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• W2989476985 abstract "Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods References Decision letter Author response Article and author information Metrics Abstract The different segments of the nephron and glomerulus in the kidney balance the processes of water homeostasis, solute recovery, blood filtration, and metabolite excretion. When segment function is disrupted, a range of pathological features are presented. Little is known about nephron patterning during embryogenesis. In this study, we demonstrate that the early nephron is patterned by a gradient in β-catenin activity along the axis of the nephron tubule. By modifying β-catenin activity, we force cells within nephrons to differentiate according to the imposed β-catenin activity level, thereby causing spatial shifts in nephron segments. The β-catenin signalling gradient interacts with the BMP pathway which, through PTEN/PI3K/AKT signalling, antagonises β-catenin activity and promotes segment identities associated with low β-catenin activity. β-catenin activity and PI3K signalling also integrate with Notch signalling to control segmentation: modulating β-catenin activity or PI3K rescues segment identities normally lost by inhibition of Notch. Our data therefore identifies a molecular network for nephron patterning. https://doi.org/10.7554/eLife.04000.001 eLife digest The main function of the kidney is to filter blood to remove waste and regulate the amount of water and salt in the body. Structures in the kidney—called nephrons—do much of this work and blood is filtered in a part of each nephron called the glomerulus. The substances filtered out of the blood move into a series of ‘tubules’, another part of the nephrons, from where water and soluble substances are reabsorbed or excreted as the body requires. If the nephrons do not work correctly, it can lead to a wide range of health problems—from abnormal water and salt loss to dangerously high blood pressure. For organs and tissues to develop in an embryo, signalling pathways help cells to communicate with each other. These pathways control what type of cells the embryonic cells become and also help neighbouring cells work together to form specialised structures with particular functions. Much is unknown about how the nephron develops, including how its different structures coordinate their development with each other so that they form in the right position in the nephron. A protein called beta-catenin was already known to play an important role in the signalling pathways that trigger the earliest stages of nephron formation. Lindström et al. further investigated how this protein helps the nephron to develop by using a wide range of techniques, including growing genetically altered mouse kidneys in culture and capturing images of the developing nephrons with time-lapse microscopy. The combined results reveal that the levels of beta-catenin activity coordinate the development of the different structures in the nephron. The beta-catenin protein is not equally active in all parts of the nephron; instead, it forms a gradient of different activity levels. The highest levels of beta-catenin activity occur in the tubules at the furthest end of the developing nephron; this activity gradually decreases along the length of the nephron, and the glomerulus itself lacks beta-catenin activity altogether. Experimentally manipulating the levels of beta-catenin at different points along the nephron caused those cells to take on the wrong identity, causing parts of the nephron to form in the wrong place. Lindström et al. were also able to establish that the signalling pathway controlled by beta-catenin activity interacts with three other well-known signalling pathways as part of a network that controls nephron development. More research is required to find out which signal activates beta-catenin in the first place and from where in the kidney this signal comes. It also remains to be discovered how a particular cell in the tubule interprets the exact activities of the different signals to give the cell its specific identity for that place in the nephron. A better understanding of these sorts of processes will eventually help build new kidneys for people with kidney failure. https://doi.org/10.7554/eLife.04000.002 Introduction All adult vertebrates depend on renal nephrons accurately performing a diverse set of roles that protect and maintain blood homeostasis. The diversity of roles that the nephron performs is reflected in the range of symptoms of abnormal nephron function and the consequent diseases range from, for example, Bartter syndrome (abnormal water and salt loss), acidosis, and rickets to essential hypertension (Simon et al., 1996; Schedl, 2007). In spite of the importance of proper nephron function and segmentation, little is known regarding how the nephron patterns and how different specialised segments form during nephrogenesis. Thus far, only a handful of genes have been shown to control the development of specific nephron segments of which none have been explicitly connected to an explanation for how patterning is regulated along the whole length of the nephron. Nephrons form during embryonic development, when Wnt9b, secreted by the ureteric bud, activates a canonical β-catenin-mediated pathway in a population of overlying Six2+ mesenchymal nephron progenitor-cells (Kobayashi et al., 2008; Karner et al., 2011; Park et al., 2012; Das et al., 2013). In the canonical WNT pathway, WNT signalling results in the destabilisation of the GSK-3β/CK1α/AXIN2/APC complex and prevents the normal tagging of cytosolic β-catenin for degradation. Stabilised β-catenin translocates to the nucleus and together with members of the TCF family of transcription factors controls the expression of a wide range of target genes (Clevers and Nusse, 2012). One of these β-catenin target genes, Wnt4, triggers a mesenchymal-to-epithelial transition (MET) (Stark et al., 1994; Kispert et al., 1998) in the nephron progenitor cells and these reconfigure into an epithelial renal vesicle (RV). Following the MET, the RV becomes polarised and connects to the ureteric bud at its distal end (Georgas et al., 2009), and during subsequent developmental steps, the nephron starts to display additional signs of pattern-formation along its proximal–distal axis. Several distinct cell-populations form and these produce the different segments of the adult nephron (Saxen, 1987); a Wt1+ cell population gives rise to proximal structures, a Jag1+ population to the medial part and Lgr5+ cells generate the distal nephron segments (Armstrong et al., 1993; Cheng et al., 2003; Chen and Al-Awqati, 2005; Cheng et al., 2007; Kreidberg, 2010; Barker et al., 2012). These segments are in turn further subdivided into functionally specialised portions, which express specific combinations of transmembrane transporters/channels for salts, glucose, and metals (Raciti et al., 2008). How the differentiation of these segments is regulated remains unknown. The initiation of the nephron MET is driven by β-catenin signalling (Kobayashi et al., 2008; Karner et al., 2011; Park et al., 2012), but the Wnt4 driven MET is most likely mediated by the non-canonical Ca2+–NFAT pathway (Burn et al., 2011; Tanigawa et al., 2011). It remains uncertain by what mechanism and at what precise stage the Six2+ cells or the RV develop distinct nephron segment lineages (Lindstrom et al., 2013). Post-MET, Wnt9b acts via the planar cell polarity pathway and controls the orientation of cell division and the elongation of collecting tubules (Karner et al., 2009). Wnt7b also has a role as it controls the development of the medulla and papilla of the kidney (Yu et al., 2009). Notch signalling has previously been identified as being important for the formation of the proximal tubule (Cheng et al., 2003, 2007). Notch2−/− nephrons form no proximal tubules or glomeruli (Cheng et al., 2007). However, ectopic expression of the intracellular and active Notch1-domain (N1ICD) in nephrons blocks glomerular development (Cheng et al., 2003, 2007; Boyle et al., 2011). N1ICD expression in Six2+ cells can actually substitute for Wnt9b and trigger nephron induction and MET (Boyle et al., 2011). Whether Notch or Wnt is important for the initial patterning of the nephron immediately post-MET remains to be determined. Using in vivo and ex vivo techniques we demonstrate that a gradient of β-catenin activity, along the proximal–distal nephron axis, controls the differentiation of segment-specific cell fates. We further investigate how β-catenin activity is prevented in the proximal and medial segments and show that BMP/PTEN/PI3K signalling in the medial nephron actively promotes the medial segment identity whilst blocking β-catenin activity. In addition, we show that modulating β-catenin or PI3K activity partially rescues the nephron segment defect phenotypes associated with the loss of Notch function. Our findings provide a model where multiple signalling pathways are integrated to control nephron segment-identity specification. Results A β-catenin activity gradient is generated along the nephron axis Regulation of β-catenin activity is essential for nephron induction and MET (Davies and Garrod, 1995; Kuure et al., 2007; Park et al., 2007). To determine whether β-catenin is involved in post-MET stages of nephron development, we tracked its activity in embryonic kidney organ cultures using a β-catenin signalling reporter mouse strain (TCF/Lef::H2B-GFP; Ferrer-Vaquer et al., 2010). Confocal and time-lapse microscopy indicated strong activity of the reporter in the ureteric bud as described before (Ferrer-Vaquer et al., 2010; Burn et al., 2011). Importantly, we also detected different GFP intensities, reporting β-catenin activity, along the proximal–distal axis of the nephron. The nomenclature we use to describe the domains of the proximal–distal axis is as defined by the GenitoUrinary Development Molecular Anatomy Project (gudmap.org) for S-shaped body nephrons. Confocal z-projections (Figures 1A, 3D rendering Video 1) of whole nephrons at an early stage of development show the signal being highest at the distal end of the nephron, where it connects to the ureteric bud, and gradually decreasing towards the proximal end. Time-lapse imaging of developing TCF/Lef::H2B-GFP expressing nephrons showed that the different GFP signal intensities propagated in a distal-to-proximal direction over time alongside the normal nephron growth and segmentation (Figure 1—figure supplement 1A and Video 2). Confocal imaging confirmed different GFP intensities in nephrons at later stages: S-shaped bodies (Figure 1B and Figure 1—figure supplement 1B) and more mature nephrons (data not shown), and we consistently found that the podocytes and their precursors at the extreme proximal end of the nephrons were almost completely devoid of β-catenin activity (Figure 1A,B, Figure 1—figure supplement 1B; Video 1). We quantified the TCF/Lef::H2B-GFP signal in cells located in the distal, medial, and proximal segments of nephrons and plotted their intensities against their position. The segments were defined with antibodies for Jag1 (medial segment; Chen and Al-Awqati, 2005; Georgas et al., 2009), Cdh1 (distal segment; Cho et al., 1998), and by morphology. The TCF/Lef::H2B-GFP signal intensities showed an exponentially decreasing gradient (R2 = 0.999; n = 11 nephrons) suggestive of a single source and first-order decay of the activating signal (Figure 1C). Jag1 immunofluorescent intensity data were used to indicate positions within nephrons. The GFP intensities measured in the distal segments differed from those in proximal segments by a minimum of a 15-fold difference to a maximum of a 72-fold difference (mean = 39-fold difference; n = 10 nephrons; Figure 1C). Figure 1 with 1 supplement see all Download asset Open asset β-catenin activity levels form a reversed gradient along the axis of the nephron. (A–B) TCF/Lef::H2B-GFP expression in nephrons: (A) late renal vesicle/early comma-shaped body nephron, (B) S-shaped body nephron, lines: white–nephron axis, purple–ureteric bud, green–distal nephron, red–medial nephron, blue–proximal nephron/glomerular precursors. Heat-maps display signal intensity in different nephron segments. (C) Quantification of nuclear H2B-GFP and cell-membrane Jag1 antibody stain signal-intensity along the proximal–distal axis. Error bars represent SEM of pixels representative of 10 µm segments. Right-hand side graph shows mean values for segments, as identified by H2B-GFP and Jag1 profiles (n = 11 nephrons), error bars indicate SEM. p-values derived from t-tests. (D) Antibody stains against total β-catenin and phosphorylated β-catenin in S-shaped body nephron—Jag1 marking the medial segment. White dashed line indicating nephron axis. https://doi.org/10.7554/eLife.04000.003 Video 1 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg 3D reconstructions of nephrons. 3D reconstruction of renal vesicle (top), S-shaped body (middle), and more mature nephron (bottom). Nephrons are positive for TCF/Lef::H2B-GFP, Jag1-red, Cdh1-blue (left panel) and the TCF/Lef::H2B-GFP reporter is shown in a heat-map overlay (right panel). https://doi.org/10.7554/eLife.04000.007 Video 2 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Time-lapse capture of nephron. The nephron is the same as shown in Figure 1—figure supplement 1A (from a TCF/Lef::H2B-GFP reporter kidney) is shown during the earliest stages of nephron development. Segments and stages are annotated based on the brightfield channel (not shown). https://doi.org/10.7554/eLife.04000.008 To further assess the observed gradient in β-catenin activity, we analysed the expression and activity of the β-catenin protein directly. Using pan-β-catenin antibodies, we found β-catenin to be expressed throughout the developing nephrons (Figure 1D). Antibodies for β-catenin that was already tagged for degradation (phosphorylated at Ser33/Ser37/Thr41), most strongly labelled the proximal domain (twofold higher compared to medial and distal, p = 6.2 × 10−5 and 1.2 × 10−4) (Figure 1—figure supplement 1C). Target genes of β-catenin (Lef1 and Ccnd1) were also expressed along the gradient (Figure 1—figure supplement 1D–F). The distribution of β-catenin protein and the expression of direct β-catenin target genes support the existence of the activity gradient. Proximal and distal positional identities are controlled by different β-catenin activity levels To investigate whether the β-catenin activity gradient has functional implications for the development of the nephron proximal–distal axis, we used inhibitors of GSK-3β (CHIR99021) and Tankyrase (IWR1) to positively and negatively modify β-catenin signalling ex vivo in whole organ cultures. We extensively characterised these small molecule inhibitors to ensure that they had their expected effect on the β-catenin signalling pathway; these data can be found in Figure 2—figure supplements 1–4 and Videos 3–4 and are briefly described here. First, we used the β-catenin reporter TCF/Lef::H2B-GFP and qRT-PCR analyses and confirmed that the inhibitors acted as expected (Figure 2—figure supplement 1). Second, because maximal activation of β-catenin has previously been suggested to be incompatible with MET (Kuure et al., 2007; Park et al., 2007, 2012), we identified CHIR concentrations that induced nephron differentiation (PAX2 and PAX8 expression) but still permitted epithelialisation (CDH1 expression; Figure 2—figure supplement 2). Increased β-catenin signalling induced ectopic nephrons to form at the periphery of the kidneys and large nephron structures developed within the ureteric tree (Figure 2—figure supplements 1,2). Third, we demonstrated that these ectopic structures, just like the tree-bound structures, were derived from Six2+ nephron progenitors by fate-mapping these using a Six2-CreGFP (Dolt et al., 2013) and a Rosa26tdRFP Cre reporter mouse model (Luche et al., 2007) (Figure 2—figure supplement 3 and Video 3). Fourth, since pharmacological inhibitors can have multiple targets, we confirmed their specificity by combining activators and inhibitors of the WNT-pathway. Pax8-Cre lineage tracing and immunofluorescent analyses allowed us to show that by blocking β-catenin signalling downstream of GSK-3β (by administering ICG001 which binds CBP and prevents β-catenin/CBP interaction), but not upstream (IWR1), CHIR-induced ectopic nephron formation was inhibited (Figure 2—figure supplement 4A and Video 4). Fifth, additional inhibitors against components of the β-catenin/Canonical WNT-pathway also confirmed our findings: (BIO (GSK3β inhibitor) induced effects similar to CHIR; salinomycin (LRP6 inhibitor) induced effects similar to IWR1 (Gandhirajan et al., 2010; Lu et al., 2011); Figure 2—figure supplement 4B). Video 3 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Time-lapse capture of Six2GFPCre/Rosa26tdRFP kidneys. Kidneys cultured in control medium and CHIR medium. Timing and conditions as shown in videos. https://doi.org/10.7554/eLife.04000.009 Video 4 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Time-lapse capture of Pax8Cre YFPlox-stop kidneys. Kidneys cultured in control medium, IWR1 and CHIR medium, or ICG001 and CHIR medium. Timing and conditions as shown in videos. https://doi.org/10.7554/eLife.04000.010 Using these inhibitors we modulated β-catenin signalling in Wt1+/GFP nephrons, where GFP labels the proximal cell population, allowing maturing podocytes within glomeruli to be recognized by their bright concentrated GFP signal (Figure 2A, Video 5). The Wt1+/GFP also labels the nephron progenitor cells and the whole of the pre-tubular aggregate during the MET, but at lower intensities, in accordance with the known expression pattern of Wt1 (Kreidberg, 2010). Time-lapse analysis showed that, by decreasing β-catenin activity, we favoured the differentiation of the proximal cell identity as was seen by an increase in maturation rate of glomeruli; in contrast, increased β-catenin activity blocked proximal identity formation and the formation of glomeruli (Figure 2A–C). Podxl antibody staining (Figure 2D), qRT-PCR analysis of additional markers for proximal differentiation (Nphs2, Synpo, Nphs1, Podxl), and Wt1+/GFP reporter kidneys (Figure 2—figure supplement 5A,B) confirmed that these markers were expressed earlier when β-catenin activity was decreased and that they were repressed when β-catenin activity was increased. We consistently noticed a small number of individual cells in CHIR conditions that remained positive for Wt1 or Podxl (Figure 2A,D). Releasing these cells from increased β-catenin activity, by removing CHIR, allowed them to resume their development (Figure 2E). The proximal identity almost fully recovered and glomerular structures formed at almost the same size as those found in controls, show that the ectopically increased β-catenin activity had been actively suppressing the proximal identity (Figure 2—figure supplement 5C). In contrast, removing IWR1 did not result in the reversal of the phenotype (Figure 2—figure supplement 5D). Figure 2 with 5 supplements see all Download asset Open asset Pharmacological modulation of β-catenin signalling alters proximal segment development. (A) Time-lapse analysis of treated Wt1+/GFP kidneys—same as shown in Video 5. (B) Quantification of mean time taken for first glomeruli to mature to crescent-shaped stage where glomeruli are tightly packed and exhibit a bright signal. (C) Mean number of mature glomeruli after 3800 min of culture. (D) Kidneys stained for podocyte marker Podxl, β-catenin target Lef1, and epithelial marker Cdh1. Arrowheads indicating structures positive for Podxl. (E) The proximal identity resumed its formation when CHIR was removed after 48 hr—white arrowheads indicate larger Podxl positive structures, blue arrowheads indicate very small Podxl positive structures, dashed line separates ectopic nephron zone (e.n.z.) nephrons from those inside the ureteric tree (UB). https://doi.org/10.7554/eLife.04000.011 Video 5 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Time-lapse capture of Wt1+/GFP kidneys. Kidneys cultured in control conditions or treated with IWR1 or CHIR. Timing, conditions, and scale as specified. https://doi.org/10.7554/eLife.04000.017 Distal cells express the epithelial stem-cell marker and β-catenin target Lgr5 (Barker et al., 2012). To test whether the β-catenin gradient also controls the formation of the distal identity, we monitored Lgr5-EGFP expression in kidneys at different β-catenin activity levels using the Lgr5+/EGFP-IRES-CreERT2 mouse model (Barker et al., 2012). Lgr5 was expressed as previously described (Barker et al., 2012), although we primarily detected strong Lgr5-EGFP expression in a subsection of the distal domain adjacent to the medial segment (Figure 3A). Lgr5 expression levels were increased at high levels of β-catenin activity. When β-catenin activity was decreased, the expression domain extended longer although the actual GFP signal was at lower levels compared to controls and samples where β-catenin activity was increased (Figure 3A). We analysed the dynamics of Lgr5 expression under different β-catenin activity levels using time-lapse imaging of Lgr5+/EGFP-IRES-CreERT2 kidneys. The number of Lgr5 positive nephrons increased significantly at higher β-catenin activity levels (3.0×; p = 0.05) compared to controls or kidneys with decreased β-catenin activity (Figure 3B,C). However, in samples with decreased β-catenin activity, Lgr5 positive nephrons emerged at an earlier time-point and these nephrons again appeared to be more elongated, but the GFP signal was lower compared to controls (Figure 3B and Video 6). Compared to controls, the actual number of Lgr5 positive nephrons remained unchanged in IWR1 treated samples (Figure 3C). Lgr5 is also an R-spondin receptor and mediates β-catenin signalling (de Lau et al., 2011). To test whether Lgr5 was functionally modulating the β-catenin signalling gradient, and thereby controlling nephron patterning, we intercrossed the Lgr5+/EGFP-IRES-CreERT2 knockin mice to homozygosity and analysed these for segmentation defects. Homozygous mutants displayed no obvious phenotype in the developing kidney (Figure 3—figure supplement 1 and Video 7). Figure 3 with 1 supplement see all Download asset Open asset Pharmacological modulation of β-catenin signalling alters distal segment development. (A) Lgr5-EGFP expression in treated nephrons with segmentation markers. (B) Time-lapse analysis of treated Lgr5+/EGFP-IRES-CreERT2 kidneys–arrowheads indicate developing nephrons, red-dashed line indicates ureteric bud (UB). (C) Mean number of Lgr5-EGFP positive nephrons per kidney. https://doi.org/10.7554/eLife.04000.005 Video 6 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Time-lapse capture of Lgr5+/EGFP-IRES-CreERT2 kidneys. Kidneys cultured in control conditions or treated with IWR1 or CHIR. Timing, conditions, and scale as specified. https://doi.org/10.7554/eLife.04000.018 Video 7 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Time-lapse capture of Lgr5+/EGFP-IRES-CreERT2 and Lgr5 EGFP-IRES-CreERT2/EGFP-IRES-CreERT2 kidneys. Kidneys cultured in control conditions over 6 days. Timing and scale as specified. https://doi.org/10.7554/eLife.04000.019 Collectively, these data confirm that the cell populations along the proximal–distal axis of the nephrons are responsive to changes in β-catenin signalling and respond positively and negatively as would be predicted from the proposed β-catenin activity gradient. Modulating β-catenin activity shifts positional identities along the nephron Having shown that the proximal and distal cell populations are both affected by increased β-catenin activity and the proximal is affected by decreased β-catenin activity, we hypothesized that the different levels of β-catenin signalling within the gradient specify the positions of identities in the nephron. If so, modulating β-catenin signalling might impose abnormal distal or proximal identities along the axis of the developing nephron (Figure 4A). We immunostained inhibitor treated nephrons with markers for proximal, medial, and distal identities (Wt1, Jag1, Cdh1) and measured the size of the domains in which each was expressed (Figure 4B). Increasing β-catenin did mildly increase total nephron lengths (1.17×, p = 0.017) compared to controls (Figure 4C), but the distal segment was significantly longer (1.8×, p = 0.0003) and the proximal segment was severely reduced (0.2×, p = 1.5 × 10−6). The medial segment showed no significant change in length (1.3×, p = 0.067). These data confirm that both the distal and proximal nephron segments respond to increased β-catenin signalling according to our hypothesis, but the medial remained unchanged in size. Decreasing β-catenin signalling, on the other hand, resulted in much elongated nephrons (Figure 4B,C: 1.7×, p = 1.8 × 10−14), as we had previously noticed (Figure 3A). Here, the length of the proximal segment was unchanged compared to controls (1.0×, p = 0.93), but as we showed above, this segment was more mature in appearance and gene expression (Figure 2). The elongation of the nephron was primarily due to the increases in both the distal (2.5×, p = 0.0002) and the medial segments (1.7×, p = 0.0007) and the nephrons appeared thinner. Although nephron identities responded to increased β-catenin as our model predicted, the large morphological changes obscured any subtle changes in segmentation in those nephrons with decreased β-catenin activity. To address this, we tested whether a gradual increase in β-catenin activity, which only mildly shifted activity levels away from the normal, would give a gradual decrease in proximal identity as would be expected if identities are β-catenin dosage-dependent. This was observed in nephrons that we exposed to different incremental concentrations of CHIR (Figure 4—figure supplement 1). qRT-PCR analysis of RNA from whole treated kidneys confirmed that a mild increase in β-catenin activity promoted expression of most distal segment genes (Wnt4, Pax8, Fgf8, Lhx1, Lgr5) (Figure 4D) thus mirroring the effect of decreasing β-catenin activity; surprisingly, Pax2 did not respond similarly to Pax8. Medial segment control genes (Jag1, Dll1, Heyl, and Irx2) did not increase in response to CHIR as distal genes did. At the highest β-catenin activity levels used [6 µM], nephron formation was reduced (Figure 2—figure supplements 1–2) and the expression profile thus dropped as would be expected (Figure 4D). To test if the observed phenotypes were due to a direct effect on the nephrogenic lineage or an indirect effect via the ureteric bud, we isolated metanephric mesenchyme away from the ureteric bud, induced it to form nephrons with spinal cord (Grobstein, 1953, 1955), and modulated β-catenin activity therein. Comparable to the phenotype observed in whole kidney rudiments, we found that inhibition of β-catenin activity favoured patterning towards the proximal fate, whereas increasing its activity had the opposite effect (Figure 4E). Figure 4 with 2 supplements see all Download asset Open asset Shifts in positional identity by altered β-catenin activity. (A) Model of predicted changes in segmentation if the gradient of β-catenin activity specifies positional identities in the nephron. Nephrons depicted as spheres representing renal vesicle stage. Dashed line indicates nephron segments. Gradient bar indicates β-catenin activity. (B) Antibody stains against segment specific markers in nephrons with different β-catenin signalling conditions. (C) Proximal, medial, and distal nephron domain-sizes in Control, CHIR, and IWR1 treated kidneys. Mean values and SEMs indicated within bars on graph. (D) qRT-PCR analysis of markers for nephron induction displayed as a heat-map with information displayed in figure. The RNA was isolated after 48 hr of culture from whole kidneys. (E) Antibody stains on nephrons developed i" @default.
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• W2989476985 title "Author response: Integrated β-catenin, BMP, PTEN, and Notch signalling patterns the nephron" @default.
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