Development of epithelial characteristics and nephron segmentation.

<p>(A) A diagram showing the morphological progression of nephron development ex vivo, and the corresponding culture time. B) Immunofluorescent image of a 2 day (early) nephron culture. The tight junction marker ZO-1 is shown in red, and the adherens junction and marker E-cadherin is shown in green (panels B-D). C) Immunofluorescent image of 3 day (early) nephron culture. D) Immunofluorescent image of the convoluted late tubule (5 days and beyond). E) Immunofluorescent image of a 5 day (late) nephron culture. Peanut lectin marks developing podocytes (red), DAPI (blue). The late nephron begins segment specific differentiation. Bar is 50 µm (B, C, E) or 100 µm (D).</p

In silico promoter analysis of Oat1, Oat3, and Oct1 transporters predicts regulation by HNF4α.

<p>Diagram of the promoters of the human, rat, and mouse Oct1, Oat1 and Oat3 promoters. Under stringent settings only a single TRANSFAC binding motif remained - V$NR2F. Six transcripts had a DR-1 binding element within the proximal promoter region, and one had a DR-2 binding element, resulting in 7 of 9 transcripts predicted to have the potential to recruit Hnf4α directly. Green markers depict binding sites for members of the Nuclear Receptor 2 family as a function of position relevant to known transcription start sites. DR-1 elements are circled, the single DR-2 element is marked by a square. Yellow shading denotes a direct call of HNF4α by Genomatix.</p

Expression and protein localization of Oat1, Oat3, Oct1, and Hnf4α in the proximal tubule.

<p>Evidence from existing literature validates the result of microarray analysis. The genes and protein products of Hnf4α, Oat1, Oat3, and Oct1 are present in the proximal tubule during organogenesis and adulthood.</p

Hepatocyte nuclear factors are associated with Slc22 genes in silico and in vitro.

<p>Evidence from the literature demonstrates the potential for functional regulation of the SLC22 family of genes by hepatocyte nuclear factors.</p

ChIP-qPCR confirms Hnf4α binds the proximal promoters of transporters in the in vivo maturing nephron.

<p>A) ChIP qPCR of Oct1 (Slc22a1), Oat1 (Slc22a6) and Oat3 (Slc22a8) promoters using an HNF4α antibody. Two loci outside of annotated coding or transcribed regions on chromosome 1 and 4 were used as negative controls. B) Schematic workflow of the chromatin immunoprecipitation qPCR experiment.</p

Informatic analysis of cultured nephrons highlights global stages of nephron development.

<p>A) self organizing map (SOM) featuring meta-meta representations of four points in nephron culture. Increasing distance between the samples on the map corresponds to increasing difference in the abstracted transcriptome. B) Non-negative matrix factorization (NMF) of the samples represented in panel A. The arrow highlights metagene F1, which comprises genes highly expressed only after 5 days (120 hours) in culture.</p