Protein Kinase 2Β Is Expressed in Neural Crest-Derived Urinary Pacemaker Cells and Required for Pyeloureteric Contraction

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Protein Kinase 2b Is Expressed in Neural Crest-Derived Urinary Pacemaker Cells and Required for Pyeloureteric Contraction

Samir M. Iskander,1,2 Meghan M. Feeney,1,2 Kirby Yee,1 and Norman D. Rosenblum1,2,3,4

1Program in Developmental and Stem Cell Biology and 3Division of Nephrology, The Hospital for Sick Children, Toronto, Ontario, Canada; and 2Departments of Laboratory Medicine and Pathobiology and 4Paediatrics, University of Toronto, Toronto, Ontario, Canada

ABSTRACT Nonobstructive hydronephrosis, defined as dilatation of the renal pelvis with or without dilatation of the ureter, is the most common antenatal abnormality detected by fetal ultrasound. Yet, the etiology of nonobstructive hydronephrosis is poorly defined. We previously demonstrated that defective development of urinary tract pacemaker cells (utPMCs) expressing hyperpolarization-activated cyclic nucleotide-gated channel 3 (HCN3) and the stem cell marker cKIT causes abnormal ureteric peristalsis and nonobstructive hydronephrosis. However, further investigation of utPMC development and function is limited by lack of knowledge regarding the embryonic derivation, development, and molecular apparatus of these cells. Here, we used lineage tracing in mice to identify cells that give rise to utPMCs. Neural crest cells (NCCs) indelibly labeled with tdTomato expressed HCN3 and cKIT. Furthermore, purified HCN3+ and cKIT+ utPMCs were enriched in Sox10 and Tfap-2a, markers of NCCs. Sequencing of purified RNA from HCN3+ cells revealed enrichment of a small subset of RNAs, including RNA encoding protein kinase 2b (PTK2b), a Ca2+-dependent tyrosine kinase that regulates ion channel activity in neurons. Immunofluores- cence analysis in situ revealed PTK2b expression in NCCs as early as embryonic day 12.5 and in HCN3+ and cKIT+ utPMCs as early as embryonic day 15.5, with sustained expression in HCN3+ utPMCs until postnatal week 8. Pharmacologic inhibition of PTK2b in murine pyeloureteral tissue explants inhibited contraction frequency. Together, these results demonstrate that utPMCs are derived from NCCs, identify new markers of utPMCs, and demonstrate a functional contribution of PTK2b to utPMC function.

J Am Soc Nephrol 29: 1198–1209, 2018. doi: https://doi.org/10.1681/ASN.2017090951

Coordinated pyeloureteric peristalsis is critical to Significance Statement the transport of urine from the kidney to the urinary bladder.1,2 Periodic coordinated sequential con- Abnormalities in urinary tract pacemaker cells have tractions of the pelvis and ureter are controlled been associated with abnormalities such as non- fl by two distinct populations of urinary tract obstructive hydronephrosis, vesico-ureteral re ux, and primary obstructive megaureter in both patients and animal models. The development and function of the pacemaker cell is poorly understood, partly due to Received September 1, 2017. Accepted January 10, 2018. limited knowledge about the developmental lineage S.M.I. and M.M.F. contributed equally to this work. and molecular markers expressed in these cells. Here, we identified the neural crest origin of pacemaker cells, Published online ahead of print. Publication date available at and identified markers highly enriched in this cell type www.jasn.org. using whole transcriptome sequencing. Our analysis fi b Correspondence: Dr.NormanD.Rosenblum,TheHospitalfor identi ed PTK2 as a potential ureter pacemaker Sick Children, Peter Gilgan Centre for Research and Learning, marker that controls pyeloureteric contraction. This 686 Bay Street, Toronto, ON M5G 0A4, Canada. Email: norman. provides a basis for studies investigating pacemaker [email protected] cell function and development in normal conditions, and in kidney–urinary tract disease. Copyright © 2018 by the American Society of Nephrology

1198 ISSN : 1046-6673/2904-1198 JAmSocNephrol29: 1198–1209, 2018 www.jasn.org BASIC RESEARCH pacemaker cells (utPMCs).3–5 utPMCs at the pelvic-kidney cells. Here, we identified the neural crest origin of HCN3+ and junction (PKJ) are characterized by expression of hyperpolar- cKIT+ utPMCs, isolated single cell type populations from tis- ization-activated cyclic nucleotide-gated channel 3 (HCN3) sue, characterized the molecular signatures of both cell pop- and low-voltage–gated T-type calcium channel TTC (also ulations, and identified PTK2b as a novel early expressed termed CaV 3.1).4,5 utPMCs located adjacent to the smooth marker that contributes to pacemaker function. muscle layer in the proximal ureter are termed interstitial cells of Cajal–like cells (ICC-LCs) and express cKIT.3,6 ICC-LCs are readily distinguished from cKIT+;CD45+ mast cells also pre- RESULTS sent in the ureter by their spindle-shaped and long cytoplasmic processes as well as the absence of CD45 expression.3,6 utPMCs Originate from the Neural Crest Cell Lineage The functional contribution of utPMCs to coordinated pye- To define the developmental origin of utPMCs, we used lineage loureteric contraction was demonstrated by studies in which tracing in mice. We generated transgenic mice in which the chemical inhibition or antibody-mediated inhibition of cKIT, expression of Rosa26tdTomato red fluorescent protein (RFP) HCN3, or TTC in pyeloureteric explant cultures disrupted (TOMATO) indelibly marked cells in one of five cell lineages organized ureteric peristalsis.3–5 Although the physiologic that contribute to the kidney and ureter: metanephric mesen- mechanisms by which utPMCs function are poorly studied, chyme, Rarb2-Cre35,36; tailbud mesenchyme, Tbx18-Cre37; Ca2+ signaling and an internal Ca2+ clock are thought to reg- neural crest, Wnt1-Cre38,39; renal stroma, Foxd1-Cre40;and ulate their activity in a manner similar to that of heart and gut ureteric bud, Hoxb7-Cre.41 E18.5 frozen kidney sections, gen- PMCs.7–17 erated from each of the five mouse models, were analyzed for Disruption of coordinated ureteric contraction causes non- colocalization of TOMATO and cKIT/HCN3 using fluores- obstructive hydronephrosis, defined as dilatation of the renal cence microscopy. First, we imaged tissue sections isolated pelvis and ureter without physical obstruction to urinary flow. from mice in which TOMATO is expressed in the neural crest Previously, we demonstrated that decreased Hedgehog signal- lineage (Wnt1-Cre;RosatdTomato)usingfluorescence micros- ing during mouse kidney development results in nonobstruc- copy. Analysis of lower and higher power images of the area tive hydronephrosis.18 Remarkably, mutant mice are also of pacemaker cell localization generated by overlay of TO- characterized by absent expression of both HCN3 and MATO (red color, Figure 1, Ai and Bi) and HCN3 (green color, cKIT.18 These observations suggest that disruption of utPMC Figure 1, Aii and Bii) imaging channels demonstrated coloc- development underlies human congenital hydronephrosis.18 alization (yellow color) in cells adjacent to the renal artery in However, further analysis of the mechanisms that cause failure the PKJ (Figure 1A [103], 1B [203]). Colocalization was also of utPMC marker expression in mutant mice with deficient observed between TOMATO and cKIT (Figure 1C). The co- Hedgehog signaling has been limited by lack of knowledge localization was observed in elongated, thin cells adjacent to regarding the developmental mechanisms by which utPMCs smooth muscle cells in the ureter, consistent with the spatial are generated and the absence of molecular markers that char- pattern of each of TOMATO and cKIT (Figure 1C). In con- acterize these cells before the midpoint of mouse kidney–uri- trast, parallel analyses in mice expressing TOMATO in the nary tract development (embryonic day [E] E15.5).18 Foxd1-derived renal stroma (Figure 2, A and B), tailbud mes- Nonobstructive hydronephrosis constitutes the most com- enchyme (Figure 2, C and D), metanephric mesenchyme mon cause of congenital hydronephrosis but has no defined (Figure 2, E and F), and ureteric bud (Figure 2, G and H) failed etiology.19–22 Although the molecular and cellular mecha- to reveal any colocalization between TOMATO and cKIT or nisms that underlie hydronephrosis are largely unknown, per- HCN3 (Figure 2, A–H). Taken together, these data indicate turbations of utPMCs have been associated with human that utPMCs arise from the neural crest but not from meta- congenital disease. Whereas the expression of HCN3 has not nephric or tailbud mesenchyme, renal stroma, or ureteric bud. been analyzed, cKIT expression is altered in ureteric tissue isolated from humans and experimental animals with congen- FACS-Isolated utPMCs Express Markers Characteristic ital kidney diseases, including vesicoureteral reflux, uretero- of Developing Neural Crest Cells pelvic junction (UPJ) obstruction, primary obstructive To further study the development and function of utPMCs, we megaureter, and nonobstructive hydronephrosis.23–34 Analy- developed a protocol to isolate pure populations of HCN3+ or sis of proximal ureter tissue sections from children with UPJ cKIT+ utPMCs from embryonic tissue. PKJs (for HCN3+ obstruction undergoing pyeloplasty demonstrated decreased cells) or proximal ureters (forcKIT+ cells) were microdissected cKIT expression,24,29,31 although primary obstructive mega- from E18.5 wildtype mouse embryos. Tissue, so isolated, was ureter tissues also exhibit significantly fewer cKIT+ cells com- digested and incubated with anti-HCN3 antibodies (to isolate pared with control samples.30,33 HCN3+ cells), or both anti-cKITantibodies (to isolate cKIT+ The critical contribution of HCN3+ and cKIT+ utPMCs to cells) and anti-CD45 antibodies (to select out cKIT+, CD45+ urinary tract health and perturbation of these cells in congen- mast cells). Using FACS, HCN3+ cells were isolated with ital kidney diseases provides an impetus to elucidate mecha- .96% purity, whereas cKIT+ utPMCs were isolated with nisms that control the specification and differentiation of these .94% purity (Figure 3, A and B). FACS indicated that

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was conﬁrmed using RT-PCR. Hcn3 was expressed .12-fold higher in FACS-sorted HCN3+ cells compared with HCN32 cells (Figure 3C). cKit expression in cKIT+, CD452 FACS-sorted cells was .35-fold higher compared with that in double negative (cKIT2, CD452) FACS-sorted cells (Figure 3D). These results provided a basis to investigate the expression of genes characteristic of neural crest in utPMCs compared with sorted HCN3– and cKIT2 cells. Sox10 is a transcription factor gene that is expressed in migratory neural crest cells and remains active in neural and melanocyte lineages later in development.42 tfAP-2a is a transcription factor gene required for correct development of multiple neural crest cell de- rivatives and is also used as a common neural crest marker.43 Our results indicate that Sox10 is highly enriched in HCN3+ utPMCs compared with HCN32 cells (Figure 3C) and that both Sox10 and tfAP-2a are highly enriched in cKIT+ utPMCs compared with double negative cells (Figure 3D).

RNA Sequencing Identiﬁes Genes Highly Enriched in HCN3+ and cKIT+ utPMCs The number of genes that are known to identify utPMCs is very small and includes only HCN3, cKIT, and TTC.3–5 Moreover, the expression of these genes is limited to later stages of embryonic development (E15.5 and later in the mouse). As a step toward identifying novel markers of utPMCs, we sought to perform genome-wide RNA sequencing in HCN3+ and cKIT+ cells. First, RNA was isolated from HCN3+ or cKIT+ cells isolated from E18.5 WT urinary tract tissue. Abundant high-quality RNA could be isolated from HCN3+ cells but not from cKIT+ cells, likely due to the difﬁculty in Figure 1. HCN3+ and cKIT+ utPMCs colocalize with Wnt1-Cre;tdTomato labelled NCCs. isolating large numbers of viable cKIT+ cells. (A and B) Lower and higher power images are shown, respectively. (A and B) TOMATO (red Analysis of HCN3+ cell RNA by RNA se- color, Ai and Bi) colocalizes with HCN3 (green color, Aii and Bii) generating orange/yellow quencing revealed enrichment of a number color in the merged image in PKJ cells adjacent to the renal artery (A and B, arrows). (C) of novel genes (Gene Ontogeny Consortium TOMATO (red color, Ci) colocalizes with cKIT (green color, Cii) generating orange/yellow [GEO] accession number GSE103400). We color in the merged image in thin, elongated cells adjacent to the ureteric wall (C, arrows). selected a subset of these genes for further cKIT, KIT Proto-Oncogene Receptor Tyrosine Kinase; DAPI, 49,6-Diamidino-2-Phenylindole; study on the basis of the following selection HCN3, Hyperpolarization Activated Cyclic Nucleotide Gated Potassium Channel 3; NCC, criteria: (1) high fold change compared with neural crest cell; SMA, smooth muscle actin; utPMCs, urinary tract pacemaker cells. HCN32 cells (.4-fold), (2) not expressed in other kidney cell types, and (3)published HCN3+ cells constitute approximately 1%, whereas cKIT+ data demonstrating function in non–urinary tract pacemaker cells utPMCs constitute approximately 0.25% of the total population and/or function in neurons, and/or expression in neural crest–- of sorted cells (Figure 3, A and B). The identity of isolated cells derived cells (Table 1). The expression of these selected genes was

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we hypothesized that they share the expression of a subset of molecular markers. To test this hypothesis, we utilized RT-qPCR on FACS- sorted cKIT+ cells. Our results reveal that two of the markers for HCN3+ cells (Ptk2b and Prkcb) are enriched in cKIT+ utPMCs when compared with cKIT2cells (Figure 4B). We analyzed the expression of genes highly expressed in HCN3+ and/or cKIT+ cells at the protein level (Figure 5). First, we analyzed the expression of PTK2b and PRKCb because they are both expressed in HCN3+ and cKIT+ utPMCs (Figure 5, A–D). The PKJ was imaged at lower (Figure 5, A and C) and higher (Figure 5, B and D) power using antibodies specific for HCN3 (Figure 5, Ai, Bi, Cii, and Dii) and either PTK2b (Figure 5, Aii and Bi) or PRKCb (Figure 5, Cii and Di). Both PTK2b (Figure 5, A and B) and PRKCb (Figure 5, C and D) colocalized with HCN3. Next, we analyzed the expression of PLEXINC1 (PLXNC1) because it is expressed in neural crest cells early during development and is a cell surface receptor, suggesting a possible functional role in pacemaker cells.44–47 Expression of PLXNC1, imaged at lower and higher power, was observed in the PKJ, albeit with variable intensity (Figure 5, Eii and Fi), and colocalized with HCN3 (Figure 5, Ei and Fii). Co- localization of PLXNC1 and HCN3 was most obvious in areas in which both proteins are highly expressed (Figure 5, E and F). The specificity of antibody-mediated signal was demonstrated using nonspecific IgG instead of antibody specific for HCN3 and specific putative markers (Figure 5G). In these images, SMA is shown via antibody labeled with Alexa Flour 647 (far red) and control antibodies consist of goat or sheep IgG (green Figure 2. No colocalization is observed between HCN3+ and cKIT+ utPMCs and color in Figure 5Gii) and rabbit IgG (red color tdTomato-labeled renal stroma, tailbud mesenchyme, metanephric mesenchyme, or in Figure 5Gi). To confirm that the observed ureteric bud. (A and B) TOMATO-labeled Foxd1-Cre+ renal stromal cells (red color, A protein colocalization was due to signal from and B) do not colocalize with (A) HCN3 or (B) cKIT (green color). (C and D) TOMATO- the same cells, confocal microscopy was used labeled Tbx18-Cre+ renal tailbud mesenchyme cells (red color, C and D) do not co- m localize with (C) HCN3 or (D) cKIT (green color). (E and F) TOMATO-labeled Rarb2-Cre to image 0.2- m optical sections (Supplemen- b + metanephric mesenchyme cells (red color, E and F) do not colocalize with (E) HCN3 tal Figure 1). HCN3 and marker (PTK2 , or (F) cKIT (green color). (G and H) TOMATO-labeled Hoxb7-Cre+ ureteric bud cells PRKCb, PLXNC1) signals were observed in (red color, G and H) do not colocalize with (G) HCN3 or (H) cKIT (green color). cKIT, KIT the same cells, but PTK2b and PRKCb were Proto-Oncogene Receptor Tyrosine Kinase; DAPI, 49,6-Diamidino-2-Phenylindole; localized to different intracellular compart- HCN3, Hyperpolarization Activated Cyclic Nucleotide Gated Potassium Channel 3; ments than HCN3. utPMCs, urinary tract pacemaker cells. Next, we analyzed the expression of PTK2b,PRKCb,andPLXNC1incKIT+ validated by RT-qPCR using RNA isolated from FACS-isolated utPMCs (Figure 5, H–K). cKIT expression was observed in HCN3+ and HCN2 cells (Figure 4A). Because HCN3+ and thin, elongated cells located in the adventitial layer of the ureter cKIT+ utPMCs both arise from the NCC developmental lineage, (Figure 5, Hii–Jii). Expression of PTK2b (Figure 5Hi) and

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Figure 3. Pure populations of utPMCs can be isolated using FACS. (A) HCN3+ utPMCs were isolated from 18.5 embryos using anti- HCN3 antibody. (B) cKIT+ utPMCs were isolated from 18.5 embryos using anti-cKIT antibody and anti-CD45 antibody. (C) FACS- isolated HCN3+ utPMCs are enriched in HCN3 and Sox10 mRNA when compared with HCN3-negative neighboring cells via RT-qPCR. (D) FACS-isolated cKIT+ utPMCs are enriched in cKIT, Sox10, and Tfap-2a mRNA when compared with cKIT-negative neighboring cells via RT-qPCR. *P,0.05; **P,0.01; CD45-APC 780, Allophycocyanin-eFluor 780; cKIT, KIT Proto-Oncogene Receptor Tyrosine Kinase; FITC, Fluorescein Isothiocyanate; HCN3, Hyperpolarization Activated Cyclic Nucleotide Gated Potassium Channel 3; utPMCs, urinary tract pacemaker cells.

PRKCb (Figure 5Ii) was observed in a similar spatial domain. control sections stained with control IgG instead of the pri- Image overlay revealed colocalization of both PTK2b and mary antibody conﬁrming the speciﬁcity of the observed signal PRKCb with cKIT (Figure 5, H and I). In contrast, PLXNC1 (Figure 5K). Taken together, these data demonstrate that was detected with weak expression in ureteric tissue not includ- PTK2b and PRKCb are coexpressed with HCN3 and cKIT ing cKIT+ cells (Figure 5Ji). No signal was observed in negative whereas PLXNC1 is only expressed in HCN+ utPMCs.

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Table 1. Selected genes identiﬁed by RNAseq as highly enriched in HCN3+ cells and with a possible role in utPMC function Fold Adjusted Gene Full Name RefSeq Gene Description27 Change P Value Mag Myelin associated 26.316 1.98E205 Member of the Ig-like superfamily expressed in myelinating glial cells. glycoprotein Prkcb Protein kinase C b 25.492 5.06E228 Member of PKC family that may regulate neuronal functions. Ptk2b Proline rich tyrosine 17.04 8.11E207 Cytoplasmic protein tyrosine kinase which is involved in calcium-induced regulation of ion kinase 2 b channels. Glipr1 GLI pathogenesis 15.92 1.06E205 Protein with similarity to both the pathogenesis-related protein (PR) superfamily and the cysteine- -related 1 rich secretory protein (CRISP) family and expressed in neural crest–derived tissues. Rtn1 Reticulon 1 12.26 3.36E209 Member of the family of reticulon encoding genes and a marker of neuroendocrine cells and degenerative neuronal tissue. Tnni2 Troponin I FS 12.21 ,0.001 Fast-twitch skeletal muscle protein, a member of the troponin I gene family, a component of the troponin complex which is responsible for the calcium-dependent regulation of striated muscle contraction. Sema4dSemaphorin 4d 5.92 2.99E209 Member of the Semaphorin gene family and a ligand for PlexinB1.28 The Sema4d-PlexinB1 interaction is involved in neural development in the hippocampus.29,30 PlxnC1 Plexin C1 4.128 ,0.001 A member of the Plexin family, which are transmembrane receptors for Semaphorins, that regulate axon guidance, cell motility, and migration.

PTK2b: A Developmental Marker of utPMCs that inhibitor for 1 hour and then imaged again for 5 minutes. Re- Controls Pelvic Contraction corded contractions were analyzed for frequency. Results PTK2b, a protein tyrosine kinase, regulates ion channel activ- demonstrated a 50% reduction in contraction frequency in ex- ity in neurons, senses Ca2+ levels, and interacts with proteins plants treated with either PTK2b inhibitor compared with con- that regulate HCN protein in non–urinary tract tissues.48–52 trol with no apparent change in the spatial origin of contraction We investigated the possibility that PTK2b marks utPMCs at (arrows, Figure 6E, Supplemental Movies 1–3). stages of development earlier than those during which HCN3 and cKIT are expressed. PTK2b was identified in E15.5 embryonic mouse kidney and ureter by immunofluorescence DISCUSSION staining in situ. Results demonstrate that PTK2b is localized in both HCN3+ utPMCs and cKIT+ utPMCs in the PKJ and The developmental and molecular mechanisms that underlie proximal ureter, respectively (Figure 6A). Next, we studied utPMCfunctioninkidney–urinary tract health and disease are PTK2b expression at time points earlier than E15.5. Because poorly understood. HCN3+ and cKIT+ populations of utPMCs HCN3 and cKITare not expressed in utPMCs before E15.5, we function sequentially to initiate and propagate pyeloureteric con- colocalized PTK2b with neural crest cells, which give rise to tractions.2,3,5 Although the presence and spatial organization of utPMCs, using TOMATO in Wnt1-Cre ROSAtdTomato mice utPMCs has been shown to be perturbed in congenital urinary (Figure 6B). Consistent with prior published data, NCCs tract malformations and particularly in hydronephrosis,18,23–34 were identified adjacent to the developing kidney and ure- little is known regarding the direct contribution of utPMCs to ter.39 PTK2b colocalized with a subset of TOMATO-positive the pathobiology of these disorders. Lack of knowledge regarding cells surrounding the developing ureter and kidney at both the origin and development of utPMCs limits the ability to in- E13.5 and E12.5 (Figure 6B, arrows). vestigate these issues. Here, we identify the embryonic origin of We investigated the functional contribution of PTK2b in utPMCs, showing that HCN3+ and cKIT+ utPMCs arise from urinary tract contraction using an established assay of pelvic- Wnt1+ cells. Our results demonstrate that utPMCs arise outside ureteric contraction.4,5 This assay was performed in tissue prep- of the nephrogenic field and migrate to take up residence in the arations isolated from 8-week-old mice. At postnatal 8 weeks, PKJ and ureter. Further, using methods we developed to isolated PTK2b expression was sustained in HCN3+ utPMCs but lost pure populations of HCN3+ and cKIT+ cells, we identified RNAs from cKIT+ utPMCs (Figure 6C). PTK2b function was investi- that are enriched in these cell populations. In so doing, we iden- gated using two pharmacologic inhibitors, PF (a selective PTK2b tified PTK2b as a novel marker of both HCN3+ and cKIT+ cells inhibitor) and leflunomide (a broader tyrosine kinase inhibitor and show that PTK2b colocalizes with migrating WNT1+ cells as that is associated with antenatal nonobstructive hydronephro- early as E12.5, when these cells are still migrating toward the sis).53–57 Pyeloureteric tissue preparations were placed on kidney–urinary tract.39,58 Finally, we demonstrate that PTKb con- porous filters, incubated at 37°C for 1 hour, and imaged for trols pyeloureteric contraction. These findings are consistent with contractile activity. Tissues displaying the expected rate of ten previous observations that WNT1+ neural crest cells give rise to contractions/minute were treated with vehicle or PTK2b cells that reside in the PKJ and ureteric wall.39 Further, the

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findings extend our understanding of the neural crest lineage by showing that the neural crest contributes functionally to the kidney–urinary tract. The developmental origin of utPMCs thus differs from ICCs in the gut and HCN4+ pacemakers of the heart, both of which are derived from gut and heart mesenchyme, respectively.61–65 Our findings that HCN3 and cKIT colocalize with Wnt1-driven TOMATO and express Sox10 and Tfap2a, neural crest markers, are consistent with the previous demonstration that cKIT colocalizes with PGP9.5, which is expressed in gut developing neural crest cells.42,43,66–68 Our results are also consistent with previous LacZ lineage-tracing studies, which demonstrated neural crest cells in the metanephros, the proximal ureter, the pelvic-kidney junction, and sparsely in the kidney cortex.39 In these studies, time-lapse analysis of murine neural crest cells marked with Wnt1-dependent LacZ expression demonstrated that neural crest cells surround the metanephric mesenchyme at E11.5.39 By E12.5, fewer neural crest cells border the metanephric mesenchyme and, at E14.5, they are confined to the ureter and renal pelvis.39 Considered in the context of our results, these studies suggest that utPMC precursors migrate into the developing ureter between E11.5 and E14.5 and subse- quently undergo their final differentiation. The finding that neural crest cells give rise to utPMCs suggests that they are essential for the functional development of the ureter. Finally, our results provide a basis to re-examine utPMC function in splotch mu- tants, in which neural crest cells fail to migrate to the developing kidney–urinary tract and in which analysis was limited to embryonic stages before utPMC development.39 Lineage tracing of Wnt1-marked neural crest cells39 in combination with our results shown here also inform our previous published work showing that Hedgehog-GLI signaling in the metanephric mesenchyme of mice controls utPMC development.18 Results now showing that utPMCs are derived from neural crest suggest that HH-GLI signaling in the metanephric mesen- Figure 4. Eight novel utPMC markers are enriched in HCN3+ chyme acts in a non–cell autonomous fashion to control the spec- utPMCs and two are shared with cKIT+ utPMCs. (A) RT-qPCR on ification and differentiation of utPMCs as they migrate into and/or fi FACS-isolated HCN3+ utPMCs con rms the enrichment of novel differentiate within the ureter/PKJ. This is consistent with the find- marker mRNA when compared with HCN3-negative neighboring ing that neural crest cell progenitors that form the cardiac outflow cells. (B) RT-qPCR on FACS-isolated cKIT+ utPMCs reveals the enrichment of Prkcb and Ptk2b mRNA when compared with cKIT- tract are not responsive to Hedgehog directly, but Hedgehog affects – 69 negative neighboring cells. *P,0.05; **P,0.01; cKIT, KIT Proto- their development in a non cell autonomous manner. Oncogene Receptor Tyrosine Kinase; HCN3, Hyperpolarization Activated Cyclic Nucleotide Gated Potassium Channel 3. Molecular Markers of utPMCs The embryonic development and physiology of utPMCs is largely undefined, in part by a lack of knowledge of the mo- identification of PTK2b,aCa+2-dependent tyrosine kinase, is lecular constituents that endow these cells with their particular consistent with prior experiments showing that Ca+2 signaling functions. Here, we used cell purification followed by RNAseq is critical to pyeloureteric contraction.11 to identify RNAs enriched in HCN3+ cells. Cell isolation and qPCR demonstrated which of these identified RNAs are either A Neural Crest Cell Origin for utPMCs shared with cKIT+ cells or are unique to HCN3+ cells. Each of The neural crest originates at the boundary between the ecto- three utPMC markers identified in HCN3+ cells (PLXNC1, derm and the neural tube in vertebrates.59 Neural crest cells SEMA4d,MAG)hasbeenshowntoplayaroleinaxonalguidance, migrate throughout the embryo and contribute to a diverse set of neuronal migration, or neural crest cell migration.44,70,71 differentiated cell types including craniofacial cartilage and PLXNC1 and MAG are membrane receptors that engage repul- bone, cardiac blood vessels, melanocytes, and peripheral neu- sive or attractive signals (context- and ligand-dependent) to rons, including the enteric nervous system of the gut.60 Our guide cell migration.44,70 SEMA4d is a secreted ligand for Plexin

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Figure 5. Novel utPMC markers are enriched in HCN3+ and cKIT+ utPMCs at the protein level in situ. Immunoﬂuorescence analysis revealed colocalization between PTK2b (green, A–Aii; red B–Bii), PRKCb (green, C–Cii; red, D–Dii), PLXNC1 (green, E–Eii; red, F–Fii), and HCN3 (green, A, C, and E; red, B, D, and F). Colocalization was observed in cells adjacent to the renal artery (RA) outside the pelvis (P) consistent with the location of utPMCs. (G–Gii) Control IgG sections revealed weak/no signal in the red and green channels. Analysis revealed colocalization between cKIT (green, H–Jii), and PTK2b (red, H–Hii) or PRKCb (red, I–Iii), but not with PLXNC1 (red, J–Jii). cKIT staining was observed in elongated cells in the adventitial layer adjacent to the ureteric muscular layer consistent with the localization of cKIT+ utPMCs. (K–Kii) Control IgG sections revealed weak/no signal in the adventitial layer in red and green channels. Scale bars represent 200 mm (A, C, and E), 100 mm (B, D, F, and G), or 20 mm(H–K). cKIT, KIT Proto-Oncogene Receptor Tyrosine Kinase; DAPI, 49,6-Diamidino-2-Phenylindole; HCN3, Hyperpolarization Activated Cyclic Nucleotide Gated Potassium Channel 3; PLXNC1, Plexin C1; PRKCb, Protein Kinase C Beta; PTK2b, Protein Tyrosine Kinase 2 Beta; SMA, smooth muscle actin; utPMCs, urinary tract pacemaker cells.

B1 that is released by different tissues to restrict or promote cell signaling has been described as a major signaling pathway in migration.71 The demonstrated properties of PLXNC1, PMC in the heart, gut, and the urinary tract.8,11,12,14–17 SEMA4d, and MAG in nonrenal tissues suggest functional con- PTK2b has also been shown to interact with SRC kinase, a tributions for these proteins in neural crest migration and con- known regulator of HCN channels.49–51 Our results indicate tribution to the kidney–urinary tract. Three other genes, the that pharmacologic inhibition of PTK2b in pyeloureteric ex- expression of which is enriched in HCN3+ cells, display func- plant tissue significantly reduces ureteric contraction frequency, tions distinct from cell migration. RTN1 is a neuroendocrine suggesting that PTK2b controls PMC function. Future studies marker known to regulate protein secretion by neuroendocrine which identify the mechanism by which PTK2b regulates pace- cells.72–74 TNNI2 is involved in muscle cell contractile func- maker activity will shed insight into the physiologic regulation of tion.75 Although GLIPR1 function is poorly characterized, it is pacemaker activity. enriched in some NCC-derived tissues.76 The functions of these proteins in utPMC-mediated functions remain to be defined. Clinical Implications The functions of both PRKCb and PTK2b in regulating elec- The etiology of human nonobstructive hydronephrosis is trical activity in nonrenal tissues suggests that both of these pro- poorly defined.82 Prior investigation of genetic defects associ- teins may perform similar functions in both HCN3+ and cKIT+ ated with Congenital Anomalies of the Kidney and Urinary utPMCs, in which they are enriched. PRKCb is an isoform of Tract using karyotyping and SNP microarrays identified sev- protein kinase C. Protein kinase C, a Ca2+-dependent kinase, eral microdeletions and duplications associated with hydro- regulates heart and gut PMCs, as well as neuron and muscle cell nephrosis.83,84 However, these studies were limited in that actions.7,77–81 PTK2b is a Ca2+-dependent tyrosine kinase in- they did not identify individual genes but rather defective volved in linking Ca2+ signaling to ion channel activity.48 Ca2+ chromosomal regions.83,84 The etiology of nonobstructive

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studies to identify the contribution of utPMC dysfunction to congenital urinary tract disease. Furthermore, because the outcome of nonobstructive hydronephrosis is heterogeneous with the majority of cases stabilizing postna- tally and a minority requiring surgery, those markersmightserveasprognosticmarkersto aid in the clinical management of nonobstructive hydronephrosis or therapeutic targets in cases of hydronephrosis.20–22,82

CONCISE METHODS

Mice Rarb2-CremicewerematedwithRosa26-tdTomato mice (Gt(ROSA)26Sortm9(CAG-tdTomato)Hze/J; The Jackson Laboratory, Bar Harbor, ME) to generate Rarb2-Cre;Rosa26-tdTomato embryos in which the metanephric mesenchyme was indelibly labeled with RFP in Cre-positive mice. In the same way, Tbx18-Cre, Hoxb7-Cre, Wnt1-Cre,andFoxd1-Cre mice were each mated with Rosa26-tdTomato mice to generate embryos in which the tailbud mesenchyme, ureteric bud, neural crest cells, and renal stroma, respectively, were indelibly labeled with Figure 6. PTK2b is an early expressed utPMC marker that controls ureteric contraction. RFP in Cre-positive mice. PCR genotyping for Cre b (A) PTK2 (red color) colocalizes with HCN3 and cKIT (green color) to generate orange/ recombinasewasperformedusingthefollowingpri- b yellow color in the merged images in E15.5 murine renal tissue. (B) PTK2 (green color) mers. For Rarb2-cre, FoxD1-Cre, HoxB7-Cre,and colocalizes with a subset of TOMATO-labeled Wnt1-Cre+ NCCs (arrows, red color) sur- Wnt1-Cre:CREFwd:59-AGCGCGATCA- rounding the developing kidney and ureter in E13.5 and E12.5 murine tissue. (C) PTK2b CATGGTCCTG-39;CRERev:59-ACGATCCTGA- expression (red color) colocalizes with HCN3 but not cKIT (green color) in adult 8-week- 9 old murine renal tissue. (D) PTK2b inhibitors reduce ureteric contraction frequency in an GACTTCCACACT-3 , which generates a 230-bp 9 ex vivo assay for pelvis-ureter contractions. (E) Representative still-frames from time-lapse product. For Tbx18-cre: TCT Fwd: 5 -CCATC- imaging of ex vivo pelvis-ureter explants illustrate the origin (x and arrows) of pye- CAACAGCACCTGGGC-39;TCTRev:39-CCAC- loureteric contractions at the pelvis in DMSO control as well as in explants treated with CATCGGTGCGGGAGATGTCCTTCACT-39, PTK2b inhibitors (PF431396andleflunomide). Representative time-lapse imaging is which generates a 313-bp product. C57BL/6 wild- presented in Supplemental Movies 1–3. **P,0.01; NS indicates P.0.05. cKIT, KIT Proto- type mice were mated to generate wildtype embryos Oncogene Receptor Tyrosine Kinase; DAPI, 49,6-Diamidino-2-Phenylindole; DMSO, Di- for pacemaker cell isolation experiments. methyl sulfoxide; HCN3, Hyperpolarization Activated Cyclic Nucleotide Gated Potassium Channel 3; NCC, neural crest cell; PF431396, PTK2b inhibitor; PTK2b, Protein Tyrosine Histologic Analysis Kinase 2 Beta; SMA, smooth muscle actin; utPMCs, urinary tract pacemaker cells. Embryos were collected at E18.5, determined by the number of days after the observation of a hydronephrosis is hypothesized to include defects in ureteric peri- copulation plug, with the day of observation corresponding to day 0.5. stalsis which could involve defective contractile smooth muscle cell Experiments using mice were approved by the Animal Ethics Com- action or defective regulatory utPMC action.18,82,85 Previously, a mittee at the Hospital for Sick Children and carried out in accordance mouse model with defective smooth muscle cell development with the Canadian Council of Animal Care. Immunofluorescence was linked Tshz3 with defective smooth muscle cell development and performed on 4% PFA-fixed, OCT-embedded frozen sections using nonobstructive hydronephrosis.85 Our published mouse model for primary a-CD117 rabbit-monoclonal (1:50 dilution; DAKO), rabbit nonobstructive hydronephrosis showed that Smo deficiency in the polyclonal anti-HCN3 (1:200 dilution; Abcam), goat polyclonal anti- metanephric mesenchyme causes defective utPMC development PTK2b (1:300dilution;SCBT),orgoatpolyclonalanti-TNNI2 and nonobstructive hydronephrosis.18 Furthermore, a reduced (1:150 dilution; SCBT); the appropriate Alexa Flour conjugated sec- number of cKIT+ utPMCs has been associated with congenital ondary antibodies were used at a dilution of 1:1,000 (Thermofisher). kidney–urinary tract defects including UPJ obstruction, vesicoure- Nuclei were visualized using DAPI (1:1,000; Thermo Scientific). Sec- teral reflux, and primary obstructive megaureter.23–30,32–34 Novel tions were washed and mounted using glass coverslips and hard-set utPMC markers described in this study can be utilized in future Vectashield (Vector Labs). Images for colocalization experiments

1206 Journal of the American Society of Nephrology J Am Soc Nephrol 29: 1198–1209, 2018 www.jasn.org BASIC RESEARCH were captured with a fluorescence microscope (Zeiss Axio Observer) contained PTK2b inhibitors (PF431396 or leflunomide,16–18,26 or or an Olympus confocal microscope. DMSO as a control). The plate was then returned to the incubator for 60 minutes with upper media changes every 20 minutes before a Fluorescence-Activated Cell Sorting second set of time-lapse images was captured. The time lapse images Wildtype C57B6 E18.5 ureters and pelvic-kidney junctions were mi- were compiled to create videos using Zeiss software (Axiovision or crodissected using a three-dimensional dissecting microscope and Zen Blue). The number of contractions per minute was quantified pooled into one sample for cell sorting. Tissue was dissociated manually on the basis of the videos for each group. using a 1 mg/ml collagenase suspension. Cells were resuspended in sorting buffer with the following antibodies. cKIT: CD117 (cKIT) Statistical Analysis anti-mouse PE (1:200; eBioscience) to positively select cKIT+ PMC Statistical analysis was performed using GraphPad Prism Software and CD45 anti-mouse APC-eFluor 780 (1:200; eBioscience) to neg- (version 6.0f). Data were analyzed using two-tailed paired t test. Sta- atively select cKIT+, CD45+ mast cells. Hcn3: a-Hcn3 rabbit-poly- tistical significance was indicated by a probability of ,0.05. Values are clonal (1:400) and secondary Alexa Fluor 488 goat-a-rabbit (1:1,000; given as mean6SEM. Life Technologies). Propidium iodide was used as a dead cell marker (10 mg/ml). Cell sorting was conducted using either MoFlo XDP or MoFlo Astrios Fluorescence Activated Cell Sorting machines. Cells were collected into sorting buffer, centrifuged at 4°C, and resuspen- ACKNOWLEDGMENTS ded in 150 ml of buffer RLT. This work was supported by grants from the Canadian Institutes of RNA Isolation, Real Time-PCR, and RNAseq Health Research and the Kidney Foundation of Canada (to N.D.R.), RNAwas extracted using an RNEasy Micro Kit (QIAGEN) and cDNA and a Tier I Canada Research Chair in Developmental Nephrology was generated using First Strand cDNA Synthesis (Invitrogen) from (to N.D.R.). total RNA. Real-time PCR was conducted with 2–4 ml of cDNA, SYBR Green PCR Mix (Applied Biosystems), and 5 mMofeachprimerina total volume of 20 ml. Primers were designed using NCBI Primer3 DISCLOSURES software. The sequences of the primers used are attached in Supple- None. mental Table 1. Real-time PCR amplification was conducted using the Applied Biosciences Viia7 Real-Time PCR System. Relative levels of mRNA expression were determined using the DDCT method and REFERENCES normalized by comparison to expression of Glyceraldehyde 3-phos- phate dehydrogenase (GAPDH). RNAseq was performed by the Illu- 1. 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