Receptor protein tyrosine phosphatases are novel components of the polycystin complex

Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutation of PKD1 and PKD2 that encode polycystin-1 and polycystin-2. Polycystin-1 is tyrosine phosphorylated and modulates multiple signaling pathways including AP-1, and the identity of the phosphatases regulating polycystin-1 are previously uncharacterized. Here we identify members of the LAR protein tyrosine phosphatase (RPTP) superfamily as members of the polycystin-1complex mediated through extra- and intracellular interactions. The first extracellular PKD1 domain of polycystin-1 interacts with the first Ig domain of RPTPσ, while the polycystin-1 C-terminus of polycystin-1 interacts with the regulatory D2 phosphatase domain of RPTPγ. Additional homo- and heterotypic interactions between RPTPs recruit RPTPδ. The multimeric polycystin protein complex is found localised in cilia. RPTPσ and RPTPδ are also part of a polycystin-1/E-cadherin complex known to be important for early events in adherens junction stabilisation. The interaction between polycystin-1 and RPTPγ is disrupted in ADPKD cells, while RPTPσ and RPTPδ remain closely associated with E-cadherin, largely in an intracellular location. The polycystin-1 C-terminus is an in vitro substrate of RPTPγ, which dephosphorylates the c-Src phosphorylated Y4237 residue and activates AP1-mediated transcription. The data identify RPTPs as novel interacting partners of the polycystins both in cilia and at adhesion complexes and demonstrate RPTPγ phosphatase activity is central to the molecular mechanisms governing polycystin-dependent signaling. This article is part of a Special Issue entitled: Polycystic Kidney Disease

Ablation of the renal stroma defines its critical role in nephron progenitor and vasculature patterning

The renal stroma is an embryonic cell population located in the cortex that provides a structural framework as well as a source of endothelial progenitors for the developing kidney. The exact role of the renal stroma in normal kidney development hasn't been clearly defined. However, previous studies have shown that the genetic deletion of Foxd1, a renal stroma specific gene, leads to severe kidney malformations confirming the importance of stroma in normal kidney development. This study further investigates the role of renal stroma by ablating Foxd1-derived stroma cells themselves and observing the response of the remaining cell populations. A Foxd1cre (renal stroma specific) mouse was crossed with a diphtheria toxin mouse (DTA) to specifically induce apoptosis in stromal cells. Histological examination of kidneys at embryonic day 13.5-18.5 showed a lack of stromal tissue, mispatterning of renal structures, and dysplastic and/or fused horseshoe kidneys. Immunofluorescence staining of nephron progenitors, vasculature, ureteric epithelium, differentiated nephron progenitors, and vascular supportive cells revealed that mutants had thickened nephron progenitor caps, cortical regions devoid of nephron progenitors, aberrant vessel patterning and thickening, ureteric branching defects and migration of differentiated nephron structures into the medulla. The similarities between the renal deformities caused by Foxd1 genetic knockout and Foxd1DTA mouse models reveal the importance of Foxd1 in mediating and maintaining the functional integrity of the renal stroma. © 2014 Hum et al

Genome Mining for Radical SAM Protein Determinants Reveals Multiple Sactibiotic-Like Gene Clusters

Thuricin CD is a two-component bacteriocin produced by Bacillus thuringiensis that kills a wide range of clinically significant Clostridium difficile. This bacteriocin has recently been characterized and consists of two distinct peptides, Trnβ and Trnα, which both possess 3 intrapeptide sulphur to α-carbon bridges and act synergistically. Indeed, thuricin CD and subtilosin A are the only antimicrobials known to possess these unusual structures and are known as the sactibiotics (sulplur to alpha carbon-containing antibiotics). Analysis of the thuricin CD-associated gene cluster revealed the presence of genes encoding two highly unusual SAM proteins (TrnC and TrnD) which are proposed to be responsible for these unusual post-translational modifications. On the basis of the frequently high conservation among enzymes responsible for the post-translational modification of specific antimicrobials, we performed an in silico screen for novel thuricin CD–like gene clusters using the TrnC and TrnD radical SAM proteins as driver sequences to perform an initial homology search against the complete non-redundant database. Fifteen novel thuricin CD–like gene clusters were identified, based on the presence of TrnC and TrnD homologues in the context of neighbouring genes encoding potential bacteriocin structural peptides. Moreover, metagenomic analysis revealed that TrnC or TrnD homologs are present in a variety of metagenomic environments, suggesting a widespread distribution of thuricin-like operons in a variety of environments. In-silico analysis of radical SAM proteins is sufficient to identify novel putative sactibiotic clusters

Kidney Development in the Absence of Gdnf and Spry1 Requires Fgf10

GDNF signaling through the Ret receptor tyrosine kinase (RTK) is required for ureteric bud (UB) branching morphogenesis during kidney development in mice and humans. Furthermore, many other mutant genes that cause renal agenesis exert their effects via the GDNF/RET pathway. Therefore, RET signaling is believed to play a central role in renal organogenesis. Here, we re-examine the extent to which the functions of Gdnf and Ret are unique, by seeking conditions in which a kidney can develop in their absence. We find that in the absence of the negative regulator Spry1, Gdnf, and Ret are no longer required for extensive kidney development. Gdnf−/−;Spry1−/− or Ret−/−;Spry1−/− double mutants develop large kidneys with normal ureters, highly branched collecting ducts, extensive nephrogenesis, and normal histoarchitecture. However, despite extensive branching, the UB displays alterations in branch spacing, angle, and frequency. UB branching in the absence of Gdnf and Spry1 requires Fgf10 (which normally plays a minor role), as removal of even one copy of Fgf10 in Gdnf−/−;Spry1−/− mutants causes a complete failure of ureter and kidney development. In contrast to Gdnf or Ret mutations, renal agenesis caused by concomitant lack of the transcription factors ETV4 and ETV5 is not rescued by removing Spry1, consistent with their role downstream of both RET and FGFRs. This shows that, for many aspects of renal development, the balance between positive signaling by RTKs and negative regulation of this signaling by SPRY1 is more critical than the specific role of GDNF. Other signals, including FGF10, can perform many of the functions of GDNF, when SPRY1 is absent. But GDNF/RET signaling has an apparently unique function in determining normal branching pattern. In contrast to GDNF or FGF10, Etv4 and Etv5 represent a critical node in the RTK signaling network that cannot by bypassed by reducing the negative regulation of upstream signals

Branching morphogenesis of the ureteric epithelium during kidney development is coordinated by the opposing functions of GDNF and Sprouty1

AbstractBranching of ureteric bud-derived epithelial tubes is a key morphogenetic process that shapes development of the kidney. Glial cell line-derived neurotrophic factor (GDNF) initiates ureteric bud formation and promotes subsequent branching morphogenesis. Exactly how GDNF coordinates branching morphogenesis is unclear. Here we show that the absence of the receptor tyrosine kinase antagonist Sprouty1 (Spry1) results in irregular branching morphogenesis characterized by both increased number and size of ureteric bud tips. Deletion of Spry1 specifically in the epithelium is associated with increased epithelial Wnt11 expression as well as increased mesenchymal Gdnf expression. We propose that Spry1 regulates a Gdnf/Ret/Wnt11-positive feedback loop that coordinates mesenchymal–epithelial dialogue during branching morphogenesis. Genetic experiments indicate that the positive (GDNF) and inhibitory (Sprouty1) signals have to be finely balanced throughout renal development to prevent hypoplasia or cystic hyperplasia. Epithelial cysts develop in Spry1-deficient kidneys that share several molecular characteristics with those observed in human disease, suggesting that Spry1 null mice may be useful animal models for cystic hyperplasia

Kidney development in the absence of Gdnf and Spry1 requires Fgf10.

GDNF signaling through the Ret receptor tyrosine kinase (RTK) is required for ureteric bud (UB) branching morphogenesis during kidney development in mice and humans. Furthermore, many other mutant genes that cause renal agenesis exert their effects via the GDNF/RET pathway. Therefore, RET signaling is believed to play a central role in renal organogenesis. Here, we re-examine the extent to which the functions of Gdnf and Ret are unique, by seeking conditions in which a kidney can develop in their absence. We find that in the absence of the negative regulator Spry1, Gdnf, and Ret are no longer required for extensive kidney development. Gdnf-/-;Spry1-/- or Ret-/-;Spry1-/- double mutants develop large kidneys with normal ureters, highly branched collecting ducts, extensive nephrogenesis, and normal histoarchitecture. However, despite extensive branching, the UB displays alterations in branch spacing, angle, and frequency. UB branching in the absence of Gdnf and Spry1 requires Fgf10 (which normally plays a minor role), as removal of even one copy of Fgf10 in Gdnf-/-;Spry1-/- mutants causes a complete failure of ureter and kidney development. In contrast to Gdnf or Ret mutations, renal agenesis caused by concomitant lack of the transcription factors ETV4 and ETV5 is not rescued by removing Spry1, consistent with their role downstream of both RET and FGFRs. This shows that, for many aspects of renal development, the balance between positive signaling by RTKs and negative regulation of this signaling by SPRY1 is more critical than the specific role of GDNF. Other signals, including FGF10, can perform many of the functions of GDNF, when SPRY1 is absent. But GDNF/RET signaling has an apparently unique function in determining normal branching pattern. In contrast to GDNF or FGF10, Etv4 and Etv5 represent a critical node in the RTK signaling network that cannot by bypassed by reducing the negative regulation of upstream signals