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

Abstract

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

Fig. 1.

Identification of novel Wnt9b target genes in kidney progenitor cells. (A-O) In situ hybridization evaluating the expression of C1qdc2 (A-C), Cited1 (D-F), Pla2g7 (G-I), Tafa5 (J-L) or Uncx4.1 (M-O) in wild-type (A,D,G,J,M), Wnt9b^−/− (B,E,H,K,N) or Wnt4^−/− (C,F,I,L,O) animals at E11.5. In all images, the pretubular aggregates are located on the left, whereas the progenitor cells are on the right. The ureteric bud tips are outlined.

Fig. 2.

β-Catenin signaling is sufficient and necessary for Wnt9b progenitor target gene expression. (A-R) In situ hybridization evaluating the expression of C1qdc2 (A,B,G-I), Pla2g7 (C,D,J-L) and Tafa5 (E,F,M-O) and Uncx4.1 (P-R). A-F represent E11.5 metanephric mesenchyme cultured for 48 hours in media alone (A,C,E) or in media containing 15 mM LiCl (B,D,F). (G-R) Embryonic kidneys grown in the presence of DMSO (G,J,M,P), 5 μM IWP2 (H,K,N,Q) or 100 μM IWR1 (I,L,O,R).

Fig. 3.

β-Catenin signaling regulates Wnt9b progenitor target gene expression in vivo. (A-L) In situ hybridization evaluating the expression of C1qdc2 (A-D), Tafa5 (E-H) and Pla2g7 (I-L) in wild-type (A,E,I), Rarb2Cre;Catnb^ex3flox (B,F,J), Wnt9b^−/− (C,G,K) and Rarb2Cre;Catnb^ex3flox;Wnt9b^−/− (D,H,L) animals at E11.5. In all images, the Wolffian duct is located towards the left while the progenitor cells are towards the right. The ureteric bud tips are outlined. Direct genetic stabilization of β-catenin in the mesenchymal progenitors is sufficient to induce expression of Wnt9b targets, even in the absence of Wnt9b. (M-O) Quantification of ChIP with β-catenin antibody on E15.5 kidneys. Enriched enhancer sequences containing Lef/TCF sites are indicated by red ovals. Non-enriched sites are indicated by blue ovals. The graph shows fold increase relative to anti-IgG control. Data are mean±s.e.m. β-Catenin protein is associated with conserved Lef/TCF binding sites in the enhancers of at least three of the Wnt9b target genes.

Fig. 4.

β-Catenin is required cell-autonomously for the expression of progenitor markers. (A-J) Expression analysis of Wnt9b target genes in wild-type (A,B,E-G) or Rarb2Cre;catnb^−/flox mutant (C,D,H-J) E12.5 kidneys. A and C are images of whole-mount kidneys hybridized with antisense probes to Tafa5 (A,C) or Pla2g7 (B,D). E-J are images of sections stained with antibodies to β-catenin (red), Six2 (green in E,H), Cited1 (green in F,I) and amphiphysin (green in G,J). All sections were counterstained with the nuclear marker To-Pro 3 (blue). Arrows indicate progenitor cells. Removal of β-catenin from the progenitors results in cell-autonomous loss of Wnt9b target genes.

Fig. 5.

Wnt9b and Six2 cooperate to regulate target gene expression. (A-P) In situ hybridization evaluating the expression of C1qdc2 (A,B), Cited1 (C,D), Pla2g7 (E,F), Tafa5 (G,H), Eya1 (I-L) and Gdnf (M-P) in E11.5 wild-type (A,C,E,G,I,M), Six2^−/− (B,D,F,H,K,O), Wnt9b^−/− (J,N) and Wnt9b^−/− Six2^−/− (L,P) animals at E11.5. In all images, the Wolffian duct is located towards the left while the progenitor cells are towards the right. The ureteric bud tips are outlined. Six2 is necessary for the expression of Class II Wnt9b target genes (D,F,H) but represses Class I targets (B).Wnt9b and Six2 genetically interact to regulate expression of Class II genes (M-P), and the loss of Class II gene expression in Wnt9b and Six2 mutants is not caused by loss of the progenitor population (I-L).

Fig. 6.

Removal of Wnt9b results in a premature loss of progenitor cells. (A-P) In situ hybridization evaluating the expression of Six2 (A,B,I,J), Pla2g7 (C,D,K,L), Tafa5 (E,F,M,N) and Uncx4.1 (G,H,O,P) in wild-type (A,C,E,G,I,K,M,O) or KspCre;Wnt9b^c/− (B,D,F,H,J,L,N,P) kidneys at E17.5 (A-H) or P1 (I-P). Wnt9b is necessary for the maintenance of the progenitor population at later stages.