Myc cooperates with β-catenin to drive gene expression in nephron progenitor cells

Abstract

For organs to achieve their proper size, the processes of stem cell renewal and differentiation must be tightly regulated. We previously showed that in the developing kidney, Wnt9b regulates distinct β-catenin-dependent transcriptional programs in the renewing and differentiating populations of the nephron progenitor cells. How β-catenin stimulated these two distinct programs was unclear. Here, we show that β-catenin cooperates with the transcription factor Myc to activate the progenitor renewal program. Although in multiple contexts Myc is a target of β-catenin, our characterization of a cell type-specific enhancer for the Wnt9b/β-catenin target gene Fam19a5 shows that Myc and β-catenin cooperate to activate gene expression controlled by this element. This appears to be a more general phenomenon as we find that Myc is required for the expression of every Wnt9b/β-catenin progenitor renewal target assessed as well as for proper nephron endowment in vivo This study suggests that, within the developing kidney, tissue-specific β-catenin activity is regulated by cooperation with cell type-specific transcription factors. This finding not only provides insight into the regulation of β-catenin target genes in the developing kidney, but will also advance our understanding of progenitor cell renewal in other cell types/organ systems in which Myc and β-catenin are co-expressed.

Keywords: Fam19a5; Mouse; Myc; Nephron progenitors; Stem cells; Wnt; β-Catenin.

Fig. 1.

A 2.9-kb fragment in intron 2 of Fam19a5 activates transcription in the renewing nephron progenitor cell population. (A) Schematic of intron 2 of mouse Fam19a5. Peaks represent areas of homology between mouse, possum or human. The boxed area indicates the position of the fragment used to engineer the reporter. The 2.9-kb fragment was cloned into the pOTV8 plasmid containing a minimal promoter and a β-galactosidase cDNA. The relative positions of the five consensus Lef/Tcf-binding sites are indicated by red ovals. (B-F) Whole embryos (E12.5; B) and kidney sections from Fam19a5-GRE-stained 11.5 (C), E12.5 (D), E14.5 (E) and P1 (F) kidneys. For C-F, the cortical zone is to the left. (G-H‴) Sections of P1 Fam19a5-GRE kidneys stained with antibodies to Six2 (red in G,G′) or Cited1 (red in H,H′), β-gal (Bgal, green), the collecting duct marker DBA (blue in G,G‴,H,H‴). G′-G‴ and H′-H‴ are single-channel images; G and H are merged images. Arrows indicate the nephron progenitor cells. Scale bars: 500 μm (B); 50 μm (C-F); 30 μm (G-H‴).

Fig. 2.

The Fam19a5-GRE is regulated by Wnt9b and β-catenin. (A-D) Sections of E11.5 (A,B) or E12.5 (C,D) Fam19a5-GRE kidneys from wild-type (A,C), Wnt9b^−/− (B) and Six2Cre;catnb^flox/− (D) kidneys stained with antibodies to β-gal (bgal, green), Six2 (red) and TO-PRO-3 (blue). Arrows indicate the nephron progenitor cells. Scale bars: 30 µm (A,B); 100 µm (C,D).

Fig. 3.

A poorly conserved 774-bp element is sufficient to drive expression within the NPCs. (A) Schematic of three different transgenic constructs used to generate transgenic lines. Red ovals represent consensus Lef/Tcf-binding sites. (B) Sections of stained kidneys from embryos injected with each construct whose DNA was scored as positive for carrying the lacZ transgene. The right-hand image shows a P1 Fam19a5-GRE-2161-2934 section stained with β-gal (bgal, green), Six2 (red) and DAPI (blue). Arrows indicate the nephron progenitor cells. Scale bars: 50 μm.

Fig. 4.

c-Myc promotes Fam19a5-GRE reporter activity in vitro. (A) Sequence fragments from the Fam19a5-GRE highlighting the consensus Lef/Tcf (red)- and Myc (blue)-binding sites. (B) Graph of luciferase reporter activity from HEK 293 cells transfected with a Fam19a5-GRE luciferase reporter and treated with LiCl at different concentrations. (C) Graph of luciferase activity from HEK 293 cells transfected with wild-type or various mutant Fam19a5-GRE reporters. Some cells were co-transfected with a full length c-Myc expression plasmid as indicated. The media was replaced with fresh media (black bars) or media supplemented with 10 mM LiCl (gray bars) 24 h after transfection and luciferase activity was measured 18 h later. Data are represented as mean+s.e.m.

Fig. 5.

Myc is necessary for Fam19a5 reporter activity and Wnt9b target gene expression in organ culture. E11.5 Fam19a5-GRE kidneys were dissected out and cultured for 48 h in media supplemented with DMSO (A,F-H), the Wnt production inhibitor IWP2 (B), a Myc inhibitor (C,I-K), a STAT1 inhibitor (D) or a STAT3 inhibitor (E). Cultured kidneys were fixed and stained with X-gal (A-E), antisense mRNA for Cited1 (F,I) or Fam19a5 (G,J), or sectioned and stained with antibodies to β-gal (bgal, green), Six2 (red) and TO-PRO-3 (blue) (H,K). Arrows indicate the nephron progenitor cells and ureteric bud is outlined. Scale bars: 30 µm.

Fig. 6.

c- and N-Myc play partially redundant roles in nephron progenitor renewal and nephron endowment. Whole-mount images (A-D) or sections through P1 urogenital systems/kidneys of wild-type (c-Myc^flox/flox;N-Myc^flox/flox; A,E,I,M,Q), N-Myc (Six2Cre;N-Myc^flox/flox; B,F,J,N,R), c-Myc (Six2Cre;c-Myc^flox/flox; C,G,K,O,S) or c/N-Myc double mutant (Six2Cre;c-Myc^flox/flox;N-Myc^flox/flox; D,H,L,P,T) kidneys. E-L show low (E-H) or high (I-L) magnification images of H&E-stained kidneys. M-T show sectioned P1 kidneys stained with antibodies to the proximal tubule marker lotus tetragonolobus lectin (LTL, red) and DAPI (blue) (M-P) or Six2 (green), the collecting duct marker cytokeratin (CK, red) and DAPI (blue) (Q-T). Arrows indicate cap mesenchyme and ureteric bud is outlined. Scale bars: 1 mm (A-H); 100 μm (I-L); 200 µm (M-P); 30 µm (Q-T).

Fig. 7.

c-Myc and N-Myc are necessary for expression of Wnt/β-catenin progenitor renewal targets. Sections of E13.5 wild-type (c-Myc^flox/flox;N-Myc^flox/flox; A-E) and mutant (Six2Cre;c-Myc^flox/flox;N-Myc^flox/flox; F-J) kidneys hybridized with antisense in situ probes for the Wnt9b-independent nephron progenitor marker Six2 (A,F), the Wnt9b-dependent progenitor renewal targets Cited1 (B,G) and Fam19a5 (C,H), or the Wnt9b-dependent nephron progenitor differentiation targets C1qdc2 (D,I) and Pax8 (E,J). Arrows indicate the nephron progenitor cells and ureteric bud is outlined. Scale bars: 50 μm.