Courtney M. Karner

Wnt9b signaling regulates planar cell polarity and kidney tubule morphogenesis

Although many vertebrate organs, such as kidneys, lungs and liver, are composed of epithelial tubules, little is known of the mechanisms that establish the length or diameter of these tubules. In the kidney, defects in the establishment or maintenance of tubule diameter are associated with one of the most common inherited human disorders, polycystic kidney disease. Here we show that attenuation of Wnt9b signaling during kidney morphogenesis affects the planar cell polarity of the epithelium and leads to tubules with significantly increased diameter. Although previous studies showed that polarized cell divisions maintain the diameter of postnatal kidney tubules, we find that cell divisions are randomly oriented during embryonic development. Our data suggest that diameter is established during early morphogenetic stages by convergent extension processes and maintained by polarized cell divisions. Wnt9b, signaling through the non-canonical Rho/Jnk branch of the Wnt pathway, is necessary for both of these processes.

Canonical Wnt9b signaling balances progenitor cell expansion and differentiation during kidney development

The mammalian kidney is composed of thousands of individual epithelial tubules known as nephrons. Deficits in nephron number are associated with myriad diseases ranging from complete organ failure to congenital hypertension. A balance between differentiation and maintenance of a mesenchymal progenitor cell population determines the final number of nephrons. How this balance is struck is poorly understood. Previous studies have suggested that Wnt9b/β-catenin signaling induced differentiation (mesenchymal-to-epithelial transition) in a subset of the progenitors but needed to be repressed in the remaining progenitors to keep them in the undifferentiated state. Here, we report that Wnt9b/β-catenin signaling is active in the progenitors and is required for their renewal/proliferation. Using a combination of approaches, we have revealed a mechanism through which cells receiving the same Wnt9b/β-catenin signal can respond in distinct ways (proliferate versus differentiate) depending on the cellular environment in which the signal is received. Interpretation of the signal is dependent, at least in part, on the activity of the transcription factor Six2. Six2-positive cells that receive the Wnt9b signal are maintained as progenitors whereas cells with reduced levels of Six2 are induced to differentiate by Wnt9b. Using this simple mechanism, the kidney is able to balance progenitor cell expansion and differentiation insuring proper nephron endowment. These findings provide novel insights into the molecular mechanisms that regulate progenitor cell differentiation during normal and pathological conditions.

Vertebrate kidney tubules elongate using a planar cell polarity-dependent, rosette-based mechanism of convergent extension

Cystic kidney diseases are a global public health burden, affecting over 12 million people. Although much is known about the genetics of kidney development and disease, the cellular mechanisms driving normal kidney tubule elongation remain unclear. Here, we used in vivo imaging to show for the first time that mediolaterally oriented cell intercalation is fundamental to vertebrate kidney morphogenesis. Unexpectedly, we found that kidney tubule elongation is driven in large part by a myosin-dependent, multicellular rosette–based mechanism, previously only described in Drosophila melanogaster. In contrast to findings in Drosophila, however, non-canonical Wnt and planar cell polarity (PCP) signaling is required to control rosette topology and orientation during vertebrate kidney tubule elongation. These data resolve long-standing questions concerning the role of PCP signaling in the developing kidney and, moreover, establish rosette-based intercalation as a deeply conserved cellular engine for epithelial morphogenesis.

WNT-LRP5 Signaling Induces Warburg Effect through mTORC2 Activation during Osteoblast Differentiation

WNT signaling controls many biological processes including cell differentiation in metazoans. However, how WNT reprograms cell identity is not well understood. We have investigated the potential role of cellular metabolism in WNT-induced osteoblast differentiation. WNT3A induces aerobic glycolysis known as Warburg effect by increasing the level of key glycolytic enzymes. The metabolic regulation requires LRP5 but not β-catenin and is mediated by mTORC2-AKT signaling downstream of RAC1. Suppressing WNT3A-induced metabolic enzymes impairs osteoblast differentiation in vitro. Deletion of Lrp5 in the mouse, which decreases postnatal bone mass, reduces mTORC2 activity and glycolytic enzymes in bone cells and lowers serum lactate levels. Conversely, mice expressing a mutant Lrp5 that causes high bone mass exhibit increased glycolysis in bone. Thus, WNT-LRP5 signaling promotes bone formation in part through direct reprogramming of glucose metabolism. Moreover, regulation of cellular metabolism may represent a general mechanism contributing to the wide-ranging functions of WNT proteins.

Stromal–epithelial crosstalk regulates kidney progenitor cell differentiation

Present models suggest that the fate of the kidney epithelial progenitors is solely regulated by signals from the adjacent ureteric bud. The bud provides signals that regulate the survival, renewal and differentiation of these cells. Recent data suggest that Wnt9b, a ureteric-bud-derived factor, is sufficient for both progenitor cell renewal and differentiation. How the same molecule induces two seemingly contradictory processes is unknown. Here, we show that signals from the stromal fibroblasts cooperate with Wnt9b to promote differentiation of the progenitors. The atypical cadherin Fat4 encodes at least part of this stromal signal. Our data support a model whereby proper kidney size and function is regulated by balancing opposing signals from the ureteric bud and stroma to promote renewal and differentiation of the nephron progenitors.

Molecular regulation of kidney development: is the answer blowing in the Wnt?

Development of the metanephric kidney is a complicated process regulated by reciprocal signals from the ureteric bud and the metanephric mesenchyme that regulate tubule formation and epithelial branching morphogenesis. Over the past several years, several studies have suggested that Wnt signaling is involved in multiple aspects of normal kidney development as well as injury response and cancer progression. We will review these data here.

Diverse chemical scaffolds support direct inhibition of the membrane bound O-acyltransferase Porcupine

Background: The acyltransferase Porcupine (Porcn) is essential for active Wnt ligand production and is chemically tractable. Results: Novel small molecules targeting Porcn enables interrogation of Wnt signaling in vitro and in vivo. Conclusion: Porcn is highly druggable and supports diverse cellular responses in embryonic development and regeneration. Significance: Porcn inhibitors represent versatile chemical probes for Wnt signaling in vivo and are potential anti-cancer therapeutic agents. Secreted Wnt proteins constitute one of the largest families of intercellular signaling molecules in vertebrates with essential roles in embryonic development and adult tissue homeostasis. The functional redundancy of Wnt genes and the many forms of cellular responses they elicit, including some utilizing the transcriptional co-activator β-catenin, has limited the ability of classical genetic strategies to uncover their roles in vivo. We had previously identified a chemical compound class termed Inhibitor of Wnt Production (or IWP) that targets Porcupine (Porcn), an acyltransferase catalyzing the addition of fatty acid adducts onto Wnt proteins. Here we demonstrate that diverse chemical structures are able to inhibit Porcn by targeting its putative active site. When deployed in concert with small molecules that modulate the activity of Tankyrase enzymes and glycogen synthase kinase 3 β (GSK3β), additional transducers of Wnt/β-catenin signaling, the IWP compounds reveal an essential role for Wnt protein fatty acylation in eliciting β-catenin-dependent and -independent forms of Wnt signaling during zebrafish development. This collection of small molecules facilitates rapid dissection of Wnt gene function in vivo by limiting the influence of redundant Wnt gene functions on phenotypic outcomes and enables temporal manipulation of Wnt-mediated signaling in vertebrates.

Mutations of HNF-1β inhibit epithelial morphogenesis through dysregulation of SOCS-3

Hepatocyte nuclear factor-1β (HNF-1β) is a Pit-1, Oct-1/2, Unc-86 (POU) homeodomain-containing transcription factor expressed in the kidney, liver, pancreas, and other epithelial organs. Mutations of HNF-1β cause maturity-onset diabetes of the young, type 5 (MODY5), which is characterized by early-onset diabetes mellitus and congenital malformations of the kidney, pancreas, and genital tract. Knockout of HNF-1β in the mouse kidney results in cyst formation. However, the signaling pathways and transcriptional programs controlled by HNF-1β are poorly understood. Using genome-wide chromatin immunoprecipitation and DNA microarray (ChIP-chip) and microarray analysis of mRNA expression, we identified SOCS3 (suppressor of cytokine signaling-3) as a previously unrecognized target gene of HNF-1β in the kidney. HNF-1β binds to the SOCS3 promoter and represses SOCS3 transcription. The expression of SOCS3 is increased in HNF-1β knockout mice and in renal epithelial cells expressing dominant-negative mutant HNF-1β. Increased levels of SOCS-3 inhibit HGF-induced tubulogenesis by decreasing phosphorylation of Erk and STAT-3. Conversely, knockdown of SOCS-3 in renal epithelial cells expressing dominant-negative mutant HNF-1β rescues the defect in HGF-induced tubulogenesis by restoring phosphorylation of Erk and STAT-3. Thus, HNF-1β regulates tubulogenesis by controlling the levels of SOCS-3 expression. Manipulating the levels of SOCS-3 may be a useful therapeutic approach for human diseases induced by HNF-1β mutations.

Increased glutamine catabolism mediates bone anabolism in response to WNT signaling

WNT signaling stimulates bone formation by increasing both the number of osteoblasts and their protein-synthesis activity. It is not clear how WNT augments the capacity of osteoblast progenitors to meet the increased energetic and synthetic needs associated with mature osteoblasts. Here, in cultured osteoblast progenitors, we determined that WNT stimulates glutamine catabolism through the tricarboxylic acid (TCA) cycle and consequently lowers intracellular glutamine levels. The WNT-induced reduction of glutamine concentration triggered a general control nonderepressible 2-mediated (GCN2-mediated) integrated stress response (ISR) that stimulated expression of genes responsible for amino acid supply, transfer RNA (tRNA) aminoacylation, and protein folding. WNT-induced glutamine catabolism and ISR were β-catenin independent, but required mammalian target of rapamycin complex 1 (mTORC1) activation. In a hyperactive WNT signaling mouse model of human osteosclerosis, inhibition of glutamine catabolism or Gcn2 deletion suppressed excessive bone formation. Together, our data indicate that glutamine is both an energy source and a protein-translation rheostat that is responsive to WNT and suggest that manipulation of the glutamine/GCN2 signaling axis may provide a valuable approach for normalizing deranged protein anabolism associated with human diseases.

Physiological Notch Signaling Maintains Bone Homeostasis via RBPjk and Hey Upstream of NFATc1

Notch signaling between neighboring cells controls many cell fate decisions in metazoans both during embryogenesis and in postnatal life. Previously, we uncovered a critical role for physiological Notch signaling in suppressing osteoblast differentiation in vivo. However, the contribution of individual Notch receptors and the downstream signaling mechanism have not been elucidated. Here we report that removal of Notch2, but not Notch1, from the embryonic limb mesenchyme markedly increased trabecular bone mass in adolescent mice. Deletion of the transcription factor RBPjk, a mediator of all canonical Notch signaling, in the mesenchymal progenitors but not the more mature osteoblast-lineage cells, caused a dramatic high-bone-mass phenotype characterized by increased osteoblast numbers, diminished bone marrow mesenchymal progenitor pool, and rapid age-dependent bone loss. Moreover, mice deficient in Hey1 and HeyL, two target genes of Notch-RBPjk signaling, exhibited high bone mass. Interestingly, Hey1 bound to and suppressed the NFATc1 promoter, and RBPjk deletion increased NFATc1 expression in bone. Finally, pharmacological inhibition of NFAT alleviated the high-bone-mass phenotype caused by RBPjk deletion. Thus, Notch-RBPjk signaling functions in part through Hey1-mediated inhibition of NFATc1 to suppress osteoblastogenesis, contributing to bone homeostasis in vivo.