Andrew T. Dudley

New role of bone morphogenetic protein 7 in brown adipogenesis and energy expenditure

Adipose tissue is central to the regulation of energy balance. Two functionally different types of fat are present in mammals: white adipose tissue, the primary site of triglyceride storage, and brown adipose tissue, which is specialized in energy expenditure and can counteract obesity. Factors that specify the developmental fate and function of white and brown adipose tissue remain poorly understood. Here we demonstrate that whereas some members of the family of bone morphogenetic proteins (BMPs) support white adipocyte differentiation, BMP7 singularly promotes differentiation of brown preadipocytes even in the absence of the normally required hormonal induction cocktail. BMP7 activates a full program of brown adipogenesis including induction of early regulators of brown fat fate PRDM16 (PR-domain-containing 16; ref. 4) and PGC-1α (peroxisome proliferator-activated receptor-γ (PPARγ) coactivator-1α; ref. 5), increased expression of the brown-fat-defining marker uncoupling protein 1 (UCP1) and adipogenic transcription factors PPARγ and CCAAT/enhancer-binding proteins (C/EBPs), and induction of mitochondrial biogenesis via p38 mitogen-activated protein (MAP) kinase-(also known as Mapk14) and PGC-1-dependent pathways. Moreover, BMP7 triggers commitment of mesenchymal progenitor cells to a brown adipocyte lineage, and implantation of these cells into nude mice results in development of adipose tissue containing mostly brown adipocytes. Bmp7 knockout embryos show a marked paucity of brown fat and an almost complete absence of UCP1. Adenoviral-mediated expression of BMP7 in mice results in a significant increase in brown, but not white, fat mass and leads to an increase in energy expenditure and a reduction in weight gain. These data reveal an important role of BMP7 in promoting brown adipocyte differentiation and thermogenesis in vivo and in vitro, and provide a potential new therapeutic approach for the treatment of obesity.

Convergent extension movements in growth plate chondrocytes require gpi-anchored cell surface proteins

Proteins that are localized to the cell surface via glycosylphosphatidylinositol (gpi) anchors have been proposed to regulate cell signaling and cell adhesion events involved in tissue patterning. Conditional deletion of Piga, which encodes the catalytic subunit of an essential enzyme in the gpi-biosynthetic pathway, in the lateral plate mesoderm results in normally patterned limbs that display chondrodysplasia. Analysis of mutant and mosaic Piga cartilage revealed two independent cell autonomous defects. First, loss of Piga function interferes with signal reception by chondrocytes as evidenced by delayed maturation. Second, the proliferative chondrocytes, although present, fail to flatten and arrange into columns. We present evidence that the abnormal organization of mutant proliferative chondrocytes results from errors in cell intercalation. Collectively, our data suggest that the distinct morphological features of the proliferative chondrocytes result from a convergent extension-like process that is regulated independently of chondrocyte maturation.

Calcium/calmodulin-dependent protein kinase II activity regulates the proliferative potential of growth plate chondrocytes

For tissues that develop throughout embryogenesis and into postnatal life, the generation of differentiated cells to promote tissue growth is at odds with the requirement to maintain the stem cell/progenitor cell population to preserve future growth potential. In the growth plate cartilage, this balance is achieved in part by establishing a proliferative phase that amplifies the number of progenitor cells prior to terminal differentiation into hypertrophic chondrocytes. Here, we show that endogenous calcium/calmodulin-dependent protein kinase II (CamkII, also known as Camk2) activity is upregulated prior to hypertrophy and that loss of CamkII function substantially blocks the transition from proliferation to hypertrophy. Wnt signaling and Pthrp-induced phosphatase activity negatively regulate CamkII activity. Release of this repression results in activation of multiple effector pathways, including Runx2- and β-catenin-dependent pathways. We present an integrated model for the regulation of proliferation potential by CamkII activity that has important implications for studies of growth control and adult progenitor/stem cell populations.

Role of Mammalian Ecdysoneless in Cell Cycle Regulation

The Ecdysoneless (Ecd) protein is required for cell-autonomous roles in development and oogenesis in Drosophila, but the function of its evolutionarily conserved mammalian orthologs is not clear. To study the cellular function of Ecd in mammalian cells, we generated Ecdlox/lox mouse embryonic fibroblast cells from Ecd floxed mouse embryos. Cre-mediated deletion of Ecd in Ecdlox/lox mouse embryonic fibroblasts led to a proliferative block due to a delay in G1-S cell cycle progression; this defect was reversed by the introduction of human Ecd. Loss of Ecd led to marked down-regulation of E2F target gene expression. Furthermore, Ecd directly bound to Rb at the pocket domain and competed with E2F for binding to hypophosphorylated Rb. Our results demonstrate that mammalian Ecd plays a role in cell cycle progression via the Rb-E2F pathway.

Cell polarity: The missing link in skeletal morphogenesis?

Despite extensive genetic analysis of the dynamic multi-phase process that transforms a small population of lateral plate mesoderm into the mature limb skeleton, the mechanisms by which signaling pathways regulate cellular behaviors to generate morphogenetic forces are not known. Recently, a series of papers have offered the intriguing possibility that regulated cell polarity fine-tunes the morphogenetic process via orienting cell axes, division planes and cell movements. Wnt5a-mediated non-canonical signaling, which may include planar cell polarity, has emerged as a common thread in the otherwise distinct signaling networks that regulate morphogenesis in each phase of limb development. These findings position the limb as a key model to elucidate how global tissue patterning pathways direct local differences in cell behavior that, in turn, generate growth and form.

A re-evaluation of two key reagents for in vivo studies of Wnt signaling.

Conditional mutations and transcription‐based reporters are important new tools for exploring the dynamic functions of biological pathways in vivo. While studying the role of the Wnt signaling pathway in cartilage, we observed that the β‐catenin‐dependent reporter TOPGAL was expressed in chondrocytes in which β‐catenin was conditionally inactivated using a Col2a1::cre driver. Here we show that in these embryos recombination is complete and full‐length β‐catenin protein is absent in chondrocytes. Although a null allele in this context, the recombined β‐catenin locus produces a stable transcript that encodes a truncated protein. The truncated protein alone fails to activate TOPFLASH, but strongly potentiates reporter activity in the presence of expressed β‐catenin or Tcf4. Together, these data show that each mouse model exhibits specific undesirable properties, findings that strongly suggest the need for specific standards to ensure proper validation of this new generation of genetic tools. Developmental Dynamics 240:2060–2068, 2011.

Chemical Pretreatment of Growth Plate Cartilage Increases Immunofluorescence Sensitivity

Immunofluorescence detection of proteins in growth plate cartilage is often unsuccessful because of innate autofluorescence, fixative-induced fluorescence, and dense cartilage matrix, which can inhibit antibody penetration. To overcome these limitations, the authors have tested various chemical pretreatments, including the autofluorescence quencher sodium borohydride, the antigen retrieval method of boiling sodium citrate, sugar-degrading enzymes (hyaluronidase, heparinase, and chondroitinase), and the proteolytic enzyme protease XXIV. Here the authors show that, in most cases, background fluorescence in cartilage is the primary obstacle to high-quality imaging. Blocking intrinsic fluorescence of the specimen in combination with specific pretreatments allows visualization using antibodies that previously did not generate a robust signal in the growth plate. Each antibody requires a specific combination of chemical pretreatments that must be empirically determined to achieve optimal staining levels. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.

A Tunable, Three-Dimensional In Vitro Culture Model of Growth Plate Cartilage Using Alginate Hydrogel Scaffolds

Defining the final size and geometry of engineered tissues through precise control of the scalar and vector components of tissue growth is a necessary benchmark for regenerative medicine, but it has proved to be a significant challenge for tissue engineers. The growth plate cartilage that promotes elongation of the long bones is a good model system for studying morphogenetic mechanisms because cartilage is composed of a single cell type, the chondrocyte; chondrocytes are readily maintained in culture; and growth trajectory is predominately in a single vector. In this cartilage, growth is generated via a differentiation program that is spatially and temporally regulated by an interconnected network composed of long- and short-range signaling mechanisms that together result in the formation of functionally distinct cellular zones. To facilitate investigation of the mechanisms underlying anisotropic growth, we developed an in vitro model of the growth plate cartilage by using neonatal mouse growth plate chondrocytes encapsulated in alginate hydrogel beads. In bead cultures, encapsulated chondrocytes showed high viability, cartilage matrix deposition, low levels of chondrocyte hypertrophy, and a progressive increase in cell proliferation over 7 days in culture. Exogenous factors were used to test functionality of the parathyroid-related protein-Indian hedgehog (PTHrP-IHH) signaling interaction, which is a crucial feedback loop for regulation of growth. Consistent with in vivo observations, exogenous PTHrP stimulated cell proliferation and inhibited hypertrophy, whereas IHH signaling stimulated chondrocyte hypertrophy. Importantly, the treatment of alginate bead cultures with IHH or thyroxine resulted in formation of a discrete domain of hypertrophic cells that mimics tissue architecture of native growth plate cartilage. Together, these studies are the first demonstration of a tunable in vitro system to model the signaling network interactions that are required to induce zonal architecture in growth plate chondrocytes, which could also potentially be used to grow cartilage cultures of specific geometries to meet personalized patient needs.