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Synopsis
Nutritional status regulates thyroid morphogenesis in vivo through glycolysis-associated suppression of cell adhesion, providing a link between metabolic state and epithelial tissue architecture during organogenesis.
Cell adhesion-related genes are upregulated in unfed Xenopus tadpoles during thyroid morphogenesis.
Thyroid follicle cells frequently establish multiple apical domains during follicle formation.
Glycolysis-associated reduction of cell adhesion promotes thyroid morphogenesis. |
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Figure 1: Feeding alters gene expression in the thyroid region.
(A) Ventral view of a fixed Xenopus laevis tadpole showing the location of the thyroid glands. Blue dotted lines outline the lower jaw cartilage, and solid pink ovals mark the left and right thyroid glands. The black rectangle indicates the region isolated for RNA-seq analysis. Both the left and right thyroid glands are included in the isolated tissue piece, along with surrounding lower jaw cartilage. (B) Timing of RNA isolation from fed tadpoles after the onset of feeding and from unfed tadpoles at the matched time points. Day 0 is defined as stage 46. (C) Principal component analysis (PCA) plot of RNA-seq data from isolated tissue piece containing the thyroid. (D) Clustering analysis based on gene expression dynamics using Day 0, Day 6, and Day 9 samples. Day 3 samples were excluded from the analysis because biological replicates showed relatively inconsistent expression patterns. Red lines indicate gene sets from fed tadpoles, and blue lines indicate those from unfed tadpoles. The vertical axis represents expression levels, and the horizontal axis represents the time points of RNA isolation. Center lines indicate the medians; box limits indicate the 25th and 75th percentiles; whiskers indicate the most extreme values within 1.5 × the interquartile range; points beyond the whiskers are plotted as outliers. All numerical values are provided in Source Data file. (E, F) KEGG pathway analysis of genes categorized in Group 1, 7, and 11. The horizontal axis represents enrichment significance, shown as −log10(BH-adjusted P values). Source data are available online for this figure. |
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Figure EV1: Related to Fig. 1. (A) Summary of RNA-seq quality metrics generated in this study. The listed parameters include total reads, total alignments, mapped alignments, the percentage of mapped alignments, the percentage of reads with mapping quality 0 (MQ0), RNA concentration, and RNA integrity number (RIN). (B) The thyroid or cartilage region used for the RNA-seq analysis. The table shows RNA-seq quality metrics used for comparison between two different regions, thyroid with cartilage and cartilage. (C) The table displaying the overlap of expressed genes in the thyroid region (red) and cartilage region (blue) at Days 0, 3, and 6. RNA was extracted from unfed larvae, and genes with raw counts ≤5 were excluded from the analysis. Percentages represent the proportion of genes in each category relative to the total number detected at each time point. |
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Figure 2: Feeding and starvation diff erentially infl uence the expression of genes related to cell adhesion, extracellular matrix,and cytoskeleton.
(A) Gene ontology (GO) analysis of genes categorized in Group 1. Actin and ECM-associated terms are labeled in red. (B) GO analysis of genes categorized in Group 2. ECM-associated term is labeled in red. (C, C’) Microtubule-associated terms in Group 3. (D) Gene names detected in each GO term shown in (A). (E) Gene names detected in each GO term shown in (B). (F) Gene names detected in GO term shown in (C). (G, H) Gene names detected in cell–cell adhesion and GO terms in Group 11. Cell adhesion-related terms are labeled in blue. (I) Gene names detected in the cell adhesion-related GO term shown in Group 4. (J) Gene name detected in the cell adhesion-related GO term shown in Group 2 and 3. |
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Figure EV2: Related to Figs. 1 and 2.
KEGG pathway analysis for each group in Fig. 1D. Bar plots show enriched pathways (x axis: −log10(BH-adjusted P values) for representative groups. Selected pathways are color-coded to highlight categories related to cell behaviors (red), metabolism (blue), and starvation-related genes (green). |
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Figure EV3: Related to Figs. 1 and 2. GO Biological Process (BP) analysis for each gene expression group shown in Fig. 1D. Genes are grouped based on their temporal expression patterns, and GO analysis is performed for the BP category. Tables show selected BP terms for each group, based on statistical ranking and biological relevance, together with the number and percentage of genes associated with each term, and the corresponding −log10(Benjamini-adjusted P value). Representative expression patterns for each group are shown above the tables. |
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Figure EV4: Related to Figs. 1 and 2. (A) Additional graphs are provided for the cell adhesion molecules, showing temporal changes in gene expression as relative expression levels (FPKM, Day 0 = 1). (B) Thyroid-related genes and their corresponding groups are summarized. |
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Figure 3: E-cadherin accumulate more at cell–cell junctions in the thyroid of unfed tadpoles than in fed tadpoles. (A–D”) Immunostaining of the thyroid in unfed (A–A”, C–C”) and fed (B–B”, D–D”) tadpoles at Day 4 (A–B”) and Day 8 (C–D”). Magenta indicates E-cadherin (Cdh1), and green indicates thyroglobulin (Tg). (A”, B”) Show cell outlines and the location of lumina labeled by Tg. Scale bar = 10 µm. (E–H) Magnified views of single cell–cell junctions detected in (A’–D’), respectively. Arrowheads indicate accumulation of E-cadherin along the junction. Scale bar = 2 µm. (I, J) Quantification of E-cadherin accumulation at cell–cell junctions located 5–10 µm beneath the surface of the thyroid. ****P < 0.0001. Mann–Whitney U test. (I: Unfed, n = 462 cells from 7 tadpoles; Fed, n = 455 cells from 7 tadpoles; J: Unfed, n = 358 cells from 5 tadpoles; Fed, n = 553 cells from 8 tadpoles.) Each dot represents one individual cell junction (biological replicate). Experiments were performed using independent clutches. No technical replicates were included. Error bars indicate s.e.m. Source data are available online for this figure. |
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Figure 4: Inhibition of E-cadherin promotes thyroid follicle formation and alters cell polarity. (A, B) Immunostaining of the thyroid in unfed tadpoles injected with IgG2b (A) or ECCD1, an inhibitory antibody against E-cadherin (B). Magenta indicates E-cadherin. Asterisk indicates lumen, and blue dots label the cell surrounding lumen. Scale bar = 5 µm. (C) Distribution of the number of cells surrounding a single lumen (labeled with blue dots in (A, B)) in IgG2b-injected control and ECCD1-injected tadpoles (200 µg/ml and 400 µg/ml). Two-sample Kolmogorov–Smirnov tests: IgG vs ECCD1-200, D = 0.2532, P < 0.0001; control vs ECCD1-400, D = 0.3271, P < 0.0001; Bonferroni corrected. (IgG: n = 170 from 9 tadpoles; ECCD1-200 µg/ml: n = 173 from 6 tadpoles; ECCD1-400 µg/ml: n = 111 from 10 tadpoles). (D) Thyroid volume of IgG2b- or ECCD1-injected tadpoles [IgG2b: n = 30, ECCD1 (200 µg/ml): n = 35, ECCD1 (400 µg/ml) = 37]. One-way ANOVA test. n.s., not significant. Each dot represents one individual thyroid. Experiments were performed using independent clutches. No technical replicates were included. Error bars indicate s.e.m. (E) Correlation between the thyroid volume and the average of cell number surrounding a lumen in each thyroid. Each dot represents one individual thyroid. Experiments were performed using independent clutches. (F–G’) Immunostaining of the thyroid in fed tadpoles injected with IgG2b (F) or ECCD1 (G). Magenta indicates Par3, and green indicates thyroglobulin. Asterisks mark cells facing two lumina. (H) Quantification of the number of cells attached multiple lumina in each thyroid. *P = 0.0469, Mann–Whitney U test. (IgG: n = 15 thyroids from 8 tadpoles; ECCD1: n = 12 thyroids from 7 tadpoles.) Each dot represents one individual cell. Experiments were performed using independent clutches. Error bars indicate s.e.m. Source data are available online for this figure. |
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Figure 5: Glycolysis reduces E-cadherin enrichment at cell–cell junctions and enlarges follicles in the thyroid. (A–B”) Immunostaining of the thyroid in fed tadpoles injected with water (A–A”) or 2-DG, a glucose analog that suppresses glycolysis (B–B”). Magenta indicates E-cadherin, and green indicates thyroglobulin (Tg). Scale bars = 20 µm in (A, B); 10 µm in (A’, A”, B’, B”). (C) Quantification of lumen volume of each follicle in the thyroid. ****P < 0.0001, Mann–Whitney U test. (Control: n = 278 lumina from 5 tadpoles; 2-DG: n = 241 lumina from 6 tadpoles). Each dot represents one individual lumen. Experiments were performed using independent clutches. Error bars indicate s.e.m. (D) Quantification of E-cadherin accumulation at cell–cell junctions located 10–15 µm beneath the thyroid surface. ****P < 0.0001, Mann–Whitney U test. (Control: n = 183 junctions from 5 tadpoles; 2DG: n = 140 junctions from 5 tadpoles). Each dot represents one individual cell junction. Experiments were performed using independent clutches. Error bars indicate s.e.m. (E, F) Body size of water- or 2-DG–injected tadpoles. Body length shows no significant differences (E), while interocular distance is shorter in 2-DG–injected tadpoles (F). **P = 0.0026, Mann–Whitney U test. (Control: n = 11; 2-DG: n = 10.) Each dot represents one individual tadpole. Experiments were performed using independent clutches. Error bars indicate s.e.m. Source data are available online for this figure. |
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Figure 6: Double-apical cells increase following glucose-enriched feeding or cell adhesion inhibition. (A, B) Body length (A) and interocular distance (B) of tadpoles fed with control or glucose-enriched gel for 10 days. (Control: n = 11; Glucose: n = 12.) Each dot represents one individual tadpole. Experiments were performed using independent clutches. Error bars indicate s.e.m. n.s.: not significant (Mann–Whitney U test). (C) Quantification of E-cadherin accumulation at cell–cell junctions located 5–10 µm beneath the thyroid surface. ****P < 0.0001, Mann–Whitney U test. (Control: n = 361 junctions from 9 tadpoles; Glucose: n = 241 junctions from 6 tadpoles.) Each dot represents one individual cell junction. Experiments were performed using independent clutches. (D) Quantification of lumen volume of thyroid follicles after 14 days of control gel or glucose-enriched gel feeding. n.s., not significant. (Control: n = 435 lumina from 8 tadpoles; Glucose: n = 404 lumina from 6 tadpoles.) Each dot represents one individual lumen. Experiments were performed using independent clutches. Error bars indicate s.e.m. n.s.: not significant (Mann–Whitney U test) *P = 0.0336 (Kolmogorov–Smirnov test). (E–F’) Immunostaining of the thyroid in tadpoles fed with normal agarose gel (E, E’) or agarose gel containing 10% glucose for 14 days (F, F’). Blue-colored cells in (E’, F’) indicate cells facing multiple lumina. (G, H) Quantification of the ratio of double-luminal cells in control or glucose-enriched gel feeding. The number of cells contacting multiple lumina per lumen is increased in the glucose-enriched group (G), while the number of lumina per thyroid shows no significant differences (H). **P = 0.0037, Mann–Whitney U test (Control: n = 15, Glucose: n = 13.) Experiments were performed using independent clutches. Error bars indicate s.e.m. Source data are available online for this figure.
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