Fig. 8: Expression of BrafV600E results in upregulation of cell cycle inhibitors p57Kip2, p27Kip1 and the senescence markers p16INK4a and p21.
a–h In situ hybridisation of sagittal sections through embryonic pituitary gland of Wt (a–d) and Prop1:Cre;BrafV600E/+ mutant pituitaries (e–h) at E16.5 reveals significantly increased p57Kip2, p16INK4a, p21 and p27Kip1 mRNA transcripts in mutant pituitaries. p57Kip2 transcripts were upregulated and its expression ___domain was expanded ventrally (arrows in e). p16INK4a mRNA transcripts were upregulated in the ventral portion of the AL (arrows in f). p21 transcripts were located in the AL in mutant pituitaries (arrows in g) and absent in Wt (c), although p21 was expressed in the basisphenoid bone (bb, arrowheads in c, g) in Wt. Expression of p27Kip1 was significantly upregulated in the ventral side of the AL (arrows in h) compared to Wt (arrows in d). The IL was negative for p27Kip1 (arrowheads in d). i–p Representative coronal sections at P1 of Wt i–l and Prop1:Cre;BrafV600E/+ mutant pituitaries m–p. p57Kip2 mRNA transcripts were localised mainly in the IL and the MZ (arrowheads in i) while in the mutants expression was found ectopically throughout the AL (arrows in m). Expression of p16INK4a (arrows in n), p21 (arrows in o) and p27Kip1 (arrowheads in p) was upregulated compared to the corresponding Wt pituitaries (j–l). Images are representative of five embryos per genotype. Asterisks indicate tissue cavities within the AL. q Quantitative RT-qPCR from P1 pituitary glands revealed increased mRNA expression of p57Kip2 (4.6-fold increase), p16INK4a (17.81-fold increase), p21 and p27Kip1 compared to Wt (****p < 0.0001; **p < 0.01; *p < 0.05 unpaired two-tailed Student’s T-test. Data represented as mean ± SEM from n = 4 pituitaries or 5 pituitaries for p16INK4a per genotype). AL anterior lobe, IL intermediate lobe, MZ marginal zone, PL posterior lobe. Scale bars in h, n & p represents 200 µm.
