a) Tree view of ontology for TS23 demonstrating the utility of the ontology for both high and low resolution annotation. b) Tree view at TS23 demonstrating the subcomponents of the maturing renal corpuscle (renal corpuscle of Stage IV nephron) in the cortex and the subdivision of the renal tubules between renal cortex and renal medulla (left) compared to the group term for maturing nephron (Stage IV nephron) (right). The remainder of the ontology is fully documented at http://www.gudmap.org/Resources/Ontologies.html.

A. Diagrammatic representation of the TS17 urogenital system accompanied by the complete ontology for this stage. TS17 embryos were dissected transversely below the forelimbs and longitudinally down the midline to expose the urogenital system. The caudal end is on the right and the hindlimb is visible. B. Representative examples of whole mount RNA in situ hybridisation at TS17 showing expression of Lim1 in the nephric duct (double arrowhead, EMAP:28426) and mesonephric glomeruli (arrowhead, EMAP:30569), Lamb1-1 in the nephric duct (double arrowhead, EMAP:28426) and mesonephric tubules (arrowhead, EMAP:27589), Crym in the metanephric mesenchyme (arrow, EMAP:29991) and mesonephric tubules (double arrowhead, EMAP:27589), Oct4 in the primordial germ cells (arrow, EMAP:2574) and Wnt4 in the gonad (arrowhead, EMAP:2572), mesonephric mesenchyme (double arrowhead, EMAP:27586), mesonephric tubules (EMAP:27589) and nephric duct, mesonephric portion (EMAP:27592). Scale bar = 50μm. C. Representative examples of RNA in situ hybridisation of TS20 urinary system, TS20 metanephroi explanted and cultured for 2 days and TS23 metanephros sections. The same genes (Wnt7b, Spint2, Wnt4, Col9a1, Crym) have been compared between these different stages to demonstrate the patterns seen for genes with specific expression in distinct compartments. Two images are shown at TS23, a global view of the metanephros with a high magnification image below (the area magnified is outlined). Wnt7b: TS20 and explanted metanephroi showed Wnt7b expression in the ureteric trunk (arrowheads, EMAP:27698) and in the ureter (double arrowheads, EMAP:27701). Expression was not detected in the ureteric tips. At TS23, the global view shows Wnt7b expression in the cortical collecting ducts (arrowheads, EMAP:28132) and high magnification of the medulla shows expression in medullary collecting ducts (double arrowhead, EMAP:28063) and immature loop of Henle (arrowhead, EMAP:28173). Spint2: Spint2 was expressed in the ureteric tip (double arrowhead) and ureteric trunk (arrowhead) of TS20 and explanted metanephroi and in the ureter of explants. At TS23, Spint2 expression continued in the ureteric tip (arrowhead in global image) and cortical and medullary collecting ducts (double arrowhead in high magnification) and was also seen in cortical and medullary renal tubules, including the immature loop of Henle (arrowhead in high magnification). Wnt4: In TS20 and explanted metanephroi, Wnt4 was expressed in early nephrons (arrowheads, EMAP:29339). Later, in explants and at TS23, Wnt4 was detected in cortical and medullary collecting ducts and the cortical and medullary interstitium immediately surrounding the collecting ducts (double arrowhead in explant, arrowhead in global image TS23) and in the pelvic urothelial lining (TS23) and ureter (explant). Expression persisted in early nephrons at TS23 with expression in pretubular aggregates, renal vesicles (arrowhead in high magnification), comma-shaped bodies and S-shaped bodies (double arrowheads in high magnification). Col9a1: Col9a1 was expressed in the renal interstitium at all stages (arrowheads), including the perihilar interstitium (double arrowheads, explant). Crym: Crym was strongly and specifically expressed in the cap mesenchyme at all stages presented (arrowheads). We have previously described an ontology for explanted kidneys (Martinez et al, 2005). Scale bar = 200μm. Images and diagrams courtesy of Kylie Georgas, Bree Rumballe, Hansheng Chiu, Grant Challen, Michael Lusis and Melissa Little.

A. Diagram and histological sections of the complete murine metanephros at TS23 annotated using terms from the ontology or their corresponding EMAP identification numbers. The inset diagram (left) shows the broad regions of the metanephros defined in the ontology and indicates the wedge of metanephros enlarged in Figure 3B. The histology sections (haematoxylin and eosin) show sagittal sections through the metanephros and ureter (left) and just beyond the midline of the metanephros (right).
B. Diagrammatic representation of a wedge of the TS23 metanephros, from renal capsule to pelvis, annotated using terms from the ontology along with their corresponding EMAP numbers. Dashed boxes indicate the location of Stage I to IV nephrons that have been annotated in detail along the left and right sides of the diagram.
C. Series of matched-pair histological images from a TS23 metanephros colouring specific anatomical terms present within the ontology. a,a′) nephrogenic zone and cortex highlighting ureteric tip (EMAP:27752, red) and renal cortical collecting duct (EMAP:28132, red), cap mesenchyme (EMAP: 27738, green), comma-shaped body (EMAP: 27837, light blue) and s-shaped body (syn: Stage II nephron, EMAP:27855, dark blue). b,b′) nephrogenic zone and cortex showing the ureteric tip (EMAP:27752, red) and renal cortical collecting duct (EMAP:28132, red), pretubular aggregate (EMAP:27745, yellow), renal vesicle (syn: Stage I nephron, EMAP:27831, dark blue), capillary loop stage renal corpuscle (Stage III renal corpuscle, EMAP: 27909, green) and renal tubules (EMAP: 27999, light blue). c,c′) Segments of the s-shaped body (Stage II nephron) including the visceral epithelium (EMAP:27897, red), parietal epithelium (EMAP: 27903, dark blue), medial segment (EMAP: 27879, green), distal segment (EMAP:27873, yellow) and renal junctional tubule (EMAP:27867, light blue). d,d′) Segments of the capillary loop stage renal corpuscle (Stage III renal corpuscle) including the visceral epithelium (EMAP:27915, red), parietal epithelium (EMAP:27921, dark blue), presumptive mesangium (EMAP:27933, green) and renal tubule (EMAP:27999, light blue). e,e′) Cortex of the TS23 metanephros showing different stages of renal corpuscle development including s-shaped body (syn: Stage II nephron, EMAP:27855, dark blue), capillary loop stage (syn: Stage III, EMAP:27909, green) and maturing (syn: Stage IV, EMAP:27945, yellow) renal corpuscles. f,f′) Medulla and pelvis of the TS23 metanephros showing the renal medullary interstitium (EMAP:8241, yellow), medullary collecting ducts (EMAP:28063, red), immature loop of Henle (EMAP:28173, dark blue), renal medullary vasculature (EMAP:28158, green) and pelvic urothelial lining (EMAP:28079, light blue). Histological images courtesy of Luise Cullen-McEwen and Melissa Little. Diagrams courtesy of Kylie Georgas.

Analysis of whole mount in situ hybridisation (WISH) of kidney and ureter at TS23 reveal 8 readily annotated patterns corresponding to the ureteric tip (A, Wnt11), ureteric trunk (B, Wnt7b), cap mesenchyme (C, Cited1), early tubule (D, Wnt4), late tubule (E, Slc12a3), renal vasculature (F, Sox18), renal interstitium (G, Foxd1) and ureter (H, Foxa1). Section in situ hybridisation of neonatal kidneys (I-P) for the same genes confirms the accuracy of these earlier annotations. Expression in deeper or more mature structures within the kidney revealed by SISH is not obvious from TS23 whole mount images either due to lack of expression of mature structures at an earlier stage or masking of deeper signal by strong superficial signals. Scale bar = 40μM. Many of the terms used to annotate whole mount material at TS23 are group terms. Images courtesy of Jing Yu.

A. Low resolution diagrammatic representation (left) and histological section (right, haematoxylin and eosin) of a TS23 metanephros identifying structures around the pelvis and pelvic-ureter junction. Scale bar = 100μm. B. Temporal panel of transverse sections of the ureter at differing distances from the metanephros (proximal, middle and distal) demonstrating the histology of the developing ureter from TS20 to adult (haematoxylin and eosin). Scale bar = 100μm. C. Annotated section through a TS25 ureter middle distance between the metanephros and bladder demonstrating the cellular layers (Massons Trichrome, which stains the lamina propria blue). Scale bar = 50μm. D. Myh11 RNA in situ hybridisation of TS23 and adult ureter. TS23 Pelvis & ureter: Myh11 showed specific expression in the pelvic smooth muscle (single arrowhead, EMAP:28121) and ureteral smooth muscle layer of the ureter (double arrowhead, EMAP:28812). Inset image shows the entire metanephros section and outlines the region enlarged in the main image. TS23 Ureter (distal): Myh11 expression in the ureteral smooth muscle layer (single arrowhead, EMAP:28812) of the ureter located close to the primitive bladder. Adult Ureter (proximal): Myh11 expression in the ureteral smooth muscle layer of the ureter (arrowhead, EMAP:29488) located close to the metanephros. Adult Ureter (distal): Myh11 expression in the ureteral smooth muscle layer of the ureter (arrowhead, EMAP:29488) located close to the bladder. Scale bar = 200μm. Images courtesy of Eleanor Mitchell, Kylie Georgas, Hansheng Chiu and Bree Rumballe. Line diagram courtesy of Kylie Georgas.

(A) Histological section of the TS23 bladder identifying terms present in the ontology at this stage and their corresponding EMAP identification numbers (haematoxylin and eosin). Scale bar in (A) = 200μm. (B) RNA in-situ hybridisation of TS20 whole mount and TS23 transverse sections of the bladder for five genes and low and high magnification transverse sections of the adult bladder for Myh11. Col16a1 (Procollagen, type XVI, alpha 1) expression was seen in the muscularis mucosa of the bladder (arrowheads TS23 EMAP:28621). Expression of Myh11 was seen in the detrusor muscle of the bladder (arrowheads, TS23 EMAP:28639). Wnt4 expression was seen in the epithelium of the bladder (arrowheads, syn: urothelium; TS20 EMAP:30874; TS23 EMAP:28603). Shh was also expressed in the epithelium of the bladder (arrowheads). Kpna2 expression was seen in a scattered pattern of single cells in all layers of the bladder (arrowhead in TS20). Myh11 expression in the adult bladder: low magnification (a) and high magnification (b) images show strong expression in the detrusor muscle of the bladder (arrowhead in B) Boxed area in (a) outlines the region enlarged in (b) Myh11 was also expressed in the vasculature of the embryonic and adult bladder. Scale bar in TS20 = 50 μm, TS23 = 200 μm. Images courtesy of Kylie Georgas, Bree Rumballe and Hansheng Chiu.

A. Example whole mount in situ hybridisation of the TS20 reproductive system demonstrating the expression of 5 genes in both male (M) and female (F) reproductive systems. Wnt7b, Spint2, Lamb1-1 and Wnt4 are ventral views of the gonad-mesonephros. The dorsal view of Shh is shown to visualise the mesonephric tubules. Inset images in Wnt7b, Spint2 and Lamb1-1 are dorsal views of the rostral end of the samples to visualise the mesonephric tubules. Mesonephros: Black double arrowheads indicate expression seen in the nephric duct, mesonephric portion in both sexes, for Wnt7b, Spint2, Lamb1-1 and Shh (syn: Wolffian duct; EMAP:28925 in F; EMAP:29154 in M). Black single arrowheads indicate expression seen in the paramesonephric duct in both sexes for Wnt7b, Spint2 and Lamb1-1 (syn: Müllerian duct; EMAP:28938 in F; EMAP:29159 in M). White arrowheads indicate expression in the mesonephric tubules for Wnt7b, Spint2, Lamb1-1 and Shh in both sexes (EMAP:38920 in F; EMAP:29149 in M). Expression in the mesonephric mesenchyme was seen for Lamb1-1 and Wnt4 (EMAP:28915 in female, EMAP:29144 in male). The white outlined arrowheads in Wnt4 M and F indicate the strongest Wnt4 signal seen in the mesonephric mesenchyme, at the junction where it contacts the gonad in both sexes and in two lines of mesonephros running medial to and parallel with both the nephric duct and paramesonephric duct. Sectioning of the hybridised whole mount tissue confirmed that the Wnt4 expression was restricted to the mesonephric mesenchyme immediately surrounding the nephric duct and not in the duct epithelium. Gonad: In the male gonads, Spint2 was expressed in the primary sex cords of the testis (EMAP:29099) and Lamb1-1 in the interstitium of the testis (EMAP:29075). In the female gonads, Spint2, Lamb1-1 and Wnt4 showed expression in the ovary (EMAP:28876). Scale bar=25μm. B. Panel B shows the temporal gene expression analyses of three genes in both sexes. These are presented as TS20 whole mount, TS23 section and adult section in situ hybridisation patterns. At TS20, Col6a1 (Procollagen, type VI, alpha 1) was detected in the mesonephric mesenchyme (arrowheads, EMAP:29144 in M; EMAP:28915 in F), nephric duct (syn: Wolffian duct; EMAP:28925 in F; EMAP:29154 in M) and paramesonephric duct (syn: Müllerian duct; EMAP:28938 in F; EMAP:29159 in M) of both sexes, the interstitium of the testis (double arrowhead, EMAP:29075) and the mesenchymal stroma of the ovary (EMAP:28881). At TS23, expression persisted in the mesonephric mesenchyme of both sexes (arrowheads, EMAP:28918 in F, EMAP:29147 in M), the interstitium of the testis (double arrowhead, EMAP:29078) and both the cortical (EMAP:29010) and medullary (EMAP:29015) components of the mesenchymal stroma of the ovary. In the adult female ovary, Col6a1 (Procollagen, type VI, alpha 1) expression was seen in the corpora lutea (arrowhead) and theca cells of pre-antral and antral follicles (double arrowhead). No expression was seen in the adult male testis (t) or epididymis (e). Kpna2 was detected in a spotted pattern in all embryonic tissue types, with strongest signal detected in the primary sex cord of the testis at TS20 (arrowhead, EMAP:29099), which was maintained in the seminiferous cords of the TS23 and adult testes (arrowhead, EMAP:29230 in TS23). No Kpna2 expression was detected in the adult epididymis (e). The ovary showed Kpna2 expression at all stages (EMAP:28876: at TS20 and EMAP:8251 at TS23). In the adult ovary, strongest Kpna2 signal was detected in the granulosa cells of all follicle states and weaker signal in the oocytes of follicles (arrowhead). Myh11, a marker of smooth muscle cells, was strongly expressed in the developing aorta and common iliac arteries adjacent to the TS20 male and female reproductive tracts. Expression was also detected in the developing vasculature of the testis and ovary at TS20 and TS23 (arrowheads; EMAP:29115 (TS20) EMAP:29118 (TS23) in M; EMAP:28889 (TS20) EMAP:28892 (TS23) in F). Expression was seen in the coelomic vessel (EMAP:29123) of the testis at TS20 (arrowhead). In the adult, Myh11 expression was detected in the vasculature of the testis, epididymis, vas deferens in the male and ovary and oviduct in the female. Adult expression was also seen in the peritubular myoid cells of the seminiferous cords, the muscle layer of the epididymis (e, arrowhead) and the muscle layer of the ductus deferens (not shown) in the male and in the theca cells surrounding the follicles of the ovary (arrowhead) and in the circular and longitudinal muscle of the oviduct (not shown) in the female. Scale bar TS20=25μm, TS23=200μm, adult=500μm. Images courtesy of Kylie Georgas, Hansheng Chiu and Bree Rumballe.