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There is growing recognition that the organization of nucleic acids and regulatory proteins is functionally linked to the assembly, localization and activity of gene regulatory machinery. Cellular, molecular, biochemical and in-vivo genetic evidence support an obligatory relationship between nuclear microenvironments where regulatory complexes reside and fidelity of transcriptional control. Perturbations in mechanisms governing the intranuclear trafficking of transcription factors and the temporal/spatial organization of regulatory proteins within the nucleus occur with compromised gene expression that abrogates skeletal development and mediates leukemogenesis.

Acute leukemias arise secondary to chromosomal aberrations that cause dysfunctions in gene regulation and regulatory factors. Significant differences in morphology between acute leukemic and nonleukemic hematopoietic cells are readily observed. How morphologic changes of the nuclei of acute leukemic cells relate to the underlying functional alterations of gene expression is minimally understood. Spatial modifications in the representation and/or organization of regulatory factors may be functionally linked to perturbations of gene expression in acute leukemic cells. Using in situ immunofluorescence microscopy, we addressed the interrelationships of modifications in nuclear morphology with the intranuclear distribution of leukemia-related regulatory factors (including ALL-1, PML, and AF-9) in cells from patients with acute leukemia. We compared the localization of leukemia-associated proteins with various factors involved in gene transcription and RNA processing (e.g., RNA polymerase II and SC-35). Our findings suggest that there are leukemia-associated aberrations in mechanisms that direct regulatory factors to sites within the nucleus. This misplacement of key cognate factors may contribute to perturbations in gene expression characteristic of leukemias.

Evidence is accruing that the architectural organization of nucleic acids and regulatory proteins within the cell nucleus support functional interrelationships between nuclear structure and gene expression. The punctate distribution of several transcription factors has provided several paradigms for pursuing mechanisms that direct these regulatory proteins to subnuclear sites where gene activation or suppression occurs. Sequences that are necessary and sufficient to direct regulatory proteins to transcriptionally active nuclear domains have been identified. Mutations that disrupt intranuclear targeting signals lead to modified subnuclear distribution of transcription factors and aberrant expression in tumor cells. J. Cell. Biochem. Suppls. 32/33:24-31, 1999.

Functional interrelationships between components of nuclear architecture and control of gene expression are becoming increasingly evident. There is growing appreciation that multiple levels of nuclear organization integrate the regulatory cues that support activation and suppression of genes as well as the processing of gene transcripts. The linear organization of genes and promoter elements provide the potential for responsiveness to physiological regulatory signals. Parameters of chromatin structure and nucleosome organization support synergism between activities at independent regulatory sequences and render promoter elements accessible or refractory to transcription factors. Association of genes, transcription factors, and the machinery for transcript processing with the nuclear matrix facilitates fidelity of gene expression within the three-dimensional context of nuclear architecture. Mechanisms must be defined that couple nuclear morphology with enzymatic parameters of gene expression. The recent characterization of factors that mediate chromatin remodeling and intranuclear targeting signals that direct transcription factors to subnuclear domains where gene expression occurs, reflect linkage of genetic and structural components of transcriptional control. Nuclear reorganization and aberrant intranuclear trafficking of transcription factors for developmental and tissue-specific control that occurs in tumor cells and in neurological disorders provides a basis for high resolution diagnostics and targeted therapy. J. Cell. Biochem. Suppls. 30/31:220-231, 1998. © 1998 Wiley-Liss, Inc.

Fetal rat calvarial-derived osteoblasts, in vitro, undergo a developmental sequence of events leading to bone tissue-like organization and osteoblast differentiation. Previous studies have documented temporal expression of genes reflecting stages of osteoblast phenotype development in relation to tissue organization. Two steroid hormones are known to modify the developmental sequence; 1,25(OH)2D3 can block differentiation when added to proliferating cells, while glucocorticoid addition to proliferating cultures increases the population of cells competent to produce a bone-like matrix and accelerates the differentiation time course. We have addressed the mechanisms contributing to these observations at the single cell level by analysis of a growth-related gene (H4 histone which is coupled with DNA synthesis) and matrix-associated genes (collagen, osteopontin, and osteocalcin) in hormone-treated cells. Our results demonstrate (1) a window of responsiveness for modifications in phenotype development; (2) distinct morphological changes and selective modifications in gene expression in response to both hormones as a function of whether the cell is proliferating or differentiated; and (3) location of the cell with respect to the mineralized nodule was a contributing factor to the levels of gene expression and hormonal responses. In response to vitamin D, surface osteoblasts associated with the nodules became flattened, elongated, and aligned, reminiscent of a bone lining cell. In glucocorticoid-treated cultures, proliferating cells became cuboidal and nodule-associated differentiated cells were approximately one-third the size of control osteoblasts. We also find subsets of hormone-responsive cells in the proliferating cultures in response to glucocorticoid but not vitamin D. In postproliferative cultures, both hormones increased osteocalcin mRNA in the more differentiated osteoblasts associated with the mineralized matrix but no induction occurred in monolayer internodular cells. Osteopontin was induced by glucocorticoid in a larger population of cells. Thus, our studies at the single cell level show selective morphological changes and changes in the level of gene expression supporting the hypothesis that hormones have differential effects on osteoblasts in relation to their stage of phenotype development.

Nuclease hypersensitive sites were mapped in the proximal promoter of the osteocalcin gene, which is expressed only in bone cells exhibiting the mature osteoblast phenotype. Nuclei from proliferating and confluent rat osteosarcoma (ROS) 17/2.8 cells were subjected to DNase I digestion, and hypersensitivity was assayed by the indirect end-labeling method, using osteocalcin gene probes. Hypersensitive sites were detected in two promoter domains: -590 to -390, which spans the vitamin D responsive element, and -170 to -70, which spans the TATA box and the CCAAT-containing OC box domain. Together, these elements regulate basal and vitamin D enhanced osteocalcin gene transcription. We observed a parallel relationship between the intensity of bands representing the hypersensitive sites and the extent to which the osteocalcin gene is transcribed. Both in confluent cultures and in response to vitamin D, when osteocalcin transcription was upregulated, the hypersensitive bands were significantly intensified. Additionally, the bands were decreased under conditions that downregulate osteocalcin gene transcription. A functional relationship between the presence of hypersensitive sites and osteocalcin gene transcription is further supported by the absence of hypersensitivity in nonosseous cells that do not express osteocalcin, although these proliferating cells exhibited hypersensitivity in a cell cycle regulated histone gene promoter. Our results suggest the involvement of chromatin structure in transcriptional responsiveness of the osteocalcin gene to physiologic modulation.

In order to investigate the ultrastructure of bone resorptive cells in the two types of adult benign human osteopetrosis, iliac crest biopsies were obtained from 11 patients and 10 normal males, who served as a control group. Six patients had the radiological type I (4 women, 2 men, aged 23-58 years, mean = 36.5 years), and 5 type II disease (5 men, aged 20-48 years, mean = 29.8 years). The normal controls (aged 23-48 years, mean 34.1 years) were recruited from the medical staff. The biopsies were immediately divided. From each patients, half was embedded in paraffin for histochemistry and light microscopy, and half in epon for transmission electron microscopy. The osteoclasts were markedly reduced in number and size in Type I disease (0.2 +/- .7 cells vs. 2.9 +/- 1.0 cells per 2.7 mm2 of bone area, p < 0.01) compared to controls, and stained only weakly for tartrate-resistant acid phosphatase (TRAP). At the ultrastructural level, no signs of active bone resorption were identified, whereas numerous mononuclear cells were observed at the bone surfaces. In type II disease, the osteoclasts were large and highly multi-nucleated, with an increased number (8.3 +/- 2.3 cells vs. 2.9 +/- 1.0. cells per 2.7 mm2 of bone area, p < 0.01) compared to controls. In all patients with this type, but never in type I or in the controls, a smooth, TRAP-positive substance was seen between the osteoclasts and the bone surface. Ultrastructurally, this substance was amorphous, with a condensation along the cell membrane.(ABSTRACT TRUNCATED AT 250 WORDS)

The nuclear matrix bound transcription factor RUNX2 is a lineage-specific developmental regulator that is linked to cancer. We have previously shown that RUNX2 controls transcription of both RNA polymerase II genes and RNA polymerase I-dependent ribosomal RNA genes. RUNX2 is epigenetically retained through mitosis on both classes of target genes in condensed chromosomes. We have used fluorescence recovery after photobleaching to measure the relative binding kinetics of enhanced green fluorescent protein (EGFP)-RUNX2 at transcription sites in the nucleus and nucleoli during interphase, as well as on mitotic chromosomes. RUNX2 becomes more strongly bound as cells go from interphase through prophase, with a doubling of the most tightly bound "immobile fraction." RUNX2 exchange then becomes much more facile during metaphase to telophase. During interphase the less tightly bound pool of RUNX2 exchanges more slowly at nucleoli than at subnuclear foci, and the non-exchanging immobile fraction is greater in nucleoli. These results are consistent with a model in which the molecular mechanism of RUNX2 binding is different at protein-coding and ribosomal RNA genes. The binding interactions of RUNX2 change as cells go through mitosis, with binding affinity increasing as chromosomes condense and then decreasing through subsequent mitotic phases. The increased binding affinity of RUNX2 at mitotic chromosomes may reflect its epigenetic function in "bookmarking" of target genes in cancer cells.

Nuclear microenvironments are architecturally organized subnuclear sites where the regulatory machinery for gene expression, replication, and repair resides. This compartmentalization is necessary to attain required stoichiometry for organization and assembly of regulatory complexes for combinatorial control. Combined and methodical application of molecular, cellular, biochemical, and in vivo genetic approaches is required to fully understand complexities of biological control. Here we provide methodologies to characterize nuclear organization of regulatory machinery by in situ immunofluorescence microscopy.

An in vitro model system is described, using metatarsal explants from 15-day mouse embryos (E15) cultured in serumless chemically defined medium, to study fetal skeletal development with particular emphasis on de novo osteoclast formation. The normal pattern of growth and differentiation observed in vitro, assessed by ultrastructure and morphometry, demonstrate a permissive local environment which replicates physiologic temporal and spatial relationships which exist in vivo. The population of committed osteoclast progenitors present in E15 metatarsals form osteoclasts and precursors which have cytochemical and ultrastructural features, as well as kinetics of formation, that are similar to that which occurs in vivo. The responsiveness of osteoclast formation to the effects of added 1,25(OH)2D3 illustrates that controlled manipulation enables one to exploit the system for investigating the role of cytokines, growth factors and osteotropic hormones in skeletal development and osteoclast ontogeny.

Regulatory machinery for gene expression, replication, and repair are architecturally organized in nuclear microenvironments. This compartmentalization provides threshold concentrations of macromolecules for the organization and assembly of regulatory complexes for combinatorial control. A mechanistic under standing of biological control requires the combined application of molecular, cellular, biochemical, and in vivo genetic approaches. This chapter provides methodologies to characterize nuclear organization of regulatory machinery by in situ immunofluorescence microscopy.

Regulatory machinery for gene expression, replication, and repair are architecturally organized in nuclear microenvironments. This compartmentalization provides threshold concentrations of macromolecules for the organization and assembly of regulatory complexes for combinatorial control. A mechanistic under standing of biological control requires the combined application of molecular, cellular, biochemical, and in vivo genetic approaches. This chapter provides methodologies to characterize nuclear organization of regulatory machinery by in situ immunofluorescence microscopy.

The relationship of cell proliferation to the temporal expression of genes characterizing a developmental sequence associated with bone cell differentiation was examined in primary diploid cultures of fetal calvarial derived osteoblasts by the combined use of autoradiography, histochemistry, biochemistry, and mRNA assays of osteoblast cell growth and phenotypic genes. Modifications in gene expression define a developmental sequence that has 1) three principle periods--proliferation, extracellular matrix maturation, and mineralization--and 2) two restriction points to which the cells can progress but cannot pass without further signals--the first when proliferation is down-regulated and gene expression associated with extracellular matrix maturation is induced, and the second when mineralization occurs. Initially, actively proliferating cells, expressing cell cycle- and cell growth-regulated genes, produce a fibronectin/type I collagen extracellular matrix. A reciprocal and functionally coupled relationship between the decline in proliferative activity and the subsequent induction of genes associated with matrix maturation and mineralization is supported by 1) a temporal sequence of events in which there is an enhanced expression of alkaline phosphatase immediately following the proliferative period, and later, an increased expression of osteocalcin and osteopontin at the onset of mineralization; 2) increased expression of a specific subset of osteoblast phenotype markers, alkaline phosphatase and osteopontin, when proliferation is inhibited by hydroxyurea; and 3) enhanced levels of expression of the osteoblast markers as a function of ascorbic acid-induced collagen deposition, suggesting that the extracellular matrix contributes to both the shutdown of proliferation and the development of the osteoblast phenotype.

RUNX/AML transcription factors are critical regulators of cell growth and differentiation in multiple lineages and have been linked to human cancers including acute myelogenous leukemia (RUNX1), as well as breast (RUNX2) and gastric cancers (RUNX3). RUNX proteins are targeted to gene regulatory micro-environments within the nucleus via a specific subnuclear targeting signal. However, the dynamics of RUNX distribution and compartmentalization between the cytoplasm and nucleus is minimally understood. Here we show by immunofluorescence microscopy that RUNX2 relocates from the nucleus to the cytoplasm when microtubules are stabilized by the chemotherapeutic agent taxol. The taxol-dependent cytoplasmic accumulation of RUNX2 is inhibited by leptomycin B, which blocks CRM-1 dependent nuclear export, and is not affected by the protein synthesis inhibitor cycloheximide. Using biochemical assays, we show that endogenous RUNX2 associates with stabilized microtubules in a concentration-dependent manner and that the RUNX2 amino terminus mediates the microtubule association. In soluble fractions of cells, RUNX2 co-immunoprecipitates alpha tubulin suggesting that microtubule binding involves the alpha/beta tubulin subunits. We conclude that RUNX2 associates with microtubules and shuttles between the nucleus and the cytoplasm. We propose that nuclear-cytoplasmic shuttling of RUNX2 may modulate its transcriptional activity, as well as its ability to interface with signal transduction pathways that are integrated at RUNX2 containing subnuclear sites. It is possible that taxol-induced acute depletion of the nuclear levels of RUNX2 and/or other cell growth regulatory factors may represent an alternative pathway by which taxol exerts its biological effects during cancer chemotherapies.

We present an overview of Runx involvement in regulatory mechanisms that are requisite for fidelity of bone cell growth and differentiation, as well as for skeletal homeostasis and the structural and functional integrity of skeletal tissue. Runx-mediated control is addressed from the perspective of support for biological parameters of skeletal gene expression. We review recent findings that are consistent with an active role for Runx proteins as scaffolds for integration, organization and combinatorial assembly of nucleic acids and regulatory factors within the three-dimensional context of nuclear architecture.

The temporal and spatial organization of transcriptional regulatory machinery provides microenvironments within the nucleus where threshold concentrations of genes and cognate factors facilitate functional interactions. Conventional biochemical, molecular, and in vivo genetic approaches, together with high throughput genomic and proteomic analysis are rapidly expanding our database of regulatory macromolecules and signaling pathways that are requisite for control of genes that govern proliferation and differentiation. There is accruing insight into the architectural organization of regulatory machinery for gene expression that suggests signatures for biological control. Localized scaffolding of regulatory macromolecules at strategic promoter sites and focal compartmentalization of genes, transcripts, and regulatory factors within intranuclear microenvironments provides an infrastructure for combinatorial control of transcription that is operative within the three dimensional context of nuclear architecture.

Postmitotic gene expression requires restoration of nuclear organization and assembly of regulatory complexes. The hematopoietic and osteogenic Runx (Cbfa/AML) transcription factors are punctately organized in the interphase nucleus and provide a model for understanding the subnuclear organization of tissue-specific regulatory proteins after mitosis. Here we have used quantitative in situ immunofluorescence microscopy and quantitative image analysis to show that Runx factors undergo progressive changes in cellular localization during mitosis while retaining a punctate distribution. In comparison, the acetyl transferase p300 and acetylated histone H4 remain localized with DNA throughout mitosis while the RNA processing factor SC35 is excluded from mitotic chromatin. Subnuclear organization of Runx foci is completely restored in telophase, and Runx proteins are equally partitioned into progeny nuclei. In contrast, subnuclear organization of SC35 is restored subsequent to telophase. Our results show a sequential reorganization of Runx and its coregulatory proteins that precedes restoration of RNA processing speckles. Thus, mitotic partitioning and spatiotemporal reorganization of regulatory proteins together render progeny cells equivalently competent to support phenotypic gene expression.

Mechanisms that coordinate the spatial organization of genes and regulatory proteins within the three-dimensional context of nuclear architecture contribute to the sorting of regulatory information as well as the assembly and activity of sites within the nucleus that support gene expression. In this article we will present an overview of experimental approaches that provide insight into the trafficking of the hematopoietic and bone-specific AML/CBF family of regulatory factors to transcriptionally active subnuclear sites.

Components of nuclear architecture are functionally interrelated with control of gene expression. There is growing appreciation that multiple levels of nuclear organization integrate the regulatory cues that support activation and suppression of genes as well as the processing of gene transcripts. The linear representation of genes and promoter elements provide the potential for responsiveness to physiological regulatory signals. Parameters of chromatin structure and nucleosome organization support synergism between activities at independent regulatory sequences and render promoter elements accessible or refractory to transcription factors. Association of genes, transcription factors, and the machinery for transcript processing with the nuclear matrix facilitates fidelity of gene expression within the three-dimensional context of nuclear architecture. Mechanisms must be defined that couple nuclear morphology with enzymatic parameters of gene expression. The recent characterization of factors that mediate chromatin remodeling and identification of intranuclear targeting signals that direct transcription factors to subnuclear domains where gene expression occurs link genetic and structural components of transcriptional control. Nuclear reorganization and aberrant intranuclear trafficking of transcription factors for developmental and tissue-specific control occurs in tumor cells and in neurological disorders. Compromises in nuclear structure-function interrelationships can occur as a consequence of microgravity-mediated perturbations in cellular architecture.

Functional interrelationships between components of nuclear architecture and control of gene expression are becoming increasingly evident. There is growing appreciation that multiple levels of nuclear organization integrate the regulatory cues that support activation and suppression of genes as well as the processing of gene transcripts. The linear organization of genes and promoter elements provide the potential for responsiveness to physiological regulatory signals. Parameters of chromatin structure and nucleosome organization support synergism between activities at independent regulatory sequences and render promoter elements accessible or refractory to transcription factors. Association of genes, transcription factors, and the machinery for transcript processing with the nuclear matrix facilitates fidelity of gene expression within the three-dimensional context of nuclear architecture. Mechanisms must be defined that couple nuclear morphology with enzymatic parameters of gene expression. The recent characterization of factors that mediate chromatin remodeling and intranuclear targeting signals that direct transcription factors to subnuclear domains where gene expression occurs, reflect linkage of genetic and structural components of transcriptional control. Nuclear reorganization and aberrant intranuclear trafficking of transcription factors for developmental and tissue-specific control that occurs in tumor cells and in neurological disorders provides a basis for high resolution diagnostics and targeted therapy. J. Cell. Biochem. Suppls. 30/31:220-231, 1998. © 1998 Wiley-Liss, Inc.

The AML/CBFα runt transcription factors are key regulators of hematopoietic and bone tissue-specific gene expression. These factors contain a 31-amino acid nuclear matrix targeting signal that supports association with the nuclear matrix. We determined that the AML/CBFα factors must bind to the nuclear matrix to exert control of transcription. Fusing the nuclear matrix targeting signal to the GAL4 DNA binding domain transactivates a genomically integrated GAL4 responsive reporter gene. These data suggest that AML/CBFα must associate with the nuclear matrix to effect transcription. We used fluorescence labeling of epitope-tagged AML-1B (CBFA2) to show it colocalizes with a subset of hyperphosphorylated RNA polymerase II molecules concentrated in foci and linked to the nuclear matrix. This association of AML-1B with RNA polymerase II requires active transcription and a functional DNA binding domain. The nuclear matrix domains that contain AML-1B are distinct from SC35 RNA processing domains. Our results suggest two of the requirements for AML-dependent transcription initiation by RNA polymerase II are association of AML-1B with the nuclear matrix together with specific binding of AML to gene promoters.

The cell cycle regulating Cdc2 protein kinase helps orchestrate cell cycle dependent changes in cell structure and function. This report shows that Cdc2 is localized to the centrosome region and is tightly bound to the nuclear matrix-intermediate filament scaffold. Antibodies to Cdc2 and to the centrosome-specific protein, pericentrin, label the centrosome in an apparently cell cycle independent manner. Isolated centrosomes also label similarly with both antibodies. Essentially, all cells show Cdc2 labeling of the centrosomes, implying an independence of the stage in the cell cycle, a conclusion supported by studies of synchronized cells. In contrast to the labeling of every cell with the Cdc2 monoclonal antibody, fewer centrosomes were labeled with an antibody to the PSTAIRE domain of Cdc2. Embedment-free, immunogold electron micrographs of extracted cell whole mounts show the centrioles and a pericentriolar network of filaments. Both Cdc2 and pericentrin antibodies decorate the amorphous pericentriolar material, while the Cdc2 antibodies also decorate the centrioles themselves. The constitutive presence of Cdc2 at the centrosome suggests a continuing role in the dynamics of centrosome function throughout the cell cycle.

The development of implantable cardiac assist devices for prolonged circulatory support has been impeded by the problem of excessive thrombogenesis on the blood-prosthetic interface, with subsequent embolization. To overcome this obstacle, a ventricular assist device has been developed with textured blood-contacting surfaces to encourage the formation of a tightly adherent, hemocompatible, biological lining. In this study, we applied molecular biological techniques, in conjunction with conventional ultrastructural and biochemical techniques, to characterize the biological linings associated with the blood-contacting surfaces of 11 of these devices, which had been clinically implanted for durations ranging from 21 to 324 days. No clinical thromboembolic events or pump-related thromboembolism occurred. Biological linings developed on the textured surfaces composed of patches of cellular tissue intermingled with areas of compact fibrinous material. In addition, islands of collagenous tissue containing fibroblast-like cells appeared after 30 days of implantation. Many of these cells contained microfilaments with dense bodies indicative of myofibroblasts. RNA hybridization analyses demonstrated that the colonizing cells actively expressed genes encoding proteins for cell proliferation (histones), adhesion (fibronectin), cytoskeleton (actin, vimentin) and extracellular matrix (types I and III collagen). Linings, which never exceeded 150 microns in thickness, remained free of pathological calcification. Textured blood-contacting surfaces induced the formation of a thin, tightly adherent, viable lining which exhibited excellent long-term hemocompatibility.

A variant human H2B histone gene (GL105), previously shown to encode a 2300 nt replication independent mRNA, has been cloned. We demonstrate this gene expresses alternative mRNAs regulated differentially during the HeLa S3 cell cycle. The H2B-Gl105 gene encodes both a 500 nt cell cycle dependent mRNA and a 2300 nt constitutively expressed mRNA. The 3' end of the cell cycle regulated mRNA terminates immediately following the region of hyphenated dyad symmetry typical of most histone mRNAs, whereas the constitutively expressed mRNA has a 1798 nt non-translated trailer that contains the same region of hyphenated dyad symmetry but is polyadenylated. The cap site for the H2B-GL105 mRNAs is located 42 nt upstream of the protein coding region. The H2B-GL105 histone gene was localized to chromosome region 1q21-1q23 by chromosomal in situ hybridization and by analysis of rodent-human somatic cell hybrids using an H2B-GL105 specific probe. The H2B-GL105 gene is paired with a functional H2A histone gene and this H2A/H2B gene pair is separated by a bidirectionally transcribed intergenic promoter region containing consensus TATA and CCAAT boxes and an OTF-1 element. These results demonstrate that cell cycle regulated and constitutively expressed histone mRNAs can be encoded by the same gene, and indicate that alternative 3' end processing may be an important mechanism for regulation of histone mRNA. Such control further increases the versatility by which cells can modulate the synthesis of replication-dependent as well as variant histone proteins during the cell cycle and at the onset of differentiation.

Primary cultures of calvarial derived normal diploid osteoblasts undergo a developmental expression of genes reflecting growth, extracellular matrix maturation, and mineralization during development of multilayered nodules having a bone tissue-like organization. Scanning electron microscopy of the developing cultures indicates the transition from the uniform distribution of cuboidal osteoblasts to multilayered nodules of smaller cells with a pronounced orientation of perinodular cells towards the apex of the nodule. Ultrastructural analysis of the nodule by transmission electron microscopy indicates that the deposition of mineral is confined to the extracellular matrix where cells appear more osteocytic. The cell body contains rough endoplasmic reticulum and golgi, while these intracellular organelles are not present in the developing cellular processes. To understand the regulation of temporally expressed genes requires an understanding of which genes are selectively expressed on a single cell basis as the bone tissue-like organization develops. In situ hybridization analysis using 35S labelled histone gene probes, together with 3H-thymidine labelling and autoradiography, indicate that greater than 98% of the pre-confluent osteoblasts are proliferating. By two weeks, both the foci of multilayered cells and internodular cell regions have down-regulated cell growth associated genes. Post-proliferatively, but not earlier, initial expression of both osteocalcin and osteopontin are restricted to the multilayered nodules where all cells exhibit expression. While total mRNA levels for osteopontin and osteocalcin are coordinately upregulated with an increase in mineral deposition, in situ hybridization has revealed that expression of osteocalcin and osteopontin occurs predominantly in cells associated with the developing nodules. In contrast, proliferating rat osteosarcoma cells (ROS 17/2.8) concomitantly express histone H4, along with osteopontin and osteocalcin. These in situ analyses of gene expression during osteoblast growth and differentiation at the single cell level establish that a population of proliferating calvarial-derived cells subsequently expresses osteopontin and osteocalcin in cells developing into multilayered nodules with a tissue-like organization.

The role of the extracellular matrix of specialized tissues in promoting cellular differentiation has long been recognized (1–2). Our studies have utilized rat osteoblast cultures (3–5) as a model system to examine a well defined extracellular matrix (ECM) and the coordinate regulation of the changes in cell structure and gene expression as related to its formation. These cells produce a mineralized ECM having a bone tissue-like organization analogous to embryonic bone (4–6). In contrast to tumor-derived or transformed osteosarcoma cell lines (7), those normal diploid bone derived cells in culture exhibit normal cell cycle regulated expression of genes which is functionally coupled to expression of differentiation specific genes (4). As the ECM develops, osteoblasts differentiate, progressing through three stages of development, a proliferation period, a period of matrix maturation, and a mineralization period (4). In the previous chapter, temporal expression of genes characterizing the osteoblast phenotype associated with this differentiation in vitro has been described in detail and is summarized in Figure 1.

In this chapter and in the one which follows we will present concepts and experimental approaches associated with the relationship of proliferation to differentiation with emphasis on the contribution of cell structure to the regulation of cell growth and tissue specific gene expression. While these relationships are of broad biological relevance, we will focus primarily on development of the osteoblast phenotype with the understanding that analogous principles apply to the regulation of phenotype expression in general.