Embryonic stem cells and induced pluripotent stem cells represent potentially important therapeutic agents in regenerative medicine. Complex interlinked transcriptional and signaling networks control the fate of these cells towards maintenance of pluripotency or differentiation. In this study we have focused on how mouse embryonic stem cells begin to differentiate and lose pluripotency and, in particular, the role that the ERK MAP kinase and GSK3 signaling pathways play in this process. Through a genome-wide siRNA screen we have identified more than 400 genes involved in loss of pluripotency and promoting the onset of differentiation. These genes were functionally associated with the ERK and/or GSK3 pathways, providing an important resource for studying the roles of these pathways in controlling escape from the pluripotent ground state. More detailed analysis identified MAP kinase phosphatases as a focal point of regulation and demonstrated an important role for these enzymes in controlling ERK activation kinetics and subsequently determining early embryonic stem cell fate decisions. Embryonic stem cells and induced pluripotent stem cells represent potentially important therapeutic agents in regenerative medicine. Manipulation of these cell types could allow us to replace dead or diseased cells in our bodies and hence potentially provide a solution to a wide range of medical problems. However, before we can perform such manipulations, we need to understand how the stem cells are wired so that we are able to re-wire them in a logical way to produce the desired cell types. Here we have attempted to understand this wiring by using an RNAi screen in which each individual component of the cell is systematically removed and the consequences on cellular fate determined. We have identified hundreds of genes that are required for efficient loss of stem cell characteristics and hence conversion into other cell types. By studying a subset of these genes, we have been able to show that many converge on two related negative regulators of one of the key pathways that act to promote loss of stem cell identity. These negative regulators, Dusps, normally limit the ability of stem cells to change their function and hence be converted to different cell types. Embryonic stem cells and induced pluripotent stem cells (iPS cells) are currently generating intense interest due to their potential therapeutic roles in regenerative medicine  locus. This reporter gene is regulated in an analogous manner to endogenous rex1Rex1GFPd2 ES cells were maintained in media containing MEK and GSK inhibitors (2i) to maintain their ES cell status and treated with siRNAs pools targeting ∼17,000 individual genes. After 24 hrs, cells were exchanged into fresh media lacking these inhibitors and the levels of GFP in each cell were assessed over time  (Rex1GFPd2) or ES cells containing an alternative reporter gene, where GFP is instead driven by the oct4  promoter, thereby providing an independent readout for the loss of pluripotency (tcf7l1(tcf3), jarid2, and dpy30jun and mbd3 which were both identified in two of these screens in addition to our own  . As our ipotency . These sipotency . These 3 our own . However our own . Thus cenras, dmbx1, jun, gli3, Having established the core network of genes working in concert with the GSK3 and ERK pathways we wanted to discover the relative contributions of these genes to the actions of the individual pathways. First we performed a counter screen in the presence of both pathway inhibitors (“+2i”) to eliminate siRNAs which promoted accumulation of GFP in the cells irrespective of the activity of the ERK and GSK3 pathways . This elWe then created a network out of the genes from the high confidence dataset identified in the “2i” screen based on previous knowledge of physical and functional interactions. Functionally related subnetworks could be identified, two of the most prominent of which are composed of genes encoding proteins associated with regulating chromatin modifications and sequence-specific DNA binding transcription factors . These gIn summary, by comparing single inhibitor assays, we have been able to subcategorise the genes required for embryonic stem cell differentiation and tentatively assign them to mediating or regulating the effects of either the ERK pathway, or the GSK3 pathway or both. Each pathway appears to require genes associated with overlapping and yet distinct biological processes.rex1 and nanog and the appearance of the early differentiation marker fgf5. The majority of the siRNAs tested showed increased rex1 and nanog expression relative to control siRNAs upon “2i” withdrawal (rex1 and nanog expression (2 = 0.81). Conversely, more than half of the siRNAs tested reduced the accumulation of fgf5 mRNA , and reduced levels of nestin after 5 days  or alternatively the genes might encode proteins which are directly phosphorylated by ERK. For example, it is known that transcriptional regulators such as Ets1, Jun and FoxO1 can all be phosphorylated by ERK in other situations Another key issue to address is whether the ERK and GSK3 pathways work together or in parallel manner, to target different substrates and ultimately control different gene expression programmes and biological functions in ES cells. The two pathway inhibitors have both distinct and overlapping affects on ES cells . The ES cells cultured under “+2i” conditions were adapted in serum/LIF culture conditions for at least 8 passages before the experiments were performed. Cells were stained for alkaline phosphatase expression using an alkaline phosphatase detection kit as described by the manufacturer's (Millipore).ES cells were generally maintained in NDiff N2B27 media  in the presence of the GSK3 inhibitor CHIR99021  and MEK inhibitor PD0325901  (“+2i” media) and were routinely passaged using Accutase  every other day. For differentiation, the media containing inhibitor was removed and replaced with NDiff N2B27 media. Where indicated, ES cells were maintained in serum/LIF conditions in media containing knockout DMEM (Invitrogen 10829-018), 15% heat inactivated FBS (Invitrogen 10082-147), 2 mM of Glutamax-1 supplement (Invitrogen 35050-038), 1% non-essential amino acids (Invitrogen 11140-035), 50 µM 2-mercaptoethanol (Invitrogen 31350-010) and 5×104/cm2 cells (ie 1.28×104 cells/well of 96 well plate) were plated out into a mixture of 0.3 µl of RNAi Max (Invitrogen) and 100 nM siRNA in 100 µl of “+2i” media for 24 hrs. All validation experiments used ON-TARGETplus siRNA SMART pools from Dharmacon.For RNAi, 4×10gapdh, hmbs and tbp. The primer-pairs used for RT-PCR experiments are listed in Real time RT-qPCR was carried out as described previously Western blotting was carried out with the primary antibodies; Erk2 , phospho-ERK , Dusp1 , Dusp6  and Pou5f1 . All experiments were carried out in 96-well plates. The lysates were directly harvested in the 2×SDS sample buffer followed by sonication . The proteins were detected using infrared dye-conjugated secondary antibodies , and the signal was collected with a LI-COR Odyssey Infrared Imager and quantified using Odyssey software .The Ras activities were examined using Ras activation ELISA assay kit (Millipore) as described in the manufacturers' instructions. The total lysates used in the ELISA assay was normalised with the quantity of the proteins assayed by the BCA protein assay kit (ThermoScientific).Flow cytometric analysis was carried out using a LSRII flow cytometer and samples were loaded using HTS loader (BD Biosciences). For sampling, media was removed from each well. Single cell suspensions were generated by treating cells with accutase at 37°C for 7 mins followed by resuspendion in 0.03% BSA/PBS. Dead cells were stained by Sytox Red dead cell stain . The cells were analysed immediately after sampling. Each sample was analysed with 10,000 event counts with the flow rate at 1 µl/s. The resulting GFP profile (green channel) was created by gating with the right ranges of cell sizes based on forward and reverse scatter plot  and dead cells were gated away based on the Sytox Red stain profile (red [APC] channel).Rex1GFPd2 ES were grown in 96 well plates in the presence of “2i” and reverse transfection was performed using siGENOME siRNA pools . 24 hrs later, the “2i” media was removed and replaced with fresh NDiff N2 B27 media. After 28 hrs, the levels of GFP in the cells were determined by flow cytometry as described above. Each plate contained 8 control non-targeting siRNAs, and the positive control siRNAs against gsk3β and fgf4. To take into account slight variations in the timing of pluripotency loss, the ratio of high GFP to low GFP expressing cells was established on each plate based on the non-targeting controls, allowing a threshold to be set as 1 (ie 50% high GFP and 50% low GFP). This threshold was used to determine the ratio of high to low GFP expressing cells in the other wells. The mean plus/minus standard deviation (SD) was calculated for each plate, and individual wells were scored positive if they exceeded 2×SD above or below this mean. The screen was performed in duplicate, with duplicate plates being analysed on different days. A final list of positive hits was determined by taking siRNAs which scored an average of 2×SD across both plates (or on a single plate where the duplicate well was defective in the case of 30 siRNAs), generally with both plates scoring >1.5×SD above the mean. However, an additional small number of siRNAs were scored as positive where the average score was >2.5×SD above the mean where only one plate had to score >1.5×SD above the mean, and also for seven siRNAs where the average score was >1.9×SD above the mean and both plates scored >1.9×SD above the mean.All liquid handling processes were performed using Biomek robotic system (Beckman Coulter). For the primary screen, Rex1GFPd2 or Oct4GFP ES cells were used and screens were performed as above except that ON-TARGETplus siRNA duplexes were used and GFP levels in Oct4GFP ES cells were determined 72 hrs after release from “2i”. Individual wells in each screen were scored as positive if the average GFP(+)/GFP(−) ratio exceeded 1.25×SD above the non-targeting controls across both duplicate plates. Additional hits were considered as positive if they scored >0.8×SD above the mean in one validation screen and also scored >1.0×SD above the mean in the other.For the validation screens, either Rex1GFPd2 were used as for the validation screens but only one inhibitor (ie either CHIR99021 or PD0325901) was withdrawn. Wells were scored as positive if the average GFP(+)/GFP(−) ratio exceeded 1.5×SD above the mean of the non-targeting controls.For the “1i” screens, For constructing networks, lists of gene names were uploaded into STRING GO term analysis was carried out using DAVID Bioinformatics Resources 6.7 (NIH) Figure S1Rex1GFPd2 ES cells grown for the indicated times in the presence of the inhibitors CHIR99021 and PD0325901 (“2i”) or upon removal of the inhibitors (“−2i”). The profiles corresponding to high GFP expressing cells are shown in dark green in (A). The effect of pre-treating cells with control non-targeting siRNAs  or siRNAs against fgf4 (blue) or gsk3 (red) on the GFP expression profile is shown in (B). The dashed line shows the position of the centre of the peak of the starting population of cells. (C) FACS profiles of GFP expression in Oct4GFP ES cells grown for the indicated times following removal of the inhibitors (“−2i”) in the presence of the indicated siRNA constructs (as labelled in B). Clear shifts in the distributions of high versus low GFP expressing cells can be observed between 27 and 30 hr for the Rex1GFPd2 ES cells and at 72 hrs for the Oct4GFP cells after removal of “2i” in the presence of fgf4 or gsk3 siRNAs.Kinetics of GFP expression level changes in the primary and validation library screens. (A and B) FACS profiles of GFP expression in (PDF)Click here for additional data file.Figure S2Rex1GFPd2 ES cells; column 2, validation “2i” screen using Rex1GFPd2 ES cells; column 3, validation “2i” screen using Oct4GFP ES cells; column 4, secondary “1i” screen using Rex1GFPd2 ES cells upon removal of CHIR99021; column 5 secondary “1i” screen using Rex1GFPd2 ES cells upon removal of PD0325901. Examples are shown for siRNAs which affect the ratio of high/low GFP expression (A) specifically when only the ERK pathway inhibitor is removed (ERK only), (B) only the GSK3 inhibitor is removed (GSK only), (C) either the ERK pathway or GSK inhibitors are removed (ERK/GSK) or (D) only when both the ERK pathway and GSK inhibitors are removed (ERK & GSK). (E) Example FACS profiles of GFP expression in the presence of control non-targeting siRNAs or siRNAs against the indicated genes from the primary “2i” screen, for hits which demonstrate enhanced loss of GFP expression. .Representative FACS profiles in each of the library screens. (A–D) FACS profiles of GFP expression in the presence of control non-targeting siRNAs or siRNAs against the indicated genes. The numbers next to each gene name indicate plate and well numbers and the numbers above each graph are the corresponding GFP high/GFP low ratios. Example profiles from the following screens are provided; column 1, primary “2i” screen using (PDF)Click here for additional data file.Figure S3Enriched functional categories of genes in the high confidence dataset. Enriched classes of genes amongst the 292 high confidence hits resulting from the validation screens [depicted in (A)] were determined by searching for Gene Ontology (GO) terms using DAVID.  Data are shown graphically according to their relative P-values or  data are visualized using REVIGO. GO terms are grouped according to the level of complexity of the terms; (B and C) molecular function, (D and E) biological process and (G and H) cellular component.  data are plotted according to the size of the GO term category  and the significance of the association with the category (x-axis). The identities of the most significant categories are indicated. (F and I) Analysis of interacting networks of enriched GO terms from the molecular function (F) and the cellular component (I) categories, depicted using the “interactive graph” view of REVIGO.(PDF)Click here for additional data file.Figure S4Rex1GFPd2 ES cells grown for the indicated times in the presence of the inhibitors PD0325901 and CHIR99021 (“2i”), upon removal of both of the inhibitors (“−2i”), or removal of either the PD0325901 or the CHIR99021 inhibitors (“−1i”). The data are shown graphically (A) and as FACS profiles of GFP expression 60 hrs after inhibitor removal (B).Kinetics of GFP expression level changes in the “1i” screens. GFP expression profiles of (PDF)Click here for additional data file.Figure S5Heatmap summary of the hits from the “1i” screens. The heatmap summary depicts the stratification of the hits according to their effects on the GFP(+)/GFP(−) ratio upon withdrawal of the GSK3 inhibitor (GSK3) or MEK inhibitor (ERK). Red indicates an increased ratio and blue represents a decreased ratio, and intermediate colours given according to the scale bar. Hits are grouped as specific to the ERK or GSK3 pathways (left side map), related to either the ERK or the GSK3 pathways  or only functional when both pathways are active (PDF)Click here for additional data file.Figure S610, and coloured according to the bar shown below the figure. Terms corresponding to distinct functional groups are manually clustered (indicated on the left), and are then ranked according to their significance scores in the “ERK only” dataset.Heatmaps of the enriched GO terms identified for genes corresponding to high confidence hits from the validation screens. The heatmap distribution of the full list of significant GO terms identified specifically associated with the ERK (133), or GSK3 (168) pathways and the total 274 validated hits. Each GO term is scored by −log(PDF)Click here for additional data file.Figure S7Heatmaps of the specific subsets of enriched GO terms identified for genes corresponding to high confidence hits from the validation screens. Heatmap distributions of specific subsets of significant GO terms identified specifically associated with the categories of hits described in (PDF)Click here for additional data file.Figure S8Interaction networks of genes associated with high confidence hits from the validation and secondary screens. STRING network analysis of the core network formed by the 274 genes associated with signal-dependent loss of pluripotency and promoting early differentiation processes in the mouse embryonic stem cells. Genes are grouped according to common biological processes. (A) In addition to a highly connected central network Click here for additional data file.Figure S9nanog and rex-1 (B), and the early differentiation marker, fgf5 (C). The expression levels were normalised by the average of three reference genes  or gapdh (C). Data are shown for each siRNA duplex relative to the maximal expression exhibited in the presence of an siRNA pool (taken as 100). The blue dashed lines indicate the threshold level (>1.5 SD above (B) or below (C) the mean of the control [ctr] siRNAs). Genes which show changes exceeding (B) or below (C) this level, are indicated by red bars. Data are presented as means ± SEM and are the average of three biological replicates (n = 3). Heatmap summaries (see rex1 (y-axis) and the reciprocal of fgf5 (x-axis) mRNA expression levels upon 2i withdrawal for 36 hrs and 48 hrs, respectively, are plotted following knockdown of individual genes (see B and C for details). Data are shown for each siRNA duplex relative to the maximal expression exhibited in the presence of an siRNA pool (taken as 100). Dotted lines represent the expression values >2 standard deviations above the mean of the negative control siRNAs. Red dots represent siRNA duplexes which promote elevated expression of nanog and lower levels of fgf5 (quadrant 1), whereas green (quadrant 3) and black (quadrant 4) dots represent siRNAs that cause changes at or below this threshold cut-off value for only one gene. The brown dot represents the negative control siRNAs. Blue dots (quadrant 2) represent siRNAs that cause elevated nanog expression but fail to show reductions in fgf5 expression.The effects of depletion of genes identified in the validation screen on the expression of markers of pluripotency and early differentiation. (A) Venn diagram (top) illustrating the number and a list of selected screen hits from each category used in the subsequent studies. (B and C) RT-qPCR analysis of the effects of depletion of the indicated genes on the mRNA expression levels of the ES-specific genes, (PDF)Click here for additional data file.Figure S10Western blot analysis of phospho-ERK levels upon depletion of genes identified in the secondary siRNA screens. ERK activation levels following 2i withdrawal (−2i) for 20 mins were determined by western blotting using a phospho-ERK-specific antibody  and normalized against total ERK levels (bottom panels). siRNAs against the indicated genes from the “1i” screens  or control non-targeting siRNAs (ctrl) are indicated. The presence or absence of inhibitors in the control samples is indicated (+/−2i). Dotted lines indicate where gels have been cut to remove irrelevant lanes and rejoined. The data shown are from one of three biological replicate experiments.(PDF)Click here for additional data file.Figure S11Summary of the effects of each screen hit on the activity of Ras and ERK. A heatmap summary see . The col(PDF)Click here for additional data file.Figure S12Rex1GFPd2 ES cells grown in the presence of LIF and released for 28 hrs. The profiles corresponding to high GFP expressing cells are shown in dark green. The effect of pre-treating cells with control non-targeting siRNAs or siRNAs against the indicated genes on the GFP expression profile under these conditions is shown. The ratio of high to low GFP expressing cells is shown quantitatively on the right as means ± SEM; n = 2–3). The vertical lines represent the mean of the non-targeting control (ctrl) (solid line) and two standard deviations above this mean (dotted line).GFP expression level changes upon gene depletion under LIF conditions. FACS profiles of GFP expression in (PDF)Click here for additional data file.Figure S13dusp1, dusp5, dusp6 and dusp14 mRNA expression in mouse ES cells. Data are normalised by the average values of three reference genes (ref) and are presented as means ± SEM and are the average of three biological replicates (n = 3). (A) The kinetics of dusp5 and dusp14 expression at the indicated times following 2i withdrawal. (B) The effects of the indicated inhibitors, either alone or in combination, on the expression of dusp5 and dusp14 at the indicated times following inhibitor withdrawal. (D and E) The effects of depletion of the indicated genes on dusp5 mRNA expression in the presence, (D) or dusp1, dusp5, or dusp5 mRNA expression in the absence, (E) of 2i. The blue dashed lines indicate the threshold level (2× SD above/below the mean of the negative controls) and levels below and above this are indicated by red and pink bars, respectively. The average activity in the presence of control siRNA (ctrl) is shown by the solid grey line. (C and F) Dusp1 and Dusp6 protein expression measured by western blot analysis (bottom panels) was quantified and normalized by Pou5F1 levels . (C) The kinetics of Dusp1 and Dusp6 expression at the indicated times following 2i withdrawal. (F) The effects of depletion of the indicated genes on Dusp1 and Dusp6 expression in the presence of 2i. Data are the average of two experiments. (G) Active ERK levels were determined by the ratio of phospho-ERK (pERK)/total ERK (ERK) levels at the indicated times following 2i release for the indicated times in the presence of the indicated siRNAs (red lines) or control siRNA (blue lines). The data are plotted relative to maximal levels with the control siRNA (taken as 100) and are presented as means ± SEM from the average of two biological replicates (n = 2).The regulation of Dusp activity and ES cell differentiation (A–B and D–E) RT-qPCR analysis of (PDF)Click here for additional data file.Figure S14The role of Dusps in MAP kinase activation in ES cells. (A) Active ERK levels were determined by the ratio of phospho-ERK (pERK)/total ERK (ERK) levels at the indicated times following 2i release in the presence of the indicated siRNAs (red lines) or control siRNA (blue lines). The data are plotted relative to maximal levels with the control siRNA (taken as 100) and are presented as means ± SEM from the average of two biological replicates (n = 2). (B) Active levels of the indicated MAPKs were determined by the ratio of phosphorylated (p) to non-phosphorylated forms at the indicated times following 2i release in the presence of the indicated siRNAs (red lines) or control siRNA (blue lines). The data are plotted relative to basal levels with the control siRNA in the presence of 2i (taken as 1) and are presented as means ± SEM from the average of two biological replicates (n = 2).(PDF)Click here for additional data file.Table S1Rex1GFPd2 cells. (B) Positive hits from the validation screen in Rex1GFPd2 cells. (C) Positive hits from the validation screen in Oct4GFP cells. See Positive hits identified in the primary and validation siRNA screens for genes involved in escape from pluripotency. (A) Positive hits from the primary siRNA screen in (XLSX)Click here for additional data file.Table S2Rex1GFPd2 and Oct4GFP cells compared to negative controls. Hits were scored as positive according to the same criteria as those which scored positive for retention of pluripotency.Hits identified in the primary and validation siRNA screens for genes involved in maintaining pluripotency. Genes are listed when their depletion results in more loss of GFP-positive cells with both (XLSX)Click here for additional data file.Table S3Overlaps with other siRNA screens. Overlaps between genes identified in this paper as important for promoting loss of pluripotency and onset of differentiation  or the m(PDF)Click here for additional data file.Table S4Rex1GFPd2 and Oct4GFP cells and were considered as a high confidence group for further analysis.Positive hits identified in both of the validation siRNA screens for genes involved in escape from pluripotency. These hits scored positive in validation screens with both (XLS)Click here for additional data file.Table S5Rex1GFPd2 cells in the presence of the MEK and GSK3 inhibitors (“+2i”). These were not taken forward for further analysis due to the lack of a potential link to active ERK or GSK3 signalling.Hits eliminated in the counterscreen. Validated hits from the primary screens which also scored as positive in a screen with (XLS)Click here for additional data file.Table S6Summary of the point of action of siRNAs with respect to the ERK signaling pathway. Genes targeted by the siRNA constructs are grouped according to whether they act to potentiate from Ras activity, act downstream of Ras to activate ERK or play a role downstream from, or in parallel to, ERK signalling. Each column indicates the association of the genes with the ERK, GSK or both pathways, as determined by the “1i” screens see .(PDF)Click here for additional data file.Table S7PCR primers used in RT-qPCR. The sequences of both forward (F) and reverse (R) primers are provided.(PDF)Click here for additional data file.


Typical flow velocities in the extraparenchymal pulmonary veins demonstrate two major antegrade flow waves: a biphasic systolic wave S1 and S2) and a monophasic early diastolic wave (D)  with RV-independent pulmonary circulation . Phase-contrast imaging of the pulmonary veins was performed on a 1.5 T MR scanner with velocity encoding set at 120 cm/s. We compared these flow patterns with the flow patterns of a control group of 10 patients (15 pulmonary veins) who had RV-dependant pulmonary circulation and underwent CMR due to other reasons .In all patients with RV-independent pulmonary circulation pulmonary vein flow curve showed a single systolic peak in early systole with the S2 peak consistently absent Figure .This study further supports the concept that the late systolic pulse (S2) of the pulmonary vein flow curve is caused by forward propagation of the right ventricular systolic pressure pulse. It also demonstrates that the early systolic pulse (S1) is independent of the right ventricle but dependent on the left atrial and left ventricular events.


In contrast, the proliferating non-glial populations of the dorsal telencephalon and hypothalamus rarely express notch3 and about half express notch1a/1b. In the non-proliferating radial glia notch3 is the predominant receptor throughout the brain. In the ventral telencephalon and in the mitotic area of the optic tectum, where cells have neuroepithelial properties, notch1a/1b/3 are expressed in most proliferating cells. However, in the cerebellar niche, although progenitors also have neuroepithelial properties, only notch1a/1b are expressed in a high number of PCNA notch3 expression is mostly in Bergmann glia and at low levels in few PCNA The adult zebrash brain has a remarkable constitutive neurogenic capacity. The regulation and maintenance of its adult neurogenic niches are poorly understood. In mammals, Notch signaling is involved in stem cell maintenance both in embryonic and adult CNS. To better understand how Notch signaling is involved in stem cell maintenance during adult neurogenesis in zebrafish we analysed Notch receptor expression in five neurogenic zones of the adult zebrafish brain. Combining proliferation and glial markers we identified several subsets of Notch receptor expressing cells. We found that 90  Teleost fish, like many non-mammalian vertebrates, display widespread neurogenesis in adulthood (see review(s) In the zebrafish dorsal telencephalon, the cellular composition of progenitors is mixed, with a fraction of cells that do not display glia characteristics intermingled with others that show markers and morphology typical of radial glia The Notch pathway is a conserved pathway throughout the animal kingdom and has been intensely studied for its crucial role in cell fate decision, proliferation and cell death during embryonic neural development  and notch3 (previously notch5) notch2 expression is excluded from the nervous system during embryonic development Multiple Notch receptors are present in the zebrafish genome, namely in situ hybridization and co-stained them with glial, neuronal, oligodendrocytic, endothelial and proliferation markers. In the adult zebrafish brain, we find all known Notch receptors to be expressed, except for notch2. Our analysis reveals that notch1a, notch1b and notch3 are expressed in all five neurogenic regions of the adult zebrafish brain and that they localize with the majority of the proliferating cells. Similar to embryonic stages, we also find overlapping and complementary expression patterns of Notch receptors in different adult neurogenic niches. The expression of Notch receptors in proliferating cells suggests that Notch signaling might be required at a basal level for maintaining proliferation of neural progenitors.Because the cellular composition of progenitor cells in the adult zebrafish brain shows great diversity and different responses of progenitors to Notch signaling has been reported in mammals, we analysed in detail the expression of the four Notch receptors in five adult zebrafish neurogenic niches. To this end, we characterized the expression pattern of Notch receptors by notch1a, notch1b and notch3 but not notch2 was detected in the adult brain. Thus notch2 is not further mentioned.We focused our analysis on five neurogenic zones: the dorsal and ventral telencephalon, the hypothalamus, the optic tectum, and the cerebellum. It has been shown that proliferating cells in these areas are periventricularly located notch1a, notch1b and notch3 are mostly expressed in cells lining the ventricular surface  for the receptors and olig2, a marker for oligodendrocytes. This revealed that all three Notch receptors are expressed in a subpopulation of olig2Tg(fli1:gfp) notch1a and notch3 localize in a subset of these GFP In the adult zebrafish telencephalon,  surface . In the  surface  –C . Inrea (Dm) , B and ea (Dll) , B . Inobserved , B . In and Dll . These T and vT . To asse2 cells . Using tP  cells .In the dorsal telencephalon different cell populations have been previously characterized based on their proliferative status and presence of glial markers Fig , 3, .in situ hybridization analysis, cells positive for notch1a and notch1b are scattered along the ventricular surface of the dT  notch1a and notch1b are often co-expressed in proliferating cells, iv) notch3 is expressed in very few S100notch1a and notch1b which are present in roughly more than half of this population.All together, these results show that i) all three Notch receptors are expressed in approximately the same number of proliferating glial cells, ii) notch1a and notch1b are expressed in some, but not all of the S100notch3 is found throughout the ventricular zone of Vd  and the ventral nucleus of the ventral telencephalon (Vv) al cells , while nne of Vd .notch3 than for notch1a/1b  in groups of cells with different expression intensities , we detected single scattered notch1a , A  andnotch1b , B  celentricle . Moreovecaudally .notch1a and notch1b are arranged in a pattern that resembles the distribution of proliferating cells in this zone as previously shown notch3 is expressed in a very similar fashion to notch1b , at the level of the posterior recess, positive cells for  notch1b .notch3 covers most of the S100notch3 is also expressed in most of the S100notch3notch1a/3 shows that notch1a is expressed in part of the notch3 domain  of adult teleost fish, active proliferating cells are located in the dorsomedial area of the periventricular gray zone (PGZ) notch1a, notch1b and notch3 in dPGZ, mPGZ and PML  . notch3  as well . We thengenitors . A few cL and GL . Co-stais notch3 . Also nonotch1a is present predominantly in the progenitor niche, whereas notch1b is also strongly expressed in a few Bergman glia cells. notch3 is mostly excluded from the niche but expressed in a high proportion of the Bergman glia , a notch3 homologue has been identified, and the expression of this receptor was also found in ventricularly located cells of the adult brain et al.notch3 is expressed in most of the radial glia population of the dorsal telencephalon and hypothalamus neurogenic niches, but also that it is associated, together with notch1a and notch1b, with proliferating cells throughout the adult brain. A possible explanation for these differences might be that we have preformed the in situ hybridization on 12 notch2 expression in the adult brain, opposed to what has been previously reported Our expression pattern analysis is consistent with what had been observed in zebrafish larval brains, where In the mammalian brain several studies support the idea that NSCs present high Notch activity, essential for their maintenance and proliferation In the dorsal telencephalon we observed that approximately 20 notch1a, notch1b and notch3 are expressed in proliferating glial cells, which form the majority of the constitutively proliferating neural progenitor population . Considering that the cellular composition of the ventricular zone cells is very diverse, different cell types could be more or less responsive to variations in Notch signaling activity, and the outcome of such variations may be very different depending on the cell type/state and neurogenic niche. It could also well be that combinatorial signaling mediated by the activation of different Notch receptors triggers distinct responses in different cells or at different stages.Here we show that pulation . However is rare . As the notch3 is strongly expressed in the majority of S100notch1a and notch1b expressed in a subset of notch3notch3 might have a more prominent role in the glial lineage, whereas notch1a/1b expression might be related to their progenitor character  of the adult zebrafish brain show a pseudostratified neuroepithelial-like morphology and interkinetic nuclear migratio In chick spinal cord, a study reports that Notch activity increases at a higher frequency prior to mitosis notch3, and partially notch1b, are expressed in Bergmann glia in the adult zebrafish. Additionally, Notch1 receptor is expressed in mature neurons in the rodent cortex notch1a is expressed both in neural precursors and in differentiating neurons whereas notch1b and notch3 are found only in neural precursors notch1b in a HuC/D notch1b expression that we observed in neurons could indicate that also in the adult zebrafish brain Notch signaling might serve later stages of neuronal differentiation and/or neuronal activity.Notch signaling is not only active in germinal zones but also in non-proliferative areas. Similarly to the adult mouse cerebellum olig2 is a marker for both oligodendrocyte precursor cells (OPCs) and, in combination with other markers, for more mature oligodendrocytes olig2olig2olig2olig2, promotes the specification of OPCs olig2 subpopulation could represent immature oligodendrocytes.Oligodendrocytes are another cell type produced in neurogenic regions of the adult zebrafish brain notch3 expression is associated with the arterial fate during arterial-venous differentiation notch1a and notch3. This suggests that also in the adult zebrafish brain, Notch signaling might be important for vessel homeostasis or for the maintenance of arterial phenotype in some cells.Notch signaling is also involved in the development of the vascular system (see review(s) notch3 might be important for the glial properties of progenitors as well as other glial cells; ii) notch1a/1b might modulate progenitors proliferative status in a level-dependent manner Our results showed that Notch receptor expression domains overlap to a great extent in the neurogenic areas, but also that regional and cellular heterogeneity in the expression of different receptors exists in the adult zebrafish brain. The differences observed between receptor expression in certain niches could indicate that they are involved in distinct cellular processes. Therefore, we hypothesise that: i) predominantly All procedures were in accordance with the live animal handling and research regulations of the University and State of Saxony, Germany, review boards, the Regierungspräsidium Dresden (permit AZ 24D-9168.11-1/2008–1 and −4). This institutional review board specifically approved this study.gol-b1 line in the AB genetic background fli1:GFP) Fish were kept under standard conditions as previously described in situ hybridization experiments, brains were cryosectioned in 14 in situ hybridization (FISH) stainings, brains were cryosectioned in 12 Whole heads with the brain exposed were fixed in 4notch1anotch1bnotch2notch3olig2in situ hybridization was done as previously described in situ reaction, with better sensitivity for detecting cells expressing lower levels of notch transcripts. All in situ hybridizations were done on at least five individuals. All probes were tested on embryos to confirm probe specificity. We have also perfomed negative controls: 1) with probe and without anti-DIG antibody, and 2) without probe and with anti-DIG antibody to assess for unspecific signal (antibody or tyramid trapping). In these tests no signal was detected under the fluorescence microscope.Riboprobe preparation was done as previously described Immunostainings for S100http://fiji.sc), CombineZP v7.0 (http://www.hadleyweb.pwp.blueyonder.co.uk/CZP/News.htm) and Adobe Photoshop CS4. Figures were assembled using Adobe Illustrator CS4. Confocal images shown are single planes or maximum projections of up to 3 planes . Confocal images were taken on a Leica TCS-SP5, using the Leica HCX PL APO 40×/0.75 Dry and Leica HCX PL APO 63×/1.2 Water objectives. Image acquisition for the quantifications was done using the Leica HCX PL APO 40×/0.75 Dry objective. To minimize cross talk between the channels, sequential image acquisition was performed. Images were processed using Fiji (http://www.r-project.org/) using ANOVA followed by a post-hoc Tukey's HSD test.The quantifications of co-localization between the FISH, S100notch1a (n = 4), 24 for notch1b (n = 4) and 26 for notch3 (n = 5). For data visualization and median comparison we used notched boxplots in R and modified them with Adobe Illustrator CS4. To confirm statistically significant differences in expression of each gene between apical and basal or between genes in the same nuclei position we applied the Wilcoxon rank-sum test or ANOVA followed by a post-hoc Tukey's HSD test, respectively. Fluorescence profile analysis was done using Fiji Plot Profile. Fluorescence values for each gene expression were normalized for background signal in the tissue and their corresponding maximum. Plots were done in Microsoft Ofice Excel 2007 and modified in Adobe Illustrator CS4.To analyse Notch receptor expression levels in proliferating cells of the ventral telencephalon we measured the mean grey value (MGV) of the FISH signal for each receptor. We selected the regions of interest based on the position of the PCNA Figure S1Notch receptor expression in telencephalic oligodendrocytes. Confocal images of double FISH showing the localization of Notch receptor (white) and olig2 (green) in the dorsal telencephalon parenchyme. A–C, notch1a, notch1b and notch3 are expressed in a subpopulation of parenchymal olig2 olig2notch1bolig2(TIF)Click here for additional data file.Figure S2Notch receptor expression in fli1:gfp endothelial cells in the telencephalic parenchyme. Confocal images showing localization of notch1a and notch3 and gfp A–Bnotch1a and notch3 expression in a few endothelial cells (white arrows); unfilled arrow in B indicates a notch3(TIF)Click here for additional data file.Figure S3Apical to basal gradient of Notch receptor expression in the ventral telencephalic niche. Fluorescence profile measurements of Notch receptor expression in the two indicated areas (dark grey rectangle in schematics) of the Vv proliferation zone. Stronger fluorescence signals are detected in proliferating cells Click here for additional data file.Figure S4notch3 expression in glia and proliferating cells of the adult zebrafish hypothalamus. Confocal images showing localization of notch3 mRNA by FISH (white), radial glia labelled with S100A, B, notch3 is expressed in most PCNA notch3A–F, notch3 localizes with most S100notch3notch3 expression in the S100notch3. Abbreviations: Hc, caudal zone of the periventricular hypothalamus; Hd, dorsal zone of the periventricular hypothalamus; Hv, ventral zone of the periventricular hypothalamus: PR, posterior recess of the diencephalic ventricle. Scale bar  = 100 (TIF)Click here for additional data file.Figure S5Overlapping and complementary notch1a/3 expression in the adult zebrafish hypothalamus. Confocal images of double FISH showing the localization of notch1a (white), notch3 (red), and PCNA (green). Cross-sections at the indicated level through the diencephalon; hypothalamic area shown in the micrographs is indicated in the cross-section schematics. A–C, notch1a is expressed in a subpopulation of notch3notch3notch1a expression partially overlaps with the notch3notch1anotch3(TIF)Click here for additional data file.Figure S6notch1a and notch1b expression in olig2  Confocal images of double FISH showing the localization of notch1a/1b (white), olig2 (green) and HuC/D (red) in the superficial layer of the optic tectum. Cross-sections at the indicated level through the mesencephalon; tectal area shown in the micrographs is indicated in the cross section schematic in A. A–B, notch1a and notch1b are expressed in a subpopulation of olig2olig2B, notch1b is also expressed in olig2(TIF)Click here for additional data file.Table S1Localization of Notch receptor positive cells with glial and proliferation markers. Number of cells counted in adult zebrafish Dm and Dl telencephalic areas according to their notch expression and co-label with radial glial (S100(PDF)Click here for additional data file.Video S13D animation showing notch1a expression in proliferating and non-proliferating glial cells of the dorsal telencephalon. This animation corresponds to the z-stack containing the images shown in (AVI)Click here for additional data file.Video S23D animation showing notch1b expression in proliferating and non-proliferating glial cells of the dorsal telencephalon. This animation corresponds to the z-stack containing the images shown in (AVI)Click here for additional data file.Video S33D animation showing notch3 expression in proliferating and non-proliferating glial cells of the dorsal telencephalon. This animation corresponds to the z-stack containing the images shown in (AVI)Click here for additional data file.


Tbx5 and Tbx4, two genes expressed in forelimb and hindlimb-forming regions respectively, play crucial roles in the initiation of limb outgrowth. Evolution of regulatory elements that activate Tbx5 in rostral LPM was essential for the acquisition of forelimbs in vertebrates. We identified such a regulatory element for Tbx5 and demonstrated Hox genes are essential, direct regulators. While the importance of Hox genes in regulating embryonic development is clear, Hox targets and the ways in which each protein executes its specific function are not known. We reveal how nested Hox expression along the rostro-caudal axis restricts Tbx5 expression to forelimb. We demonstrate that Hoxc9, which is expressed in caudal LPM where Tbx5 is not expressed, can form a repressive complex on the Tbx5 forelimb regulatory element. This repressive capacity is limited to Hox proteins expressed in caudal LPM and carried out by two separate protein domains in Hoxc9. Forelimb-restricted expression of Tbx5 and ultimately forelimb formation is therefore achieved through co-option of two characteristics of Hox genes; their colinear expression along the body axis and the functional specificity of different paralogs. Active complexes can be formed by Hox PG proteins present throughout the rostral-caudal LPM while restriction of Tbx5 expression is achieved by superimposing a dominant repressive (Hoxc9) complex that determines the caudal boundary of Tbx5 expression. Our results reveal the regulatory mechanism that ensures emergence of the forelimbs at the correct position along the body. Acquisition of this regulatory element would have been critical for the evolution of limbs in vertebrates and modulation of the factors we have identified can be molecular drivers of the diversity in limb morphology.Tight control over gene expression is essential for precision in embryonic development and acquisition of the regulatory elements responsible is the predominant driver for evolution of new structures.  Tbx5. Activation of Tbx5 in the forelimb-forming region of the developing embryos is essential for forelimbs to form and disruption of human TBX5 causes limb abnormalities. We show that activation of Tbx5 in a restricted territory is achieved through a combination of activation inputs that are present broadly throughout the embryo flank and dominant, repressive inputs present only in more caudal regions of the flank. The sum of these inputs yields restricted activation in the rostral, forelimb-forming flank. Our results explain how the regulatory switches that were harnessed for the acquisition of limbs during evolution operate and how they can be turned off during the evolution of limblessness in species such as the snake.The acquisition of limbs during vertebrate evolution was a very successful innovation that enabled this group of species to diversify and colonise land. It has become clear recently that the primary driver behind the evolution of new structures, such as limbs, is the acquisition of novel regulatory elements that control when and where genes are activated rather than the proteins encoded by the genes themselves acquiring novel functions. We have identified the regulatory element from a gene,  Tbx5 in nascent forelimbs and Tbx4 in hindlimbs Fgf10 in the limb mesenchyme Fgf8 expression in the apical ectodermal ridge (AER) and Fgf8 produced from the AER, in turn, maintains Fgf10 expression in mesenchyme to establish a positive feedback loop of Fgf signalling that maintains limb growth. Mutations in human TBX5 cause Holt-Oram Syndrome (HOS OMIM142900), a disorder characterised by upper limb and heart abnormalities TBX4 cause Small Patella Syndrome (SPS OMIM 147891), a disorder characterised by knee, pelvis and toe defects Tbx5 is the earliest marker of presumptive forelimb mesenchyme and because activation of this factor within a defined region of the LPM ultimately dictates the position at which the forelimbs will arise, identifying the factors that control activation of this Tbx5 expression domain will reveal the mechanisms employed that allowed the acquisition of limbs in vertebrates and that dictate forelimb position in the embryo.Forelimbs and hindlimbs are derivatives of the lateral plate mesoderm (LPM) that arise at fixed positions along the vertebrate body axis. Limb formation is initiated by limb induction signals from axial tissues Tbx5 is initially expressed in the forelimb-forming region of LPM prior to the emergence of a bud and it is subsequently restricted to the forelimb region as development proceeds. Tbx5 is essential for forelimb formation and this exclusive requirement is limited to a short time window when limb bud initiation occurs Tbx4, the paralog of Tbx5, is able to rescue forelimb formation following conditional deletion of Tbx5Tbx4/5 gene represented by AmphiTbx4/5 of the limbless cephalochordate, amphioxus, can fully compensate for the loss of Tbx5 in the mouse Tbx5 expression in the LPM was a critical step in the acquisition of limbs during vertebrate evolution.Hox genes are conserved homeodomain-containing transcription factors that are arranged in clusters in the genome. The chromosomal organization of the genes in the complex reflects their expression pattern along the rostro-caudal body axis to determine positional identity Tbx5 regulatory element sufficient for early forelimb expression Tbx5. However, since the ability to activate Tbx5 is not strictly restricted to Hox genes expressed only at forelimb level, the mechanism by which a rostro-caudal Hox code establishes forelimb-restriction of Tbx5 remained unknown. Here, we demonstrate how Hox paralogous group members act cooperatively to restrict expression of Tbx5 in the LPM, which ultimately determines the positions the forelimbs will emerge from the flank of the embryo. We show that mutations of a single Hox binding site in the Tbx5 forelimb regulatory element cause expanded reporter gene expression in caudal LPM. Rostral restriction in Tbx5 expression through repression in the caudal LPM is mediated by Hoxc8/9/10 genes and this repressive function is limited to Hox genes that are expressed in Tbx5-negative caudal LPM. We further map the Hoxc9 protein domains required to confer transcriptional repression that distinguishes these paralogs from other Hox proteins expressed throughout the flank of the embryo. Our results demonstrate how a nested, combinatorial code of Hox protein transcriptional activation and repression along the rostro-caudal embryo axis restricts Tbx5 expression to the forelimb and ultimately determines forelimb position.We have previously identified a Tbx5 gene that recapitulates the dramatic forelimb-restricted expression of this gene Tbx5 expression, we generated a series of constructs in which each individual Hbs site1-6 is mutated and tested their ability to activate a LacZ reporter gene in transgenic mice. While the Tbx5 int2(361) reporter construct drove forelimb-restricted expression of LacZ (Tbx5 int2(565)) . To distinguish the requirement for Hbs2 and 6bp3′, we next mutated either of these sites )  that cont2(565)) . We haveess Tbx5 . These rse sites . Mutatioreporter , while mreporter  equivalereporter . These rThese results demonstrate that the binding sites within this regulatory element can be divided into 2 distinct functional groups. Hbs1 and 3-6 act as ‘on’ switches important for the amplitude of activation, whereas Hbs2 determines spatial resolution by hosting repressive complexes that restrict the domain of activation. This element can therefore have a binary function, serving as a site for the formation of transcriptional activation or repression complexes.Hox genes can activate this regulatory element Hox genes in chick and mouse embryos at stages when Tbx5 is first expressed in the forelimb-forming region. Tbx5 is first expressed at the level of somites 13–20 in chick and somites 4–11 in mouse embryos. As previously reported Hox4 and Hox5 paralogs overlap with that of Tbx5 in both mouse and chick embryos   construct  reporter, LacZ expression is repressed in the forelimb region   LacZ repficiency ), β-gal ficiency  indicatireporter  demonstrTbx5 expression, we co-electroporated Hoxc9 with a Tbx5 int2(361) reporter in which Hbs2 is mutated  showed clear repression of the reporter demonstrating that within the limits of this assay the entire N-terminus or the domain overlapping the junction between N1 and N2 is required for repressive activity  can reduce LacZ expression , suggestTbx5 as an assay, we have been able to distinguish the opposing transcriptional activities of different Hox paralogous group proteins. Hoxc9, as well as Hoxc8 and Hoxc10, that are normally expressed in the LPM caudal to the forelimb, can repress Tbx5 to restrict its expression to the forelimb level region of the LPM  is required for its repression in caudal LPM (Our results demonstrate that five of the six Hox binding sites (namely Hbs1 and Hbs3-6) within the udal LPM . One posudal LPM , suggesthttp://www.genomatix.de) is the presence of a 6-bp sequence named Pbx1-Meis1 complexes site located 3′ of Hbs2 (6bp3′). Pbx is a Hox co-factor that can attenuate Hox-mediated gene transcription by recruiting histone deacetylases (HDACs) Tbx5 forelimb regulatory element.An alternative model is that the transcriptional activity of the Hox complex bound at Hbs2 is determined by a co-factor(s). The sequence of Hbs2 is identical to the sequences of Hbs1 and Hbs3, therefore we compared the sequences surrounding these Hox binding sites. One distinguishing feature of Hbs2 identified using Mat Inspector  in the protein. The hexapeptide of AbdA represses dpp expression by inhibiting the function of a glutamine (Q)-rich C-terminal activation domain Tbx5 transcription, we suggest the model that Hoxc9 supresses Tbx5 expression by interaction with co-repressor(s) (in vitro translated proteins or nuclear extract from LPM (data not shown). Other potential collaborators are Smad proteins. In the Drosophila haltere, a Mad/Med/Shn complex works in combination with Ubx to repress Sal expression Tbx5 expression . Other candidate repressors are engrailed (En) and sloppy paired (Slp) since, in Drosophila, they form a complex with Hox, Exd and Hth to repress transcription Engrailed1 and Engrailed2, are expressed in LPM at pre-limb bud stages As it is unlikely that Hox protein itself directly represses essor(s) . One canTbx5 expression or that they share similar 3D structure domains in spite of their distinct amino acid sequences.We have shown that Hoxc8 and Hoxc10 have transcriptional repression ability similar to Hoxc9 . To gainTbx5 forelimb regulatory element reveals a direct link between patterning of the rostro-caudal axis of the embryo by Hox genes and the programme that controls positioning of the forelimb forming territory. A clear correlation between Hox expression and establishment of the forelimb territory of the LPM has previously been suggested Tbx5Hoxc9 is reduced Tbx5 (and the subsequent forelimb programme) being essential for emergence of an ectopic wing bud from this region. In the limbless python, Hoxc8 expression is rostrally expanded to the anterior limit of the trunk Tbx5-negative caudal LPM at pre-limb bud stages in chick and mouse , however, we did not detect expression of Hoxc9 in the forelimb-forming region, in contrast to the strong staining in caudal tissues. We therefore conclude that Hoxc9 is not present in the forelimb-forming region at stages when Tbx5 expression is first initiated. Later expression of Hoxc9 is not sufficient to cause detectable repression of the domain of Tbx5 already activated by Hox4/5 paralogous genes.A previous study has demonstrated the presence of and a function for Tbx5 expression, our study does not exclude the possibility that other caudally-expressed Hox genes have a similar repressive ability. We favour a model in which other caudally-expressed Hox paralogs have redundant functions in repression of Tbx5. Hoxc cluster null mice have no defects in the limb skeleton Tbx5 in these mutants have not been reported and we predict that the ectopic expansion of Tbx5 in caudal LPM would not cause any skeletal defects. Further analysis will be required to uncover the requirement of caudally–restricted Hox paralogs, such as Hox8, Hox9 and Hox10 for Tbx5 repression in caudal LPM.While we have shown that Hoxc8, Hoxc9 and Hoxc10 can repress Tbx5 in the LPM, a similar Hox protein code is present in axial tissues  that do not express Tbx5. The activity of the forelimb regulatory element of Tbx5 is restricted to LPM and this LPM restriction is maintained following mutation of Hbs2 that leads to caudal expansion in expression. One possible explanation for LPM restriction is the presence of unknown repressors in axial tissues or alternatively additional factors, which are active exclusively in LPM, are required for Tbx5 expression. Odd-skipped related (Osr) genes are candidates as they are expressed in LPM, but excluded from axial tissues such as neural tube and somites Tbx5 forelimb regulatory element to test if reporter activity was lost. The activity of the element was unaffected, however, suggesting Osr genes are not required for Tbx5 LPM expression  complex that ultimately determines the caudal boundary of Tbx5 expression. Thus, the regulation of Tbx5 expression in the LPM represents an excellent system to understand the interactions between neighbouring Hox binding sites and how the consequent output is integrated.Our analysis of the http://www.genomatix.de). Transgenic embryos were generated by the Procedural Service section, NIMR by standard pronuclear microinjection techniques. Mouse embryos were staged according to LacZ transgene were identified by PCR using specific primers . Sequences surrounding putative Hox binding sites and the mutations induced are as followings, binding sites are shown in bold; Hbs1, ATTATTGGAAC; mut Hbs1, ATGCTTGGAAC; Hbs2, ATTATCGACTCTCA; mut Hbs2, ACGATCGACTCTCA; mut 6bp3′, ATTATCGACTGCAA; mut Hbs2+6bp3′, ACGATCGACGCTTA; Hbs3, TAATTCAGA; mut Hbs3, TCGTTCAGA; Hbs4, TTATTAAGGCC; mut Hbs4, TTGGCAAGGCC; Hbs5, TTATCTTGCCAT; mut Hbs5, TCGTCTTGCCAT; Hbs6, TTATTTTG; mut Hbs6, TCGTTTTG.For reporter analysis in chick and mouse, we used the BGZA reporter vector in situ hybridizations were carried out essentially as previously described Pitx1, Tbx5 and mouse Hox genes have been described previously Whole mount Fertilized chick embryos  were incubated at 38°C and staged according to Hamburger Hamilton (HH) In vitro translated proteins were produced using a TnT Coupled Reticulocyte Lysate System (Promega). Proteins were labelled with 35S-Methionine (PerkinElmer) to verify and quantify translation. LPM strips adjacent to somites 5–10  and lateral to somite 14 to its caudal extreme  were dissected from E9 mouse embryos. Nuclear extracts were prepared using the NE-PER Nuclear and Cytoplasmic Extraction Kit (Pierce) following manufacturers instructions.32P by incubating with T4 polynucleotide kinase (NEB) for 30 minutes. 2 µl of in vitro translated protein or nuclear extract were blocked with 200 ng poly-dIdC, 2 µg of poly-dGdC or 2 µg of poly-dAdT in binding buffer  in a total volume of 22 µl for 15 minutes on ice. For super-shift, 2 µl of the antibody recognising flag epitope  was added to the binding reaction and incubated for a further 15 minutes. Then, 1 µl of 32P -labelled double-stranded oligonucleotides were mixed and incubated for 30 minutes. The protein∶DNA hybrids were resolved on 6% PAGE in 0.5xTBE.Double-strand oligonucleotides were labelled with Figure S1Tbx5 forelimb regulatory element. A–C. The Tbx5 int2(361) LacZ reporter construct was electroporated with pCAβ-dsRed-Express in the presumptive forelimb region of HH14 chick embryos. 24 hours later, the efficiency of electroporation was assessed by dsRed expression (A–C) and embryos were stained for LacZ to analyze the enhancer activity (A′–C′). Co-electroporation of pcDNA-mHoxc8 (B and B′) and pcDNA-mHoxc10 (C and C′) reduced LacZ expression.Hoxc8 and Hoxc10 can repress activity of the (TIF)Click here for additional data file.Figure S2LacZ reporter expression. A, Schematic representation of the Tbx5 intron2 regulatory element. A putative smad binding site and Hox binding sites are shown as green and blue boxes, respectively. B–C, E9.5 embryos were stained for β-galactosidase for a mutated construct of Tbx5 int2(565) reporter.Disruption of a putative smad binding site does not expand (TIF)Click here for additional data file.Figure S3LacZ reporter expression. A, Schematic representation of the Tbx5 intron2 regulatory element. A putative Osr binding site (purple box) locates proximal to Hbs5 (blue box). B–C, E9.5 embryos were stained for β-galactosidase for mutated constructs of Tbx5 int2(565) reporter.Disruption of a putative Osr binding site does not affect (TIF)Click here for additional data file.


Activation of neutrophils sequestered in the alveolar milieu can cause the release of reactive oxygen species (ROS), increasingly regarded as key substances modulating epithelium dysfunction and disruption. These oxidants are generated by the neutrophil respiratory burst oxidase system that reduces molecular oxygen (O2) to superoxide (O2-). Alpha-1 antitrypsin (AAT) deficiency (AATD) provides us with the most definitive evidence for the physiological and clinical importance of AAT and in this study we examined the immunomodulatory activity of AAT and investigated whether neutrophil ROS production was regulated by AAT.Neutrophil O2- production in response to fMLP (10-6M) and IL-8 (10ng) was measured by a cytochrome C reduction assays respectively. Analysis of ERK phosphorylation in response to fMLP ± AAT was carried via Western blot analysis of neutrophil whole cell lysates. To verify fMLPs interaction with its cognate receptors fMLP R1 (FPR1) and fMLP R2 (FPR2), flow cytometry was used to measure levels of FITC labeled fMLP binding to the membranes of neutrophil.In this study we demonstrate using in vitro models that AAT modulates neutrophil O2- production elicited by fMLP and IL-8 in a dose dependant manner (P<0.05). Mechanisms of inhibition were investigated and in vitro studies revealed that AAT functions to inhibit fMLP signaling through inhibiting its interaction with its receptors FPR1 and FPR2 on the neutrophil plasma membrane.The potential of AAT as a regulator of neutrophil ROS production adds a new understanding to the role of AAT in health and disease.


These characteristics make members of the transient receptor potential (TRP) superfamily likely candidates for this role. One of these candidates is the transient receptor potential melastatin 1 protein (TRPM1), which is expressed in various cells types within the cochlea of the mouse including the hair cells. Recent studies demonstrate that mutations in the TRPM1 gene underlie the inherited retinal disease complete congenital stationary night blindness in humans and depolarizing bipolar cell dysfunction in the mouse retina, but auditory function was not assessed. Here we investigate the role of Trpm1 in hearing and as a possible hair cell MET channel using mice homozygous for the null allele of Trpm1 (−/−Trpm1) or a missense mutation in the pore domain of TRPM1 (tvrm27/tvrm27Trpm1). Hearing thresholds were evaluated in adult (4–5 months old) mice with auditory-evoked brain stem responses. Our data shows no statistically significant difference in hearing thresholds in −/−Trpm1 or tvrm27/tvrm27Trpm1 mutants compared to littermate controls. Further, none of the mutant mice showed any sign of balance disorder, such as head bobbing or circling. These data suggest that TRPM1 is not essential for development of hearing or balance and it is unlikely that TRPM1 is a component of the hair cell MET channel.Sound and head movements are perceived through sensory hair cells in the inner ear. Mounting evidence indicates that this process is initiated by the opening of mechanically sensitive calcium-permeable channels, also referred to as the mechanoelectrical transducer (MET) channels, reported to be around the tips of all but the tallest stereocilia. However, the identity of MET channel remains elusive. Literature suggests that the MET channel is a non-selective cation channel with a high Ca The mechanotransduction function of hair cells occurs within the set of stereocilia on each hair cell. The hair cell stereocilia are coupled to one another by intercilliary links including tip links, which are protein structures that connect the tip of each stereocilium to the side of its tallest neighboring stereocilium TRP channels are tetramers comprised of six transmembrane polypeptide subunits that form permeable ion channels. TRP channels play prominent roles in calcium signaling and are ubiquitously expressed and participate in the perception of a wide variety of sensory stimuli including taste , temperature and light Another potential candidate from the TRP family is the transient receptor potential channel melastatin 1 (TRPM1). The TRPM family contains eight channels expressed in the stria vascularis, organ of Corti, outer and spiral ganglion cells TRPM1 causes the complete form of congenital stationary night blindness (cCSNB), an autosomal recessive disease that is characterized by depolarizing bipolar cell (DBC) dysfunction Trpm1 (−/−Trpm1) lacked the b-wave of the electroretinogram (ERG) tvrm27Trpm1, was recently identified in a chemical mutagenesis screen based on a similar ERG b-wave reduction tvrm27/tvrm27Trpm1 mutants showed an eye phenotype consistent with that reported for −/−Trpm1 mice with one important distinction: tvrm27/tvrm27Trpm1 mice retain expression of the TRPM1 protein on the dendritic tips of DBCs Trpm1 mouse mutants.In humans, a mutation in tm1Lex/tm1LexTrpm1, hereafter referred to as Trpm1−/− mice, were generated by Lexicon Genetics and acquired from the European Mouse Mutant Archive (www.emmanet.org). tvrm27/tvrm27Trpm1 mice were derived from a mutagenesis program tvrm27Trpm1 changes a highly conserved alanine at position 1068 to threonine (p.A1068T) Trpm1−/− and tvrm27/tvrm27Trpm1 mice were tested. The genotype of each mouse used in this study was confirmed by PCR (for the knockout and sibling controls) or DNA sequence analysis (for the point mutation tvrm27).All mice were cared for and treated in accordance to the Institutional Animal Care and Use Committee of Case Western Reserve University (CWRU). This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and animal welfare guidelines at CWRU, USA. The protocol was approved by the Institutional Animal Care and Use Committee at CWRU (Protocol Number: 2012-0068).To evaluate hearing, auditory-evoked brain stem response (ABRs) was recorded following presentation of pure tone stimulus to the mouse ear at frequencies of 8, 16 and 32 kHz in accordance with previous descriptions Trpm1−/− or tvrm27/tvrm27Trpm1 mice to controls (Trpm1+/− or Trpm1+/+ mice).The data were collected in Excel® ; statistical analysis was performed using GraphPad Prism® . A Mann-Whitney test was used to compare thresholds from Trpm1−/− allele: Details of the targeted deletion of Trpm1 was reported previously GGCATGTGTAGCTACCACAG and KA1216: GCATAGTCCATGGACCTAGC or KA1215 and KA1217: GCAGCGCATCGCCTTCTATC), platinum Taq polymerase and PCR buffer as suggested by the manufacturer . The reaction mix was cycled 35 times at 94°C, 20 seconds, 55°C, 20 seconds and 72°C, 60 seconds. The PCR products were resolved on 2% agarose gel. An 840 base pair fragment for the wild-type and 280 base pair fragment for the knockout allele are expected.tvrm27Trpm1 allele: The genotype of the tvrm27Trpm1mice was confirmed by amplification of the target sequence by PCR (primers used KA1220: ATTCAGGGAGTGCTTGGTTG and KA1221: GGTATCTGCCACCCTCTCAG) followed by DNA sequence analysis. Genomic DNA (∼500 ng/reaction) obtained from tail biopsies were amplified using Platinum Taq DNA polymerase and reaction buffer supplied by the manufacturer . PCR was carried out for 35 cycles . The 639 base pair PCR product was resolved by agarose gel electrophoresis (2% agarose gel). Primer KA1220: ATTCAGGGAGTGCTTGGTTG was used to sequence the PCR product.Trpm1 mRNA is expressed in the cochlea of −/−Trpm1 mice, RT-PCR analysis was carried out as described previously Trpm1 message was detected by amplification of the target cDNA by PCR (primers used KA1223: ACCTCATGGTGAAGGACTGG and KA1224: TTCCTTTGAGCAAGGCAGTT). PCR amplification of β-actin cDNA  from each RT reaction served as a control for RNA  and RT-PCR protocol. RNA isolated from C57BL/6J (B6) mice cochlea served as a reference for the expected PCR product size for Trpm1 and β-actin from a wild-type tissue. PCR was carried out for 35 cycles . The PCR products (847 base pairs for Trpm1 and 932 base pairs for beta-actin) were resolved using agarose gel (2%) electrophoresis.To determine if TRPM1 and the fact that TRPM1 is expressed in hair cells Trpm1.Trpm1−/− or tvrm27/tvrm27Trpm1 mice and age-matched controls. The ABR waveforms recorded from the Trpm1 mutants were comparable to those obtained from control mice. Trpm1 mutants, and ABR thresholds did not differ from control at any frequency examined .Physiological findings associated with mutations in examined . The gen− allele  or DNA smutation . We conf/− mouse . The dat−/−Trpm1 mice indicates that TRPM1 is not essential for the development of inner ear (vestibular and cochlear) structure or function. More specifically, adult mice display normal inner ear function indicating that hair cell MET function in the mouse inner ear is not dependent on TRPM1. Mice carrying a missense mutation in the pore domain of TRPM1, where the highly conserved alanine at position 1068 is converted to threonine (p.A1068T), also displayed no inner ear phenotype: The tvrm27/tvrm27Trpm1 mice (n = 4) showed hearing thresholds in the normal range  consistent with that reported for −/−Trpm1 mice with an important difference: tvrm27/tvrm27Trpm1 mice retain expression of the TRPM1 protein on the dendritic tips of DBCs tvrm27/tvrm27Trpm1 mice to retain expression of the mutant TRPM1 protein in hair cells. Since comparable ABR thresholds were obtained in the −/−Trpm1 mutant and the loss of function mutant tvrm27/tvrm27Trpm1, we believe the knockout mutation in Trpm1 is less likely to trigger a compensatory response. Further, if TRPM1 mediated its function as a heteromer with a related channel protein, then we would expect to observe loss of hair cell function to some degree in the tvrm27/tvrm27Trpm1 mutants due to a dominant negative effect. Our data suggests that TRPM1 is less likely to function as heteromers in the ear.The second possibility for the lack of an auditory phenotype in TRPM1Trpm1 mouse mutants with different effects on gene expression and trafficking provides important tools with which to understand TRPM1 function in the body.The data presented in this report demonstrates that TRPM1 is not essential for development of hearing or balance and it is unlikely that TRPM1 is the mechanotransduction channel in mouse auditory hair cells. This conclusion is consistent with the lack of auditory involvement in patients with cCSNB due to mutations in TRPM1 is not essential for the development of hearing or balance in the mouse model and it is unlikely that TRPM1 is the hair cell mechanotransduction channel.


The physical state of the patient before surgery is defined by the American Society Anesthesiology (ASA) physical status classification system. The Simplified Acute Physiology Score (SAPS II) provides an estimate for the risk of intrahospital death for ICU patients. The Sequential Organ Failure Assessment (SOFA) score is used to monitor the patient's condition during his/her stay in the ICU, assessing the extent of organ dysfunction or failure. Is the ASA physical status classification system a good prognostic index for determining postsurgical patient's admittance to the ICU? What is the evolution of these patients? Could we predict the outcome of these patients?A retrospective analysis of the ASA, SAPS II and SOFA of all postsurgical patients admitted to an ICU, between 1 May and 31 October 2010.Total ICU admissions: 323 patients, 118 being postsurgical patients . Maximum patient SOFA: between 0 and 19. Patient SAPS II: between 8 and 99. Of the 118 patients, five had ASA 5, a mortality of 100% being expected but only three died. The expected mortality rate of the three deceased  was 5.2%, 92.5%, 98.4%, respectively. The two patients who got better had a SAPS II of 21 and 56 with a maximum SOFA of 4 and 16, which means that they improved significantly, against all odds. Most ICU admitted patients were ASA 3 and ASA 4. Fifty per cent of ASA 3 patients presented a maximum SOFA between 0 and 5; maximum SOFA was higher in 34% of ASA 3 patients (5 to 10) with predicted ICU mortalities of up to 7% and 46%, respectively. Four patients of the ASA 3 group died. Of the ASA 4 patients, 43% had a maximum SOFA between 5 and 10, and 34% presented a lower maximum SOFA (0 to 5). In 10 (26%) ASA 4 patients, maximum SOFA exceeded 11 with a mortality ICU predicted rate of 56%. In fact, five died. The reason for admission to the ICU of the 20 patients with lower ASA  was a need for tighter monitoring or stabilization of postsurgical complications. Indeed, all deaths in the ASA 2 (1/17) and ASA 3 (4/38) groups were related to complications from co-morbidities.ASA 3 and ASA 4 patients are those who benefit the most from a stay in an ICU, enabling one to reduce mortality predicted by SAPS II and SOFA scores. The ASA physical status classification system is not a good indicator of mortality, but its association with SAPS II and maximum SOFA scores define more effectively the severity and prognosis of the postsurgical patient.


Vitamin B12 is an essential micronutrient required for optimal hemopoetic, neuro-cognitive and cardiovascular function. Biochemical and clinical vitamin B12 deficiency has been demonstrated to be highly prevalent among patients with type 1 and type 2 diabetes mellitus. It presents with diverse clinical manifestations ranging from impaired memory, dementia, delirium, peripheral neuropathy, sub acute combined degeneration of the spinal cord, megaloblastic anemia and pancytopenia. This review article offers a current perspective on the physiological roles of vitamin B12, proposed pathophysiological mechanisms of vitamin B12 deficiency, screening for vitamin B12 deficiency and vitamin B12 supplementation among patients with diabetes mellitus. Vitamin B12 or cobalamin is a water soluble vitamin that plays a very fundamental role in DNA synthesis, optimal haemopoesis and neurological function. The clinical picture of vitamin B12 deficiency hence, is predominantly of features of haematological and neuro-cognitive dysfunction > 100 fl) with/without anaemia, ovalocytes, hyper segmented white blood cells (i.e. >5% of neutrophils with ≥5 lobes) and pancytopenia . Due to Several cross sectional studies -12 and c<150 pmol/l or total serum vitamin B12of 150–250 pmol/l and holotranscobalamin ≤37 pmol/l and homocysteine ≥15 μmol/l.Due to the diverse definitions of vitamin B12 deficiency used in most studies and the cultural and religious beliefs in different regions of the world, comparison of the prevalence of vitamin B12 deficiency among T2DM patients and healthy general populations is difficult. In one population based study among 1048 elderly Finnish subjects aged 65–100 years, the total prevalence of definite vitamin B12 deficiency was 12.1% . PreviouIn India, a country with a large proportion of vegetarians due to cultural and religious beliefs, very high prevalence of vitamin B12 deficiency among the general population has been reported. In one study by Yajnik et al. to determine the frequency of vitamin B12 deficiency and hyperhomocysteinemia among 441 healthy middle aged Indian men, vitamin B12 deficiency as defined by vitamin B12 concentrations <150 pmol/L was reported among 67% of the study participants . VegetarIn another cross sectional study among 175 healthy elderly Indian subjects aged >60 years, vitamin B12 deficiency also defined as vitamin B12 concentrations <150 pmol/L was reported among 16% of the study participants . ElevateIn the absence of contraindications like renal and hepatic dysfunction, recent guidelines advocate for the use of metformin as the first line glucose lowering agent concurrently with life style modification approaches ,25. DespThe risk of developing metformin associated vitamin B12 deficiency is greatly influenced by increasing age, metformin dose and duration of use ,18. In ath month [Decrease in vitamin B12 absorption and levels following metformin use typically starts as early as the 4th month . Clinicath month .The proposed mechanisms to explain metformin induced vitamin B12 deficiency among patients with T2DM include: alterations in small bowel motility which stimulates bacterial overgrowth and consequential vitamin B12 deficiency, competitive inhibition or inactivation of vitamin B12 absorption, alterations in intrinsic factor (IF) levels and interaction with the cubulin endocytic receptor . MetformType 1 DM (T1DM) is an auto immune condition that results from auto immune destruction of insulin secreting beta cells of the pancreas. It is invariably associated with other organ and non organ specific auto immune and endocrine conditions leading to development of autoimmune polyglandular syndromes .Pernicious anemia resulting from chronic autoimmune gastritis is highly frequent among patients with T1DM. Chronic autoimmune gastritis and pernicious anemia occurs in about 2% and up to 1% of the general population respectively. Among patients with T1DM, the prevalence is increased by 3 to 5 folds .Vitamin B12 deficiency due to pernicious anemia occurs frequently among patients with T1DM. In one cross sectional study done in South India among 90 patients with T1DM, low vitamin B12 levels were noted among 45.5% of the study subjects as defined by the manufactures’ cut off point of <180 pg/ml and among 54% using the published cut off point of <200 pg/ml . No posiPatients with T1DM actively exhibit auto antibodies to intrinsic factor (AIF) type 1 and 2  and pariThe PCA inhibit secretion of intrinsic factor resulting into pernicious anemia, a condition which is 10 times more prevalent among type 1 diabetic patients than non diabetic patients. Type 1 AIF result into vitamin B12 deficiency by blocking the binding of vitamin B12 to IF. This prevents its transportation to its absorption site, the terminal ileum. These auto antibodies are found in 70% of patients with pernicious anemia .Primary autoimmune hypothyroidism and celiac disease are frequent co morbidities among patients with T1DM -38 and dVitamin B12 deficiency among patients with autoimmune hypothyroidism could be explained by the presence of antibodies to the gastric parietal cells and intrinsic factor, reduced oral intake, dyserythropoesis due to thyroid hormone deficiency and defective absorption due to reduced bowel motility, bowel wall oedema and bacterial overgrowth .Celiac disease which is a highly prevalent autoimmune mediated gastrointestinal condition occurs in 1-16% of T1DM patients compared to 0.3-1% in the general population . IngestiCurrently, there are no published guidelines advocating for routine screening for vitamin B12 deficiency among patients with T2DM. However among type 2 diabetic patients, it is clinically plausible to screen for vitamin B12 deficiency prior to initiation of metformin and later annually among elderly patients with history of long term use of metformin (≥3-4 years), use of high doses of metformin (≥2 g/day), clinically worsening diabetic distal polyneuropathy in the presence or absence of the discussed haematological abnormalities .The screening approach for vitamin B12 deficiency among diabetic patients and the general population is similar. Measurement of the serum vitamin B12 concentrations should be the preliminary screening step for vitamin B12 deficiency among patients with T2DM. Concentrations <200 pg/ml are usually diagnostic of vitamin B12 deficiency while concentrations >400 pg/ml confirm absence of vitamin B12 deficiency .Measurement of serum MMA or homocysteine concentrations is a more sensitive and specific approach for screening especially among type 2 diabetic patients with borderline serum vitamin B12 concentrations of 200-400 pg/ml and subtle haematological manifestations. Serum homocysteine and MMA concentrations of 5-15 μmol/l and <0.28 μmol/l are considered within the normal range respectively ,44.Among patients with T1DM, there are no clear guidelines regarding screening for vitamin B12 deficiency. However, due to the high prevalence of pernicious anaemia and subsequent vitamin B12 deficiency among T1DM patients reported in most cross sectional studies, it would be pragmatic to screen at diagnosis and then later yearly for 3 years, then five yearly thereafter or in presence of any clinical indication since vitamin B12 deficiency can develop at anytime . ScreeniTreatment of vitamin B12 deficiency does not differ regardless of the aetiology. All patients deficient of vitamin B12 should receive replacement therapy with either oral or parenteral vitamin B12 ,47. BothAmong young patients with T1DM and co-existing vitamin B12 deficiency, replacement therapy with daily intra muscular or oral vitamin B12 in the dose of 100μg for a week and then monthly is satisfactory. In severe cases, parenteral or oral administration of 1000 μg/day of vitamin B12 for a week, followed by the similar dose given every week for 1 month and then later monthly is advised .Concomitant folate deficiency should be treated with oral folate replacement in doses of 5 mg daily for 1–4 months. Folate administration prior to correcting vitamin B12 deficiency should be avoided because it results into progression and / worsening of the associated neurological manifestations .Vitamin B12 deficiency and the accompanying hyperhomocysteinemia and elevated MMA levels have been documented to cause a distinct sensory polyneuropathy that closely mimics diabetic neuropathy. Worsening of diabetic neuropathy is also noted among patients with co-existing vitamin B12 deficiency .Vitamin B12 replacement has been shown to cause symptomatic improvement among patients with severe diabetic neuropathy. One meta-analysis showed that if used either alone or in combination with vitamin B complex, there was a significant improvement in the somatic symptoms like pain and paraesthesias. Three included studies also noted an improvement in autonomic symptoms with use of vitamin B12 alone .Similar superior positive findings of reduction in pain and paraesthesias were also noted with use of vitamin B12 as compared to nortriptyline in a randomized, single-blind clinical trial done in Iran among 100 patients with diabetic neuropathy. This study was approved by the Ethics review board of the Isfahan University of Medical Sciences, Iran .19. The doses of vitamin B12 in the multivitamin formulations used by the study subjects in this survey were probably inadequate to correct vitamin B12 deficiency.There are no guidelines to address how often patients with T1DM and T2DM should be supplemented with vitamin B12. The optimal supplementation dose of vitamin B12 is also unknown. A recently published follow-up study from the United States of America showed that administration of oral vitamin B12 among type 2 DM patients on long term use of metformin was ineffective in correcting biochemical vitamin B12 deficiencyThis stresses the need of further studies to determine the optimal vitamin B12 supplementation dose and frequency of supplementation among patients with DM. To avert vitamin B12 deficiency especially among adult type 2 diabetic patients on long term use of metformin, it is plausible to adopt a simple and cost effective supplementation approach in diabetes care. A 1000 μg dose of vitamin B12 given annually would be sufficient to replenish the body’s vitamin B12 stores among this category of patients .Clinical and biochemical vitamin B12 deficiency is highly prevalent among patients with both types 1 and 2 DM. Future large and well designed studies on screening for vitamin B12 deficiency, vitamin B12 supplementation and optimal supplementation dose among type 1 and type 2 diabetic patients are warranted to help guide formulation of guidelines in diabetes clinical care. Annual screening for vitamin B12 deficiency using more sensitive methods like serum homocysteine and methylmalonic acid concentrations  and supplementation should be adopted among diabetic patients with specific risk factors of vitamin B12 deficiency.The authors declare no competing interests.Both authors equally contributed to the development of the concept and manuscript, critically read and approved the final manuscript.