Green Fluorescent Protein Transgene Research Articles

Longitudinal in vivo imaging of retinal gliosis in a diabetic mouse model

In this study, we visualize and quantify retinal gliosis in vivo for monitoring early diabetic retinopathy (DR) in a transgenic mouse model. Onset of diabetes was triggered via intraperitoneal injection of streptozotocin (STZ) into transgenic F1 hybrid (FVB/N × C57BL/6J) mice expressing green fluorescent protein (GFP) under the control of glial fibrillary acidic protein (GFAP) promoter. Retinal glial cells are imaged once pre-STZ treatment followed by weekly post-STZ imaging for five weeks using a confocal scanning laser ophthalmoscope. Mice develop diabetes one week after STZ induction as confirmed from the high blood glucose levels (>13.9 mmol/L). A significant increase is observed in the GFAP–GFP transgene expression from astrocytic cell bodies and processes as early as week 5 for the STZ-treated mice. Retinal astrocytes also undergo hyperplasia progressively from week 0 to 5. This precedes any structural abnormalities to the retinal vasculature. Immunohistochemistry (IHC) on retinal sections as well as quantitative RT-PCR of endogenous and transgene GFAP mRNA supports our in vivo observation. Our in vivo data correlates with clinical reports with regards to retinal gliosis-related inflammatory response during early diabetic retinopathy. This opens up the possibility of using in vivo molecular imaging of retinal glial cells as a platform for monitoring the efficacy of anti-DR drug candidates which intervene at an early stage.

Use of the parabiotic model in studies of cutaneous wound healing to define the participation of circulating cells

Previous experimental studies to assess the contribution of blood-borne circulating (BBC) cells to cutaneous wound healing have relied on discontinuous pulsing of labeled BBC elements or bone marrow transplant protocols. Such approaches do not allow the examination of stable BBC cells that have matured in a physiologically normal host. We have used a parabiotic murine model for cutaneous wound healing to evaluate the relative contribution of stable populations of peripheral blood cells expressing the green fluorescent protein (GFP) transgene in otherwise normal animals. Circulating cells (mature and immature) expressing the GFP transgene were easily detected and quantified in wounds of GFP- parabiotic twins during all evaluated stages of the healing response. Using multiple antibody probes, the relative contribution of various subsets of BBC cells could be comparatively assessed. In early wounds, some cells expressing mesenchymal epitopes were documented to be of hematopoietic origin, indicating the utility of this model in assessing cell plasticity in the context of tissue regeneration and repair. Application of this approach enables further investigation into the contribution of peripheral blood in normal and abnormal healing responses.

Letting the Animals Teach

Letting the Animals Teach

Genetic Modification of Airway Progenitors after Lentiviral Gene Delivery to the Amniotic Fluid of Murine Fetuses

Lentiviral vectors with the firefly luciferase or enhanced green fluorescent protein (EGFP) transgenes were delivered to the amniotic fluid of murine fetuses at Embryonic Day (E) 14.5 or E16.5. Whole-body imaging of luciferase recipients after birth demonstrated transgene expression in the peritoneal and thoracic regions. Organ imaging showed luciferase expression in lung, skin, stomach, and/or intestine. Histological immunofluorescence analysis of EGFP recipients demonstrated that small clusters (≤ three cells) of EGFP-positive epithelial cells were present in the large and small airways of recipients at up to 7 months (n = 11). There was no difference in the frequency of transgene expression in mice injected at E14.5 or E16.5 in respiratory or nonrespiratory organs. Analysis of the bronchoalveolar duct junctions on tissue sections of recipient mice identified multiple EGFP-positive epithelial cells. Cells coexpressing EGFP, Clara cell 10-kd protein, and surfactant protein C (SPC) were also found in lungs, consistent with the transduction of bronchoalveolar stem cells. Next, naphthalene lung injury in both luciferase and EGFP recipients was performed to determine whether transduced cells could contribute to tissue repair. In luciferase recipients, the whole-body luciferase signal increased 2- to 20-fold at 2 weeks after naphthalene treatment. Remarkably, immunohistological analysis of the lungs of EGFP recipients after lung injury repair demonstrated repopulation of airways with long stretches of EGFP-positive epithelial cells (n = 4). Collectively, these data demonstrate that lentiviral gene delivery to the amniotic fluid of murine fetuses genetically modifies long-lived epithelial progenitors capable of contributing to lung injury repair.

Reporter-Based Isolation of Induced Pluripotent Stem Cell– and Embryonic Stem Cell–Derived Cardiac Progenitors Reveals Limited Gene Expression Variance

Induced pluripotent stem (iPS) cells can differentiate into multiple cell types, including cardiomyocytes and have tremendous potential for drug discovery and regenerative therapies. However, it is unknown how much variability exists between differentiated lineages from independent iPS cell lines and, specifically, how similar iPS cell-derived cardiomyocytes (iPS-CMs) are to embryonic stem (ES) cell-derived cardiomyocytes (ES-CMs). We investigated how much variability exists between differentiated lineages from independent iPS cell lines and how similar iPS-CMs are to ES-CMs. We generated mouse iPS cells in which expression of NKX2-5, an early cardiac transcription factor, was marked by transgenic green fluorescent protein (GFP). Isolation of iPS- and ES-derived NKX2-5-GFP(+) cardiac progenitor pools, marked by identical reporters, revealed unexpectedly high similarity in genome-wide mRNA expression levels. Furthermore, the variability between cardiac progenitors derived from independent iPS lines was minimal. The NKX2-5-GFP(+) iPS cells formed cardiomyocytes by numerous induction protocols and could survive upon transplantation into the infarcted mouse heart without formation of teratomas. Despite the line-to-line variability of gene expression in the undifferentiated state of ES and iPS cells, the variance narrows significantly in lineage-specific iPS-derived cardiac progenitors, and these progenitor cells can be isolated and used for transplantation without generation of unwanted cell types.

Acid‐sensing ion channels in rat hypothalamic vasopressin neurons of the supraoptic nucleus

Body fluid balance requires the release of arginine vasopressin (AVP) from the neurohypophysis. The hypothalamic supraoptic nucleus (SON) is a major site of AVP synthesis, and AVP release is controlled somatodendritically or at the level of nerve terminals by electrical activities of magnocellular neurosecretory cells (MNCs). Acid-sensing ion channels (ASICs) are neuronal voltage-insensitive cationic channels that are activated by extracellular acidification. Although ASICs are widely expressed in the central nervous system, functional ASICs have not been assessed in AVP neurons. ASICs are modulated by lactate (La(-)), which reduces the extracellular calcium ion concentration. We hypothesize that ASICs modify neuronal function through La(-) that is generated during local hypoxia resulting from osmotic stimulation in the SON. In the present study, we used the whole-cell patch-clamp technique to show that acid-induced ASIC current is enhanced by La(-) in isolated rat SON MNCs that express an AVP-enhanced green fluorescent protein (eGFP) transgene. Immunohistochemistry and multi-cell reverse transcriptase-polymerase chain reaction experiments revealed that these neurons express the ASIC1a and ASIC2a subunits. In addition, increased La(-) production was specifically observed in the SON after osmotic stress. These results suggest that interaction between ASICs and La(-) in the SON plays an important role in the regulatory mechanism of body fluid homeostasis.

Gene Correction in Human Embryonic and Induced Pluripotent Stem Cells: Promises and Challenges Ahead

The sequencing of the human genome has greatly facilitated our ability to identify the gene candidates that are critical for developmental regulation and disease progression. However, investigators seeking to use such data to gain a better understanding of the developmental biology and therapeutic potential of pluripotent stem cell types may be frustrated by the limitations of current genetic engineering techniques. Targeted genomic manipulation of mammalian cells is inefficient, and unintended effects of such manipulation on the cells’ developmental and proliferative potential remain uninvestigated. To this end, the recent demonstration of targeted gene correction in human induced pluripotent stem cells (hiPSCs) before and after reprogramming, as reported in this issue of Molecular Therapy, warrants special mention.1

Efficient Isolation and Chondrogenic Differentiation of Adult Mesenchymal Stem Cells with Fibrin Microbeads and Micronized Collagen Sponges

Mesenchymal stem cells (MSCs) have been demonstrated to potentially undergo chondrogenic differentiation. We propose a new matrix for stem cell-based chondrogenesis using dense fibrin microbeads (FMBs) combined with grounded dehydrothermally crosslinked collagen sponges (micronized collagen). In this study, MSCs were isolated from bone marrow of transgenic green fluorescent protein C57/Bl mice by FMBs in high yield. After 48 h in slowly rotating suspension culture, micronized collagen was added. The cells on the FMBs migrated to the collagen pieces and formed aggregates that developed into cartilage-like structures. Following chondrogenic differentiation, alcian blue staining and collagen type II immunohistochemistry demonstrated the presence of chondrocytes in the 3D structures. PCR for the expression of aggrecan and collagen type II genes supported these findings. The in vitro structures that formed were used for ectopic subdermal implantation in wild-type C57/Bl mice. However, the chondrogenic markers faded relative to the pre-implant in vitro structures. We propose that FMBs with micronized collagen could serve as a simple technology for MSC isolation and chondrogenesis as a basis for implantation.

ORIGINAL ARTICLE: The Persistence of Paternal Antigens in the Maternal Body is Involved in Regulatory T‐Cell Expansion and Fetal‐Maternal Tolerance in Murine Pregnancy

Citation Zenclussen ML, Thuere C, Ahmad N, Wafula PO, Fest S, Teles A, Leber A, Casalis PA, Bechmann I, Priller J, Volk H‐D, Zenclussen AC. The persistence of paternal antigens in the maternal body is involved in regulatory T‐cell expansion and fetal‐maternal tolerance in murine pregnancy. Am J Reprod Immunol 2010; 63: 200–208Problem Mammalian pregnancy is a state of immunological tolerance and CD4+ CD25+ regulatory T cells (Treg) contribute to its maintenance. Knowing that Treg act in an antigen‐specific way during pregnancy, we hypothesized that they are generated after maternal immune cells encounter paternal antigens.Method of study We mated wild type females with transgenic green fluorescent protein (GFP) males in an allogenic setting and killed them on different days of pregnancy.Results Presence of paternal and maternal MHC class II+ cells in vaginal lavage on day 0.5 of pregnancy was confirmed. Thus, antigen presentation may take place early during pregnancy in the periphery either by the direct or indirect pathways. Foxp3+ cells known to have regulatory activity could be detected on day 2 of pregnancy in lymph nodes and shortly after implantation at the fetal‐maternal interface.Conclusion Our data suggest that paternal antigens are processed early during pregnancy, which leads to the generation of Treg. The continuous release of placental antigens into the maternal circulation allows the maintenance of a Treg population which is specific for paternal antigens and mediates tolerance toward the semi‐allogeneic fetus until the time point of birth.

Intravital imaging of DSS-induced cecal mucosal damage in GFP-transgenic mice using two-photon microscopy

Two-photon laser-scanning microscopy (TPLSM) is a powerful diagnostic tool for real-time, high-resolution structural imaging. However, obtaining high-quality in vivo TPLSM images of intra-abdominal organs remains technically challenging. An organ-stabilizing system was applied to high-quality TPLSM imaging. Real-time imaging of visceral organs, such as the liver, spleen, kidney and intestine, of transgenic green fluorescent protein (GFP) mice was performed in vivo using TPLSM. The bacterial translocation model using dextran sodium sulfate (DSS)-induced colitis was also investigated in prepared GFP mice following simple surgery. This allowed the capture of morphological real images using in vivo TPLSM. Immunohistochemical analysis of ZO-1 was performed to support the morphological findings of TPLSM. We established an organ-stabilizing system to evaluate the real-time imaging of visceral organs in actin-GFP transgenic mice using in vivo TPLSM. DSS-induced colitis showed irregularity of crypt architecture, disappearance of crypts, inflammatory cell infiltration and increased rolling of white blood cells along the vasculature. In addition, the intercellular distance of mucosal cells in the crypt and vascular endothelial cells in the intestinal wall was increased in the intestinal mucosa during DSS colitis. In DSS colitis, there was remarkable loss of mucosal and vascular endothelial ZO-1 expression, as could be seen by a decrease in ZO-1 staining. In conclusion, our observations suggested the possibility that our TPLSM imaging system can be used to clarify the pathophysiological changes in various diseases using longitudinal studies of microscopic changes in the same animal over long periods of time.

Efficient phagosomal maturation and degradation of Plasmodium-infected erythrocytes by dendritic cells and macrophages

Dendritic cells (DC) and macrophages phagocytose pathogens and degrade them in their phagosomes to allow for proper presentation of foreign antigens to other cells of the immune system. The Plasmodium parasite, causative agent of malaria, infects RBC that are phagocytosed by DC and macrophages during the course of infection. Under specific conditions, the functionality of these cells can be affected by phagocytosis of Plasmodium-infected RBC. We investigated whether phagosomal maturation and degradation of Plasmodium yoelii-infected RBC in phagosomes is affected in DC and macrophages. We show that recruitment of the phagolysosomal marker Lamp-1 and of MHC-II, as well as acidification of phagosomes, was achieved in a timely manner. Using P. yoelii-infected RBC labelled with a fluorescent dye or transgenic green fluorescent protein (GFP)-expressing parasites, we found a gradual, rapid decrease in the phagosome fluorescence signal, indicating that P. yoelii-infected RBC are efficiently degraded in macrophages and DC. We also observed that pre-incubation of DC with infected RBC did not affect phagosomal maturation of newly internalized P. yoelii-infected RBC. In conclusion, after phagocytosis, Plasmodium-infected RBC are degraded by DC and macrophages, suggesting that the process of phagosomal maturation is effectively completed in malaria.

423 PRODUCTION OF CLONED PIGS EXPRESSING APOLIPOPROTEIN E-SPECIFIC SMALL HAIRPIN (shRNA)

Apolipoprotein E (apo E) is a known risk factor for developing premature atherosclerosis and Alzheimer’s syndrome. The aim of this study was to create a pig model with reduced apo E levels using RNA interference (RNAi) and somatic cell nuclear transfer (SCNT) technologies. Three synthetic small interfering RNA targeting the porcine apo E mRNA were designed, and the knockdown efficiency was assessed in cultured porcine granulosa cells by real-time PCR. The observed apo E knockdown efficiency ranged from 45 to 82% compared with control cells, indicating the targeted degradation of apoE mRNA.A small hairpin RNA (shRNA) expressing vector was constructed in PRNAT.U6.Neo (Genscript Corp., Piscataway, NJ, USA) based on the most effective apo E RNAi sequence under the control of polymerase III (U6) promoter, and then introduced into fetal porcine fibroblast cells. Clones were selected by neomycin treatment and green fluorescent protein (GFP) expression. SCNT was performed using IVM oocytes collected from prepubertal gilts. Oocyte maturation and activation and embryo culture were performed as previously described (Nascimento et al. 2009 Reprod. Domest. Anim. in press). Embryos were cultured in vitro for 5 to 6 days, briefly exposed to fluorescent light to confirm GFP expression, and then surgically transferred into the uterus of recipient gilts. The recipient gilts were synchronized by daily oral administration of altrenogest (20 mg day-1; Regu-Mate®, Intervet, Millsboro, MD, USA) for 12 or 13 days, followed by 1000 IU of eCG injected in the last day of altrenogest treatment and 500 IU of hCG 72 h later. Pregnancy diagnosis was performed by ultrasonography at Day 20 to 25 after embryo transfer, and parturition was induced by injecting PGF2? (10 mg of dinoprost tromethamine; Lutalyse®, Pfizer Canada Inc., Montreal, QC, Canada) at Day 115 of pregnancy. Rates of cleavage (74.7%) and development to the blastocyst stage (37.2%) were comparable with that of embryos reconstructed with nontransfected cells from the same cell line. A total of 309 embryos were transferred to 5 recipients, of which 3 became pregnant and farrowed. Seven live and 1 stillborn piglets were delivered naturally. The presence of the introduced plasmid and the expression of the GFP transgene tag were confirmed by PCR in placental and umbilical tissues of all the piglets. Six cloned pigs have survived after weaning and exhibit no obvious morphological defects. The status of apo E gene expression is currently under investigation. Supported by a NSERC Discovery Grant to VB.

Generation of Novel AAV Variants by Directed Evolution for Improved CFTR Delivery to Human Ciliated Airway Epithelium

Recombinant adeno-associated virus (AAV) vectors expressing the cystic fibrosis transmembrane conductance regulator (CFTR) gene have been used to deliver CFTR to the airway epithelium of cystic fibrosis (CF) patients. However, no significant CFTR function has been demonstrated likely due to low transduction efficiencies of the AAV vectors. To improve AAV transduction efficiency for human airway epithelium (HAE), we generated a chimeric AAV library and performed directed evolution of AAV on an in vitro model of human ciliated airway epithelium. Two independent and novel AAV variants were identified that contained capsid components from AAV-1, AAV-6, and/or AAV-9. The transduction efficiencies of the two novel AAV variants for human ciliated airway epithelium were three times higher than that for AAV-6. The novel variants were then used to deliver CFTR to ciliated airway epithelium from CF patients. Here we show that our novel AAV variants, but not the parental, AAV provide sufficient CFTR delivery to correct the chloride ion transport defect to ~25% levels measured in non-CF cells. These results suggest that directed evolution of AAV on relevant in vitro models will enable further improvements in CFTR gene transfer efficiency and the development of an efficacious and safe gene transfer vector for CF lung disease.

Transcriptional Changes in Somatic Cells Recovered From Embryonic Stem–Somatic Heterokaryons

Following fusion, embryonic stem cells (ESCs) are capable of reprogramming somatic cells in cell hybrids. It has also been shown that transcriptional changes can occur in a heterokaryon, without nuclear hybridization. However, it is unclear whether these changes can be sustained after the removal of the dominant nucleus. In this study, we analyze the changes in embryonic stem (ES)-somatic heterokaryons following the removal of the ESCs nucleus. We also show that after ES-somatic cell fusion using tetraploid ESCs, a heterokaryon can be reverted to an autologous diploid state by differential enucleation of the denser tetraploid ES nucleus. To recover somatic cells from ES-somatic heterokaryons, we fused tetraploid ESCs containing the thymidine kinase (TK) suicide gene with mesenchymal stem cells containing a green fluorescent protein (GFP) transgene under the control of the OCT4 promoter. Following post-fusion enucleation (PFE), negative selection against the tetraploid ES genome was achieved using ganciclovir. The resulting GFP-positive clones were analyzed and shown to have undergone changes in growth characteristics, alkaline phosphatase activity, and gene expression using RT-PCR and microarray analysis. These results demonstrate that a change in transcriptional expression can be detected in somatic cells after the removal of the ES nucleus from ES-somatic heterokaryons.

Exaggerated response of a vasopressin-enhanced green fluorescent protein transgene to nociceptive stimulation in the rat.

Nociceptive stimulation elicits neuroendocrine responses such as arginine vasopressin (AVP) release as well as activation of the hypothalamo-pituitary-adrenal axis. We have generated novel transgenic rats expressing an AVP-enhanced green fluorescent protein (eGFP) fusion gene, and we examined the effects of nociceptive stimulation on transgene expression in the hypothalamus after subcutaneous injection of saline or formalin into the bilateral hindpaws in these rats. We have assessed (1) AVP levels in plasma and the changes of eGFP mRNA and AVP heteronuclear RNA (hnRNA) in the supraoptic nucleus (SON) and the paraventricular nucleus (PVN) using in situ hybridization histochemistry, (2) gene expression changes in distinct magnocellular and parvocellular divisions of the PVN, (3) eGFP fluorescence in the SON, the PVN, the median eminence (ME), and the posterior pituitary gland (PP). Plasma AVP levels were significantly increased 15 min after formalin injection. In the same time period, the AVP hnRNA levels in the PVN were increased, especially in the parvocellular division of the PVN in formalin-injected rats. In the same region, eGFP mRNA levels after formalin injection were also significantly increased to a much greater extent than those of AVP hnRNA. The eGFP fluorescence in the SON, the PVN, the ME, and the PP was markedly increased in formalin-injected rats and especially increased in the parvocellular divisions of the PVN. Together, our results demonstrate robust and rapid changes in the expression of the AVP-eGFP transgene in the rat hypothalamus after acute nociceptive stimulation.

Tissue-Specific Activities of an Immune Signaling Module Regulate Physiological Responses to Pathogenic and Nutritional Bacteria in C. elegans

Microbes represent both an essential source of nutrition and a potential source of lethal infection to the nematode Caenorhabditis elegans. Immunity in C. elegans requires a signaling module comprised of orthologs of the mammalian Toll-interleukin-1 receptor (TIR) domain protein SARM, the mitogen-activated protein kinase kinase kinase (MAPKKK) ASK1, and MAPKK MKK3, which activates p38 MAPK. We determined that the SARM-ASK1-MKK3 module has dual tissue-specific roles in the C. elegans response to pathogens--in the cell-autonomous regulation of innate immunity and the neuroendocrine regulation of serotonin-dependent aversive behavior. SARM-ASK1-MKK3 signaling in the sensory nervous system also regulates egg-laying behavior that is dependent on bacteria provided as a nutrient source. Our data demonstrate that these physiological responses to bacteria share a common mechanism of signaling through the SARM-ASK1-MKK3 module and suggest the co-option of ancestral immune signaling pathways in the evolution of physiological responses to microbial pathogens and nutrients.

Construction of a binary transgenic gene expression system for recombinant protein production in the middle silk gland of the silkworm Bombyx mori

To construct an efficient system for the production of recombinant proteins in silkworm (Bombyx mori), we investigated the promoter activity of the silkworm sericin 1, 2, and 3 genes (Ser1, Ser2, and Ser3) using a GAL4/UAS binary gene expression system in transgenic silkworm. The promoter activity of the upstream region of Ser1 was strong, yielding high expression of an enhanced green fluorescent protein (EGFP) transgene in the middle and posterior regions of the middle silk gland (MSG) after day 2 of the fifth instar. The Ser3 upstream region exhibited moderate promoter activity in the anterior MSG, but the Ser2 upstream region did not exhibit any promoter activity. Since the strongest promoter activity was observed for Ser1, we devised a system for the production of recombinant proteins using a GAL4-Ser1 promoter construct (Ser1-GAL4). Transgenic silkworms harboring both the Ser1-GAL4 construct and the previously reported upstream activating sequence (UAS)-EGFP construct, which contains the TATA box region of the Drosophila hsp70 gene, yielded approximately 100 microg EGFP per larva. When we then analyzed the TATA box region, signal peptide, and intron sequences for their effects on production from the UAS-EGFP construct, we found that the optimization of these sequences effectively increased production to an average of 500 microg EGFP protein per transgenic larva. We conclude that this binary system is a useful tool for the mass production of recombinant proteins of biomedical and pharmaceutical interest in silkworm.

Unconditioned adult-derived neurosphere cells mainly differentiate towards astrocytes upon transplantation in sclerotic rat hippocampus

Cell transplantation is being investigated as an alternative treatment for medically refractory temporal lobe epilepsy (TLE). In this study the fate of adult-derived neurosphere cells was evaluated after transplantation in the lesioned hippocampus of the intrahippocampal kainic acid (KA) model for TLE. Neurosphere-forming cells were derived from the subventricular zone (SVZ) of transgenic green fluorescent protein (GFP) reporter mice and expanded in culture. After 10 passages in vitro neurosphere-derived cells were transplanted in the hippocampus three days (KA3d group) and three weeks (KA3w group) after intrahippocampal KA injection. Survival and differentiation of neurosphere cells were evaluated three and six weeks after transplantation. A fraction (about 1%) of GFP-expressing neurosphere cells survived for at least six weeks after transplantation with a higher and more robust survival rate in the KA3d compared to the KA3w group. Although a small fraction of the cells expressed the neuronal marker NeuN, neurosphere cells mainly differentiated towards astrocytes. Our results indicate that adult-derived neurosphere cells are able to survive upon transplantation in the sclerotic hippocampus. The transplanted cells do not or hardly contribute to neuronal replacement and mainly adopt an astrogliotic fate.

Green fluorescent protein has no effect on blastocyst development in C57BL/6-Tg(ACTB-EGFP)1Osb/J mice

The changes in the state of long-term culture of blastocysts derived from female C57BL/6 mice after crossing with C57BL/6-Tg(ACTB-EGFP)1Osb/J males with a green fluorescent protein transgene EGFP on chromosome 15 were studied. Possible causes of different culture results were analyzed: the preservation of undifferentiated cells as dense clusters in the inner cell mass or their differentiation into extraembryonic endoderm. Comparison of the events going in blastocysts with the -/- or -/EGFP genotypes demonstrated that the GFP presence has no effect on cell processes. This allows us to use embryonic material from this mouse line in experiments that require long-term vital observation of embryonic cells.

Neuropeptide Y is expressed by osteocytes and can inhibit osteoblastic activity

Osteocytes are the most abundant osteoblast lineage cells within the bone matrix. They respond to mechanical stimulation and can participate in the release of regulatory proteins that can modulate the activity of other bone cells. We hypothesize that neuropeptide Y (NPY), a neurotransmitter with regulatory functions in bone formation, is produced by osteocytes and can affect osteoblast activity. To study the expression of NPY by the osteoblast lineage cells, we utilized transgenic mouse models in which we can identify and isolate populations of osteoblasts and osteocytes. The Col2.3GFP transgene is active in osteoblasts and osteocytes, while the DMP1 promoter drives green fluorescent protein (GFP) expression in osteocytes. Real-time PCR analysis of RNA from the isolated populations of cells derived from neonatal calvaria showed higher NPY mRNA in the preosteocytes/osteocytes fraction compared to osteoblasts. NPY immunostaining confirmed the strong expression of NPY in osteocytes (DMP1GFP(+)), and lower levels in osteoblasts. In addition, the presence of NPY receptor Y1 mRNA was detected in cavaria and long bone, as well as in primary calvarial osteoblast cultures, whereas Y2 mRNA was restricted to the brain. Furthermore, NPY expression was reduced by 30-40% in primary calvarial cultures when subjected to fluid shear stress. In addition, treatment of mouse calvarial osteoblasts with exogenous NPY showed a reduction in the levels of intracellular cAMP and markers of osteoblast differentiation (osteocalcin, BSP, and DMP1). These results highlight the potential regulation of osteoblast lineage differentiation by local NPY signaling.