Vandenberg LN , Lemire JM , Levin M .

F32GM087107 NIGMS NIH HHS , R01-GM077425 NIGMS NIH HHS , F32 GM087107 NIGMS NIH HHS , R01 GM077425 NIGMS NIH HHS

Fig. 1. Theory-neutral experiments indicate that serotonin has early, non-ciliary roles in LR patterning. (A) For experiments where gain-of-function or loss-of-function reagents were examined, organ situs was examined to determine the effects of treatments on LR patterning. In stage 45 tadpoles, the position of three organs was examined: the heart, normally positioned on the animal’s left with the apex on the animal’s right (red arrowhead); the gall bladder, normally positioned on the animal’s right (green arrowhead); and the stomach, normally coiled toward the animal’s left (yellow arrowhead). Inversions of one or more of these organs were scored as heterotaxia. (B) Ectopic serotonin induces heterotaxia, with the greatest effects observed when injections were targeted to left blastomeres. Serotonin (15 or 30 ng) was injected into one blastomere at the four-cell stage, together with a lineage label to verify blastomere targeting. At the lower dose of serotonin, only injections in the ventral left (VL) or dorsal left (DL) blastomere induced significant amounts of heterotaxia. At the higher dose, injections into any blastomere including the ventral right (VR) or dorsal right (DR) induced heterotaxia, but left-sided injections were most effective. *P<0.01 compared with uninjected controls (2.5% heterotaxia, not shown). For all treatments, at least 100 embryos were included for analysis. (C) Expression of mRNA encoding dominant-negative (DN) serotonin receptor 3A (R3A) induces heterotaxia. Injection of this construct into either the DL or VR blastomeres was sufficient to induce significant levels of heterotaxia, but expression in the VR blastomere produced more than twice as many heterotaxic embryos compared with DL expression. *P<0.01, **P≪0.001 compared with embryos injected with β-gal lineage label alone. For all treatments, at least 75 embryos were included for analysis. (D) Left lateral plate mesoderm (LPM) explants were isolated prior to ciliary flow (stage 13), after ciliary flow (stage 18) or at the time that Xnr-1, lefty and pitx2 begin to be expressed asymmetrically in a robust manner (stage 22/23). Explants and unmanipulated siblings were developed to stage 23 (Xnr-1), stage 25/26 (lefty) or stage 29/30 (pitx2), fixed and probed for expression of the appropriate mRNA. Arrows indicate areas with positive expression of the indicated mRNA. (E) Quantification of expression of laterality markers in left LPM explants. For all three markers assessed, the vast majority of explants were positive for expression of each of the three left-side marker genes. #P<0.01 comparing heterotaxia incidence between treatment groups.

Fig. 2. Analysis of cilia number, cilia length and ciliary flow in published studies indicates large variability and lack of consistency in these parameters. (A–C) To analyze the consistency of ciliary parameters in an in vivo model, data on ciliary length, cilia number and ciliary flow parameters were collected from published studies (Oishi et al., 2006; Shu et al., 2007; Okabe et al., 2008; Ferrante et al., 2009; Hatler et al., 2009; Lin and Xu, 2009; Neugebauer et al., 2009; Francescatto et al., 2010; Gao et al., 2010; Lopes et al., 2010; Kim et al., 2011; Liu et al., 2011; Wang et al., 2011; Bisgrove et al., 2012; Caron et al., 2012; Chen et al., 2012). Zebrafish studies were selected because, in contrast to Xenopus studies, a significant number measured the same parameters and reported data in a consistent manner (including means, s.e.m. or s.d., and sample sizes). (A) Cilia length was reported in 13 studies examining wild-type zebrafish at somite stage 10 (control samples C1–C13). ANOVA and Bartlett values indicate significant differences between groups and significantly different variances. Posthoc analyses were not performed because of the large number of groups examined. Six treatments (mutations, knockdowns via morpholinos, etc.) that were shown to affect cilia length (T1–T6), either by decreasing or increasing the length of cilia, are also shown on the graph. These treated groups cannot be statistically distinguished from wild-type groups in at least one other study. (B) Cilia number was reported in eight studies examining wild-type zebrafish at somite stage 10 (control samples C1–C8). ANOVA values indicate significant differences between groups; Bartlett’s values indicate no significant differences in variance between groups. Additionally, three conditions or treatments that were shown to affect cilia number (T1–T3) are displayed. Again, these treated groups cannot be statistically distinguished from one or more wild-type control groups. (C) Ciliary flow rate was reported in six zebrafish studies (C1–C6). ANOVA and Bartlett’s tests indicate significant differences between groups and significant differences in variance. Because there were fewer studies included in this measure, Bonferroni posthoc analyses were performed. Lowercase letters on the graph indicate significant differences between groups of controls. For all three graphs, data shown are means ± s.d., as collected from original publications or calculated from the reported s.e.m. and sample size.

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