1. Introduction. 1 Page 1

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1 1. INTRODUCTION Osteoblast differentiation is the key component of the bone formation, growth and remodeling. During this incompletely understood process a subpopulation of multipotential mesenchymal stem cells (MSCs) undergoes osteoblast lineage commitment and matures through a series of well-orchestrated differentiation steps (Mödder and Khosla, 2008; Kassem et al., 2008). Each step involves distinct changes in the expression of bone-related genes and transcription factors accompanied by a specific phenotypic (morphological and functional) alteration of corresponding cells. In response to coordinate interaction among diverse paracrine, autocrine and endocrine signals the induced osteoprogenitor cells first proliferate, then secrete proteinaceous extracellular matrix (osteoid) that will then progressively mineralize. By the end of osteoblast lineage differentiation (paralleled by the process of bone formation and growth) some of the cells become completely buried in mineralized bone matrix while others persist on a bone surface as quiescent osteoblasts or bone lining cells. The osteocytes, cells engulfed in mineralized bone matrix, represent the terminal differentiation stage of osteoblast lineage (Bonewald, 2011). They are the most abundant and longest-living cellular component of mature mammalian bones constituting as much as 95% of bone cells that cover 94% of all bone surfaces (Frost, 1960; Marotti, 1996). Although classically being viewed as passive cellular elements entrapped in their cage-like lacunae, osteocytes are actually multifunctional cells and one of the major regulators of bone metabolism. They are able to modify their local environment and have important roles in maintaining structural and functional integrity of bone tissue and adaptation to various mechanical or chemical stimuli impose on it (Bonewald, 2011). Due to their overall morphology, regular distribution throughout the bone matrix and high degree of interconnectivity and communications (through a delicate meshwork of their dendritic cellular processes) with other cells of osteoblast lineage as well as with osteoclasts and bone lining cells, osteocytes are ideally suited to act as a so called "bone nerve cells". They represent key responders to various signaling molecules and local factors that initiate bone formation and remodeling executed by osteoblasts and osteoclasts (Marotti, 2000). Furthermore, increasing amount of experimental data suggest that they act as mechanosensors that register spatial changes in intensity and distribution of mechanical strain and translate these impulses into biochemical signals providing yet another regulatory signaling network 1 Page 1

2 that coordinates the processes of bone turnover and promotes the adaptation of osteoprogenitor lineage to a new conditions (Chen at al., 2010; Jacobs et al., 2010). Comprehensive analysis of gene expression patterns and elucidation of spatiotemporal genetic cascades involved in commitment, differentiation and functional maturation of osteogenic cells responsible for bone development, growth and remodeling is a prerequisite not only for thorough deciphering of the multiple factors that control these processes at a molecular level. It also has the crucial role for better understanding of physiological bone structure and function and for the development of more suitable therapeutic approaches to various debilitating bone-related diseases and inherited disorders affecting the skeleton. Although the recent advances in molecular and genetic studies have enabled a better understanding of some of the key molecular players, regulatory networks, signaling events as well as humoral and transcriptional factors that control the establishment and maintenance of differentiation and maturation of osteoblast cell lineage, our current picture of this process is still quite partial and elusive. There is especially limited knowledge about the underlying gene expression pattern that is responsible for the terminal osteoblast to osteocytes transition (osteocytogenesis), determination of the osteocytes morphology and regulation of their important multifaceted cellular functions. Up to now, osteogenic commitment of multipotential MSCs, lineage progression, differentiation and function of mature bone cells has been studied in various genetically modified animal models, avian, rodent and human derived bone tissue, and numerous in vitro cell lines and primary cell cultures (Kartsogiannis and Wah Ng, 2004). The most extensively studied osteogenic primary cell cultures are murine derived calvarial bone cells obtained by successive enzyme digestion of neonatal bone tissue. Primarily, the attention has been given to the expression of specific gene products, but up to now only a handful of them have been fully characterized within the in vivo context of osteoblast lineage differentiation. Obtaining a comprehensive view of the spatiotemporal changes in gene expression profile as osteoblasts differentiate to their mature phenotype (osteocytes and bone lining cells) was not feasible until recently with the advent of microarray technology and the use of other genomewide approaches. However, the variety of cell sources and in vitro models, and the use of different array platforms that are also accompanied with various methods of microarray data analysis 1 Page 2

3 underscore a potential pitfalls and difficulties with comparing results and extracting the biologically meaningful and generally applicable informations from the so far published studies that are using this approach. The limited numbers of cells that become mature osteoblasts and/or osteocytes after osteogenic induction (factors used to stimulate matrix production and mineralization) of isolated osteogenic cells and the heterogeneity of obtained primary cultures represent additional obstacles in their analysis. Moreover, used osteogenic inducers (e.g. ascorbic acid, beta-glycerophosphate, and dexamethasone used for induction of calvarial cell cultures) have specific and often diverse modulatory effects on gene expression profile of targeted cells. That complicates the possible distinction between the specific and unspecific effects caused by inducers and changes caused solely by physiological process of osteoblast lineage differentiation. In addition, utilization of heterogeneous in vitro systems makes unclear whether the observed changes in gene expression profile originate mainly from a population of fully differentiated osteoblasts and/or osteocytes or from other cell populations present in the analyzed samples. Furthermore, the results obtained through in vitro studies represent only an approximation of changes occurring in the true physiological setting of isolated cells and have to be confirmed in appropriate in vivo models. Unfortunately, the inaccessibility of the mature bone forming cells due to the stony hard and highly mineralized nature of bone tissue, its inherent cellular heterogeneity and the lack of appropriate molecular and cell surface markers able to identify and distinguish terminal stages of osteoblast lineage prevents the isolation of homogeneous cell populations. Therefore, the experiments performed on enzymatically-isolated osteoblasts and osteocytes still have to deal with substantial heterogeneity of obtained primary cultures. That obscures the underlining gene expression profiles, complicates the interpretation of microarray studies design to identify genes associated with terminal stages of osteoblast lineage differentiation and hinders the attempts to gain a comprehensive understanding of their biology. To overcome the aforementioned obstacles mature osteoblasts and osteocytes cells have to be clearly identified and distinguish from the other, heterogeneous cell population of murine neonatal calvarial or other targeted bone tissue, uncontaminated with other cell fractions (various pre-osteoblast cell stages and non-osteoblast lineage cells). Selective identification and differentiation of terminal stages of osteoblast lineage may be performed by utilization of recently developed and characterized specific green fluorescence 1 Page 3

4 protein (GFP) based bone-directed visual transgenic markers that drive GFP expression in specific subpopulations of the osteoblast lineage (Rowe, 2005). The GFP-expressing cells present either in heterogeneous osteogenically-induced transgenic in vitro cell lines or enzymatically released from targeted bone tissue of transgenic animal models may be subsequently successfully phenotypized sorted from other GFP negative cells, and harvested as more homogeneous cell populations that are more appropriate for subsequent usage in microarray gene expression studies or some other experimental approaches necessitating the homogeneous cell samples (Kalajzic et al., 2005) OBJECTIVE AND GOALS OF THE STUDY In this PhD study terminal stages of osteoblast lineage differentiation were obtained through a novel transgenic approach using dual GFP-reporter mice in which mature osteoblast and osteocyte cells were identified by expression of different, cell-type specific transgenic GFPvariants. That allowed their clear distinction and separation as more homogeneous cells population obtained by fluorescence activating cell sorting (FACS) of enzymatically digested neonatal calvarial bone tissue. Utilization of this novel experimental approach in isolation of mature osteoblasts and osteocytes combined with microarray technology and bioinformatics analysis of obtained gene expression data allowed us to define their distinct, cell type specific expression profiles representative of their true in vivo settings SPECIFIC AIMS 1. Generation of experimental double-transgenic mice and selective identification of terminal cell stages (osteoblasts and osteocytes) of osteoblast lineage differentiation That was achieved through a novel transgenic approach generating a dual GFPreporter mouse (pobcol2.3gfpcyan/dmp1gfptopaz) characterized by expression of previously developed and characterized bone-directed promoter-gfp transgenic constructs. Osteocytes were detected by a GFP (topaz) fluorescent marker directed by the dentin matrix protein 1 (DMP1) promoter, while osteoblasts were identified by expression of GFP (cyan) fluorescence marker driven by 2.3 kilo base (kb) fragment from the type I collagen gene (Col1a1) promoter. 1 Page 4

5 Histological evaluation of cell type specific expression of used GFP-variants was performed on a longitudinal cryosections of calvarial bones isolated from neonatal mice harboring dual GFP-reporter constructs. 2. Isolation of pure population of osteoblasts and osteocytes from neonatal murine calvarial bone tissue uncontaminated with other cell fractions (various preosteoblast and non-osteoblast lineage cells). Enzymatically released calvarial cells of neonatal double GFP-transgenic mice were subjected to flow cytometry phenotypization, cell sorting and samples preparation using FACS-Vantage BD fluorescence activated sell sorting machine. 3. Identification of differentially expressed genes in osteoblasts and osteocytes by genome-wide expression analysis of murine calvarial tissue Comprehensive analysis of gene expression profiles of homogeneous cell populations of osteoblasts and osteocytes and heterogeneous population of GFP negative cells obtained from neonatal murine calvarial bone tissue was performed by Mouse-WG6 v.1 expression BeadChip arrays using Illumina microarray platform. Analysis, filtering (presence/absence call), normalization and annotation of microarray scan data were performed using Illumina BeadStudio microarray analysis software (lumi and nulid package) followed by selection of differentially expressed genes performed with SAM (Significance Analysis of Microarray data) and LIMMA (Linear Modeling of Microarray data Analysis) statistical methods for data analysis. 4. Bioinformatic analysis of differentially expressed genes and identification of genes that define some specific morphological and functional aspects of osteocytes in their true in vivo settings. Bioinformatic analysis and characterization of gene expression patterns between osteocytes and osteoblasts were performed using a proprietary FlowGram bioinformatic software for the analysis of microarray data [developed by Dr. Don-Guk Shin at Department of Computer Science, University of Connecticut, Storrs, Connecticut, USA] and the open web based DAVID Bioinformatic resource 2008 [The Database for Annotation, Visualization and Integrated Discovery (DAVID ) v6.7] used to explore pathways, gene ontology (GO) and other functional classification and annotation tools in order to identify significantly up-regulated or down-regulated osteocytes genes (compared to osteoblasts gene expression profile) 1 Page 5

6 that are significantly enriched in specific biological pathways, GO terms or functional classes. 5. Real time PCR analysis for evaluation of obtained microarray expression data was performed on a subset of differentially expressed genes using a TaqMan Gene Expression Assay WORKING HYPOTHESIS Rationale: Each step of osteoblast lineage differentiation involves distinct changes in the expression of bone-related genes and transcription factors that are accompanied by a specific phenotypic (morphological and functional) alteration of corresponding cells. Osteocytes, the terminal differentiation stage of osteoblast lineage have unique gene expression profile which determines their overall morphology and governs their important, multifaceted cellular functions. Hypothesis: Identification of differential gene expression pattern of adequately isolated pure population of osteocytes versus homogeneous osteoblast cell population and a heterogeneous cell population composed of different preosteoblast cell stages and other bone-related cells of various nonosteoblast lineages present in the neonatal murine calvarial bone tissue will reveal the genes that are specific to the osteocyte phenotype in its true in vivo settings. 1 Page 6