Fig. 1: 5HT-sensitive “bridge” cells and 5HT-producing chromaffin cells are present together in developing adrenal glands.

a, b Transversal section of adrenal gland of Htr3a^EGFP+/− embryos immunostained for TH (marker chromaffin cells and sympathoblasts), EGFP (indicating expression of Htr3a), and 5HT (5-hydroxytryptamine, serotonin) (a), and for SOX10 (marker of SCPs) (b) at E12.5. White arrows point at TH⁺/Htr3a^EGFP+/5HT⁺ (a) and SOX10⁺/Htr3a^EGFP+/TH⁻ (b) cells, indicating formation of “bridge” cells and their differentiation into 5HT⁺ early chromaffin cells. c Cell numbers at E12.5. d Venn diagrams of Htr3a^EGFP+, TH⁺, SOX10⁺ (left), and 5HT⁺ (right) cells at E12.5. e Proportions of TH⁺ cells also positive for Htr3a^EGFP and 5HT (left), and proportions of Htr3a^EGFP+ cells with TH and 5HT signal (right). f, g Immunohistochemistry on the transversal section of adrenal glands of Htr3a^EGFP+/− embryos stained for TH, EGFP, 5HT (f) and for SOX10 (g) at E13.5. White arrows point at TH⁺/Htr3a^EGFP+/5HT⁺ cells in (f) and SOX10⁺/Htr3a^EGFP+/TH⁻ cells in (g) at E13.5. h Cell numbers at E13.5. i Venn diagrams of Htr3a^EGFP+, TH⁺, SOX10⁺ (left), and 5HT⁺ (right) cells at E13.5. j Proportions of TH⁺ cells positive for Htr3a^EGFP, 5HT (left) and proportions of Htr3a^EGFP+ cells positive for TH, 5HT (right). k, l Transversal sections of adrenal glands from Htr3a^EGFP+/− embryos stained for TH, EGFP, and 5HT in (k), and for SOX10 in (l) at E14.5. Yellow arrows point at TH⁻/Htr3a^EGFP+/5HT⁻ cells (k) and SOX10⁺/Htr3a^EGFP−/TH⁻ cells (l) indicating the end of “bridge” differentiation, SOX10⁺ represent supporting glial cells. m Cell numbers at E14.5. n Venn diagrams of Htr3a^EGFP+, TH⁺, SOX10⁺ (left), and 5HT⁺ cells (right) at E14.5. o Proportions of TH⁺ cells positive for Htr3a^EGFP, 5HT (left), and proportions of Htr3a^EGFP+ cells positive for TH and 5HT (rigth). p Schematic representation of the proposed paracrine/autocrine regulation: chromaffin cells release 5HT activating HTR3A receptors on the surface of “bridge” cells (solid lines). Note that few “bridge” cells produce 5HT and can stimulate “bridge” population in an autocrine mode as well as some chromaffin cells can sense 5HT with other 5HT receptors (dashed lines). Scale bars for the insets are 10 µm. Quantification is presented as mean ± SD, biological n = 3. Adrenal medulla (AM) is outlined by the dashed line in all sections. SRG: suprarenal ganglion, SCG: sympathetic chain ganglion.