Vil-Cre specific Schlafen 3 knockout mice exhibit sex-specific differences in intestinal differentiation markers and Schlafen family members expression levels

Abstract

The intestinal epithelium requires self-renewal and differentiation in order to function and adapt to pathological diseases such as inflammatory bowel disease, short gut syndrome, and ulcers. The rodent Slfn3 protein and the human Slfn12 analog are known to regulate intestinal epithelial differentiation. Previous work utilizing a pan-Slfn3 knockout (KO) mouse model revealed sex-dependent gene expression disturbances in intestinal differentiation markers, metabolic pathways, Slfn family member mRNA expression, adaptive immune cell proliferation/functioning genes, and phenotypically less weight gain and sex-dependent changes in villus length and crypt depth. We have now created a Vil-Cre specific Slfn3KO (VC-Slfn3KO) mouse to further evaluate its role in intestinal differentiation. There were increases in Slfn1, Slfn2, Slfn4, and Slfn8 and decreases in Slfn5 and Slfn9 mRNA expression that were intestinal region and sex-specific. Differentiation markers, sucrase isomaltase (SI), villin 1, and dipeptidyl peptidase 4 and glucose transporters, glucose transporter 1 (Glut1), Glut2, and sodium glucose transporter 1 (SGLT1), were increased in expression in VC-Slfn3KO mice based on intestinal region and were also highly female sex-biased, except for SI in the ileum was also increased for male VC-Slfn3KO mice and SGLT1 was decreased for both sexes. Overall, the variations that we observed in these VC-Slfn3KO mice indicate a complex regulation of intestinal gene expression that is sex-dependent.

Fig 1. Gene targeting scheme for generating VC-Slfn3KO mice.

The Slfn3^βgeo mice had a knockout first, lacZ-tagged insertion, and conditional allele of Slfn3. These mice were bred with B6.129S4-Gt(ROSA)26Sor^tm2(FLP*)Sor/J (Flpo) mice, resulting in Slfn3^flox mice. The Slfn3^flox mice were then bred with B6.Cg-Tg(Vil1-cre)1000Gum/J (VilCre) mice to create VC-Slfn3KO mice.

Fig 2. Confirmation of Slfn3 knockout in the Vil-Cre intestinal epithelial specific cells.

RNA in situ hybridization experiments were performed by Advanced Cell Diagnostics, Inc. using RNAscope technology. (A) Images of Ctrl and VC-Slfn3KO villi with arrows indicating Slfn3+ intestinal epithelial cells. (B) Bar graph quantification of the average number intestinal epithelial cells that were Slfn3+ per villi (n = 3 mice/group, average of 22 villi per sample, *p ≤ 0.05).

Fig 3. Villi length and crypt depth in VC-Slfn3KO mice.

VC-Slfn3KO mice do exhibit (A) villus length and (B) crypt depth that is different from Ctrl mice. (n = 4 mice/group, average of 13–21 villi per sample, average of 14–24 crypts per sample).

Fig 4. Slfn family expression changes based on intestinal epithelial specific loss of Slfn3, intestinal region, and sex.

Total RNA was isolated from intestinal mucosa of the duodenum, jejunum, and ileum of control (Ctrl) and VC-Slfn3KO (KO) mice. The mRNA expression of (A) Slfn1, (B) Slfn2, (C) Slfn4, (D) Slfn5, (E) Slfn8, and (F) Slfn9 was analyzed by qPCR using RPLP0 as a reference control gene. (n = 9–48, *p<0.05 to respective Ctrl).

Fig 5. Intestinal differentiation markers SI, Vil1, and Dpp4 vary expression due to intestinal epithelial specific loss of Slfn3, intestinal region, and sex.

Total RNA was isolated from intestinal mucosa of the duodenum, jejunum, and ileum of control (Ctrl) and VC-Slfn3KO (KO) mice. The mRNA expression of (A) SI, (B) Vil1, and (C) Dpp4 was analyzed by qPCR using RPLP0 as a reference control gene. (n = 9–50, *p<0.05 to respective Ctrl). Single cell suspension of intestinal cells from the duodenum, jejunum, and ileum were analyzed by flow cytometry in Ctrl and VC-Slfn3KO mice. (D) Representative dot plot gating on CD3^- SI⁺ from jejunal intestinal cells were then further gated on EpCam⁺ cells by histogram. (E) CD3^- SI⁺ EpCam⁺ cells were normalized to control duodenum cell numbers within each experiment and by sex. (male n = 14–15, female n = 9–14; *p<0.05 to respective Ctrl).

Fig 6. Glucose transporters SGLT1, Glut1, and Glut2 change expression due to intestinal epithelial specific loss of Slfn3, intestinal region, and sex.

Total RNA was isolated from intestinal mucosa of the duodenum, jejunum, and ileum of control (Ctrl) and VC-Slfn3KO (KO) mice. The mRNA expression of (A) SGLT1, (B) Glut1, and (C) Glut2 was analyzed by qPCR using RPLP0 as a reference control gene. (n = 9–41, *p<0.05 to respective Ctrl).