Phase relationship between co-cultured fibroblasts and tissues.

(A) Experimental schema. The medium of the Bmal1-RED fibroblasts was replaced with luciferin medium, resetting the phase. Lung tissue, adrenal gland tissue, and thyroid gland tissue were isolated from Bmal1^ELuc-A1 mice and cultured in luciferin medium until start of co-culture. At various time points after the medium change on the Bmal1-RED fibroblast culture, a tissue explant on a culture insert was added to the fibroblasts and bioluminescence recordings were started. Open bar indicates light period or projected light period. Black bar indicates dark period or projected dark period. (B) Medium change synchronized the phase of Bmal1-RED fibroblasts and induced a small amplitude rhythm in the cell population. (C–E) Co-culture with adrenal gland tissue induced a large amplitude rhythm in fibroblasts. The peak times of fibroblasts induced by the adrenal gland tissue were independent of the phase of the adrenal gland tissue or the time after the medium change, but were dependent on the co-culture start time. The time of application of adrenal gland tissue is shown by an arrowhead. Bioluminescence recordings of Bmal1-RED fibroblasts and adrenal gland tissue are plotted by red lines and green lines, respectively. Shadings show projected dark periods for animals. (F) Relationship between co-culture start time and phases of tissues. Peak times of adrenal gland tissue (green circles) or lung tissue (green squares) are plotted against co-culture start time. Peak times of both tissues were not affected by the co-culture start time. The co-culture start time after the medium change of Bmal1-RED fibroblasts is shown on the x axis. Peak times of tissues after the last lights on for animals are shown on the y axis. (G) Relationship between the co-culture start time and phases of Bmal1-RED fibroblasts. Peak times of Bmal1-RED fibroblasts co-cultured with adrenal gland tissue (red circles) or lung tissue (red squares) are plotted against co-culture start time. Co-culture start time on the x axis and peaks of Bmal1-RED fibroblasts on the y axis were measured relative to the time of the medium change of fibroblasts. Adrenal, adrenal gland. Thyroid, thyroid gland.