Ccl21a gene expression in the neonatal thymus is restricted to mTECs. (A) Flow cytometric analysis and quantitation of tdTomato expression in total CD45⁺ thymocytes and the indicated thymic stromal populations in postnatal day–10 (P10) heterozygous Ccl21a^tdTom mice (n = 8). Thymic mesenchyme was identified as CD45⁻CD31⁻EpCAM1⁻ cells. Red lines indicate tdTomato staining levels in heterozygous Ccl21a^tdTom mice; gray histograms indicate nonfluorescent control cells. Flow cytometric plots and analysis representative of 3 independent experiments. (B) Analysis of Ccl21a^tdTom expression in P10 EpCAM1⁺Ly51⁺UEA1⁻ cTECs and EpCAM1⁺Ly51⁻UEA1⁺ mTECs. (C) Ccl21a^tdTom expression after subdivision of total mTECs into MHCII^lowCD80^low (mTEC^lo) and MHCII^hiCD80^hi (mTEC^hi) subsets (n = 10). In all cases, error bars represent mean ± SEM. For analysis of data in panel A, multiple comparison analysis was achieved by a 1-way analysis of variance followed by Tukey’s posttest in GraphPad Prism to determine statistical differences. In panel D, confocal microscopy was used to show tdTomato expression in heterozygous Ccl21a^tdTom P10 mice alongside identification of cTECs with either anti-CD205 (green) or mTECs with UEA1 (green); dotted line denotes the CMJ. Scale bars represent 50 μm. Images shown are representative of 3 mice. ****P < .0001. C, cortex; M, medulla; MHC, major histocompatibility complex.