Figure 1.
Development of epithelial characteristics and nephron segmentation.
(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).
Figure 2.
Maturation of the tight junction occurs as the nephron expresses markers of epithelium.
A) A diagrammatic representation of the columnar epithelium of the proximal tubule. B) Western blot of early (0 day) and late (7 day) nephron cultures. Intercellular junction proteins specific to the tight and adherens junctions (Including the nephron specific KSP-Cadherin) denote the mesenchymal to epithelial transition during development of the nephron. C) Western blot showing detergent extraction of nephron cultures from 3 to 5 days. GRP94 was used as a control for extraction.
Figure 3.
Informatic analysis of cultured nephrons highlights global stages of nephron development.
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.
Table 1.
Literature based “legacy” genes provide a core to build a SS to PT development network.
Figure 4.
Putative proximal tubulogenesis gene network shows high connectivity of Hnf4α.
A) The genes that significantly differ from 72 to 120 hours in ex vivo nephron culture and from S-shaped body to proximal tubule in vivo represented in an interaction network based on the proximal tubule “legacy” genes shown in Table 1. B) Quantitative representation of the number of connections (edges) each gene (node) has to others in the proximal tubulogenesis network shown in panel A. Hnf4α is the most influential (largest number of edges) node in the network.
Figure 5.
qPCR validation of Oat and Hnf4α expression in cultured nephrons.
Expression of Oat1 (A), Oat3 (B), and Hnf4α (C) show change in expression in developing nephrons and are expressed as normalized expression, relative to GAPDH. D) The fold change in each transcript.
Figure 6.
Functional transport occurs simultaneously with tight junction maturation and Oat expression.
A) Immunofluorescent image of late nephron cultures incubated with the Oat-selective organic anion 6-carboxy fluorescein (6-cf). B) Immunofluorescent image of late nephron culture incubated with 6-cf and the Oat inhibitor probenecid. The bar (A and B) is 100 µm.
Figure 7.
In silico promoter analysis of Oat1, Oat3, and Oct1 transporters predicts regulation by HNF4α.
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.
Table 2.
Expression and protein localization of Oat1, Oat3, Oct1, and Hnf4α in the proximal tubule.
Table 3.
Hepatocyte nuclear factors are associated with Slc22 genes in silico and in vitro.
Figure 8.
ChIP-qPCR confirms Hnf4α binds the proximal promoters of transporters in the in vivo maturing nephron.
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.