DEV006544 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig S1. Comparative analysis of genes upregulated by different inducers. The Venn diagram displays percentages of genes upregulated by either Lif, or NGAL, or NHBF (>8-fold compared to freshly isolated metanephric mesenchyme at one or more time points) and the overlap of these gene sets (all affected genes=100%). Note the substantial subgroup of genes induced by all three inducers (24.4%). Also note that each of the inducers activates distinct sets of genes.

• Supplemental Figure S2 -

  Fig. S2. Temporal sequence of gene expression during epithelial differentiation. Heatmap representations of microarray data indicate expression level for a given gene at the indicated time points (day 0-7) with inducers (Lif, NGAL, NHBF) or at baseline (CON). The corresponding class (B or C) is indicated in blue. Bar graphs display triplicate real-time RT-PCR confirmation of microarray data on biologically distinct metanephric mesenchymes after induction with Lif (20-30 pooled samples). Class B genes represent transcripts expressed in pretubular aggregates in vivo, whereas class C genes include multiple transcripts expressed in polarized renal epithelia.

• Supplemental Figure S3 -

  Fig. S3. In situ hybridizations for class B genes Wnt4, Pax8 and Frzb reveal localization in pretubular aggregates in kidneys from E15.5 rat embryos. UB, ureteric bud; PA, pretubular aggregates; SB, S-shaped bodies.

• Supplemental Figure S4 -

  Fig. S4. Efficient gene transfer using adenovirus expressing GFP (Ad-GFP) into rat metanephric mesenchyme. (A) Following adenoviral infection and culture for 14 hours, ubiquitous GFP fluorescence (green) is detected inside and outside progenitor clusters staining positive for Pax2 (red). (B) Confocal microscopy reveals GFP expression in a subset of Pax2-expressing epithelial progenitors.

• Supplemental Table S1 -

  Table S1. Upregulated genes. The Excel table contains a list of all probe sets that were significantly regulated over time (Q<0.1) and upregulated more than 2-fold during epithelial differentiation when compared with freshly isolated metanephric mesenchyme (baseline) independent of which inducer was used.

• Supplemental Table S2 -

  Table S2. Downregulated genes. The Excel table contains a list of all probe sets that were significantly regulated over time (Q<0.1) and downregulated to less that 0.5-fold during epithelial differentiation when compared with freshly isolated metanephric mesenchyme (baseline) independent of which inducer was used.