Elevated levels of Sox9 and Angptl1 levels along with Plods and Lox are normalized in Tbx1 ^neo2/neo2 thymic lobes following minoxidil treatments. (A) E13.5 thymuses were processed for immunofluorescence using wild-type (Tbx1^+/+, n = 2), Tbx1^neo2/neo2-carrier (n = 4), and Tbx1^neo2/neo2-minoxidil treatment groups. The drug treatment is listed under the genotype name, and the embryo identifier is shown with a # sign. Staining was done with antibodies detecting Sox9 (green), Pdgfra (red), and DAPI (purple), the latter used to detect the nucleus. Confocal microscopy was performed to more precisely delineate Sox-9 expression in the indicated embryonic thymuses. Some of the images are the same as those in Fig. 4 B, with individual staining shown in this Supplemental figure. The composite was provided in Fig. 4 B. (B) The indicated embryos were done with antibodies specific for Angptl1 (red) and Nrp1 (purple), along with a nuclear marker (DAPI, blue), to compare the levels of Angptl1. Immunofluorescence was used to visualize these levels. Sections are representative of five independent samples/genotype. (C) A heat map was established comparing the transcript levels in mesenchymal cells for genes coupled to Plods and lox, enzymes involved in collagen cross-linking. These were separated into four clusters. The relative z-score and identification of the mesenchymal subclusters are illustrated. Genes with P < 0.00001 and logbase₂ differences >1.5 are shown. (D) Quantitation of Angptl1 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.