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Notch signaling in thyrocytes is essential for adult thyroid function and mammalian homeostasis

Nature Metabolism volume 5, pages 2094–2110 (2023)Cite this article

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Abstract

The thyroid functions as an apex endocrine organ that controls growth, differentiation and metabolism1, and thyroid diseases comprise the most common endocrine disorders2. Nevertheless, high-resolution views of the cellular composition and signals that govern the thyroid have been lacking3,4. Here, we show that Notch signalling controls homeostasis and thermoregulation in adult mammals through a mitochondria-based mechanism in a subset of thyrocytes. We discover two thyrocyte subtypes in mouse and human thyroids, identified in single-cell analyses by different levels of metabolic activity and Notch signalling. Therapeutic antibody blockade of Notch in adult mice inhibits a thyrocyte-specific transcriptional program and induces thyrocyte defects due to decreased mitochondrial activity and ROS production. Thus, disrupting Notch signalling in adult mice causes hypothyroidism, characterized by reduced levels of circulating thyroid hormone and dysregulation of whole-body thermoregulation. Inducible genetic deletion of Notch1 and 2 in thyrocytes phenocopies this antibody-induced hypothyroidism, establishing a direct role for Notch in adult murine thyrocytes. We confirm that hypothyroidism is enriched in children with Alagille syndrome, a genetic disorder marked by Notch mutations, suggesting that these findings translate to humans.

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Fig. 1: Mouse and human thyroids are composed of a heterogeneous population of thyrocytes with distinct Notch activity profiles.
Fig. 2: Notch activity in adult thyrocytes is essential for their function.
Fig. 3: Notch blockade induces mitochondrial defects in thyrocytes.
Fig. 4: Notch-induced hypothyroidism triggers a cascade of thermoregulation defects.

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Data availability

All RNA-seq data and single-cell RNA-seq data are publicly available as of the date of this publication. The datasets produced in this study are available in the following databases: SuperSeries: Notch Signaling in the Thyroid is Essential for Adult Homeostasis GSE200268. SubSeries: Notch Signaling in the Thyroid is Essential for Adult Homeostasis (RNA-seq thyroid) GSE200265. SubSeries: Notch Signaling in the Thyroid is Essential for Adult Homeostasis (RNA-seq BAT) GSE200266. SubSeries: Notch Signaling in the Thyroid is Essential for Adult Homeostasis (scRNA-seq thyroid) GSE200267. The primary human single-cell sequencing is deposited in the Sequence Read Archive under BioProject ID PRJNA1006400. The reference genome used in this study was the Mus musculus (GRCm38) RefSeq assembly and the Homo sapiens (GRCh38) RefSeq assembly, deposited and publicly available in the NCBI BioProject databank under accession code GCF_000001635.20 and GCF_000001405.26. All raw data in this manuscript are available from the corresponding author upon reasonable request.

Code availability

This paper does not report original code. Any additional information required to reanalyze the data reported in this paper is available from the corresponding author upon request.

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Acknowledgements

We are grateful to K. Seidel, C. Cottonham, P. Himmels and A. Lun for experimental support and discussion, M. Chen and M. Stolzenberg for helping score BAT activity. We thank the Genentech Research Pathology, Necropsy, Histology and Next-Generation Sequencing laboratories for their experimental contributions and B. Bolon and D. Wilson (GEMpath) for their assessment of thyroid histology. We thank S. Kajimura, A. Zorzano and N. Chandel for sharing methods and for expert feedback on metabolism and mitochondria results. We thank E. Andersson (Karolinska Institutet) and N. Van Hul (Karolinska Institutet) for providing serum samples from Jag1-mutated mice (data not included). We appreciate the insightful feedback and comments on the paper from F. de Sousa e Melo, C. Metcalfe, W. Ye and H. Jasper. The scRNA-seq work in Barcelona was funded by Instituto de Salud Carlos III, Madrid, Spain, grant PI1700324 and co-financed by the European Regional Development Fund.

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Authors and Affiliations

  1. Department of Discovery Oncology, Genentech, South San Francisco, CA, USA

    Lluc Mosteiro, Simona Hankeova & Christian W. Siebel

  2. Department of Oncology Bioinformatics, Genentech, South San Francisco, CA, USA

    Thi Thu Thao Nguyen & Robert Piskol

  3. Translational Immunology Group, Vall d’Hebron Institut de Recerca (VHIR), Campus Vall Hebron, Barcelona, Spain

    Daniel Alvarez-Sierra & Ricardo Pujol-Borrell

  4. Department of Cell Biology, Physiology and Immunology, Universitat Autònoma de Barcelona, Bellaterra, Spain

    Daniel Alvarez-Sierra & Ricardo Pujol-Borrell

  5. Department of Research Pathology, Genentech, South San Francisco, CA, USA

    Mike Reichelt & Alexis Scherl

  6. Division of Gastroenterology, Hepatology and Nutrition, Hospital for Sick Children, University of Toronto, Toronto, Ontario, Canada

    Shannon M. Vandriel & Binita M. Kamath

  7. Department of Drug Metabolism and Pharmacokinetics, Genentech, South San Francisco, CA, USA

    Zijuan Lai, Feroza K. Choudhury & Dewakar Sangaraju

  8. Vall Hebron Institute of Oncology (VHIO), Campus Vall Hebron, Barcelona, Spain

    Ricardo Pujol-Borrell

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  1. Lluc Mosteiro
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Contributions

L.M. designed and performed the experiments described below, analyzed and discussed data throughout the manuscript and co-wrote the manuscript. T.T.N. and R.P. analyzed and discussed the mouse and human scRNA-seq and bulk RNA-seq data. S.H. provided experimental assistance with the genetic mouse model. D.A.-S. and R.P.-B. provided the human thyroids sequencing dataset. M.R. performed the cryo-EM experiments and analysis. S.M.V. and B.M.K. collected the thyroid data from ALGS patients. Z.L., F.K.C. and D.S. performed the thyroid metabolomics experiment and analysis. A.S. provided histopathological analysis and discussion. C.W.S. supervised the study, provided feedback on experimental design and data interpretation and co-wrote the manuscript. All authors discussed the results and commented on the manuscript.

Corresponding authors

Correspondence to Lluc Mosteiro or Christian W. Siebel.

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Competing interests

L.M., T.T.T.N., S.H., M.R., Z.L., F.K.C., D.S., A.S., R.P. and C.W.S. are or were employed by Genentech, which has commercial interest in some of the molecules described. The remaining authors declare no competing interests.

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Peer review information

Nature Metabolism thanks Luca Persani, Sabine Costagliola and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editor: Isabella Samuelson, Yanina-Yasmin Pesch, in collaboration with the Nature Metabolism team.

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Extended data

Extended Data Fig. 1 Mouse and human thyroids are composed of a heterogeneous population of thyrocytes.

A. Schematic representation of the mouse scRNA sequencing. Dissected thyroids were combined into a pool of six per sample and digested into single cells. Libraries were prepared using the Chromium platform and sequenced in single lanes using the HiSeq400 (Illumina). Sample demultiplexing, barcode processing and single cell 3’ gene counting were performed using 10x CellRanger. After assessing the percentages of mitochondrial reads and doublets, only cells with a mitochondrial UMI fraction <0.25 were analyzed. Dataset alignment, cell clustering, visualization and differential expression analysis were performed according to “Orchestrating Single-Cell Analysis with Bioconductor” (OSCA, (Amezquita et al., 2020)). The pipeline merged single-cell data generated from multiple batches, and batch effects (observed in two processing dates) were corrected. B. Heatmap of the z-scores of the expression of published cell-specific markers used for cluster identification and annotation in mouse single cell sequencing analysis. The cluster identity is shown on the X axis and the gene names on the Y axis. C. Uniform Manifold Approximation and Projection for dimension reduction (UMAP) plot corresponding to Fig. 1a. Colors highlight the 16 clusters identified: endothelial cells, EC; Thyroid Follicular Cells or thyrocytes, TFC; erythrocytes, Eryth; myeloid cells, M; T cells, T; fibroblasts, F; smooth muscle cells, S; parathyroid cells, P; Schwann cells, Sw. D. t-distributed stochastic neighbor embedding (t-SNE) plot for the mTFC and mTFC2 clusters, with blue intensities showing expression of the indicated genes. n = 3 samples. E. Representative immunofluorescent and -histochemical staining images of PAX8 and NKX2-1 in thyroid sections. 40X objective; scale bars = 20 μm; n = 5 mice. F. Heatmap of the z-scores of the expression of published cell-specific markers used for cluster identification and annotation in human single cell sequencing analysis. The cluster identity is shown on the X axis and the gene names on the Y axis. G. UMAP plot for all sequenced cells isolated from the thyroid of patient samples. Colors highlight the 7 clusters identified: endothelial cells, EC and Thyroid Follicular Cells or thyrocytes, TFC. H. Single cell net mapping of mouse TFC1 and TFC2 (yellow and blue, respectively) signatures onto the UMAP of human single cell thyroid data. Graphs indicate the number of human cells onto which the mTFC1 and mTFC2 cells mapped. Extended Data Fig. 1a reprinted from BioRender.com (2023). https://app.biorender.com/biorender-templates.

Extended Data Fig. 2 TFC1 express Notch ligands, receptors and target genes.

A. t-SNE plots for the mouse TFC clusters (top) and human TFC clusters (bottom) showing the expression levels (blue intensities) of the indicated Notch ligands and receptors. n = 3 mice and n = 4 human. B. t-SNE plots for the mouse TFC clusters (top) and human TFC clusters (bottom) showing the expression levels (blue intensities) of the indicated Notch target genes. n = 3 mice and n = 4 human. C. Expression of Jag1, Jag2, Notch1 and Notch2 mRNAs detected by RNAscope (amplified in situ hybridization) in control thyroid sections, n = 3 mice. Scale bars = 100μm.

Extended Data Fig. 3 N2 activity is detected in thyrocytes.

A. Representative double immunofluorescent detection of gamma-secretase-cleaved (active) NICD1* plus PAX8 or NICD1* plus CALCITONIN in thyroid sections of control mice (n = 5). 40X objective; n = 5 mice. B. Representative images of immunohistochemistry or -fluorescent detection of NICD1*, NICD1* plus the endothelial cell marker ENDOMUCIN, NOTCH2 (N2), and cleaved (active) NICD2*. 40X objective; scale bars = 100μm; n = 5 mice. C. Representative image of dual staining using amplified in situ hybridization (RNAscope) and immunostaining to detect Notch2 mRNA (red, Opal 690) and NKX2-1 protein (yellow, Opal 570), respectively. n = 3 mice. White arrows: thyrocytes co-stained for Notch2 mRNA and NKX2-1 protein; orange arrows: thyrocytes negative for NKX2-1 protein with low or no Notch2 mRNA. Scale bars = 50 μm.

Extended Data Fig. 4 Notch inhibition decreases the expression thyrocyte markers.

A. NICD2*, in thyroid sections from mice (n = 6 and 9 controls, n = 9 aJ12) treated as in Fig. 2a. 40X objective. Mean ± s.d. B. Quantification of Heyl, Hey1 and Nrarp mRNA expression detected using RNAscope probes in thyroid sections from mice (n = 4) treated as in Fig. 2b. Percentage of positive cells and H score. H score is calculated by totaling the percentage of cells in each bin, according to the number of dots/cell (0, 0 dots; 1, 1-3 dots; 2, 4-9 dots; 3, 10-15 dots and 4, >15 dots). Mean ± s.d. C. Notch3 mRNA expression detected using RNAscope in thyroid sections from mice (n = 3) treated as in (B), showing decreased levels in aJ12 treated thyroids. Scale bars = 100μm. Mean H score ± s.d. D. Normalized Enrichment Score (NES) of a Notch target gene set (Hey1, Nrarp, HeyL, Notch3) from thyroids isolated at the indicated times after treating mice (n = 5) as in (A). E. Top 30 pathways downregulated by aJ12 treatment (relative to aRW), identified using GSEA of differentially expressed genes in total thyroids (bulk RNA-seq) from mice treated as in (A) (n = 5 mice). P values were derived by comparing calculated ES to ES values calculated from 10000 permutations of the gene rank list followed by Benjamini-Hochberg correction. F. Fold change of thyrocyte and parafollicular (PFC) marker gene expression induced by aJ12 treatment and determined by bulk RNA sequencing of thyroids isolated from mice (n = 5 thyroids) treated as in (A). y axis, nRPKM fold change relative to aRW isotype control. Mean ± s.d. G. Fold change of mRNA expression of thyrocyte and parafollicular (PFC) marker genes and Notch target genes (normalized to actin mRNA expression), induced by aJ12 treatment and determined by qRT-PCR of thyroid mRNA isolated from mice (n = 8 aRW thyroids and 6 aJ12) treated as in (A) for 5 days. y axis, fold change relative to aRW isotype control. Mean ± s.d.

Extended Data Fig. 5 Notch inhibition induces defects in thyrocytes.

A. Representative immunohistochemical detection of CALCITONIN and calcitonin gene-related protein (CGRP) in thyroid sections from mice (n = 5) treated as in (A) for 5 days. Scale bars = 100μm. B. Percentage of PAX8+ cells assessed by immunofluorescence in the thyroids of mice (n = 8 aRW and n = 10 aJ12) treated as in (A). Mean ± s.d. C. Distribution of expression of the indicated genes in mTFC2 cells from mice (n = 3) treated as in (B). Boxes represent interquartile ranges and medians are shown as lines. Whiskers above and below the boxes are the lowest and highest values within 1.5 times IQR. Outliers are shown as circles beyond the whiskers. D. Representative immunofluorescent detection of PAX8 in FRTL5 cells (n = 4 wells) treated for 2 days with aRW (25 μg/ml), aJ12 (25 μg/ml each) or aN123 (25 μg/ml each). DAPI was used for nuclear staining. 20X objective. E. Representative immunofluorescent images of the thyroid organoids derived from thyrocytes isolated from mice treated as in (A) and stained for NKX2-1 (red), CALCITONIN (green) and PAX8 (white). Scale bars = 100 μm. F. Number of organoids generated from 10000 single cells isolated from thyroids of mice treated as in (A). Single cells were either isolated from 7 thyroids, digested in a pool, and plated into 3 wells, before counting organoids/well on day 6 (D6) or isolated from 8 thyroids, pooled into 2 thyroids/pool, and plated into single wells before counting organoids/well on day 7 (D7). Mean ± s.d.; n = 3 wells. G. Characterization of organoids formed 10 days after their isolation from mice treated in vivo. Size (small<20 cells/organoid≤big); development (developed, > 1 follicle formed; underdeveloped, no follicles, unorganized cell mass). n = 49 aRW and 33 aJ12 organoids. H. Top 200 pathways (gray dots) identified using Gene Set Enrichment Analysis (GSEA) that are deregulated following treatment as in Fig. 1b with aN12 (Y axis) and aJ12 (X axis) in mTFC2 cells. Yellow dots represent pathways associated with endoplasmic reticulum. I. Akaike Information Criterion (AIC) and Bayesian Information Criterion (BIC) statistics per number of topics represented in the single cell data.Y axis= AIC and BIC scores; X axis= number of topics. Seven topics yielded a low AIC score and was the smallest number that minimized the BIC score, which otherwise increased with topic numbers > 8 because of over-fitting. J. Boxplots showing the topic scores in each treatment group. Boxes represent interquartile ranges and medians are shown as lines. Whiskers above and below the boxes are the lowest and highest values within 1.5 times IQR. Outliers are shown as circles beyond the whiskers. n = 3 thyroids/group. Statistical significance was assessed using the unpaired two-tailed Student’s t-test with Welch’s correction: p < 0.05, *; p < 0.01, **; p < 0.001, ***; p < 0.0001, ****.

Extended Data Fig. 6 Notch blockade induces mitochondrial defects in thyrocytes.

A. Distribution of expression of the indicated mitochondrial genes in mTFC1 cells isolated from mice (n = 3) treated for 7 days with aRW (40 mg/kg), aJ12 (20 mg/kg each) or aN12 (5 and 10 mg/kg, respectively), assessed by scRNA-seq (Fig. 2b). Boxes represent interquartile ranges and medians are shown as lines. Whiskers above and below the boxes are the lowest and highest values within 1.5 times IQR. Outliers are shown as circles beyond the whiskers. B. Representative electron microscopy images of mitochondria from non-thyrocyte cells from thyroids of mice (n = 3) treated as in (A). Scale bars = 100 nm. C. Fluorescent intensity (Y axis) of TMRM in primary thyrocytes from mice (n = 5) treated as in (A). TMRM fluorescence intensity decreases after aJ12 (red) or aN12 treatment (blue), compared to control aRW (gray dotted). D. Quantification of COX staining using the VitroViewTM kit in thyroid cryosections of mice from Fig. 3c (n = 6 aRW; n = 8 aJ12; n = 6 aN12) treated as in (A). Mean ± s.d. E. Cytochrome c oxidation kinetics of primary thyrocytes isolated from mice (n = 5) treated as in (A). Mean ± s.d. F. Ratio of mitochondrial DNA (mDNA, Nd1 and 16 S) versus genomic DNA (gDNA, HK and 18 S) in thyroids from mice (n = 9) treated as in (A). Mean ± s.d. G. Seahorse assay of ATP production rates in the FRTL5 thyrocyte cell line cultured for 2 days as in Fig. 3e. Mean ± s.d.; n = 13 wells. H. Gating strategy used in Fig. 3f. Data represents one aRW sample and the same gates were applied to all samples.

Extended Data Fig. 7 Notch inhibition decreases mitochondrial activity.

A. Percentages of thyrocytes with high ( ≥ 2500 MFI); low mitoTracker ( < 2500 MFI) intensities isolated from mice treated for 7 days with aRW (40 mg/kg), aJ12 (20 mg/kg each) or aN12 (5 and 10 mg/kg, respectively). Percentages of mitoSOX positive thyrocytes ( ≥ 450 MFI) in the same samples. Mean fluorescence intensities (MFI) of mitoTracker and mitosox in cells with low mitoTracker staining. Mean ± s.d.; n = 5 mice. B. Mean fluorescence intensity following DCFDA staining to measure total ROS levels as in (A). C. Principal component analysis (PCA) of thyroid metabolites from mice (n = 4) treated as in (A). Samples group together according to their treatments. D. Quantification of glycolysis and TCA cycle metabolites of samples in (C). Succinate, Citrate, Lactate and Pyruvate were quantified using targeted analysis (ng/g), and glucose-6-phosphate levels are reported as MS peak area units. Mean ± s.d.; n = 4 thyroids. E. Quantification in MS peak area units of urea cycle and glutamine cycle metabolites of samples in (C). Mean ± s.d.; n = 4 thyroids. F. Quantification of ketogenesis metabolites of samples in (C). 3-OH-butyrate and 2-OH-3-methylbutyric acid were quantified using targeted analysis or with MS peak area units, respectively. Mean ± s.d.; n = 4 thyroids. G. Quantification of catalase activity in thyrocytes isolated from mice (n = 5) treated as in (A). Mean ± s.d. H. Quantification of glutathione disulfide (GSSG) in thyrocytes isolated from mice (n = 5) treated as in (A). Mean ± s.d. Statistical significance was assessed using the unpaired two-tailed Student’s t-test with Welch’s correction: p < 0.05, *; p < 0.01, **; p < 0.001, ***; p < 0.0001, ****.

Extended Data Fig. 8 Notch inhibition induces hypothyroidism.

A. Quantification of thyroid histological changes from male (n = 4) and female (n = 5) mice treated as in Fig. 4a. Thyroid sections estimated w > 40% of peripheral follicles inactive ( < 4 vacuoles) were interpreted as having reduced activity. Flattening epithelium in follicles: 0% = normal, <10% = ‘minimal’, 10-40%= ‘mild’, 40-75% = ‘moderate’ and >80% follicles = ‘marked’. Percentage of follicles with decreased thyroid activity ( < 4 resorption vacuoles) or lined with cuboidal epithelium (as indicated) in thyroids from different female mice (dots). B. Thyroxine (T4) serum levels from female mice (n = 5) treated as in (A) for 7 days and measured by ELISA. Mean ± s.d. C. T4 and triiodothyronine (T3) in male mice (n = 6) treated as in (A) over the indicated time course. Mean ± s.d. n = 11 aRW and n = 6 aJ12 mice at day 1; n = 7 mice at day 5; n = 10 mice at day 7. D. Serum levels of thyroxine (T4) measured at 7 days post treatment. Male mice were treated with a single i.p. dose of aRW (40 mg/kg); aJ12 (20 mg/kg each); aN123 (5, 10 and 20 mg/kg); aN1 (5 mg/kg); aN2 (10 mg/kg); aN3 (20 mg/kg); aJ1 (20 mg/kg) or aJ2 (20 mg/kg); aN12 (5,10 mg/kg), aN23 (10, 20 mg/kg, aN3 twice a week) and aN13 (5, 20 mg/kg, aN3 injected twice a week). Mean ± s.d; n ≥ 5 mice. E. Serum levels of Thyrotropin Releasing Hormone (TRH), secreted by the hypothalamus, and Thyrotropin Secreting Hormone (TSH), secreted by the pituitary gland, in male mice treated as in (A) for the indicated days. Mean ± s.d.; n = 6 mice. F. Serum levels of calcitonin in male mice treated as in (A) for 7 days. Mean ± s.d.; n = 11 aRW treated mice; n = 11 aJ12 treated mice and n = 3 aN123 treated mice. G. Body temperatures measured using a rectal probe at the same time on each of the indicated days in male mice treated as in (A) and housed at 22 °C. Data from 5 experiments. Mean ± s.d.; n = 22 mice (D1-7) and n = 6 (D12−16). H. Body temperatures measured using a rectal probe at the same time on each of the indicated days in female mice treated as in (A) and housed at 22 °C. Mean ± s.d.; n = 5 aRW, 4 aJ12, 5 aN12 mice. I. T4 serum levels assessed by ELISA in Ob/Ob or WT male mice housed at the indicated temperatures and treated for 7 days as in (A). Mean ± s.d.; n = 6 Ob/Ob mice; n = 5 mice at 4 °C; n = 6 mice at 20 °C.

Extended Data Fig. 9 Exogenous administration of thyroid hormones rescues the hypothyroidism effects induced by Notch blockade.

A. T4 levels following T3 and T4 administration (1 µg/g and 0.05 µg/g, respectively) according to the schematic. T3 and T4 administration significantly increase the levels of T4 in serum. Mean ± s.d.; n = 4 mice. B. Body temperatures measured using a rectal probe at the same time on each of the indicated days in male mice treated as in (A) and housed at 22 °C or at 4 °C. The dotted line indicates when the T3T4 treatment starts. Mean ± s.d.; n = 5 mice. C. Electron microscopy images of mitochondria of thyrocytes from male mice treated as in (A) for 7 days, n = 3 mice. Arrows = aberrant mitochondria. Scale bars = 400 nm. D. Representation of the temperature dissipated from the tail measured by infrared thermography in female and male mice treated as in (A) during 6 days. Mean ± s.d.; n = 5 mice. E. Score of the BAT activity, assessed blindly in the BAT sections from male mice treated as in (A). Score of 1 is white adipose tissue phenotype and 10, BAT at its maximum activity. Mean ± s.d.; n = 5 mice. Representative picture of the interscapular brown adipose tissue (BAT) of male mice treated with aRW or aJ12 antibodies. n = 5 mice. F. Wet weight of brown adipose tissue (BAT) in female mice treated as in (A), normalized to the average wet weight measured in the control group. Scoring done as in (E). Mean ± s.d.; n = 5 mice. G. RNA sequencing (nRPKM fold compared to aRW) of the indicated genes in the BAT of male mice treated as in (A). Mean ± s.d.; n = 5 mice. H. Representative images of immunohistochemistry against UCP1 in BAT from male mice treated for 5 days as in (A). n = 5 mice. Scale bars = 100μm. I. BAT temperature in male mice treated as in (A). Temperature was measured by IPT transponders implanted in the interscapular region. Data were collected from 3 independent experiments. Mean ± s.d.; n = 12 aRW mice; n = 12 aJ12 mice; n = 10; aRW+T3T4-treated mice; aJ12 + T3T4-treated mice. Statistical significance was assessed using the unpaired two-tailed Student’s t-test with Welch’s correction: p < 0.05, *; p < 0.01, **; p < 0.001, ***; p < 0.0001, ****.

Extended Data Fig. 10 Genetic deletion of Notch1 and Notch2 in the thyroid induces hypothyroidism.

A. Serum levels of glucose, cholesterol, triglycerides and NEFAs of male mice treated for 7 days after a single i.p. injection of aRW (40 mg/kg), aJ12 (20 mg/kg each) or aN12 (5 and 10 mg/kg, respectively) and daily dose of T4 and T3 (1 and 0.05 µg/g, respectively). Mean ± s.d.; n = 5 mice. B. Energy expenditure in mice (n = 5) treated as in (A). Black and white bars represent daily night and day cycles, respectively. Oxygen and carbon dioxide levels were measured every 15 min for 3 days before and 11 days after treatment, as indicated. Mean ± s.d. C. Scheme of tamoxifen administration in N1fl/fl/N2fl/fl; Tg-CreERT2pos (+/+) mice and the respective Tg-CreERT2neg (-/-) controls. Analysis started at day 7 after the first tamoxifen administration and tissues were collected at day 14. Agarose gel electrophoresis showing PCR products of genomic DNA isolated from the thyroids and amplified using primers to detect recombined Notch1 (478 bp band), WT Notch2 (1485 bp band) or recombined Notch2 (480 bp band). The experiment was performed 2 times. Immunohistochemistry against CRE (upper) or NOTCH2 (middle) or dual RNAscope (bottom) against Notch1 (red) and Notch2 (green) on thyroid sections, n > 8 mice. Scale bars = 50μm. D. Infrared thermography pictures of mice treated as in (C) and measured at day 13 after tamoxifen, showing increased heat dissipation in N1fl/fl/N2fl/fl; Tg-CreERT2pos (+/+) mice compared to their Tg-CreERT2pos (-/-) counterparts. The bar on the right shows the maximum and minimum temperature registered in each picture with the corresponding gradient of colors, n = 5 mice. E. Score of the BAT activity, assessed blindly in the BAT sections from mice treated as in (C), showing increased BAT activity in N1fl/fl/N2fl/fl; Tg-CreERT2pos (+/+) thyroids compared to their Tg-CreERT2pos (-/-) counterparts. Score of 1 is white adipose tissue phenotype and 10, BAT at its maximum activity. F. Serum levels of glucose, cholesterol and triglycerides of mice treated as in (C). Mean ± s.d.; n = 8 mice. Statistical significance was assessed using the unpaired two-tailed Student’s t-test with Welch’s correction: p < 0.05, *; p < 0.01, **; p < 0.001, ***; p < 0.0001, ****. Extended Data Fig. 10c reprinted from BioRender.com (2023). https://app.biorender.com/biorender-templates.

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Supplementary Fig. 1 (gating strategy) and Tables 1 and 2.

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Mosteiro, L., Nguyen, T.T.T., Hankeova, S. et al. Notch signaling in thyrocytes is essential for adult thyroid function and mammalian homeostasis. Nat Metab 5, 2094–2110 (2023). https://doi.org/10.1038/s42255-023-00937-1

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