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Figure 1. Abnormal craniofacial phenotypes caused by xMLTK-MO, thioridazine and ethanol treatments can self-correct during pre-metamorphic stages. Craniofacial phenotypes of control, xMLTK-MO-, thioridazine-, ethanol- and ICI 118,551-treated tadpoles were observed at early and late pre-metamorphic stages. (A,B) Images of the same set of tadpoles at the early and late pre-metamorphic stages, dorsal (A) or anterior (B) views. xMLTK-MO, thioridazine, ethanol and ICI 118,551 treatments induce craniofacial defects that are visible by NF stages 43-45. These phenotypes improve in the case of the xMLTK-MO-, thioridazine- and ethanol-treated tadpoles by NF stages 49-50. Arrowheads indicate common craniofacial abnormalities present in treatment groups, such as abnormal eye shape, eye size, branchial arch or mouth. White asterisks indicate the injected side of xMLTK-MO tadpoles. Scale bars: 500 μm.
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Figure 2. Geometric morphometric analysis of corrected abnormal craniofacial morphology. Morphological metrics for (A,B) eye ratios, (C,D) nostril-midline angles and (E,F) mouth corner-midline angles were quantified for control, xMLTK-MO-, thioridazine- and ethanol-treated tadpole groups at NF stage 45 and 49. Measurements are presented as the mean difference between the experimental and control value±s.e.m. ns=P>0.05, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 (two-tailed Mann-Witney U-test after Bonferroni adjustment); N=3-6 biological replicates, n=10-20 tadpoles per group per stage. Total n of 120 for controls, 60 for ethanol, 76 for thioridazine and 62 for xMLTK-MO.
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Figure 3. Alcian Blue staining reveals corrective remodeling of cartilage in xMLTK-MO-, thioridazine- and ethanol-treated tadpoles between NF stages 45 and 50. (A,B) Representative images of bleached and Alcian Blue stained control, xMLTK-MO-, thioridazine-, ethanol- and ICI 118,551-treated specimens at NF stage 45-46 and NF stage 49-50, with corresponding cartilage schematics. MPC, Meckel's and palatoquadrate cartilage; CC, ceratohyal cartilage; BC, branchial cartilage. (C) Craniofacial cartilage phenotypes were scored as normal, moderate or severe (see Fig. S3 for scoring reference). Goodness of fit χ2 tests for each group were performed with the expected distributions set to the mean percentage distributions of stage 45; ns=P>0.05, *=P<0.05, **=P<0.01, ***=P<0.001 after Bonferroni adjustment; N=2-4 biological replicates, n=15-33 tadpoles per group per stage; total n is shown in the graphs. Scale bars: 500 μm.
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Figure 4. Candidate gene expression profiles of tadpoles experiencing adaptive tissue craniofacial remodeling are not consistent with precocious metamorphosis. Expression profiles for mmp2, rarα, rxrα, thrα and thrβ were analyzed at NF stages 45, 47, 49 and 50 in T3-, thioridazine-, ethanol-, ICI 118,551- and xMLTK-MO-treated tadpoles. The T3-treated group serves as a positive control for mild thyroid hormone induction of the TH pathway for NF stages 47+. Upregulated expression of mmp2, thrα and thrβ is indicative of thyroid hormone pathway induction. Upregulated expression of rxrα and rarα is indicative of endocrine hormone pathway induction. Mean log10 fold candidate gene expression is relative to the wild-type control group (control log10 fold change=0); mean gene expression levels for controls and experimental tadpoles were first normalized to the eukaryotic elongation factor 1a (eef1a) gene. Box and whisker plots: median values (middle bars) ± a quartile (boxes); whiskers indicate 1st and 4th quartile; any outliers are >1.5× the interquartile ranges. The red dashed line indicates a conservative biological significance threshold (1.5× fold change). N=3-5 biological replicates, n=10 tadpoles per group per stage. *P<0.05, **P<0.001 (Kruskal-Wallis). Total n of 50 for controls, 30 for ethanol, 50 for ICI 118,551, 30 for T3, 50 for thioridazine and 30 for xMLTK-MO.
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Figure 5. Thyroid hormone inhibition does not block normalization of abnormal craniofacial morphologies in ethanol-, thioridazine- or xMLTK-MO-treated tadpoles. Morphological metrics (e.g. eye ratio, nostril-midline angle and mouth corner-midline angle) were quantified at NF stages 45 and 49 for wild-type control, ethanol-, thioridazine- and xMLTK-MO-treated tadpoles that were either left in untreated media or exposed to methimazole. Thus, the eight groups are designated wild type±methimazole, ethanol±methimazole, thioridazine±methimazole and xMLTK-MO±methimazole. Measurements are presented as the mean difference between the experimental and control value±s.e.m. For wild-type control, ethanol and thioridazine: N=3 biological replicates, n=6-18 tadpoles, for a total n of 29-48 per group/stage. Total n of 47 for wild type±methimazole, 30 for ethanol±methimazole, 35 for thioridazine±methimazole and 13 xMLTK-MO±methimazole. Two-tailed Mann-Witney U test applied to all but xMLTK-MO groups due to the total n<25 for each xMLTK-MO group, which is below our set value for adequate statistical power. ns=P>0.05, *P<0.05, **P<0.01, ***P<0.001 after Bonferroni adjustment.
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Figure 6. Increased dopamine signaling hinders normalization of abnormal craniofacial morphologies in thioridazine-treated tadpoles. Morphological metrics (e.g. eye ratio, nostril-midline angle and mouth corner-midline angle) were quantified at NF stages 45 and 49 for wild-type control and thioridazine-treated tadpoles that were either left in untreated media or exposed to pergolide mesylate (PM). Thus, the four groups are designated wild type±PM and thioridazine±PM. Measurements are presented as the mean difference between the experimental and control value±s.e.m. Two-tailed Mann-Witney U test: ns=P>0.05, *P<0.05, **P<0.01, ***P<0.001 after Bonferroni adjustment. N=3 biological replicates, n=14-18 tadpoles per group per stage. Total n of 47 for wild type−PM, 48 for wild type+PM, 48 for thioridazine−PM and 45 for thioridazine+PM.
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Figure 7. Differential gene expression of mmp1, mmp13 and mmp7 in control, ethanol-, thioridazine-, ICI 118,551- and xMLTK-MO-treated tadpoles detected through RT-qPCR. Expression profiles for mmp7, mmp1 and mmp13 were analyzed at NF stages 47 and 49 in ethanol-, ICI 118,551-, thioridazine- and xMLTK-MO-treated tadpoles. Upregulated expression of Mmp genes is indicative of tissue remodeling pathway induction. Mean log10 fold candidate gene expression is relative to the wild-type control group (control log10 fold change=0); mean gene expression levels for controls and experimental tadpoles were first normalized to the eukaryotic elongation factor 1a (eef1a) gene. Box and whisker plots: median values (middle bars) ± a quartile (boxes); whiskers indicate 1st and 4th quartile; any outliers are >1.5× the interquartile ranges. The red dashed line indicates a conservative biological significance threshold (1.5× fold change). N=3 biological replicates, n=10 tadpoles per group per stage. *P<0.05 (Kruskal-Wallis U test).
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Figure 8. Model for the adaptive TH-independent tissue remodeling response seen in tadpoles with self-correcting malformed craniofacial features. In both wild-type control and abnormal tadpoles, craniofacial morphology is monitored and adaptively regulated through innate growth and tissue remodeling mechanisms. Tadpoles with abnormal craniofacial morphology shown to self-correct prior to metamorphosis (middle) trigger a more-robust tissue remodeling and growth phase through the upregulation of endocrine hormone genes (e.g. prolactin.2.S) relative to controls. Tadpoles with abnormal craniofacial morphology that do not self-correct prior to metamorphosis (right) are either unable to increase endocrine hormone signaling or have missing craniofacial features.
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Figure S1. Exposure to xMLTK-MO, thioridazine, ethanol, and ICI 118,551 produces consistent craniofacial phenotypes. Typical range of craniofacial phenotypes observed with these perturbations. For experimental groups the phenotypes are arranged from most abnormal on the left to least abnormal on the right. Morpholino-based knockdown of xMLTK and finite pharmacological (e.g. thioridazine, ethanol, and ICI 118,551) exposures during neurula stages produce abnormal phenotypes with little variation, relative to most teratogens. White asterisks denote the injected side of xMLTK-MO tadpole. Scale bars: 500μm.
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Figure S2. Whole-mount in situ hybridization for sox9 on embryos following
exposure to ethanol, ICI 118,551, and thioridazine highlight their effects on neural
crest cell localization.
A) Representative images showing the range of sox9 spatial patterning in control and experimental tadpoles. Grey arrowheads point to regions of reduced sox9 expression, red arrowheads point to mispatterned sox9 expression.
B) Quantification of scored sox9 phenotypes in control and experimental tadpoles. N = 2, n = 25-29, for a total n of 54 for controls, 56 for ethanol, 51 for ICI 118,551, and 56 for thioridazine. Scale bars: 500μm.
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Figure S3. Schematics for cartilage phenotype scoring categories, based on Alcian blue staining. Bleached and Alcian blue stained NF stg. 45-50 specimen, with corresponding WT cartilage schematics. Craniofacial cartilage phenotypes were qualitatively scored as normal, moderate, or severe based on the shape and presence of these major craniofacial cartilaginous features. MPC = Meckel’s and Palatoquadrate cartilage, CC = Ceratohyal cartilage, BC = Branchial cartilage. Schematics for severe phenotypes leave uncolored, but outlined, the features commonly missing entirely from the most abnormal specimen. Scale bars:500μm.
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Figure S4. Geometric morphometric analysis in Control, thioridazine, and ethanol
tadpoles at pre-metamorphic stages 46-50/51. Morphological metrics (e.g. left eye ratio and right mouth corner-midline angle) were quantified at NF stg 45 through 50 for wildtype control and thioridazine tadpoles. Measurements were taken from Individually tracked tadpoles across several stages, light blue and red lines represent individual experimental tadpoles and control tadpoles, respectively. N = 1 biological replicate, n = 8-15 tadpoles. Opaque blue and red lines represent experimental and control group means, no error bars or statistical tests were applied because data are from a single biological replicate.
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Figure S5. T3 exposure leads to precocious limb bud and barbel growth by NF
stages 49 and 50. Representative images of control, +T3 (thyroid hormone), and +Met (methimazole) tadpoles at NF stage 49 and 50. The yellow and green arrow heads point to precocious growth of barbels and limb buds, respectively. The white dashed outline highlights premature or accelerated fusion of the olfactory bulbs in +T3 tadpoles. Scale bars: 500μm.
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Figure S6. Prolactin.2.S is differentially expressed in ethanol and thioridazine
tadpoles at NF stage 45 and 47. A,B) Expression of prolactin.2.S and prolactin receptor was quantified in ethanol, thioridazine, and ICI 118,551 tadpoles at NF stage 45 and 47, respectively. Mean log10 fold candidate gene expression is relative to the WT control group (control log10 fold change = 0); mean gene expression levels for controls and experimental tadpoles were first normalized to the eukaryotic elongation factor 1a (eef1a) gene. The red dashed line denotes a conservative biological significance threshold (1.5x fold change). N = 2-3 biological replicates, n = 10 tadpoles per group per stage. * = KW test, p < 0.05.
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Figure S7. Pergolide mesylate exposure affects the growth and morphology of
anterior craniofacial structures and branchial arches. Representative images of wildtype control and thioridazine tadpoles at NF stg 50 that were either left in untreated media (-PM) or exposed to pergolide mesylate (+PM) from NF stg 45 to 50. Pergolide mesylate exposure, reduces branchial arches and anterior CF features, altering the overall head morphology of +PM specimen. Blue arrowhead denotes underdeveloped anterior craniofacial region and green arrowhead denotes reduced branchial arch width, relative to -PM control. Scale bars: 500μm.
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