Fig. 7
Deletion of the tumour suppressor Apc results in β-catenin-accumulating senescent clusters with reduced SASP. a Brightfield and haematoxylin–eosin pictures of wild-type and Hesx1Cre/+;Apcfl/fl pituitaries, and Hesx1Cre/+;Ctnnb1lox(ex3)/+ pituitary tumours. Note that the Apc-deficient pituitary is enlarged but no tumours are apparent and it is histologically normal. The age of the mice is indicated in weeks. Scale bar: 1 mm. b Double immunostaining revealing the expression of p21, phospho-IκBα and phospho-DNA-PKcs in the β-catenin-accumulating clusters, indicating cell cycle inhibition and activation of the NFκB and DNA damage response pathways, in both Hesx1Cre/+;Apcfl/fl and Hesx1Cre/+;Ctnnb1lox(ex3)/+ pituitaries at 18.5 dpc. Note that the clusters are smaller in the Hesx1Cre/+;Apcfl/fl compared with the Hesx1Cre/+;Ctnnb1lox(ex3)/+ pituitaries. Scale bar: 50 μm. c qRT-PCR analysis showing the relative expression of several genes in Hesx1Cre/+;Apcfl/fl relative to Hesx1Cre/+; Ctnnb1lox(ex3)/+ pituitaries at 18.5 dpc. The expression of the WNT pathway targets Axin2 and Lef1 are similar between the two genotypes, but several senescence and SASP factors show reduced expression levels in the Hesx1Cre/+;Apcfl/fl compared with the Hesx1Cre/+;Ctnnb1lox(ex3)/+ pituitaries. Bars represent the mean, and error bars represent standard error of the mean (SEM) of three biological replicates. *p < 0.05, **p < 0.01, Student’s t test. d In situ hybridisation on histological sections of embryos at 14.5 dpc showing the expression of Axin2 in small cell clusters and very reduced or absent detection of Bmp4, Fgf3 and Shh in the Apc-deficient pituitaries. Scale bar: 100 μm. e Triple immunostaining revealing the expression of β-catenin, SOX9 and EMCN. Note that the large areas of EMCN+;SOX9+ cells surrounding the β-catenin-accumulating clusters are not present in the Hesx1Cre/+;Apcfl/fl pituitaries. Scale bar: 50 μm. f Quantification of the EMCN+ areas demonstrating that wild-type and Apc-deficient pituitaries show similar levels, which are significantly increased in the Hesx1Cre/+;Ctnnb1lox(ex3)/+ pituitaries. Bars represent the mean, and error bars represent standard error of the mean (SEM) of three biological replicates. ***p < 0.001, one-way ANOVA. All results are representative of at least three biological replicates
