Fig. 5.

Disruption of the Sall1-NuRD interaction causes accelerated differentiation of renal progenitor cells. (A) Sections of wild-type and mutant kidney at E13 and E18 stained for Six2 and cytokeratin, and with DAPI. The number of Six2⁺ caps surrounding UB tips looks similar in the wild type and mutant at E13. However, by E18 the number of Six2⁺ caps is reduced and Six2⁺ cells are in structures that resemble renal vesicles. (B) Quantification of the number of Six2⁺ caps/UB tip in E13, E15 and E18 kidney. The number of Six2⁺ caps/tip is reduced by E18 in the ΔSRM homozygous mutant (*P<0.05). Quantification of the number of renal vesicles (RVs)/UB tip in wild-type and mutant kidney at E13 and E15 (see C for E18). There are significantly more RVs/tip in the mutant at E13, E15 and E18 (*P<0.05, two-tailed t-test). Quantification performed on 10 non-sequential sections for each stage and genotype; E13, n=3; E15, n=2; E18 wild type, n=2, E18 mutant, n=4. (C) E18 wild-type and mutant kidney sections stained for Six2 and NCAM, and with DAPI. In the wild-type kidney, Six2⁺ cells are in caps surrounding the ureter. In the mutant, there are RV structures that are Six2⁺ NCAM⁺ towards the periphery of the kidney. (D) E18 kidney sections stained for Six2 and Rcdh, and with DAPI (left). Rcdh is found in the luminal side of RVs as well as in further differentiated structures. Examples of RVs are marked by asterisks in the wild-type and mutant kidney, although the Rcdh⁺ vesicles in the mutant kidney are also Six2⁺. E18 kidney sections stained for WT1 and Lhx1, and with DAPI (right). Properly polarized RVs are marked by asterisks in the wild-type and mutant kidney. The majority of RVs are properly polarized; however, we observed some toward the periphery of the kidney that do not properly polarize (arrow). (E) In situ hybridization for Pax8 at E13, E15 and E18. Pax8 mRNA expression is increased in developing differentiating structures at three developmental stages. Arrows at E18 indicate RVs in the wild type and mutant, with the RV in the mutant expressing strong Pax8 towards the periphery of the kidney. n=2 for each embryonic stage. (F) Immunofluorescence staining of E18 kidneys for Pax8 and NCAM, and with DAPI. At E18 in the wild type, Pax8 is detected in NCAM/Pax8 double-positive pre-tubular aggregates, developing RVs and comma/S-shaped bodies, with low expression in the UB; it is undetectable in the cap mesenchyme (arrowheads). In the mutant, Pax8 is detectable in the region of the cap mesenchyme ventral to the UB. These Pax8/NCAM double-positive structures are forming aggregates or RV-like structures toward the periphery of the kidney, indicative of induced mesenchyme and epithelial differentiation (four examples show the variation in phenotypes observed, with arrows indicating Pax8-positive aggregates in the first example). n=4 sections from three different embryos for each genotype. Scale bars: 50 µm in A,C,D,F; 25 µm in E.