Fig. 4: Prenatal 5HT influences cell cycle length of the “bridge” progenitors in embryonic adrenal glands.

a Pregnant Htr3a^EGFP+/− females were administered 5HTP at E11.5; at E12.5, females received 5HTP together with 5-Ethynyl-2′-deoxyuridine (EdU); in 4 h females received 5-Chloro-2′-deoxyuridine (CldU); embryos were harvested in 4 h after CldU injection at E12.5. This allows identifying the proportions of cells, which incorporated EdU⁺ only, CldU⁺ only, or both thymidine analogues (EdU⁺CldU⁺). b Numbers of all cells (left) and numbers of proliferated cells (right) in populations of SCPs, “bridge” cells, early chromaffin cells, and mature chromaffin cells at E12.5 under the influence of 5HTP in adrenal medulla. Shapiro–Wilk test for normality and unpaired double-sided t-test p-value ns > 0.05, ** < 0.002, biological n = 3 (control SCPs), 4 (control other cell types), 4 (5HTP SCPs), 5 (5HTP other cell types). c Proportions of EdU⁺ only, CldU⁺ only, and EdU⁺CldU⁺ cells in populations of SCPs, “bridge” cells, and early chromaffin cells at E12.5 under the influence of 5HT in adrenal medulla. Note that the cell cycle lengthened in “bridge” cells in the treated condition, as there are more EdU⁺CldU⁺ “bridge” cells. Shapiro–Wilk test for normality and unpaired double-sided t-test p-value ns > 0.05, * < 0.05, biological n = 3 (control SCPs), 4 (control other cell types), 4 (5HTP SCPs), 5 (5HTP other cell types). d Transversal sections of adrenal glands from Htr3a^EGFP+/− embryos immunostained for TH, EGFP (indicating the expression of Htr3a), EdU, and CldU. Arrowheads point at EdU⁺ only, CldU⁺ only, and EdU⁺CldU⁺ cells. e Numbers of cleaved CASP3⁺ cells in adrenal medulla and Organ of Zuckerkandl. Note that the number of cleaved CASP3⁺ cells is exceptionally low. Cell numbers are presented in violin plots, the green line indicates median and the red lines are quartiles. Shapiro–Wilk test for normality and unpaired double-sided t-test (AM) and Mann–Whitney test (ZO), p-value ns > 0.05, biological n = 5. f Proposed mechanism of the paracrine/autocrine regulation of a cell cycle length in HTR3A⁺ “bridge” precursors by 5HT-releasing chromaffin cells in the developing adrenal medulla.