Excision of exon 3 from Ctnnb1tm1Mmt by Vil1-Cre leads to embryonic lethality
To further understand the role CTNNB1 plays during intestinal cancer development, mice carrying a Ctnnb1tm1Mmt, a conditional, stabilizing mutation in Ctnnb1tm1Mmt, were crossed with mice carrying Vil1-Cre. Mice carrying both Vil1-Cre and Ctnnb1tm1Mmt alleles were not recovered at birth suggesting that they were embryonic lethal (Table 1).
Table 1
Genotypes resulting from Ctnnb1tm1Mmt by Vil1-Cre crosses.
Days Post Coitus | Number of Total Embryos/Mice | Ctnnb1tm1Mmt % of Total | Ctnnb1tm1Mmt, Vil1-Cre % of Total | Ctnnb1tm1Mmt, Vil1-Cre Viable % of Total |
E10.5 | 29 | 52 | 57 | 100 |
E11.5 | 9 | 56 | 44 | 100 |
E12.5 | 7 | 43 | 57 | 43 |
E13.5 | 52 | 54 | 46 | 17 |
PND21 (Weaning) | 27 | 27 | 0 | 0 |
To further investigate the lack of viable offspring carrying Vil1-Cre and Ctnnb1tm1Mmt, embryos were collected between E10.5 and E13.5 and visually analyzed for aberrant development and reabsorption. Double mutants showed dramatic differences in development by E12.5 (Fig. 1A), with hemorrhaging and visible size differences. By E13.5 most of the embryos were at various stages of reabsorption.
Vil1 promoter expresses CRE in the yolk sac epithelium
In order to identify possible causes of the embryonic lethality, the mice carrying the (ROSA)26Sortm1Sor reporter were crossed with mice carrying Vil1-Cre. Embryos and extraembryonic tissues were collected and stained for beta-galactosidase activity, which showed beta-galactosidase activity in the yolk sac epithelium (Fig. 1B). No expression was observed in embryos. This result is consistent with lethality at E12.5 being caused by abnormal function of extraembryonic tissues. Consistent with these results, previous studies have shown that CTNNB1 signaling in the yolk sac epithelium plays an essential role in the activation and maintenance of embryonic hematopoiesis [9, 10].
Inflammation pathways are altered by stabilization of CTNNB1 in the yolk sac epithelium
To identify functional changes in the yolk sac epithelium, RNAseq was performed on RNA isolated from yolk sac membranes from wildtype and double mutant Ctnnb1tm1Mmt, Vil1-Cre embryos at E11.5 - E12.5. Hierarchical clustering of differentially expressed genes separated the mutant from wildtype samples with two mutant samples appearing to be outliers (Fig. 2A), which was confirmed by PCA (Fig. 2B). After the outliers were removed, PCA and hierarchical clustering showed clustering by genotype (Fig. 2C, D).
The functional categories of the differentially expressed genes were analyzed using Ingenuity Pathway Analysis (IPA). The differentially expressed genes were enriched for pathways connected to inflammatory responses (Fig. 3A). The top affected pathways were Granulocyte Adhesion and Diapedesis and Agranulocyte Adhesion and Diapedesis, both of which are associated with inflammation and macrophage activity. Inflammatory response was the top functional category (p = 1.84 × 10− 30, Fig. 3B), and the top predicted upstream regulator of the differentially regulated genes was tumor necrosis factor (TNFA) (p = 2.27 × 10− 41, Fig. 3C).