Detection of YS‐derived ATMs in mouse

To label YS‐derived Mϕs, we adapted the lineage‐tracing protocol described elsewhere [11, 18]. In brief, we crossed the Cx3cr1^{tm21.(cre/ERT2)Jung} (The Jackson Laboratory, Bar Harbor, ME, USA) mouse line, which carries a tamoxifen inducible Cre recombinase under the control of the Cx3cr1 promoter, with the Gt(ROSA)26Sor tm1^{(CAG−tdTomato−EGFP*)Etes} (The Jackson Laboratory) reporter line. The latter mouse line expresses red fluorescent Tomato protein, along with a stop codon–blocked eGFP sequence. When Cre recombinase is active, the sequence that encodes red fluorescent Tomato protein is excised, along with the stop codon that blocks eGFP expression. As a result, cells with an active Cx3cr1 promoter at the time of tamoxifen injection will express eGFP, whereas other cells maintain the expression of the red fluorescent Tomato protein. To avoid contamination with maternal Mϕs, we used mothers that lacked Cre recombinase, and, thus, maternal Mϕs remained fluorescent red. Both mouse lines were kindly provided by Dr. Bernd Baumann and Prof. Dr. Jan Tuckermann (University of Ulm, Ulm, Germany).

To measure ATM proliferation and to characterize CX₃CR1⁺ ATMs, we used C57/BL6 male mice at age postnatal d 7 and 56 (The Jackson Laboratory). All mouse strains were kept under specific pathogen–free conditions, and experiments were carried out according to local legislation.