K

K., Peng H. experiments designed to distinguish between these models. The results uniformly support a role for serotonin in the cleavage-stage embryo, long before the appearance of cilia, in ventral right blastomeres that do not contribute to the ciliated organ. INTRODUCTION Understanding how consistent left-right (LR) asymmetry of the body-plan is made is important for developmental biology and medicine. Individuals with LR patterning problems, including heterotaxia (the loss of concordance among the visceral organs), and isomerisms (loss of asymmetry), suffer grave medical effects (Hackett, 2002; Peeters and Devriendt, 2006; Zhu et al., 2006). The frog offers proven to be an excellent model for the study of LR asymmetry because of the vast number of developmental, molecular-genetic, physiological and pharmacological tools that are available for use in this organism. Specific benefits of this model system also include the large number of eggs available for study at the earliest of phases NCH 51 (which allowed the finding of many very early asymmetry-causing mechanisms that have so far been too hard to study in mice), and a well-defined fate-map that allows focusing on of remaining- and right-side cells at will (which is NCH 51 not possible in zebrafish for example). Therefore, the frog embryo is definitely a uniquely powerful model in which to study the earliest events that link biophysical chirality to asymmetric gene manifestation and subsequent organ situs. There are currently two competing paradigms of how the LR axis is made. One model proposes that symmetry is definitely 1st broken during neurulation, when cilia localized to a node-like structure generate a chiral fluid circulation that asymmetrically distributes a morphogen, or causes asymmetric bending of sensory cilia (examined by Basu and Brueckner, 2008; Hashimoto and Hamada, 2010). This model indicates substantial evolutionary divergence among phyla. Because many different model systems (including amniotes such as the chick and pig) orient their asymmetry without the benefit of cilia (Levin and Palmer, 2007; Spder et al., 2007; Gros et al., 2009), it is unclear which varieties is the best model for human being disease-relevant symmetry breaking. The additional model proposes that a highly conserved chiral intracellular cytoskeleton component drives asymmetric localization of ion transporters, therefore creating a biased voltage gradient that results in asymmetric localization of a charged molecule that is instructive for LR identity (examined by Aw and Levin, 2009; Vandenberg and Levin, 2010). This model is definitely supported by molecular-genetic gain- and loss-of-function data that show that asymmetry is made during the 1st cell cleavages (Levin et al., 2002; Aw et al., 2008) and determine the neurotransmitter serotonin as the small molecule that is redistributed at cleavage phases to provide LR identity to blastomeres (Fukumoto et al., 2005a; Fukumoto et al., 2005b; Adams et al., 2006; Carneiro et al., 2011). A recent study (Beyer et al., 2012) suggests a permissive role for serotonin in the specification of the gastrocoel roof plate (GRP; the node) and cilia-dependent fluid flow. Thus, the two main LR asymmetry models now converge on a common molecule, the neurotransmitter serotonin. The question of timing, i.e when serotonin actually functions during LR patterning, is crucial not only because it impacts NCH 51 the plausibility of the late origin versus early origin models of asymmetry but also because it identifies the embryonic stages that would be most sensitive to the serotonergic compounds in common medical use today (Shuey et al., 1992; Alwan et al., 2007; Noorlander et al., 2008; Sadler, 2011). Here, we statement the results of experiments that allowed us to resolve the role of both the timing and the location of serotonin in LR patterning of the frog embryo. These studies were designed to experimentally distinguish between two hypotheses: one that suggests a role for serotonin in right-sided ventral blastomeres during cleavage stages (i.e. the EARLY model) (Levin and Palmer, 2007), and one that requires a role for serotonin late, in the left side (Vick et al., 2009) of the dorsally derived GRP cells (i.e. the LATE model). The results of these experiments support a role for serotonin during early cleavage stages and exclude a role for serotonin signaling in the GRP. TRANSLATIONAL IMPACT Clinical.PLoS ONE 5, e9610. to specify the dorsal region of the embryo made up of chiral cilia that generate asymmetric fluid circulation during neurulation, a much later process. We performed theory-neutral experiments designed to distinguish between these models. The results uniformly support a role for serotonin in the cleavage-stage embryo, long before the appearance of cilia, in ventral right blastomeres that do not contribute to the ciliated organ. INTRODUCTION Understanding how consistent left-right (LR) asymmetry of the body-plan is established is important for developmental biology and medicine. Individuals with LR patterning defects, including heterotaxia (the loss of concordance among the visceral organs), and isomerisms (loss of asymmetry), suffer grave medical effects (Hackett, 2002; Peeters and Devriendt, 2006; Zhu et al., 2006). The frog has proven to be an excellent model for the study of LR asymmetry because of the vast number of developmental, molecular-genetic, physiological and pharmacological tools that are available for use in this organism. Specific benefits of this model system also include the large number of eggs available for study at the earliest of stages (which allowed the discovery of many very early asymmetry-causing mechanisms that have so far been too hard to study in mice), and a well-defined fate-map that allows targeting of left- and right-side cells at will (which is not possible in zebrafish for example). Thus, the frog embryo is usually a uniquely powerful model in which to study the earliest events that link biophysical chirality to asymmetric gene expression and subsequent organ situs. There are currently two competing paradigms of how the LR axis is established. One model proposes that symmetry is usually first broken during neurulation, when cilia localized NCH 51 to a node-like structure generate a chiral fluid circulation that asymmetrically distributes a morphogen, or causes asymmetric bending of sensory cilia (examined by Basu and Brueckner, 2008; Hashimoto and Hamada, 2010). This model implies considerable evolutionary divergence among phyla. Because many different model systems (including amniotes such as the chick and pig) orient their asymmetry without the benefit of cilia (Levin and Palmer, 2007; Spder et al., 2007; Gros et al., 2009), it is unclear which species is the best model for human disease-relevant symmetry breaking. The other model proposes that a highly conserved chiral intracellular cytoskeleton component drives asymmetric localization of ion transporters, thus establishing a biased voltage gradient that results in asymmetric localization of a charged molecule that is instructive for LR identity (examined by Aw and Levin, 2009; Vandenberg and Levin, 2010). This model is usually supported by molecular-genetic gain- and loss-of-function ATV data that show that asymmetry is established during the first cell cleavages (Levin et NCH 51 al., 2002; Aw et al., 2008) and identify the neurotransmitter serotonin as the small molecule that is redistributed at cleavage stages to provide LR identity to blastomeres (Fukumoto et al., 2005a; Fukumoto et al., 2005b; Adams et al., 2006; Carneiro et al., 2011). A recent study (Beyer et al., 2012) suggests a permissive role for serotonin in the specification of the gastrocoel roof plate (GRP; the node) and cilia-dependent fluid flow. Thus, the two main LR asymmetry models now converge on a common molecule, the neurotransmitter serotonin. The question of timing, i.e when serotonin actually functions during LR patterning, is crucial not only because it impacts the plausibility of the late origin versus early origin models of asymmetry but also because it identifies the embryonic stages that would be most sensitive to the serotonergic compounds in common medical use today (Shuey et al., 1992; Alwan et al., 2007; Noorlander et al., 2008; Sadler, 2011). Here, we statement the results of experiments that allowed us to resolve the role of both the timing and the location of serotonin in LR patterning of the frog embryo. These studies were designed to experimentally distinguish between two hypotheses: one that suggests a role for serotonin in right-sided ventral blastomeres during.