Journal: Development (Cambridge, England)

Article Title: Revising the embryonic origin of thyroid C cells in mice and humans

doi: 10.1242/dev.126581

Figure Lengend Snippet: Tracing Sox17 + progeny in embryonic thyroid in Sox17-2A-iCre;R26R mice. Immunofluorescence images of single and merged channels of the indicated markers from serial sections of the same specimen; merged images additionally show 4′,6-diamidino-2-phenylindole (DAPI) nuclear stain. (A) Coexpression of β-galactosidase (β-gal) and Nkx2-1 in follicular cells (upper panel), β-gal and CD31 in endothelial cells (middle panel), and β-gal and calcitonin in C cells (lower panel). (B) Colocalization of β-gal and calcitonin as revealed by confocal laser scan microscopy. Arrows indicate β-gal+ C cells. Arrowheads indicate β-gal– stromal cells. Scale bars: 100 µm (A); 25 µm (B).

Article Snippet: Cryosections (10 µm thick) collected on glass slides were subjected to pairwise double immunolabeling as indicated with the following antibodies: rabbit anti-Nkx2-1 (Biopat, Italy; PA0100, 1:1000), rabbit anti-calcitonin (DAKO; A0576, 1:500), guinea pig anti-Foxa1 (kindly provided by Jeffrey Whitsett, Cincinnati Children's Hospital, OH, USA; 1:2000; applied for mouse tissue only), mouse anti-Foxa1 (Seven Hills Bioreagents; WMAB-2F83, 1:1000; applied for human specimens only), rabbit anti-Foxa2 (Seven Hills; WRAB-FOXA2, 1:2000), rat anti E-cadherin (ECCD2; Calbiochem; 205604, 1:1000), rat anti-CD31/Pecam-1 (BD Biosciences; 557355, 1:250), chicken anti-β-gal (Abcam; ab9361, 1:2000) and rabbit anti-Ki67 (Abcam; ab16667, 1:100).

Techniques: Immunofluorescence, Staining, Microscopy

Journal: Development (Cambridge, England)

Article Title: Revising the embryonic origin of thyroid C cells in mice and humans

doi: 10.1242/dev.126581

Figure Lengend Snippet: Expression of forkhead box transcription factors Foxa1 and Foxa2 in mouse ultimobranchial bodies, C cell precursors and embryonic C cells. Analysis of wild-type embryos with in situ hybridization (A-C) and immunofluorescence (D-N), respectively. Endoderm and derivatives thereof were identified with E-cadherin (E-cad) as indicated. (A) Overview of Foxa2 expression in midline structures. (B) Foxa2 expression in the ultimobranchial body (image from a more posterior section to that shown in A). (C,D) Foxa2 at fusion of ultimobranchial body with midline thyroid as identified by Nkx2-1 expression (data from serial sections of the same specimen). (E) Loss of Foxa2 expression in the prospective ultimobranchial body; arrow indicates transition between fourth pharyngeal pouch and endoderm proper. (F) Nkx2-1+ cells (arrowhead) exclusively present in the fourth pouch of lateral pharyngeal endoderm. (G) Differential expression of Foxa1 (arrowhead) in the emerging Nkx2-1+ ultimobranchial body. (H-J) Distribution of Foxa1+ (arrowheads) and Foxa2+ (arrows) cells in the developing ultimobranchial body from the time of delamination (H) to fusion with midline thyroid (J). Inset (in H) outlines the ultimobranchial body epithelium co-stained with DAPI. Small arrow (in I) indicates a single Foxa1+/Foxa2+ cell. (K,L) Distribution of Ki67+ ultimobranchial body cells before (K) and at fusion with midline thyroid (L). Arrow and arrowhead in K correspond to the same labels in I for comparison with Foxa1/Foxa2 expression pattern. (M) Coexpression of Foxa1 and Foxa2 in C cell precursors dispersed in the thyroid lobe after fusion of primordia. Arrowheads indicate follicular parenchyma with DAPI staining only. (N) Coexpression of Foxa1 and calcitonin in embryonic thyroid C cells. Note separate staining of markers (arrows) confined to nucleus and cytoplasm, respectively. e, pharyngeal endoderm; es, esophagus; fp, floor plate; pp, pharyngeal pouch; t, embryonic thyroid; t(m), midline thyroid primordium; tr, trachea; ub, ultimobranchial body. Scale bars: 100 µm (C,D,G,H); 50 µm (A,B,E,F,I-L); 25 µm (M,N).

Article Snippet: Cryosections (10 µm thick) collected on glass slides were subjected to pairwise double immunolabeling as indicated with the following antibodies: rabbit anti-Nkx2-1 (Biopat, Italy; PA0100, 1:1000), rabbit anti-calcitonin (DAKO; A0576, 1:500), guinea pig anti-Foxa1 (kindly provided by Jeffrey Whitsett, Cincinnati Children's Hospital, OH, USA; 1:2000; applied for mouse tissue only), mouse anti-Foxa1 (Seven Hills Bioreagents; WMAB-2F83, 1:1000; applied for human specimens only), rabbit anti-Foxa2 (Seven Hills; WRAB-FOXA2, 1:2000), rat anti E-cadherin (ECCD2; Calbiochem; 205604, 1:1000), rat anti-CD31/Pecam-1 (BD Biosciences; 557355, 1:250), chicken anti-β-gal (Abcam; ab9361, 1:2000) and rabbit anti-Ki67 (Abcam; ab16667, 1:100).

Techniques: Expressing, In Situ Hybridization, Immunofluorescence, Staining

Journal: Development (Cambridge, England)

Article Title: Revising the embryonic origin of thyroid C cells in mice and humans

doi: 10.1242/dev.126581

Figure Lengend Snippet: Expression of Foxa1 and Foxa2 in neuroendocrine thyroid cancer. (A) Comparison of microarray data sets from human medullary thyroid carcinoma (MTC; n =9) and follicular variant of papillary thyroid cancer (FPTC; n =7). FOXA1 / FOXA2 expression levels relative to mRNAs of biomarkers for the respective tumor type (CALC, calcitonin; CHGA, chromogranin A; TG, thyroglobulin; TPO, thyroid peroxidase) are shown as the difference from the mean of all genes in each data set. Asterisks indicate P <0.001. Error bars represent s.d. (B-F) Immunofluorescence staining of Foxa1 and Foxa2 in tumor tissues from an MTC patient. Sections were co-stained for calcitonin to distinguish tumor from stromal cells. (B) Nodules from primary tumor (P) and lymph node metastasis (M) at low magnification. (C,D) Detailed views of tumor tissues with different cell densities. Arrows indicate accumulation of Foxa1 predominantly in nuclei (in C) or cytoplasm (in D) associated with low and high cellularity, respectively. (E) Nuclear accumulation of Foxa1 differs between peripheral (p) and central parts (c) of tumor nodule. (F) Expression levels of Foxa2 in peripheral (p) versus central parts (c) of tumor nodule. (G) Distinct expression pattern of Foxa1 and Foxa2 in MTC related to tumor tissue organization (depicted from data shown in E,F). Increase/decrease refers to nuclear accumulation. (H) Distribution of proliferating cells in tumor nodule. Arrows indicate clusters of Ki67+ cells in the peripheral zone (p) facing the tumor stroma. Ki67+ cells are not present in the nodule center (c). Scale bars: 100 µm (B); 25 µm (C,D); 50 µm (E,F,H).

Article Snippet: Cryosections (10 µm thick) collected on glass slides were subjected to pairwise double immunolabeling as indicated with the following antibodies: rabbit anti-Nkx2-1 (Biopat, Italy; PA0100, 1:1000), rabbit anti-calcitonin (DAKO; A0576, 1:500), guinea pig anti-Foxa1 (kindly provided by Jeffrey Whitsett, Cincinnati Children's Hospital, OH, USA; 1:2000; applied for mouse tissue only), mouse anti-Foxa1 (Seven Hills Bioreagents; WMAB-2F83, 1:1000; applied for human specimens only), rabbit anti-Foxa2 (Seven Hills; WRAB-FOXA2, 1:2000), rat anti E-cadherin (ECCD2; Calbiochem; 205604, 1:1000), rat anti-CD31/Pecam-1 (BD Biosciences; 557355, 1:250), chicken anti-β-gal (Abcam; ab9361, 1:2000) and rabbit anti-Ki67 (Abcam; ab16667, 1:100).

Techniques: Expressing, Microarray, Variant Assay, Immunofluorescence, Staining

Journal: Development (Cambridge, England)

Article Title: Revising the embryonic origin of thyroid C cells in mice and humans

doi: 10.1242/dev.126581

Figure Lengend Snippet: Altered Foxa2 expression in invasive MTC tumor cells. Sections were co-stained for E-cadherin (E-cad) to evaluate concurrent epithelial-to-mesenchymal transition. (A) Overview of tumor invasive zone. Large arrows indicate clusters of infiltrating tumor cells close to a large tumor nodule (asterisk); small arrows indicate distanced single tumor cells. Note that Ki67+ cells are not enriched in the tumor stroma compared with solid tumor nodule. (B) Redistribution of E-cadherin from the surface of single tumor cells (arrows). (C) Coordinated loss of Foxa2 in invasive tumor cells. Three areas (dashed boxes) of motif are shown in C′-C‴ with alternating channels for improved resolution: Foxa2 and E-cad (upper panel); Foxa2 and DAPI (middle panel); and E-cad and DAPI (lower panel). Arrowheads indicate different cell phenotypes further commented on in the Results. (D) Preserved expression of Foxa1 confined to the cytoplasm in infiltrating tumor cells. Note variable expression of calcitonin in both single tumor cells (arrow) and small aggregates (arrowhead). Scale bars: 50 µm (A); 25 µm (B-D).

Article Snippet: Cryosections (10 µm thick) collected on glass slides were subjected to pairwise double immunolabeling as indicated with the following antibodies: rabbit anti-Nkx2-1 (Biopat, Italy; PA0100, 1:1000), rabbit anti-calcitonin (DAKO; A0576, 1:500), guinea pig anti-Foxa1 (kindly provided by Jeffrey Whitsett, Cincinnati Children's Hospital, OH, USA; 1:2000; applied for mouse tissue only), mouse anti-Foxa1 (Seven Hills Bioreagents; WMAB-2F83, 1:1000; applied for human specimens only), rabbit anti-Foxa2 (Seven Hills; WRAB-FOXA2, 1:2000), rat anti E-cadherin (ECCD2; Calbiochem; 205604, 1:1000), rat anti-CD31/Pecam-1 (BD Biosciences; 557355, 1:250), chicken anti-β-gal (Abcam; ab9361, 1:2000) and rabbit anti-Ki67 (Abcam; ab16667, 1:100).

Techniques: Expressing, Staining