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. 2011 May 17;20(5):583-596.
doi: 10.1016/j.devcel.2011.03.013.

The WTX Tumor Suppressor Regulates Mesenchymal Progenitor Cell Fate Specification

Free PMC article

The WTX Tumor Suppressor Regulates Mesenchymal Progenitor Cell Fate Specification

Annie Moisan et al. Dev Cell. .
Free PMC article

Erratum in

  • Dev Cell. 2012 May 15;22(5):1109-17


WTX is an X-linked tumor suppressor targeted by somatic mutations in Wilms tumor, a pediatric kidney cancer, and by germline inactivation in osteopathia striata with cranial sclerosis, a bone overgrowth syndrome. Here, we show that Wtx deletion in mice causes neonatal lethality, somatic overgrowth, and malformation of multiple mesenchyme-derived tissues, including bone, fat, kidney, heart, and spleen. Inactivation of Wtx at different developmental stages and in primary mesenchymal progenitor cells (MPCs) reveals that bone mass increase and adipose tissue deficiency are due to altered lineage fate decisions coupled with delayed terminal differentiation. Specification defects in MPCs result from aberrant β-catenin activation, whereas alternative pathways contribute to the subsequently delayed differentiation of lineage-restricted cells. Thus, Wtx is a regulator of MPC commitment and differentiation with stage-specific functions in inhibiting canonical Wnt signaling. Furthermore, the constellation of anomalies in Wtx null mice suggests that this tumor suppressor broadly regulates MPCs in multiple tissues.


Figure 1
Figure 1. Germline Inactivation of Wtx Leads to Defects in Multiple Mesenchyme-Derived Tissues
(A) Gross images of E18.5 wild-type (WT) and Wtx null (KO) embryos. (B) Weight of neonatal organs; organs showing significant differences are indicated in bold, *p < 0.01. 1Wtx null kidneys were either absent or heavier than wild-type counterparts. (C) Left: Representative gross images of neonatal kidneys (K) and adrenals (A) showing unilateral agenesis in a Wtx null mouse. Right: Immunohistochemistry (IHC) showing increased numbers of Six2+ cells in neonatal Wtx null kidneys; quantification of the Six2+ aggregates is shown at the bottom (p < 0.001). (D) Hematoxylin and eosin (H&E) staining of the interscapular region at E18.5 showing paucity of brown adipose tissue (BAT, arrow) in Wtx KO mice. (E) Alizarin red/Alcian blue stained neonatal skeletal preparations. Wtx KO mice show: (a) enlargement of the skull, (b) dysplasia of the sternum, and (c) enlarged and bowed radius and ulna (arrowhead) and absence of the deltoid tuberosity (arrow). (F) Von Kossa staining of mineralized bone matrix in neonatal femurs; cortical bone (CB). (G) PET-CT images of skull (left) and femur (right) of 4-month-old wild-type and Wtx heterozygous (HET) mice. (H) Bone mineral density (BMD) of adult skulls. See also Figures S1 and S2.
Figure 2
Figure 2. Targeted Inactivation of Wtx in Mesenchymal Progenitors Causes Osteosclerosis and Impaired Adipogenesis
(A) Wtxlox mice were crossed with the Prx1-Cre, Col2a-Cre, or Ocn-Cre strains to inactivate Wtx at different stages of skeletal development (schematic diagram). Alizarin red/Alcian blue stained neonatal sternums (left) and femurs (center), and H&E stained adult femurs (right) from the indicated models. Prx1-Cre;Wtxlox/Y mice show dysplasia of sternum and femur (arrows) and marked osteosclerosis. (B–E) Analysis of the Prx1-Cre model at age 6 weeks. Von Kossa staining of tibiae is shown in (B). Histomorphometry showing bone volume/tissue volume (BV/TV), trabecular thickness (Tb.Th), osteoblast number/tissue surface (N.Ob/T.Ar), bone formation rate per tissue volume (BFR/TV), mineralized surface per bone surface (MS/BS), and mineral apposition rate (MAR) is shown in (C). ***p < 0.001, **p < 0.01, *p < 0.05, N.S., not significant. In (D), upper panel is of H&E showing stromal cells (*) in trabecular bone; the numbers of stromal cells per field are indicated. Lower (D) is an alkaline phosphatase staining (ALP) of trabecular bone. H&E staining of distal tibiae is shown in (E) (arrows: adipocytes). See also Figures S3 and S4.
Figure 3
Figure 3. Wtx Regulates Osteoblastogenesis and Adipogenesis in BMPCs
(A–C) Wild-type and Wtx KO BMPCs were grown in noninducing (ni) and osteogenic (osteo) conditions for 6 days or in adipogenic (adipo) conditions for 12 days, respectively and analyzed by western blot for Wtx expression (A), ALP (B, top and middle) and oil red O staining (B, bottom; % stained cells are indicated), and qRT-PCR analysis for expression of osteoblast-selective genes and adipocyte-selective genes (C). (D–F) ST2 cells expressing shGFP (control) or shWtx were grown under noninducing (ni) and osteogenic (osteo) conditions for 6 days, or in adipogenic (adipo) conditions for 12 days; cells were analyzed for Wtx expression by western blot (D), ALP and oil red O staining as indicated (E), and expression of osteoblast-selective (F, top) and adipocyte-selective genes (F, bottom). *p < 0.05. See also Figure S5.
Figure 4
Figure 4. Wtx Acts Upstream of Runx2 and Pparγ to Control Osteogenic and Adipogenic Fate Determination
(A) Runx2 expression detected by western blot in ST2 cells expressing shGFP or shWtx. (B) ST2 cells expressing combinations of shRNAs targeting control (GFP), Wtx, and Runx2 were grown under noninducing (ni) and osteogenic (osteo) conditions for 6 days, or in adipogenic (adipo) conditions for 12 days, and examined for ALP staining (top and center rows) and oil red O (bottom row). (C) ST2 cells expressing shGFP or shWtx were grown under adipogenic conditions and analyzed for adipogenic differentiation markers. *p < 0.05. (D) ST2 cells overexpressing either control GFP or PPARγ, in combination with either shGFP or shWtx, were grown under indicated conditions and analyzed for oil red O staining (top), and ALP staining (middle and bottom). Oil red O staining was performed on adipogenic day 12 in the eGFP-expressing cells and on day 6 in PPARγ-expressing cells. (E) IHC in E15.5 humerus showing increases in Runx2 and Osx staining in Wtx KO mice compared to WT controls. HC, hypertrophic chondrocytes; PC, perichondrium. (F) Osteogenic (Runx2 and Osterix), chondrogenic (Sox9), and adipogenic (Pparγ) lineage marker expression detected by western blot of neonatal WT and Wtx KO femur. (G) WT and Wtx KO neonatal long bones were analyzed for osteoblast-selective gene expression by qRT-PCR. See also Figure S6.
Figure 5
Figure 5. Wtx-Mediated Control of β-Catenin Levels in BMPCs Determines Osteoblast and Adipocyte Specification
(A and B) Prx1-Cre;Wtx+/Y and Prx1-Cre;Wtxlox/Y BMPCs were grown under the indicated conditions for 4 days and analyzed for expression of Wtx, active β-catenin (ABC), and total β-catenin (β-cat) by western blot (A) and the β-catenin target genes, Axin2 and Tcf1, by qRT-PCR (B). (C and D) ST2 cells expressing shGFP or different shRNAs targeting Wtx (Wtx-1 and Wtx-2) were grown under the indicated conditions for 4 days and analyzed for expression of total β-catenin by western blot (C) and Axin2 and Tcf1 by qRT-PCR (D). (E–G) ST2 cells expressing shGFP or shWtx in combination with shControl or sh-β-catenin were grown under noninducing (ni) and osteogenic (osteo) conditions for six days or in adipogenic (adipo) conditions for 12 days and analyzed for ALP and oil red O staining (E); Wtx, β-catenin, and Runx2 expression by western blot (F); and expression of Pparg and Fabp4 by qRT-PCR (G). β-catenin knockdown rescues both the increased in ALP staining and reduction in oil red O staining caused by Wtx knockdown, and also restores normal Runx2 and Pparg expression. See also Figure S7.
Figure 6
Figure 6. Wtx Modulates β-Catenin Activity during Bone Development
(A) IHC for Tcf1 and Lef1 in E14.5 wild-type and Wtx null humerus. HC, hypertrophic chondrocytes. (B) Western blot analysis for total β-catenin protein levels in wild-type and Wtx null limbs at E15.5 and time of birth. (C) Mice with the Prx1-Cre, Wtxlox, and β-cateninlox alleles were crossed to generate compound mutants with the indicated genotypes. Skeleton preparation (top) and Von Kossa staining (bottom) of neonates show, respectively, that dysplasia of the sternum and accumulation of femoral cortical bone (CB) in Prx1-Cre; Wtxlox mice is suppressed by concurrent β-catenin hemizygosity. See also Figure S7.
Figure 7
Figure 7. Wtx Inactivation Impairs Maturation of Committed Osteoblasts
(A and B) Delayed ossification in Wtx null embryos. Wtx null embryos (E18.5) and neonates (P0) were characterized by wide cranial fontanelles (F) as revealed by skeleton preparation (A, first and third columns), Von Kossa staining (second column), and H&E staining (fourth column). OF, ossification front. Staining of femurs with Von Kossa showing that Wtx-deficient mice have delayed mineralization of cortical bone (CB) at E15.5 and reduced formation of trabecular bone (TB) at E17.5 and E19.5 (B). (C and D) Wtx knockdown impairs osteoblast differentiation of MC3T3 pre-osteoblasts as determined by ALP staining (C) and Alizarin red staining (D) at the indicated time points. DAPI (C) and Coomassie blue staining (D) indicate that the total number of cells were unaffected. See also Figure S8.
Figure 8
Figure 8. Wtx Inactivation Impairs Osteoblast Maturation Independent of β-Catenin Deregulation
(A) Wtx inactivation in committed osteoblasts is associated with reduction in activated β-catenin (ABC) and total β-catenin (β-cat) as shown in the Prx-Cre BMPCs and MC3T3-E1 models at the indicated time points. (B) Expression of β-catenin target genes in Prx1-Cre;Wtxlox/+ and control BMPCs. Cells were noninduced (ni) or grown 10 days under osteogenic (osteo) conditions. (C and D) ALP staining of MC3T3-E1 cells grown under noninducing (ni) or osteogenic conditions (osteo) for 6 days and expressing shβ-catenin and shWtx singly or in combination shows that β-catenin knockdown fails to rescue the loss of ALP staining caused by Wtx inactivation (C). BIO enhances ALP staining in control shGFP-expressing cells but fails to rescue the defective ALP staining of Wtx-knockdown cells (D). MeBIO is an inert control compound. (E) Model of Wtx function. In MPCs, Wtx decreases β-catenin levels thereby calibrating lineage commitment decisions, ensuring the appropriate balance between osteoblastogenic and adipogenic determination. Wtx also inhibits β-catenin stability in Osx+ osteoblast precursors, thereby restraining the osteoblastic lineage expansion. Subsequently, Wtx has a distinct function directing the terminal differentiation of committed osteoblasts, a role not associated with inhibition of canonical Wnt/β-catenin signaling.

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