Eya1 interacts with Six2 and Myc to regulate expansion of the nephron progenitor pool during nephrogenesis

Abstract

Self-renewal and proliferation of nephron progenitor cells and the decision to initiate nephrogenesis are crucial events directing kidney development. Despite recent advancements in defining lineage and regulators for the progenitors, fundamental questions about mechanisms driving expansion of the progenitors remain unanswered. Here we show that Eya1 interacts with Six2 and Myc to control self-renewing cell activity. Cell fate tracing reveals a developmental restriction of the Eya1(+) population within the intermediate mesoderm to nephron-forming cell fates and a common origin shared between caudal mesonephric and metanephric nephrons. Conditional inactivation of Eya1 leads to loss of Six2 expression and premature epithelialization of the progenitors. Six2 mediates translocation of Eya1 to the nucleus, where Eya1 uses its threonine phosphatase activity to control Myc phosphorylation/dephosphorylation and function in the progenitor cells. Our results reveal a functional link between Eya1, Six2, and Myc in driving the expansion and maintenance of the multipotent progenitors during nephrogenesis.

Figure 1. The Eya1⁺ IM Contributes to Caudal Mesonephric and Metanephric Nephrons

(A–C and E) β-gal staining of an Eya1^LacZ/+ embryo at E8.75 (A), E9.25 (B), E10.5 (C), and E11.5 (E). The arrow points to the Eya1⁺ IM. (D and F) Sections of β-gal⁺ embryos at E10.5 (D) and E11.5 (F). (G and H) β-gal staining on E11.5 (shorter staining) (G) and E12.5 (H) kidney sections. The arrows in (H) point to weaker activity of the LacZ⁺ subregion. (I and J) Immunostaining for Eya1/Six2on anE13.5 kidney section, showing Eya1 alone (I) and a merged image for both Eya1 (green) and Six1 (red) (J). The arrows point to lower levels of the Eya1/Six2 subregion. (K–V) Fate mapping of Eya1⁺ cells in Eya1^CreERT2/+; R26R^LacZ/+ embryos at E10.5–E12.5 after injection of oil (K, O, Q, and U) or 2–3 mg Tm (L–N, P, R–T, and V) at E8.5–E8.75. (K–O and S) show whole-mount lateral or (P) ventral views. The arrows point to the anterior limit of the Eya1⁺ IM. The arrowhead points to the forelimb region. (Q, R, and T–V) show sections counterstained with hematoxylin. cmt, caudal mesonephric tubule; fl, forelimb; hl, hindlimb; k, kidney; nd, nephric duct; nm, nephrogenic mesoderm; rmt, rostral mesonephric tubule; sg, sympathetic ganglion; so, somite. See also Figure S1.

Figure 2. Eya1⁺ Cells Contribute Continuously to Nephron Tubules throughout Kidney Development

(A–E) β-gal-stained kidneys from Eya1^CreERT2/+; R26R^LacZ/+ embryos at E18.5 (oil or 2 mg Tm at E8.5). (A and B) show a whole-mount view. (C) shows a section view. (D) shows a kidney section costained for β-gal/cytokeratin. (E) shows a higher magnification of (C). (F–K) Sections costained for β-gal/Wt1 (F), PDGFRb (G), PECAM1 (H), uromodulin (I), αSMA (J), or cytokeratin (K). (L–Q) β-gal-stained kidneys at P0 after injection of oil (L, N, and P) or 1.5 mg Tm (M, O, and Q) at E15.5. (L and M) show a whole-mount view. (N–Q) show a section view. (P and Q) show a higher magnification of (N and O). (R and S) β-gal-stained kidney sections at P0 injected with oil (R) or 0.5 mg Tm (S) at E12.5. a, adrenal gland; c, comma-shaped body; cd, collecting duct; cm, cap mesenchyme; g, glomerulus; nt, nephron tubule; pa, pretubular aggregate; sb, S-shaped body. See also Figure S2.

Figure 3. Clonal Tracing of Individually Labeled Eya1⁺ Cells

(A and B) β-gal-stained kidney sections from P0 Eya1^CreERT2;R26R^LacZ/+ mice injected with oil (A) or 0.1–0.2 mg Tm (B) at E12.5). (C–H) Higher magnification showing three different clusters on serial sections (C–E) or three clones (red) on serial sections (F–H) from P0 Eya1^CerERT2, R26R^Rainbow mice (0.2 mg Tm at E12.5). cb, Comma-shaped body; cm, cap mesenchyme; dt, distal tubule; pt, proximal tubule. See also Figures S2C–S2H.

Figure 4. Temporal Deletion of Eya1 in the MM Progenitors Results in Depletion and Premature Differentiation of the Progenitors

(A, A′, B, and B′) Kidneys at E17.5 (A and A′) and E14.5 (B and B′) of wild-type and Eya1^CreERT2/+;Eya1^Flox/Flox (Cko/Cko) embryos (Tm at ~E10.75). (C and C′) Hematoxylin and eosin (H&E)-stained kidney section of the wild-type (C) and Eya1^CreERT2/+;Eya1^Flox/Flox (C′) at E11.75. The arrows point to ectopic vesicles. (D, D′, E and E′) H&E-stained section of wild-type (D and E) and Eya1^CreERT2/+;Eya1^Flox/Flox (D′ and E′) kidneys at E12.5 (D and D′) and E14.5 (E and E′). The arrows point to ectopic vesicles. (F and F′) Immunostaining with anti-Wt1 on wild-type (F) and Eya1^CreERT2/+;Eya1^Flox/Flox (F′) kidney sections at E14.5. The arrows point to the depletion of nephron progenitors. (G–J″) In situ hybridization showing Wnt4 (G–G″ and H–H″) and Pax8 expression (I–I″ and J–J″) in PAs at E11.5 (G–G″ and I–I″) and RVs at E12.5 (H–H″ and J–J″) in wild-type and CKO embryos. (K–K″) Six2 expression in the MM in E11.5 wild-type and CKO embryos. (L–L″) Wnt9b expression in the UB in wild-type and CKO embryos. See also Figures S3 and S4.

Figure 5. Eya1 Interacts with Six2 and Myc and Regulates Myc Postphosphorylation Modification in Nephron Progenitors

(A) CoIP analysis. Antibodies for IP and western blot are indicated. (B–D) Immunostaining of E13.5 kidney sections for pT58 (B), C-Myc (C), and N-Myc/pHH3 (D). (E) Western blot of cell extracts from E12.5 wild-type or CKO kidneys (Tm at E11.0) with the indicated antibodies. The membrane was stripped and reprobed. (F–J′) Immunostaining of E12.5 kidney sections for pT58 (Fand F′), N-Myc/pT58(G and G′), C-Myc/pT58 (Hand H′), PCNA/γH2AX (I and I′), and p27Kip1 (J and J′) in the wild-type (F–J) and mutant (F′–J′). See also Figure S5.

Figure 6. Eya1 Requires Six2 for Its Nuclear Localization, and Reduction of N-Myc Causes Elevation of p27Kip1

(A–B′) Immunostaining for Six2 (A and A′) and p27Kip1/PCNA (B and B′) on kidney sections of E14.5 wild-type and N-Myc^9a/9a embryos. The arrows point to increased p27Kip1⁺ cells. (C) Quantification of p27Kip1⁺ and PCNA⁺ cells per UB tip. P27Kip1⁺ or PCNA⁺ cells were counted in the mesenchyme surrounding the UB tip from a total of 25 tips on 10 μm sections and quantified using a StatView t test. Error bars indicate SD. p Values were between 0.0297 and 0.0318. (D) Immunoblot of cell extracts from wild-type or Six2^−/− kidneys at E11.75–E12.0 with the indicated antibodies. The membrane was stripped and reprobed. (E and E′) Immunostaining for Eya1/pT58 on sections of E11.25 wild-type (E) and Six2^−/− (E′) kidneys. The bottom panels show a higher magnification of the boxed areas. (F and F′) Coimmunostaining for N-Myc/pT58 on sections of E11.5 wild-type (F) and Six2^−/− (F′) kidneys. (G) Model of the combined effects of Eya1-Six2-Myc on nephron progenitor cell proliferation. After receiving a growth-stimulatory signal, Myc gene transcription is induced, and newly synthesized Myc is phosphorylated on S62, which is necessary for the subsequent Gsk3-mediated phosphorylation at T58. Previous work demonstrated that S62 phosphate is removed by PP2A in the process of Myc ubiquitination. Our findings indicate that the maintenance of Myc protein is regulated in an Eya1/Six2-dependent manner. In the absence of Eya1 or Six2, pT58 levels are accumulated and targeted for degradation, which causes cell cycle/growth arrest as well as cell death. Eya1-Six2-Myc may also form a complex to activate target genes to control the timing of cell cycle exit. p, phosphorylation at S62; pp subsequent phosphorylation at T58. See also Figure S6.

Figure 7. Eya1 Stabilizes Myc

(A) Immunoblots (IB) of cytoplasmic (C) and nuclear (N) extracts from 293 cells transfected with FLAG-Eya1, FLAG-Eya1/His-Six2, or FLAG-Eya1/FLAG-Myc. (B) Immunostaining with anti-FLAG of 293 cells transfected with FLAG-Eya1 or FLAG-Eya1/His-Six2. (C) CoIP analysis. 293 cells were transfected with the plasmids indicated on the left or for lanes (lane 1, FLAG-Eya1/His-Six2/GFP-N-Myc; lane 2, empty lane; lane 3, His-Six2/FLAG-N-Myc; lane IgG for IP. Anti-FLAG was used for IP. Input was ~5% of the amount of proteins used for IP. (D–F) Immunoblots with anti-N-Myc (D), anti-FLAG (D–F), anti-His (D), anti-Eya1 (E and F), anti-pT58 (E and F), or β-actin (loading control). Twenty-four hours posttransfection with the indicated plasmids, the cells were treated with CHX and lysed at the indicated times. Experiments were performed in triplicate, and graphs show quantification of the average results (t_1/2, half-life). Myc levels were normalized to β-actin. See also Figure S7.

Figure 8. Eya1 Targets the T58 Phosphate of Myc

(A) In vitro phosphatase assay. Purified Myc protein was phosphorylated by GSK3β with ³²P-γATP. The phosphorylated proteins were incubated with FLAG-Eya1, analyzed on SDS-PAGE, and exposed by phosphorimager. (B) Eya1 dephosphorylates Myc at T58 but not S62. Purified Myc was incubated with either buffer (−) or FLAG-Eya1 at 30°C for 15 or 30 min. Samples were run in triplicate and immunoblotted with anti-pT58, anti-pS62, or anti-FLAG as indicated. See also Figure S8.a