Fig 2: Unbekandt-culture-derived ureteric bud spheres respond to BMP4 to express uroplakin. (A) and (B) Ureteric bud structures (outlined by dashed lines), isolated from Unbekandt orgranoids7 were transplanted into host metanephric (A) or peri-Wolffian (B) mesenchyme of E11.5 kidneys and cultured for 5 days. Induction of uroplakin is seen when transplanted into peri-Wolffian mesenchyme. (C) Generation and bead treatment of Ganeva kidneys: ureteric spheres are obtained from 1-day Unbekandt organoids then isolated and surrounded by e11.5 metanephric mesenchyme to form Ganeva kidneys, which were treated 2 days later with BMP4 soaked beads changed daily for up to three days. The dashed box highlights differences between the Ganeva and Grobstein method. (D) Ganeva kidney with no treatment; (E) treatment with a BSA-soaked bead; (F) A BMP4-soaked bead induces the nearby tubule to become an unbranched, uroplakin-positive, nephron-free tubule. (G) Nephron patterning of natural cultured kidneys is comparable to BMP4-treated Ganeva kidneys (H). Outlines of developing nephrons has been drawn on and approximate sections annotated (Dist, Distal tubule; Med, Medial tubule; and Prox, Proximal tubule). UPK, Uroplakin. PCK, Pan-cytokeratin, Col4, Collagen Type IV. Arrows indicate induced uroplakin. Scale bar = 200 µm.

Fig 3: BMP4 induces expression of uroplakin in the developing collecting ducts. Developing collecting ducts were treated globally with BMP4 and uroplakin expression was investigated after 5 days of culture. (A) E11.5 kidney rudiments were cultured in media containing vehicle only (BSA), 25 ng/ml of BMP4 or 100ng/ml of BMP4. (B) Globally BMP4-treated E11.5 kidney rudiments were cultured in the presence of a Gremlin soaked bead or vehicle only soaked bead. *Bead location, n = a few examples of developing nephrons. Scale (unless otherwise stated) = 100 µM.

Fig 4: Local administration of BMP4 induces expression of Uroplakin in targeted developing collecting duct. Developing collecting ducts were exposed to BMP4 beads. (A) Illustration of the treatment method (*Bead). (B) Nomenclature of branches. (C) and (D) E11.5 kidneys cultured for 24hrs then given a bead soaked in either BSA (Vehicle: C) or BMP4 (D) at a secondary branch point. (E) Application of the beads to Grobstein culture; (F) Untreated; (G) BSA-treated; (H) BMP4 treated. Inserts: isolated uroplakin channel. UPK, Uroplakin. PCK, Pan-cytokeratin. Lam, Laminin. Arrows indicate induced uroplakin expression. Scale bar = 200 µm.

Fig 5: Wnt-dependent Fendrr target gene regulation. (A) Schematic of the Foxf1 and Fendrr promoter region with the indication of the location of the 3 gRNAs used for specific Fendrr endogenous activation. Sanger sequencing of NIH3T3 and MLg genomic DNA verified absence of mutations in gRNA binding sites. (B) Expression changes of Fendrr and Foxf1 after Fendrr CRISPRa (FendrrSAM) in MLg and NIH3T3 cells. Note that only in NIH3T3 cells upregulation of Fendrr could be detected. (C) Fendrr triplex containing Fendrr target genes expressed in NIH3T3 cells after 48 h of FendrrSAM transfection. (D) Expression changes after 48 h of co-stimulation with FGF. (E) Expression changes after 48 h of co-stimulation with BMP-4. (F) Expression changes after 48 h of co-stimulation of Wnt-signalling. (G) Schematic of the generation of NIH3T3 FendrrBox deletion mutants. (H) Expression changes of NIH3T3 FendrrBox deletion mutants upon FendrrSAM and co-stimulation with Wnt. Each dot represents an independent clone. Expression changes after treatment are normalised to untreated cells transfected with control gRNA (set to 1), represented by the dashed line. (D–F, H) Statistics are given when significant by t-test analysis.