Changes in global 5hmC and 5mC levels and gene expression of cytosine modifying enzymes within the bursa of Fabricius during embryonic and neonatal development. A. Immunofluorescent staining of sections through embryonic day (ED) 16, ED18, one (D1) and seven days post hatch (D7) bursa of Fabricius. White dashed outlines delineate an individual follicle and arrows indicate epithelial structures. Scale bar = 100 μM. B. Dot blot assay using ten-fold dilutions of bursa genomic DNA from various developmental stages probed with an antibody directed to either 5hmC or 5mC. C. Real time analysis of cytosine modifiying enzymes AID, Tet 1 and Tet 2 mRNA expression levels during the development of the bursa of Fabricus. n.s. =non-significant, *p < 0.05, **p < 0.01, ***p < 0.005 as determined by ANOVA and post-hoc tests. n = 4-5 birds per group.