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. 2023 Oct 4;11:1265407. doi: 10.3389/fcell.2023.1265407

FIGURE 1.

FIGURE 1

Transcription loops. (A) Schematics of a transcription loop (TL) formed by RNAPIIs moving along a gene and carrying nascent RNA transcripts. (B) Visualization of four thyroid-specific genes by RNA-FISH. In contrast to the lowly expressed genes Tshr and Mct8 (B1, B2), the other two genes, Tpo and Tg (B3, B4), exhibit larger TLs with sizes correlated to their expression levels. Length and expression level of the genes are indicated on the left and right of the panels, respectively. For better comparison, TLs are shown as grey scale images in the insertions. Note, that in case of the Mct8 gene located on X chromosome, there is only one signal, because the tissue originated from a male mouse. For the number of analysed nuclei, see Table 1. (C) RNA-FISH with an oligoprobe for Tg mRNA labels TLs (green arrows), single mRNAs in the nucleoplasm (green arrowheads) and in the cytoplasm (red asterisk). (D,E) Immunostaining of TG (green) highlights the cytoplasm of thyrocytes (D), which is densely packed with remarkably hypertrophic cisterns (C) of endoplasmic reticulum (E). (F) Histogram showing comparative Tg transcript levels in thyroids of young mice (P1 and P14) in comparison to adult mice. Since thyroid tissue includes 40% of non-thyrocyte cells, as well as chunks of practically inseparable parathyroid gland, the qPCR values were normalized to the transcription level of the thyroid specific transcription factor Pax8 to avoid erroneous results. Error bars are SDM, three biological replicates were analyzed per developmental stage The observed changes were not significant (p > 0.05) as determined by one-way ANOVA with Tukey’s multiple comparisons post hoc. (G) Examples of thyrocytes from P1 (top) and P14 (bottom) thyroids. The left column shows follicles formed by thyrocytes with various degree of Tg TL development; the right and middle columns show thyrocytes at a higher magnification with fully or underdeveloped Tg TLs. Arrows point at nuclei with underdeveloped loops. Note that in some nuclei only one allele is active. For P1 and P14, image stacks of about 50 and 70 nuclei, respectively, were acquired. Images on B-D and G are projections of 3–5 µm confocal stacks; RNA-FISH and immunostaining signals are green; nuclei are counterstained with DAPI (red). Scale bars: B, C, E, 2 μm; D, 70 μm; G, follicle overviews on the left, 10 μm, zoomed in nuclei, 5 µm.