IFIH1, one common and four rare SNPs have been associated with lower risk for type 1 diabetes. Our aim was to test whether these type 1 diabetes-associated IFIH1 polymorphisms are associated with the occurrence of enterovirus infection in the gut of healthy children, or influence the lack of association between gut enterovirus infection and islet autoimmunity.Interferon induced with helicase C domain 1 (IFIH1) senses and initiates antiviral activity against enteroviruses. Genetic variants of HLA-DR4-DQ8/DR3-DQ2) as well as 375 children without this genotype were included for monthly fecal collections from 3 to 35 months of age, and genotyped for the IFIH1 polymorphisms. A total of 7,793 fecal samples were tested for presence of enterovirus RNA using real time reverse transcriptase PCR.After testing of 46,939 Norwegian newborns, 421 children carrying the high risk genotype for type 1 diabetes  as compared to wild-type homozygotes ; odds ratio 2.5, p = 0.06. The association was stronger when infections were restricted to those with high viral loads . The lack of association between enterovirus frequency and islet autoimmunity reported in our previous study was not materially influenced by the IFIH1 SNPs.We found no association with frequency of enterovirus in the gut for the common IFIH1 polymorphisms have no, or only minor influence on the occurrence, quantity or duration of enterovirus infection in the gut. Its effect on the risk of diabetes is likely to lie elsewhere in the pathogenic process than in the modification of gut infection.We conclude that the type 1 diabetes-associated  Picornaviridae) cause mostly inapparent or subclinical infections; acute disease may range from minor illness to paralytic disease Enteroviruses (family IFIH1 (interferon induced with helicase C domain 1), also known as MDA5 (melanoma differentiation associated gene 5), encodes a cytoplasmic sensor that recognizes certain types of double stranded RNA (dsRNA) molecules, which are commonly produced during the replication of some RNA viruses. Signaling via IFIH1 triggers activation of NF-κB and interferon regulatory pathways, and induces antiviral interferon responses in vivo effects of IFIH1 in mda5 knockout mice infected with coxsackievirus IFIH1Enteroviruses have been considered as possible environmental triggers or accelerators of islet autoimmunity leading to type 1 diabetes IFIH1 gene revealed that lower risk of type 1 diabetes is also associated with two rare variants with a presumed loss of function (nonsynonymous SNPs rs35744605 (E627X) and rs35667974 (I923V)), and with two noncoding variants affecting conserved splice sites (rs35337543 (1641+1G>C)) and rs35732034 (2807+1G>A)), odds ratios of about 0.5–0.7 IFIH1 polymorphisms to type 1 diabetes, and if enterovirus is involved here, remains unclear and paradoxical. Presumably, variants associated with reduced IFIH1 function would confer the host with a mild antiviral response to enterovirus infection. If so, this would influence the observed association between enterovirus frequency and type 1 diabetes in the population, and failure to account for IFIH1 SNPs in population studies could be hypothesized to “conceal” relations between enterovirus and islet autoimmunity or type 1 diabetes.Recently, deep sequencing of exons and splice sites in the IFIH1 polymorphisms associated with type 1 diabetes can predict frequency, viral load, or duration of gut infections with enterovirus. We further aimed to test whether our previously reported lack of association between enterovirus and islet autoimmunity was modified by taking these IFIH1 polymorphisms into account.The primary aim of the present study was to assess in healthy children whether the DRB1*04:01-DQA1*03-DQB1*03:02/DRB1*03-DQA1*05-DQB1*02 (DR4-DQ8/DR3-DQ2) which confers high risk for type 1 diabetes HLA DR4-DQ8/DR3-DQ2 genotype and 375 children without this genotype, as described in detail in The participants were identified and recruited in the MIDIA-study that during 2001–2007 screened 46,939 newborns from the Norwegian general population for the HLA genotype IFIH1 polymorphisms modified the risk of type 1 diabetes-related islet autoimmunity among children with the HLA-DR4-DQ8/DR3-DQ2 genotype  and the Norwegian Data Inspectorate.Fecal samples were tested for enterovirus as described earlier IFIH1 SNP , and four rare SNPs rs35744605 (E627X), rs35667974 (I923V), rs35732034  and rs35337543 . SNP genotyping of rare variant SNPs was performed with the MassARRAY system using iPLEX chemistry . The common SNP rs1990760 was genotyped using TaqMan technology with predesigned primer-probe mixes . Among children with at least one fecal sample tested for enterovirus (n = 808), genotyping with the MassARRAY system failed in 5–8 DNA samples (<0.1%), and with TaqMan only two samples failed. Duplicate genotyping was performed in 5% randomly chosen samples, with 100% concordance.Subjects were genotyped for one common 2 test) in either of the polymorphisms. Haploview 4.2 was used to assess linkage disequilibria between the alleles of the typed SNPs. Association analyses were performed using logistic regression models with enterovirus infection (yes or no) as the response, individual SNP genotypes as predictors, and a random intercept to account for potential intra-individual correlation (clustering) of infections .The final sample for analysis included 796 children and their 7,793 fecal samples . There wApart from taking enterovirus positivity as an outcome, in further sensitivity analysis we restricted infections to those with above median quantity of enterovirus RNA among positive samples , or to the first enterovirus RNA positive samples among series of two or more consecutively positive samples. Prolonged infection episodes with at least two consecutively positive samples were assessed by excluding other positive samples.IFIH1 SNPs evaluated. This was modeled similarly as for the IFIH1 SNP-enterovirus relation, but with autoimmunity status as a covariate and an additional random intercept to specify matched set .The frequencies of enterovirus in fecal samples by genotypes of the five SNPs are shown in  the gut , we obse the gut . Here thP = 0.01, P = 0.09) (2<0.04), the above association is unlikely to be explained by linkage disequilibrium to other known diabetes-associated variants. The association of each IFIH1 SNP with enterovirus was similar among children with and without the type 1 diabetes-associated HLA DR4-DQ8/DR3-DQ2 high-risk genotype, respectively (data not shown).This suggestive association was strengthened when the analysis was restricted to infections with high viral loads  . As therIFIH1 polymorphisms into account would modify our previously published observation of no association between enterovirus frequency and islet autoimmunity IFIH1 rs1990760  IFIH1 SNP our data suggest that the per allele odds ratio is highly unlikely to be greater than about 1.2 . We cannot exclude weak to moderate effects of the rare variants because of limited power, but the possible existence of such weak effects of rare IFIH1 variants on enterovirus infections would have limited population impact on the epidemiology of enterovirus infections, if any at all.We are aware of only two previous studies relating mda5 knockout mice mda5 knockout mice with coxsackievirus have produced partially conflicting results, but one group showed significantly decreased production of type I IFN, but this was not correlated with increased virus titers If the suggestive association between the rare A variant of rs35732034 and frequency of high quantity enteroviral infections is true, this indicates a dominant or dose-dependent effect of the predicted interference with splicing of the intron enterovirus genus in type 1 diabetes pathogenesis seem likely, yet the limited numbers of available longitudinal studies of islet autoimmunity where enteroviral RNA have been tested as a risk factor have been inconsistent between populations and sources of the sample The evidence for the involvement of the IFIH1 alleles that had been previously associated with reduced type 1 diabetes risk were predicted to be loss of function variants, and may thus confer increased risk for enterovirus infection. We therefore reasoned that our previously published lack of association between enterovirus RNA and islet autoimmunity IFIH1 SNPs. However, adjusting for IFIH1 (rs1990760) and the exclusion of children with rare IFIH1 alleles did not influence the lack of association. A prospective cohort study recently reported lack of association with islet autoantibody development for genotypes of the IFIH1 SNP rs2111485, a SNP in close proximity and in strong linkage disequilibrium with rs1990760. While this may partially be explained by limited statistical power, their study showed an association with faster progression from autoimmunity to type 1 diabetes The IFIH1 variants previously associated with type 1 diabetes are loss of function variants, and experimental data showing an important role of IFIH1 in antiviral immunity to enteroviruses, out data suggests that these variants have no or just minor impact on the frequency and duration of gut infection with enterovirus in healthy infants. Its effect on the risk of diabetes is likely to lie elsewhere in the pathogenic process than in the modification of gut infection frequency.In conclusion, despite the predictions that rare Table S1The effect of IFIH1 genotypes on different measures of enterovirus infection in longitudinal fecal samples.(DOC)Click here for additional data file.


Uncoupling protein 3 (UCP3) is primarily expressed in the inner membrane of skeletal muscle mitochondria. It has been proposed that UCP3 reduces production of reactive oxygen species (ROS) and oxidative damage. However, the mechanisms by which UCP3 attenuates ROS production are not well understood. Here we report that UCP3 interacts with the non-processed form of thioredoxin 2 (Trx2), a redox protein that is localized in mitochondria, but not processed Trx2, which is involved in cellular responses to ROS. The hydrophilic sequences within the N-terminal tail of UCP3, which faces the intermembrane space, are necessary for binding to Trx2. In addition, Trx2 directly associated with UCP3 through a mitochondrial targeting signaling sequence, was processed in the intermembrane space, and thereby allowing redox reactions. A bimolecular fluorescence complementation analysis demonstrated that the interaction of these proteins occurs in the mitochondrial intermembrane space. Furthermore, increased UCP3 expression significantly attenuated ROS production in isolated mitochondrial without effects on membrane potential, however this effect is lost by Trx2 knock down. These results suggest that UCP3 binds to Trx2 in the mitochondrial intermembrane space and attenuates ROS production.


A transcription factor called Promyelocytic Leukemia Zinc Finger (PLZF) calibrates the balance between spinal cord progenitor maintenance and differentiation by enhancing their sensitivity to mitogens that are present in developing embryos. Distinct classes of neurons and glial cells in the developing spinal cord arise at specific times and in specific quantities from spatially discrete neural progenitor domains. Thus, adjacent domains can exhibit marked differences in their proliferative potential and timing of differentiation. However, remarkably little is known about the mechanisms that account for this regional control. Here, we show that the transcription factor Promyelocytic Leukemia Zinc Finger (PLZF) plays a critical role shaping patterns of neuronal differentiation by gating the expression of Fibroblast Growth Factor (FGF) Receptor 3 and responsiveness of progenitors to FGFs. PLZF elevation increases FGFR3 expression and STAT3 pathway activity, suppresses neurogenesis, and biases progenitors towards glial cell production. In contrast, PLZF loss reduces FGFR3 levels, leading to premature neuronal differentiation. Together, these findings reveal a novel transcriptional strategy for spatially tuning the responsiveness of distinct neural progenitor groups to broadly distributed mitogenic signals in the embryonic environment. The embryonic spinal cord is organized into an array of discrete neural progenitor domains along the dorsoventral axis. Most of these domains undergo two periods of differentiation, first producing specific classes of neurons and then generating distinct populations of glial cells at later times. In addition, each of these progenitors pools exhibit marked differences in their proliferative capacities and propensity to differentiate to produce the appropriate numbers and diversity of neurons and glia needed to form functional neural circuits. The mechanisms behind this regional control of neural progenitor behavior, however, remain unclear. In this study, we identify the transcription factor Promyelocytic Leukemia Zinc Finger (PLZF) as a critical regulator of this process in the chick spinal cord. We show that PLZF is initially expressed by all spinal cord progenitors and then becomes restricted to a central domain, where it helps to limit the rate of neuronal differentiation and to preserve the progenitor pool for subsequent glial production. We also demonstrate that PLZF acts by promoting the expression of Fibroblast Growth Factor (FGF) Receptor 3, thereby enhancing the proliferative response of neural progenitors to FGFs present in developing embryos. Together, these findings reveal a novel developmental strategy for spatially controlling neural progenitor behavior by tuning their responsiveness to broadly distributed growth-promoting signals in the embryonic environment. The formation of neural circuits within the developing central nervous system (CNS) depends upon the spatially and temporally ordered generation of distinct classes of neurons and glia from multipotent neural stem and progenitor cells (NPCs). An essential feature of this progression is the ability of NPCs to self-renew in a manner that permits early-born cells such as neurons to form while maintaining a sufficient progenitor pool to generate later-born cell types such as glia. At the heart of this process is the interplay between mitogenic signals from the extracellular environment and cell intrinsic factors, which integrate this information to permit either progression through the cell cycle or the onset of terminal differentiation These features of NPCs are exemplified in the developing spinal cord, where many extrinsic and intrinsic factors regulating progenitor maintenance and differentiation have been characterized. In the early neural plate and tube, NPCs are organized in a proliferative neuroepithelial sheet and sustained by the mitogenic actions of several growth factors, particularly Fibroblast Growth Factors (FGFs). FGFs are broadly present in neural tissues and the surrounding mesoderm and act through receptor tyrosine kinases (FGFRs) expressed by NPCs throughout the course of neural development As development proceeds, NPCs become increasingly poised to undergo terminal differentiation through the actions of retinoids, which activate the expression of homeodomain and bHLH transcription factors such as PAX6 and OLIG2. These factors participate in the dorsoventral patterning of NPCs and promote the accumulation of proneural bHLH proteins needed to trigger cell cycle exit and neuronal differentiation The period of neurogenesis in the spinal cord is relatively brief, lasting for only a few days in chick and mouse development, after which time undifferentiated NPCs up-regulate expression of the pro-glial transcription factors SOX9 and NF1A, and begin to give rise to astrocyte and oligodendrocyte precursors Olig2 mutant mice. Olig2+ NPCs exhibit limited capacity for self-renewal, suggesting that Olig2 represses genes that promote NPC proliferation Zbtb16, which encodes the Promyelocytic Leukemia Zinc Finger (PLZF) transcription factor, as one of the most prominently elevated genes in the absence of Olig2 function  and e3 (HH stage 17)  stage 10] in a subset of SOX2tage 17) –C, G–H. + progenitors and is down-regulated as cells express the proneural transcription factor NEUROG2 and begin to form differentiated interneurons marked by LHX1/5 and NEUN staining , progenitors in the intermediate spinal cord up-regulate the expression of the early glial fate determinants SOX9 and NF1A, and begin to transform into astrocyte progenitors s emerge . Togethes emerge .Since PLZF is associated with stem and progenitor cell maintenance in other tissues, we sought to examine whether its function plays a comparable role in the developing spinal cord. We first investigated the consequences of elevating PLZF activity on NPC maintenance and neuronal differentiation. Expression vectors encoding PLZF and an IRES-nuclear Enhanced Green Fluorescent Protein (nEGFP) reporter cassette under the control of the cytomegalovirus enhancer-chick beta actin promoter were electroporated into the chicken spinal cord, and embryos collected 2 d later to assess changes in neuronal differentiation. In spinal cords electroporated with the empty vector, ∼60% of transfected cells expressed NPC markers such as SOX2 while the remaining ∼40% expressed neuronal markers such as NEUN . When PLASCL1 and NEUROG1 mRNA and a ∼28% decrease in the number of NPCs expressing NEUROG2 protein relative to spinal cords transfected with the control vector  along with an IRES-nEGFP reporter cassette to identify the transfected cells. Electroporation of this construct into the spinal cord reduced PLZF levels to nearly background staining levels (HES5-2 and ID2 (+ or TUJ1+ neurons formed after PLZF knockdown (unpublished data). Perhaps accounting for this result, we found that PLZF loss was associated with a ∼2-fold increase in the frequency of cells undergoing apoptotic cell death measured by activated CASPASE3 staining , which possess a nonsense mutation in the PLZF coding sequence that ablates its DNA binding function Lu/LuZbtb16 mutant mice (unpublished data). However, using a panel of lineage-restricted makers on the spinal cords from Lu/LuZbtb16 mutant and control littermates, we found that differentiation was increased among some interneurons whose progenitors normally express high levels of PLZF  and p2  central progenitor domains were reduced by ∼12% in the Lu/LuZbtb16 mutants, and this decrease was mirrored by a >14% rise in the number of dI6, V1, V2a, and V2b interneurons distinguished by their expression of specific transcription factors including Bhlhe22 (Bhlhb5), Foxp2, Vsx2 (Chx10), and Gata3  or PLZF-transfected (∼5%) embryos  form of FGFR1 that forms nonfunctional heterodimers with FGFR3 and other FGFRs and blocks their downstream signaling activity ed cells . In conted cells .+ cells seen after electroporation with shPLZF alone  and ETV4 (PEA3), as well as feedback inhibitors of the pathway such as STAT3 is a noncanonical effector of FGF signaling +highFGFR3 intermediate region of the spinal cord compared to the PLZF−lowFGFR3 dorsal spinal cord. For this analysis, PAX6 was used to monitor NPCs in place of SOX2. The extent of PAX6 expression completely overlaps with SOX2, and the high versus low levels of PAX6 in the intermediate and dorsal spinal cord served as a convenient proxy for assessing the presence or absence of PLZF  that can override the endogenous transcriptional repressor functions of PLZF and potentially other BTB/POZ proteins than with shRNA constructs selectively targeting PLZF. To date, we have identified five additional family members with expression in the developing spinal cord, suggesting there may be complementary functions with PLZF (Z.B.G. and B.G.N. unpublished observations). A comparable situation exists within the hematopoietic lineage where the lack of a prominent phenotype in either PLZF-null mice or in cell lines transfected with PLZF targeting shRNA is attributed to the presence of other BTB/POZ proteins such as the closely related FAZF The FGF pathway also receives inputs from other signaling networks and has extensive feedback regulatory mechanisms The growth and morphogenesis of the nervous system depends upon the ability of the FGFs to promote the rapid proliferation of NPCs and block neuronal differentiation highFGFR3 NPCs in the intermediate spinal cord display a heightened response to ectopically expressed FGF8 compared to their lowFGFR3 dorsal counterparts  In summary, PLZF and FGFR3 work in parallel with other FGFR, mitogen signaling pathways, and, most likely, other members of the BTB/POZ family, to modulate proliferation in the spinal cord and thereby permit NPCs to differentiate at characteristic rates and times in development. PLZF facilitates the mitogenic activity of the FGFs, which act in a STAT3-dependent manner to maintain a specific population of NPCs in a proliferative state and ensure that the necessary number of progenitors is available for the transition from neurogenesis to gliogenesis. Aberrant activation of FGFR3 and STAT3 has been observed in a multitude of human cancers Drosophila Engrailed repressor domain 5′-cgcagctgagatcctagaaata-3′ and 5′-ttcagcctgaagcaccagctgg-3′) into the plasmid pCIG-shRNA Plasmid expression vectors were generated by cloning cDNAs of interest into a Gateway cloning-compatible variant of the vector pCIG CreOlig2GFPOlig2Fertilized chicken eggs were acquired from AA Lab Eggs, Inc. and McIntyre Poultry and Fertile Eggs. Eggs were incubated at 37°C and 60% humidity, staged, and electroporated with plasmid vectors as previously described 2, equilibrated overnight in 30% sucrose, frozen on crushed dry ice in OCT mounting media (Sakura Tissue-Tek), and cryosectioned. Prior to staining, slides were fixed in 4% paraformaldehyde for an additional 10 min at 4°C and then rinsed twice in PBS containing 2 mM MgCl2, for 10 min per wash. Slides were overlaid with 1 mL of X-Gal Staining Buffer  and placed in a humidified chamber at 37°C for several hours to overnight. Once signal had developed, slides were repeatedly rinsed in PBS with 2 mM MgCl2, coverslipped, and imaged using brightfield microscopy.Dissected tissue was briefly fixed in 4% paraformaldehyde at 4°C, rinsed repeatedly in PBS containing 2 mM MgClt test using GraphPad Prism 5.0 software.All images were collected using either a Zeiss Observer D1 microscope equipped with an Apotome optical imaging system or a Zeiss LSM5 Exciter confocal imaging system. Images were processed using Zeiss Axiovision and LSM Exciter software suites and Adobe Photoshop. Pixel intensity analysis of mRNA and protein expression was performed using NIH ImageJ software. Cell counts were performed manually and in most cases represented as the mean value of multiple images of tissue sections collected from several independent specimens as indicated in the figure legends. For in ovo electroporation experiments, all transfected cells were counted for each image analyzed, with, on average, 100 cells per image in experiments focusing on the ventral region of the spinal cord at e4 (HH 21), more than 350 transfected cells per image in experiments with an e5 (HH 25) endpoint, and over 900 cells per image in experiments with an e15 (HH 41) endpoint. Unless stated otherwise, results are expressed as fractional changes normalized to electroporation with empty vector controls set to a value of 1.0. The statistical significance of differences observed between experimental and control groups were assessed with the Student's Figure S1Olig2 mutant mice and demarcates neural progenitors in the developing mouse spinal cord.  Expression of Zbtb16 mRNA and PLZF protein in wild-type e10.5 mice. PLZF is broadly expressed by Sox2+ progenitors, including Olig2+ motor neuron progenitors, but absent from differentiated Isl1/2+ motor neurons. (C) PLZF expression is elevated in the ventral spinal cord of e10.5 Cre/CreOlig2 mice. Microarray expression profiling revealed that Zbtb16 mRNA levels were 2.86-fold elevated in Olig2 mutants, p = 0.00126 (unpublished data), and comparable changes in PLZF protein staining are seen using immunohistochemistry. (F–O) Analysis of wild-type mouse embryos at e9.5 and e11.5 shows that the pattern of PLZF expression is similar to that observed in chicken embryos. PLZF is initially expressed by all Sox2+ progenitors and then becomes restricted to a central domain bordered by Msx1 and Olig2 expression. PLZF is subsequently down-regulated as cells differentiate into TuJ1+ neurons. (P–U) PLZF and Fgfr3 mRNA expression are highly overlapping at multiple stages of mouse development. Serial sections of e9.5, e11.5, and e12.5 spinal cords are shown.PLZF is increased in (TIF)Click here for additional data file.Figure S2GFAP expression.  Stable electroporation of chick embryos at e3 with CMV::PLZF expression vectors using the Tol2 transposon system does not lead to any change in the expression of either SOX9, NF1A, or GFAP when analyzed at e7.PLZF expression precedes the appearance of early glial progenitor markers, but its misexpression does not alter the normal course of their onset. (A–J) Antibody costaining analysis of PLZF and two early markers of glial progenitor fate, SOX9 and NF1A. SOX9 expression in the chick commences on e4, whereas NF1A appears later at e5–e6 (TIF)Click here for additional data file.Figure S3HES gene expression or dorsoventral pattern. (A–B) Spinal cords transfected with PLZF did not exhibit any significant alteration in the mRNA expression of two of the principal Notch effector genes, HAIRY1 and HES5-2. Insets show the extent of transfection in the corresponding sections marked by the presence of nEGFP protein. (C–D) PLZF+ cells in the intermediate spinal cord of e5 (HH 25) chick embryos express high levels of PAX6 protein (blue brackets). However, PLZF is largely absent from dorsal progenitors that express low levels of PAX6 protein (yellow brackets). (E–H) PLZF misexpression does not alter the expression of the homeodomain proteins IRX3, PAX6, PAX3, or PAX7 that demarcate the boundaries of progenitor domains in the developing spinal cord. All electroporations were carried out at e3 (HH 17) and collected for analysis on e5 (HH 25).PLZF misexpression does not lead to changes in (TIF)Click here for additional data file.Figure S4Zbtb16) gene, which lacks the sites targeted by the shPLZF construct. (M) Chart displays the mean ventricular zone area ± SEM for embryos electroporated with the indicated plasmids relative to the untransfected contralateral sides of the spinal cord. Blue dotted lines demarcate the border of the contralateral VZ in each image. (N) Chart displays the mean number of transfected NPCs expressing NEUROG2 ± SEM, relative to empty vector controls. All electroporations were performed at e2 (HH 10) and collected at e4 (HH 21). In all panels, ***p<0.001 and ****p<0.0001. Counts were based on at least 12 images taken from ≥8 electroporated embryos.PLZF knockdown can be rescued by the coexpression of human PLZF. (A–C) Electroporation of e3 (HH 17) chick spinal cords with a vector encoding PLZF shRNAs and an IRES-nEGFP transfection marker reduced endogenous PLZF protein expression at e5 (HH 25) by 93.7±1.29%. Chart displays the mean pixel intensity of PLZF antibody staining ± SEM for spinal cords electroporated with the control or PLZF shRNA constructs, relative to PLZF expression on the nontransfected contralateral control sides. (D–F) Electroporation with a vector producing a nontargeting control shRNA does not alter PLZF, SOX2, or NEUROG2 expression. (G–L) The effects of PLZF knockdown on SOX2 and NEUROG2 expression are rescued by coelectroporation with an expression construct encoding the human PLZF ((TIF)Click here for additional data file.Figure S5Lu/LuZbtb16 (PLZF) mutant mice. The number of cells found in the PLZF-expressing p1 and p2 domains, though not the p0 domain, were significantly decreased in Lu/LuZbtb16 mutants, while progenitors in the adjacent pMN domain that does not normally express high levels of PLZF were unaffected. Each of these progenitor pools was identified by both the absence of the early neuronal differentiation marker Sox11  and the presence of specific patterning markers (AA). The pMN was distinguished by the expression of Olig2, the p2 by the expression of Nkx6.1 dorsal to Olig2+ cells, the p1 domain as being situated between zones of Nkx6.1 (p2) and Dbx1 (p0) expression, and p0 by the expression of Dbx1. (M–X) The number of dI6, V1, V2a, and V2b neurons, which are normally derived from PLZF+ progenitors (Lu/LuZbtb16) mice. However, neurons that are not associated with PLZF+ progenitors, such as dI3 interneurons and motor neurons, are not changed.  Charts displaying the mean number of cells expressing the indicated progenitor or neuronal markers ± SEM relative to WT and Lu/+Zbtb16 littermate controls. Results are representative of >10 images collected from at least five embryos of each genotype. In all panels, *p<0.05, **p<0.01, and ***p<0.001. (AA) Summary of the transcription factors that define specific progenitor (p) pools and their neuronal progeny in the developing spinal cord.Reduced progenitor pools and excessive neuronal production in PLZF deficient mice. (A–L) Several progenitor pools that express PLZF are reduced in genitors , are inc(TIF)Click here for additional data file.Figure S6FGFR and SPROUTY genes in the wild-type and PLZF-electroporated spinal cord. (A–F) Analysis of FGFR1, FGFR2, and FGFR3 mRNA expression in e4 (HH 21) and e5 (HH 25) chick spinal cords. FGFR4 was not present in the spinal cord at any stage examined (unpublished data). (G–I) PLZF misexpression at e3 (HH 17) increases FGFR3 expression in the e5 (HH 25) dorsal spinal cord, but does not alter the expression of either FGFR1 or FGFR2.  At e5 (HH 25), neither SPRY1 nor SPRY2 are expressed in the intermediate spinal cord where FGFR3 levels are normally high (C), nor were they elevated following PLZF misexpression at e3 (HH 17). (L) FGF2 mRNA is expressed by scattered cells throughout the e5 (HH 25) chick spinal cord.Expression of (TIF)Click here for additional data file.Figure S7STAT3 is expressed throughout the VZ of the e5 (HH 25) chick spinal cord. (F–I) Both PLZF and FGFR3 misexpression at e3 (HH 17) increase the activity of a cotransfected STAT3 responsive-LacZ reporter construct when assessed at e4 (HH 21), suggesting that elevated FGF signaling can stimulate the activity of the STAT3 pathway. Results in (I) are represented as the mean activity of the STAT3-LacZ reporter ± SEM seen following PLZF or FGFR3 misexpression, relative to the activity of the reporter transfected with control plasmids. Counts were based on at least 10 images taken from 8–10 electroporated embryos. ****p<0.0001.PLZF and FGFR3 promote NPC maintenance through the STAT3 pathway. (A–D) ERK1/2 phosphorylation is not observed in the central spinal cord of wild-type embryos or those electroporated with expression constructs producing constitutively active (ca) FGFR1, caFGFR3, or PLZF. (E) (TIF)Click here for additional data file.Figure S8p<0.0001.Coexpression of PLZF and FGF8 disrupts neuronal differentiation in a manner that recapitulates the expression of a constitutively activated form of FGFR3. (A–O) The coexpression of PLZF with FGF8 leads to a significant expansion in the VZ marked by PAX6 expression. Effects were seen in both the dorsal spinal cord (yellow brackets and associated panels) and intermediate spinal cord (blue brackets and associated panels). This phenotype was fully penetrant and ranged from moderate (A–E) to extremely severe (K–O).  Misexpression of caFGFR3 increases the proportion of transfected cells expressing SOX2, similar to the effects seen with the concomitant misexpression of PLZF and FGF8 (A). (R) Chart displays the mean number of caFGFR3-transfected cells expressing SOX2 ± SEM, relative to transfection with an empty control vector. All electroporations were performed at e3 (HH 17) and collected at e5 (HH 25). Counts were based on at least 10 images taken from ≥8 embryos. ****(TIF)Click here for additional data file.Table S1Antibodies used for immunohistochemistry.(DOCX)Click here for additional data file.Table S2PCR primers used to create in situ probes.(DOCX)Click here for additional data file.


Histone modification plays an important role in cell differentiation and tissue development. A recent study has shown that the dimethylation of lysine 4 residue on histone 3 (H3K4me2) marks the gene body area of tissue specific genes in the human CD4+ T cells and neural cells. However, little is known of the H3k4me2 distribution dynamics through the cell differentiation and tissue development.We applied several clustering methods including K-means, hierarchical and principle component analysis on H3K4me2 ChIP-seq data from embryonic stem cell, neural progenitor cell and whole brain of mouse, trying to identify genes with the H3K4me2 binding on the gene body region in different cell development stage and study their redistribution in different tissue development stages.A cluster of 356 genes with heavy H3K4me2 labeling in the gene body region was identified in the mouse whole brain tissue using K-means clustering. They are highly enriched with neural system related functions and pathways, and are involved in several central neural system diseases. The distribution of H3K4me2 on neural function related genes follows three distinctive patterns: a group of genes contain constant heavy H3K4me2 marks in the gene body from embryonic stem cell stage through neural progenitor stage to matured brain tissue stage; another group of gene have little H3K4me2 marks until cells mature into brain cells; the majority of the genes acquired H3K4me2 marks in the neural progenitor cell stage, and gain heavy labeling in the matured brain cell stage. Gene ontology enrichment analysis also revealed corresponding gene ontology terms that fit in the scenario of each cell developmental stages.We investigated the process of the H3K4me2 mark redistribution during tissue specificity development for mouse brain tissue. Our analysis confirmed the previous report that heavy labeling of H3K4me2 in the downstream of TSS marks tissue specific genes. These genes show remarkable enrichment in central neural system related diseases. Furthermore, we have shown that H3K4me2 labeling can be acquired as early as the embryonic stem cell stage, and its distribution is dynamic and progressive throughout cell differentiation and tissue development. Post-translational modifications of histones play important roles in regulating DNA activities and gene expressions in eukaryotic cells -4. In thth position on the N-terminal tail of histone 3) is an important histone mark and has been shown to bind to both gene promoter regions and enhancer regions and its binding is often associated with gene activation. A recent study demonstrated that in human CD4+ T cell and neural tissue, H3K4me2 shows specific binding patterns on the tissue-specific genes in their transcribed regions [H3K4me2  or group 3 (with heavy H3K4me2 acquired in NP stage). In addition, GO function analysis also suggests that groups 1 and 3 genes are more involved in essential neural functions such as transmission of nerve impulse and synaptic transmission while the group 2 (with heavy H3K4me2 only acquired in the latest stage) are more involved in developing and maintain synapse structures as a mature brain function. These observations imply that these disease-related genes are more involved in the essential neural tissue functions than in the advanced brain activities.Besides H3K4me2, many other histone marks as well as epigenetic events such as DNA-methylation are also involved and more insight about the differentiation process can be gained with more data available. In addition, the brain tissue data in this study is obtained from whole brain sample even though it is well known that different anatomical and functional regions of the brain have distinctive gene expression patterns and more refined analysis of region and function specific genes could be identified using similar approach as in this paper.In summary, we investigated the process of the H3K4me2 mark redistribution during tissue specificity development for mouse brain tissue. Our analysis confirmed the previous report that heavy labeling of H3K4me2 in the downstream of TSS marks tissue specific genes. These genes are not only highly enriched with neural system functions and pathways, but also highly involved in CNS diseases. Furthermore, we have shown that such labeling can be acquired as early as the embryonic stem cell stage, and its distribution is dynamic throughout cell differentiation and tissue development. The distribution of H3K4me2 on neural function related genes follows three distinctive patterns: a group of genes contain constant heavy H3K4me2 marks in the gene body from ES through NP to matured brain tissue stage; another group of gene have little H3K4me2 marks until cells mature into brain cells; the majority of the genes acquired H3K4me2 marks in the NP stage, and gain heavy labeling in the matured brain cell stage. GO enrichment analysis also revealed corresponding GO terms that fit in the scenario of each cell developmental stages.Three H3K4me2 ChIP-seq datasets are downloaded from NCBI Gene Expression Omnibus with accession number GSE11172 including murine embryonic stem (ES) cells, ES-derived neural progenitor (NP) cells, and whole brain (WB) tissues . The 36-In WB tissue, the cluster with an elevated H3K4me2 marking downstream of TSS was further divided into four clusters using K-means  to obtain a cluster of 356 genes with highly elevated H3K4me2 labeling downstream of TSS. This set of genes was compared with clusters obtained from ES and NP samples, and largely overlapped genes and clusters were further subject to GO enrichment analysis using the MetaCore software as well as the NIH DAVID tool.http://www.genego.com/metacore.php), as well as Database for Annotation, Visualization and Integrated Discovery . Principal Component Analysis (PCA) was performed on the same combined data using Matlab code.At the same time, the combined TSS profiles of the 356 genes from all three origins were clustered using hierarchical clustering in Matlab and three clusters were selected based on visual inspection: the cluster with constant H3K4me2 labeling from ES through NP to matured brain; the cluster with minimal H3K4me2 labeling in ES and NP stage, but gets heavy H3k4me2 labeling in brain tissue; and the cluster with minimal H3K4me2 labeling in ES stage, but gradually gains H3K4me2 labeling as the cells differentiate into NP cells and mature as brain tissue. Each cluster of genes was subject to gene set enrichment and network analysis using bioinformatics software MetaCore™ by GeneGo (H3K4me2: dimethylation of lysine 4 residue on histone 3; ES: embryonic stem; NP: neural progenitor; WB: whole brain; GO: gene ontology; TSS: transcription start site; GnRH: Gonadotropin-releasing hormone; ChIP-seq: chromatin immunoprecipitation sequencing; CNS: central nerve system; PCA: principal component analysisThe authors declare that they have no competitng interests.JZ performed all the analysis and wrote the manuscript. JDP participated in the discussion and manuscript editing. KH designed the study and participated in the writing of the manuscript.


There was a formatting error in Table 5 in the PDF. On page 8, an incorrect heading was given for the second page of Table 5. "(A) Comparison of sequencing based point variants and variants between references" should be "(B) Coding SNP summary".


Nosocomial infections in patients admitted in coronary intensive care unit (CCU) are frequently caused by potentially pathogen microorganism (PPM). The aim of the present study is 1) to determine the incidence of PPM in patients admitted in our CCU the last year 2) to identify the risk factors for colonization with PPM.Electronic medical records of all patients without previous infection who hospitalized in CCU unit from January since December 2010 were reviewed. During hospitalization, specimens were taken from the nasopharynx, blood and urine cultures and if applicable from the central or peripherals lines.49 patients were included in the study with mean age 63,73yrs (SD=15.45). 64% of the participants were colonized with PPM. The most common isolated pathogens were Staph.Epidermidis (36.7%), Klebsiella Pneumoniae (32.6%), Pseudomonas Aeruginosa (10.2%), Candida Albicans (8.2%) και MRSA (4.1%). Risk factors for colonization with PPM were found the duration of stay in CCU  and the high levels of urea and creatinine .The rates of PPM were significant high. Proper attention should be given in the risk factors that were found to be correlated.None declared.


Cigarette smoking has been shown as major environmental risk factor for rheumatoid arthritis (RA). Epidemiological studies indicate an association of cigarette smoking with development of RA ,2, althoRASF obtained from patients undergoing joint replacement surgery were stimulated with freshly prepared cigarette smoke extract (CSE) for 24 hours. Expression of HDACs was measured at the mRNA level by Real-time TaqMan and SYBR green PCR and at the protein level by immunoblot analysis. Global histone 3 (H3) acetylation was analyzed by immunoblot.Stimulation of RASF (n = 8-10) with CSE significantly enhanced the expression of HDAC1 , HDAC2  and HDAC3  at the mRNA level while the expression of HDAC 4-11 remained unchanged. On the protein level, expression of HDAC1 and HDAC3 were not altered, whereas the expression of HDAC2 protein was decreased in CSE stimulated RASF. No measurable changes in global acetylation of H3 were induced by CSE in RASF (n = 6).CSE specifically downregulates the expression of HDAC2 in RASF. Differential regulation of HDAC2 at the mRNA and protein level points to post-transcriptional degradation mechanisms induced by smoking. Even though global H3 acetylation was not changed by CSE, decreased HDAC2 levels might be associated with hyper-acetylation and thus increased expression of specific HDAC2 regulated genes.


Oligomannose sugars on gp120 are of significance to HIV vaccine design because of the description of the broadly neutralizing mAb 2G12 and, more recently, of the potent and broadly neutralizing ‘PGT’ series of mAbs, which bind with high affinity to clusters of oligomannose moieties on gp120. Attempts to elicit oligomannose-specific antibodies have focused mainly on immunizing with antigenic clusters of synthetic oligomannose or natural oligomannose. These strategies have had limited success, suggesting that alternative approaches are needed. Here, we present the surface lipo-oligosaccharide (LOS) of Rhizobium radiobacter Rv3 that antigenically mimics the 2G12 epitope, as one potential new avenue of exploration.The chemical structure of the Rv3 OS was determined by NMR and its antigenic similarity to the 2G12 epitope determined by ELISA and X-ray crystallography. Immunogenicity of the Rv3 OS and its ability to elicit antibodies of 2G12-like specificity was assessed by immunizing mice with heat-killed Rv3 bacteria.The detailed chemical analysis of the Rv3 OS revealed that its carbohydrate backbone consists of a unique tetramannose backbone that is analogous to the D1 arm of mammalian oligomannose. 2G12 bound with at least similar affinity to purified Rv3 OS as reported for oligomannose. The 2.4 Å-structure of the 2G12:Rv3 OS complex shows that 2G12 contacts all four mannosyl moieties in the Rv3 backbone that mimic the D1 arm, with the majority of contacts occurring with the terminal mannose disaccharide. Antibodies elicited by immunizing with heat-killed bacteria bound a synthetic tetramannose epitope and monomeric gp120.Although the elicited antibodies failed to exhibit the desired neutralizing activity, our data suggest that presentation of an antigenic analog of mammalian oligomannose in a bacterial context presents a novel avenue for pursuing immunogens to elicit oligomannose-specific HIV neutralizing antibodies. The development of Rv3-based bioconjugates using human carbohydrate vaccine carriers is ongoing.


In postnatal skin the transcription factor Sox2 is expressed in the dermal papilla (DP) of guard/awl/auchene hair follicles and by mechanosensory Merkel cells in the touch domes of guard hairs. To investigate the consequences of Sox2 ablation in skin we deleted Sox2 in DP cells via Blimp1Cre and in Merkel cells via K14Cre. Loss of Sox2 from the DP did not inhibit hair follicle morphogenesis or establishment of the dermis and hypodermis. However, Sox2 expression in the DP was necessary for postnatal maintenance of awl/auchene hair follicles. Deletion of Sox2 via K14Cre resulted in a decreased number of Merkel cells but had no effect on other epithelial compartments or on the dermis. The reduced number of Merkel cells did not affect the number or patterning of guard hairs, nerve density or the interaction of nerve cells with the touch domes. We conclude that Sox2 is a marker of two distinct lineages in the skin and regulates the number of differentiated cells in the case of the Merkel cell lineage and hair follicle type in the case of the DP. •Sox2 is a marker of two distinct lineages in the skin.•Sox2 is required for postnatal maintenance of awl/auchene hair follicles.•Loss of Sox2 results in a reduction in Merkel cells. The transcription factor Sox2 is involved in maintenance of the early, pluripotent stem cells of the eipiblast  and in rSox2 is expressed in the dermal papilla cells of guard/awl/auchene hair follicles  and in tWithin the epidermis Sox2 is expressed in a small population of mechanosensory cells known as Merkel cells . These nIn view of the key contributions of DP cells and Merkel cells to skin function and the observation that Sox2 is a marker of SKPs, we have investigated the consequences of deleting Sox2 in the DP and Merkel cell compartments.All experiments were approved by King's College London, Cambridge University and Cancer Research UK local ethics committees and performed under the terms of a UK government Home Office licence. Sox2fl/fl mice, in which flox sequences flank the Sox2 locus , were kiFlow cytometry was performed on dermal preparations as described previously  using a Gating criteria were as follows. Debris was gated out using forward and side scatter plots. Doublets and dead cells were also gated out and analysis was performed on live cells using GFP and APC channels. Gating for positively labelled cells was performed against negative control samples to less than 0.5% background.Preparation and immunostaining of conventional cryosections (5–30 μm thick) and whole mounts of tail epidermis, back skin and whisker pad were performed as described previously . Back skThe following primary antibodies were used at the dilutions indicated: Sox2 1:100 (R&D Systems), CD133 1:50 (eBioscience), Itga8 1:200 (R&D Systems), Dcc 1:100 (R&D Systems), K14 1:1000 (Covance), Blimp1 1:50 (eBioscience), Corin 1:100 (R&D Systems), K8 1:100 , NF-H 1:100 (Millipore), PGP9.5 1:100 (Dako), V-GLUT2 1:500 (Invitrogen), Rab3c 1:3000 (Abcam), synapsin II 1:200 (Abcam), Cav2.1 1:100 (Millipore), piccolo 1:2000 (SYSY) and GFP 1:500 (Invitrogen).Images of back skin whole mounts immunostained for PGP9.5 were acquired from the epidermal side using a confocal microscope. The Metamorph software tube formation application was used to quantitate the percentage of total skin area covered by nerves.Back skin was collected from E18.5 mice. Sample processing and image capture were carried out by Dr Jeremy Skepper, Anatomy Department, Multi-Imaging Centre, University of Cambridge. Tissues were fixed in 4% glutaraldehyde in 0.1 M HEPES buffer at pH 7.4 for 12 h at 4 °C and rinsed 5 times in 0.1 M HEPES buffer. Tissues were then treated with 1% osmium ferricyanide at room temperature for 2 h, rinsed 5 times in distilled water and treated with 2% uranyl acetate in 0.05 M maleate buffer at pH 5.5 for 2 h at room temperature. Tissues were rinsed in distilled water and dehydrated in an ascending series of ethanol solutions from 70% to 100%. This was followed by treatment with two changes of dry acetonitrile and infiltration with Quetol epoxy resin. Images were taken with a FEI Tecnai G2 operated at 120 Kv using an AMT XR60B digital camera running Deben software.E18.5 embryos were harvested from Blimp1Cre+/Sox2fl/wt×Sox2fl/fl intercrosses. Skin was dissected from the embryos and surgically sutured onto the backs of adult NSG mice. Sutures were removed after 1 week and grafts were harvested after 10 weeks.The number of Merkel cells per touch dome was quantitated in 7 mice per genotype. For each biological replicate 3 areas were analysed, corresponding to a total of 30 touch domes per mouse. Nerve density was analysed in 8 biological replicates, with 3 areas per mouse. Innervation of Merkel cells , touch domes per unit area and guard hairs per unit area were analysed in 4 biological replicates per genotype. In all cases data from the biological replicates were pooled. The D’Agostino and Pearson omnibus test was used to examine normality of data distribution. Since none of the data followed a Gaussian distribution, a non-parametric Mann-Whitney test was applied to examine the significance of differences between datasets.In order to delete Sox2 in the DP we required a promoter that would drive Cre expression in the dermis at the appropriate stages of development. One candidate was the promoter of the transcription factor B lymphocyte induced maturation protein (Blimp1). Blimp1 has previously been shown to be required for maintenance but not initial specification of DP progenitors .To examine Blimp1 expression in developing skin we performed immunostaining with Blimp1 antibodies and also examined expression of GFP under the control of the Blimp1 transcriptional regulatory regions . Blimp1 Analysis of Blimp1 mRNA expression levels in previously published microarray datasets  revealedBlimp1+ cells have previously been shown to give rise to mature DP, the dermal sheath, and the upper (papillary) dermis . We therFlow cytometry showed that 67–75% of CD133+ cells in E18.5 and P2 dermis were also GFP+ H and I, The lineage relationship between dermal papilla, dermal sheath, and cells in the papillary region has not been clearly defined. Our results suggest that these cell types share a common embryonic origin and may be distinct from other fibroblast populations in the dermis.Our data on Blimp1 expression indicated that expression of Cre under the control of Blimp1 transcriptional regulatory elements could be used to ablate gene expression in all DP at the earliest stages of dermal papilla morphogenesis. Blimp1Cre mice were therefore crossed with Sox2fl/fl mice. Blimp1Cre+/Sox2fl/fl mice were born at the expected Mendelian ratio but died shortly after birth, probably because of Sox2 deletion in the brain. Analysis of Sox2 expression in Blimp1Cre/Sox2fl/fl wholemounts and thick cryosections of skin confirmed efficient ablation of Sox2 in the DP of E16.5 follicles, in contrast to control littermates A–D.Expression of the DP markers CD133, Itga8, Corin and two specific markers of guard/awl/auchene DP, Dcc and Sox10 , was unaTo investigate whether Sox2 plays a role in the postnatal maintenance of hair follicles we grafted E18.5 Blimp1Cre/Sox2fl/fl and control littermate skin onto NSG immunodeficient mice. At the macroscopic level, 10 week old wild type and Sox2 null grafts were indistinguishable A and B. Merkel cells reside in the epidermal basal layer, directly linked to nerve cells containing NF-H positive neurofilaments A and B. Immunostaining for Sox2 and the Merkel cell marker Keratin 8 (K8)  revealed−, littermates had Sox2+ Merkel cells  in K14CreSox2fl/fl skin  did not reveal a significant difference (We next prepared horizontal whole mounts  of E18.5fference F–H. TherMerkel cells are excitatory cells that carry out synaptic processes . TransmiSox2 has long been recognised as a marker of embryonic pluripotency , and recCurrent approaches to modulating gene expression in the dermal papilla include driving Cre expression via dermal papilla specific or pan-fibroblast promoters . We founAblation of Sox2 in the DP did not affect hair follicle morphogenesis but did modulate hair follicle type. Since the decrease in awl/auchene follicles was correlated with an increase in zigzag hairs, rather than a decrease in overall follicle number, it is likely that Sox2 specifies follicle subtype, rather than being required for maintenance of awl/auchene follicles. We have previously shown that Sox2-expressing cells are required for formation of awl/auchene follicles in skin reconstitution assays  and our Our results highlight a distinction between those genes that are required for core DP functions and those that specify the identity of the dermal papillae of different types of follicle . ExampleWhen we ablated Sox2 in the epidermis via K14Cre, the number of Merkel cells per touch dome was reduced, confirming the recent studies of It has been suggested previously that Merkel cells play a role in attracting nerve growth into the skin during embryonic development . In suppSox2 was not required for expression of synaptic and voltage-gated signalling markers or formation of dense core synaptic vesicles in Merkel cells. Given that Sox2 is a well-established neural transcription factor, we speculate that other neural transcription factors are capable of compensating for the loss of Sox2 and maintaining expression of sufficient levels of synaptic proteins for synaptic vesicles to form. Indeed Sox2, which is repressed by the Polycomb complex , acts asIn conclusion, we have shown that Sox2 plays a role in Merkel cell differentiation and in specifying dermal papilla subtypes. Sox2 thus plays a role in lineage commitment of both epithelial and mesenchymal cells within the skin.


PLoS Genetics sheds light onto how Cdk9 function is interconnected with the monoubiquitination of histone H2B in the fission yeast Schizosaccharomyces pombeTranscriptional regulation in eukaryotes requires the transcriptional machinery to negotiate the complexities of DNA packaged into chromatin. Specific modifications of the core histone proteins serve to regulate transcription and ensure that genes are expressed at the right place and the right time CDK9 controls transcriptional elongation, transcription-coupled mRNA processing, and histone modification One post-translational histone modification emerging as a key player in numerous processes is histone H2B monoubiquitination (H2Bub1). In mammals this modification appears to serve as an important tumor suppressor The new study by Sansó et al. has unraveled additional details about the complex dance between the fission yeast ortholog of CDK9  and H2Bub1 S. pombe H2B ubiquitin ligase Brl2 both lead to septation defects, these can be rescued by blocking spCdk9. Furthermore, compound mutants of Spt5 and Set1 suggest that the phenotypic effects of H2Bub1 loss may be due to multifaceted downstream effects of H2Bub1 on both Spt5 (through spCdk9 recruitment) and H3K4me3 (through Set1). Further support for a homeostatic feedback mechanism is provided by studies of RNAPII occupancy in fission yeast with reduced spCdk9 activity. Consistent with opposing roles of H2Bub1 and spCdk9, the authors observed increased RNAPII occupancy in transcribed regions and decreased occupancy at gene 3′ ends. Importantly, the combined mutation of spCdk9 and H2Bub1 rescued the H2B K119R mutation and displayed RNAPII occupancy similar to mutant spCdk9 alone.Does this mean that spCdk9 and H2Bub1 play opposing roles in fission yeast? Phenotypic studies suggest that this may be at least partially true. For example, while H2B K119R mutation or deletion of the These results demonstrate the intricacy and complexity of the choreographed tango between CDK9 orthologs and chromatin, and illustrate significant differences between yeast and metazoans. This study shows that although H2Bub1 requires Cdk9 activity in fission yeast, Bur1 in budding yeast


The paternal 5mC is converted in fertilized oocytes to 5hmC allowing for epigenetic reprogramming of sperm DNA. Human tumors contain much lower levels of 5hmC compared to normal tissue. Proliferating cells in a tumor have drastically reduced levels of 5hmC, suggesting that this stable base modification is lost during DNA replication. 5hmC is most abundant in cells of the nervous system. We investigated patterns of 5mC and 5hmC during neurogenesis in the embryonic mouse brain. 5hmC levels increase during neuronal differentiation and this modification associates preferentially with gene bodies of activated neuronal function-related genes, in which gain of 5hmC is often accompanied by loss of H3K27me3. Importantly, gain of 5hmC is rarely associated with DNA demethylation suggesting that 5hmC is a rather stable epigenetic mark. Functional perturbation of the H3K27 methyltransferase Ezh2 or of Tet proteins leads to defects in neuronal differentiation suggesting that formation of 5hmC and loss of H3K27me3 cooperate to promote brain development. We propose that the function of 5hmC in promoter regions is to “repair” inappropriate de novo DNA methylation but its exact mechanistic role in gene bodies is still unknown.


IGF2 has multiple promoters to maintain its complex tissue- and developmental stage-specific imprinting and expression. IGF2 is also imprinted in marsupials, but little is known about its promoter region. In this study, three IGF2 transcripts were isolated from placental and liver samples of the tammar wallaby, Macropus eugenii. Each transcript contained a unique 5' untranslated region, orthologous to the non-coding exons derived from promoters P1–P3 in the human and mouse IGF2 locus. The expression of tammar IGF2 was predominantly from the P2 promoter, similar to humans. Expression of IGF2 was higher in pouch young than in the adult and imprinting was highly tissue and developmental-stage specific. Interestingly, while IGF2 was expressed throughout the placenta, imprinting seemed to be restricted to the vascular, trilaminar region. In addition, IGF2 was monoallelically expressed in the adult mammary gland while in the liver it switched from monoalleleic expression in the pouch young to biallelic in the adult. These data suggest a complex mode of IGF2 regulation in marsupials as seen in eutherian mammals. The conservation of the IGF2 promoters suggests they originated before the divergence of marsupials and eutherians, and have been selectively maintained for at least 160 million years.In mice and humans,  Human IGF2 has five promoters (huP1 and P0–P3) and transcription start sites (TSS) each adjoining distinct 5′ non-coding exons, while rodent IGF2 has only 4 (P0–P3) Monodelphis domestica, detected a single product from total neonatal RNA using 5′ RACE and identified only one 5′ non-coding exon IGF2 isotype from a single promoter.Insulin-like growth factor 2 IGF2 is imprinted in the fetal liver and is paternally expressed from P1, P2 and P3 (orthologous to mouse P1–P3 respectively) with dominant expression from the P2 promoter in the majority of tissues tested IGF2 has continued expression from P1 and P3 in addition to biallelic expression from huP1 (previously reported as P1) Human Igf2 is paternally expressed in the embryo, but biallelically expressed in the choroid plexus and leptomeniges, which are the only major sites of Igf2 gene expression in the adult rodents IGF2-P0 transcript is paternally expressed at high levels in fetal skeletal muscle, in the term placenta and at low levels in all adult tissues except the brain In the mouse P0-derived transcripts are only expressed in the labyrinthine layer of the placenta, whereas transcripts from P1–P3 are found throughout the developing embryo and placenta with dominant expression from the P3 promoter Igf2 domain has 3 differentially methylated regions (DMRs), DMR0 surrounding the area that codes for the placental specific promoter P0, DMR1 located 5′ of P1 and DMR2 located at the second coding exon IGF2 in mesoderm-derived tissues  without disrupting H19 imprinting, whilst maintaining IGF2 imprinting in the endoderm-derived tissues (liver and placenta) IGF2, therefore DMR2 is an activation site The mouse IGF2 control mechanisms amongst the eutherian mammals. Marsupials diverged from the eutherian mammals around 160 million years ago IGF2 is highly conserved and imprinted. However, very little is known about the regulation of this gene in marsupials. Most marsupials have a short pregnancy supported by a much less invasive placenta than typically seen in eutherian mammals, and all give birth to a highly altricial neonate IGF2 gene.The presence of a potential functional equivalent of mouse DMR2 IGF2 is imprinted in the whole bodies of opossum neonates IGF2 shows complete imprinting in the fetal liver, but has paternally biased expression in the placenta and brain and is biallelically expressed adult liver IGF2 in the neonatal opossum IGF2 gene that may account for the biased expression observed in the head and the placenta IGF2. This study investigates the evolution of the IGF2 regulatory regions in a marsupial, the tammar wallaby Macropus eugenii, and identified 3 promoters in the tammar IGF2 region.All experiments and wild animal collection were approved by the University of Melbourne Animal Experimentation Ethics Committee and the animal handling and husbandry procedures were in accordance with the National Health and Medical Research Council of Australia (2004) guidelines. Animals were collected under approval from the South Australian Department of Environment and Natural Resources.Tammar wallabies of Kangaroo Island, South Australia origin were held in our breeding colony in Melbourne. Pregnancy was initiated in females carrying an embryo in diapause by the removal of their pouch young (RPY) 2O  in a dilution of 1/24. Total RNA was DNase treated  to remove contaminating DNA and tested by performing a housekeeping gene PCR. Total RNA quality was assessed by running samples on a gel  and quantified with a nano-spectrometer . cDNA was synthesised using Roche Transcriptor High Fidelity cDNA Synthesis Kit (Roche). Typically 4000 ng of total RNA was used in each cDNA synthesis reaction, with 1.1 µl of Oligo (dT)18 (50 µM), trilaminar yolk sac samples for qPCR analysis were amplified using random hexamer primers. All cDNA samples were diluted to 40 uL for a final concentration of 100 ug/ul. cDNA integrity was immediately assessed with GAPDH PCR.Genomic DNA (gDNA) was extracted from approximately 20 mg of snap frozen tissue using a Wizard Genomic DNA Purification Kit (Promega). Total RNA was extracted from liver, placenta and brain tissues using Tri-Reagent (Ambion) as described by the manufacturer, and from mammary glands using RNeasy Lipid Tissue Mini Kit (QIAGEN), with a final elution of RNA in 50–80 µl of RNAsecure HAll primers were designed using Primer3 (v. 0.4.0) IGF2 transcription start sites (TSS) we performed 5′RACE, using both the 5′ RACE System for Rapid amplification of cDNA ends, Version 2.0, (Invitrogen) and SMARTer RACE cDNA Amplification Kit (Clontech). Liver and placental RNA was used. Gene specific primers were designed in the first translated exon (To identify tammar ted exon  and werePCR products were subcloned in to the pGEM-T Easy vector and transformed in to JM109 competent cells (Promega). Plasmids were purified using the Wizard Plus SV Minipreps DNA Purification System (Promega) and sequences were obtained according to standard methods using ABI3130xl capillary genetic analysers, with BDV3.1 terminators.IGF2 transcripts in the brain, liver, mammary gland and placenta. Each sample was extracted and prepared as described above. In the brain expression profile, 1 female and 3 male samples were used in the day 10 pouch young (PY) group and all 3 of the day 50 PY group were male. In the liver, 1 female and 3 male samples were used in the day 10 PY group and 2 female and 2 male samples were used in the day 75 PY group. All of the adult samples were female. As there were no obvious outliers in the data, we decided it was acceptable to use both sexes.Quantitative real-time polymerase chain reaction (qPCR) was used to quantify the relative expression from each of the IGF2 qPCR primers were designed so the reverse primer encompassed the intron exon boundary between exon 2 and 3 and was used with the transcript specific primers  using the following conditions: 95°C for 10 min, followed by 45 cycles at 95°C for 15 sec and 61°C for 30 min and 72°C for 30 sec. A liver sample triplicate and a negative template triplicate were included on each plate as a calibrator and negative control respectively for each primer combination.PCRs were carried out in triplicates in 20-µl volumes consisting of FastStart Universal SYBR Green Master (Rox) (Roche), forward and reverse In addition to using the dissociation curve, PCR products from the first plate were run on a gel to confirm amplification of a single band and that the NTC wells were blank. The data was analysed in Microsoft Excel and R statistical package IGF2 allelic expression sample specific genotypes were identified using PCR and direct sequencing of gDNA. Two single nucleotide polymorphisms IGF2 primers for gDNA and transcript specific primers for cDNA  was used to optimise reaction efficiency and only 30–35 cycles of PCR were used to prevent saturation. For expression analysis GoTaq Green Master Mix (Promega) was used. PCR cycles consisted of 94 or 96°C for 2 min, followed by a maximum of 35 cycles of 30 sec at 94 or 96°C, 30 sec-1 min at 58–63°C, and 30 sec-1 min at 72°C, and a final extension at 72°C for 5 min.IGF2 snps. Of these samples 2 adult livers, 3 mammary glands, 4 placentas and 2 pouch young brains and livers were analysed for IGF2 imprinting. PCR products from cDNA and gDNA were resolved by gel electrophoreses and bands extracted . The purified product was then sequenced. Sequences were assessed by eye using the FinchTV (v.1.3.1) DNA sequence chromatogram trace viewer software. The relative peak height for each allele indicates biallelic  or imprinted  expression.Approximately 9 pouch young, 13 adults and 17 placental samples were screened for Using MethPrimer IGF2 gene is highly complex in eutherian mammals. This study confirmed that marsupials also have complex regulation of IGF2, and identified three evolutionarily conserved promoters, each with three distinct non-coding exons that showed tissue specific imprinting and expression in the tammar wallaby.The regulation of the imprinted IGF2, 5′ RACE was performed on mRNA derived from two tissues, liver and placenta. Three distinct bands were amplified from the liver and placental samples, representing three distinct TSS (IGF2 may not have a liver specific promoter orthologous to human HuP1 or a placenta specific promoter orthologous to mouse P0.To determine how many different transcription start sites (TSS) there are in the tammar inct TSS . As addiinct TSS . Each bainct TSS . Three a+1 nucleotide in the Inr motif (where A+1 is the transcription start site) . A TATA-like sequence at -27 relative to the P3 A+1 nucleotide in the Inr motif, similar to the TATA-box observed at the mouse P3 At the mouse and rat P1 there are no TATA, CAT or GC boxes, but there are direct and inverted repeats of various length in the region upstream of the P1 non-coding exon IGF2, pairwise alignments using a 100 bp sliding window were performed in VISTA Using 20 kb genomic sequences of IGF2The opossum non-coding exon was highly conserved in the tammar genome and aligned to the tammar with 92% sequence identity approximately 300 bp upstream of the first tammar exon 1B TSS . This suIGF2 transcription in the liver peaks shortly after birth IGF2 in the tammar is similarly developmental stage-specific; expression is higher in the pouch young, during the time of rapid growth and nutrient transfer from the mother, compared to both fetal and adult stages IGF2 is synthesized in most tissues and its production declines rapidly in rodents soon after birth IGF2 mRNA peaks approximately 10 days after birth IGF2 expression peaks in the male pouch young between 70 and 100 days after birth and in females around 150 days IGF2 in liver was predominantly monoallelic from each of the 3 promoters in pouch young aged 12 and 72 days, and biallelic in both adults tested  identified by 5′RACE are indicated by the arrows. ggaggag repeats are in boxes, CTCCCAAG repeats are highlighted in yellow and TGTCCC repeats are highlighted in green. TATA-boxs (TATAWAAR) are emboldened and red, CCATT and GC-box/Sp1-sites are underlined, Inr elements (YYANWYY) are emboldened and blue and DPE (RGWYV) sequences are emboldened and green. Exon sequences are in capitals. P1 is characterised by multiple repeat sequences. P2 has multiple TSSs and a high number of CCATT and GC-box/Sp1-sites. A TATA-Inr core promoter was identified around the fourth TSS. P3 is an Inr-DPE core promoter. Degenerate nucleotides represented using IUPAC codes.(PDF)Click here for additional data file.Figure S2Alignment of tammar exon 1B region with opossum exon 1 region. Opossum non-coding exon = 1–420 Tammar exon 1B = 725–1104. Alignments were performed using ClustalW and were highlighted using BOXshade 3.31.(PDF)Click here for additional data file.Table S1Primers used in this study.(PDF)Click here for additional data file.


Msx1 and Msx2, as well as that of Dlx5 and Dlx6, lead to limb patterning defects with anomalies in digit number and shape. Msx1;Msx2 double mutants are characterized by the loss of derivatives of the anterior limb mesoderm which is not observed in either of the simple mutants. Dlx5;Dlx6 double mutants exhibit hindlimb ectrodactyly. While the morphogenetic action of Msx genes seems to involve the BMP molecules, the mode of action of Dlx genes still remains elusive. Here, examining the limb phenotypes of combined Dlx and Msx mutants we reveal a new Dlx-Msx regulatory loop directly involving BMPs. In Msx1;Dlx5;Dlx6 triple mutant mice (TKO), beside the expected ectrodactyly, we also observe the hallmark morphological anomalies of Msx1;Msx2 double mutants suggesting an epistatic role of Dlx5 and Dlx6 over Msx2. In Msx2;Dlx5;Dlx6 TKO mice we only observe an aggravation of the ectrodactyly defect without changes in the number of the individual components of the limb. Using a combination of qPCR, ChIP and bioinformatic analyses, we identify two Dlx/Msx regulatory pathways: 1) in the anterior limb mesoderm a non-cell autonomous Msx-Dlx regulatory loop involves BMP molecules through the AER and 2) in AER cells and, at later stages, in the limb mesoderm the regulation of Msx2 by Dlx5 and Dlx6 occurs also cell autonomously. These data bring new elements to decipher the complex AER-mesoderm dialogue that takes place during limb development and provide clues to understanding the etiology of congenital limb malformations.The Dlx and Msx homeodomain transcription factors play important roles in the control of limb development. The combined disruption of  The developing vertebrate limb is widely adopted as a model to study cell-cell signaling, pattern formation and morphogenesis, and has provided a wealth of knowledge of the function and regulation of specific transcription factors and signaling molecules Dlx and Msx homeobox transcription factors, expressed in the AER and the mesoderm of the limb buds, play an important morphogenetic role, although their functions and regulations are yet to be defined at the molecular level. Dlx5;Dlx6 double knock-out (DKO) mice are characterized by loss of the central digit(s) and/or fusion with the lateral ones DLX5;DLX6 region, and for which point mutations in the DNA-binding domain of DLX5 have recently been found Msx1 and Msx2 genes results in moderate-penetrance polydactyly of the forelimbs (FL) and oligodactyly of the hindlimbs (HL) (loss of the anterior digits and part of the tibia in the zeugopod) Proximo-distal limb development and digit extension is directly controlled by the signaling activity of the AER via expression of morphogens of the FGF family Dlx5, Dlx6, Msx1 or Msx2 shows evident limb defects in vitro the Dlx and Msx proteins compete for the same DNA binding sites, form heterodimers via their homeodomain and reciprocally inhibit their transcriptional activities Dlx genes are implicated in the differentiation of specific cell lineages, such as forebrain interneurons Dlx and Msx genes have been shown to cooperate only in specific cases Msx2 transcription in the AER, via homeodomain binding elements present in the gene’s promoter Studies on the cellular and molecular functions of Dlx and Msx proteins during limb development have met with difficulties owing to several reasons. First, none of the single knockout for Dlx5;Dlx6 and Msx1 or Msx2 in limb development, by generating double and triple Dlx;Msx compound mutant mice and analyzing their limb phenotypes. The limb phenotype of Msx1;Dlx5;Dlx6 triple knock-out (TKO) mice shows features of the Msx1;Msx2 DKO phenotype Dlx5;Dlx6 control the expression of Msx2, but not of Msx1. In contrast, the limb phenotype of Msx2;Dlx5;Dlx6 TKO mice is consistent with a severe aggravation of the ectrodactyly defect. We also re-examine the spatio-temporal expression of Dlx and Msx genes in FLs and HLs and observe that in the AER these genes are co-expressed whereas in the anterior mesenchyme Msx expression precedes that of Dlx, ruling out a direct regulation of Msx by Dlx in this territory. Combining these findings with qPCR expression analyses on the limb buds of different compound embryos, and with ChIP data, we propose that two modes of regulation coexist during limb development: 1) a direct transcriptional regulation of Msx2 by Dlx proteins in the AER and, later on, in the limb mesoderm, and 2) a Bmp2- and Bmp4-mediated non-cell autonomous regulatory loop between the AER and the anterior limb mesoderm.We investigate possible interactions between lacZ/+Dlx5 (hereafter named Dlx5+/−); neo/+Dlx5;Dlx6 lacZ/+; Msx1 and lacZ/+Msx2  have been previously reported +/−Msx1 or the +/−Msx2 single heterozygous parents with the +/−Dlx5;Dlx6 double heterozygous ones, and then crossing the triple heterozygotes. Following mating, the day of the vaginal plug was considered as embryonic age 0.5 (E0.5).All animal procedures were reviewed and approved by the Ethical Committee of the University of Torino and University of Genova, the Italian Ministry of Health and the French Ministère de l’Enseignement supérieur et de la Recherche. No surgery or other manipulation on adult animals was used in this study. All efforts were made to minimize suffering. Generation and genotyping of the lacZ expression analysis E10.5 embryos were fixed for 15–30 min in 2% paraformaldehyde in PBS, while E14.5 embryos were fixed for 15–30 min in 4% PFA. X-gal staining was performed as described Cartilage staining (with Alcian Blue) of E14.5 embryos as well as bone and cartilage staining (with Alcian Blue and Alizarine Red) of E18.5 embryos were carried out as previously described in situ hybridization (WMISH), embryos at the desired age were dissected in cold RNAse-free PBS, fixed in cold 4% PFA for 12–16 hrs, rinsed with PBS, permeabilized by treatment with proteinase-K, prehybridized and hybridized as previously published Dlx5 probe comprised 780 bp and was linearized with EcoRI and transcribed with T7 RNA polymerase Dlx6 probe is a 350 bp fragment spanning exons 3–4 Msx1 probe was a 550 bp 3′ spanning the homeodomain, containing exon 1; the Msx2 probe corresponded to 378 bp in the first exon of Msx2 cDNA, the Fgf8 probe corresponded to most of the mouse coding sequence, the Bmp4 probe (a kind gift from B. Hogan) contains the 3′ UTR and most of the coding sequence from a mouse cDNA Gremlin corresponded to the entire murine coding sequence (a kind gift from R. Zeller). After hybridization, the signal was revealed with the NBT-BCIP chromogenic reaction.For RNA:RNA whole-mount +/−;Dlx5−/−;Dlx6Msx1−/−) biological triplicates could be analyzed. Primer sequences were designed with the Primer Express online tool. RNA quality, primer efficiency and correct size were tested by RT-PCR and agarose gel electrophoresis. Standard curve were performed using WT cDNA with four calibration points: 1∶10; 1∶40; 1∶160; 1∶640. Specificity and absence of primer dimers was controlled by denaturation curves. Rps9 mRNA abundance was used for normalization . Pools of two half limbs from the same embryos were used to extract total RNA, using the Tissue Lyser II reagent followed by elution through RNA micro-kit plus (Qiagen). cDNA synthesis was done using standard conditions, 3 ng of each cDNA sample were used to carry out qPCRs on a CFX96 equipment (Biorad) using the SybrSafe system (Invitrogen). Samples were analyzed in technical triplicates, and for each genotypes (except for the We used the Position-Weight matrix (PWM) provided by JASPAR under accession PH0024.1. The score of a site was computed with standard log-likelihood ratios, using as null model the nucleotide frequencies computed over the whole intergenic fraction of the mouse genome. We considered for further analysis putative sites scoring 50% of the maximum possible score or better. We selected among the sites identified above the ones that are conserved in at least two of 8 vertebrate species (genome sequence version in parenthesis): mouse (mm9), human (hg19), cow (bosTau4), opossum (monDom5), platypus (ornAna1), chicken , frog (xenTro2), zebrafish (danRer6) and lamprey (petMar1). A site is defined as conserved with species S if it lies in a region of the mouse genome which is aligned with a region of the S genome and the aligned sequence in/S/is a site according to the same definition used for mouse sites. All genomic sequences and pre-computed “Net” alignments were obtained from UCSC.A ranked list of putative Dlx target genes was obtained from the sites determined above by associating each site conserved in at least one species to its closest Refseq mRNA, and then selecting the sites located either within 10 kb upstream of the TSS, or within the non-coding portion of the first exon, or in the first intron. We then associated to each putative target a score equal to the sum of the conservation scores (number of species) of its associated sites.DLX5-myc expression vectors  containing the full-length human DLX5 cDNA with an in-frame insertion of the myc-TAG at the C-terminus, was used as described DLX5-myc expression vector indicated above, by site directed mutagenesis and sequence verified .A DLX5 mRNA endogenously, but have been shown to respond to Dlx expression with activation of the p63 promoter DLX5-myc expression vectors were used for transfections, which yielded an efficiency of 35% . Chromatin was crosslinked, sonicated, immunoprecipitated with either the anti-myc TAG  or the anti-acetyl-Histone H4  antibodies and de-crosslinked according to instructions . Fragments of the human BMP2 and BMP4 loci spanning the identified conserved regions were PCR-amplified and analysed by gel electrophoresis .Previous evidences indicate that Dlx5 binds to homeodomain-responsive elements in a proximal region of the Dlx5;Dlx6 intergenic region Msx2 promoter Next, we screened conserved regions of the vertebrate genome for the presence of consensus Dlx DNA-binding sites, as defined by the Dlx5 PWM Msx1 and Msx2 genes were found in the top 10% of the list of putative Dlx targets, strengthening the possibility that Dlx proteins might directly regulate Msx expression.We then generated a ranked list of putative Dlx targets, based on the position of predicted conserved Dlx sites in the genome, as indicated in the Methods sections. The ranked list contains 3,051 Refseq mRNAs associated to 2,412 unique Entrez gene IDs (available upon request). The Dlx5, Dlx6, Msx1 and Msx2 in the FL and HL of normal embryos, at E10.5 and E11.5, by X-gal staining of heterozygous embryos carrying an allele with inserted lacZ reporter , and by WMISH (Dlx6)  of both the FL and the HL  and in the anterior HL mesoderm .We examined expression of H (Dlx6) . WMISH fd the HL , whereas compare  with O–Pis stage . In the r margin . At this the AER -AB. HistMsx expression precedes that of Dlx in the HL anterior mesoderm, consequently in this location Dlx genes are unlikely to regulate Msx gene transcription cell-autonomously and Dlx and Msx proteins are unlikely to interact in the anterior limb mesoderm, at early stages.In summary, Dlx and the Msx genes during limb development, we used animals DKO for Dlx5;Dlx6 and analyzed the expression of the Msx-lacZ reporter in this genetic background. To do this, we compared the expression patterns of the lacZ reporter between +/−Msx1 and +/−Msx1;−/−;Dlx6−/−Dlx5 mice  mutant embryos, as compared to WT. In the −/−Msx2 mutant the expression level of Dlx5, Dlx6 and Msx1 was unchanged, whereas in the −/−Msx1 mutant the expression levels of Dlx5, Dlx6 and Msx2 were reduced by 65%, 40% and 15%, respectively. Moreover, in the posterior half of the HLs from the −/−Msx1 mutant we observed a reduction of 40% in the abundance of Dlx5 and Msx2 mRNA, whereas Dlx6 did not change  to quantify the reduction of  changed . We thent change .−/−;Dlx5−/−;Dlx6−/−Msx1 or −/−;Dlx5−/−;Dlx6−/−.Msx2 TKO newborns were obtained at a frequency lower than expected, and all died shortly after birth, due to severe craniofacial malformations and consequent breathing impairment. Quadruple knock-out embryos were never obtained in spite of several attempts.To reveal possible functional interactions between the Dlx and the Msx gene products during limb development, we generated TKO mice with genotype Msx2;Dlx5;Dlx6 TKO animals, the HLs were severely affected  and E18.5/birth . In affected  whereas affected . The lim2 allele .Msx1;Dlx5;Dlx6 TKO mice the FL were usually normal, although in one case (1/6) anterior polydactyly was observed (data not shown). Of note, polydactyly has been reported for the FLs in Msx1;Msx2 DKO mice with a moderate penetrance Msx1;Msx2 DKO usually exhibit loss of 1–2 anterior digits and of the tibia Dlx5;Dlx6Msx1;Msx2In Msx1;Msx2 DKO phenotype in the Msx1;Dlx5;Dlx6 TKO animals, which retain two functional Msx2 alleles, argues in favor of a severe reduction of Msx2 expression in the TKO limbs, as compared to the Dlx5;Dlx6 DKO.The appearance of features of the Msx1 allele in the context of Dlx5;Dlx6 DKO background  results in a 50% reduction of Msx2 mRNA in both the anterior and the posterior half of the embryonic HLs, as compared to WT. Moreover, expression of Msx2 in the anterior and posterior halves of Msx1 homozygus mutant HLs was reduced by 30% compared to Msx1 heterozygous HLs (data not shown). This indicates that reduced Msx2 expression may result from the combination of (1) loss of Dlx5 and Dlx6  the loss of Msx1, with the consequent decrease of Msx2 expression  but not significantly in the anterior mesoderm .We carried out WMISH for mesoderm . Bmp4 ex embryos , as expes region . We also embryos . Thus, wBmp4 expression is unchanged in the anterior mesoderm of the limbs; however this technique is poorly quantitative and cannot reveal minor changes in gene expression level. To further investigate the mechanism that might induce and/or sustain Msx2 expression in the anterior half of the HLs, we determined the relative abundance of Bmp2 and Bmp4 mRNAs by qRT-PCR in the anterior and posterior halves of the embryonic HLs (E11) with different genotypes. Expression of Bmp2 is mainly decreased in the anterior (about 50%), and minimally in the posterior half (20%) of the HLs from Dlx5;Dlx6 DKO embryos  than the posterior half (50%) (data not shown). Strikingly we observed a strong synergy between Msx1 and Dlx5;Dlx6 in controlling BMP expression. When combined with the Dlx5;Dlx6 DKO mutation, the loss of a single Msx1 allele was sufficient to reduce Bmp2 and Bmp4 expression to levels lower than 10% of that of control samples, both in the anterior and in the posterior halves of the HLs. Considering that Msx2 is a known target of Bmp2 and Bmp4 in several tissues Msx2 expression is reduced in the Msx1;Dlx5;Dlx6 TKO, and how this mutant shows a limb phenotype with similarities to the Msx1;Msx2 DKO.The WMISH results reported above suggest that  embryos . Bmp2 exr halves . Of noteFgf8 and Shh mRNAs in HL samples from the relevant genotypes (Fgf8 mRNA was slightly reduced in the absence of Dlx5 and Dlx6 (DKO samples), and was further drastically reduced when one Msx1 allele was eliminated in the absence of Dlx5;Dlx6. Similarly, the abundance Shh mRNA  was slightly reduced in the absence of Dlx5 and Dlx6, and was further reduced when one Msx1 allele was simultaneously eliminated. While reduced Fgf8 expression is not surprising Shh expression is novel and may contribute to the altered digit number and morphogenesis seen in the Msx1,Dlx5;Dlx6 TKO embryos.Finally, we determined the abundance of enotypes . The AbuBmp2 and Bmp4 loci by Dlx5, we first exploited the genome-wide predictions of conserved Dlx sites, described above. In the proximity of the Bmp2 locus we identified three Dlx consensus binding sequences, named B2-RE1, located 30 kb upstream of the transcription start site (TSS), B2-RE2 and B2-RE3, located respectively 3.5 kb and 18 kb downstream of the end of the Bmp2 transcript. All these Dlx binding elements are conserved, although not identical, in at least two vertebrate species, and fall within stretches of conserved genomic sequence , as well as an irrelevant sequence (IL10) did not show any enrichment (not shown). These results reinforce the notion that DLX proteins physically interact with four (out of five) conserved elements close to the BMP2 and the BMP4 loci, and might exert a direct transcription regulatory activity, relevant for normal limb development.To demonstrate that the Dlx5 protein physically binds to these putative responsive elements, we transfected a human Dlx5, Dlx6, Msx1 and Msx2 genes encode homeodomain transcription factors involved in limb development. The targeted disruption of each of these genes, individually, does not lead to limb defects, however double Msx1/Msx2Dlx5/Dlx6in vitroMsx2;Dlx5;Dlx6 TKO mice show an aggravation of the ectrodactyly phenotype, as compared to Dlx5;Dlx6 DKO, they do not present any obvious additional defect. Furthermore, inactivation of either Dlx5;Dlx6 or Msx2 does not modify the level of Msx1 expression, suggesting that the Msx2;Dlx5;Dlx6 TKO phenotype represents the exclusive contribution of the absence of Msx2 in the Dlx5;Dlx6 mutant context. This leads us to conclude that either Msx2 has a rather minor function in limb development, or that its function is largely compensated by Msx1, in agreement with the limited defects observed in +/−;Msx2−/−Msx1 compound mutant animals Msx1;Dlx5;Dlx6 TKO mice display a limb defect which can be interpreted as the sum of the limb anomalies found in the Msx1;Msx2 DKO and in the Dlx5;Dlx6 DKO. These results strongly suggest that the expression of Msx2 is suppressed in this genetic context, and that therefore Dlx5;Dlx6 are genetically upstream of Msx2.Although Dlx5;Dlx6 DKO limbs Msx2 expression is diminished in the central sector of the AER. As starting at E10.5 Dlx and Msx genes are co-expressed in the AER, it is possible to hypothesize that in this territory Dlx proteins bind directly on the Msx2 promoter, as previously reported Msx1 and Msx2 precedes that of Dlx5 and Dlx6. This precludes the possibility of a direct regulation of Msx genes by Dlx proteins. Nevertheless, our qRT-PCR analyses show that Msx2 expression is reduced in the anterior limb mesoderm of Dlx5;Dlx6 DKO limbs, implying the existence of a non cell-autonomous mode of regulation between the AER and the anterior limb mesoderm. It is possible, therefore, that a diffusible protein, expressed by AER cells in a Dlx-dependent fashion, is required to initiate and/or sustain Msx2 expression in the anterior limb mesoderm.We show that in Msx genes are well documented downstream effectors of BMP signaling in several developing structures Gremlin, results in upregulation of both Msx1 and Msx2 expression Msx2 expression Msx1, in the dorsal ectoderm, induces the formation of ectopic AERs Msx1 and Msx2 leads to a phenotype that mimics, in some aspects, the loss of BMP signaling Msx genes are also upstream of Bmp4 in several developmental systems. During tooth germ development, mesodermal Msx1 is needed for efficient Bmp4 expression Msx1−/− mice, can be rescued by a Bmp4-expressing transgene Msx genes are required in the mesoderm for maintenance of Bmp4 expression Msx genes, depending on the context and the developing structure. The possibility that Bmp2 and Bmp4 participate in a Dlx-Msx signaling loop between the limb bud AER and mesoderm is clearly in line with previously identified roles of these molecules.Noticeably, Dlx5;Dlx6 and mesodermal Msx2. Indeed, we find that Bmp2 and Bmp4 expression is significantly reduced in Dlx5;Dlx6 DKO hindlimbs, at E11 when no evident defect is yet visible. Reduction for Bmp2 and Bmp4 in the anterior part of the HL is too high  to be accounted for by the ectoderm alone, and rather indicates a downregulation of these Bmps in the mesoderm, too. This could mean that Bmps produced in the ectoderm activate Bmps also in the mesoderm, either directly or via Msx1. Indeed, further inactivation of one Msx1 allele in the context of the loss of Dlx5;Dlx6 nearly abolishes Bmp2 and Bmp4 expression, necessarily implying both the ectoderm and the mesoderm  accompanied, however, by less severe zeugopod defect. Thus, it appears that both AER- and mesenchyme-derived Bmps are involved in the Msx;Dlx defects. The possibility that misexpression of AER-Bmps alone directly cause the TKO defects is unlikely. Rather, in light of the well known self-regulatory system of signalling loops comprising FGFs, SHH and BMPs Fgf8 and Shh upon loss of Msx1, Dlx5 and Dlx6 genes, most likely the overall nature of the limb defects in TKO mutant embryos is a quantitative misregulation of the “slow module” of this loop. Dlx and Msx genes can be regarded as new players in this complex regulation. Since Shh participates in the “slow module”, a more critical role for it could be envisioned, as suggested by resemblance of the phenotype of Msx1;Dlx5;Dlx6 TKO limbs with that exhibited by Shh KO embryos The loss of mesenchymal In conclusion, we propose a model that involves a complex epithelial-mesodermal dialogue between Dlx and Msx , entailiDlx5;Dlx6 DKO vs. +/−;Dlx5−/−;Dlx6−/−Msx2 vs. Msx2;Dlx5;Dlx6 TKO  the genetic inactivation of one allele, and b) the lack of Dlx5;Dlx6 genes.The importance of allelic dosage, hence quantitative gene expression, is increasingly being recognized, even when phenotypes are not evident see . As a fuMsx1;Dlx5;Dlx6 TKO animals, Msx2 expression is further reduced. A residual level is nonetheless observed; however, this is not sufficient to compensate for the loss of Msx1. In conclusion, allelic dosage and quantitative gene expression are crucial factors to be considered in the interpretation of a series of phenotypes, especially when related genes are involved.Likewise, in DLX5 and DLX6 genes cause the SHFM-type-1 congenital malformation when lost or mutated, while the MSX1 and MSX2 genes cause cleft palate and tooth agenesis. By crossbreeding mutant mouse strains, we show that the Dlx5;Dlx6 and Msx1;Msx2 genes cooperate for normal limb development and morphogenesis. At least two modes of regulation have emerged, one in which Dlx5;Dlx6 control expression of Msx2 cell-autonomously, the other in which the AER and the anterior mesenchyme interact non cell-autonomously, entailing Bmps as signaling molecules. We further show that the BMP2 and BMP4 loci comprise Dlx5-binding elements, occupied by Dlx5. Thus, the highly related homeodomain genes Dlx and Msx are two key players of a novel set of molecular and histological interactions during limb development.In human, the Figure S1Top. Location of predicted conserved Dlx binding sites in the Dlx5-Dlx6 intergenic genomic region. Sites are indicated with colour vertical bars (asterisk) and annotated with the species conservation. The chromosomal position and coordinates are also reported. The mammalian genomic conservation is reported on the bottom. The known i56i element is correctly predicted by the PWM bioinformatic approach we have adopted. Bottom. Same as above, relative to the Msx2 proximal promoter. Two known conserved Dlx binding sites are correctly predicted.(PDF)Click here for additional data file.Figure S2Quantification of the Msx2 mRNAs by qRT-PCR in the anterior and posterior halves of HLs from Msx1+/−;Dlx5−/−;Dlx6−/− embryos, relative to the corresponding WT samples (set = 1).(PDF)Click here for additional data file.Figure S3Quantification of the Fgf8 and Shh mRNAs by qRT-PCR in the HLs from Msx1−/− (top left), Dlx5−/−;Dlx6−/− (top right), Msx1+/−;Dlx5+/−;Dlx6+/− (bottom left) and Msx1+/−;Dlx5−/−;Dlx6−/− (bottom right) embryos, relative to the corresponding WT samples (set = 1).(PDF)Click here for additional data file.Table S1Sequences of the oligonucleotides used for real-time qPCR on mouse embryonic tissues.(PDF)Click here for additional data file.Table S2Sequences of the oligonucleotides used for ChIP analysis on the predicted Dlx elements near the human BMP2 and BMP4 loci.(PDF)Click here for additional data file.Table S3The Dlx5 Position-Weight matrix and results of the prediction of Dlx5 binding sites based on genomic conservation.(PDF)Click here for additional data file.Table S4Sequences of the mouse and human conserved genomic regions containing predicted Dlx5 binding sites near the BMP2 locus.(PDF)Click here for additional data file.Table S5Sequences of the mouse and human conserved genomic regions containing predicted Dlx5 binding sites near the BMP4 locus.(PDF)Click here for additional data file.