Ift140, which causes a mid-gestation embryonic lethal phenotype in homozygous mutant mice. Mutant embryos exhibit a range of phenotypes including exencephaly and spina bifida, craniofacial dysmorphism, digit anomalies, cardiac anomalies and somite patterning defects. A number of these phenotypes can be attributed to alterations in Hedgehog signalling, although additional signalling systems are also likely to be involved. We also report the identification of a homozygous recessive mutation in IFT140 in a Jeune syndrome patient. This ENU-induced Jeune syndrome model will be useful in delineating the origins of dysmorphology in human ciliopathies.Cilia are architecturally complex organelles that protrude from the cell membrane and have signalling, sensory and motility functions that are central to normal tissue development and homeostasis. There are two broad categories of cilia; motile and non-motile, or primary, cilia. The central role of primary cilia in health and disease has become prominent in the past decade with the recognition of a number of human syndromes that result from defects in the formation or function of primary cilia. This rapidly growing class of conditions, now known as ciliopathies, impact the development of a diverse range of tissues including the neural axis, craniofacial structures, skeleton, kidneys, eyes and lungs. The broad impact of cilia dysfunction on development reflects the pivotal position of the primary cilia within a signalling nexus involving a growing number of growth factor systems including Hedgehog, Pdgf, Fgf, Hippo, Notch and both canonical Wnt and planar cell polarity. We have identified a novel ENU mutant allele of  Ift140 gene, and these mice exhibit phenotypes that are often seen in this set of human syndromes. We have complimented this study with the discovery of a patient that presents with Jeune Syndrome resulting from mutation of human IFT140. This model will allow us to explore the role of IFT140 and the primary cilia in normal human development and provide insight into the field of human skeletal ciliopathies.Skeletal ciliopathies are an emerging field of human disease in which skeletal birth defects arise due to abnormal communication between cells. This failure in communication arises following mutation in components of the primary cilia, a hair-like structure present on every cell. The skeletal ciliopathies are debilitating and in severe cases lead to death in early infancy. However, the mechanisms by which these malformations come about remains unclear. Mouse models are often used to delineate the causes of human birth defects and we have identified a model that mimics one of these conditions known as Jeune syndrome. It is the first mouse model with a mutation in the  Primary cilia provide highly regulated cellular sensory functions with diverse roles in the development and maintenance of many tissues and organs Components of the primary cilia have to be transported from their site of synthesis within the cytoplasm to the cilium itself using a microtubule motor-based system known as intraflagellar transport (IFT). The axoneme grows from the distal tip and individual axonemal subunits must be transported along the nascent axoneme by IFT The IFT-A complex consists of at least 6 subunits with a highly stable core sub-complex of three proteins, while the IFT-B complex consists of around 14 subunits with a salt-stable core of 9 Over the past decade our appreciation of the importance of primary cilia function in development and disease has grown rapidly. There are now at least 12 disorders that have been attributed to mutations in cilia proteins and collectively they impact on nearly every organ system in the body The primary cilia are endowed with an array of signal transduction components such as ion channels, receptor tyrosine kinases, Notch receptors and matrix receptors including the Hedgehog signalling components Ptch1, Smo1 and Gli, PDGFRa, IGFR1, EGFR and Tie2 Ift140. This mutation results in mid-gestation lethality, exencephaly, craniofacial dysmorphism, skeletal malformation and mixed poly- and oligodactyly. A number of these defects can be attributed to disregulated Hedgehog signalling while other defects indicate involvement of additional signalling systems. Based on the identification of an Ift140 mutation in this mouse model we also sequenced the human orthologue in a cohort of SRP and Jeune syndrome patients. We present the identification of a novel IFT140 mutation in a case of Jeune syndrome. IFT140 has recently been implicated in Mainzer-Saldino and Jeune syndromes and conditional deletion in mice causes a polycystic kidney phenotype We carried out an ENU mutagenesis screen to identify genes with critical functions during early embryonic development cauli strain was identified via a comprehensive phenotype-driven ENU screen undertaken to identify novel genes involved in embryogenesis cauli homozygous embryos survive to approximately embryonic day (E) 13.5, however a small proportion has lived beyond this age (n = 10/130). No live embryos were obtained >E16.5. Fully penetrant phenotypes identified in cauli mutants include exencephaly (n = 130/130), anopthalmia (n = 130/130) and polydactyly of the hindlimbs  identified a T to A substitution in exon 19 at position 2564  were all predicted to be ‘tolerated’ by this analysis (http://toolkit.tuebingen.mpg.de/). Interestingly, secondary structure prediction using the PSIPRED algorithm We utilized two algorithms designed to assess the potential effect of the isoleucine to lysine mutation on Ift140 function. The prediction output from PolyPhen-2 for the analysis . This re+/+Ift140 and cauli/cauliIft140 limb buds by SEM  revealed severe craniofacial defects including failure of calvarial development, cervical vertebral fusion and agenesis or hypoplasia of many facial bones , lateral bifurcation (branching) and thickened ossified protuberances  is a downstream mediator of Fgf signalling. Expression of Dusp6 is elevated across the anterioposterior axis but is anteriorly expanded  is a component of cell to cell adherens junctions and is required for the maintenance of tight intracellular junctions and oriented alignment of the cytoskeletal networks ocalised . In a prse cells .cauli missense mutation on Ift140 function by examining the distribution of Ift140 in the primary cilia of limb bud epithelial cells. In wildtype epithelium Ift140 localises to the base and tip of the primary cilium  and a short chest cage were noted on a third trimester ultrasound performed for growth assessment. No other abnormalities were observed at this time. The couple were counselled about a likely poor prognosis given the degree of thoracic hypoplasia. The child was delivered at 37 weeks of gestation with a birth head circumference of 37 cm (>98th centile) and birth length of 48 cm (50th centile). The baby was in poor condition and required high frequency ventilation with high peak pressures (32 mm Mercury) due to pulmonary hypoplasia and hypertension. On examination, the child had short square fingers and hands with five digits each, low nasal bridge, normal ears and palate, and hepatomegaly. A small muscular ventricular septal defect was found on echocardiography, and ultrasound of the enlarged liver showed normal hepatic texture. Normal liver and renal function was documented. The baby deteriorated, despite ventilation, and died aged 7 days. Post mortem radiography was obtained and revealed short long bones, with metaphyseal cupping, bowed femora and humeri, short ribs and thorax, trident appearance to the acetabular roofs, and advanced tarsal bone ossification  and Jeune (OMIM 208500) syndromes IFT80, a component of the anterograde transport complex B, has also been demonstrated to cause Jeune syndrome There are now at least 12 clinical entities defined as ciliopathies cauli/cauliIft140 mutant mouse phenocopies many of the features of Jeune syndrome although the mouse phenotype is generally more extreme than in humans. The severe dysmorphology seen in cauli/cauliIft140 embryos is incompatible with continued development beyond mid-gestation in the majority of cases. Despite considerable efforts only a very small number of embryos have been obtained beyond E15.5 and in these cases severe skeletal malformation was observed. Skeletal phenotypes include severely disorganised ribs with extensive exostoses, vertebral and palatal defects and agenesis/hypoplasia of the craniofacial skeleton. These phenotypes are broadly consistent with those observed in Jeune syndrome cases including the case presented here. The range of phenotypes observed in cauli/cauliIft140 embryos is also broadly similar to that exhibited by other IFT-A mutant mice The cauli/cauliIft140 mutant phenotype may also reflect the nature of the Ift140 mutation in this model. While the cauli strain arose following a missense mutation in Ift140 we have observed a substantial reduction in both protein and RNA levels in mutant tissues. These data together with the identical phenotype observed in cauli/cauliIft140 and −/−Ift140 embryos suggests that the cauli phenotype results from loss of Ift140 rather than altered function following amino acid substitution. Ift140 is an essential component of the IFT-A complex and retrograde transport will be severely compromised in its absence. Thus, it is likely that much of the developmentally important signalling normally mediated through the primary cilia is compromised in cauli/cauliIft140 embryos resulting in the devastating phenotype observed.The severity of the cauli/cauliIft140 mutation results in loss of one third of the Ift140 RNA population. One possible explanation for this observation is that the c2564T>A mutation, which is 17 bp upstream of the 3′ splice site in exon 19, leads to an abnormal splicing event. It is likely that this would introduce a nonsense mutation downstream of the splicing event and the aberrantly spliced RNA would then be subject to nonsense mediated decay cauli mutation disrupts predicted overlapping SRSF5 and SRSF6 binding sites raising the possibility that an exonic splice enhancer (ESE) could be impacted A missense mutation would normally not be expected to result in RNA degradation and it is not clear why the cauli/cauliIft140 mutant embryos exhibit a dramatic loss of normal patterning, exhibiting both branching and irregular distribution. There does not appear to be an obvious loss of individual somites suggesting that segmentation of the lateral plate mesoderm proceeds relatively normally. However, irregular morphology of somites suggests that some aspects of somitogenesis, such as the mesenchymal to epithelial transition, may not be regulated appropriately. These somite patterning anomalies are likely responsible for the later rib defects in cauli/cauliIft140 since the ribs are derived from the dermomyotome, a dorsolateral compartment of the somites cauli/cauliIft140 mutant ribs suggests a disregulation of osteogenesis. cauli/cauliIft140 mutants exhibit elevated Ptch1 expression within the flank indicating that rib exostoses may result from altered Hh signalling. Mutants are significantly growth retarded relative to littermates, which is most likely related to the mid-gestational demise of most mutants but may also be affected by reduced axial growth. cauli/cauliIft140 mutants exhibit a convoluted neural tube, a phenotype thought to be caused by disruption of coordination between neural tube and axial elongation. Convergent extension defects have been linked to the disruption of primary cilia formation previously cauli/cauliIft140 mutants exhibit both cranial and lumbar level neural tube closure defects and it will be interesting in future studies to examine neurulating cauli/cauliIft140 embryos for defects in convergent extension activities.The somites in cauli/cauliIft140 mutants is similar to that reported in other cilia mutant mouse lines and is highly reminiscent of patterning defects exhibited by a number of Shh signalling mutants Grem and dHand, whose expression is linked to Shh and Gli3 action. Our analysis demonstrates that expression of these genes is expanded into the anterior of the autopod in cauli embryos where Gli3 repressor normally predominates, indicating that a loss of Gli3 repression has occurred. The ectopic expression of Ptch1 in the anterior limb bud is consistent with previously described IFT-A mutants The polydactylous phenotype observed in Shh is regulated by a complex network of transcription factors. Ectopic expression of dHand similar to that observed in the anterior limb bud of cauli embryos has been shown to be sufficient to induce ectopic ShhShh expression in the anterior limb bud and Twist1 null mice have polydactyly Twist1 in cauli/cauliIft140 mutants and it is therefore possible that the reduction in Twist1 levels is responsible for releasing the repression of Shh in the anterior limb bud. Further experimentation will be required to establish if this is the case and what role these changes have on autopod morphology.The expression of cauli/cauliIft140 hindlimbs and 50% of forelimbs. On the other hand, approximately one quarter of cauli/cauliIft140 forelimbs exhibit oligodactyly while it is never seen in the hindlimbs. The variable phenotype between fore- and hindlimbs is difficult to reconcile and suggests that inherent differences in signalling or temporal differences in developmental mechanisms between fore- and hindlimbs result in disparate outcomes. While the co-existence of both oligodactyly and polydactyly within forelimb autopod appears contradictory, especially in light of the highly penetrant polydactyly in the hindlimb autopod, it is important to note that digit number is a continuum and that the final outcome is the result of a complex interplay of transcriptional influences. Mixed forelimb oligo/polydactlyly is also observed in mice with reduced Twist1 levels Twist1 is at the heart of a transcriptional network involved in the control of Fgf and Shh signalling within the developing limb Twist1 results in reduced growth of the posterior limb bud and an expansion of the anterior limb bud due to ectopic anterior Shh expression Shh and Fgf signalling. We observe anterior ectopic Shh and it is possible that a similar mechanism operates in the cauli/cauliIft140 forelimb. In particular, given the likelihood that the cauli Ift140 mutation primarily disrupts IFT rather than any specific impact on Hh signalling, differences in the level of transport during development could provide significantly variable signalling outcomes. Defects in retrograde transport have been demonstrated to result in the accumulation of large amounts of IFT cargo along the length of the axoneme Polydactyly is observed in 100% of Ift140 disrupts ciliary transport in the motile cilia of the node. The consequences of disrupted IFT for motility are unclear but 50% of mice with immotile nodal cilia are able to generate normal left-right asymmetry. This suggests a mechanism of signalling in nodal cilia that are normally motile that is independent of the directional flow of signalling components in establishing asymmetry Ift140 impacts on cardiogenesis both during the looping phase and during later septation events.Motile cilia function at the node, the embryonic organiser, and help determine cardiac left-right asymmetry by the generation of directional flow of signalling components cauli/cauliIft140 mutant embryos precludes an analysis of a number of issues that are likely to result from Ift140 mutation. A targeted deletion of Ift140 in collecting ducts results in polycystic kidneys beginning around postnatal day 5 IFT140 mutations with a range of kidney pathologies including cysts cauli/cauliIft140 mutant kidneys appear normal at E13.5 which excludes agenesis but is too early to assess more typically IFT-A associated defects. Similarly, the IFT140 mutation described here in the patient with Jeune syndrome did not result in any obvious kidney dysfunction but this may be due to the early post-natal lethality of the condition in this case. An Ift144 mutation has been shown to result in craniofacial defects such as cleft palate cauli/cauliIft140 mutants die around the stage at which palate fusion occurs. Since cauli/cauliIft140 mutants are often growth retarded relative to littermates it is likely that palatal shelf elevation and fusion could be delayed making it difficult to determine if Ift140 mutation results in cleft palate. Similarly, mid-gestational lethality of cauli/cauliIft140 mutants makes it difficult or impossible to study the impact of Ift140 mutation on a range of other organ systems. Further analysis of these issues will require the use of conditional Ift140 alleles such as the one presented here, the value of which has already been demonstrated in the study of kidney pathology The early demise of Ift140 mouse model of Jeune syndrome. We support this with the identification of a recessively inherited IFT140 mutation in a patient with Jeune syndrome. The difference in severity of the phenotype between mice and humans harbouring IFT140 mutations makes a direct comparison difficult. However, this unique ENU-induced cauli/cauliIft140 mouse model will continue to provide insights into the mechanisms controlling normal limb and skeletal development and the congenital anomalies that arise when these signalling systems are perturbed. These insights will be invaluable in increasing our understanding of the origins of Jeune syndrome and related skeletal ciliopathies and provide a foundation for further analysis of the signalling systems regulated through the primary cilium.The data presented here describes the first cauli/cauliIFT140 embryos obtained from a large-scale ENU mutagenesis program at the Australian Phenomics Facility, Canberra. Embryonic stem cell clones and chimeric mice were generated from Ift140-targeted ES cell line EPD0073_5_G01 (www.komp.org) as part of the Australian Phenome Network, “ES Cell to Mouse” initiative. −/−Ift140 embryos were generated by crossing mice heterozygous for the floxed Ift140 allele with a CMV-Cre deletor strain and then back crossing fl/−Ift140 progeny. All mouse procedures were approved by the Murdoch Children's Research Institute Ethics Committee, MCRI AEC #A647, #A684 and #728. All experiments involved harvest of embryos and mice were culled according to MCRI SOP#21. Embryos were then cooled and placed into fixative.Overt craniofacial and limb phenotypes were identified in cauli line was maintained on an inbred C57BL6/J background and outcrossed to the C3H/HeH mapping strain. Linkage analysis was carried out using 13 F2 progeny using a panel of fluorescent microsatellite markers to localise the mutation to a specific chromosomal interval. Using the UCSC genome browser cauli embryos were screened for mutations in Ift140 (NM_134126) by sequencing all exons, intron/exon boundaries and most of the 5′ and 3′ untranslated regions.The Ift140 gene and DNA amplified with AmpliTaq (Roche) DNA Polymerase using standard PCR cycling conditions with an annealing temperature of 55°C. PCR products were sequenced with a BigDye v3.1 Terminator Cycle Sequencing Kit (Applied Biosystems) and products read using an ABI 3130xl capillary genetic analyser (Applied Biosystems). Sequencing chromatograms were compared to the published gDNA sequence and any differences identified and determined for potential pathogenicity using PolyPhen-2 and SIFT Gene-specific primers were designed for amplification of all 30 exons of the murine Ift140 exon 19 using primers Ift140.genF: 5′-CCAGAACTGGAAGCCAGAGT and Ift140.genR: 5′- CTGGGACTACTCACCAGCAT. For HRM, a PCR reaction was performed in 10 µl containing 2.5 µl of 1/500 diluted gDNA, 5 µl Lightcycler480 High Resolution Melting Master Mix (Roche), 3 mM MgCl2 and 0.2 µM of each primer. DNA was amplified for 45 cycles of 95°C for 10 sec, 62°C for 15 sec, 72°C for 5 sec. HRM melting curve data were obtained by slowly increasing the temperature from 75°C to 90°C at a rate of 0.02°C per second. Results were analysed by comparing each melt curve to control genotype DNA. Tissue from Ift140 null mice was extracted and amplified using a common forward PCR primer and two different allele specific reverse primers to produce unique products for each genotype. Primers are detailed in Adult mouse ear clips/tails and embryonic sacs were digested for genotyping in 0.4 mg/ml Proteinase K in Proteinase K buffer  overnight at 55°C. DNA was then purified by isopropanol extraction for amplification and High Resolution Melt (HRM) analysis of cauli p.I855K mutation was estimated using PolyPhen-2 (http://genetics.bwh.harvard.edu/pph/) where structural query options were set to default. A PSIC score above 0.85 identifies that the particular mutation was never or almost never observed in that protein family and would be classified as ‘probably-damaging’, scores of 0.15 to 0.85 classified as ‘possibly damaging’, and scores of <0.15 as ‘benign http://sift.jcvi.org/) was used to support the predicted effect of the cauli p.I855K amino acid substitution on protein function. A SIFT BLink analysis was performed using Ift140 protein ID NP_598887.3. Exonic splice enhancer analysis was performed using ESEfinder v3.0 (http://rulai.cshl.edu/cgi-bin/tools/ESE3/esefinder.cgi?process=home).Pathogenicity of the Adult female mice were anaesthetised with isoflurane and culled by cervical dislocation according to the National Health and Medical Research Council Australian code of practice for the care and use of animals for scientific purposes . Embryos were dissected free from all extra embryonic membranes in Phosphate Buffered Saline (PBS) and processed dependent on the subsequent application. Between 2–6 independent embryos were processed for each analysis. Heterozygous embryos were indistinguishable to wild-type.Embryos were fixed in 4% PFA for up to several weeks then transferred to 70% Ethanol prior to processing through xylene and paraffin using an Excelsior ES tissue processor (Thermo Scientific). Tissues were embedded in the required orientation using a Histocentre 2 Embedding Station (Shandon Life Sciences) and 10 µM sections cut on an MR-2 microtome (RMC products). A standard H&E protocol was followed with 5 min staining in hematoxylin, 1 min staining in eosin and mounted with Entellan (Merck). Images were taken on a Nikon Eclipse 80i microscope (Pathtech).Embryos >E16.5 were processed for skeletal preparations to visualize bone (Alizarin red) and cartilage (Alcian blue) as previously described E10.5 limb buds were dissected from wild-type, heterozygous and homozygous embryos, fixed and processed for scanning electron microscopy as previously described myogenin, Sox9 and Twist1 were amplified from RNA and ligated into pCRII-TOPO using the TOPO TA cloning kit (Invitrogen). Plasmids containing Msx1, Shh, Ptch1, Grem1, Gli3, dHand, Dusp6 and Fgf8 were obtained from C.Wicking, IMB, University of Queensland. RNA probes were generated by plasmid amplification with M13 primers, synthesised with T7 or SP6 RNA polymerase using a MAXIscript kit (Ambion), integrating dig-UTP (Roche). Probes were treated with Turbo DNase (Ambion) and unincorporated nucleotides removed using NucAway Spin Columns (Ambion). A sense probe was always generated for analysis of first-use probes.Total RNA was isolated from adult mouse brain tissue using a Qiagen RNeasy Midi Kit (Qiagen) or whole E11.5 wild-type embryos in Trizol (Invitrogen) by phenol/chloroform extraction. cDNA was generated from 1 µg RNA using random hexamers (Sigma) and BioScript reverse transcriptase (Bioline). cDNA fragments specific for the 3′UTR sequences of cauli embryos were processed for whole mount in situ hybridisation as previously described in situ riboprobe. After adequate washing to remove unbound probe, embryos were blocked with 10% sheep serum and incubated with an anti-digoxigenin-AP antibody . After additional washing, colour was developed with nitroblue tetrazolium (NBT) and 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and the reaction stopped with EDTA buffer. The colour was fixed with 4% PFA, background staining reduced by storage in 100% methanol and images taken using a Nikon Eclipse 80i microscope (Pathtech).E10.5–E13.5 Embryos at E10.5 were processed for immunohistochemistry by fixation in 4% paraformaldehyde (PFA)×30 min, permeabilised/blocked in 0.1% Triton X-100/2% bovine serum albumin (BSA) in PBS, and washed for several hours/overnight in PBS. Embryos were incubated with a rabbit anti-Ift140 polyclonal antibody  and mouse acetylated α-tubulin antibody  overnight at 4°C, washed for several hrs/overnight in PBS and incubated overnight in the dark with Alexa Fluor 488-conjugated goat anti rabbit IgG and Alexa Fluor 594-conjugated goat anti mouse IgG secondary antibodies . Following several washes in PBS, embryos were post fixed in 4% PFA×5 min and re-washed in PBS. Embryo limb tips were dissected off and mounted with Vectasheild (Vector Laboratories).For analysis of cellular architecture, E10.5 embryos were processed as above, incubated with a mouse monoclonal anti-E-cadherin , secondary Alexa Fluor 594-conjugated goat anti mouse IgG secondary antibody  and incubated with Alexa Fluor 488 phalloidin .Immunofluorescence was analysed using a Leica TCS SP2 laser scanning confocal microscope (Leica Microsystems).Independently, whole E11.5 wild-type, heterozygous and homozygous embryos were lysed by sonication  in Laemlli SDS Sample Buffer plus 0.1 M DTT. A western blot was performed using standard procedures. Briefly, 100 µg of protein from each genotype was run through a 6 and 10% Tris-Glycine gel and transferred onto PDVF membrane  for 1 hour at 100 V. Membranes were preblocked with 5% non-fat milk and incubated with a rabbit anti-Ift140 polyclonal  or mouse anti-Human Transferrin Receptor monoclonal  antibody. Protein was detected with rabbit  or mouse  HRP-linked secondary antibodies, respectively. Blots were processed using the Amersham ELC Prime detection kit . Results are representative of at least 3 independent immunoblots, with protein from a minimum of 2 mutant embryos. Protein levels were calculated using ImageQuant TL, v7 software .Whole E13.5 embryos were collected in Trizol reagent (Invitrogen) and sonicated for RNA extraction. Total RNA was extracted from embryos of each genotype using standard chloroform extraction procedures. First-strand cDNA synthesis was performed on 1 µg RNA from each genotype using BioScript reverse transcriptase (BioLine) using random primers. Quantitative real-time PCR reactions were performed in triplicate on a Lightcycler 480 System (Roche) using GoTaq qPCR Master Mix (Promega). Data were normalised to the housekeeping gene TBP (TATA Binding Protein). Primer sequences are detailed in IFT140 (NM_014714.3) was sequenced, including all 31 exons, intron/exon boundaries and most of the 5′ and 3′ untranslated regions and any variations to the GRCh37 human reference assembly identified. Alterations in the nucleotide sequence of JS 1-1 were analysed using the following criteria: 1) segregation was consistent with recessive inheritance; 2) PCIS score >0.15 by Polyphen-2; 3) SNP allele frequency <0.005 in public databases dbSNP All human analyses were approved by the Royal Children's Hospital Human Research Ethics Committee project 31190. Genomic DNA was isolated from blood samples received from the proband (JS 1-1), his parents (JS 1-2 and JS 1-3) and saliva from the unaffected sibling (JS 1-4). The candidate gene


Circulating tumour cells (CTC) have a crucial role in metastasis formation and can consistently provide information on patient prognosis. Epithelial-mesenchymal transition (EMT) is considered as an essential process in the metastatic cascade, but there is currently very few data demonstrating directly the existence of the EMT process in CTCs.CTCs were enriched by blood filtration using ISET , triply labelled with fluorescent anti-vimentin, anti-pan-keratin antibodies and SYTOX orange nuclear dye, and examined by confocal microscopy in six patients with metastatic non-small cell lung cancer (NSCLC). In parallel, CTCs were morphocytologically identified by an experienced cytopathologist.Isolated or clusters of dual CTCs strongly co-expressing vimentin and keratin were evidenced in all patients (range 5–88/5 ml). CTCs expressing only vimentin were detected in three patients, but were less frequent (range 3–15/5 ml). No CTC expressing only keratin was detected.We showed for the first time the existence of hybrid CTCs with an epithelial/mesenchymal phenotype in patients with NSCLC. Their characterisation should provide further insight on the significance of EMT in CTCs and on the mechanism of metastasis in patients with NSCLC. Circulating tumour cells (CTCs) have an active role in the formation of metastasis and their detection can provide information on patient prognosis and treatment efficacy. Current clinical results rely exclusively on the detection and enumeration of CTCs expressing epithelial-lineage markers  in the peripheral blood . HoweverThe present study was approved by our institutional review board and local ethics committee. Informed and written consent was obtained from all patients. Peripheral blood samples were collected from six patients with histologically or cytologically confirmed non-small cell lung cancer (NSCLC). For each patient, 10 ml of blood was collected in EDTA tubes  for CTC enumeration by isolation by size of epithelial tumour cells (ISET). Peripheral blood samples were collected from six healthy volunteers for control experiments.μM , a Gaussian blur (radius=0.10 μm) was applied, and contrast was slightly enhanced in every fluorescent channels of acquisitions. CTCs were selected, digitised and validated by a senior and experienced cytopathologist (PV) as cells presenting all the following criterias: (i) nuclear size equal or larger than two pores (16 μM); (ii) irregularity of the nuclear contour; (iii) presence of a visible cytoplasm; (iv) high nuclear-to-cytoplasmic ratio (greater than 0.8). A size threshold of 16 μM was chosen to exclude most normal cells.Within 4 h after venopunction, blood samples were diluted 1 : 10 in an erythrocyte lysis buffer containing paraformaldehyde, filtered on the ISET device using filters with calibrated pores of 8 μM . Filters2 at 37 °C. For control experiments, 25 cells from each cell line were precisely micro-manipulated under an inverted microscope before being spiked into blood from healthy donors. Blood samples containing spiked cell lines were then diluted using the erythrocyte lysis buffer, filtered and stained as described for patient samples.The SKBR3 (breast cancer), MDA-MB-435S (breast cancer) and A549 (lung adenocarcinoma) cultured cell lines were used as controls for markers expression. All cell lines were cultured in 1640 RPMI medium supplemented 10% fetal calf serum and maintained in a humidified incubator in 5% COImmunohistochemical stainings were performed on sections of formalin-fixed paraffin-embedded samples, using cytokeratin 7 (Dako) and vimentin (Santa Cruz Biotechnology) according to standard procedures.Blood samples from six patients with metastatic NSCLC were processed on the ISET device, which allows CTC enrichment by blood filtration through filters with calibrated pores . Triple Our exploratory study demonstrates for the first time that the majority of isolated or clusters of CTCs in patients with advanced metastatatic NSCLC harbour a dual epithelial–mesenchymal phenotype, suggesting that EMT is a relevant process for invasion and metastatasis in these patients. CTCs expressing only vimentin  were also observed. These data underscore the reality of EMT in CTCs and provide experimental evidence to the paradigm of a phenotypical continuum between epithelial and mesenchymal states in CTCs (


Uterine leiomyomas (ULs) are benign tumors occurring in the majority of reproductive aged women. Despite the high prevalence of these tumors, little is known about their etiology. A hallmark of ULs is the excessive deposition of extracellular matrix (ECM), primarily collagens. Collagens are known to modulate cell behavior and function singularly or through interactions with integrins and growth factor-mediated mitogenic pathways. To better understand the pathogenesis of ULs and the role of ECM collagens in their growth, we investigated the interaction of leiomyoma smooth muscle cells (LSMCs) with two different forms of collagen, non-polymerized collagen (monomeric) and polymerized collagen (fibrillar), in the absence or presence of platelet-derived growth factor (PDGF), an abundant growth factor in ULs. Primary cultures of human LSMCS from symptomatic patients were grown on these two different collagen matrices and their morphology, cytoskeletal organization, cellular proliferation, and signaling pathways were evaluated. Our results showed that LSMCs had distinct morphologies on the different collagen matrices and their basal as well as PDGF-stimulated proliferation varied on these matrices. These differences in proliferation were accompanied by changes in cell cycle progression and p21, an inhibitory cell cycle protein. In addition we found alterations in the phosphorylation of focal adhesion kinase, cytoskeletal reorganization, and activation of the mitogen activated protein kinase (MAPK) signaling pathway. In conclusion, our results demonstrate a direct effect of ECM on the proliferation of LSMCs through interplay between the collagen matrix and the PDGF-stimulated MAPK pathway. In addition, these findings will pave the way for identifying novel therapeutic approaches for ULs that target ECM proteins and their signaling pathways in ULs. Uterine leiomyomas (ULs) are one of the most common pelvic neoplasms in reproductive aged women with a reported prevalence of 25-70% depending on age –3. TheseThe most distinct feature of ULs is the excess synthesis and deposition of ECM proteins, mainly collagens type I and III –10. EarlECM collagens are known to both maintain cellular morphology and act as conduits between extracellular stimuli and cells by regulating proliferation, migration, differentiation, and survival . The ultProposed mechanisms that may explain the growth modulatory effects of different forms of ECM collagens include interaction through integrins which are the main collagen receptors. Clustering and activation of integrin receptors induces cytoskeletal reorganization and formation of focal adhesions followed by activation of specific focal adhesion kinases (FAK). Activation of FAK then activates signaling pathways such as mitogen activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) pathways, altering the expression of cell cycle regulatory proteins and promoting proliferation –37. Collin vitro model system of ECM collagen, we examined the interaction of cultured LSMCs with monomeric unpolymerized collagen films and fibrillar polymerized collagen gels in modulating cellular morphology, cell proliferation, cell cycle progression, and the associated signaling pathways.The fact that ULs are fibrotic tumors containing an abundance of disorganized ECM collagen ,17 led u2.Leiomyoma samples were obtained from premenopausal women undergoing hysterectomy at either Carle Foundation Hospital  or Feinberg School of Medicine, Northwestern University . Permission to use these samples was approved by the Institutional Review Board at the University of Illinois at Urbana-Champaign and Northwestern University. All samples were obtained after receiving written informed consent from the patients. Tissue samples were manually minced and then digested in Dulbecco Modified Eagle’s Medium  containing 1.5 mg/ml Collagenase for 4-6 hours at 37 degrees. Once digested, cells were cultured in DMEM medium containing 10% serum  at 37°C in a humidified atmosphere of 95% air and 5% COPureCol collagen solution  was diluted to 1.0 mg/ml with 0.1 M acetic acid at room temperature. For monomeric collagen coating, plastic dishes were incubated with 0.5 M acetic acid for 20 minutes at room temperature, rinsed once with distilled water and coated with diluted collagen under sterile conditions for 4-6 hours. For fibrillar collagen coating, diluted collagen was neutralized with 10x PBS, 0.1 N NaOH and 0.1 N HCl on ice. Plastic dishes were coated with fibrillar collagen and incubated at 37 degrees overnight for the gel to polymerize. Coated dishes were rinsed with PBS several times before seeding cells.5) of trypsinized LSMCs from confluent dishes were cultured in 10% serum-containing medium on different matrices. Cells were trypsinized and counted every other day using a hemocytometer. We also used thymidine incorporation assays to measure the proliferation of LSMCs on different matrices in the absence or presence of PFGF-AB . Briefly ,LSMCs were cultured at equal numbers (2000 cells) in 96-well plates coated with monomeric or fibrillar collagen. Cells from 3 patients with 6 experimental replicates per plate were used. For cell cycle synchronization, LSMCs at 90% confluence were serum starved for 24 hours and then treated with 10% serum medium, medium with no serum, or medium containing 10 ng/ml PDGF for 24 hours. Cells were labeled with 0.01 µCi/µl tritiated thymidine  for 18 hours after treatment. Cells were then trypsinized and harvested on filter papers for reading in a Wallace Microbeta liquid scintillation counter .To obtain the growth curve of LSMCs, equal numbers  and 1 mg/ml RNase A /PBS overnight at 4°C. Stained cells were taken and analyzed for cell cycle phase distributions in a FACScan flow cytometer . All data were analyzed using FCS express v4 software .2 surface area were averaged and represented as mean ± SEM.Adhesion of LSMCs to different matrices was compared in cells seeded onto either plastic, monomeric collagen or fibrillar collagen coated dishes. Three hours after seeding, dishes were gently rinsed with PBS to remove unattached cells and imaged to count the number of attached cells to each matrix. Adhesion assays were performed on cells from three different patients with two experimental replicates for each patient. The numbers of attached cells in 10 fields of 20 mmImmunofluorescence staining of cells was carried out by first fixing cells in 4% paraformaldehyde for 30 minutes at room temperature. Following three washes in PBS, cells were permeabilized in 0.1% triton X-100 for 15 minutes. Dishes were next incubated in ultra-cold methanol for 15 minutes at -20°C and washed for half an hour before blocking by Image-iT FX Signal FX Enhancer  for 30 minutes. Following a PBS wash, cells were incubated with 1:100 dilutions of primary antibodies against vinculin , pFAK , and integrin α2  for 2 hours at room temperature. A 1:200 dilution of secondary antibodies conjugated with different fluorophore antibodies  was applied to incubating cells for 2 hours. After three washes with PBS, cells were stained with 10 µg/ml DAPI  for 15 minutes. Dishes were cured in Prolong Gold  for 24 hours in darkness before imaging with a Zeiss LSM710 microscope .LSMCs were cultured on different matrices and treated with or without protein inhibitors  prior to treatment with 10 ng/ml PDGF. At the end of the treatment time, cell lysates were collected using RIPA lysis buffer containing protease cocktail inhibitors . BCA assays  were performed on cell lysates to determine protein concentrations. Proteins were loaded onto 4-20% gradient SDS-PAGE gels  under reducing conditions before being transferred to nitrocellulose membranes. Primary antibodies  were incubated at 4°C overnight. Incubation with HRP-conjugated secondary antibody  was for 60 min at room temperature  and HRP signal detection was carried out with Super Signal West Pico Chemiluminescent Substrate kit . Membranes were first probed with anti- phosphorylated protein antibody and then stripped and re-probed with anti-total protein antibody as a loading control. ImageJ software from the National Institutes of Health was used for densitometric analysis.post-hoc orthogonal comparisons were applied. Values are expressed as mean ± SEM and p< 0.05 was considered statistically significant. STATA SE (Version 11.2) software was used for conducting all statistical analyses.Regression analysis was performed to estimate the growth rate of LSMCs. Experimental variability between treatments/matrices was determined by an analysis of variance (ANOVA) model. To determine statistical differences between groups, To understand the interaction of LSMCs with different collagen matrices, we cultured primary LSMCs on plastic, monomeric collagen or fibrillar collagen-coated dishes and examined cell morphology 48 hours later. LSMCs on monomeric collagen-coated dishes displayed a spindle-like morphology typical of SMCs that was similar to LSMCs on control, plastic dishes . In contTo quantify LSMC proliferation on different collagen matrices, growth curves using individual cell counts and tritiated thymidine incorporation assays were performed. Growth curves for LSMCs cultured on collagen matrices in the presence of 10% serum showed that both monomeric and fibrillar collagen were permissive to cell proliferation. However, the rate of LSMC proliferation on monomeric collagen was significantly greater than for LSMCs cultured on fibrillar collagen . The douLSMCs within leiomyoma tissues are exposed to many growth factors including PDGF which can exist bound to the ECM. We next examined whether the interaction of LSMCs with ECM collagen alters the way these cells respond to the stimulatory effects of PDGF. Basal and PDGF-stimulated proliferation of LSMCs cultured on either monomeric or fibrillar collagen matrices for 24 hours was measured. Results showed that, similar to our growth curve analysis, both matrices were permissive to proliferation of LSMCs in the presence or absence of PDGF with monomeric collagen having a significant potentiating effect over fibrillar collagen . InteresTo determine whether the stimulatory effects of monomeric collagen on the growth of LSMCs was the result of cell cycle modulation, cells were cultured on plastic or monomeric collagen-coated dishes for 24 hours in the presence or absence of PDGF and the distribution of DNA content was measured by flow cytometry (FACS). Due to the difficulties in separating LSMCs from the fibrillar collagen gels we were unable to obtain single cell suspensions required for FACs analysis for this treatment group. In support of the proliferation studies on plastic and monomeric collagen, FACs analysis showed that treatment of LSMCs with PDGF increased the percentage of cells in the S phase of the cell cycle compared to non-stimulated cells . CultureECM exerts its effects on cells through the activation of several mediatory proteins including integrins, FAK and vinculin leading to the assembly of cytoskeletal components. To investigate whether the differential proliferative responses of LSMCs cultured on monomeric and fibrillar collagen matrices occur through such mediatory proteins, we immunostained LSMCs cultured on collagen matrices for α2β1 integrin, phospho-FAK (pY397), vinculin, and F-actin stress fibers. Results showed no difference in abundance or clustering of α2β1 integrin across matrices whereas LSMCs cultured on monomeric collagen displayed abundant activation (phosphorylation) of FAK, specifically at the sites of matrix adhesion . In contTo elucidate the signaling mechanisms involved in the interaction of collagen matrices with LSMCs, we investigated MAPK, AKT and Rho-GTPase signaling which have been shown to be associated with the effects of ECM in a variety of cell types. LSMCs were cultured on plastic, monomeric or fibrillar collagen-coated dishes and stimulated with 10 ng/ml PDGF for 0, 10, 30, and 60 minutes. Results showed that while there was no significant difference in the activation of RhoA across matrices , both AKWe next determined the specificity of PDGF activation of the MAPK signaling pathway in LSMCs. LSMCs cultured on plastic were pre-treated with inhibitors specific to PDGFR and ERK1/2, AG1296 and PD98059 respectively, for two hours followed by a 10 minute stimulation with 10 ng/ml PDGF. Results showed that both inhibitors could significantly reduce the phosphorylation of ERK1/2, suggestIn the present study, we investigated interactions of primary human uterine LSMCs with different collagen matrices representing normal and fibrotic uterine SMCs in the absence or presence of the mitogenic growth factor PDGF. In our model, we used non-polymerized monomeric collagen matrix to mimic the ECM environment in ULs where SMCs continuously synthesize nascent collagen and collagen is also subjected to MMP-mediated degradation leading to a higher proportion of non-cross linked collagen that is laid down in a disorganized manner ,17. PolyCell shape and extracellular environment have been recognized as major determinants of cell behavior and function –48. CellMechanical stress exerted upon cells through the ECM in both normal and diseased tissues is transmitted through molecular players such as integrin receptors, focal adhesions, cytoskeletal proteins, and signaling pathways including MAPK, PI3K/AKT, and Rho-GTPase –60. We oThe decreased level of activation or clustered localization of vinculin, FAK, and F-actin stress fibers in LSMCs cultured on fibrillar collagen observed in this study could explain the state of quiescence or decreased cell proliferation in LSMCs grown on this matrix. In fact, potent suppression of focal adhesion formation, as indicated by altered vinculin localization, has been linked to changes in gene expression and cell function in vascular SMCs cultured on polymerized fibrillar collagen . Cell cyIn ULs an increased presence of and response to growth factors such as PDGF have been documented –68 alongActivation of PI3K/AKT or JAK/STAT in cells grown on different matrices has been reported. In hepatocytes matrix stiffness enhanced ERK1/2, protein kinase B (PKB/AKT) and signal transducer and activator of transcription-3 (STAT3) phosphorylation and resulted in enhanced mitogenic signaling in response to hepatocyte growth factor . SimilarWe also observed different activation patterns for ERK1/2 on monomeric collagen versus fibrillar collagen matrix. Rapid activation of ERK1/2 on monomeric collagen nicely followed the activation of PDGFR in response to PDGF treatment. In contrast, we observed persistent ERK1/2 activation in LSMCs cultured on fibrillar collagen in response to PDGF stimulation. The duration of ERK1/2 activity in response to growth factors has been linked to differential regulation of cellular responses and cell fate in a cell-type dependent manner. Treatment of PC12 pheochromocytoma cells with nerve growth factor caused sustained activation of ERK1/2 for 2-3 hours leading to cell differentiation whereas epidermal growth factor induced a transient ERK1/2 activation required for cell proliferation ,79. On tIn conclusion, our significant findings on the characterization of LSMCs cultured on different forms of collagen as a simple model to better replicate the ECM environment in normal myometrium and fibrotic leiomyoma tissue were two fold. First, the findings demonstrate the importance of carefully defining the culture systems for studying specific cell functions. This study along with others, using various cell lines including LSMCs ,94, have


Usage of the enhanced green fluorescent protein (eGFP) in living mammalian cells is limited to aerobic conditions due to requirement of oxygen during chromophore formation. Since many diseases or disease models are associated with acute or chronic hypoxia, eGFP-labeling of structures of interest in experimental studies might be unreliable leading to biased results. Thus, a chromophore yielding a stable fluorescence under hypoxic conditions is desirable. The fluorescence of flavin mononucleotide (FMN)-based fluorescent proteins (FbFPs) does not require molecular oxygen. Recently, the advantages of FbFPs for several bacterial strains and yeasts were described, specifically, their usage as a real time fluorescence marker in bacterial expression studies and their ability of chromophore formation under anaerobic conditions. Our objective was to verify if FbFPs also function in mammalian cells in order to potentially broaden the repertoire of chromophores with ones that can reliably be used in mammalian studies under hypoxic conditions. In the present study, we demonstrate for the first time, that FbFPs can be expressed in different mammalian cells, among them murine neural stem cells during proliferative and differentiated stages. Fluorescence intensities were comparable to eGFP. In contrast to eGFP, the FbFP fluorescence did not decrease when cells were exposed to defined hypoxic conditions neither in proliferating nor in differentiated cells. Thus, FbFPs can be regarded as an alternative to eGFP in studies that target cellular structures which are exposed to hypoxic conditions. Aequorea victoriaBacillus subtilisPseudomonas putidain vivo or in vitro models of these diseases or conditions are connected to primary hypoxia or secondary ischemic hypoxia, reporter proteins should also reliably function during hypoxic conditions. For instance, several studies report hypoxia-induced changes in proliferation and differentiation of tissue specific stem cells and human embryonic stem cells directly illustrating the necessity for stable reporter proteins also in stem cell research if those populations are supposed to be investigated under hypoxic conditions in vivo in viable human tumors Since its first description and purification, the green fluorescent protein (GFP) from Cellular structures of interest, although potentially surviving a situation of critical oxygen supply, would stand to lose their accessibility to analyses when the intensity of the eGFP signal decreases under hypoxic conditions. We therefore investigated if FbFPs can be used in mammalian cells as suitable reporter proteins under oxygen limitation.To evaluate, if PpFbFP (PP1) and EcFbFP (BS2) can be used as fluorescent reporter proteins in mammalian cells, we first generated recombinant derivatives of plasmid vectors pcDNA3.1 or pEF6 carrying the respective reporter genes . For effTo verify if the intensity of FbFP fluorescence is independent from molecular oxygen, we quantified the FbFP  and, for control, eGFP fluorescence under normoxic and hypoxic conditions. For this purpose, first, the average grey values as indicators for fluorescence intensities of HT22 cells, undifferentiated or differentiated msNSCs (each stably tranfected with eGFP or FbFP PP1 or BS2 under CMV or EF1α promoter) under normoxic conditions were determined. Afterwards, the average grey values of each group were determined after 24 or 72 hours under hypoxic conditions .The presented values under normoxic conditions are relative average grey values, different intensities in the three examined cell types arise from their different protein expression levels. Notably, the baseline fluorescence intensities of eGFP and FbFPs under normoxic conditions nearly reached the same relative levels in stably transfected cell lines, whereas the baseline fluorescence intensity of FbFPs in transiently transfected cell lines was lower in comparison to eGFP.After tracing the baseline fluorescence intensities, we exposed all cell types to hypoxia for 24 or 72 hours and again recorded fluorescence intensities . As expeMurine ES cell-derived neural populations showed a much more sensitive reaction to hypoxia than tumor cells. After more than 24 hours of hypoxia, murine ES cell-derived neural populations began to die off. This phenomenon was observed by visual inspection and DAPI nuclei staining. Therefore, we were only able to study fluorescence stability of differentiated and undifferentiated msNSCs during a time interval of 24 hours of hypoxia. Like in the tumor cell lines, we were able to detect a significant decrease in eGFP fluorescence intensities after 24 hours of hypoxia in the undifferentiated neural stem cell stage as well as in mature cell populations whereas the fluorescence intensities of FbFPs remained unchanged .To investigate the homogeneity of the FbFP signal within different parts of individual cells, we verified morphological aspects of neurons, whose small and ramified neurites are sometimes poorly labelled by the eGFP signal. For that, we performed immunocytochemical stainings against βIII-tubulin, a pan-neuronal marker, on stably transfected, differentiated msNSCs. We found that both types of fluorescent proteins showed a uniform distribution within the cytoplasm and neurites under normoxic conditions . HoweverThen, to verify possible negative effects of FbFPs on the survival or proliferation of cells, we measured the size of the populations that were either stably transfected with one of the two different FbFPs (PP1 or BS2) under the CMV promoter in comparison to wild type cells or stably eGFP transfected cells under the CMV promoter. For this purpose, CHO cells or undifferentiated msNSCs were plated at the same densities and MTT-assays were performed 1 day, 4, 7 or 9 days after plating. CHO cells were only examined until day 7 due to their excessive proliferation. Notably, 9 days after seeding, FbFP PP1 transfected msNSC cells showed a reduced population size in comparison to wild type as well as FbFP BS2 or eGFP transfected cells. In CHO cells this phenomenon was not detectable .B. subtilis and P. putida were genetically modified to increase the fluorescence in comparison to the wild type photoreceptors Porphyromonas gingivalis and as gene expression reporters in Bacteroides fragilis, Roseobacter clade bacteria, Candida albicans, Saccharomyces cerevisiae and E. coliThe light oxygen voltage domains (LOV) were described as blue-light sensitive photoreceptor domains in plants and prokaryotes. All LOV proteins bind FMN as chromophore and exhibit very low autofluorescence when excited by blue light at 450 nm et al.We were able for the first time to show, that oxygen independent FbFPs are suitable fluorescent reporter proteins in mammalian cells as well. After genetic modification of plasmid vectors and the usage of strong eukaryotic promoters, comparative expression studies revealed that the resulting visible fluorescence intensity of PP1 and BS2 after transfection and expression into mammalian cells was comparable to that of the widely used eGFP, although another study by Drepper Most notably, we found that FbFPs are the more suitable fluorescence reporter under defined oxygen deprived conditions, in comparison to eGFP in mammalian cells. We were able to show that the fluorescence intensity of FbFPs remained stable under hypoxic conditions in comparison to that of eGFP which showed a reduction of up to 40%. We also investigated the fluorescence within neurites under hypoxic conditions in eGFP- or FbFP – transfected neurons and found that FbFPs more clearly label these structures. This directly illustrates that FbFPs can help to analyse such structures under hypoxic conditions. But also other applications of FbFPs in biomedical research during situations of critical oxygen supply are imaginable. In particular, cells that indeed suffer from low oxygen conditions finally being able to recover are now detectable during that critical period. For instance, in cancer research, transgenic labelling of the inner parts of solid tumors that are indeed at risk for necrotic transformation but still vital yet might be difficult as oxygen supply here is critical B. fragilis and P. gingivalis localized within human murine macrophages J774.1 and primary gingival epithelial cells, respectively without influencing the life cycle and the proliferative activity of these organisms In the past, FbFPs were successfully used to visualize the anaerobic pathogens However, as FbFP PP1 transfected msNSC showed a reduced population size, anti-proliferative and/or toxic properties of FbFP PP1 might be a shortcoming if mammalian neural stem cells are targeted by this fluorescent protein.Another possible application is tracing the integration of experimentally transplanted cells into animal models for human diseases. For instance, stem cell-derived progeny is actually under investigation in the field of reconstructive neurobiology. As neovascularisation often lags behind proper graft integration, improved visualization of transplanted cells under situations of critical oxygen supply might be of substantial importance. And this might be true not only for studies in the field of stroke research.Spinoloricus nov. sp., Rugiloricus nov. sp., and Pliciloricus nov. sp., that were recently described to spend their entire live cycle under permanently anoxic conditions in the deep hypersaline anoxic basins of the Mediterranean Sea It is further thinkable to use FbFPs as fluorescence reporter proteins in the species Taken together, our findings might pave the way for a completely new field of FbFP application in mammalian and other eukaryotic cells, specifically in biomedical research. We believe that after possible enhancements like improvement of photobleaching and quantum yields, as well as the generation of colour variants of FbFPs could enrich the field of molecular cell biology.Tumor cell lines like human embryonic kidney cells (HEK cells), chinese hamster ovarial cells (CHO cells) or neuro 2a cells (N2A cells) and the murine, immortalized, hippocampal tumor cells line (HT22) were cultured in D-MEM High Glucose media  with 10% fetal calf sera (FCS) . Mouse embryonic stem cell-derived (ES-derived) undifferentiated neural stem cells (msNSC) where cultured in D-MEM/F12 supplemented with N2-supplement , bovine serum albumin (BSA)  and 20 ng epidermal growth factor and basic fibroblast growth factor (EGF and FGF) , respectively. Cell culture media contained Glutamax 20 mM , Penicillin 100 U/ml and Streptomycin 100 µg/ml . For differentiation of msNSC, the growth factors were withdrawn from the cell culture media. Cultivation of mouse embryonic stem cells and the generation of undifferentiated neural stem cells are described elsewhere 2 gas was applied. The flow rate was 20 litres per minute for 15 minutes and resulted in an oxygen concentration of approximately 1 mg per litre, 0.01–0.02% or an oxygen partial pressure of 0.1–0.2 mmHg in the cell culture medium measured with a colorimetric test . After flushing the chamber it was sealed and placed in a regular cell culture incubator for the given time points . To avoid acidification of cell culture media under hypoxic conditions due to CO2 withdrawal in the sealed hypoxia chamber, we used CO2 independent cell culture media  in all experiments (as well under hypoxic as under control conditions).Hypoxia was generated by using a hypoxia incubator chamber  regarding to previous studies BamHI and EcoRI, which were used to clone the corresponding reporter genes into pCDNA3.1  under the control of the CMV promoter, yielding pCDNA3.1-Bs2 and pCDNA3.1-Pp1, respectively. Afterwards, the genes encoding the FbFP proteins were excised from pCDNA3.1-BS2 and pCDNA3.1-PP1 by usage of BamHI and NotI and subsequently cloned into pEF6/Myc-His-lacZ under control of the EF1α promoter, yielding pEF6-Bs2 and pEF6-Pp1. For each step, successful cloning was verified by plasmid preparation, restriction analysis and subsequent sequence analyses. EGFP control vector (pEGFP-C1) is commercially available at BD-Biosciences . FMN-based fluorescent proteins (FbFPs) are commercially available under the trademark evoglow at evocatal GmbH .Codon-optimized genes encoding EcFbFP and PpFbFP respectively, were obtained with an upstream Kozak sequence from GeneArt AG . Genes were synthesized with flanking restriction sites for Cells were seeded on glass cover slips. After the experimental procedure, cells were fixed with Roti Histofix 4%  and blocked with 1-fold Roti Immuno Block/0.01% Triton X-100 . For visualisation of neurons, an antibody against βIII-tubulin  was added and incubated over night at 4°C in 1-fold Roti Immuno Block. For detection of the primary antibody, an indocarbocyanine coupled secondary antibody  was used in 1-fold Roti Immuno Block.For visualization of transgene expression, cells were fixed with Roti Histofix 4% . All slides were mounted with fluorescent mounting media . EGFP expression was visualized with the following filter set: excitation wavelength BP 460–490 nm, emission wavelength BA 510 IF, dichroic mirror 505 nm . For better visualization of FbFPs, a fluorescence filter set with excitation wavelength 450–500 nm, emission wavelength 510–560 nm and a long pass dichroic mirror 470 nm wavelength was used .Average grey values were used as indicators for fluorescence intensities in stable cell lines. Therefore, all experiments with stably transfected cell lines were performed under the same conditions . After the indicated time points of incubation under hypoxic or normoxic conditions, cells were fixated in Roti Histofix 4%  and microscope images at the same magnification and exposure time were taken with a black and white camera. Then every cell in the resulting visual field was marked by a ROI (region of interest) and the average grey values were determined by CellR software . For this calculation each pixel is assigned to a grey value. The lowest grey value (zero) is assigned to black whereas the highest value is assigned to pure white. Afterwards grey values of all pixels in the ROI are summed up and the sum is divided by the amount of pixels in the ROI. The resulting value is the average grey value. Each visual field contained in between 30–100 cells. Grey values of 5–10 visual fields were averaged for calculation of the final average grey values that are displayed in To analyze the population extent of msNSC and CHO cells, the optical density, indicative of conversion of 3--2, 5-diphenyltetrazolium bromide  into formazan crystals which takes place in live cells only, was determined after the indicated time points. Therefore, cells were incubated with MTT  for 3 hours. Afterwards cells were lysed in DMSO  and the optical density was measured at 560 nm in a spectrophotometer. Cell culture plates used for neural stem cell culture were precoated with poly-L-ornithine  and fibronectin .


Individuals differ in their cognitive resilience. Less resilient people demonstrate a greater tendency to vigilance decrements within sustained attention tasks. We hypothesized that a period of sustained attention is followed by prolonged changes in the organization of “resting state” brain networks and that individual differences in cognitive resilience are related to differences in post-task network reorganization. We compared the topological and spatial properties of brain networks as derived from functional MRI data (N = 20) recorded for 6 mins before and 12 mins after the performance of an attentional task. Furthermore we analysed changes in brain topology during task performance and during the switches between rest and task conditions. The cognitive resilience of each individual was quantified as the rate of increase in response latencies over the 32-minute time course of the attentional paradigm. On average, functional networks measured immediately post-task demonstrated significant and prolonged changes in network organization compared to pre-task networks with higher connectivity strength, more clustering, less efficiency, and shorter distance connections. Individual differences in cognitive resilience were significantly correlated with differences in the degree of recovery of some network parameters. Changes in network measures were still present in less resilient individuals in the second half of the post-task period (i.e. 6–12 mins after task completion), while resilient individuals already demonstrated significant reductions of functional connectivity and clustering towards pre-task levels. During task performance brain topology became more integrated with less clustering and higher global efficiency, but linearly decreased with ongoing time-on-task. We conclude that sustained attentional task performance has prolonged, “hang-over” effects on the organization of post-task resting-state brain networks; and that more cognitively resilient individuals demonstrate faster rates of network recovery following a period of attentional effort. Many daily activities require the maintenance of attention over long periods of time which causes behavioural performance to deteriorate There has been recent progress to describe the human brain as a complex network of interconnected processing nodes Previous studies documented the flexibility of the brain network topology We acquired fMRI data during RS periods before and after a vigilance task and used a graph analytical approach to investigate long-lasting changes in functional brain topology induced by sustained attention. We assumed that (i) prolonged attentional performance leads to less integrated networks with more clustered brain regions and a change from long-distance to short-distance connections. Furthermore, we expected (ii) that altered network attributes recover slowly after task processing and that (iii) network recovery and individual task performance are correlated.To further investigate possible mechanisms underlying these long-lasting changes in network topology we also analyzed changes in network topology during task performance. Our own work suggests that cognitive fatigue and prolonged attentional performance affect endogenous neural resting state activations Twenty healthy, right-handed subjects  participated in the experiment.http://www.dgps.de/, PI: Christiane Thiel, registration number: CT05022008DGPS) and subjects signed written informed consent.The study was approved by the ethics committee of the German Psychological Association (Each scanning session was divided into different time periods in which either resting state (RS) or BOLD activations during task processing were measured see . There wDuring the task period, subjects performed a visual vigilance task in which a display with a central black fixation cross (the same as used during the RS condition) and two additional flanker windows were presented . SubjectTo investigate changes in network topology during task processing fMRI scans acquired during task performance were divided into blocks of 256 scans each, building five different task blocks (T1 to T5). Within these task blocks subjects processed the same trials with the same number of targets and the same number of alternating “motion on” and “motion off” phases in the flanker windows. The analysis of local task activations related to specific task conditions were published previously The individual vigilance decrement of each subject was estimated by the slope of a robust linear regression analysis with the individual response latencies or reaction times (RTs) as dependent variable and the series of attentional trial numbers as independent variable. In contrast to standard linear regression analysis in which each data point has equal influence on the least square curve fit, robust regression analysis uses a bisquare weighting function so that data points further apart from the expected line get reduced weight. Robust fits are less affected by outliers The Stanford Sleepiness Scale (SSS) 2, relaxation time (TR) = 1500 ms, echo time (TE) = 50 ms and 90° flip angle). EPI data were continuously measured with the same sequence in all RS and task periods. Structural T1-weighted images were obtained after the fMRI experiment, using magnetization frequency pulse and rapid gradient-echo (MP RAGE) sampling: 1 mm isotropic voxels, 176 slices, FoV = 256×256 mm2, TR = 2130 ms, TE = 3.93 ms and 15° flip angle.Functional and structural images were acquired on a 1.5 Tesla MRI scanner . Functional images were obtained using multislice T2*-weighted gradient echo planar imaging (EPI). Each volume consisted of 17 axial slices  = 200×200 mmhttp://www.fil.ion.ucl.ac.uk/spm/). The functional images were spatially normalized to standard stereotaxic MNI space  and regionally parcellated using a template image comprising 442 cortical and subcortical brain regions to estimate the mean fMRI time series for each region for each participant during each RS and task period 3;) than the range in size of the 90 nodes of the template  was used to ensure that each node was connected with at least one other node even at the sparsest connection density. Following the MST the algorithm adds the highest correlations of each node in an iterative way see  we stoppBased on these adjacency matrices we calculated the central graph theoretical measures of global, nodal and local efficiency following the formulas of Latora and Marchiori Local efficiency is closely related to the clustering coefficient The physical distances were based on the Euclidean distances between the centres of coordinates (MNI-space) of all functionally connected brain seed regions. The averaged physical distances of all nodes built the measure of mean distances. Further, the number of connections/edges in each of several distance bins in the histogram of connection distances was counted  at the cost level of 50%. Differences between the numbers of edges within each bin at different RS periods were tested with two-sided paired t-tests (Bonferroni corrected).first mixed-effects model we compared the resting state data before and following task processing and modelled the data with the factor RS period , the behavioural performance as continuous factor , and the interaction between both factors. In the second analysis we compared the change in topology during task processing. Therefore we modelled the network metrics as linear function of time (factor ‘task period’). In a third analysis we analyzed the change in network topology from rest to task performance in the beginning of the task period and from task to rest following the task period. The mixed-effects models included the factor ‘task switch’ (the change from RS to task period), the factor ‘time point’ (task switch at the beginning or following the task period), and their interactions.For each network metric we used three different linear mixed-effects models to analyse the data. All models included an intercept term for each subject to model a random offset The network metrics global efficiency, clustering, and mean distances were averaged over the a-priori-defined cost range for both models in order to generalize the results across cost level (see above). In contrast to the normal analyses of variance, linear mixed-effects models can handle data with unequal variances. Thus, before significance testing, variances of data between RS periods were compared and significant in-homogeneities of variances were explicitly modelled within the data analysis.Pair-wise differences between RS periods were tested post-hoc by two-sided one-sample t-tests. Pearson’s correlations were used to test whether participants with higher or lower vigilance decrements show different changes in network metrics. To simplify this analysis, we also divided the participants into the two subgroups of “attentionally impaired”  and “attentionally resilient” subjects  and compared both groups with two-sample t-tests.To control type 1 error in the context of the multiple comparisons entailed by analysis of nodal efficiency and clustering, we used p≤0.0025 (1/N) as the threshold for significance, so that less than one false positive test is expected for each whole brain analysis of nodal network properties.Subjects generally performed the task well and showed low rates of misses . Therefore the analysis of the behavioural data focused on reaction times (RT) only. Reaction times showed a significant vigilance decrement over all subjects ; whereas the other ten participants had no significant change in response time to targets over the course of the task  B. We reThe ratings in the Stanford Sleepiness Scale before and after the experiment showed that after task performance participants felt more fatigued (t(19) = 7.89, p≤0.001, mean increase = 2.23, STD = ±1.25). There was no correlation between individual RT decrements and sleepiness ratings.Before the connectivity strength and network metrics were compared in different experimental conditions it was tested whether their variances were significantly different  the data analysis revealed a significant main effect for the factor RS period (S period . ImmediaS period : ConnectS period . For theS period  in whichThese changes immediately following the task persisted in the post-task phase (RS2 to RS3) when averaged over the whole group (see relation to performance below) and were still significantly different when compared to the RS period previous to the task (RS1 to RS3) .For physical distances, the analyses of the distribution of the physical lengths of the connected regions (edges) showed a significant shift from long distance connections (distances in the range from 78 to 108 mm) towards shorter distance connections (distances in the range from 8 to 53 mm) following task processing  showed a significant reduction in connectivity strength and clustering in the last RS period (RS3) in the direction of pre-task values. For attentionally impaired subjects (subjects with significant reaction time increase during task performance) connectivity strength and clustering showed no such recovery effects; their numerical values further increased until the end of the experiment. This correlation between clustering and performance was significant at all investigated cost levels .A further interesting finding is that the connectivity strength of RS1 (RS period before the task has started) showed a trend for a significant correlation with subsequent task performance . Regions that correlated with individual performance were localized in the left and right thalamus, right supramarginal gyrus, right precuneus, right amygdala, left insular cortex and right middle occipital cortex  for all investigated metrics . In otheFurthermore, measures of network topology were significantly affected by the change from resting state towards task processing : 9/30 were in the group of ‘attentionally resilient’ subjects, and 14/30 were in the groups of ‘attentionally impaired’ subjects. Second, we observed that “scrubbing” did not change the relationship between functional connectivity and physical distances when the “unscrubbed” correlation matrix is subtracted from the “scrubbed” correlation matrix (ΔR). Thus, “scrubbing” time points  provide no additional benefit to our data  that prolonged attentional task performance led to less brain network integration following task processing, (ii) that this reduction in network integration already started during task processing and (iii) that network integration remained diminished for long periods of time. Task performance initially increased network integration, but with ongoing time-on-task functional brain topology became less integrated, less efficient, more clustered and showed less long-distance connections. Following task performance these changes in network integration could be still detected after 6 minutes of resting state.As a second major finding, we could show that late changes in network topology (more than 6 minutes after the task) were correlated with individual differences in cognitive resilience during task performance. Attentionally resilient participants showed a larger reduction of network clustering and disintegration, i.e., a greater degree of recovery towards pre-task network topology during the post-task period.network topology and configuration.Several studies reported changes in resting state connectivity in low frequency ranges (<0.1 Hz) subsequent to task performance. Changes in connectivity strength were evident after motor As a further new aspect we found that changes in network topology persist for at least 6 minutes following task performance. Long-lasting effects after task performance on RS oscillations have been infrequently reported in studies to date. One study by Barnes et al. During task performance, two different processes seem to influence functional network topology. As first process, task performance increased network integration and induced higher global efficiency and less clustering. Network integration remained higher during task processing in comparison to resting state periods, but with ongoing time-on-task brain networks showed the opposite effects and became more fragmented with more clustering, and showed less global efficiency and shorter distances between brain nodes. This on-going process of network fragmentation over time was also evident when data around the switch between rest and task condition at the beginning and the end of the experiment were analysed; at the end of the task networks were less efficient, more clustered and showed more short distance connections. In summary, within the current study our results showed that declines in network integration started during task performance, progressed with on-going task performance, and that networks remained fragmented for a long time period.While measures of network topology were significantly changed from rest towards task processing, ‘whole brain’ functional connectivity seemed to be less sensible towards direct effects of task performance, and showed no general offset with the onset of task performance and continuously increased with on-going time of task.during task performance  showed shorter path lengths and higher global efficiency values in brain network topology Interestingly, mean connectivity strength was significantly correlated with declines of behavioural performance. Reaction times and connectivity strength continuously increased with time-on-task and the mean connectivity strength over all task periods significantly correlated with individual vigilance decrements. In addition, subjects with higher connectivity strength during the resting state period before task performance showed a tendency for higher vigilance decrements (p = 0.06). Thus, subjects already showed differences in brain networks before task performance which might have influenced their cognitive resilience during task processing see also . A relatWith respect to network topology, we found that individual behavioural differences did not predict task induced changes in network topology following the end of the task (i.e. RS1 vs. RS2), but were highly correlated with changes in topology in the post-task or recovery phase (RS2 and RS3). Only few studies have investigated recovery effects after tasks on endogenous dynamics so far. Barnes et al. Beside the analysis of the averaged clustering values over all nodes (seed regions of the template), we further investigated regional changes in clustering before and following the task and in the post-task phase. On nodal level, effects of increased clustering immediately following the task (from RS1 to RS2) were, among others, evident bilaterally in the visual cortices, the superior temporal cortices and the basal forebrain  and the Significant correlations between behaviour and nodal clustering were only observed in the post-task phase (RS2 to RS3) and among others evident in the thalamus, a key structure of the arousal system Previous studies have shown that learning-induced plasticity can have a long-lasting impact on subsequent RS periods On molecular level metabolic costs may account for long-lasting effects of task performance. Mental tasks are accompanied by an increased level of glycolysis in the brain Most graph analyses ignore that correlations between two brain regions can also be influenced by a common covariate that induces indirect correlations. Even though indirect correlations are a general problem that also affects the analyses of resting state data they become more obvious during the analysis of task data in which the experimental stimulation might have simultaneously influenced two brain regions. Therefore we focussed in the interpretation of our results mainly on changes between conditions with the same experimental stimulation (comparisons between rest conditions or within task blocks).Even though previous results revealed that functional connectivity do not significantly change if participants are instructed to fixate or to keep their eyes open without fixation over a time period of 5 minutes In many studies investigating neural correlates of task-related processing  spontaneous signal fluctuations were only considered as noise factor. Over the last years this perspective has changed, since previous studies have shown that, for example, spontaneous blood oxygen level dependent (BOLD) signal fluctuations contribute to the prediction of variability in behavioural performance within and between subjects In real world, economical reasons often force researchers to present several tasks within one fMRI scanning or MEG session, neglecting or underestimating changes in functional brain networks resulting from a previous task. Our data indicate that 12 minutes of rest period between each task are not enough to fully recover functional brain networks from previous task processing. Our data strongly encourage to take into account “hang-over” effects on the organization of post-task brain networks that possibly interact with the processing of new tasks. These “hang-over” effects introduce a specific brain state, which will impact on subsequent neural processing and behavioural performance. In future, the individual capability of brain network recovery might be an additional measure to obtain cognitive resilience of patients and to detect early symptoms of potential neurological disorders. Our data support that the recovery of connectivity strength and network topology may serve as biomarkers of cognitive demand and resilience that significantly contribute to the understanding of inter-subject differences in the recovery from challenging tasks.File S1Alternative parcellation templates.(PDF)Click here for additional data file.File S2Graph metrics at different cost levels.(PDF)Click here for additional data file.File S3Pre-task level on connectivity strength and performance.(PDF)Click here for additional data file.File S4Analyses of suspect motion-corrupted frames.(PDF)Click here for additional data file.


Emerging evidence suggests that connexin mediated gap junctional intercellular communication contributes to many aspects of bone biology including bone development, maintenance of bone homeostasis and responsiveness of bone cells to diverse extracellular signals. Deletion of connexin 43, the predominant gap junction protein in bone, is embryonic lethal making it challenging to examine the role of connexin 43 in bone in vivo. However, transgenic murine models in which only osteocytes and osteoblasts are deficient in connexin 43, and which are fully viable, have recently been developed. Unfortunately, the bone phenotype of different connexin 43 deficient models has been variable. To address this issue, we used an osteocalcin driven Cre-lox system to create osteoblast and osteocyte specific connexin 43 deficient mice. These mice displayed bone loss as a result of increased bone resorption and osteoclastogenesis. The mechanism underlying this increased osteoclastogenesis included increases in the osteocytic, but not osteoblastic, RANKL/OPG ratio. Previous in vitro studies suggest that connexin 43 deficient bone cells are less responsive to biomechanical signals. Interestingly, and in contrast to in vitro studies, we found that connexin 43 deficient mice displayed an enhanced anabolic response to mechanical load. Our results suggest that transient inhibition of connexin 43 expression and gap junctional intercellular communication may prove a potentially powerful means of enhancing the anabolic response of bone to mechanical loading. Normal remodeling of bone requires synchronized activity between bone resorbing osteoclasts and bone forming osteoblasts, as well as the coordination of this process by osteocytes, by far the most abundant bone cell. An abundance of data generated over the last several years suggest that connexins and gap junctional intercellular communication (GJIC) play a critical role in coordinating synchronized bone cell activity, especially in response to extracellular signals including those induced by mechanical load Most of the data supporting a role for Cx43 and GJIC in osteoblastic cell differentiation, coordinated cell responsiveness, and mechanotransduction comes from in vitro studies. For instance, studies from our laboratory While these studies strongly suggest that Cx43 and GJIC are critical to normal bone development and remodeling, they are limited by their in vitro nature or the fact that they reflect the consequence of global Cx43 deficiency during development only, as global Cx43 deletion is embryonic lethal To more closely examine this issue we selectively deleted the Cx43 gene in mouse osteoblasts and osteocytes using Cre recombinase driven by the human osteocalcin promoter, as previously reported by Plotkin et al flx/flx, designated wild type), and conditional Cx43 deficient equivalent  mice.Using the protocol described in The breeding strategy we used is more efficient for breeding Cx43 deficient mice than the method used by Plotkin et al flx/flx, and deletion of Cx43 results in expression of β-galactosidase flx/flx) mice displayed positive X-gal staining but osteoblastic cells from wild type (Cx43flx/flx) mice did not . μCT analysis of the midshaft of the femur revealed periosteal and endosteal expansion in Cx43 deficient mice relative to wild type mice . The femFemurs from 8 week old Cx43 deficient mice displayed a 35% and 36% decrease in whole bone strength and material strength, respectively, compared to wild type mice .Since bone mass is balanced by bone formation and resorption, we next examined, using dynamic histomorphometry and quantification of a serum bone formation marker, whether the phenotype in Cx43 deficient mice is due to decreased bone formation. Eight week  and 6 moCx43 deficient mice displayed more TRAP positive cells than did wild type mice in femur cortical and trabecular areas . We quanThe RANKL-OPG axis is a major pathway through which osteoblasts and osteocytes regulate osteoclastic bone resorption. To examine the mechanism by which Cx43 deficiency in osteoblasts and osteocytes leads to increased osteoclastogenesis and bone resorption, we quantified the ratio of RANKL mRNA levels to OPG mRNA levels (RANKL/OPG ratio), using real time RT-PCR, in primary osteoblastic cells from Cx43 deficient and wild type mouse long bones. Steady state Cx43 protein and mRNA levels in osteoblastic cells from Cx43 deficient mice were significantly decreased relative to levels in osteoblastic cells from wild type mice, confirming Cx43 deficiency in Cx43 deficient mice . HoweverWe next examined the RANKL/OPG mRNA ratio in osteocytic cells. Because it is challenging to isolate homogeneous populations of primary osteocytic cells, making interpretation of results difficult, we used MLO-Y4 as model osteocytic cells. MLO-Y4 cells were rendered Cx43 deficient by transfection with siRNA directed against Cx43. As controls we examined MLO-Y4 cells transfected with scrambled siRNA. We previously demonstrated that MLO-Y4 cells transfected with Cx43 siRNA, identical to the cells we used in this study, display significantly lower Cx43 protein levels compared to MLO-Y4 cells transfected with scrambled siRNA At 6 months of age, tibiae of Cx43 deficient mice dispalyed p.MS, p.MAR, and p.BFR values that were not statistically significantly different than wild type mice . In wildEmerging data suggest that Cx43 mediated GJIC is critical to normal bone development. Unfortunately, examination of the role of Cx43 post development is challenging because mice with global Cx43 deficiency die at birth To further address this issue we deleted Cx43 in osteoblasts and osteocytes using the osteocalcin driven Cre and floxed approach taken by Plotkin et al One possible explanation as to why our results differ from those previously reported is that we used μCT and dynamic histomorphometry to access overall adult bone morphology, as well as bone formation and resorption, whereas previous studies used DEXA alone or DEXA combined with dynamic histomorphometry, which may be less sensitive approaches than μCT. Furthermore, in the case of the murine model developed by Chung et al Since deleting Cx43 in osteoblasts and osteocytes did not affect mouse ambulation (data not shown), our data indicate that functional Cx43 in osteoblasts and osteocytes is required for normal bone mass acquisition and maintenance. Our finding that MAR and BFR are not reduced, and if anything are increased, in Cx43 deficient mice, together with those of Plotkin, Chung and Grimston showing no, or only slight, increases in MAR and BFR in a similar mouse model, suggests that a decrease in osteoblastic activity does not contribute to the bone phenotype we observed in osteoblast and osteocyte specific Cx43 deficient mice.Because our data suggest that decreased osteoblastic function was not mediating the cortical bone phenotype in Cx43 deficient mice we examined another possible explanation, increased osteoclastic bone resorption. Interestingly, TRAP staining revealed that Cx43 deficient mice have more osteoclasts than wild type mice . Cx43 deWe next sought to determine how Cx43 deficiency could result in increased osteoclast differentiation or activity. A major regulator of osteoclast formation and activity is the RANKL/OPG system. RANKL, a ligand expressed on the surface of marrow stromal cells and osteoblastic precursors, binds to its receptor found on the surface of osteoclastic precursor cells and thereby activates osteoclastogenesis. OPG serves as a soluble decoy molecule and binds with RANKL thus blocking its activity and inhibiting osteoclastogenesis. The ratio of RANKL to OPG, therefore, provides a reliable indicator of relative osteoclastogenesis. However, we found the RANKL/OPG mRNA ratio in osteoblastic cells isolated from Cx43 deficient mice was similar to that in osteoblastic cells isolated from wild type mice. Therefore, it is unlikely that Cx43 regulation of the RANKL/OPG pathway in osteoblastic cells contributed to altered osteoclastogenesis.We next examined whether Cx43 deficiency in osteocytic cells could alter the RANKL/OPG ratio and thereby contribute to decreased osteoclastogenesis. Osteocytes are the most abundant cells in bone The mechanism by which Cx43 and GJIC interacts with the RANKL/OPG pathway is unclear. However, published reports suggest that the activity of SP-1/SP-3 transcription factors is regulated by Cx43 mediated GJIC in osteoblastic cells in vitroIn vivo, Lozupone et al Emerging data suggest that Cx43 and GJIC contribute to mechanotransduction in bone In contrast, our in vivo mechanical loading data clearly demonstrate that deletion of Cx43 in osteoblasts and osteocytes results in an enhanced periosteal response to physiological mechanical loading. That these data contrast with those of Grimston et al may be due to differences in basal levels of periosteal osteoblast function, magnitude of induced strains  flx/flx) flx/+ mice. Next, we crossed OC-Cre; Cx43flx/+ with Cx43flx/flx to generate OC-Cre; Cx43flx/flx. We then back bred OC-Cre; Cx43flx/flx mice with Cx43flx/flx mice to generate equal number of OC-Cre; Cx43flx/flx  and Cx43flx/flx (wild type) littermate mice. Mice derived from this breeding strategy were bred with C57BL/6 for 3 generations resulting in mice with a C57BL/6 background. Genotyping was performed by PCR using genomic DNA isolated from mouse ear pieces and primers listed in Mice expressing Cre recombinase under the control of the human osteocalcin promoter , was cultured on type I collagen (BD Laboratory) coated plates in Modified Eagle Medium Alpha  supplemented with 5% fetal bovine serum (FBS), 5% calf serum (CS), and 1% penicillin and streptomycin (P/S). The human fetal osteoblastic progenitor cell line hFOB 1.19 cells (kindly provided by Dr. Steven Harris) was maintained using Dulbecco's modified Eagle's medium (DMEM)–Ham's F-12 1∶1 media (GIBCO) supplemented with 10% FBS and 1% P/S.Primary osteoblastic cells were isolated from explant cultures of 3 week old Cx43 deficient or wild type femurs and tibias which were digested in 1 mg/mL collagenase  for 75–120 min at 37°C in a shaking water bath. The bones were cut into small pieces and the pieces from one femur and one tibia placed into one well of a 6 well plate in 600 µL of αMEM, with 10% FBS, 1%PS, 1% antimycotic , 50 µg/mL ascorbic acid phosphate  for 5 days. The media was replaced with fresh media without antibiotics for one week. Cells that grew out from the bone fragments were then sub-cultured into 100 mm dishes. When the cells were 90% confluent gap junctional communication, protein and mRNA levels were quantified.GJIC was quantified using a dual label fluorescent dye transfer technique and flow cytometry as previously described Femurs from the right side of 8 week and 6 month old mice were harvested for μCT analysis. The diaphyses were scanned starting at the midpoint of the bone and acquiring 76 slices distally using a Scanco vivaCT 40  with scan settings of 55 KVp, 145 µA, and 200 ms integration time. Images were reconstructed as a matrix of 2048×2048×76 isotropic voxels measuring 10.5 µm. Images were gaussian filtered  and a threshold  was applied to remove the surrounding soft tissue. The periosteal and endosteal boundaries of the cortical bone were segmented using the Scanco semi-automated edge detection algorithm. Periosteal volume, endosteal volume, bone porosity, cortical bone thickness, and BMD were calculated for the diaphysis of each femur using the Scanco Image Processing Library routines.Femurs from 8 week old mice were stored in PBS at −80°C before being mechanically tested to failure in three-point bending using an MTS MiniBionix 858 testing apparatus  Using the midshaft μCT image to calculate the moment of inertia, the ultimate tensile stress was calculated using Euler-Bernoulli beam theory. The ultimate tensile stress was calculated using the flexural formula (stress = FLc/4I), where F is the ultimate load, L is the support span length, c is the perpendicular distance from the neutral axis to the most lateral point on the bone surface, and I is the bending moment of inertia calculated about the neutral axis.3/µm2 per year) For examination of 8 week and 6 month old mice, male mice were given an intraperitoneal injection of calcein (Sigma) at 10 mg/kg body weight and 6 days later injected with Alizarin red (Sigma) at 30 mg/kg body weight. Seven days after the second injection mice were sacrificed and ulnae were dissected and embedded using an Osteo-Bed bone embedding kit  following the manufacturer's protocol. The embedded ulnae midshafts were then cut into approximately 100 µm thick discs followed by abrasive polishing to a thickness of about 50 µm with lapping films . Digital images were obtained using a confocal microscope . The following periosteal and endcortical bone parameters were quantified with ImageJ : total perimeter (B.Pm); single label perimeter (sL.Pm); double label perimeter (dL.Pm), and double label area (dL.Ar). The following values were then calculated: mineralizing surface ; mineral apposition rate  and bone formation rate  and C-terminal telopeptides (CTX) were measured with EIA or ELISA kits from Immunodiagnostic Systems Inc  according to manufacturer's protocols.Right femurs from 6 month old mice were fixed in 4% paraformaldehyde for 3 days prior to decalcification with 10% EDTA (pH 7.4) for 5 days. These specimens were embedded in paraffin and cut into 5 µm sections then subjected to TRAP staining using a technique modified from Erlebacher and Derynck Osteocytic MLO-Y4 cells were transfected with either Cx43 or scramble control siRNA as previously described Initial studies indicated that Cx43 deficient mice demonstrated an osteopenic cortical bone phenotype with expanded endocortical and periosteal surfaces compared to wild type littermates. This morphology suggests an accumulation of elevated periosteal osteoblast function and elevated endocortical osteoclast function through development. We confirmed this morphologic phenotype in 17 week old mice via high resolution μCT. Concurrent bone labeling indicated that the periosteal surface was highly active in the majority of Cx43 deficient mice at this age.As an active periosteum has the potential to confound assessment of bone's response to physiologic loading Data are expressed as means ± SD. Statistical significance was assessed by Student's t-test when comparing two groups. One way ANOVA was used to assess multiple group comparisons and a Tukey post-hoc test to assess differences between groups. p<0.05 was considered significant.