The Wnt/β-Catenin Pathway Interacts Differentially with PTHrP Signaling to Control Chondrocyte Hypertrophy and Final Maturation

Sequential proliferation, hypertrophy and maturation of chondrocytes are required for proper endochondral bone development and tightly regulated by cell signaling. The canonical Wnt signaling pathway acts through β-catenin to promote chondrocyte hypertrophy whereas PTHrP signaling inhibits it by holding chondrocytes in proliferating states. Here we show by genetic approaches that chondrocyte hypertrophy and final maturation are two distinct developmental processes that are differentially regulated by Wnt/β-catenin and PTHrP signaling. Wnt/β-catenin signaling regulates initiation of chondrocyte hypertrophy by inhibiting PTHrP signaling activity, but it does not regulate PTHrP expression. In addition, Wnt/β-catenin signaling regulates chondrocyte hypertrophy in a non-cell autonomous manner and Gdf5/Bmp signaling may be one of the downstream pathways. Furthermore, Wnt/β-catenin signaling also controls final maturation of hypertrophic chondrocytes, but such regulation is PTHrP signaling-independent

Yap1 Regulates Multiple Steps of Chondrocyte Differentiation during Skeletal Development and Bone Repair

Hippo signaling controls organ size and tissue regeneration in many organs, but its roles in chondrocyte differentiation and bone repair remain elusive. Here, we demonstrate that Yap1, an effector of Hippo pathway inhibits skeletal development, postnatal growth, and bone repair. We show that Yap1 regulates chondrocyte differentiation at multiple steps in which it promotes early chondrocyte proliferation but inhibits subsequent chondrocyte maturation both in vitro and in vivo. Mechanistically, we find that Yap1 requires Teads binding for direct regulation of Sox6 expression to promote chondrocyte proliferation. In contrast, Yap1 inhibits chondrocyte maturation by suppression of Col10a1 expression through interaction with Runx2. In addition, Yap1 also governs the initiation of fracture repair by inhibition of cartilaginous callus tissue formation. Taken together, our work provides insights into the mechanism by which Yap1 regulates endochondral ossification, which may help the development of therapeutic treatment for bone regeneration

Functional Role of Mst1/Mst2 in Embryonic Stem Cell Differentiation

<div><p>The Hippo pathway is an evolutionary conserved pathway that involves cell proliferation, differentiation, apoptosis and organ size regulation. Mst1 and Mst2 are central components of this pathway that are essential for embryonic development, though their role in controlling embryonic stem cells (ES cells) has yet to be exploited. To further understand the Mst1/Mst2 function in ES cell pluripotency and differentiation, we derived <i>Mst1/Mst2</i> double knockout (<i>Mst-/-</i>) ES cells to completely perturb Hippo signaling. We found that <i>Mst-/-</i> ES cells express higher level of Nanog than wild type ES cells and show differentiation resistance after LIF withdrawal. They also proliferate faster than wild type ES cells. Although <i>Mst-/-</i> ES cells can form embryoid bodies (EBs), their differentiation into tissues of three germ layers is distorted. Intriguingly, <i>Mst-/-</i> ES cells are unable to form teratoma. <i>Mst-/-</i> ES cells can differentiate into mesoderm lineage, but further differentiation to cardiac lineage cells is significantly affected. Microarray analysis revealed that ligands of non-canonical Wnt signaling, which is critical for cardiac progenitor specification, are significantly repressed in <i>Mst-/-</i> EBs. Taken together our results showed that Mst1/Mst2 are required for proper cardiac lineage cell development and teratoma formation.</p> </div

Isolation of <i>Mst-/-</i> ES cells.

<p>(A) Genotyping of wild type (WT) ES cells and <i>Mst-/</i>- ES cells derived from blastocysts by PCR amplification of genomic DNA. Wild type ES cells showed a larger band while <i>Mst-/</i>- ES cells displayed a smaller band. <i>Actin</i> was used as an internal control. (B) Phase contrast microscopy of wild type (WT) and two independent <i>Mst-/</i>- knockout ES cell lines (<i>Mst-/-</i>1 and <i>Mst-/-</i>2) grown on 0.2% gelatin in 2i+LIF medium (Upper). These cells were stained for alkaline phosphatase (Lower). Scale bar, 200 μm. (C) mRNA level of <i>Mst1</i> and <i>Mst2</i> in wild type ES cells and <i>Mst-/</i>- ES cells examined by quantitative real-time PCR using primers flanking the deleted region of <i>Mst</i>1 and <i>Mst</i>2. The data are shown as the mean ± S.D (n=3). <i>Actin</i> was normalized as an internal control. Statistically significant differences are indicated (*, P<0.05; **, P<0.01; ***, P<0.001). (D) Immunoblotting analysis of the expression of Mst1 and Mst2 in wild type ES cells and <i>Mst-/</i>- ES cells. Gapdh1 was used as a loading control.</p

Characterization of Mst-/- ES cells.

<p>(A) Quantitative real-time PCR to examine the mRNA level of pluripotent markers <i>Pou5f1</i>, <i>Sox2</i> and <i>Nanog</i> in wild type ES cells and <i>Mst-/</i>- knockout ES cells. <i>Actin</i> was analyzed as an internal control. The data are shown as the mean ± S.D (n=3). Statistically significant differences are indicated (*, P<0.05; **, P<0.01; ***, P<0.001). (B) Immunofluorescence staining of the pluripotent protein Oct4 and SSEA1 expression in wild type ES cells and <i>Mst-/</i>- knockout ES cells. Neuclei were stained with DAPI. Scale bar, 200μm. (<i>C</i>) Immunoblotting and densitometric analysis of Nanog and Oct4 in wild type ES cells and <i>Mst-/</i>- ES cells. Gapdh1 was analyzed as an internal control. The data are shown as the mean ± S.D (n=2). Statistically significant differences are indicated (*, P<0.05; **, P<0.01; ***, P<0.001). (D) Immunoblotting and densitometric analysis of the expression of Yap and phosphorylated Yap (YapS127) in wild type ES cells and <i>Mst-/</i>- ES cells. Gapdh1 was analyzed as an internal control. The data are shown as the mean ± S.D (n=2). Statistically significant differences are indicated (*, P<0.05; **, P<0.01; ***, P<0.001).</p

<i>Mst-/-</i> ES cells proliferate faster than wild type ES cells.

<p>(A) Morphology of 1x10⁵ wild type ES cells or <i>Mst-/</i>- ES cells grown in 2i+LIF ES medium for 2 days, 3 days and 4 days respectively. Scale bar, 200 μm. (B) Statistical analysis of the growth rate of wild type ES cells and <i>Mst-/</i>- ES cells on day 3 and day 4 culture. The data were shown as the mean ± S.D (n=3). Statistically significant differences are indicated (*, P<0.05; **, P<0.01; ***, P<0.001). (C) Immunofluorescence staining with BrdU antibodies to examine BrdU integration in wild type ES cells and <i>Mst-/</i>- ES cells after serum starvation for 12 hours. Cells are pulsed labeled with BrdU for 45 minutes. The nuclei were stained with DAPI. Scale bar, 200 μm. (D) Representative histograms of cell cycle distribution in <i>Mst-/</i>- ES cells and wild type ES cells. (E) Table of the cell cycle distribution in <i>Mst-/</i>- ES cells and wild type ES cells from two independent experiments. (F) Statistical analysis of cell cycle distribution in <i>Mst-/</i>- ES cells and wild type ES cells from two independent experiments. (*, P<0.05).</p