stimulate inward calcium movement (Derkach et al., 1989Go; Hargreaves et al., 1994Go); that the 5-HT3 agonist decreased Cai2+ implicates the former mechanism. Insights may come from the cation ATPase contributions to lateral identity (Levin et al., 2002Go; Adams et al., 2006Go; Raya et al., 2004Go; Ellertsdottir et al., 2006Go; Shu et al., 2007Go). In zebrafish, NCX4a and Na,K-ATPase {alpha}2 affect laterality by controlling blastomere Cai2+ levels (Shu et al., 2007Go); morphants had elevated Cai2+, a situation that parallels the avian loss of 5-HT3 and H+K+-ATPase. Because NCX4a and its related transporters are unique to fish, it was unclear whether similar mechanisms governed calcium asymmetry in other vertebrates. Our data suggest that the H+K+-ATPase and Na+K+ exchange activity of 5-HT3 could have analogous roles in chick to keep right Cai2+ low.

The H+K+-ATPase prevents cell depolarization on the right side of the chick embryo (Levin et al., 2002Go). As depolarization would favor calcium influx and stimulate CICR/RyR, this may be a key H+K+-ATPase role following midline establishment. That H+K+-ATPase inhibitors reduce external calcium (Raya et al., 2004Go) and increase Cai2+ is consistent with this mechanism. Conversely, H+V-ATPase activity (Adams et al., 2006Go) was essential to induce and sustain the left-side Cai2+ elevation. How it regulates Cai2+ is unknown. Protonation is a potent inhibitor of both RyR (Zucchi and Roncha-Testoni, 1997Go) and membrane calcium channels, such as polycystin-2 (Gonzalez-Perrett et al., 2002Go), and thus proton export could control CICR/RyR activity directly, or indirectly via voltage-gated calcium channels. Vacuolar ATPases also govern vesicular trafficking and, thus, could regulate membrane calcium channel levels (Jarvis and Zamponi, 2007Go). Taken together, our findings affirm the importance of proton ATPases to LR asymmetry through their effects on Cai2+. Their disparate effects upon Cai2+ induction and maintenance indicate that they serve multiple roles. It is unknown whether their activity might be coupled with that of the Na+K+-ATPases, which, in zebrafish, govern laterality in a calcium-dependent manner (Ellertsdottir et al., 2006Go; Shu et al., 2007Go). Additional studies should clarify these issues.

Retinoic acid and lateral identity
Retinoids mediate LR identity: retinoid receptor antagonists randomize laterality (Tsukui et al., 1999Go), whereas VAD quail frequently have left-sided hearts (~70%) (Zile et al., 2000Go). Retinoids also affect the bilateral symmetry of the somites (Kawakami et al., 2005Go; Sirbu and Duester, 2006Go), and act in parallel with Shh and upstream of Nodal, Lefty1 and Pitx2 to affect left identity (Zile et al., 2000Go; Tsukui et al., 1999Go). However, their precise contributions remain elusive. We found that a majority of VAD quail had reversed LR Cai2+ asymmetry, an outcome consistent with their high frequency of reversed heart laterality. Right Cai2+ levels were not enriched; rather, left Cai2+ enrichment was absent, which suggests that the latter process requires retinoids. An obvious explanation is a transcriptional role for retinoids in the gene(s) that governs Cai2+ elevation, such as the proton ATPases or a serotonergic repressor. Alternatively, retinoids may sustain the midline boundary that enables left and right to function as separate compartments; for example, through induction of the Nodal inhibitor Lefty (Tsukui et al., 1999Go; Tabin, 2006Go). RA also regulates Cxn43 expression (Clairmont and Sies, 1997Go), and thus could affect gap junctions that might transmit and amplify a laterality signal, such as Cai2+ or an ion balance (Levin and Mercola, 1999Go). These mechanisms could be explored in future studies.


   ACKNOWLEDGMENTS
 
Supported by NIH MERIT Award R37 AA11085 to S.M.S. M.H.Z. is supported by NIH award R01 HL61982, USDA NRI 2005-35200-15257 and the Michigan Agricultural Experiment Station. We thank HyLine International for the generous donation of our chickens and John Fallon for helpful discussions.


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 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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