Role for compartmentalization in nephron progenitor differentiation

Abstract

Embryonic nephron progenitor cells are segregated in molecularly distinct compartments of unknown function. Our study reveals an integral role for bone morphogenetic protein-SMAD in promoting transition of progenitors from the primitive Cbp/p300-interacting transactivator 1 expressing (CITED1+) compartment to the uniquely sine oculis-related homeobox 2 expressing (SIX2-only) compartment where they become inducible by wingless-type mouse mammary tumor virus integration site family member (WNT)/β-catenin signaling. Significantly, CITED1⁺ cells are refractory to WNT/β-catenin induction. We propose a model in which the primitive CITED1⁺ compartment is refractory to induction by WNT9b/β-catenin, ensuring maintenance of undifferentiated progenitor cells for future nephrogenesis. Bone morphogenetic protein 7-SMAD is then required for transition to a distinct compartment in which cells become inducible by WNT9b/β-catenin, allowing them to progress toward epithelialization.

Metanephric kidney development has been intensely studied since the 1950s when Clifford Grobstein used this system to reveal many of the fundamental principles of epithelial–mesenchymal interaction in organogenesis (1–3). Knockout and transgenic mouse models have clarified the genetic basis for many of these principles, and gene expression analyses have uncovered unanticipated distinctions between cell types in the developing kidney that were not apparent from morphological studies such as the arrangement of nephron progenitor cells in a series of molecularly distinct compartments (4, 5). The significance of these compartments is unclear, and understanding their functions is important for our basic knowledge of kidney development and for ongoing efforts to differentiate kidney tissue from pluripotent stem cells.

Formation of a sufficient number of nephrons during kidney development relies on a continuous supply of nephron progenitor cells. As the collecting system branches, progenitor cell aggregates at collecting duct tips known as cap mesenchymes are induced to form nephrons. The collecting duct provides survival, proliferation, and differentiation signals to the cap mesenchyme, which ultimately gives rise to all epithelial components of the nephron (6–8). In turn, cap mesenchyme provides signals for growth and branching of the collecting duct. These events take place within the nephrogenic zone, a progenitor cell niche where bone morphogenetic proteins (BMPs), fibroblast growth factors (FGFs), and wingless-type mouse mammary tumor virus integration site family members (WNTs) maintain the balance between renewal and differentiation (9–12). Studies of WNT signaling in particular have demonstrated bimodal effects on cap mesenchyme cells, with WNT9b-activated β-catenin signaling (canonical WNT signaling) causing both renewal of undifferentiated cells and epithelial differentiation (13). The sine oculis-related homeobox 2 (SIX2) transcription factor blocks the inductive activities of WNT signaling, and it has been suggested that the level of SIX2 expression in progenitor cells determines responsiveness to WNT9b, indicating that contradictory effects of Wnt9b may be explained by differential responsiveness of cells within cap mesenchyme (13, 14). Using in vitro nephrogenic zone culture, we have studied the responsiveness of individual cap compartments to inductive signaling and defined signals that drive the transition of cells through sequential compartments toward nephron differentiation. We find that BMP7-induced tran-sition between compartments is required for cells to acquire responsiveness to WNT/β-catenin signaling. This unanticipated function of BMP7 is mediated through the SMAD signaling pathway, in contrast to the progenitor renewal effect that we have shown operates through mitogen activated protein kinase (MAPK) (15). Based on these findings, we propose a model whereby compartmentalization is necessary to prevent Cbp/p300-interacting transactivator 1 (CITED1)-expressing cells from premature induction, allowing maintenance of undifferentiated cells within the cap mesenchyme for future rounds of nephrogenesis.

Results and Discussion

BMP7 Promotes Transition of Cap Mesenchyme Cells Between Early Progenitor Compartments.

The earliest progenitor cells associated with the capsular aspect of the collecting duct tip express CITED1 (Fig. 1A), whereas the slightly more differentiated population associated with the internal aspect of the collecting duct tip loses expression of CITED1 but maintains expression of SIX2 (Fig. 1B). Lymphoid enhancer binding factor 1 (LEF1) expression is limited to the more differentiated pretubular aggregate that is primed for epithelial conversion (Fig. 1C). Immunofluorescence staining of CITED1, SIX2, and LEF1 proteins shows that a population of “SIX2 only” progenitors resides between the CITED1⁺ and LEF1⁺ cells (Fig. 1D). A comparison of green fluorescent protein (GFP) expression within cap mesenchymes of Cited1-creERT2-Gfp and Six2-creERT2-Gfp strains reveals a similar discrepancy, strongly arguing for a genuine difference in expression domains rather than distinct half-lives of the RNAs or proteins (7, 8). These results suggest an arrangement of distinct cell states, or compartments, in which CITED1 expression is lost as cells differentiate to a SIX2-only compartment before entering the LEF1⁺ compartments after which they epithelialize in the renal vesicle (Fig. 1E). Having previously established that CITED1⁺ nephron progenitors depend on FGF/receptor tyrosine kinase signaling for maintenance of their phenotype, we sought to understand which signals push CITED1⁺ cells to the LEF1⁺ pretubular aggregate compartment (16).

Fig. 1.

BMP7 promotes transition of CITED1⁺ progenitors to the SIX2-only compartment. (A) CITED1 (red) localizes to the capsular aspect of the collecting duct (green). (B) Progenitors in the cap lose CITED1 (red) but retain SIX2 (green, arrows). (C) LEF1 (red) is limited to the pretubular aggregate and downstream compartments. (D) A “SIX2 only” progenitor population (green, arrows) resides between the CITED1⁺ (orange/yellow) and LEF1⁺ compartments (red). (E) Schematic representation of compartments showing loss of CITED1 as cells differentiate to SIX2-only before entering the LEF1⁺ pretubular aggregate and renal vesicle compartments. (F) BMP7 treatment results in a loss of CITED1 (red), maintenance of SIX2 (green), and does not promote LEF1 (red, absent). E14.5 Bmp7^−/− kidneys are hypomorphic, have a reduced number of CITED1⁺ progenitors (G and H), and a substantial decrease of the SIX2-only population (I and J, arrows). (K) Quantitation of SIX2-only clusters in E14.5 Bmp7^−/− (n = 4, 130 total tips) and wild-type kidneys (n = 6, 510 total tips). Error bars represent average values ± SEM, and the P value is derived from Student t test.

To screen for pathways that promote CITED1⁺ progenitor differentiation, we used the primary nephrogenic zone culture system (15, 17). This monolayer culture of nephrogenic zone cells (NZCs) essentially lacks inductive cells of the ureteric bud (less than 0.04% contamination). We screened signaling pathways with known effects in kidney development for their ability to extinguish CITED1⁺ expression, while maintaining proliferation and growth of the culture (Fig. S1). Surprisingly, stimulating β-catenin signaling with the glycogen sythase kinase 3β (GSK3β) inhibitor 6-bromoindirubin-3′-oxime (BIO) caused only a modest effect (Fig. S1). This finding was unanticipated because β-catenin activation promotes epithelial differentiation of nephron progenitor cells in cultured metanephric mesenchymes (18). However, BMP7 treatment reduced CITED1 protein expression drastically compared with all other recombinant proteins as determined by immunofluorescence and Western blot (Fig. 1F and Fig. S1). BMP7 increased proliferation and significantly expanded the total number of progenitors while maintaining more than 95% viability (Fig. S2). When BMP7 is replaced with vehicle control after 24 h, expression of CITED1 is not reacquired, showing an irreversible exit of cells from the CITED1⁺ state (Fig. S2). Although BMP7 treatment clearly causes loss of CITED1 and maintenance of SIX2, it does not promote LEF1 expression, showing that cells do not differentiate to the pretubular aggregate but simply transition to the SIX2-only compartment (Fig. 1F). To understand whether this unanticipated effect of BMP7 is a feature of the culture system, or if it occurs in vivo, we analyzed CITED1 and SIX2 expression in kidneys from the Bmp7^−/− mouse. At embryonic day (E)14.5, Bmp7^−/− kidneys are hypomorphic yet the nephrogenic zone can still be studied. Numbers of CITED1⁺ cells are reduced in the mutant kidney, and they are linearly arranged around the periphery in contrast to wild type in which they wrap around collecting duct tips (Fig. 1 G–J). However, although CITED1⁺ cells are present in mutant kidneys, loss of Bmp7 causes a substantial decrease of the SIX2-only population (Fig. 1 I–K). These results strongly support a role for BMP7 in promoting transition of nephron progenitor cells from the CITED1⁺ compartment to the SIX2-only compartment in vivo.

SMAD-Dependent Signaling Is Required for Transition of Progenitors from the CITED1⁺ Compartment.

We have shown that BMP7 promotes nephron progenitor proliferation through TAK1-MAPK, explaining the reduction of CITED1⁺ cells seen in the E14.5 Bmp7^−/− (Fig. 1H) (15). However, we also confirmed that SMAD1/5 is phosphorylated in nephron progenitors in response to BMP7 treatment (Fig. 2A). Interestingly, tissue staining reveals nuclear phospho-SMAD1/5 (pSMAD1/5) in a subset of progenitors underneath the collecting duct tip, at the junction between CITED1⁺ and CITED1⁻ cells (Fig. 2B). To determine which BMP7-initiated signaling branch is responsible for the transition of progenitors from the CITED1⁺ to the SIX2-only compartment, NZCs were treated with BMP7 with or without the addition of dorsomorphin, a small molecule BMP-SMAD (pSMAD1/5) inhibitor, or TAK1 inhibitor (Fig. 2C) (19, 20). BMP7 treatment with dorsomorphin, but not TAK1 inhibitor, blocked the ability of BMP7 to promote transition out of the CITED1⁺ compartment, showing that SMAD signaling mediates this effect. Mesenchyme homeobox 1 (Meox1) and D4, zinc and double PHD fingers, family 3 (Dpf3), which are coexpressed with Cited1 in the cap mesenchyme (5), also show coregulated expression with Cited1 indicating that BMP7-SMAD mediated transition out of the CITED1⁺ compartment represents a change of cellular state rather than simply a change of Cited1 expression (Fig. 2D). Furthermore, tissue staining revealed a loss of pSMAD1/5 in the distal cap of the Bmp7 null kidney at E14.5, confirming that SMADs are activated in the distal cap specifically by BMP7 in vivo (Fig. 2E and Fig. S3). In summary, BMP7 treatment promotes transition of progenitors from the CITED1⁺ to the SIX2-only compartment by SMAD activation, and the finding that pSMAD1/5 is regionalized in the distal cap mesenchyme supports a role for this process in vivo. SMAD4 is required for nuclear translocation of pSMAD1/5, and its conditional inactivation in nephron progenitor cells results in partial recapitulation of the Bmp7^−/− phenotype with premature cessation of nephrogenesis, but with retention of PAX2 and WT1 expressing nephron progenitor cells within the nephrogenic zone (6). This phenotype is recapitulated by inactivation of Cv2, which acts as an amplifier of BMP7-induced SMAD signaling in cap mesenchyme (21). These genetic findings strongly support the concept that BMP7/SMAD signaling is essential for progenitor cell differentiation.

Fig. 2.

BMP7 promotes SMAD-mediated signaling in nephron progenitors. (A) BMP7 treatment results in pSMAD1/5 (green) activation in PAX2 progenitors (red); costaining is yellow. (B) Immunofluorescence of E17.5 kidney sections shows nuclear pSMAD1/5 in the distal cap underneath collecting duct tips (arrows). Inset shows CITED1 (red) and pSMAD1/5 (green) at the CITED1⁺/CITED1⁻ border. (C) CITED1 staining (red) of NZCs pretreated with vehicle, BMP7, BMP7 + dorsomorphin (DM), or BMP7 + TAK1 inhibitor (TAKi) shows that SMAD1/5 inhibition blocks the ability of BMP7 to reduce CITED1 expression. (D) Quantitative PCR analysis shows that inhibition of SMAD-dependent signaling by DM, compared with TAKi, blocks the ability of BMP7 to reduce transcription (48 h) of a group of early progenitor markers (Cited1, Meox1, Dpf3). Raw data are normalized to β-actin expression, and fold changes are relative to the vehicle control. (E) pSMAD1/5 in the distal cap mesenchyme is lost in the Bmp7^−/−.

SMAD-Dependent BMP Signaling Primes CITED1 Progenitors for Induction by WNT/β-catenin.

WNT9b/β-catenin signaling is required for differentiation of cap mesenchyme cells (12, 22). However, in our hands, activation of the WNT pathway directly in CITED1⁺ progenitor cells using BIO or recombinant WNT9b had only a mild effect on differentiation from the CITED1⁺ to the LEF1⁺ compartment (Fig. 3A and Fig. S1). We reasoned that progression through sequential cap mesenchyme compartments might be required for acquisition of susceptibility to differentiating signals. We therefore tested whether BMP7-induced SIX2-only cells gain the potential for β-catenin–mediated differentiation to the LEF1⁺ stage. We stimulated freshly isolated NZC cultures with BMP7 for 24 h to promote their transition from the CITED1⁺ to the SIX2-only compartment and added BIO during the remaining 16 h (Fig. 3A). Under these conditions, LEF1⁺ cells became clearly abundant, demonstrating that BMP7-induced transition from the CITED1⁺ to the SIX2-only compartment primes cells to respond to WNT/β-catenin induction. In line with our earlier results, treatment with dorsomorphin completely abrogated the capacity of BMP7 treatment to sensitize the culture to BIO-mediated differentiation. Similar results were seen at the transcriptional level (Fig. 3B), and we also demonstrated that BMP7 and BMP4 could function redundantly in this capacity (Fig. S4). Interestingly, increased Wnt4 transcription accompanies LEF1 expression upon BMP7 and BIO treatment, indicating that the cells are entering the renal vesicle stage (Fig. 3B). The β-catenin dependence of the response to BIO was verified by using an inhibitor of WNT/β-catenin (FH535), which completely abrogated Wnt4 and Lef1 transcription responses (Fig. 3C and Fig. S4). Lithium chloride (LiCl) has been more commonly used as an inducer of nephrogenesis, and to verify that it functions interchangeably with BIO with respect to differentiation of NZCs, we repeated the experiment with LiCl (Fig. 3C). Indeed, a similar effect was observed with LiCl, but the amplitude of Wnt4 activation was lower. These results indicate that SMAD mediated differentiation from the CITED1⁺ to the SIX2-only compartment is required for β-catenin–mediated differentiation. Cells of the cap mesenchyme express Bmp7, and to test whether endogenously produced growth factor sensitizes cells to the differentiating effect of canonical WNT signaling, we measured Wnt4 expression in BIO-treated NZCs after BMP-SMAD inhibition (Fig. 3D). We found that the small increase in Wnt4 expression seen after BIO treatment can be reduced by dorsomorphin, suggesting signaling by endogenously produced BMP7 in this culture system. Also, immunoblotting for pSMAD1/5 reveals a modest signal in untreated NZCs that can be quenched by dorsomorphin (Fig. S4). We hypothesize that the large volume of the monolayer system dilutes endogenously produced BMP7 to a level that is inadequate to fully prime cells for BIO induction.

Fig. 3.

BMP-SMAD signaling primes nephron progenitors for induction by WNT/β-catenin. (A) LEF1 expression (red) in NZCs treated with the indicated factors. Pretreatment with BMP7 is required for BIO-induced transition to the LEF1 compartment and this effect is reversed by dorsomorphin. (B) Quantitative RT-PCR (RT-qPCR) of NZCs primed with BMP7 before 8-h BIO treatment showing a synergistic induction of Lef1 and Wnt4 and reduction in Cited1. Results are derived from three independent replicates per treatment. Error bars represent average values ± SEM, and P values are derived from the Student t test. *P < 0.03, **P < 0.002 when BMP7⁺ BIO treatment is compared with either BMP7 or BIO alone. (C) RT-qPCR of NZCs showing a synergistic induction of Wnt4 with BMP7 plus BIO or LiCl. Pretreatment with the WNT/β-catenin inhibitor FH535 abrogates the increase in Wnt4 expression. Error bars represent average values ± SD. (D) RT-qPCR of NZCs shows a small increase in Wnt4 after BIO treatment that can be quenched by dorsomorphin, indicating a low level of endogenous BMP activity in the monolayer culture system. Error bars represent average values ± SD.

Effects of BMP7 on Compartment Transition Are Inherent to CITED1⁺ Progenitors.

To mimic signaling events in the nephrogenic zone, our studies have used primary NZCs. This culture is a mixed population, and there is a possibility that the synergistic effects of SMAD and β-catenin on CITED1⁺ cells are not the result of direct signaling to these cells. To test whether SMAD and β-catenin act directly on CITED1⁺ cells, we developed a method for their purification. We have been unable to identify a surface marker specific to CITED1⁺ cells, so we developed an antibody-based protocol to deplete cells other than CITED1⁺. Major populations identified in the mixed culture are shown in Fig. S5. Using antibody-based magnetic depletion of CD105⁺, CD140a⁺, TER-119⁺, and CD326⁺ cells, we were able to remove more than 99% of these cells (Fig. 4A). Immunostaining demonstrates an enrichment of CITED1⁺ progenitors from 55 to more than 96% (Fig. 4 B and C). Similar to NZCs, pure CITED1⁺ cells require BMP7 priming for a vigorous differentiation response to β-catenin signaling (Fig. 4 D and E), indicating that niche cells within the nephrogenic zone are neither required for CITED1⁺ cells to maintain their refractory state to WNT/β-catenin induction, or for their susceptibility to induction after BMP7 treatment.

Fig. 4.

Effects of BMP-SMAD are inherent to CITED1⁺ progenitors. (A) Flow cytometry showing that CD105, CD140a, TER-119, and CD326 mark specific subpopulations of NZCs (Upper) and that antibody based magnetic depletion efficiently removed these populations (Lower). PE-high labeled TER-119⁺ cells are distinguished from PE-low labeled CD105⁺ cells as verified by additional FITC staining (Fig. S5B). (B) CITED1 staining of depleted cells demonstrates an enrichment of CITED1⁺ progenitors (red) from 55 to greater than 96%. (C) Quantified results from B. Error bars represent average values ± SEM, and P values are derived from Student’s t test. (D) RT-qPCR showing synergistic induction of Lef1 and Wnt4 in CITED1 pure cultures primed with BMP7 before 8 h of BIO treatment. (E) LEF1 (red) in CITED1 pure cultures after treatment with the indicated factors as described in Fig. 3A.

CITED1⁺ Progenitors Require BMP and WNT Signals to Form Epithelial Tubules.

To test the capacity of CITED1⁺ cells for epithelial induction, we modified our culture system for 3D growth, spotting densely packed cellular aggregates on Nuclepore filters and culturing under serum-free organ culture conditions. E11.5 kidney mesenchyme cells undergo epithelialization when induced with LiCl, and we therefore tested the differentiation capacity of our culture system by using aggregated single-cell suspensions of E11.5 mesenchymes (18). Immunostaining for E-cadherin revealed that LiCl-treated aggregates form epithelia (Fig. 5 A and B). Surprisingly, aggregates of E17.5 CITED1⁺ cells do not epithelialize, although LiCl promotes their survival and adherence (Fig. 5 C and D). Thus, although epithelia can be differentiated from E11.5 mesenchyme cells by using a standard LiCl differentiation procedure, E17.5 nephron progenitors are refractory. LiCl elicits a weaker differentiation response than BIO (Fig. 3C), and we therefore tested the ability of BIO to promote epithelialization in E17.5 aggregates. We found that 2 µM BIO causes vigorous epithelial tubule formation in E17.5 CITED1⁺ aggregates (Fig. 5 E–G). We conclude that our 3D culture system can be used to study epithelialization of E17.5 nephron progenitors and that pure CITED1⁺ cells are competent to differentiate into epithelial tubules. However, the capacity to induce differentiation directly with BIO differs from our monolayer system, in which BMP7 pretreatment is required to prime cells for this response. Aggregate culture differs from monolayer in that endogenously produced growth factors can be trapped in the matrix between cells, resulting in high local concentrations. CITED1⁺ cells produce endogenous BMP (Fig. 3D), and we hypothesized that ligand might be sufficiently locally concentrated in 3D culture to prime cells for BIO induction, obviating the need for BMP7 addition. Loss of expression of the BMP response gene Cv2 in cultures treated either with dorsomorphin or with noggin indicates active endogenous signaling, and treatment of aggregates with dorsomorphin prevents epithelial induction by BIO (Fig. 5 H and I and Fig. S6). We conclude that BMP-SMAD signaling is required to prime cells for induction by BIO in 3D culture and that adequate BMP signal intensity is achieved from endogenously produced ligand. In agreement with previous studies, inhibition of the planar cell polarity and Ca²⁺ release pathways in CITED1⁺ progenitors treated with BIO prevents tubulogenesis, demonstrating a requirement for noncanonical WNT signaling (Fig. 5 J and K). Noncanonical WNT signaling is likely mediated through induction of WNT4, because tubulogenesis with BIO is inhibited with the WNT secretion inhibitor IWP2 (Fig. S6). E11.5 cells are more sensitive to LiCl induction than E17.5 cells, and we therefore asked whether there is the same requirement for BMP-SMAD priming at these two stages. Using dorsomorphin, we found that BMP-SMAD signaling is dispensable for the differentiating effect of BIO in E11.5 aggregates (Fig. 5 L–N). This observation suggests that the early metanephric mesenchyme may be highly responsive to β-catenin induction, whereas later nephron progenitors require BMP7 priming. This feature could be an adaptation to the thinning of cap mesenchyme seen from E11.5–E13.5 and a requirement for maintenance of undifferentiated CITED1⁺ cells at the rapidly growing collecting duct tips. Intriguingly, nephron formation occurs in both Bmp7 null and Bmp7cre;Smad4 conditional mutant kidneys up to approximately E13.5, supporting the idea that BMP-SMAD signaling is required for cap differentiation at later stages of nephrogenesis.

Fig. 5.

CITED1⁺ progenitors form tubules directly in response to BMP and WNT signaling. (A and B) E-cadherin staining (green) in single-cell aggregates from E11.5 mesenchymes epithelialize after 5 d in culture on Nuclepore filters when treated with LiCl (B), but do not survive in the absence of LiCl (A). (C and D) E17.5 CITED1⁺ progenitors do not undergo epithelialization when treated with LiCl (D). (E–G) E17.5 CITED1⁺ progenitors undergo expansion and tubulogenesis when cultured with BIO. DAPI is blue, and lotus lectin is red. G shows 40× enlarged image of epithelialized tubule from boxed area in F. (H) BMP antagonists dorsomorphin and noggin block endogenous BMP activity in E17.5 CITED1⁺ culture as determined by RT-qPCR of the BMP response gene Cv2. (I) Dorsomorphin blocks tubulogenesis in BIO-treated CITED1⁺ progenitors. Negative and positive controls shown in E and F. (J and K) Canonical BMP and WNT mediated tubulogenesis of CITED1⁺ progenitors is extinguished by inhibitors of noncanonical WNT signaling, including Ca²⁺ release (cyclosporin A) and JNK (SP600125) pathways. (L–N) Dorsomorphin treatment does not block tubulogenesis in single-cell aggregates from E11.5 mesenchymes treated with BIO. Insets show lotus lectin staining (red) from the boxed regions. (O–T) E17.5 CITED1⁺ progenitors transfected with Wnt9b, but not Gfp or Wnt4, undergo tubulogenesis. Inset in Upper shows GFP expression after 24 h culture. Lower shows lotus lectin staining (red) with DAPI counterstain (blue), after 14 d of culture. One of three independent replicates is shown for each condition. (U) Semiquantitative PCR demonstrating increased Wnt9b and Wnt4 expression in progenitors transfected with Wnt9b and Wnt4 constructs, respectively. (V) Model for compartmentalization of the cap mesenchyme.

CITED1⁺ Progenitors Must Be Programmed Before Induction by WNT4.

Previous studies have shown that WNT4 can directly induce the differentiation of E11.5 metanephric mesenchymes (23). WNT4 is known to elicit a noncanonical response, and there is a possibility that this mode of signaling may be exempt from the requirement for transition to the SIX2-only compartment for induction. Recently, a method for induction of rat metanephric mesenchyme by WNT4 soaked beads was described (24). Using this method, we were unable to provoke epithelial induction in E17.5 CITED1⁺ 3D cultures (Fig. S7), indicating that WNT4 is not capable of inducing cells in the CITED1⁺ compartment. To confirm this finding, we developed a method to transfect CITED1⁺ 3D aggregate cultures (Fig. 5 O–T). Transfection of a GFP control construct resulted in protein expression, but aggregates show no evidence of epithelialization up to 14 d in culture. As expected, transfection with a construct encoding WNT9b resulted in vigorous epithelialization and expression of the induction markers Lef1, jagged 1 (Jag1), and Wnt4 after 3 d (Fig. 5 Q and R and Fig. S7). Transfection with WNT4, however, did not result in any noticeable epithelialization and actually decreased expression of Lef1, Jag1, and endogenous Wnt4 (Fig. 5 S and T and Fig. S7). From these studies we find no evidence that WNT4 has the capacity to directly induce CITED1⁺ cells, and we propose that orderly progression through progenitor compartments is required to acquire susceptibility to WNT4 epithelialization.

CITED1⁺ progenitor maintenance is driven by FGFs 9 and 20 and low-level WNT9b/β-catenin signaling (13, 16, 25). β-catenin–mediated signaling elicited by ureteric bud derived WNT9b directs progenitors to express WNT4 that, in turn, stimulates noncanonical WNT signaling, inducing formation of epithelia from nephron progenitors in an autocrine manner (12, 24). We therefore propose a model for epithelial induction of cap mesenchyme in which cells of the CITED1⁺ compartment are refractory to the inductive action of WNT9b until they have undergone BMP7-SMAD signaling and transitioned to the SIX2-only compartment (Fig. 5V). An intriguing molecular distinction between early progenitor compartments has previously been observed: complexes of SIX2 and β-catenin can be isolated from SIX2⁺ but not CITED1⁺ cells, suggesting significant differences in canonical WNT-induced transcriptional responses between SIX2-only and CITED1⁺ compartments (27). Within the SIX2-only compartment progenitors become susceptible to WNT9b/β-catenin induction, leading to expression of LEF1 and WNT4. WNT4 then promotes epithelialization of the LEF1⁺ pretubular aggregate through autocrine noncanonical WNT signaling. This orderly progression through a series of functionally distinct progenitor cell compartments ensures that the CITED1⁺ population is sequestered from induction and retained for future rounds of nephrogenesis. Further studies should be performed to test critical aspects of this model such as how the SMAD versus TAK1-MAPK response to BMP7 is regulated at the molecular level and how the CITED1⁺ population is carried forward to the next collecting duct tip to seed a new nephron.

Materials and Methods

Cell Culture.

NZCs were extracted from 20 to 24 embryonic kidneys and cultured in monolayer in keratinocyte serum-free medium (Gibco) supplemented with 50 ng/mL FGF2 (R&D Systems) as described (15–17). Cultures were treated with 50 ng/mL BMP7 (R&D Systems), 500 nM BIO (Calbiochem), 15 mM LiCl (Sigma), or 10 µM FH535 (Calbiochem). TAK1 inhibitor (AnalytiCon Discovery) concentration was empirically determined as the amount necessary (50 nM) to reverse BMP7-induced proliferation of nephron progenitors. Dorsomorphin (Sigma) concentration (2.5 µM) was determined as the amount necessary to block BMP7-induced phosphorylation of SMAD1/5 in nephron progenitors. Three-dimensional aggregates containing 250,000 progenitors were spotted from single-cell suspensions in a 1-µL volume on floating 0.1-μm pore-size VCTP membrane filters (Millipore) and cultured for 5 d in DMEM/F12 supplemented with FGF2 (200 ng/mL), 5% (vol/vol) KnockOut Serum Replacement (Invitrogen) and additional factors as described (26). For CITED1⁺ progenitor transfection experiments, freshly purified cells were incubated at 37 °C in BSA-coated 1.5-mL Eppendorf tubes on a nutator for 1.5 h. One hundred microliters of prepared lipofectamine/DNA reagent (4 μg of DNA to 2 μL of lipofectamine) was added to 1 mL of cells in DMEM/F12 medium. Cells were spun at 300 × g and resuspended in DMEM/F12 for 3D aggregate spotting.

Immunofluorescence and Microscopy.

Cells and sections were immunostained as described (15). Primary antibodies were incubated overnight at 4 °C: CITED1 1:100 (NeoMarkers); LEF1 1:100 (Cell Signaling Technology); PAX2 1:100 (Invitrogen); phistone-H3 1:100; pSMAD1/5 1:100 (both Cell Signaling Technology); SIX2 1:100 (Santa Cruz Biotechnology); DBA lectin 1:200 (Vector Laboratories); LT lectin 1:200 (Vector Laboratories) and E-cadherin 1:100 (BD Transduction Laboratories). PAX2 and pSMAD1/5 or CITED1 and LEF1 were costained by converting rabbit pSMAD1/5 or LEF1 antibodies to goat by using anti-goat FAB fragment according to the manufacturer’s instructions (Jackson ImmunoResearch) followed by 5-min fixation with 4% (wt/vol) paraformaldehyde, before incubation with PAX2 or CITED1 antibodies for 24 h at 4 °C. Immunofluorescent images were quantified by using ImageJ, and percentages were normalized to the total number of DAPI⁺ cells per field. P values were calculated by using a two-tailed homoscedastic Student t test.

Quantitative PCR.

RNA purification, cDNA synthesis, and quantitative PCR were performed as described (16). Average values (±SD) of three technical replicates from NZCs of 20–24 pooled embryonic kidneys are shown. P values were calculated by using a heteroschedastic two-tailed Student t test with P < 0.05 considered significant.

Magnetic Bead Depletion and Flow Cytometry.

Total NZCs were isolated and CITED1⁺ cells were enriched by negative depletion with magnetic activated cell sorting (MACS)-phycoerythrin (PE)–conjugated antibodies (Fig. S5A) and anti-PE MicroBeads by following the manufacturer’s protocol (Miltenyi Biotec). Cells were purified by passing NZCs twice through an autoMACS separator by using the “Depletes” program setting. Total and CITED1 enriched cell populations were stained by using PE-, allophycocyanin-, and fluorescein-conjugated antibodies, collected on a FACSCalibur (BD), and data were analyzed by using FlowJo software.

Mouse Strains.

Animal care was performed in accordance with the National Research Council Guide for the Care and Use of Laboratory Animals. Animal protocols were approved by the Institutional Animal Care and Use Committee of Maine Medical Center. Institute for Cancer Research stock was used for all NZC harvests, and Bmp7^−/− was maintained on an Institute for Cancer Research background.