Sox9-expressing mesenchymal cells accumulate in the capsular region of embryonic thymus from mouse models of 22q11.2DS. (A) RT-qPCR was performed with RNA isolated from the indicated embryonic thymuses obtained from the carrier or minoxidil-treated pregnant dams. Probes specific for Col2a1, Col9a2, Col11a1, Acan, and Sox9 were used. (B–D) Fetal thymuses were isolated from Tbx1^+/+ and Tbx1^neo2/neo2 E13–13.5 embryos from carrier and minoxidil-treated pregnant mice and used for immunofluorescence. (B) Antibodies against Sox9 (green), Pdgfra (red), and a nuclear marker (DAPI, blue) were used to compare Sox9 expression in embryonic thymic lobes. Sections are representative of five to eight independent samples/genotype with individual colors provided per sample. Note that the samples in Fig. 4 B are duplicated in the Fig. S5 B for comparative purposes. (C) Quantitation of the number of Sox9-expressing cells per total DAPI-positive cells was determined using one-way ANOVA. The embryonic thymuses included Tbx1^+/+-carrier (n = 5), Tbx1^neo2/neo2-carrier (n = 6), and Tbx1^neo2/neo2-minoxidil (n = 9) treatment groups. (D) Confocal microscopy was used to visualize the Sox9⁺ cells in the thymic capsule using the same sections as in B. Sections shown are representative of five to eight independent samples/genotype. (E) The number of Sox9⁺ cells in the capsular region was enumerated using a defined area of focus for all the immunohistochemistry (IHC) images. Statistical significance was determined as in C. (F) Antibodies against Col2a1(purple), along with Pdgfra (red) and a nuclear marker (DAPI, blue), were used to compare the levels of Col2a1. Sections are representative of five independent samples/genotype. (G) Quantitation of Col2a1 levels was performed by comparing the mean fluorescent intensity of a defined area among all the groups analyzed. Statistical analyses were done using one-way ANOVA. This was determined with thymuses from Tbx1^+/+-carrier (n = 3), Tbx1^neo2/neo2-carrier (n = 6), and Tbx1^neo2/neo2-minoxidil (n = 5) groups. Statistically significant differences were established by one-way ANOVA (Brown–Forsythe and Welch tests). (H) Postnatal human thymuses from non-22q and 22q11.2DS patients were processed and visualized with H&E staining. (I and J) The human thymuses were also analyzed by immunofluorescence with an antibody specific for human COL2a1 along with a nuclear marker (DAPI, blue). (J) Confocal microscopy was used to visualize the exact location of the elevated COL2a1 expression. The different quadrants were stitched to create a composite image.