Eight characteristic and readily discernible gene expression patterns are identified in the E15.5 kidney and ureter by in situ hybridization and associated with specific annotation terms (groups). Whole-mount in situ hybridization analysis (left panels) of E15.5 whole-mount kidneys and ureters reveals anatomically distinct expression profiles for eight kidney genes that are further resolved through high-resolution section in situ hybridization analysis (center panels). Together, these eight probes mark many of the major cell types/structures of the kidney and ureter (schematized in the right panels). When a gene is expressed in both superficial and internal structures, signals from superficial structures may mask internal signals in whole-mount in situ hybridization analysis. Arrows indicate expression associated with the respective annotation terms. Scale bar: 200 μm.

A selection of expression patterns potentially marking new compartment boundaries within the developing nephron, the ureteric epithelium and ureter. Primary whole-mount in situ hybridization analysis is shown in the left panels and high- and low-magnification section in situ hybridization in right panels. (A) Molecular subdivision of the S-shaped body from genes showing an early tubule annotation. Within the medial segment of the S-shaped body (SSB), Osr2 and Irx1 appear to display non-overlapping expression domains. The proximal boundary of Tcfap2b expression in the distal segment of the SSB (line) appears to lie slightly distal to that of Pou3f3 (line). The S-shaped bodies with their attaching ureteric epithelium are outlined in the high-magnification images and the expression domains of the genes are illustrated in the schematic drawings (far right). (B) Comparative expression of Tcfap2b and Pou3f3 in the loop of Henle (LOH) anlage suggests that Tcfap2b expression is restricted to a single arm of the LOH: the distal ascending limb (red arrows). (C) Differential expression of Sox8 and Emx2 in the bifurcating ureteric epithelium. (D) Ureteric trunk patterning and differentiation, as illustrated by the divergent expression domains of Foxi1 (intercalated cells of the collecting duct epithelium) and Foxa1 (urothelial lining of the pelvis and medullary collecting duct). (E) Prrx1 labels outer layers of loose ureteral mesenchyme. Scale bars: 200 μm.

Prediction and validation of transcriptional targets using synexpression and bioinformatics. (A) Genespring histogram representing all genes predicted to show a pattern of synexpression in comparison with the transcription factor Tcfap2b when compared with the 15 developing kidney subcompartments previously profiled by Brunskill et al. (Brunskill et al., 2008). (B) List of the most statistically robust putative targets for each of five transcription factors as assessed using Monkey. (C) An example of validation of such predicted targets. The transcription factor Tcfap2b displays a complex pattern of expression during kidney development, initiating at the ureteric tip (UT) and in the distal renal vesicle (RV) (top panels), distal comma-shaped body, and distal and medial S-shaped body (SSB, middle panels), then later in connecting ducts, distal tubules and the loop of Henle (LoH, lower panels). The same patterns of expression are observed for two predicted targets, Wfdc2 and Pou3f3. CD, collecting duct; CM, cap mesenchyme; CSB, comma-shaped body; CT, connecting tubule; PA, pretubular aggregate; PT, proximal tubule; RC, renal corpuscle; Scale bars: 25 μm.

Expression of genes encoding transcriptional regulators within each annotation group. Low- and high-magnification whole-mount in situ hybridization images (left panels) display transcriptional regulators whose expression is restricted to one of the eight characteristic expression domains: the number of transcriptional regulators showing unique expression in each of these domains versus the total number of genes with a specific annotation to each term is shown (far right). Complementary low- and high-magnification section in situ hybridization images for each gene of interest provide cellular resolution of expression domains (right panels). ‘Unique’ genes may also be expressed in extrarenal compartments within the urogenital system. Most genes showing regionally restricted expression are expressed in more than one annotation group. Arrows indicate expression in ureteric tips. Scale bars: 200 μm (low magnification); 20 μm (high magnification).

Vasculature-associated gene expression and expression of genes with broad sporadic expression in the developing kidney. (A) Selected genes with vasculature-associated expression patterns. (a-f) Bcl6b is weakly expressed in the cortex and medulla. (c-f) High magnification images showing its expression in individual cells in the renal cortex (red arrow) and cells invading the lower cleft of the S-shaped body (red arrow) (c), the glomerular capillary of the capillary loop stage renal corpuscle (d, red arrow), and the peritubular capillaries (e, red arrow). Bcl6b is also expressed in the endothelial cells of the arterioles and renal arteries (red arrows) but not that of the renal veins (yellow arrow) (f). Panels c and f are from a different tissue section from that of panel b. (g-l) Heyl is expressed in the renal arterial (h, red arrow) and arteriolar (i, red arrows) smooth muscles, but not the arterial and arteriolar endothelium (h,i, yellow arrows). It is also expressed in the mesangium of the capillary loop stage renal corpuscle (j, red arrows) but decreased to almost undetectable levels in that of the immature renal corpuscle (i, yellow arrow). Double in situ hybridization studies shows that Heyl (blue) and Wt1 (brown, podocytes) do not overlap (k), whereas Heyl (blue) overlaps with Pdgfrb (brown) in glomerular mesangium (l). Heyl is also expressed in non-vascular locations, in early nephrons (g,h). Panels j, k and l are from different tissue sections from that of panel h. (m-o) Hopx shows very strong and specific expression in the glomerular and extraglomerular mesangium (o, red arrows), whereas Nkx3-1 expression (p-r) is restricted to smooth muscles of renal arteries and arterioles (r, red arrow). (Ba-g) Sfpi1 and Egr1 displayed punctate expression throughout the kidneys, the former reflecting interstitial macrophages and the latter potentially reflecting dynamic signaling responses. Scale bar: 200 μm.

Foxi1 is negatively correlated with its predicted target gene Gsdmc. (A) Section in situ hybridization analysis of four predicted Foxi1 targets, Cldn8, Ehf, Gsdmc and Rnf186, showing expression in a region of the cells within the ureteric trunk epithelium. p, pelvis. Scale bar: 50 μm. (B) Two-color single molecule fluorescent in situ hybridization showing the negative correlation of expression patterns between Foxi1 and Gsdmc. Fixed E15.5 kidney sections were simultaneously hybridized with two differentially labeled probe libraries (Cy5 and Alexa594). Single mRNA molecules appear as diffraction-limited spots in an epifluorescence microscope. Transcripts were automatically detected and assigned to individual cells based on membrane staining via myristoylated GFP. Images show detected dots for Foxi1/Gsdmc in seven z-sections at 0.3 μm intervals and a correlation plot for Foxi1/Gsdmc. Each dot in the correlation plot denotes the absolute transcript density of a single cell for both genes. (C) Model of transcriptional role for Foxi1 in the switch between multi-potential progenitor and principal cell versus intercalated cell (IC) fate.