Journal: Scientific Reports

Article Title: β-catenin regulates the formation of multiple nephron segments in the mouse kidney

doi: 10.1038/s41598-019-52255-w

Figure Lengend Snippet: ( A – C ) In the β-catenin loss-of-function mutant kidney by Osr2Cre , the podocytes, proximal tubules, and loops of Henle show little β-catenin staining, suggesting that Ctnnb1 , the gene encoding β-catenin, was deleted by Osr2Cre . Cells lacking β-catenin are able to form Wt1+ podocytes ( A ), LTL + proximal tubules ( B ), and Slc12a1+ loops of Henle ( C ). Note, however, that the β-catenin mutant kidney shows aberrant arrangement of Wt1+ podocytes, weaker LTL staining, and thinner Slc12a1+ tubules, suggesting developmental defects. ( D ) In the β-catenin mutant kidney, β-catenin is still present in Slc12a3+ distal tubules, consistent with the fact that Osr2Cre does not target distal tubules. Images are representative of three independent experiments. Stage E18.5. Scale bar: 100 μm.

Article Snippet: Embryonic kidneys at E18.5 were fixed with 4% paraformaldehyde in PBS at room temperature for 10 min, incubated in 10% sucrose in PBS at 4 °C overnight, and imbedded in OCT. Cryosections of 10 or 12 μm were incubated in PBS containing 0.1% Triton x-100, 5% heat-inactivated sheep serum, and the following antibodies: Jag1 (TS1.15 H, rat, 1:20, Developmental Studies Hybridoma Bank), Wt1 (sc-7385, mouse IgG1, 1:100, Santa Cruz), GFP (GFP-1020, chick IgY, 1:500, Aves Labs), Slc12a3 (HPA028748, rabbit, 1:300, Sigma), LTL (FL-1321, FITC, 1:200, Vector Laboratories), Slc12a1 (18970–1-AP, rabbit, 1:200, Proteintech), β-catenin (71–2700, rabbit, 1:500, Invitrogen), β-catenin (sc-7963, mouse IgG1, 1:50, Santa Cruz), Pecam1 (sc-18916, rat, 1:300, Santa Cruz), Mafb (HPA005653, rabbit, 1:300, Sigma), Akap12 (25199–1-AP, rabbit, 1:300, Proteintech), Cdh6 (HPA007047, rabbit, 1:300, Sigma), Hnf4a (ab41898, mouse IgG2a, 1:500, Abcam), Pax2 (21385–1-AP, rabbit, 1:200, Proteintech), Slc5a2 (HPA041603, rabbit, 1:100, Sigma) Fluorophore-conjugated secondary antibodies (Thermo Fisher Scientific or Jackson ImmunoResearch Laboratories) were used at a 1:500 dilution.

Techniques: Mutagenesis, Staining

Journal: Scientific Reports

Article Title: β-catenin regulates the formation of multiple nephron segments in the mouse kidney

doi: 10.1038/s41598-019-52255-w

Figure Lengend Snippet: β-catenin is required for the formation of differentiated proximal tubule cells with strong LTL staining. Hnf4a marks both presumptive and differentiated proximal tubules. In the control kidney, presumptive proximal tubules show strong Cdh6 signal and weak LTL staining while differentiated proximal tubules show weak Cdh6 signal and strong LTL staining. In the β-catenin loss-of-function mutant kidney by Osr2Cre , all Hnf4a+ cells show strong Cdh6 signal, failing to form differentiated proximal tubules with strong LTL staining. Images are representative of three independent experiments. Stage E18.5. Scale bar: 100 μm.

Article Snippet: Embryonic kidneys at E18.5 were fixed with 4% paraformaldehyde in PBS at room temperature for 10 min, incubated in 10% sucrose in PBS at 4 °C overnight, and imbedded in OCT. Cryosections of 10 or 12 μm were incubated in PBS containing 0.1% Triton x-100, 5% heat-inactivated sheep serum, and the following antibodies: Jag1 (TS1.15 H, rat, 1:20, Developmental Studies Hybridoma Bank), Wt1 (sc-7385, mouse IgG1, 1:100, Santa Cruz), GFP (GFP-1020, chick IgY, 1:500, Aves Labs), Slc12a3 (HPA028748, rabbit, 1:300, Sigma), LTL (FL-1321, FITC, 1:200, Vector Laboratories), Slc12a1 (18970–1-AP, rabbit, 1:200, Proteintech), β-catenin (71–2700, rabbit, 1:500, Invitrogen), β-catenin (sc-7963, mouse IgG1, 1:50, Santa Cruz), Pecam1 (sc-18916, rat, 1:300, Santa Cruz), Mafb (HPA005653, rabbit, 1:300, Sigma), Akap12 (25199–1-AP, rabbit, 1:300, Proteintech), Cdh6 (HPA007047, rabbit, 1:300, Sigma), Hnf4a (ab41898, mouse IgG2a, 1:500, Abcam), Pax2 (21385–1-AP, rabbit, 1:200, Proteintech), Slc5a2 (HPA041603, rabbit, 1:100, Sigma) Fluorophore-conjugated secondary antibodies (Thermo Fisher Scientific or Jackson ImmunoResearch Laboratories) were used at a 1:500 dilution.

Techniques: Staining, Mutagenesis