Bone morphogenetic protein (BMP) and leukemia inhibitory factor (LIF)signaling both promote the differentiation of neural stem/progenitor cells into glial fibrillary acidic protein (GFAP) immunoreactive cells. This study compares the cellular and molecular characteristics, and the potentiality, of GFAP+ cells generated by these different signaling pathways. Treatment of cultured embryonic subventricular zone (SVZ) progenitor cells with LIF generates GFAP+ cells that have a bipolar/tripolar morphology, remain in cell cycle, contain progenitor cell markers and demonstrate self-renewal with enhanced neurogenesis - characteristics that are typical of adult SVZ and subgranular zone (SGZ) stem cells/astrocytes. By contrast, BMP-induced GFAP+ cells are stellate, exit the cell cycle, and lack progenitor traits and self-renewal - characteristics that are typical of astrocytes in the non-neurogenic adult cortex. In vivo, transgenic overexpression of BMP4 increases the number of GFAP+ astrocytes but depletes the GFAP+ progenitor cell pool, whereas transgenic inhibition of BMP signaling increases the size of the GFAP+progenitor cell pool but reduces the overall numbers of astrocytes. We conclude that LIF and BMP signaling generate different astrocytic cell types,and propose that these cells are, respectively, adult progenitor cells and mature astrocytes.

The hypothesis that BMPs (bone morphogenetic proteins), which act early in gut morphogenesis, also regulate specification and differentiation in the developing enteric nervous system (ENS) was tested. Expression of BMP-2 and BMP-4, BMPR-IA (BMP receptor subunit), BMPR-IB, and BMPR-II, and the BMP antagonists, noggin, gremlin, chordin, and follistatin was found when neurons first appear in the primordial bowel at embryonic day 12 (E12). Agonists, receptors, and antagonists were detected in separated populations of neural crestand noncrest-derived cells. When applied to immunopurified E12 ENS precursors, BMP-2 and BMP-4 induced nuclear translocation of phosphorylated Smad-1 (Sma and Mad-related protein). The number of neurons developing from these cells was increased by low concentrations and decreased by high concentrations of BMP-2 or BMP-4. BMPs induced the precocious appearance of TrkC-expressing neurons and their dependence on neurotrophin-3 for survival. BMP-4 interacted with glial cell line-derived neurotrophic factor (GDNF) to enhance neuronal development but limited GDNF-driven expansion of the precursor pool. BMPs also promoted development of smooth muscle from mesenchymal cells immunopurified at E12. To determine the physiological significance of these observations, the BMP antagonist noggin was overexpressed in the developing ENS of transgenic mice under the control of the neuron-specific enolase promoter. Neuronal numbers in both enteric plexuses and smooth muscle were increased throughout the postnatal small intestine. These increases were already apparent by E18. In contrast, TrkC-expressing neurons decreased in both plexuses of postnatal nogginoverexpressing animals, again an effect detectable at E18. BMP-2 and/or BMP-4 thus limit the size of the ENS but promote the development of specific subsets of enteric neurons, including those that express TrkC.

Heterotopic ossification (HO), the abnormal formation of true marrow-containing bone within extraskeletal soft tissues, is a serious bony disorder that may be either acquired or hereditary. We utilized an animal model of the genetic disorder fibrodysplasia ossificans progressiva to examine the cellular mechanisms underlying HO. We found that HO in these animals was triggered by soft tissue injuries and that the effects were mediated by macrophages. Spreading of HO beyond the initial injury site was mediated by an abnormal adaptive immune system. These observations suggest that dysregulation of local stem/progenitor cells could be a common cellular mechanism for typical HO irrespective of the signal initiating the bone formation. STEM CELLS 2009;27:150 -156 Disclosure of potential conflicts of interest is found at the end of this article.

Neural stem cells proliferate and maintain multipotency when cultured in the presence of FGF2, but subsequent lineage commitment by the cells is nevertheless influenced by the exposure to FGF2. Here we show that FGF2 effects on neural stem cells are mediated, in part, by beta-catenin. Conversely, the effects of beta-catenin in neural stem cells depend in part upon whether there is concurrent fibroblast growth factor (FGF) signaling. FGF2 increases beta-catenin signaling through several different mechanisms including increased expression of beta-catenin mRNA, increased nuclear translocation of beta-catenin, increased phosphorylation of GSK-3beta, and tyrosine phosphorylation of beta-catenin. Overexpression of beta-catenin in the presence of FGF2 helps to maintain neural progenitor cells in a proliferative state. However, overexpression of beta-catenin in the absence of FGF2 enhances neuronal differentiation. Further, chromatin immunoprecipitation (ChIP) assays demonstrate that both beta-catenin and Lef1 bind directly to the neurogenin promoter, and luciferase reporter assays demonstrate that beta-catenin is directly involved in the regulation of neurogenin 1 and possibly other proneural genes when neural stem cells are cultured in the presence of FGF2. We suggest that the balance between the mitogenic effects and the proneural effects of beta-catenin is determined by the presence of FGF signaling.

The effects of bone morphogenetic protein (BMP) signaling on enteric neuron development were examined in transgenic mice over expressing either the BMP inhibitor, noggin, or BMP4 under control of the neuron specific enolase (NSE) promoter. Noggin antagonism of BMP signaling increased total numbers of enteric neurons and those of subpopulations derived from precursors that exit the cell cycle early in neurogenesis (serotonin, calretinin, calbindin). In contrast, noggin overexpression decreased numbers of neurons derived from precursors that exit the cell cycle late (γ-aminobutyric acid, tyrosine hydroxylase [TH], dopamine transporter, calcitonin gene related peptide, TrkC). Numbers of TH-and TrkC-expressing neurons were increased by overexpression of BMP4. These observations are consistent with the idea that phenotypic expression in the enteric nervous system (ENS) is determined, in part, by the number of proliferative divisions neuronal precursors undergo before their terminal mitosis. BMP signaling may thus regulate enteric neuronal phenotypic diversity by promoting the exit of precursors from the cell cycle. BMP2 increased the numbers of TH-and TrkC-expressing neurons developing in vitro from immunoselected enteric crest-derived precursors; BMP signaling may thus also specify or promote the development of dopaminergic TrkC/NT-3-dependent neurons. The developmental defects in the ENS of noggin overexpressing mice caused a relatively mild disturbance of motility (irregular rapid transit and increased stool frequency, weight, and water content). Although the function of the gut thus displays a remarkable tolerance for ENS defects, subtle functional abnormalities in motility or secretion may arise when ENS defects short of aganglionosis occur during development.

We previously isolated and identified steroid receptor coactivator-1 (SRC-1) and peroxisome proliferator-activated receptor (PPAR)-binding protein (PBP/PPARBP) as coactivators for PPAR, using the ligand-binding domain of PPAR␥ as bait in a yeast two-hybrid screening. As part of our continuing effort to identify cofactors that influence the transcriptional activity of PPARs, we now report the isolation of a novel coactivator from mouse, designated PRIP (peroxisome proliferator-activated receptor interacting protein), a nuclear protein with 2068 amino acids and encoded by 13 exons. Northern analysis showed that PRIP mRNA is ubiquitously expressed in many tissues of adult mice. PRIP contains two LXXLL signature motifs. The amino-terminal LXXLL motif (amino acid position 892 to 896) of PRIP was found to be necessary for nuclear receptor interaction, but the second LXXLL motif (amino acid position 1496 to 1500) appeared unable to bind PPAR␥. Deletion of the last 12 amino acids from the carboxyl terminus of PPAR␥ resulted in the abolition of the interaction between PRIP and PPAR␥. PRIP also binds to PPAR␣, RAR␣, RXR␣, ER, and TR␤1, and this binding is increased in the presence of specific ligands. PRIP acts as a strong coactivator for PPAR␥ in the yeast and also potentiates the transcriptional activities of PPAR␥ and RXR␣ in mammalian cells. A truncated form of PRIP (amino acids 786 -1132) acts as a dominant-negative repressor, suggesting that PRIP is a genuine coactivator.

Culture of embryonic stem (ES) cells at high density inhibits bothβ-catenin signaling and neural differentiation. ES cell density does not influence β-catenin expression, but a greater proportion ofβ-catenin is targeted for degradation in high-density cultures. Moreover,in high-density cultures, β-catenin is preferentially localized to the membrane further reducing β-catenin signaling. Increasing β-catenin signaling by treatment with Wnt3a-conditioned medium, by overexpression ofβ-catenin, or by overexpression of a dominant-negative form of E-cadherin promotes neurogenesis. Furthermore, β-catenin signaling is sufficient to induce neurogenesis in high-density cultures even in the absence of retinoic acid (RA), although RA potentiates the effects of β-catenin. By contrast,RA does not induce neurogenesis in high-density cultures in the absence ofβ-catenin signaling. Truncation of the armadillo domain ofβ-catenin, but not the C terminus or the N terminus, eliminates its proneural effects. The proneural effects of β-catenin reflect enhanced lineage commitment rather than proliferation of neural progenitor cells. Neurons induced by β-catenin overexpression either alone or in association with RA express the caudal neuronal marker Hoxc4. However, RA treatment inhibits the β-catenin-mediated generation of tyrosine hydroxylase-positive neurons, suggesting that not all of the effects of RA are dependent upon β-catenin signaling. These observations suggest thatβ-catenin signaling promotes neural lineage commitment by ES cells, and that β-catenin signaling may be a necessary co-factor for RA-mediated neuronal differentiation. Further, enhancement of β-catenin signaling with RA treatment significantly increases the numbers of neurons generated from ES cells, thus suggesting a method for obtaining large numbers of neural species for possible use in for ES cell transplantation.

Fibrodysplasia ossificans progressiva (FOP) isFibrodysplasia ossificans progressiva (FOP), also known as myositis ossificans progressiva, is a autosomal dominantly inherited connective tissue disease characterized by progressive postnatal heterotopic ossification. The earliest pathological finding in FOP is perivascular lymphocytic infiltration into normal-appearing skeletal muscle, followed by muscle-cell degeneration and highly vascular fibroproliferative soft tissue swelling. The fibroproliferative lesions evolve, through an endochondral process, into mature lamellar bone with marrow elements. Heterotopic ossifications are usually first detected around the spine and proximal extremities, then at multiple other places, which leads to dysfunction of articulations and often premature death.