(D) Antibody discolorations against total -catenin and phosphorylated -catenin in S-shaped body nephronJag1 marking the medial segment
(D) Antibody discolorations against total -catenin and phosphorylated -catenin in S-shaped body nephronJag1 marking the medial segment. modulating -catenin activity or PI3K rescues segment identities normally lost by inhibition of Notch. Our data therefore identifies a molecular network for nephron patterning. DOI: http://dx.doi.org/10.7554/eLife.04000.001 cell population gives rise to proximal structures, a Jag1+ population to the medial part and Lgr5cells 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 BD-1047 2HBr segments is usually regulated remains unknown. The initiation of the nephron MET is usually driven by -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+CNFAT 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 unique nephron segment lineages (Lindstrom et al., 2013). Post-MET, Wnt9b functions 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). nephrons form no proximal tubules or glomeruli (Cheng et al., 2007). However, ectopic expression of the intracellular and active Notch1-domain name (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 usually important for the initial patterning of the nephron immediately post-MET remains to be determined. Using in vivo and ex lover vivo techniques we demonstrate that a gradient of -catenin activity, along the proximalCdistal nephron axis, controls the differentiation of segment-specific cell fates. We further investigate how -catenin activity is usually 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 -catenin activity. In addition, we show that modulating -catenin or PI3K activity partially rescues the nephron IL10A 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 -catenin activity gradient is usually generated along the nephron axis Regulation of -catenin activity is essential for nephron induction and MET (Davies and Garrod, 1995; Kuure et al., 2007; Park et al., 2007). To determine whether -catenin is usually involved in post-MET stages of nephron development, we tracked its activity in embryonic kidney organ cultures using a -catenin signalling reporter mouse strain (expressing nephrons showed that the different GFP transmission intensities propagated in a distal-to-proximal direction over time alongside the normal nephron growth and segmentation (Physique 1figure product 1A and Video 2). Confocal imaging confirmed different GFP BD-1047 2HBr intensities in nephrons at later stages: S-shaped body (Physique 1B and Physique 1figure product 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 -catenin activity (Physique 1A,B, Physique 1figure product 1B; Video 1). We BD-1047 2HBr quantified the transmission 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 transmission intensities showed an exponentially decreasing gradient (R2 =.