Col6a3), a component of extracellular matrix, is often up-regulated in tumours and is believed to play a pro-oncogenic role. However the mechanisms of its tumorigenic activity are poorly understood. We show here that Col6a3 is highly expressed in densely growing mouse embryonic fibroblasts (MEFs). In MEFs where the TAF4 subunit of general transcription factor IID (TFIID) has been inactivated, elevated Col6a3 expression prevents contact inhibition promoting their 3 dimensional growth as foci and fibrospheres. Analyses of gene expression in densely growing −/−Taf4 MEFs revealed repression of the Hippo pathway and activation of Wnt signalling. The Hippo activator Kibra/Wwc1 is repressed under dense conditions in −/−Taf4 MEFs, leading to nuclear accumulation of the proliferation factor YAP1 in the cells forming 3D foci. At the same time, Wnt9a is activated and the Sfrp2 antagonist of Wnt signalling is repressed. Surprisingly, treatment of −/−Taf4 MEFs with all-trans retinoic acid (ATRA) restores contact inhibition suppressing 3D growth. ATRA represses Col6a3 expression independently of TAF4 expression and Col6a3 silencing is sufficient to restore contact inhibition in −/−Taf4 MEFs and to suppress 3D growth by reactivating Kibra expression to induce Hippo signalling and by inducing Sfrp2 expression to antagonize Wnt signalling. All together, these results reveal a critical role for Col6a3 in regulating both Hippo and Wnt signalling to promote 3D growth, and show that the TFIID subunit TAF4 is essential to restrain the growth promoting properties of Col6a3. Our data provide new insight into the role of extra cellular matrix components in regulating cell growth.Collagen 6A3 ( In lox/−Taf4 MEFs the TFIID contains predominantly TAF4, whereas in −/−Taf4 MEFs, TAF4b replaces TAF4 to maintain TFIID integrity and cell viability −/−Taf4 MEFs display TGFβ-dependent autocrine growth and deregulated expression of more than 1000 genes.TAF4 is a subunit of the general transcription factor TFIID. In vertebrates, the TAF4 family comprises a ubiquitously expressed TAF4 protein and a tissue specific paralogue, TAF4b, required for testis and ovary function Contact inhibition is a process that arrests cell proliferation upon cellular contacts under conditions of high density. It is an important mechanism of anti-cancer defence, as tumour cells normally lose this property and grow in an uncontrolled manner. The molecular mechanisms underlying contact inhibition are still poorly understood. A number of recent studies identified the Hippo signalling pathway as a major effector of contact inhibition Loss of contact inhibition is not the only event in oncogenic transformation. Other events are required, such as the activation of the Wnt signalling pathway. This pathway regulates many biological processes, including morphology, proliferation, motility and cell fate. The canonical Wnt pathway involves binding of Wnt proteins to cell-surface receptors of the Frizzled family, causing the receptors to activate Dishevelled family proteins and resulting in stabilization and nuclear import of β-catenin. Inappropriate activation of this pathway with accumulation of nuclear β-catenin is observed in several human cancers It is becoming increasingly recognised that the extracellular matrix (ECM) not only provides a 3 dimensional (3D) matrix for cell growth and organogenesis, but that signals from the ECM play critical roles in cell fate and cell growth −/−Taf4 background, a subpopulation of MEFs loses contact inhibition, resulting in the formation of 3D foci and growth as fibrospheres. The cells forming foci are characterised by activated Wnt signalling and inhibition of Hippo signalling. Strikingly, Col6a3 silencing is sufficient to restore contact inhibition in −/−Taf4 MEFs. Our data show for the first time that Col6a3 plays a role in modulating signalling pathways involved in contact inhibition providing an explanation for the observed association between Col6a3 and cancer. It also suggests that changes in the ratio of TAF4 and TAF4b can play a role in the susceptibility of cells to Col6a3-promoted 3D growth. Finally, we also show that all-trans retinoic acid (ATRA) treatment represses Col6a3 expression thus abrogating premalignant changes in both wild-type and −/−Taf4 MEFs. Our data suggest that ATRA could be a valuable treatment for refractory Collagen VI-associated cancers.Here we show that TAF4 is able to attenuate the growth promoting activities of Collagen VI. In a Taf4lox/− and C3Taf4−/− MEFs were derived from genetically modified Taf4lox/− mouse embryos and have previously been described Taf4 allele was defloxed in the C1 MEFs by expression of the Cre recombinase and loss of TAF4 expression in the C3 MEFs was verified by PCR genotyping and by western blot analysis as described (10). Cells were cultured in Dulbecco's minimal essential media (DMEM) supplemented with 4.5 g/l glucose and 10% foetal calf serum. Cells were treated with 10−6 M ATRA dissolved in ethanol (DMSO) as indicated.The C16 of the indicated cells were inoculated and grown for 10 days.For fibrosphere assays, the indicated cells were also grown under non-adherent conditions in bacterial culture plates. 10Immunofluoressence on C1 and C3 cells was performed by standard procedure using following antibodies: COL6A3 , CTNNB1 , YAP1 , TAZ , SOX2 . Immunofluorescence was visualized using a Zeiss Axiophot  microscope equipped with epifluorescence illumination. Confocal microscopy was performed on a Leica SP2 microscope.IWR-1  and XAV939  were used at 1 µg/mL concentration.RNA-seq was performed as previously described Lentiviral shRNA expression vectors and packaging plasmids were purchased from Sigma-Aldrich. The TRC numbers are indicated in the Supplemental information. Lentiviral particles were generated by co-transfection of shRNA vector together with packaging plasmids into 293T cells. Medium was changed 24 hs after transfection and viruses harvested 48 hs after transfection and used for infection of C3 cells. Infected C3 cells were selected with puromycin resistant and silencing of targeted gene was checked by RT-qPCR.RNA was prepared using Trizol reagent  according to manufacturer's protocol. Reverse transcription was performed with Super Script II reverse transcriptase  as described in manufacture's protocol. Random hexaoligonucleotides were used as primers. qPCR was performed using LightCycler 480 SYBR Green I master mix  on the LightCycler 480 Real-Time PCR System. List of oligonucleotides is supplied in x S1.4 cells were seeded in 10 cm plates. At the indicated times the cells were trypsinised and counted. BrdU incorporation was performed using a BrdU Cell Proliferation Assay kit (QIA58) from Merck/CalBiochem as per the manufacturers instructions. Briefly, the cells were grown for the indicated times with or without RA and then BrdU incorporation for 24 hours was measured by an immuno-colometric assay.For cell counting, 5×10−/−Taf4 MEFs by defloxing of lox/−Taf4 MEFs −/−Taf4 MEFs (hereafter C3) are irregularly shaped and have lost contact inhibition as they readily form three dimensional (3D) foci that are never observed with the lox/−Taf4 MEFs (hereafter C1) (We have previously reported generation of fter C1) . We alsofter C1) .Many types of transformed cells can be grown under non-adherent conditions as spheres such as ‘mammospheres’ in the case of breast cancer cells We next used RNA-seq to profile the changes in gene expression that occur upon 3D growth. RNA was prepared from low or high-density adherent cultures of C3 cells comprising 3D foci and from fibrospheres. In comparison with non-confluent cells, 669 transcripts were-up-regulated and 714 down-regulated in dense cells . SimilarOntology analysis of the down-regulated transcripts indicated strong enrichment in those involved in cell cycle and cell division consistent with the fact that proliferation is considerably reduced in dense C3 cells or when grown as spheres compared to the rapid growth of the non-dense cultures . In contWe had previously reported that genes of the interferon response were strongly induced in the C3 cells lacking TAF4 compared to the C1 cells. We ascribed this difference to TAF4 inactivation Olfm1 and Spp1, although induced in dense C3 cells, is very much higher in fibrospheres  and laminins (a4 and a5) are strongly induced along with VCAM1, thrombospondin 2, intergrins b8, a2b and a7 and cadherins 13 and 26. Thus, 3D growth involves major changes in cell adhesion and remodelling of the ECM. On the other hand, expression of ospheres .Col6a3 strongly increases in dense C3 cells and is strongly expressed in fibrospheres  whose expression is strongly down regulated in dense cells and fibrospsheres . The ability of TAF4 to counteract the growth promoting effects of high Col6a3 expression can be evaluated by comparing the responses of the two cell types under conditions of dense growth. In TAF4-expressing C1 cells, YAP1 and TAZ are located in the nucleus as the cells proliferate at low density. At high density, Kibra remains highly expressed, while Fat4 expression is strongly induced. This suggests that Hippo signalling is maintained in dense C1 cells to mediate contact inhibition, an idea confirmed by the translocation of YAP1 from the nucleus and the overall down-regulation of TAZ expression in dense cultures.In this study, we show that −/−Taf4 cells, YAP1 accumulates in the nucleus to promote 3D growth. Kibra expression can be induced by YAP1 overexpression through an as yet unknown mechanism In contrast, in the absence of TAF4, the pathways and factors that normally maintain Kibra expression under dense conditions are no longer operative and Kibra expression is repressed attenuating Hippo signalling. Consequently, in dense −/−Taf4 MEFs. In dense −/−Taf4 MEFs, Wnt9a expression is strongly up-regulated while Sfrp2 expression is repressed. The nuclear localisation of β-catenin in the cells forming 3D foci shows enhanced Wnt signalling in these cells. Also shRNA-mediated Wnt9a silencing or use of chemical inhibitors of Wnt signalling abrogates 3D growth indicating the critical role of the pathway in this process. Interestingly, ChIP-seq has revealed SOX2 Wnt9a gene. This suggests that YAP1 and SOX2 in dense C3 cells may activate Wnt9a expression in a positive feed forward loop to promote 3D growth. This contrasts with the TAF4-expressing MEFs, where Wnt9a expression is not induced under dense conditions, but Sfrp2 expression is strongly induced. Consequently, in dense TAF4-expressing MEFs, Wnt signalling is repressed, the opposite of what is observed in Taf4−/− MEFs. The loss of TAF4 therefore modifies Wnt9a and Sfrp expression to activate Wnt signalling in conditions of high density to promote 3D growth.Several lines of evidence indicate that Wnt signalling is also involved in 3D growth of Together our results support a model where loss of contact inhibition through diminished Hippo signalling allows the cells to form dense foci, while enhanced Wnt signalling is further required for full 3D growth. It is also interesting to note that high SOX2 expression is seen already in rare nuclei of low-density C3 cells. Thus TAF4 inactivation leads to heterogeneity in the cell population suggesting that it is the SOX2 high population that is competent to generate 3D foci under dense conditions.Taf4−/− MEFs for 3D growth appears to be associated with high Col6a3 expression. The expression of several membrane and ECM components is strongly induced in dense conditions. Nevertheless, shRNA mediated silencing of Col6a3 alone is sufficient to abolish 3D growth. Further evidence for a critical role of Col6a3 in 3D growth comes from the observation that its expression is down-regulated by ATRA that restores contact inhibition and represses 3D growth. ATRA down-regulates both Col6a3 and to a lesser extent Col6a2, thereby down-regulating holo-collagen VI fibre formation. While the expression of many membrane and ECM components are strongly induced by 3D growth, Col6a3 is one of the few regulated by ATRA and is the most strongly repressed. Moreover, ATRA treatment does not affect expression of known components of the Hippo pathway and may even potentiate Wnt signalling through up-regulation of Wnt9a. These observations, together with the results of Col6a3 silencing, indicate that the major mechanism by which ATRA inhibits 3D growth is through repression of Col6a3. The growth suppressive effect of ATRA on these cells by regulation of ECM components is therefore fundamentally different from that seen in F9 embryonal carcinoma cells, HL60 myeloid cells, or mammary carcinoma cells where RA treatment induces cell cycle arrest, differentiation and under some conditions apoptosis The capacity of Col6a3 silencing stimulates expression of Kibra and re-activates Hippo signalling leading to reduced YAP1 expression. These observations show that Col6a3 plays an active role in modulating expression of growth control genes and are in line with previous results showing that high Col6a3 expression modulates cell and tumour growth. Ovarian cancer cells resistant to cisplatin show a potent induction of the Col6a3 gene and in vivo, high grade tumours express higher levels of Col6a3 than low grade tumours Col6a3 is up-regulated in the stroma of colon tumours Col6a3 can modulate expression of critical regulators of the Hippo and Wnt pathways to promote growth and how it can serve as a target for ATRA mediated suppression of growth.ECM components such as COL6A3 can provide a network that physically facilitates 3D growth. While the reduction in this network in the presence of ATRA may contribute to its ability to repress 3D growth, remodelling the ECM does not appear to be the only role of COL6A3 as it's silencing also dramatically modulates gene expression and signalling. ShTogether the results described here reveal a novel and complex interplay between at least three signalling pathways  that control 3D fibroblast growth. We describe the ability of TAF4 to control expression of critical components of the Hippo and Wnt pathways and a novel role of COL6A3 as ATRA-regulated modulator of 3D growth and as regulator of gene expression.Figure S1Expression and localisation of TAZ and SOX2 in C1 MEFs.A. Immunostaining of non-dense and dense C1 MEFs for TAZ. B. Immunostaining of non-dense and dense C1 MEFs for SOX2 (20× magnification). C. Control staining of F9 embryonal carcinoma cells and of hepatocyte cells with SOX2 antibody to demonstrate the specificity of the signal.(PDF)Click here for additional data file.Figure S2Expression and localisation of TAZ, YAP1 and SOX2 in C3 MEFs.A. Immunostaining of non-dense and dense C3 MEFs for TAZ. B. Immunostaining of low density C3 cells with YAP1 and SOX2 antibody (20× magnification). Cells expressing low or high levels of SOX2 are indicated by arrows. C Immunostaining of dense C3 MEFs for YAP1 and SOX2 (20× magnification). The location of cells growing in a 3D foci is indicated.(PDF)Click here for additional data file.Figure S3A. Effect of RA on C3 cell proliferation.A. Kinetics of cell growth in presence or absence of RA as evaluated by cell counting. B. Assessment of cell division by incorporation of BrdU on cells grown for the indicated periods in presence or absence of RA. C. Results of a representative FACS assay showing the % cells in each stage of cell cycle. D. Clonogenic assays of C3 cells in presence or absence of ATRA or shCol6a3 on wells coated with fibronectin.(PDF)Click here for additional data file.Figure S4Effect of shCol6a3 knockdown on gene expression. RT-qPCR on the indicated genes in C3 cells expressing control shRNA or shRNA directed against Col6a3 grown for 3 or 10 days as indicated.(PDF)Click here for additional data file.Figure S5Expression of TAZ in shCol6a3 knockdown cells.A. Expression of TAZ in low-density shCol6a3 knockdown cells. B Expression of TAZ in high density shCol6a3 knockdown cells.(PDF)Click here for additional data file.Table S1Sequences of primers used for qPCR of the indicated genes on the forward and reverse strands.(DOC)Click here for additional data file.Table S2Excel table of RNA-seq results. Page 1 shows transcripts induced in dense conditions. Shown are, the Ensembl gene ID, the average RPKM expression values under each condition, the fold change and Log2 change values under the indicated conditions, gene name and description. Pages 2–4 show the same information concerning transcripts induced in spheres, repressed under dense conditions and repressed in spheres respectively.(XLS)Click here for additional data file.Table S3Ontology analyses of genes whose expression is modified under conditions of dense or 3D growth. Each page shows the analysis of genes differentially regulated under the specified conditions with the indicated ontology terms.(XLS)Click here for additional data file.Table S4Genes regulated by ARTA in C3 MEFs. Pages 1 and 2 show the induced and repressed genes after 12 and 72 hours of ATRA treatment. Shown are the Ensembl gene IDs, gene name, log2 ratios −ATRA/+ATRA 12 hours, −ATRA/+ATRA 72 hours, +ATRA12 hours/+ATRA 72 hours, and gene description.(XLS)Click here for additional data file.


Central precocious puberty (CPP) could be a phenotype of pathology in the central nervous system. While it is generally accepted that all boys with CPP and girls with CPP at less than 6 years of age should undergo brain imaging as part of the workup, there have been controversies as to the use of brain imaging in girls who develop CPP between 6 to 8 years.To evaluate the prevalence and clinical characteristics of intracranial lesions in patients with central precocious puberty aged 6 to 8 years in a single centre in the past 11 years.Retrospective chart review of girls with CPP and their MRI findings between year 1999 to 2009 in a single centre.One hundred and eighty-eight girls had central precocious puberty in the study period and 157 of them (83.5%) had MRI of the pituitary done as part of the workup. The prevalence of intracranial pathology among girls with CPP aged 6 to 8 years was 20.0% while among all girls with CPP aged less than 8 years, 34 girls (21.7%) were found to have intracranial pathology. These pathologies included: pituitary adenoma (n = 16), pineal cyst (n = 8), Rathke’s cleft cysts (n = 4), arachnoid cyst (n = 1), intra-ventricular cyst (n = 1), venous angioma over the left frontal lobe (n = 1), hydrocephalus (n = 2) and an old infarct over the frontal lobe (n = 1). The two cases of hydrocephalus and the case with an old infarct were known before the onset of CPP. None of the lesions detected required further interventions with surgical removal, chemotherapy or radiotherapy within the follow-up period of 7.2 ± 3.0 years.Brain imaging the girls with CPP in our centre mainly detected benign lesions not requiring any intervention during our follow-up period. Though the current data do not justify a practice of performing routine MRI for girls diagnosed to have CPP at 6 to 8 years, longer follow-up assessment of such lesions detected in childhood may be necessary before concluding on their benign outcome.


Thymic epithelial cells (TECs) are necessary for normal T cell development. Currently, one transcription factor, Foxn1 is known to be necessary for the progression of fetal TEC differentiation. However, some aspects of fetal TEC differentiation occur in Foxn1 mutants, suggesting the existence of additional transcriptional regulators of TEC differentiation. The goal of this study was to identify some of the additional candidate transcription factors that may be involved in the specification and/or differentiation of TECs during fetal development.Foxg1, Isl1, Gata3, Nkx2-5, Nkx2-6 and Sox2 for further studies. Whole mount in situ hybridizations confirmed the expression of these transcription factors within subdomains of the third pharyngeal pouch from E9.5–E10.5. By E11.5 days Foxg1 and Isl1 transcripts were the only mRNAs from this group of genes detected exclusively within the thymus domain of the third pouch. Based on this initial in situ hybridization analysis, we focused on defining the expression of Foxg1 and Isl1 during multiple stages of thymus development and TEC differentiation. We found that Foxg1 and Isl1 are specifically expressed in differentiating TECs during fetal and postnatal stages of thymus development. In addition, we found differential expression of Islet1 and Foxn1 within the fetal and postnatal TEC population.We identified candidate fetal TEC transcriptional regulators via data and text mining. From our data mining we selected the transcription factors Foxg1 and Isl1 may play a role in the regulation of TEC differentiation during fetal and postnatal stages. Our results also demonstrate heterogeneity of TECs marked by the differential expression of transcription factors, potentially providing new insights into the regulation of TEC differentiation.Our studies have identified two developmental transcription factors that are excellent candidate regulators of thymic epithelial cell specification and differentiation during fetal development. Our results suggest that  Thymic epithelial cells (TECs) are a critical component of the thymic microenvironment. TECs are derived from the endoderm of the third pharyngeal pouch. Despite their essential role in thymus function, our current understanding of fetal TEC specification and differentiation is very limited. For example, we do not know which transcriptional regulators are necessary for the earliest specification of the thymus organ domain within the third pharyngeal pouch. In addition we have very limited knowledge about the transcription factors that regulate the differentiation and function of TECs during fetal and postnatal thymus development. Identifying the factors that regulate these key steps in the development of thymic epithelial cells is a key part of understanding the genetic pathways that regulate thymus organogenesis and function.Gcm2 at E9.5 in the parathyroid domain and Foxn1 at E11.25 in the thymus domain of the 3rd pharyngeal pouch marks the patterning of the pouch into the primordium of the two organs Foxn1 which is first detected at E11.25 Foxn1 is first expressed suggests that additional transcription factors are acting within the pouch at times prior to Foxn1 expression. These factors include the transcriptional regulators that activate Foxn1 within the thymus primoridium.Our current knowledge regarding the earliest events in the specification of the parathyroid and thymus suggests that specification occurs early in third pouch development. Localized expression of Hoxa3, Pbx1, Tbx1, Pax1, Pax9, Six1 and Eya1 as necessary for 3rd pouch development. All of these transcription factors, except for Tbx1 are expressed throughout the 3rd pouch at E10.5 prior to detectable Foxn1 expression Tbx1 is initially expressed throughout the 3rd pouch and becomes restricted to the presumptive parathyroid domain at E10.5 in the pouch endoderm Hoxa3, Eya1, Six1, Pax9, Tbx1 and Pbx1 the homozygous mutants either fail to form the 3rd pouch or exhibit very severe early defects in the formation of both the thymus and parathyroid primordia Previous studies have identified the transcription factors in situ hybridization analysis based on previous expression data, our genetic studies have shown that it is necessary for the normal development of the third pharyngeal pouch Gata3 mutants at E12.5 days indicating an early role for this gene in the development of the pouch A major goal of this screen was to identify candidate developmental transcription factors that play an important role in 3rd pharyngeal pouch endoderm development and/or the differentiation of fetal TECs into functional components of the thymus microenvironment. To enable the characterization of the genes identified in our screen we focused our screen on genes for which well-characterized knockout mice are available. In the case of Gata3, which we chose for detailed in situ hybridization data are limited in terms of resolution, they can be used to suggest candidate genes for in depth characterization. We took advantage of this information to focus our in situ hybridization analysis to a short list of candidate transcription factors that were likely to be expressed in localized domains of the 3rd pouch and/or thymic epithelium. For third pharyngeal pouch expression we visually screened data in the MGI/GXD and Emage in situ hybridization databases and examined published reports describing Cre recombinase expression patterns in situ hybridization of para-sagittal sections in the Genepaint database indicated that most of the genes we had identified as being expressed in the third pouch were also expressed within the thymus at E14.5. This suggested that these transcription factors were excellent candidates for genes involved in thymus development and differentiation. After additional literature mining, we selected Nkx2-5, Nkx2-6, Foxg1, Islet1, Gata3 and Sox2 for further analysis in this study. The screen of in situ database information allowed us to focus on performing a detailed characterization of a small group of genes rather than performing a less detailed large-scale screen.A large amount of published and unpublished mouse developmental gene expression data is available in online databases Foxn1 expression within the thymus domain of the third pouch at E11.25 days are not known. One purpose of our study was to search for transcription factors with regionalized expression patterns in the third pharyngeal pouch endoderm between E9.5–E11.25. We analyzed the expression of our candidate genes in E9.5 and E10.5 somite stage matched wild type mouse embryos by in situ hybridization and 3D reconstruction, focusing on their expression in the pharyngeal pouches. By carefully staging embryos by somite counting we found that embryos at the same somite stage had comparable pouch morphologies as determined by 3D reconstruction of paraffin sections. By comparing the expression patterns of the genes we examined to the Gcm2 expression pattern at E10.5 as a landmark for organ specific domains, we found a surprisingly diverse and complex combination of regionalized expression patterns in the third pouch endoderm.Although pouch endoderm as early as E9.5 is capable of developing into a fully functional thymus when transplanted under the kidney capsule Nkx2-5 and Nkx2-6 expression have detected expression of Nkx2-5 in the pharyngeal endoderm and Nkx2-6 in the pharyngeal pouch endoderm. However, these studies have not described the expression pattern of either gene in the 3rd pouch in detail Nkx2-5 is expressed in a localized domain of the 3rd pouch Nkx2-5 at some point in its history Nkx2-5/Nkx2-6 double mutants Previous studies of in situ hybridization analysis revealed that Nkx2-5 and Nkx2-6 are expressed in localized regions of the third pouch. At E9 days (20–23 somites) Nkx2-5 is expressed on the ventral side of the forming third pharyngeal pouch endoderm  indicated that it is expressed in the thymus domain of the E10–E10.5 (30–35 somites) 3rd pouch Gata3 are associated with a syndrome of hypoparathyroidism, sensorineural deafness and renal disease Gata3 in development of 3rd pouch derivatives.Previous studies have detected Isl1 during pharyngeal pouch development. At the 20–23 somite stage, Isl1 is expressed in the cells on the ventral side of the developing second and third pharyngeal pouches but on the dorsal portion of the first pouch , Foxg1 expression is detected at the ventral tip of the 3rd pouch endoderm .Our analysis revealed a dynamic pattern of endoderm . By 30–3al pouch , 2F. Thihe pouch . We alsoNkx2-5, Nkx2-6, Isl1, Gata3 and Foxg1 in the ventral portion of the third pharyngeal pouch suggested that these factors might be expressed in the thymic epithelial cells at later stages. To test this idea we examined the expression of these 5 transcription factors in E11.5 wild type embryos. In the E11.5 third pouch endoderm, the thymus domain can be identified by the expression of Foxn1 in the ventral posterior portion of the pouch, complimentary to the parathyroid domain which is marked by Gcm2 expression The expression of in situ hybridization analysis showed that Nkx2-5, Nkx2-6 and Gata3 are not expressed within the thymus domain of the third pouch at E11.5. In fact, at this stage Nkx2-5 and Nkx2-6 are no longer expressed in any of the pharyngeal pouches  protein, which has been shown to be expressed in medullary thymic epithelial cells and in a subset of the cortical epithelial cells at late fetal and postnatal stages In sections of E16.5 fetal thymus, ISL1 expression was detected in all FOXN1-expressing thymic epithelial cells . To confFoxg1 null embryo (data not shown) In contrast, we found that FOXG1 expression in E16.5 thymus completely co-localizes with FOXN1 in TECs . The antFoxn1 transcript and protein expression are at various levels in medullary thymic epithelial cells high; ISL1low; FOXG1low TECs and FOXN1high; ISL1high; FOXG1high TECs were widely present at these stages  embryos were dissected in DEPC-PBST  and somite number was determined. Embryos within a narrow range of somite stages were pooled in groups of 3–5 for processing. Therefore, we refer to individual embryos as being within a range of somite numbers since we cannot accurately count somites after the Nkx2-5, a cDNA plasmid clone was generated by RT-PCR using E11-day mouse total RNA (Clontech). Primers were Nkx2-5-3-F: CTACG GCGTG GGTCT CAATG C and Nkx2-5-3-R: GCGTT AGCGC ACTCA CTTTA ATGG. The transcription template was generated by PCR using the SP6 and T7 promoter primers and using our Nkx2-5 plasmid cDNA as template. The Nkx2-6 probe template was amplified from mouse genomic DNA using primers: T7-Nkx2-6-F: taata cgact cacta tagg ACTGGTACTGGACGGCAAGC and SP6-Nkx2-6-R: attta ggtga cacta taga GCACAGCATCTACGTGGCTA. Isl1 probe template was generated by PCR from an MGC cDNA (accession BC132263) using primers: Isl1F-T7: taata cgact cacta taggT CATCC GAGTG TGGTT TCAA and Isl1R-SP6: attta ggtga cacta tagaT GAATG TTCCT CATGC CTCA. Foxg1 probe template was generated by PCR from an MGC cDNA (accession BC046958) using primers: Foxg1F: AGTTACAACGGGACCACGTC and Foxg1R-T3: aatta accct cacta aagg CCCCT GATTT TGATG TGTGA. The Sox2 probe template was generated by PCR using mouse genomic DNA and primers: Sox2-F: GCCCA TGAAC GCCTT CATGG and T3-Sox2-R: aatta accct cacta aagg C ATGCT GATCA TGTCC CGGA. The Gata3 probe template was described previously Digoxygenin (DIG)-labeled antisense RNA probes were synthesized using standard procedures. All probe templates were generated by PCR reactions using either mouse genomic DNA or cDNA clones as templates. In all cases, probe templates were carefully designed to not include highly conserved sequences to eliminate the possibility of cross hybridization. In the following primer sequences the lower case letters indicate the phage promoters. For Comparisons of pouch morphology in 3D reconstructions from different embryos showed that pouch morphology is comparable between different embryos of the similar somite stage (data not shown). This similarity allowed us to make comparisons between reconstructions of different gene expression patterns. Digital images of serial sagittal paraffin sections from a single embryo were assembled into a three-dimensional (3D) image using the WinSurf 4.3 software. The gene expression positive areas and the third pharyngeal pouch endoderm were traced as separate objects.3Citrate pH 6, 0.05% Tween20) for 30 minutes. After cooling down, slides were washed once with 0.05% PBST (0.05% Triton X-100) and blocked in 10% serum in PBST at room temperature for at least 30 minutes. Primary antibodies were mixed in 1% serum/PBST and incubated at 4°C overnight. After 3 PBST washes, secondary antibodies diluted in PBST were added and incubated at room temperature for 30 minutes. Slides were then washed and mounted in FluoroGel (EMS). Images were acquired using a Zeiss LSM510 META confocal imaging system.Dissected embryos or postnatal thymus tissue were treated differently for ISL1 and FOXG1 antibody staining. For ISL1 staining, E16.5 embryos were fixed in 4% PFA for 4 hours or postnatal thymus for 1.5 hr on ice. Fixed embryos or tissue were then washed three times in PBS and dehydrated through methanol series and embedded into paraffin blocks. 8 µm sections were cut on a Leica RM2155 microtome and de-waxed and rehydrated into water. Antigen retrieval was done by boiling the slides in AR buffer . 10 µm frozen sections were then cut on a Leica CM3050 S cryostat. The sections were blocked and incubated with primary and secondary antibodies as described for ISL1 staining.Foxn1  The mouse anti-ISL monoclonal antibody was from the Developmental Studies Hybridoma Bank . This ISL1 monoclonal was developed by Dr. Thomas Jessell and has a long track record of use in mice


CDKN2A) gene that encodes the p16INK4a and p14ARF tumor suppressors, and the isocitrate dehydrogenase 1 (IDH1) gene as potential markers of survival for 40 individuals with NDTMM GBMs (telomerase negative and ALT negative by standard assays), 50 individuals with telomerase, and 17 individuals with ALT positive tumors. The analysis of CDKN2A showed NDTMM GBMs had an increased minor allele frequency for the C500G (rs11515) polymorphism compared to those with telomerase and ALT positive GBMs (p = 0.002). Patients with the G500 allele had reduced survival that was independent of age, extent of surgery, and treatment. In the NDTMM group G500 allele carriers had increased loss of CDKN2A gene dosage compared to C500 homozygotes. An analysis of IDH1 mutations showed the R132H mutation was associated with ALT positive tumors, and was largely absent in NDTMM and telomerase positive tumors. In the ALT positive tumors cohort, IDH1 mutations were associated with a younger age for the affected individual. In conclusion, the G500 CDKN2A allele was associated with NDTMM GBMs from older individuals with poorer survival. Mutations in IDH1 were not associated with NDTMM GBMs, and instead were a marker for ALT positive tumors in younger individuals.Prognostic markers for glioblastoma multiforme (GBM) are important for patient management. Recent advances have identified prognostic markers for GBMs that use telomerase or the alternative lengthening of telomeres (ALT) mechanism for telomere maintenance. Approximately 40% of GBMs have no defined telomere maintenance mechanism (NDTMM), with a mixed survival for affected individuals. This study examined genetic variants in the cyclin-dependent kinase inhibitor 2A ( Acquisition of a telomere maintenance mechanism prevents telomere attrition and is a hallmark of cancer TP53 mutations are a marker for individuals with a poorer prognosis In glioblastoma multiforme (GBM) all of the telomere maintenance scenarios outlined above occur CDKN2A) that encodes two proteins p16INK4a and p14ARFCDKN2A, a substitution in the 3′ UTR of a cytosine for a guanine base at cDNA nucleotide 500 , is associated with different cancer types. The G500 allele had an increased frequency in melanoma families, and in psoriasis patients with squamous cell carcinoma INK4ap16 expression in sporadic colorectal cancer CDKN2A expression CDKN2B) expression; a gene in close proximity to CDKN2A on chromosome 9p21 and encodes the tumor suppressor p15INK4BOther molecular characteristics of GBMs include loss of the cyclin-dependent kinase inhibitor 2A  were ALT positive by standard techniques i.e. long and heterogeneous telomere lengths by TRF length analysis, the presence of large aggregates of the promyelocytic leukemia (PML) protein and telomere DNA called ALT-associated promyelocytic leukemia (PML) bodies (APBs) in >0.5% of tumor cells, and very low or no telomerase activity in tumor protein lysates by the TRAP assay CDKN2A UTR (rs11515). The C500G genotypes are given in Individuals in each GBM telomere maintenance subgroup were genotyped for the C500G polymorphism in the 3′ CDKN2A mutations were identified in G500 heterozygotes as evident by dHPLC and sequence analysis of all CDKN2A exons and exon/intron boundaries in amplicons of tumor and blood leukocyte extracted DNA. Nine individuals  were heterozygous for the G442A polymorphism that substitutes an adenine for a tyrosine amino acid at amino acid residue 148 in p16INK4a.All individuals with the G500 allele were homozygous for the major allele at two other polymorphic sites in close proximity (C540T and C547G). No CDKN2A sequence variant examined was associated with an increased frequency in the NDTMM group. In the telomerase GBM group the G500 allele frequency was not significantly different to the control population selected with the same ethnicity, and age and sex matched to the GBM cohort.In summary, in GBM the G500 allele and no other Cox proportional hazards regression analysis was performed to test whether the GC and CC C500G genotypes in the NDTMM and telomerase positive groups were independently associated with survival. Variables included in the analysis were age, gender, treatment, and extent of surgery . The C50th to the 75th percentile), which was higher compared to a median age of 58 for C500 homozygotes , p = 0.04 .In the NDTMM tumor group survival data was available for all 40 individuals. Individuals heterozygous for the G500 allele had a median survival time of 3 months following the initial tumor diagnosis, compared to 8 months for individuals homozygous for the C500 allele . The twoIn the telomerase positive tumor group survival data was available for 44 of 50 individuals. Individuals heterozygous for the G500 allele had a median survival time of 4.9 months following the initial tumor diagnosis compared to median survival time of 6 months for CC homozygotes .INK4ap16 or ARFp14, multiplex PCR was used to estimate the gene dosage of exon 1α (INK2A)p16 and exon 1β (ARF)p14 relative to an internal β-globin gene fragment To test if the reduced survival associated with the G500 allele in the NDTMM GBM cohort was attributed to a greater loss of homozygosity or heterozygosity for In the NDTMM group, individuals heterozygous for G500 that had loss of exon 1α and 1β gene dosage had a median survival of 4 months, which was not significantly different to G500 heterozyotes that retained exon 1α and 1β homozygosity  with altered survival when adjusted for other variables . The 20 tumor sections selected included ten from G500 heterozygotes typed with loss of INK4ap16 gene dosage using the multiplex PCR analysis and ten tumors from C500 homozygotes typed with no loss in INK4ap16 gene dosage by multiplex PCR analysis. All tumors from C500 homozygotes had 2 red and 2 green signals in cellular nuclei, a result consistent with no loss in INK4ap16 gene dosage (data not shown). Seven tumors from G500 heterozygotes had 2 green signals and no red signals consistent with a loss of both INK4ap16 alleles (data not shown). Three tumors from G500 heterozygotes had 2 green signals and 1 red signal consistent with a loss of one INK4ap16 allele. The results from the FISH analysis confirmed those obtained using PCR.To verify that the NDTMM GBMs had lost the INK4a. The 20 tumor sections selected were those used for the FISH analysis described above. All tumors from C500 homozygotes had p16INK4a positive cells . For IDH2 mutations, one mutation was found. The D177A, amino acid residue substitution occurred in a NDTMM GBM.Tumor DNA from all GBMs was used as the template in PCR reactions that amplified all exons of In summary, the majority of ALT positive tumors contained IDH1 mutations. In NDTMM and telomerase positive GBMs, IDH1 mutations were rare.CDKN2A allele may be an important biomarker for NDTMM GBMs identifying individuals with a poorer prognosis. The C500G genotype was associated with survival independently of age and treatment, which are other factors associated with GBM patient survival Currently there is no prognostic marker for GBM tumors without a defined telomere maintenance mechanism. The G500 TP53 mutations compared to ALT positive tumors (unpublished observations). Moreover, the different G500 allele frequency, the absence of IDH1 mutations, and the different effect of mutant TP53 on survival  on survival mitigates against these “None” GBMs being miss-classified as telomerase or ALT tumors It is unknown why a considerable portion of GBMs have no currently defined telomere maintenance mechanism. Most tumor types documented without telomerase activity or ALT are low-grade tumors such as grade 1 astrocytomas and papillary thyroid carcinomas INK4ap16 and ARFp14 expression CDKN2A is associated with poor tumor outcome in patients and mice INK4ap16 and ARFp14 transcripts, which may give a greater selection pressure for inactivation by a later DNA deletion CDKN2A was predicted to be affected by the C500G polymorphism The G500 allele as a risk factor for cancer has been most extensively characterized for melanoma CDKN2A coding region or the 3′ UTR of CDKN2A region analyzed. The C500G alleles may affect expression of other genes in close proximity to CDKN2A. Both CDKN2B and the non-coding RNA designated ANRIL are included with CDKN2A in the susceptibility locus associated with glioma and other diseases et al. (2010), the G500 allele reduced CDKN2B and no effect on ANRIL expression The G500 allele was not associated with another sequence variant in the The causative variant may be in linkage disequilibrium with the G500 allele and in a region on chromosome 9 not analyzed in this study.INK4ap16 and ARFp14 function and poorer survival. Interestingly, this was not the case for G500 heterozygotes, as those that had loss of exon 1α and exon 1β gene dosage had no difference in survival compared to those that retained exon 1α and exon 1β. One explanation for this discrepancy is that the G500 allele may lead to loss of INK4ap16 and ARFp14 transcripts initially, followed by loss of exon 1α and exon 1β DNA at a latter stage of tumor development. The poorer survival associated with the G500 allele may be attributed in part to an increased age at initial tumor diagnosis for G500 heterozygotes Homozygotes for the C500 allele with loss of exon 1α and exon 1β gene dosage had a poorer survival compared to those that had retained exon 1α and exon 1β, consistent with an association between loss of IDH1 mutations, a marker for improved prognosis in ALT positive tumors IDH1 mutations and ALT are associated in Glioma remains to be understood.The NDTMM GBMs were not associated with New treatments targeting telomerase positive tumors are being developed CDKN2A G500 allele in the 3′ UTR was more frequent in patients with tumors that have no defined telomere maintenance mechanism and was a prognostic marker for poorer survival. The increased incidence of the G500 allele and the absence of IDH1 mutations provide justification for the NDTMM GBMs being a molecularly distinct subgroup of tumor. Further investigation is required to understand the function of the G500 allele in altered cancer risk. In younger GBM patients with ALT positive tumors, IDH1 mutations predominate. Therefore the molecular signature of telomere maintenance mechanisms may provide additional prognostic information for GBM patients in addition to that for age and treatment received.The The study had ethical approval from the Multi-region Ethics and the Upper South Regional Ethics committees in New Zealand, and written individual informed consent was obtained.The inclusion criterion for the study was a diagnosis of a GBM tumor. One hundred and seven tumors with both paraffin-embedded and frozen tissue available were analyzed. All tumors were collected in New Zealand, and all were debulking samples. Consultant pathologists performed the histopathological tumor diagnoses. All individuals were of New Zealand European ethnicity.Telomerase activity was measured in tumor lysates using the Telomeric Repeat Amplification Protocol (TRAP) method. The TeloTAGGG Telomerase PCR ELISA Plus kit  was used according to the manufacturer's instructions. For identification of ALT positive tumors, measurement of telomere length was made by the terminal restriction fragment (TRF) assay using tumor lysates. The TeloTAGGG Telomere Length Assay Kit  was used according to the manufacturer's instructions. The criteria for ALT by TRF length was that previously described 5′-GTGCCACACATCTTTGACCTCAG-3′) and an anti-sense prime in the 3′ UTR (5′-TGCTTGTCATGAAGTCGACAGCT-3′). The C500G alleles were genotyped in 150 blood leukocyte extracted DNA samples from individuals randomly selected from the general New Zealand population of European ancestry.To genotype the C500G (rs11515) polymorphism CDKN2A were amplified by PCR using blood leukocyte and tumor extracted DNA as the template. The following sets of primers were used to amplify exon 1α  and 1β  CDKN2A wild-type DNA (1∶1) and re-analyzed by dHPLC to allow non-heterozygous sequence variants to be detected.All exons of CDKN2A exon 1α (INK4Ap16) and exon 1β (ARFp14) using tumor extracted DNA Multiplex PCR as previously described for astrocytomas, was used to assay the gene dosage of In situ hybridization was used to confirm the p16INK4a gene dosage results obtained from the multiplex PCR method, and measured p16INK4a gene dosage in 20 NDTMM GBM paraffin embedded tumor sections using the P16 Detection Probe  and the control chromosome 9 probe deletion probe (D9Z3 Cytocell Aquarius). The in situ hybridization protocol was based on that used in the laboratory for FISH analysis of telomeric DNA 5′-ATGCCATCACTGCAGTTGTAGG-3′ and antisense 5′-CCTTGCTTAATGGGTGTAGAT-3′, all primers used were those described previously To identify tumors with IDH1 mutations all exons of the IDH1 gene were amplified from tumor extracted DNA and sequenced. With the exception of the PCR reaction designed to amplify exon 4, which used the following set of primers, sense INK4a epitope was detected using an anti-mouse p16INK4a monoclonal antibody , and detection of the enzyme linked conjugated system  used DAB. Detection of DAB positive cells used a Leica DM2000 light microscope at 400× magnification and Leica Version 3.5.0 Application Suite software .The p16INK4a. The analysis was made using the PHREG Procedure SAS System .The frequency of the G500 allele was compared between GBMs with different telomere maintenance mechanisms using the Fisher's exact test and GraphPad Prism software . In the NDTMM and telomerase GBM groups the age at brain tumor diagnosis was compared between G500 heterozygotes and C500 homozygotes, and used the unpaired Student's t test with 95% CI and Welch's correction to compensate for unequal variance. In the NDTMM and telomerase GBM groups, the loss of homozygosity or the loss of heterozygosity of exon 1α or exon 1β was compared to tumors that retained exon 1α or exon 1β homozygosity using the Fisher's exact test and GraphPad Prism software (GraphPad). The frequency of missense mutations in IDH1 was compared between GBMs with different telomere maintenance mechanisms using the Pearson's chi square test and SHEsis software The primary end point was overall survival. Cox proportional hazards regression analysis was performed to assess the GC and CC G500C genotypes as a prognostic marker for survival and was adjusted for other variables age (years), gender, tumor resection , and treatment , loss of p16


Drosophila, the polycomb repressive complexes (PRC) associate with defined polycomb response DNA elements (PREs). In mammals, however, the mechanisms underlying polycomb recruitment at targeted loci are poorly understood. We have used an in vivo approach to identify DNA sequences of importance for the proper recruitment of polycomb proteins at the HoxD locus. We report that various genomic re-arrangements of the gene cluster do not strongly affect PRC2 recruitment and that relatively small polycomb interacting sequences appear necessary and sufficient to confer polycomb recognition and targeting to ectopic loci. In addition, a high GC content, while not sufficient to recruit PRC2, may help its local spreading. We discuss the importance of PRC2 recruitment over Hox gene clusters in embryonic stem cells, for their subsequent coordinated transcriptional activation during development.Polycomb group (PcG) proteins are essential for the repression of key factors during early development. In  Hox genes are essential for the proper organization of structures along the developing vertebrate body axis. These genes must be activated at a precise time and their premature transcription is deleterious to the organism. Early on, Hox gene clusters are covered by Polycomb Repressive protein Complexes (PRCs), which help keep these genes silent. However, the mechanism(s) that selectively recruit PRCs to these particular genomic loci remains elusive. We have used a collection of mutant mice carrying a set of deletions inside and outside the HoxD cluster to try and detect the presence of any DNA sequence of particular importance in this mechanism. We conclude that a range of low affinity sequences synergize to recruit PRCs over the gene cluster, which makes this process very robust and resistant to genetic perturbations. Polycomb) was characterized genetically as a repressor of Drosophila homeotic genes via epigenetic mechanisms such as X-inactivation trithorax families (trxG), displaying opposing functions, was shown to maintain Hox gene expression during the entire life of DrosophilaPolycomb group (PcG) proteins are essential for proper development of most eukaryotic organisms. The founding member . PRC2 carries a methyl-transferase activity that methylates the histone H3 tail at lysine 27, a mark largely associated with gene silencing. While the initial deposition of this post-transcriptional modification is carried through by PRC2, PRC1 maintains this methylated status and compacts chromatin, largely, though not solely Drosophila, PcG group proteins are recruited to chromatin through the recognition of polycomb response elements (PREs). These DNA segments, which were generally identified by forward genetics, must satisfy three criteria: (1) they must bind polycomb when randomly inserted into the genome, (2) they must be able to induce a H3K27me3 domain and (3) they must repress a reporter construct, when associated with them Drosophila PREs are approximately 1.5 kb long in average and are usually located in proximal promoter regions. This is the case of the PREs associated with either engrailedHedgehogpolyhomeoticIn in vivo several kilobases away from their target genes and their identification may be biased by genome wide chromatin immunoprecipitation (ChIP) studies. Recently indeed, it has become clear that only a fraction of polycomb enriched regions are direct targets of PRC, whereas others are indirect, via chromatin looping polycomb enriched regions may not always reflect the genuine presence of polycomb at a particular locus. Instead, they may derive from the three-dimensional organization of the genome, which along with the technology employed, may lead to false positives. Furthermore, although high throughput studies have shown that the binding profiles of polycomb proteins correlate with both the transcription start sites (TSSs) and stalled RNA PolII However, PREs have also been mapped Pho ortholog YY1 Pho and PRC remains controversial In mammals, the understanding of the general mechanism (if any) accounting for the recruitment of PRC2 is lacking too. PRC2 recruitment has been associated with the presence of binding sites for the Hox gene clusters  cells, including the four clusters . In thisHox loci by using an in vivo approach, based on the large number of genomic re-arrangements associated with the HoxD locus Drosophila Bithorax complex where a 300 kb large domain of trimethylated histones involves only few PREs, we show that the mouse HoxD locus implements a mechanism that can compensate for large and systematic deletions within the target DNA interval. These results indicate that PRC2 recruitment at this locus must rely upon a range of cooperating binding sites, rather than upon a few nucleation sites. By using isolated transgenes, we also show that in this particular context, CpG islands (or a high GC content) are not the prime factors in PRC2 recruitment, despite their potential importance for local spreading.We have addressed the question of polycomb recruitment at in vivo, we used a genetic approach of the mouse HoxD gene cluster, which is a main target of Pc silencing in ES cells and adult tissues To study the recruitment of polycomb complexes and the resulting H3K27me3 histone modification HoxD cluster did not seem to affect the binding profiles of PRC proteins when assessed by ChIP-qPCR ) in vivo had no effect upon gene expression within the HoxD cluster del(12)). Here again, we did not score any modification of the H3K27me3 profile throughout the gene cluster. We next scanned the entire locus with a set of adjacent deletions, including either Hoxd8, Hoxd9, Hoxd10, Hoxd11, Hoxd12 or Hoxd13 and found no significant change in polycomb-mediated silencing (data not shown), using H3K27me3 as a proxy for PcG occupancy  and Del(9-12)  . These tH3K27me3 , suggestHox gene cluster were of particular importance to set up a platform for recruiting PRC2. We used a large deletion (del(1-10)), where two-thirds of the anterior part of the cluster were removed including its most anterior gene Hoxd1. Again, the remaining mini-cluster was able to compensate for this significant trimming and the H3K27me3 pattern over the remaining loci was nearly identical to that found in wild type conditions d11Lac), did not significantly affect the presence of these epigenetic marks over the remaining 5′ located Evx2 gene , even though the latter displayed massive amounts of H3K27me3 over the anterior part of the cluster , del(13) and del(10-13), which share the same breakpoints but in various configurations. In this set of deletions, the reconstitution of three different neighborhoods did not modify the methylation patterns.This result indicated that each piece of the gene cluster is rather independent in its ability to recruit PRC2, regardless of what would happen over the neighboring loci. The poor impact of the neighboring sequences upon the coverage of any given HoxD. This intergenic region ‘i’ is over 13 kb long and maps between Hoxd4 and Hoxd8. Its deletion (del(i)) results in a further concentration of genes by bringing Hoxd1, Hoxd3 and Hoxd4 closer to the centromeric (posterior) side including Hoxd8 to Hoxd13. del(i) did not show any clear difference in the H3K27me3 profile over the remaining parts of the gene cluster (del(i)).Single gene deletions did not markedly change the relative distance between transcription units and hence they may not affect PRC2 recruitment, should several PREs locate near each gene and synergize. We thus modified the distance between two genes by excising the longest gene free DNA segment within i to produce a cluster with a 26 kb large gene-free domain inside. Intriguingly, the duplicated configuration did not exhibit any change on region i, except for a weak gain of signal over the duplicated region suggesting that both copies are covered by H3K27me3. The flanking DNA segments, however, displayed the same H3K27me3 profiles as in wild type brains (del(i), dup(i)) indicating that the mechanism recruiting PRC2 over the mouse HoxD cluster can compensate for modifications in the distance between transcription units, emphasizing once more the robustness of this process.We next looked at the effect of introducing a gene free region within the cluster such as to increase the distance between neighboring genes. We duplicated region HoxD cluster, which contain numerous cis-acting sequences. We deleted a 230 kb large piece of DNA, from eight kb upstream Evx2 to a breakpoint located within the flanking centromeric gene desert (del(R1-R5)-d9Lac) HoxD gene cluster per se, it removed the border of the H3K27me3 domain and hence it reconstituted a neighborhood between heavily H3K27 tri-methylated nucleosomes and nucleosomes not methylated at all.In the absence of any strong and discrete signal for PRC2 recognition and nucleation within the cluster itself, we asked whether Pc proteins may be targeted by elements localized within the regulatory landscapes flanking the HoxD cluster in the developing brain, nor did it induce any leakage over the centromeric DNA from the gene desert (del(R1-R5)-d9Lac). Therefore, as previously reported The deletion of this ‘epigenetic border’ did not elicit any loss of H3K27me3 marks over the HoxA, HoxC and HoxD clusters. In the case of HoxD, the Evx2 gene is found ca. 10 kb upstream Hoxd13 on the opposite strand. This gene, which is covered by H3K27me3 marks and locates close to the epigenetic border, was however not removed in the del(R1-R5)-d9Lac deletion. Therefore, we analyzed a second deletion, ca. 260 kb large, with the same upstream breakpoint into the gene desert (see above), but with a telomeric breakpoint located between Hoxd10 and Hoxd11. In this del(11-R5)-d9Lac mutant, the entire posterior part of the HoxD cluster was removed including Hoxd11, Hoxd12 and Hoxd13 as well as the Evx2 transcription unit and the epigenetic border.Another mechanism to set the PcG epigenetic borders may involve transcripts encoded by the opposite DNA strand, a feature found in the del(R1-R5)-d9Lac deletion, suggesting that additional transcriptional units encoded by either DNA strands are not necessary for the recruitment of PRC2 at the extremity of the HoxD cluster, nor for the fixation of a sharp epigenetic boundary (del(11-R5)-d9Lac). In both deletions, however, a Hoxd9/Lac transgene was relocated at the breakpoint, raising the possibility that transgenic sequences would interfere with PRC2 recruitment and hence we used a final mutant configuration carrying a ca. 800 kb large deletion including the HoxD centromeric regulatory landscape. This del(Nsi-Atf2) deletion not only removes the 5′ epigenetic border, but also most of the regulatory elements that contact Hoxd genes and impose a chromatin topology to the locus In this configuration, the H3K27me3 profile remained unchanged when compared to the wild type pattern. In particular, the reconstituted epigenetic boundary was similar to that seen with the shorter HoxD cluster towards the new centromeric neighboring sequences did not exceed a 800 bp large interval, which corresponds to twice the average length of the sonicated DNA fragments (data not shown). These results further indicated that the capacity to recruit PRC2 is restricted to Hox genes themselves, without any contribution from the surrounding genomic sequences. To demonstrate this point, we produced a transgenic line containing the Hoxd10 gene, which had inserted into a genomic region of average GC density and poor in H3K27me3 marks. While H3K27me3 was scored on the entire transgene, this histone modification did not spread over flanking nucleosomes, as assessed by ChIP-seq . Therefore, when this transgenic stock was crossed back into a mouse carrying a homozygous deletion of Hoxd10 (TgN/del(10)−/−), the H3K27 trimethylation profile (or the lack thereof-) over Hoxd10 reflected that of the ectopic Hoxd10 copy. The Hoxd10 locus was selected because the CpG island located upstream the promoter (CpG32 from UCSC) could be removed by using FRT sites and the Flip recombinase in vivo, without affecting the transcription start site (TSS). To make sure that no additional CpG islands remained after deletion of CpG32, we deleted another potential short island (CpG26) from our starting transgenic construct.The Hoxd10 null mice to assess their H3K27me3 status in developing forebrains . Similar results were observed when the CpG26 sequences had been removed (TgNd10). Moreover, similar amounts of H3K27me3 were scored when the transcription start site of TgNd10 was deleted, suggesting that the recruitment of PRC2 may be independent of transcription (TgNd10∂TSS) TgNd10∂CpG).Transgenic animals were crossed with a Cre-deleter strain to adjust copy number to one and various transgenes were thus crossed over rebrains . When th removed , TgNd10.Hoxd10 was used, H3K27me3 marks were no longer detected, even though this construct still contained an annotated CpG island (TgNd10∂3) and was globally GC-rich. Of note, a transgene containing the same four kilobases together with exon 1 of Hoxd10 showed no recruitment of PRC2 either (TgNd10∂PREd10), regardless whether or not the TSS was present (TgNd10∂TSS∂PREd10), suggesting that neither the TSS, nor the CpG32 are essential for recruiting PRC2 in this configuration. Mapping the insertion sites did not reveal any correlation between the presence of H3K27me3 on the transgenes and their insertion into either a H3K27me3-rich or a GC-rich DNA region. In fact, transgenes were found integrated at least 500 kb away from H3K27me3-rich spots and into DNA segments with rather average GC contents (data not shown). These experiments thus defined a 1.4 kb large DNA segment, containing exon 2 and the 3′UTR of Hoxd10, which was necessary for the deposition of H3K27me3 marks. This DNA segments is referred to as PREd10 below.However, when a four kb large transgene containing only the 5′ sequence upstream Hox clusters are twice as dense in differentiated tissues than in ES cells  were thus used to assess the H3K27 methylation status of distinct electroporated DNA elements, overlapping with the deleted Hoxd10 DNA segment. Various portions of the TgNd10 transgene were first cloned between two homologous arms (Env) flanking the transgenes, in the hope of comparing random and targeted integration sites. However, homologous recombination events were not found.While H3K27me3 marks covering the ES cells , the extHoxd10 fragment, including the TSS and both exons, or the 1.4 kb long PREd10 were introduced into our iPSdel(Hoxd10) cells, they became H3K27 tri-methylated, in agreement with the results obtained using classical transgenesis. More surprisingly, when the 5′ sequence corresponding to that used in TgNd10∂3 was assayed, H3K27me3 was detected too, in contrast to the results obtained in transgenic mice. We checked the capacity of either the vector backbone, or the PGK-neomycin gene promoter to recruit PRC2, by electroporating the neomycin cassette alone. The PGK promoter is ubiquitous and hence neomycin transcripts were detected in all conditions tested . We verified if this recruitment was influenced by the presence of the DNA homology arms included for a potential recombination at the locus, which contained sequences from both the Hoxd11 and a portion directly 3′ to Hoxd10, which could thus initiate a ‘spreading’ of H3K27me3 marks over the Env-d10∂3 fragment. Accordingly, we electroporated d10∂3 (42% rich in GC) into iPSdel(Hoxd10) without any other surrounding DNA sequences. While H3K27me3 was detected over a multimerized version of d10∂3, this mark was lost after the CRE recombinase had reduced copy number to one (d10∂3-CRE). In contrast, when CpG-island free PREd10 (44% rich in GC) was introduced into iPSdel(Hoxd10), H3K27me3 marks were readily scored after CRE-excision of the multimers (d10-CREd10PRE). PRC1/2 subunits were also detected over this exogenous, randomly integrated sequence, suggesting it contains all proper information necessary for PcG recruitment to ectopic sites  and PREd10-600 (43% rich in GC), which were tested as individual transgenes. Unexpectedly, both fragments were decorated by H3K27me3, when introduced into iPSdel(Hoxd10) cells as single copy  displaying a temporal sequence in the establishment of their segmented body plan systematically show a complete clustering of their Hox gene complement, whereas other animals following different strategies (such as cell lineages) usually have broken Hox clusters or even Hox genes scattered throughout the genome  Hox clusters are amongst the genomic loci with the highest GC content.As for many Drosophila PREs, PREHox gene clusters, they do not rule out their potential importance for the spreading or the re-enforcement of the coverage by PRC2 (see below). The existence of CpG islands devoid of PcG, as well as PcG target DNA devoid of CpG islands, such as in the case of the first described mammalian PRE-like sequence regulating the mouse MafB/Kreisler, support this view. Moreover, sequences unable to recruit PRC when present as single copy transgenes may become H3K27me3 when concatamerized, suggesting that larger stretches of GC-high sequences can artificially recruit PRC, a possible explanation to the discrepancies observed between our results and those of others Here again however, our results do not support a high GC content as the major parameter in recruiting PRC2. We show that DNA segments with a GC content similar to the average of the mouse genome (42%) are still able to properly recruit PcG proteins and the deletion of CpG islands from our transgenic constructs did not abrogate the trimethylation of H3K27. While these results suggest that CpG islands are neither sufficient, nor required, for the tethering of PcG proteins in the context of ad minima model whereby H3K27me3 is deposited on a series of low affinity PRC2 interacting sequences, which work synergistically between themselves and together with GC-rich sequences to confer robust silencing over target genes .Hoxd1 and either the site telomeric to Hoxd8 (del(1-i)) or the site centromeric of to Hoxd4 (del(1-4)). The del(i) line was produced by TAMERE between the loxP telomeric of Hoxd8 and the one centromeric to Hoxd4. Genotyping was performed on individual yolk sacs.All stocks of mice were kept as heterozygous and bred to homozygosity. Lines were all described and can be found in previous publications of the Duboule laboratory. Two additional lines were produced by recombination between the loxP site in the second exon of Chromatin immunoprecipitation followed by quantitative polymerase chain reaction was performed as described in Mouse embryonic fibroblasts were derived from heterozygous crosses of E13.5 embryos using standard protocols. Cells were cultured in standard MEF/ES cell culture conditions. MEF/ES media contained DMEM supplemented with 10% FBS and LIF (ES media only). Isolated MEF lines were first genotyped using embryonic tissues and subsequently confirmed with DNA extraction procedures. Passage three MEFs were used for iPS derivation experiments.Oct4, Klf4 and Sox2 were cloned and separated by bacterial 2A sequences, in a single lentiviral backbone (3F). Virus was produced in 293T cells using FuGENE HD transfection reagent  and ultracentrifuged. Induced pluripotent (iPS) stem cells were derived following standard protocols SSEA1, Nanog, Oct4 and Sox2 by immunohistochemistry and western blot, standard protocols), a non-aberrant chromosome count  and re-establishment of bivalent domains .Human 6. Electroporated cells were plated on 10 cm dishes coated with DR4 resistant feeders. G148 selection (200 µg/ml) (Sigma G8168-10ML) was started 24 hours after electroporation and was continued until individual colonies were picked and genotyping. CRE treatment of iPS cells was done as follows: 3×105 cells were plated overnight and transduced with a PGK-CRE lentiviral construct at MOI 100. Individual colonies were picked 5 days post-transduction, expanded and genotyped.Electroporation of induced pluripotent stem cells was performed using an Amaxa Nucleofector I and the Lonza mouse embryonic stem cell kit (Lonza VPH-1001). Briefly, 25 µg of DNA were digested overnight, phenol-chloroform purified and resuspended in 10 µl H2O. Media was changed 4 hours before electroporation. Cells were washed twice with Mg(2+)-Ca(2+)-Free PBS, trypsinized and aliquoted to 2×10HoxD cluster were spotted with 25-mer oligonucleotides at 15b bp resolution . Fragmentation, labeling and hybridization of ChIPed DNA were done following standard protocols.Affymetrix custom-made tiling arrays covering two megabases surrounding the mouse Rps9) were used to derive mean values. Primers are given in Cells were first disrupted and homogenized using a Polytron (kinematic) before RNA was extracted using the RNeasy Microkit . qRT-PCR was performed with SYBR Green. Two biological replicates, processed in triplicates and normalized to a housekeeping gene  from Affymetrix. Data was exported as plain text using a log2 or −10log10 scale for the signal, respectively the p-value. Files were visualized in RChiV, an in-house developed genome browser, which takes into account the deleted segment and normalizes the signal using a sliding window approach.Figure S1HoxD cluster in mutant configurations. (A) ChIP-qPCR profiles of PRC1  and PRC2 (Ezh2 (middle panel) and Suz12 (lower panel)) over the HoxD cluster. The wild type values of six genes (from Evx2 to Hoxd3) found within the H3K27me3 domain are used as positive controls (black). Lnp and Mtx2 are found outside of the H3K27me3 domain and are thus used as negative controls (black). Different mutant configurations are color coded and specified on the top (del(10), del(10-11), del(9-12), del(10-13), del(11-R5)). dN stands for HoxdN. NA refers to the absence of the given DNA segment in the specified allele. NI refers to mutant alleles which where not included in the experiment (Suz12 on both del(10-11) and del(11-R5)).Binding profiles of PRC1 and PRC2 over the (TIF)Click here for additional data file.Figure S2HoxD cluster and flanking gene deserts. (B) H3K27me3 profiles of wild type and deleted animals. Genotypes are specified on the left.Effect of large deletions upon the H3K27me3 profiles. (A) Wild type genomic landscape of the murine (TIF)Click here for additional data file.Figure S3Hoxd10 specific probe . A wild type sample (+/+) was used as control (right panel). The positions of both the restriction sites for PvuII and HindIII and the probe used for southern blot are depicted on the wild type profile in Southern blot of transgenic animals. Southern blot using a robe see . Restric(TIF)Click here for additional data file.Figure S4del(10) embryo .H3K27me3 profiles in pluripotent and terminally differentiated cells. A large (A) or focused (B) view of wild type H3K27me3 profiles from differentiated cells dissected from the embryonic brain (top) compared to pluripotent cells derived from a (TIF)Click here for additional data file.Figure S5Hoxd genes in various constructs eletroporated in iPS cells carrying a deletion of Hoxd10. Lnp is located outside the HoxD cluster and is used as a control for active genes, while Hoxd13 is used as a control for silent genes. Clones and culture conditions are color coded and specified on the top. G418 stands for the presence (+) or absence (−) of the antibiotic. Vector refers to a control cell line.H3K27me3 and RNA profiles in various cell lines. ChIP-qPCR and mRNA expression of control and (TIF)Click here for additional data file.Table S1List of the primers used for RT-PCR, either for ChIP experiments (top) or for RNA dosage (bottom).(DOC)Click here for additional data file.


Irish ICUs typically have bed occupancy rates approaching 100%, with 75 to 80% being the recommended level [The GICU database was examined from 1 March 2010 to 1 March 2011. The H1N1 pandemic was recognised as a period of strain on the ICU and this period was estimated as 24 December 2010 to 21 January 2011. All ICU readmissions during the same hospital stay were noted. Transfers between GICU, cardiac ICU and theatre recovery were excluded as patients were still being treated by the intensive care team. Patients readmitted after transfer for extracorporeal membrane oxygenation (ECMO) were also excluded.The number of GICU admissions during the period was 422. There were 19 readmissions (readmission rate of 4.6%). However, this rate increased to 8.6% during the period of high activity encompassing the H1N1 pandemic (Figure The annual readmission rate for our unit was acceptable . A clear


Preclinical studies in mice and non-human primates showed that AAV serotype 5 provides efficient liver transduction and as such seems a promising vector for liver directed gene therapy. An advantage of AAV5 compared to serotype 8 already shown to provide efficient correction in a phase 1 trial in patients suffering from hemophilia B, is its lower seroprevalence in the general population. Our goal is liver directed gene therapy for Crigler-Najjar syndrome type I, inherited severe unconjugated hyperbilirubinemia caused by UGT1A1 deficiency. In a relevant animal model, the Gunn rat, we compared the efficacy of AAV 5 and 8 to that of AAV1 previously shown to be effective. Ferrying a construct driving hepatocyte specific expression of UGT1A1, both AAV8 and AAV1 provided an efficient correction of hyperbilirubinemia. In contrast to these two and to other animal models AAV5 failed to provide any correction. To clarify whether this unexpected finding was due to the rat model used or due to a problem with AAV5, the efficacy of this serotype was compared in a mouse and two additional rat strains. Administration of an AAV5 vector expressing luciferase under the control of a liver specific promoter confirmed that this serotype poorly performed in rat liver, rendering it not suitable for proof of concept studies in this species. The large variety of AAV serotypes with different tropisms provides a gene therapy platform that can be applied in different tissues. For instance AAV serotype 1 (AAV1) can be used to correct lipase deficiency in the muscle Efficacy in patients demonstrates that adeno-associated viral (AAV) vectors can be used for in vivoPresence of low levels of neutralizing antibodies (NAb) towards an AAV serotype will preclude efficient transduction of the target organ in vivo gene therapy in CN patients. In addition, the availability of several suitable vectors does allow switching to another serotype for treating patients with pre-existing NAb towards one of these serotypes Our long term goal is to treat Crigler-Najjar syndrome (CN), familial severe unconjugated hyperbilirubinemia. This recessive inherited severe liver disorder is caused by deficiency of uridine diphospho-glucuronosyl transferase 1A1 (UGT1A1) In this study we evaluate the efficacy of scAAV5 and scAAV8 to that of scAAV1 in the Gunn rat. AAV8 and AAV1 both provide efficient correction of hyperbilirubinemia. In contrast, AAV5 does not provide any detectable activity in this rat model. Subsequent studies in different mouse and rat models using other transgene cassettes confirm the lack of efficacy of AAV5 in rat liver rendering proof of concept studies in this species with this clinically relevant serotype impossible.AAV vectors were constructed by replacing the factor IX cDNA with the UGT1A1 cDNA, using the EcoR1 and Bbs1 sites of plasmid scAAV-LP1-hFIXco . This plasmid carries the AAV2 backbone with an intact 5′ terminal resolution site (trs) without the 3′trs analogous, a LP1 promoter consisting of core liver-specific elements from human apolipoprotein hepatic control region (HCR) and the human alpha-1-antitrypsin (hAAT) promoter, a modified SV40 small intron and the SV40 late polyA sequence, as previously described 11 and 9×1011 genome copies per plate. Of the average titers (genome copies/ml) of the purified batches of the vectors were 5.3×1012 for scAAV2/1, 5.3×1012 for scAAV2/5 and 1.9×1012 for scAAV2/8. Because of the double stranded genome, each scAAV vector was calculated as containing 2 copies of ss viral genomes.Recombinant AAV was produced with AAV2 Rep and pseudotyped with capsid from AAV serotype 1, 5 and 8 using the adenovirus-free method described before Gunn rats from our own breeding colony were used for all experiments and fed ad libitum. All animal experiments were performed in strict accordance with the Animal Ethical Committee guidelines of the Academic Medical Center of Amsterdam and the Animal Ethical Committee guidelines of the University of Navarra. The protocol was approved by the Animal Ethical Committee of the Academic Medical Center of Amsterdam and the Animal Ethical Committee of the University of Navarra.11 gc/kg of scAAV-LP1-UGT1A1 vector packaged with each of the viral capsids from AAV1, 5 and 8. For portal injections the rats were anesthetized with an intraperitoneal injection of KAR mix: 4 ml ketamine (100 mg/ml), 2 ml Rompun , 1 ml atropine (1 mg/ml); dose of 0.1 ml/100 g body weight. Under deep anesthesia, the peritoneal cavity was opened and AAV vector resuspended in a maximum volume of 500 µl of PBS was injected into the portal vein using a 30-gauge needle. The animals were sutured and received the analgesic Temgesic subcutaneously following recovery from KAR mix. For bile collection, rats were anesthetized by intraperitoneal injection of KAR mix as above and bile was collected by cannulation of the bile duct as described Male Gunn rats, 8 to 10 weeks of age, with a weight between 180 and 200 g, received a single intraportal injection of 3×10Luciferase activity is represented as photons/sec and RLU per miligram of total protein respectively.Blood was collected by tail vein puncture under gas anesthesia in pediatric heparin tubes. After spinning down the cells the plasma was stored at −80°C until bilirubin measurement performed at the routine clinical chemistry department.5′-GACGCCTCGTTGTACATCAG-3′; reverse, 5′-CACGCTGCAGGAAAGAATC-3′, using the following conditions 95°C for 5 min and 45 cycles of 95°C for 10 s, 60°C for 10 s, and 72°C for 12 s. The values were normalized with the rat β-actin gene, using as a forward primer: 5′ AGCCATGTACGTAGCCATCCA3′ and 5′ TCTCCGGAGTCCATCACAATG3′ as the reverse primer. A standard curve was generated by dilution in rat genomic DNA of the UGT1A1 plasmid. To determined the UGT1A1 mRNA levels in liver the qPCR was performed using the same primers for UGT1A1 as used to determine genomic copies and primers for 18S (Fw: 5′-CGAACCTCCGACTTTCGTTCT-3′ and a Rv: 5′-TTCGGAACTGAGGCCATGAT-3′). Vector genomic copies per diploid genome equivalent (vc/dGE) and UGT1A1 mRNA/18S were calculated using the LinRegPCR software High-molecular-weight DNA from tissues was isolated by a sodium dodecyl sulfate-proteinase treatment and subsequently complexed with silica particles in the presence of guanidinium thiocyanate, as described in Total bilirubin in serum was determined by the routine clinical chemistry department using a standard colorimetric assay. Unconjugated bilirubin and bilirubin conjugates in bile were analyzed and quantified by HPLC as described The results obtained in C57/BL6 mice are statistically significant compared to the obtained in Sprague Dawley and Wistar rats using the nonparametric Mann–Whitney test .11 gc/kg of scAAV-LP1-UGT1A1 vector pseudotyped with serotype 1, 5 and 8. Of these scAAV2/8 achieved the most prominent long term correction  was injected into the tail vein of eight Gunn rats. This also resulted in the presence of vector genomes mainly in liver and spleen, but not in detectable expression of GFP mRNA and protein, which renders this explanation unlikely (data not shown). In mice, the stronger liver tropism with AAV1 than AAV5 can be explained by its receptor, platelet derived growth factor receptor (PDGFR) which is expressed at high levels on hepatocytes AAV is the most promising delivery system for The large difference in efficacy of AAV5 found between mice and rat is unexpected but recently two new species of AAV’s from rat and mouse liver Recently it was reported that in rabbits AAV5 also transduced liver very poorly In conclusion, we showed that scAAV8 and scAAV1 provided an efficient liver transduction in the rat model for CN syndrome, the Gunn rat. Furthermore, our data indicated that proof of concept studies with AAV5 as a vector for correcting liver disorders are not possible in rat models.Data S1Supplementary data.(PDF)Click here for additional data file.


Expression of the sole CX3CR1 ligand, the membrane-tethered and sheddable chemokine CX3CL1/fractalkine, is restricted in the brain parenchyma to selected neurons. Here we summarize our current understanding of the physiological role of CX3CR1 for microglia function and the CX3C axis in microglial/neuronal crosstalk in homeostasis and under challenge. Moreover, we will discuss the efforts of our laboratory and others to exploit CX3CR1 promoter activity for the visualization and genetic manipulation of microglia to probe their functional contributions in the central nerve system (CNS) context.Microglial cells in brain and spinal cord are characterized by high expression of the chemokine receptor CX Under these conditions microglia might however loose much of their uniqueness and turn into prototype macrophages. This calls for the development of experimental systems that will allow the study of microglia in their unique physiological CNS environment.Microglia are members of the mononuclear phagocyte system alongside other macrophages, monocytes and dendritic cells . Of note, a potential confounding problem is the fact that when using CX3CR1gfp mice for microglia studies, animals are heterozygotes for the chemokine receptor and microglia display due to the haplo-insufficiency considerable less CX3CR1 surface expression  promoter  resulting in impaired cognitive functions  and amyothrophic lateral sclerosis (ALS), as well as neuropathological conditions, such as neuropathic pain and cerebral ischemia.The CX3CR1-deficiency has been introduced into several established murine AD models, including the hTAu  deposits surrounded by activated glia and dystrophic neurites. The CX3CR1-deficient microglia were found to overexpress IL-1β and display neurotoxic activity  was reported to depend on microglial release of the lysosomal protease cathepsin S potentially in response to ATP release and P2X7 receptor  (LysM-Cre:GRK2f/+ mice)  and are hence located behind and in front of the BBB, respectively. Spinal cord and DRG are therefore differentially accessible to the influx of monocytes and contribution of monocyte-derived macrophages (see below).One issue complicating the exact assessment of the importance of the CX3C axis in microglia biology is that constitutively expressed membrane-tethered neuronal CX3CL1 seems to provide a tonic inhibitory signal to microglia that keeps these cells in a quiescent “sampling” or surveillance mode  origin of microglia. Yet, interpretation of the results obtained was confounded by the facts that first microglia are radio-resistant and thus not replaced by a bone marrow graft, and that second the irradiation compromised the BBB allowing monocyte infiltrates. Revision of these studies taking advantage of CX3CR1gfp mice to mark either the microglia or the blood compartment and introducing cellular exchange in parabionts and CCR2 dependency as a indication of monocyte-derivation  that involves the BBB breakage and substantial monocyte recruitment  of the human estrogen receptor (ER) (Feil et al., In order to overcome the above constraints of current microglia studies, we decided to exploit the high CX3CR1Cre and CX3CR1CreER animals differ considerably with respect to the cells targeted. In CX3CR1CreER animals only cells that express CX3CR1 and hence the CreER transgene will undergo rearrangement at the time of the TAM treatment. In contrast and as best demonstrated in combination with respective reporter mouse strains (Yona et al., 3CR1Cre animals also cells that are derived from CX3CR1+ cells but subsequently silenced CX3CR1 expression will have recombined the loxP-flanked loci. CX3CR1Cre animals therefore report on the history of the cell and can be used for fate mapping studies (Yona et al., Importantly, CX3CR1Cre and CX3CR1CreER mice crossed to animals harboring a floxed YFP reporter gene (Srinivas et al., 3CR1Cre: YFP mice, and only in TAM-treated CX3CR1CreER: YFP mice, more than 95% of both brain and spinal cord microglia were found YFP labeled (Figure 3CR1+ microglia, either straight from their development onwards, using the constitutive Cre system, or in specific time windows, using the inducible Cre system.Analysis of CX3CR1. TAM treatment of CX3CR1CreER mice results accordingly also in gene rearrangement in these cells (Yona et al., 3CR1 expression, they are not targeted by the CX3CR1CreER approach in adulthood (Yona et al., 3CR1+ intestinal macrophages on the other hand loose their gene modifications, as they are progressively replaced by monocytes (Zigmond et al., 3CR1Cre and CX3CR1CreER animals provides a unique tool to probe for the involvement of microglia in CNS development, CNS maintenance and CNS responses to pathological challenges.Of note, certain lymphocyte subsets and myeloid cells, other than microglia express CX3C axis is likely to play a prominent role in these activities. Thus accumulating evidence suggest that neuronal CX3CL1 acts as a critical inhibitory signal retaining microglia in quiescent mode and preventing collateral damage due to microglia hyper-activation. Aside from its biological role, the CX3CR1 chemokine receptor provides us however also with a unique foothold to study microglia in context using state of the art imaging and gene manipulation approaches. The near future is hence likely to provide valuable insight into contributions of these intriguing cells to brain physiology and might pave the way for the development of microglia manipulation for therapeutic purposes.Microglia are the main representatives of the immune system in the healthy brain as such likely to critically contribute to the brain's resistance to pathological challenges. Moreover, recent data highlight the critical involvement of microglia in CNS development and homeostasis. Given its strategic localization at the neuronal/microglial interface the CXThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.