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Keywords:

  • type I collagen;
  • green fluorescent protein;
  • marrow stromal cell;
  • calvarial osteoblast;
  • osteoblast lineage differentiation

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. ANALYSIS OF TRANSGENE EXPRESSION
  6. RESULTS
  7. DISCUSSION
  8. Acknowledgements
  9. REFERENCES

Green fluorescent protein (GFP)-expressing transgenic mice were produced containing a 3.6-kilobase (kb; pOBCol3.6GFPtpz) and a 2.3-kb (pOBCol2.3GFPemd) rat type I collagen (Col1a1) promoter fragment. The 3.6-kb promoter directed strong expression of GFP messenger RNA (mRNA) to bone and isolated tail tendon and lower expression in nonosseous tissues. The 2.3-kb promoter expressed the GFP mRNA in the bone and tail tendon with no detectable mRNA elsewhere. The pattern of fluorescence was evaluated in differentiating calvarial cell (mouse calvarial osteoblast cell [mCOB]) and in marrow stromal cell (MSC) cultures derived from the transgenic mice. The pOBCol3.6GFPtpz-positive cells first appeared in spindle-shaped cells before nodule formation and continued to show a strong signal in cells associated with bone nodules. pOBCol2.3GFPemd fluorescence first appeared in nodules undergoing mineralization. Histological analysis showed weaker pOBCol3.6GFPtpz-positive fibroblastic cells in the periosteal layer and strongly positive osteoblastic cells lining endosteal and trabecular surfaces. In contrast, a pOBCol2.3GFPemd signal was limited to osteoblasts and osteocytes without detectable signal in periosteal fibroblasts. These findings suggest that Col1a1GFP transgenes are marking different subpopulations of cells during differentiation of skeletal osteoprogenitors. With the use of other promoters and color isomers of GFP, it should be possible to develop experimental protocols that can reflect the heterogeneity of cell differentiation in intact bone. In primary culture, this approach will afford isolation of subpopulations of these cells for molecular and cellular analysis.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. ANALYSIS OF TRANSGENE EXPRESSION
  6. RESULTS
  7. DISCUSSION
  8. Acknowledgements
  9. REFERENCES

Acquisition and maintenance of normal bone mass, irrespective of bone-resorbing cells, ultimately is dependent on an adequate number and activity of osteoblasts.(1) Increasingly, it is being recognized that impaired generation of bone cells either because of an underperforming lineage or diversion of the lineage to other cell types is a fundamental cause for diminished bone mass.(2, 3) Thus, identifying the cellular pathways and the factors that regulate this progression to full osteoblast differentiation is an important requirement for understanding diseases of bone and for designing rational therapeutic strategies.

Osteoblast precursors are recruited from a multipotential mesenchymal progenitor that can give rise to several differentiated cell phenotypes including fibroblasts, chondrocytes, adipocytes, and osteoblasts.(4–8) The existence of osteoprogenitor cells within cultured cells derived from calvaria or bone marrow is shown by their ability to produce mineralized bone nodules when the culture is grown under osteoblast inductive conditions.(9–11) During the process of osteoblastic differentiation, an osteoprogenitor proliferates and undergoes a series of maturational steps before becoming a differentiated osteoblast. For example, cell surface markers like activated leucocyte-cell adhesion molecule (ALCAM), identical to SB-10,(12) and STRO-1(13, 14) may be useful in identifying the earliest stages of the osteoblast lineage in bone marrow stromal cell (MSC) cultures. Cells at later stages are defined by morphology or by the expression of bone-associated marker genes. Preosteoblasts are characterized by a fibroblastic morphology, alkaline phosphatase (ALP), and type I collagen (Col1a1) messenger RNA (mRNA) expression. Early osteoblast stages are more cuboidal and express bone sialoprotein (BSP). Osteocalcin (OC) mRNA and mineralization of bone nodules are associated with terminal differentiation.(15) Although morphological and molecular analysis has defined stages of differentiation, the presence of the dense extracellular matrix produced by these cultures requires an endpoint assay in which the individual culture plate is harvested.(16, 17) These problems have prevented observation of the progression of differentiation in real time or isolation of relatively homogeneous populations of these cells for further analysis.

Stage-specific promoters driving easily visualized transgenes have been used successfully to study stages of lineage development in the brain and gut.(18, 19) We have identified fragments of the rat Col1a1 promoter that show preferential expression in different Col1a1-producing tissues. Studies in transgenic mice harboring a Col3.6 or Col2.3 chloramphenicol acetyltransferase (CAT) transgene showed that a 3.6-kilobase (kb) fragment directed expression to both osseous (bone and tooth) and nonosseous tissues (tendon, skin, and lung). Col2.3 showed a more restricted pattern of expression with strong activity in bones, low activity in tendons, and very low or undetectable activity in other tissues.(20) Similar observations were made by Rossert et al. in studies using mouse pro-α1(I) collagen promoter fragments.(21) Osteoblastic cell cultures derived from neonatal calvaria maintained expression of Col3.6 while Col2.3 expression is very low or undetectable.(22) However, when the cultures are grown under differentiating conditions, Col2.3 transgene expression increases sharply.(23) Marrow stromal cells derived from mice bearing these transgenes showed a similar pattern, early expression of Col3.6 and late expression of Col2.3 that is associated with differentiation to a mature osteoblast.(24)

The purpose of this study was to determine if these promoter fragments have sufficient strength and specificity to monitor the differentiation of the lineage in real time by fluorescent microscopy. We generated transgenic mice in which green fluorescent protein (GFP) expression is under the control of the 3.6 and 2.3 Col1a1 promoter fragments. Transgene expression was assessed in MSCs and mouse calvarial osteoblast cell (mCOB) cultures grown under conditions that induce osteoblastic differentiation as well as in intact bone tissue. The results indicate that distinctly different populations of cells within the osteoblastic lineage can be recognized and that this approach may be exploited to assess stages of osteoblastic differentiation in cell culture and in intact bone.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. ANALYSIS OF TRANSGENE EXPRESSION
  6. RESULTS
  7. DISCUSSION
  8. Acknowledgements
  9. REFERENCES

Generation of transgenic mice

Col1a1 fragments were used to drive the expression of GFP. Briefly, the GFP coding sequence was excised from pGFPemd-basic (Packard, Meriden, CT, USA, now licensed to Clontech, Palo Alto, CA, USA) as an NaeI and HindIII fragment and cloned into pBC KS using HincII and HindIII (pBCGFPemd) followed by destruction of the HindIII site. The receiving vector was a derivative of pOB4CAT(25) in which the XbaI site located at the 5′ end of the CAT gene was converted to BglII and the XhoI site in the SV40 promoter was changed to XbaI. The CAT gene was removed with BglII/XhoI and replaced with the BamHI/XhoI fragment from pBCGFPemd generating pOB4GFPemd. The 2.3-kb Col1a1 promoter fragment(20) was inserted as an HindIII-XbaI fragment replacing the SV40 promoter/enhancer to generate pOBColGFPemd 2.3/0. Flanking HindIII and EcoRI sites were changed into SalI using appropriate adapters. The pOBColGFPemd 2.3/0 plasmid was opened with XbaI and BamHI and a 1.6-kb XbaI-BamHI rat first intron fragment that originates from pOB253.6/1.6/0(26) was inserted to obtain pOBColGFPemd 2.3/1.6, which was renamed pOBCol2.3GFPemd.

To construct pOBCol3.6GFP, an NaeI-HindIII GFPtpz fragment was inserted into the SmaI-HindIII sites of a Cla adapter plasmid(27) into which we had inserted the bovine growth hormone polyadenylation signal. The recipient plasmid contained a 5.2-kb XbaI-BamHI fragment encoding the collagen 3.6/1.6 promoter/intron sequence in a pBC SK backbone. The GFP-bPA Cla fragment was excised from the adaptor plasmid and inserted into the recipient plasmid to create pOBCol3.6GFP.

The transgene was microinjected(28) by the Institutional Transgenic Animal Facility. CD-1 transgenic founders and F1 generation were identified by dot blot hybridization using a 0.7-kb HindIII-Xho fragment of GFP DNA as a probe. Fluorescent microscopy of tail was used to screen F1 progeny and subsequent generations of mice. Transgenic lines were bred to homozygosity and experimental mice were generated by mating homozygous transgenic with nontransgenic CD-1 mice.

ANALYSIS OF TRANSGENE EXPRESSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. ANALYSIS OF TRANSGENE EXPRESSION
  6. RESULTS
  7. DISCUSSION
  8. Acknowledgements
  9. REFERENCES

RNA extraction and Northern blot analysis

Animals were killed with CO2 asphyxiation followed by cervical dislocation and excised soft tissues were frozen immediately in liquid nitrogen in a 15-ml polypropylene tube (Falcon Cat 2059; Becton Dickinson, Franklin Lakes, NY, USA). The epiphyseal portions of the long bones (humerus, tibia, and femur) were cleaned of attached muscle and the marrow-flushed using a 25G needle before freezing. Calvaria and tail containing tendon and vertebrae were minced with scissors before freezing. Tail tendon samples were obtained by stripping tendon bundles away from their bony insertions. Frozen samples were suspended in 3 ml of TRIzol Reagent (Molecular Research Center, Inc., Cincinnati, OH, USA) and immediately homogenized with a 5-mm Polytron probe (Brinkman, Westbury, NY, USA) for 30 s. Long bone samples were crushed in TRIzol reagent in 15 ml of Falcon polypropylene tubes with a custom-fit pestle before the Polytron step. Total RNA was prepared from mouse tissues and cultured cells using TRIzol Reagent according to the manufacturer's instructions. After the isopropanol precipitation, the pellet was redissolved in 300 μl of GTC buffer (4.5 M of guanidinium isothiocyanate, 10 mM of 2-mercaptoethanol (β-MSH), 15 mM of sodium N-lauryl sarcosine, and 10 mM of sodium citrate, pH 7.0) followed by precipitation with 300 μl isopropanol. The precipitate was washed once with 80% ethanol, drained, and redissolved in 50 μl of water. RNA was separated on a 2.2 M formaldehyde/1% agarose gel and transferred onto a nylon membrane (Maximum Strength Nytran; Schleicher & Schuell, Keene, NH, USA). Membranes were probed with the 0.7-kb GFP fragment, a 900-base pair (bp) PstI fragment of rat Col1a1 (pα1R2(29)), a 440-bp PstI/EcoRI mouse OC fragment (p923(30)), and a 1000-bp EcoRI mouse BSP and osteopontin (OP) fragment.(31) Probes were radiolabeled by the random primer method using (α-32P)deoxycytosine triphosphate (dCTP; 3000 Ci/mmol; New England Nuclear, Boston, MA, USA) obtaining a probe-specific activity of approximately 1 × 109 cpm/μg. Filters were hybridized with 3 × 106 cpm/ml32P-labeled probe at 42°C in 50% formamide, 5 × SSPE (1 × SSPE = 0.149 M of NaCl, 10 mM of NaH2PO4, and 1 mM of EDTA, pH 7.4), 1.2 × Denhardt's solution, and 0.5% sodium dodecyl sulfate (SDS).(32) Filters were washed once in 6 × SSPE and 0.5% SDS for 10 minutes at room temperature, once in 0.1 × SSPE and 0.1% SDS for 10 minutes at 37°C, and once in 0.1 × SSPE and 0.1% SDS for 10 minutes at 65°C. Prior hybridization signals were removed by washing in 0.1 × SSPE and 0.1% SDS for 20-30 minutes at 80°C.

Histological evaluation of GFP expression

Femurs and calvariae from 7-day-old, 2-month-old, and 5-month-old mice were dissected free of surrounding tissue and fixed in 4% paraformaldehyde/phosphate buffered saline (PBS; adjusted to pH 7.4 with 10N of NaOH) at 4°C for 1-7 days depending on the age of the mice. Soft tissues (tendon, skin, lung, bladder, liver, fat, and brain) were dissected and placed in paraformaldehyde overnight. Following fixation, bones were decalcified in 15% EDTA (adjusted to pH 7.1 with concentrated NH4OH) for 1 week, dehydrated in progressive concentrations of ethanol, and cleared in xylene. Samples were placed in three changes of paraffin (paraplast X-TRA tissue-embedding medium; Fisher Scientific, Pittsburgh, PA, USA) at 56°C for 1 h each and embedded. Bones were sectioned longitudinally in 5-μm-thin sections. Samples were protected from exposure to daylight during the preparation process.

Preparation of MSC cultures

Six- to 8-week-old transgenic mice were killed by CO2 asphyxiation. Femurs and tibias were dissected from surrounding tissues. The epiphyseal growth plates were removed and the marrow was collected by flushing with α-modified essential medium (α-MEM) culture containing 100 U/ml of penicillin, 100 μg/ml of streptomycin, and 10% fetal calf serum (FCS; Gibco BRL, Life Technologies, Grand Island, NY, USA) with a 25G needle. Single cell suspensions were prepared by passing the cell clumps through an 18G needle followed by filtration through a 70-μm cell strainer (Falcon 2350; Fisher Scientific). An aliquot of cells was diluted 1:1 with 0.04% trypan blue in PBS and viable cells were plated at a density 2-2.5 × 106 cells/cm2 in 35-mm culture plates (Falcon 3046; Fisher Scientific). On day 4, one-half of the medium containing nonadherent cells was replaced with fresh medium. Medium was changed completely on day 7 to α-MEM supplemented with 50 μg/ml of ascorbic acid, 10 nM of dexamethasone, and 8 mM of β-glycerophosphate (Sigma Chemical Co., St. Louis, MO, USA). Medium was changed every 2 days for the duration of the experiment.

Preparation of mCOB culture

Calvarial cells were isolated from 7- to 9-day-old transgenic mice using a modification of the method described by Wong and Cohn.(33) Briefly, after removal of sutures, calvariae were subjected to four sequential 15-minute digestions in an enzyme mixture containing 0.05% trypsin (Gibco BRL, Life Technologies, Inc.) and 0.1% collagenase P (Boehringer Mannheim, Mannheim, Germany) at 37°C on a rocking platform. Cell fractions 2-4 were collected and enzyme activity was stopped by addition of an equal volume of DulbeccO's modified Eagles' medium (DMEM) containing 10% FCS, 100 U/ml of penicillin, and 100 μg/ml of streptomycin (Gibco BRL, Grand Island, NY, USA). The fractions were pooled, centrifuged, resuspended in DMEM containing 10% FCS, and filtered through a 70-μm cell strainer. Cells were plated at a density of 1-1.5 × 104 cells/cm2 in 35-mm culture plates in DMEM containing 10% FCS. Twenty-four hours later, medium was exchanged and 3 days later cultures were fed again. At 1 week of culture, the medium was changed to differentiation medium (α-MEM containing 10% FCS, 50 μg/ml of ascorbic acid, and 4 mM of β-glycerophosphate) and thereafter the medium was changed every 2 days.

Histochemical analysis of cell cultures

Histochemical staining for ALP activity was performed using a commercially available kit (86-R Alkaline Phosphatase; Sigma Diagnostics, Inc., St. Louis, MO, USA) according to the manufacturer's instructions. Mineralization was assessed using modified von Kossa silver nitrate staining method. Briefly, cells were fixed for 10 minutes in 2% paraformaldehyde in 0.1 M of sodium cacodylate, pretreated for 20 minutes with saturated lithium carbonate solution, and washed in deionized water. The plates were incubated with 5% silver nitrate solution for 30 minutes under a bright light, washed with water, and treated with a 5% sodium thiosulphate solution for 2-3 minutes followed by washing with water and air-drying. The results of both staining procedures were recorded with a Kodak DCS420 color digital camera (Kodak, Rochester, NY, USA).

Observation of GFP fluorescence

Fluorescent microscopy

GFP expression in cell culture was visualized using an Olympus IX50 inverted system microscope equipped with an IX-FLA inverted reflected light fluorescence (Olympus America, Inc., Melville, NY, USA). A specific excitation wavelength was obtained using filters for GFPtpz (exciter, D500/20; dichroic, 525DCLP; emitter, D550/40) and GFPemd (exciter, D470/40; dichroic, 495LP; emitter, D525/50) and recorded with an SPOT-camera (Diagnostic Instruments, Inc., Sterling Heights, MI, USA). Phase contrast images were converted to grayscale. Fluorescent images were taken with equal exposure times applied to cells derived from different transgenic constructs. Both 4 × and 10 × magnifications were used.

GFP expression in histological sections was observed and photographed using a Nikon microscope equipped with a fluorescein isothiocyanate (FITC) and Texas red dual-fluorescent filter cube (Chroma Technology Corp., Brattleboro, VT, USA). This cube allows the yellow-green GFP signal to be distinguished from the light red autofluorescent background located in the marrow space and minimizes the strong background fluorescence that is a characteristic of decalcified bone.

Fluorimager analysis

Distribution of GFP expression in cell culture was observed with a FluorImager SI (Molecular Dynamics, Sunnyvale, CA, USA) using a 515-nm emission spectrum at PMT settings of 800. Images were processed with ImageQuaNT software (Molecular Dynamics).

Flow cytometry

Cells were prepared by washing in PBS twice and then by digesting the matrix with 0.2% collagenase P and 0.2% hyaluronidase in 0.25% trypsin/1 mM EDTA. The process was terminated by the addition of cold medium containing serum followed by centrifugation. Cells were resuspended in PBS and filtered through a 70-μm cell strainer (Falcon, Cat 2013). Flow cytometric analysis was carried out on a FACscan/Calibur (Becton-Dickinson, San Jose, CA, USA) using a 488-nm excitation wavelength generated by a 15 mw argon ion laser. Emission was detected using a 500-nm long pass filter (GFP). Cells from nontransgenic mice were used as controls.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. ANALYSIS OF TRANSGENE EXPRESSION
  6. RESULTS
  7. DISCUSSION
  8. Acknowledgements
  9. REFERENCES

GFP expression in Col1a1 transgenic mice

Three transgenic lines with each construct (pOBCol3.6GFP and pOBCol2.3GFP) were generated (Figs. 1A and 1B). No differences in health, reproductive capacity, or development between transgenic and wild-type mice were observed. Transgenic mice were genotyped using fluorescence microscopy of tail clips (Figs. 1C-1E). pOBCol2.3GFP mice showed a strong fluorescent signal coming from the vertebral bone within the tail segments, whereas pOBCol3.6GFP mice exhibited a more uniform fluorescence because of strong transgene expression in skin. Northern blot analysis of tissues from 6-week-old pOBCol3.6GFP heterozygous males showed strong expression in long bones, calvaria, tail, and tendon and a weaker expression in other Col1a1-producing tissues such as skin, aorta, bladder, lung, muscle, and fat. Transgene expression was not detected in heart, kidney, liver, thymus, spleen, and intestine (Fig. 1F). All three lines of pOBCol2.3GFP show the strongest transgene expression in osseous tissues, weaker expression in tendon, and no expression in other Col1a1-producing tissues (Fig. 1G).

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Figure FIG. 1.. Characterization of the Col1a1GFP transgenes. Constructs used in transgenic mice. (A) pOBCol3.6GFP contains 3520 bp of 5′-flanking sequence of the rat Col1a1 promoter, first exon, and first intron(26) driving GFPtpz. “BE” refers to homeodomain binding sequence necessary for high expression of these constructs in bone.(23) (B) pOBCol2.3GFP contains a HindIII deletion of the upstream promoter sequence to −2300 bp. (C-E) Detection of transgene expression in intact mice. The upper panel represents the transmitted images mice tails derived from (C) nontransgenic (NT) and transgenic (D) pOBCol3.6 and (E) pOBCol2.3. The lower panel shows characteristic GFP signal from nontransgenic and transgenic mice (arrow indicates a strong fluorescence inside a vertebral body of pOBCol2.3 mice). (F and G) Northern blot analysis of transgene expression. Selected tissues from (F) pOBCOl3.6GFP and (G) pOBCol2.3GFP transgenic mice. RNA was extracted from 6-week-old heterozygous male transgenic animals. Ten micrograms of total RNA was gel separated and hybridized with a GFP complementary DNA (cDNA) probe (HindIII-XhoI fragment), Col1a1, OC, and 18S ribosomal RNA (rRNA) cDNA.

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Stage-specific activation of the Col1a1 promoter

MSC cultures were established from 6- to 8-week-old pOBCol3.6GFP and pOBCol2.3GFP transgenic mice. To assure that comparable levels of osteoblastic differentiation developed in these cultures, sister plates were harvested for the expression of bone-related markers of differentiation. The pattern of staining for ALP activity and mineralization was equivalent in cultures from various transgenic lines (Fig. 2A). Northern blot analysis for Col1a1, BSP, OP, and OC revealed the same temporal pattern of expression of these markers in cultures derived from pOBCol3.6GFP and pOBCol2.3GFP mice (Fig. 2B(24)). However, the pattern of GFP expression was distinctly different. pOBCol3.6GFP was detected at day 7 and increased at later time points and pOBCol2.3 appeared on day 14, before OC expression.

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Figure FIG. 2.. Osteoblastic differentiation in primary MSC culture derived from Col1a1GFP transgenic mice. (A) Staining for ALP expression in 7-, 14-, and 18-day-old cultures. Mineralization was detected by the von Kossa method on day 21. The upper panel represents cells derived from pOBCol3.6GFP and the lower from pOBCol2.3GFP transgenic mice. (B) Northern blot analysis of RNA derived from the two transgenic constructs. Line 263 and line 268 are two lines harboring pOBCol3.6GFPtpz whereas lines 442 and 428 are two transgenic lines with the pOBCol2.3GFPemd construct. Each lane contains 10 μg of total RNA prepared from RNA extracted at different time points of osteoblastic differentiation. Blots were probed for GFP, Col1a1, BSP, OP, OC, and 18S rRNA to normalize for the RNA quantity.

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Examination of the same culture dishes over time for GFP fluorescence indicated that different subsets of cells expressed the two transgenes. At day 7, MSC cultures derived from pOBCol3.6GFP mice showed a weak fluorescence in cells with spindle-shaped morphology that form early colonies. The strength of signal increased as differentiation proceeded (day 11 and day 14) and peaked by day 21 in the multilayered areas containing cuboidal cells (Fig. 3A). In contrast, the pOBCol2.3 transgenic construct was inactive in 7-day-old cultures. When it became active it produced very bright fluorescence and was localized in cuboidally shaped cells in the center of a mineralized nodule (Fig. 3B).

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Figure FIG. 3.. Expression of GFP in differentiating primary MSC cultures derived from Col1a1GFP transgenic mice. Primary MSC cells were grown under differentiating conditions and GFP expression was monitored by fluorescent microscopy. (A and B) The upper panels show phase contrast images, and the lower panels show images at the same positions under the fluorescent light. A time course of GFP expression from day 7 to day 21 is shown in which the mineralized portion of the nodule (M) and nonmineralized area (NM) can be distinguished by day 21. (A) The Col3.6 promoter showed weak expression of GFP by day 7 with increasing intensity by day 14 and further accentuation in the mineralized areas at day 21. (B) In contrast, Col2.3 promoter that was not active on day 7 is first observed in scattered cells within multilayered areas of future mineralization (day 14). Extremely strong expression developed in areas of mineralization (day 21).

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Comparable findings were observed in neonatal mouse calvarial cultures. In this system, cells that initiate the culture originate from various stages of osteoblastic differentiation as judged by the presence of GFP-negative and both pOBCol3.6- and pOBCol2.3-positive cells in the suspension of freshly isolated calvarial cells. After 7 days, Col3.6GFP expression was detected throughout the culture in cells showing a fibroblastic morphology (Fig. 5A) concomitant with the expression of Col1a1 mRNA and ALP activity (Figs. 4A and 4C). At this time only isolated cells showed expression driven from 2.3 promoter fragment (Fig. 5B). The 14-day-old culture revealed a clear difference in the spatial distribution of GFP expression. The pOBCol3.6 construct was active in both mineralized and nonmineralized areas (Fig. 5A) whereas pOBCol2.3 expression was restricted to highly dense parts of nodules in the cuboidal-shaped cells that are associated with mineralization (Fig. 5B). At this time point BSP and OC mRNA were detected in sister cultures (Fig. 4C). These findings were consistent with day 21 of culture at which time heavy mineralization of nodules was detected by the von Kossa staining (Figs. 4A and 4B).

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Figure FIG. 4.. Osteoblastic differentiation of primary mCOB cells derived from Col1a1GFP transgenic mice. Primary mCOB cultures were grown in the presence of ascorbic acid and β-glycerol phosphate to promote osteoblastic differentiation. Cultures were stained for ALP expression on days 7, 14, and 18 and mineralization was assessed by von Kossa staining on day 21. Cultures were derived from (A) pOBCol3.6GFPtpz and (B) pOBCol2.3GFPemd transgenic mice. (C) Northern blot analysis of GFP expression and markers of osteoblastic differentiation in cultures derived from nontransgenic (NT) and transgenic animals. Blots were probed for GFP, Col1a1, BSP, OC, and 18S rRNA to normalize for the RNA quantity.

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Figure FIG. 5.. Expression of GFP in differentiating primary mCOB cultures derived from Col1a1GFP transgenic mice. Primary mCOB cells were grown under differentiating conditions and GFP expression of same position in a culture plate was monitored by fluorescent microscopy. (Upper panel, phase contrast images; lower panel, fluorescent images.) (A) The pOBCol3.6 construct was expressed from the beginning of culture, increasing in number and strength by days 5-7, with a further increase in cells located within mineralized areas at late time points (days 14-21). (B) The pOBCol2.3GFP revealed the presence of isolated cells expressing GFP throughout the culture at day 2 that diminished both in number and in intensity by day 7. By days 11-14, a different population of cells began to express the transgene in regions undergoing mineralization. By day 21, an extremely strong GFP signal is associated with mineralized parts of nodules.

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One powerful application of the ColGFP transgenes in primary culture is the ability to quantitate osteoblastic differentiation either in an intact culture or as isolated cells. The FACScan Caliber can assess the proportion of the total cell population that is GFP-positive and provide a distribution of signal strength within the positive population. In the cells that initiated the mCOB culture, flow cytometry showed that the 3.6-kb promoter was expressed in 50% of the cells whereas the 2.3-kb promoter was expressed in 20% of the cells (data not shown). By day 7 of culture, 50% of the pOBCol3.6GFP cells were fluorescent, whereas only 1% of the pOBCol2.3GFP cells was active. By day 14 of culture, the pOBCol3.6GFP construct continued to be present in 38% of cells but strength of the signal had increased. In the corresponding culture, pOBCol2.3GFP was expressed in 8% of the cells with a broad distribution of signal strengths that exceeded the intensity of the pOBCol3.6 transgene (Figs. 6A and 6C).

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Figure FIG. 6.. Flow cytometry and fluorimager analysis of differentiating mCOB cultures. Cells isolated from cultures of wild-type and transgenic mice were analyzed by flow cytometry on (A) day 7 and (C) day 14. M1 parameter was used to gate cells that do not express GFP and the M2 gate was used to define GFP expressing population of cells. The percentage of cells that are GFP-positive is indicated beneath the M2 bar. pOBCol2.3 transgene contained an exceptionally strong population of cells that generated an out-of-scale peak, which is indicated by the arrow. (B and D) Sister plates were imaged repetitively at the same time points using a fluorimager scanner.

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In addition to the visualization of individual cells by fluorescent microscopy, the entire culture can be assessed for the pattern of transgene expression by fluorescent imaging. The same plate of cells can be imaged repeatedly over time by fluorescent scanning. By day 7, pOBCol3.6GFP was detected as a weak signal diffusely spread throughout the culture that increased in strength by day 14. In contrast, pOBCol2.3GFP was not evident at day 7 but by day 14 it generated a strong signal that was restricted to the developing mineralized bone nodules (Figs. 6B and 6D).

Histological evaluation of GFP expression

Decalcified and paraffin-embedded sections of femur and other connective tissues from transgenic mice of different ages were prepared for histological examination. Preliminary experiments showed that the strength of the GFP signal in paraffin-embedded tissues was comparable with the level found in cryosectioned samples (data not shown). pOBCol3.6GFP transgenic mice showed wide expression of GFP in dermal fibroblasts, bladder smooth muscle cells, and in peribronchial connective tissues whereas no GFP-positive cells were evident in the same tissues from the pOBCol2.3GFP mice (data not shown).

Our study focused on the expression pattern of Col-GFP transgenes in bone. Multiple animals from all three transgenic lines were analyzed for the pattern of GFP expression. Eight-day-old mice harboring a pOBCol3.6GFP showed a strong GFP signal in osteoblasts lining the periosteal, endosteal, and trabecular surfaces and in cortical bone that was very distinctive in the collar region of the growth plate (Fig. 7A). Fluorescence also was evident in the metaphyseal region containing periosteal fibroblasts (Fig. 7A). The intensity and number of osteoblasts expressing GFP was lower at 2 months. Only a small number of cells expressed GFP at 5 months. At this time, only an occasional bone-lining osteoblast was positive whereas faintly positive cells were still observed in the periosteal layer (high-power image).

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Figure FIG. 7.. Histology of femoral bones at different maturational stages of Col1a1GFP transgenic mice. (A) Metaphyseal regions of femurs from 8-day-, 2-month-, and 5-month-old pOBCol3.6GFP transgenic mice. The GFP signal was detected in the periosteal fibroblast (Pf), trabecular osteoblast (T), and endosteal osteoblast extending into collar region (E). A fluorescent signal was not detected in the chondrocyte lineage cells of the developing growth plate (G). At the age of 2 months, fluorescence was detected also in the osteoblasts lining the endosteal (E) and periosteal (P) surface of cortical bone. At 5 months, low-level positive cells are still visible and better appreciated at high power (boxed area) as cells lining cortical bone (Cb) and overlying periosteum (Pf). (B) Metaphyseal bones derived from pOBCol2.3GFP transgenic mice. GFP expression was more restricted to periosteal osteoblast with no expression from the periosteal fibroblast. Fluorescence within the trabecular area also had a restricted pattern of expression with no fluorescence in the growth plate. At 5 months the low-level fluorescence was detected within cortical bone but not in the periosteal fibroblast layer. (C and D) Diaphyseal region of femur from (C) pOBCol3.6GFP and (D) pOBCol2.3GFP transgenic mice at different maturational stages. Expressing osteocytes (O) can be observed deep in the cortical bone in the pOBCol2.3GFP and the pOBCol3.6 showed positive osteocytes restricted to the regions close to the endosteal surface (E). Sections derived from 2-month-old nontransgenic mice (NT) of the same areas of interest (metaphysis and diaphysis) show a representative level of bone and bone marrow autofluorescence.

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In contrast, pOBCol2.3 drove expression in a smaller number of osteoblastic cells even at the 8-day stage (Fig. 7B). Cells lining the bone had strong GFP expression, although the density of positive lining cells was less than the 3.6 cells and this number fell by 2 months and 5 months. No fluorescence was observed in periosteal fibroblasts of pOBCol2.3GFP bone. The diaphyseal region of bone of older mice revealed another feature that distinguished the expression of two transgenes. Osteocytes from pOBCol2.3GFP mice showed expression throughout cortical bone and osteocytes expressing pOBCol3.6GFP were primarily active in cortical areas adjacent to the endosteal surface (Figs. 7C and 7D).

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. ANALYSIS OF TRANSGENE EXPRESSION
  6. RESULTS
  7. DISCUSSION
  8. Acknowledgements
  9. REFERENCES

Readily visualized transgenes, primarily lacZ, have been used widely to assess complex developmental processes. Transplantation and migration studies have used cells marked with a GFP or lacZ transgene driven by a constitutive promoter. Transduction of transplanted cells(34, 35) or microinjection of resident cells(36, 37) with expressing marker transgenes has indicated that a specific cell population can be discriminated within a complex tissue. Developmental studies of solid tissues have relied on differentially regulated promoters to drive the marker transgene. The lineage of cells within the gut epithelium,(19) muscle,(38) kidney,(39) brain,(40) and neural crest tissues(41) are excellent examples of this experimental approach. Although in situ hybridization and immunological techniques can be used to appreciate the microheterogeneity in a developing or remodeling tissue, the ease and specificity of detecting a visible marker gene has great experimental appeal.

Most studies using lacZ in bone and connective tissue have been performed in embryonic samples. Less success has been found in adult tissues because of technical difficulties of infusing the lacZ substrate and the high background caused by endogenous β-galactosidase activity. For the marker gene approach to be of value in defining stages of development within the osteoprogenitor lineage, a marker needs to be developed that can be observed in real time in osteoblastic culture models and reliably interpreted in histological sections of developing and mature bone.

The use of the Col1a1 promoter fragments driving GFP appears to fulfill many of the requirements of a marker gene for cell lineage studies in bone. The fluorescent signal is observed easily in primary osteoblastic cultures derived from neonatal or adult mice allowing the progression of individual colonies to be recorded repeatedly over time. The signal is detected with filters optimized for a specific GFP and the only significant background fluorescence is located in the mineralized nodules. The progress of differentiation of the entire culture can be assessed by recording the pattern and intensity of the fluorescent signal with fluorescent imagers. The number of GFP-positive cells and their relative level of fluorescence can be assessed by flow cytometry. The ability to identify these cells by cytometric analysis raises the possibility of their isolation by cell sorting. This application could be a major contribution to the interpretation of microarray expression studies of culture models that are inherently heterogeneous in cellular composition.

The other advantage of these transgenes is the ability to retain their fluorescent property after extensive tissue preparation steps of paraformaldehyde fixation, decalcification, and paraffin embedding. In contrast, histological studies using eGFP (Clontech) call for cryosections or low temperature paraffin processing to preserve the fluorescent signal.(42) Standard paraffin processing preserves the histological architecture of bone allowing the GFP signal to be recorded in unstained sections. The GFP can be viewed by confocal or mercury lamp fluorescent microscopy. The signal observed is not dependent on diffusion of a substrate and appears to be stable indefinitely. Optimal selection of filters is necessary to distinguish the GFP signal from background fluorescence that is most pronounced in the bone marrow. Optimized GFP filters do not discriminate GFP from the background, but addition of a rhodamine or Texas red filter did separate the two as a yellow-green GFP signal and an orange-red autofluorescent background.

Perhaps one explanation for the success of using GFP in intact mice in this study relates to the strength of the promoter used to drive expression. Previous published work has used actin,(43) cardiac actin,(44) versican,(45) Tie2,(46) or Col2a1(47) promoter in connective tissues and glial fibrillary acidic protein promoter in brain.(18) Weaker promoters driving lacZ have been used successfully in developmental studies presumably because of the relatively stronger activity of the promoter during embryogenesis and the ease of diffusing the substrate. GFP has advantages here too,(48) particularly when development can progress in organ culture allowing these events to be observed over time.(49) In the case of this mouse study, it was possible to visualize the skeleton through the developing embryo in the fluorescent tissue culture microscope (data not shown) as well as genotype neonatal mice based on fluorescence of the distal tail.

In both MSC and mCOB cultures, the two promoters used to drive GFP expression had distinctly different patterns of expression during osteoblast differentiation in vitro. The pOBCol3.6GFP transgene became active coincident with Col1a1 and ALP expression in the culture and was localized in the early cluster of cells destined to become a nodule. It continued to remain active as the culture matured and developed two levels of expression. Cells with lower expression surrounded the mineralized portion of the nodule and extended into the internodular area and those with strong expression were contained within the mineralized areas. Within intact bone, two different populations of cells were observed with different levels of fluorescent intensity. Cells within the periosteal layer of metaphyseal bone, often with an elongated shape, showed a weak fluorescence. Strong fluorescence was observed in the cuboidal-shaped cells lining the surface of cortical and trabecular bone and cells within the forming bone. The number of lining cells diminished in older mice and those cells within bone that retained fluorescence were close to the bone surface. We interpret these finding to indicate that the Col3.6 promoter drives a transgene that is expressed at a lower level in preosteoblastic cells that have the potential to become fully differentiated bone cells. When osteoblastic differentiation occurs, the promoter is up-regulated and remains active for a period of time after the bone cell is embedded within the bone matrix. However, in mature mice, expression appears to fall in certain bone-lining cells that may represent quiescent bone cells that can become reactivated during bone turnover. This interpretation may explain the clusters of positive cells on the bone surface observed in mature bone. Loss of expression also is apparent in osteocytes that are buried deep within the bone matrix.

In contrast, the pOBCol2.3GFP in cultured primary cells is activated much later than pOBCol3.6GFP and is restricted to cells within the mineralized portion of the nodule. Unlike pOBCol3.6GFP, pOBCol2.3GFP was expressed at a uniform level by cells that have a cuboidal appearance. The pattern of fluorescence in intact bone is different from pOBCol3.6GFP. Only bone-lining cells and cells within the matrix were GFP-positive. The number of osteocytes continuing to express pOBCol2.3GFP is higher than pOBCol3.6GFP. We interpret this pattern to reflect a promoter fragment that marks a cell late in the osteoblast lineage that extends into mature osteocytes.

Increasingly, the modular design of promoters is becoming appreciated in which all the elements interact to define endogenous expression. Taking the element out of their full context can create a promoter activity that may be advantageous for identifying a subset of cells that can use the construct but which do not reflect the total pattern of expression of the endogenous gene. For example, it would appear that deletion of the fragment from 3.6-2.3 removes an element that prevents the pOBCol1a1 promoter fragment from expression in mature osteocytes. The pOBCol3.6GFP transgene may be useful in isolating a preosteoblastic cell but only in primary cultures during the 3-4 days preceding osteoblastic differentiation. In contrast, isolating cells expressing the pOBCol2.3GFP transgene would represent a relatively homogeneous subpopulation of highly differentiated osteoblastic cells. Although these constructs do not specifically mark cells in the osteoblastic lineage, they can be useful for identifying a subpopulation of cell in culture models that are derived from a tissue composed of the particular lineage. For example, pOBCOL3.6GFP could be used to identify preosteoblastic cells in primary bone cell cultures or subpopulation of dermal of lung fibroblasts with different levels of Col1a1 production. The value of the construct for marker studies of any tissue will have to be assessed individually. However, in the case of bone, these markers do appear to have experimental value for assessing lineage progression within the osteoprogenitor pathway.

Although this work shows the usefulness of the Col1a1 promoters for discriminating different levels of osteoblastic differentiation, the full potential of this approach requires a larger number of promoter to be investigated, which may have greater developmental specificity, assuming they have sufficient promoter strength. Use of multiple promoters and fluorescent isomers of GFP may make it possible to identify cells of the osteoblastic lineage by the combinatorial definition of color expression, tempo, and spatial localization. The transgene has proven to be extremely reliable over time both in primary culture and intact mice. Development of mice with these properties may become a valuable reagent to standardize mice and culture models in different laboratories studying the regulation of the osteoprogenitor lineage in response to exogenous perturbations and endogenous genetic manipulation.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. ANALYSIS OF TRANSGENE EXPRESSION
  6. RESULTS
  7. DISCUSSION
  8. Acknowledgements
  9. REFERENCES

This study was supported by the National Institute of Arthritis and Musculoskeletal Disease grant AR43457. I. Kalajzic holds a Michael Geisman Fellowship from the Osteogenesis Imperfecta Foundation.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. ANALYSIS OF TRANSGENE EXPRESSION
  6. RESULTS
  7. DISCUSSION
  8. Acknowledgements
  9. REFERENCES
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