During growth, individual skull bones overlap at sutures, where osteoblast differentiation and bone deposition occur. Mutations causing skull malformations have revealed some required genes, but many aspects of suture regulation remain poorly understood. We describe a zebrafish mutation in osterix/sp7, which causes a generalized delay in osteoblast maturation. While most of the skeleton is patterned normally, mutants have specific defects in the anterior skull and upper jaw, and the top of the skull comprises a random mosaic of bones derived from individual initiation sites. Osteoblasts at the edges of the bones are highly proliferative and fail to differentiate, consistent with global changes in gene expression. We propose that signals from the bone itself are required for orderly recruitment of precursor cells and growth along the edges. The delay in bone maturation caused by loss of Sp7 leads to unregulated bone formation, revealing a new mechanism for patterning the skull and sutures.

An activating bone morphogenetic proteins (BMP) type I receptor ACVR1 (ACVR1R206H) mutation enhances BMP pathway signaling and causes the rare genetic disorder of heterotopic (extraskeletal) bone formation fibrodysplasia ossificans progressiva. Heterotopic ossification frequently occurs following injury as cells aberrantly differentiate during tissue repair. Biomechanical signals from the tissue microenvironment and cellular responses to these physical cues, such as stiffness and rigidity, are important determinants of cell differentiation and are modulated by BMP signaling. We used an Acvr1R206H/+ mouse model of injury-induced heterotopic ossification to examine the fibroproliferative tissue preceding heterotopic bone and identified pathologic stiffening at this stage of repair. In response to microenvironment stiffness, in vitro assays showed that Acvr1R206H/+ cells inappropriately sense their environment, responding to soft substrates with a spread morphology similar to wild-type cells on stiff substrates and to cells undergoing osteoblastogenesis. Increased activation of RhoA and its downstream effectors demonstrated increased mechanosignaling. Nuclear localization of the pro-osteoblastic factor RUNX2 on soft and stiff substrates suggests a predisposition to this cell fate. Our data support that increased BMP signaling in Acvr1R206H/+ cells alters the tissue microenvironment and results in misinterpretation of the tissue microenvironment through altered sensitivity to mechanical stimuli that lowers the threshold for commitment to chondro/osteogenic lineages.

No abstract

Fibrodysplasia ossificans progressiva (FOP) is a rare genetic disease characterized by the formation of extraskeletal bone, or heterotopic ossification (HO), in soft connective tissues such as skeletal muscle. All familial and sporadic cases with a classic clinical presentation of FOP carry a gain-of-function mutation (R206H; c.617 G > A) in ACVR1, a cell surface receptor that mediates bone morphogenetic protein (BMP) signaling. The BMP signaling pathway is recognized for its chondro/osteogenic-induction potential, and HO in FOP patients forms ectopic but qualitatively normal endochondral bone tissue through misdirected cell fate decisions by tissue-resident mesenchymal stem cells. In addition to biochemical ligand-receptor signaling, mechanical cues from the physical environment are transduced to activate intracellular signaling, a process known as mechanotransduction, and can influence cell fates. Utilizing an established mesenchymal stem cell model of mouse embryonic fibroblasts (MEFs) from the Acvr1 R206H/+ mouse model that mimics the human disease, we demonstrated that activation of the mechanotransductive effectors Rho/ROCK and YAP1 are increased in Acvr1 R206H/+ cells. We show that on softer substrates, a condition associated with low mechanical signaling, the morphology of Acvr1 R206H/+ cells is similar to the morphology of control Acvr1 +/+ cells on stiffer substrates, a condition that activates mechanotransduction. We further determined that Acvr1 R206H/+ cells are poised for osteogenic differentiation, expressing increased levels of chondro/osteogenic markers compared with Acvr1 +/+ cells. We also identified increased YAP1 nuclear localization in Acvr1 R206H/+ cells, which can be rescued by either BMP inhibition or Rho antagonism. Our results establish RhoA and YAP1 signaling as modulators of mechanotransduction in FOP and suggest that aberrant mechanical signals, combined with and as a result of the increased BMP pathway signaling through mutant ACVR1, lead to misinterpretation of the cellular microenvironment and a heightened sensitivity to mechanical stimuli that promotes commitment of Acvr1 R206H/+ progenitor cells to chondro/osteogenic lineages. Generation of mouse embryonic fibroblasts and cell cultureKnock-in Acvr1 R206H/+ MEFs were isolated at embryonic day 13.5 (E13.5) as previously described (43) from mice described in Chakkalakal and colleagues. (41) Genotypes of cell lines (MEFs) and presence of the R206H mutation were confirmed by PCR (primers in Supplemental Table S1). Acvr1 +/+ controls were littermates that did not contain an Acvr1 R206H allele. Cells were cultured in Dulbecco's modified Eagle's medium, high glucose (DMEM, Gibco, Thermo Fisher Scientific,

Fibrodysplasia ossificans progressiva (FOP) is a rare genetic disease in which extraskeletal (heterotopic) bone forms within tissues such as skeletal muscles, often in response to injury. Mutations in the BMP type I receptor ACVR1/ALK2 cause FOP by increasing BMP pathway signaling. In contrast to the growing understanding of the inappropriate formation of bone tissue within the muscle in FOP, much is still unknown about the regenerative capacity of adult diseased muscles. Utilizing an inducible ACVR1R206H knock-in mouse, we found that injured Acvr1R206H/+ skeletal muscle tissue regenerates poorly. We demonstrated that while two resident stem cell populations, muscle stem cells (MuSCs) and fibro/adipogenic progenitors (FAPs), have similar proliferation rates after injury, the differentiation potential of mutant MuSCs is compromised. Although MuSC-specific deletion of the ACVR1R206H mutation does not alter the regenerative potential of skeletal muscles in vivo, Acvr1R206H/+ MuSCs form underdeveloped fibers that fail to fuse in vitro. We further determined that FAPs from Acvr1R206H/+ mice repress the MuSC-mediated formation of Acvr1R206H/+ myotubes in vitro. These results identify a previously unrecognized role for ACVR1R206H in myogenesis in FOP, via improper interaction of tissue-resident stem cells during skeletal muscle regeneration.

SUMMARY WNT signaling promotes the reprogramming of somatic cells to an induced pluripotent state. We provide genetic evidence that WNT signaling is a requisite step during the induction of pluripotency. Fibroblasts from individuals with Focal Dermal Hypoplasia (FDH), a rare genetic syndrome caused by mutations in the essential WNT processing enzyme PORCN, fail to reprogram using standard methods. This blockade in reprogramming is overcome by ectopic WNT signaling and by PORCN overexpression, thus demonstrating that WNT signaling is essential for reprogramming. The rescue of reprogramming is critically dependent on the level of WNT signaling: steady baseline activation of the WNT pathway yields karyotypically normal iPS cells, whereas daily stimulation with Wnt3a produces FDH-iPS cells with severely abnormal karyotypes. Therefore, although WNT signaling is required for cellular reprogramming, inappropriate activation of WNT signaling induces chromosomal instability, highlighting the precarious nature of ectopic WNT activation, and its tight relationship with oncogenic transformation.

No abstract

While current thinking posits that insulin signaling to GLUT4 exocytic translocation and glucose uptake in skeletal muscle and adipocytes is controlled by phosphorylation-based signaling, many proteins in this pathway are acetylated on lysine residues. However, the importance of acetylation and lysine acetyltransferases to insulin-stimulated glucose uptake is incompletely defined. Here, we demonstrate that combined loss of the acetyltransferases E1A binding protein p300 (p300) and cAMP response element binding protein binding protein (CBP) in mouse skeletal muscle causes a complete loss of insulinstimulated glucose uptake. Similarly, brief (i.e. 1 h) pharmacological inhibition of p300/CBP acetyltransferase activity recapitulates this phenotype in human and rodent myotubes, 3T3-L1 adipocytes, and mouse muscle. Mechanistically, these effects are due to p300/CBP-mediated regulation of GLUT4 exocytic translocation and occurs downstream of Akt signaling. Taken together, we highlight a fundamental role for acetylation and p300/CBP in the direct regulation of insulin-stimulated glucose transport in skeletal muscle and adipocytes.