Commentaries Upper urinary tract pacemaker cells join the GLI club Doris Herzlinger

Department of Physiology and Biophysics and Department of Urology, Cornell University Medical College, New York, New York, USA.

Mutations in GLI3, a component of the (Shh) signaling the presence of a patent outflow tract, dif- pathway, cause a variety of developmental syndromes. In this issue ferentiated ureteral epithelium, and well- of the JCI, Cain and colleagues show that tightly regulated GLI3 developed smooth muscle coat (6). activity is essential for Shh-dependent differentiation of upper urinary tract pacemaker cells and the efficient flow of urine from the kidney to the blad- Pacemaker cells are essential der. These results link defective pacemaker cell differentiation with hydro- for triggering outflow tract nephrosis and provide a cellular basis for one of the abnormal renal defects smooth muscle contraction observed in with the GLI3-linked disease Pallister-Hall syndrome. Fundamental scientific studies dating back to the late 1800s provided insight The kidney outflow tract is highly the ureter into the bladder, or overgrowth into a possible etiology of hydronephrotic prone to congenital defects of the outflow tract epithelium. Alterna- syndromes that could not be explained by The kidney outflow tract carries wastes tively, it can be caused by functional defects either structural occlusion or gross defect removed from the systemic circulation by in the formation of the outflow tract in ureteral epithelial or smooth muscle for- glomerular filtration and tubular secre- smooth muscle coat, leading to severely mation. These studies suggested that the tion to the bladder (1). It is composed of impaired or even absent peristalsis. Finally, efficient flow of urine from the kidney to the renal calyces, pelvis, and ureter and has hydronephrosis could be caused by defects the bladder is dependent on the presence specialized differentiated properties that in the cells that initiate and coordinate out- of specialized myocytes that trigger the mediate this essential conduit function. flow tract contraction. coordinated, proximal-to-distal peristaltic The outflow tract epithelium has an imper- waves that propagate through the outflow meable luminal surface preventing wastes Elucidating the required tract smooth muscle coat (7). These spe- carried in the urine from diffusing back for outflow tract formation cialized myocytes, called pacemaker cells, into the systemic circulation. The proxi- Over the past 10 years, several mouse mod- elicit spontaneous membrane depolariza- mal-to-distal contractions that propagate els that develop hydronephrosis during tions that provide the stimulus for trigger- through the outflow tract smooth muscle have provided ing ureteral smooth muscle contraction coat are essential for propelling urine into insight into the genetic pathways control- and play a fundamental role in controlling the lower urinary tract. ling outflow tract formation and function. the frequency and origin of contractile Despite its seemingly simple structure Targeted deletion of members of the uro- waves in the outflow tract musculature. and function, congenital defects localized plakin family, which are selectively As in the heart, the outflow tract contains to the kidney outflow tract are detected in expressed by the urothelium, result in primary pacemakers that trigger contrac- approximately 0.5% of fetuses analyzed by structural obstruction as a result of epi- tions at the site of fluid inflow as well as routine antenatal sonography (2). These thelial hyperplasia (3). Proper insertion of secondary, latent pacemaker activity that defects obstruct the flow of urine out of the ureter into the bladder wall is essential can trigger contraction in the event of pri- the kidney, causing dilations of the renal for the formation of a patent outflow tract, mary pacemaker failure. pelvis (i.e., hydronephrosis). Although a and studies by Mendelsohn’s group dem- Although the electrophysiological prop- substantial percentage of fetal hydrone- onstrated that this process is dependent erties of upper urinary tract pacemaker phrotic syndromes spontaneously resolve, on retinoic acid signaling mediated in part cells have been extensively analyzed, the if severe and persistent, hydronephro- by the tyrosine cRet (4). Finally, ion channels and receptors required for sis results in permanent kidney damage mouse models that develop hydronephro- their function have remained elusive until and is the major cause of renal failure in sis in the absence of any obvious structural recently. Our group has begun to unravel infants and children. occlusions in the outflow tract have led to the molecular mechanisms mediating The causes of congenital hydronephrosis the identification of several signaling and outflow tract pacemaker activity, recently are various (Figure 1). For example, it can factors required for outflow discovering that hyperpolarizing activated be caused by structural occlusions of the tract smooth muscle differentiation and cation channels (HCNs) play a fundamen- outflow tract as a result of compression by organization, including bone morphoge- tal role in triggering outflow tract peri- ectopic renal vessels, abnormal insertion of netic 4, Tbx18, Teashirt, and Six1, stalsis (8). The HCN gene family includes to name only a few (reviewed in ref. 5). at least 4 members, and HCN2 and HCN4 However, mice with conditional inactiva- are essential for mediating cardiac pace- Conflict of interest: The author has declared that no conflict of interest exists. tion of the calcineurin signaling pathway maker activity (9). We found that HCN3 Citation for this article: J Clin Invest. 2011; in the periureteral mesenchyme develop is selectively expressed by cells adjacent to 121(3):836–838. doi:10.1172/JCI46400. hydronephrosis and renal damage despite the renal-most edge of the outflow tract

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competent smooth muscle coat. However, contractions in this mutant outflow tract musculature were uncoordinated and did not efficiently propel urine from the kid- ney to the bladder. Further analyses dem- onstrated that -deficient mice exhibited wild-type levels of epithelial and smooth muscle differentiation markers. Strikingly, the mutant mice lacked detect- able levels of HCN and c-Kit expression. Collectively, these data indicate that the contraction defects and resulting hydrone- phrosis observed in Smoothened-deficient mice are caused by abnormal pacemaker cell differentiation. The above findings, combined with the renal defects observed in humans with Pal- Figure 1 lister-Hall syndrome, suggest that Smooth- The kidney outflow tract includes the renal calyces, pelvis, and ureter and is highly prone to congenital defects. Abnormalities that severely impair the flow of urine through the outflow tract ened may be required for the upregulated lead to hydronephrosis and often permanent kidney damage. Known causes of hydronephrosis expression of HCN and c-Kit via a GLI3- include outflow tract compression by ectopic renal vessels, structural occlusions intrinsic to the dependent process. To address this, Cain outflow tract, and aberrant outflow tract smooth muscle differentiation that results in aperistaltic et al. examined outflow tract formation in ureter segments. The report by Cain et al. (13) shows that hydronephrosis can also be caused a murine model of Pallister-Hall syndrome by inefficient, dysplastic peristalsis caused by an absence of upper urinary tract pacemakers, with a targeted mutation in Gli3 (D699) the specialized cells that trigger smooth muscle contractions. that generates truncated protein (13). Homozygous Gli3D699 mutant mice devel- oped urinary tract phenotypes that were musculature, where contractions nor- phrosis, Cain et al. disrupted this signal- nearly identical to those observed in mice mally initiate. Furthermore, optical map- ing pathway in the developing murine with conditional Smoothened inactivation, ping analyses demonstrated that HCN outflow tract by a variety of genetic including an absence of c-Kit– and HCN3- activity is essential for the spontaneous manipulations. Previous studies demon- expressing cells, uncoordinated peristalsis, membrane depolarizations that initiate strated that targeted deletion of Shh in the and hydronephrosis. Taken together, these and coordinate outflow tract peristalsis. ureter epithelia beginning at early stages data indicate that the acquisition of uni- These data indicate that HCN3 expression of urinary tract morphogenesis resulted directional, coordinated peristalsis in the marks primary outflow tract pacemaker in abnormal outflow tract smooth muscle developing outflow tract is dependent on cells and that these cells are required for formation (14). The present studies by tightly regulated GLI3 repressor activity outflow tract function. Moreover, our pre- Cain et al. demonstrate that Shh signaling and provide insight into the etiology of the vious studies indicated that c-Kit tyrosine is also crucial later in development for the hydronephrotic syndromes observed in kinase activity is also required for coor- acquisition of coordinated outflow tract Pallister-Hall patients. dinated outflow tract peristalsis in vitro smooth muscle contraction (13). In conclusion, the work of Cain et al. (10). Cells expressing c-Kit are distinct Shh signaling is mediated by transcrip- (13), as well as other fundamental scientif- from HCN3+ cells and exhibit spatial dis- tion factors belonging to the GLI fam- ic studies elucidating the genetic pathways tribution and electrophysiological prop- ily of transcriptional regulators: GLI1, mediating outflow tract formation, pro- erties consistent with secondary outflow GLI2, and GLI3. Notably, the GLIs vari- vide insight into the etiology of congenital tract pacemakers (10–12). Collectively, ably activate and repress transcription; hydronephrosis, one of the most common these results imply that abnormal expres- in the absence of secreted Shh, the cell congenital defects in humans. Results of sion of c-Kit and HCN in the developing surface receptor Patched associates with these studies are likely to lead to novel ther- outflow tract can lead to uncoordinated a second protein, Smoothened, convert- apies for the treatment of this condition and inefficient outflow tract peristalsis ing GLI3 into a transcriptional repressor. and provide the groundwork for much- and impaired urine flow into the lower In the presence of Shh, Patched dissoci- needed genetic tests that discriminate urinary tract. The manuscript included ates from Smoothened, and GLI3 — along between fetal hydronephrotic syndromes in this issue of JCI by Cain et al. provides with GLI2 — activates Shh-dependent that spontaneously resolve and those that strong support for this hypothesis (13). transcription (15, 16). result in permanent renal damage. Cain et al. first showed that disruption Pacemaker cell differentiation is of Shh signaling by conditional deletion Acknowledgments dependent on regulated Sonic of Smoothened in the urinary tract mesen- The author thanks Romulo Hurtado, Cindy hedgehog signaling chyme at midgestation resulted in hydrone- Liang, and Cathy Mendelsohn for critically To understand how abnormal Sonic phrosis and permanent renal damage (13). reading the manuscript and acknowledges hedgehog (Shh) signaling in humans Smoothened mutant outflow tracts were NIH for full support of her research pro- causes renal defects, including hydrone- patent and surrounded by a contraction- gram (grants DK45218 and DK02723).

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Address correspondence to: Doris Herzlinger, actions during ureter development. Kidney Int. cells in the mouse ureter: the interstitial cells of 2007;72(12):1459–1467. Cajal of the urinary tract. Am J Physiol Renal Physiol. Cornell University Medical College, Physiol- 6. Chang CP, et al. Calcineurin is required in urinary 2003;284(5):F925–F929. ogy and Biophysics D-501, 1300 York Ave., tract mesenchyme for the development of the 12. Lang RJ, Tonta MA, Zoltkowski BZ, Meeker WF, New York, New York 10021-4805, USA. pyeloureteral peristaltic machinery J Clin Invest. Wendt I, Parkington HC. Pyeloureteric peristalsis: Phone: 212.746.6377; Fax: 212.746.8690; 2004;113(7):1051–1058. role of atypical smooth muscle cells and interstitial 7. Santicioli P, Maggi CA. Myogenic and neurogenic cells of Cajal-like cells as pacemakers. J Physiol. 2006; E-mail: [email protected]. factors in the control of pyeloureteral motil- 576(pt 3):695–705. ity and ureteral peristalsis. Pharmacol Rev. 1998; 13. Cain JE, Islam E, Haxho F, Blake J, Rosenblum 1. Johnson LR. Essential Medical Physiology. New York, 50(4):683–722. ND. GLI3 repressor controls functional devel- New York, USA: Raven Press; 1992. 8. Hurtado R, Bub G, Herzlinger D. The pelvis-kidney opment of the mouse ureter. J Clin Invest. 2011; 2. Becker A, Baum M. Obstructive uropathy. Early junction contains HCN3, a hyperpolarization-acti- 121(3):1199–1206. Hum Dev. 2006;82(1):15–22. vated cation channel that triggers ureter peristalsis. 14. Yu J, Carroll TJ, McMahon AP. Sonic hedgehog 3. Hu P, et al. Ablation of uroplakin III gene results in Kidney Int. 2010;77(6):500–508. regulates proliferation and differentiation of small urothelial plaques, urothelial leakage, and ves- 9. Biel M, Wahl-Schott C, Michalakis S, Zong X. mesenchymal cells in the mouse metanephric kid- icoureteral reflux. J Cell Biol. 2000;151(5):961–972. Hyperpolarization-activated cation channels: from ney. Development. 2002;129(22):5301–5312. 4. Mendelsohn C. Using mouse models to understand genes to function. Physiol Rev. 2009;89(3):847–885. 15. Biesecker LG. What you can learn from one gene: normal and abnormal urogenital tract develop- 10. David SG, Cebrian C, Vaughan ED Jr, Herzlinger GLI3. J Med Genet. 2006;43(6):465–469. ment. Organogenesis. 2009;5(1):306–314. D. c-kit and ureteral peristalsis. J Urol. 2005; 16. Riobo NA, Manning DR. Pathways of signal trans­ 5. Airik R, Kispert A. Down the tube of obstructive 173(1):292–295. duction employed by vertebrate Hedgehogs. Bio- nephropathies: the importance of tissue inter- 11. Pezzone MA, et al. Identification of c-kit-positive chem J. 2007;403(3):369–379. Unraveling virus-induced lymphomagenesis Chris Boshoff

UCL Cancer Institute, University College London, London, United Kingdom.

Kaposi sarcoma herpesvirus (KSHV), a human gammaherpesvirus, is the upregulation of surface molecules required etiological agent for the endothelial-derived Kaposi sarcoma (KS) and also for KSHV infection, for example, heparin for certain lymphoproliferative disorders. In these lymphoproliferations, sulfate (8) and DC-SIGN (CD209) (7), the KSHV-infected cells carry the stigmata of B lymphocytes, with plasma- and/or whether such activation triggers blastic features. The JCI has published three manuscripts addressing key signaling pathways that encourage viral questions related to B cell infection and viral latent expression in B cells. entry and intracellular transport (9). Myoung and Ganem provide evidence that CD4+ lymphocytes suppress Myoung and Ganem showed that expo- KSHV replication, promoting latency in B cells; Hassman and colleagues sure of primary human tonsillar explants show that KSHV infection drives plasmablast differentiation in a subset to KSHV virions results in infection of of IgM+ λ light chain–expressing cells; and Ballon and colleagues describe B and T lymphocytes, with B lymphocytes the in vivo transdifferentiation of B lymphocytes by KSHV-encoded viral producing substantial amounts of infec- FLICE-inhibitory protein (vFLIP). tious virions (2). Strikingly, and in con- trast to exposure of B lymphocytes to EBV, Two lymphotropic human herpesviruses JCI has now published three papers (2–4) KSHV displays predominantly lytic infec- are linked to lymphoma development: EBV that reveal provocative findings regarding tion in tonsillar-derived B lymphocytes. and Kaposi sarcoma herpesvirus (KSHV). KSHV and B cell infection and function. This spontaneous lytic viral reactivation The mechanisms by which EBV infects The main route for infection by EBV of infected B lymphocytes was suppressed B lymphocytes and induces their differen- and KSHV is via saliva. EBV enters tonsil- when the investigators added activated tiation and proliferation are reasonably well lar B cells via the CD21 receptor and steers T lymphocytes from tonsillar explants. understood (1). In vitro, EBV infection of the differentiation of pregerminal naive However, these activated CD4+ T lympho- human primary B cells causes the establish- B lymphocytes toward memory cells by way cytes did not induce B lymphocyte cytoly- ment of latent infection in a fraction of cells of viral latent transcripts. The presence of sis and were not dependent on autologous exposed to virus, cellular transformation, KSHV in saliva (5) and in tonsillar and T lymphocytes being used. Thus, the sup- and the outgrowth of indefinitely prolifer- peripheral CD19+ B cells (6) and the inef- pression of spontaneous viral lytic cycle ating B lymphoblastoid cell lines. In con- ficient in vitro infection of primary non- entry in B lymphocytes was MHC unre- trast, the lack of B cell systems available for stimulated B lymphocytes from PBMCs stricted and not dependent on killing of tar- the study of KSHV in vitro and in vivo has prompted the groups of Don Ganem (2) get cells. Treatment of mixed cultures with hampered our understanding of the natural and Dean Kedes (3) to utilize primary ton- the T cell inhibitor, cyclosporine, abrogated life cycle of KSHV in B cells and of KSHV- sillar explants to study KSHV infection ex the inhibition of lytic replication. Myoung induced B cell lymphoproliferations. The vivo. Previously, efficient productive or lytic and Ganem found that activated viable infection of IL-4 and CD40 ligand–activated T lymphocytes require physical contact with PBMC-derived B lymphocytes and infec- the infected B lymphocytes to inhibit lytic Conflict of interest: The author has declared that no conflict of interest exists. tion of B lymphocytes from tonsils were virus replication. They therefore proposed Citation for this article: J Clin Invest. 2011; demonstrated (7). It is unclear whether that unidentified effector T cell surface 121(3):838–841. doi:10.1172/JCI46499. activation of B lymphocytes results in the ligands are responsible for T cell–target cell

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