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.

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.

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^−/−.

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.

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.

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.