Figure 1
Attenuated Runx1 P2 expression in P2neo/neo embryos impaired thymus development. (A) RT-PCR analysis of RNA from kidney and stomach of E16.5 WT and P2neo/neo embryos, and from tongue of P1.5 WT and P2neo/neo neonates. P2-mediated transcription in P2neo/neo tissues diminished, whereas P1-mediated transcription was largely unaffected. (B) IHC analysis of glandular stomach of E16.5 WT (left) and P2neo/neo (right) embryos. Runx1 expression is detected in epithelial cells of WT embryo, but missing in P2neo/neo littermate (10× magnification). (C) Reduced size of thymic lobes in P1.5 P2neo/neo mice (right) as compared to WT littermate (left). (D) RT-PCR analysis of Runx1 P1- and P2-mediated transcription in thymus of WT embryos, neonates and young mice. (E) Analysis of Runx1 expression by In-situ hybridization of E15.5 WT (left) and P2neo/neo (right) thymic sections, using the P2-5'UTR probe. P2-derived transcripts are clearly visible in the cortex of E16.5 WT, but not of P2neo/neo (10× magnification). (co) = cortex; (me) = medulla. (F) Western blot analysis of proteins extracted from thymus of E15.5–E17.5 WT and P2neo/neo embryos. Runx1 proteins were not detected in E15.5 P2neo/neo thymus, but gradually accumulated in E16.5 and E17.5 thymi. (G) Northern blot analysis of RNA from thymi of WT and P2neo/neo newborn mice. Whereas P2-mediated transcription (the 4 Kb and 8 Kb transcripts) [30, 31], in P2neo/neo thymocytes was markedly attenuated, P1-mediated transcription (the 2 Kb and 6 Kb transcripts) was apparently unaffected. (H) Reduced thymus cellularity in P2neo/neo embryos and neonates. Five mice of each genotype were analyzed. The difference between the number of thymocytes from WT and P2neo/neo thymus was significant at P < 0.0001 (*) and P < 0.05 (#) by Student's t test.