The Notch signalling pathway plays fundamental roles in diverse developmental processes in metazoans, where it is important in driving cell fate and directing differentiation of various cell types. However, we still have limited knowledge about the role of Notch in early preimplantation stages of mammalian development, or how it interacts with other signalling pathways active at these stages such as Hippo. By using genetic and pharmacological tools in vivo, together with image analysis of single embryos and pluripotent cell culture, we have found that Notch is active from the 4-cell stage. Transcriptomic analysis in single morula identified novel Notch targets, such as early naïve pluripotency markers or transcriptional repressors such as TLE4. Our results reveal a previously undescribed role for Notch in driving transitions during the gradual loss of potency that takes place in the early mouse embryo prior to the first lineage decisions. We start life as a single cell, which immediately begins to divide to form an embryo that will eventually contain all the different kinds of cells found in the adult body. During the first few rounds of cell division, embryonic cells can become any type of adult cells, but also form the placenta, the organ that sustains the embryo while in the womb. As cells keep on dividing, they lose this ability, called potency, and they take on more specific and inflexible roles.The first choice embryonic cells must make is whether to become part of the placenta or part of the future body. These types of decisions are controlled by molecular cascades known as signalling pathways, which relay information from the cells surface to its control centre. There, specific genes get turned on or off in response to an outside signal.Previous research showed that two signalling pathways, Hippo and Notch, help separate placenta cells from those that will form the rest of the body. However, it was not known whether the two pathways worked independently, or if they were overlapping. Menchero et al. therefore wanted to find out when exactly the Notch pathway started to be active, and examine how it helped cells to either become the placenta or part of the future body.Experiments with developing mouse embryos showed that the Notch pathway was activated after the very first two cell divisions, when the embryo consists of only four cells. Genetic manipulations combined with drug treatments that changed the activity of the Notch pathway confirmed that Notch and Hippo acted independently at this stage. Further, larger-scale analysis of gene activity in these embryos also revealed that Notch signalling was working in a previously unknown way: it turned off the genes that maintain potency, pushing the cells to become more specialised.Ultimately, identifying this new mode of action for the Notch pathway in the early embryo may help to understand how the signalling cascade acts in other types of processes. This knowledge could be useful, for example, to push embryonic cells grown in the laboratory towards a desired fate. The totipotent mammalian zygote has the self-organising capacity of generating embryonic and extraembryonic structures to build a complete organism . This unCdx2 and Gata3  is translocated into the nucleus where it binds to the transcription factor RBPJ to promote target gene expression. Both Notch and Hippo converge on the TEE, an enhancer upstream of Cdx2  in which we did not detect any CDX2 expression in all embryos of this genotype analysed  to inhibit the Notch pathway  showed lower CDX2 expression and a decrease in the number of CDX2 positive cells  and late (17–32 cells) morulae and analysed CDX2 expression in wildtype and  embryos . We founpression  and numbpression . In contpression . The samve cells , but latve cells . This reCdx2, and that both pathways exert non-redundant roles. Our observations are suggestive of a model where Notch regulates the onset of Cdx2 expression, and the Hippo pathway subsequently maintains its expression.These results indicate that there is an earlier requirement for Notch than for Hippo in the regulation of In light of the above findings revealing a requirement of the Notch pathway for the early stages of mouse preimplantation development, we decided to investigate when Notch is first active, using the CBF1-VENUS reporter line as a transcriptional readout of the pathway. We recovered embryos from the CBF1-VENUS line and found that the reporter was first active in 4 cell embryos, albeit at lower levels than at later stages . The numIn order to follow the dynamics of the reporter and determine how restriction of Notch activity is achieved during development, we performed live imaging for up to 24 hr of embryos from the compacted morula (16 cell) to the early blastocyst stage . After tConfirming previous findings , althougTherefore, the analysis of the CBF1-VENUS line showed that the reporter is active before the first lineage decision is taken, and that differences in the levels of pathway activation in inner or outer cells of the compacted morula correlate with the final position of their descendants in the blastocyst.Mastermind-like 1 (DN-MAML1), a transcriptional co-activator of the Notch pathway, linked to a H2B-eGFP by a cleavable 2A peptide, whose expression leads to the loss of function (LOF) of the pathway, while the third is a gain of function (GOF) cassette through the expression of a constitutively active NICD linked to an HA-H2B-Cerulean , and cells expressing the control, LOF or GOF cassette. Although the probabilities of recombination are higher when the LoxP sites are closer to one another (the control recombination event in this case), we found that most of the recombined cells were Notch GOF  while only a small proportion (11%) were Notch LOF, . If we uNext, we determined the proportion of cells from each population that were in an inner or outer position. Approximately 60% of unrecombined or wild type (red) cells, both being controls, were located at outer positions in both morula and blastocyst stage. However, Notch-LOF cells (green) were enriched at inner positons of the morula or more clearly in the inner cell mas of the blastocyst, while Notch-GOF cells (blue) tended to occupy outer positions . These dCdx2 gene expression and in determining the position of cells to inner or outer locations. To gain further insight into how Notch is acting during preimplantation development, we carried out RNA-sequencing (RNA-seq) in control and -/-Rbpj single morulae. 2028 genes were differentially expressed  and, interestingly, also genes related with the embryonic pluripotency network including Sall1, Sall4, Tbx3 or Sox21 (Dppa3 (Stella) and Prdm14, which have been characterised as naïve pluripotency markers as compared to injected embryos that had been partially  or not  edited  edited . However deleted . These a-/-Rbpj embryos identified genes related with naïve pluripotency among the upregulated genes. Naïve pluripotency corresponds to a state in which cells are not prone to differentiate, in contrast to primed pluripotency . In this study, we show that Notch signalling regulates the early expression of polarity  and it aer cells  suggestepression . Similarpression . In agrepression , is preselopment . In suchelopment .Jag1 in smooth muscle cells  and DNA methyltransferases , which are known to be repressed by PRDM14  in accordance with national and European Legislation. Procedures were approved by the CNIC Animal Welfare Ethics Committee and by the Area of Animal Protection of the Regional Government of Madrid (ref. PROEX 196/14).2, in M16 medium (Sigma) covered with mineral oil . For experiments that did not require culture, embryos were collected at morula or blastocyst stage by flushing the oviduct or the uterus with M2 medium (Sigma) and fixed.Females from the different mouse lines or outbred CD1 were superovulated as previously described , except Immunofluorescence was performed as previously described . The folhttp://katlab-tools.org/) (Images of antibody-stained embryos were acquired on glass-bottomed dishes (Ibidi or MatTek) with a Leica SP5, Leica SP8 or Zeiss LSM880 laser scanning confocal microscopes. The same parameters were used for imaging each experiment. Semi-automated 3D nuclear segmentation for quantification of fluorescence intensity was carried out using MINS, a MATLAB-based algorithm (ls.org/) , and anals.org/) . To corrFor live imaging, embryos were cultured in microdrops of KSOM on glass-bottomed dishes (MatTek) in an environmental chamber as described previously . Images Cell tracking of 3D-movies was carried out using a TrackMate plugin in Fiji . The 3D 2, containing the corresponding pharmacological inhibitor or only DMSO as control until the corresponding stage. The following inhibitors and concentrations were used: 10 or 20 μM of the γ-secretase inhibitor RO4929097  (Two-cell or morula stage embryos were cultured in drops of M16 medium (Sigma) covered with mineral oil  at 37°C, 5% COeckchem)  and 10 μeckchem) .Actin (in mESC or pools of embryos) or 18S rRNA (in single embryos). Primers used are detailed in RNA from pools of 25–30 embryos  or from single embryos (for CRISPR/Cas9 editing) was isolated using the Arcturus PicoPure RNA Isolation Kit (Applied Biosystems) and reverse transcribed using the Quantitect Kit (Qiagen). RNA was isolated from ES cells with the RNeasy Mini Kit (Qiagen) and reverse transcribed using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). cDNA was used for quantitative-PCR (qPCR) with Power SYBR Green (Applied Biosystems) in a 7900HT Fast Real-Time PCR System (Applied Biosystems). Expression of each gene was normalized to the expression of the housekeeping genes Rbpj and Neo were used to genotype the samples. Three mutant and three control (two wildtype and one heterozygote) embryos were selected for analysis. Changes in gene expression were considered significant if associated to Benjamini and Hochberg adjusted p-value<0.05.RNA-seq was performed on single morulae. cDNA synthesis was performed using SMART-Seq Ultra Low Input RNA Kit (Clontech). Library preparation and sequencing was performed by the CNIC Genomics Unit using the Illumina HiSeq 2500 sequencer. Gene expression analysis was performed by the CNIC Bioinformatics Unit. Reads were mapped against the mouse transcriptome  and quantified using RSEM v1.2.20 . Raw expRBPJ binding motifs were located according to the consensus motif from CIS-BP database (M6499_1.02 motif) using FIMO . AssociaSequencing data have been deposited at GEO under accession number GSE121979.2 until the morula stage.For the generation of transient transgenics, F1 (C57Bl/6 x CBA) females were superovulated to obtain fertilized oocytes as described . Each coctatagggcgaattggagctcTTCTTTAGAGGCACCAGTC and ggatccactagttctagagcggccgcATAAAGCCATTTTGCTTAACTG. Primers to amplify and clone the 600 bp Tbx3-i7 region are ctatagggcgaattggagctcCAAGCCAGCCTCAGTCCC and ggatccactagttctagagcggccgcCACACAAGCTTGCCAGCC. Lower case indicates sequence annealing to the plasmid and capital letters indicates sequence annealing to the genome. Constructs were linearized and plasmid sequences removed before microinjection. For H2BmRFP detection, embryos were fixed in 4% paraformaldehyde for 10 min at room temperature and immunostained. Any embryo showing at least one cell expressing the reporter was scored as positive. Due to mosaicism and variability in the amount of transgene present per cell, signal intensity of the reporter cannot be used as a reliable measure of enhancer activity in these assays. When using an empty vector containing only the minimal promoter and the reporter as a negative control, we routinely obtained H2BmRFP expression in approximately 10% of embryos (Each fragment to be tested was amplified from mouse genomic DNA using NEBuilder HiFi DNA Assembly kit (New England Biolabs) and cloned into a modified pBluescript vector  containi embryos .RBPJ) was generated by site-directed mutagenesis (Mutagenex Inc), changing the TGTGGGAAA binding motif to TGTccGAAA. Mutated version of Tbx3-i7 (Tbx3-i7 mutRBPJ) was generated using QuickChange II XL Site-Directed Mutagenesis Kit (Agilent Technologies) changing CGTGGGAAA to CGTccGAAA. Lower case indicates the altered residues. Changes that abolish RBPJ binding were based on previously described mutated versions of the binding site (Mutated version of Tle4-up (Tle4-up muting site .http://crispor.tefor.net/) . Aliquots of 10 µl were used for DNA extraction for PCR genotyping. Mosaic embryos were those where we detected both the deleted and the wildtype allele. The remaining 40 µl were used for RNA extraction for RT-qPCR. To characterize the deletions generated, after PCR genotyping deleted bands of some embryos were gel purified, cloned and sequenced.Two guide-RNAs at 60 ng/µl were incubated with tracRNA (Sigma) at 240 ng/µl for 5 min at 95°C. The hybridised gRNAs were then incubated with the Cas9 protein (PNA bio) at 30 ng/µl for 15 min at RT and microinjected into the pronuclei of (CBAxC57) F1 zygotes. sgRNAs were designed using the CRISPOR tool (or.net/) . The folMycoplasma by the CNIC Cell Culture Facility. Cells were cultured in standard ESC media  supplemented with 15% foetal bovine serum (HyClone), 1% Glutamine, 1% NEAA (Hyclone), 0.1% ß-mercaptoethanol (Sigma) and LIF (produced in-house) or 2i  in dishes seeded with a feeder layer of mouse embryonic fibroblasts (MEFs). Cells were transfected with a Cre expressing plasmid to induce recombination using Lipofectamine 2000 (Invitrogen) for 24 hr. After recombination, cells were sorted using a Becton Dickinson FACS Aria Cell Sorter. To promote spontaneous differentiation, cells were cultured on gelatine-covered dishes for 48–72 hr after LIF or 2i + LIF removal in DMEM (Gibco) supplemented with 20% serum, 1% Glutamine and 0.1% ß-mercaptoethanol (Sigma).iChr-Notch-Mosaic ES cells were generated by Rui Benedito at CNIC and have been tested negative for Statistical analyses were performed with GraphPad Prism seven or R studio. Data are presented as means ± s.d. or means ± s.e.m. as indicted in each case. Differences were considered statistically significant at p-value<0.05. Tests used to calculate p-value are detailed in the figure legends. Student’s t-test was used to compare two groups. ANOVA with Fisher or Bonferroni post-test was used to compare several groups. Fisher’s exact test was used to compare distributions. In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Kevin Struhl as the Senior Editor. The reviewers have opted to remain anonymous.Thank you for submitting your article "Transitions in cell potency during early mouse development are driven by Notch" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:Cdx2 expression in the pre-implantation mouse embryo, and the identification of Tle4 as a Notch-signalling target and agree that the work is, in principle, very suitable for publication in eLife. You will see that the reviewers require substantial additional experimental support for your conclusions.All three reviewers were enthusiastic about the findings suggesting a role for Notch signalling in regulating Essential revisions:-/-Rbpj embryos subjected to single cell sequencing 3) strengthening of the data that shows that Notch and Hippo operate in parallel and 4) a more in-depth analysis of the differentiation potential of iChr-Notch mosaic ES cell.In revising the manuscript we would ask you in particular to address the four main concerns raised during the review process, namely 1) to improve the data to substantiate your claim that Notch signalling regulates cell positioning 2) to increase the number of The issues raised with regard to these points are clearly articulated in the reviewers’ comments below.Reviewer #1:Cdx2 in mouse preimplantation embryos. CDX2 is widely acknowledged to be an important regulator of trophectoderm cell fate, and the role of Hippo signaling in regulating Cdx2 is well established. By comparison, the role of Notch signaling in regulating CDX2 and cell fate is a relatively new topic of inquiry, as is the connection between Notch and Hippo signaling.During the preimplantation period of mammalian development, cell potency is gradually diminished as cells adopt distinct fates, including inner cell mass and trophectoderm. This interesting study from Menchero and colleagues examines the role of the Notch signaling pathway in regulating expression of Cdx2 starting at the 4-cell stage. They conclude that Notch signaling is active before the stage that Hippo signaling is active in embryos. They further claim that Notch signaling dictates cell position, which is important because cell position is thought to dictate trophectoderm versus inner cell mass fate. They also identify new targets of Notch signaling by RNA-seq and evaluate the roles of these genes in ES cell differentiation.The authors employ a variety of clever and sophisticated new genetic tools to manipulate and measure Notch signaling in the embryo. From their analyses, the authors conclude that Notch signaling promotes expression of the trophectoderm gene All of these findings would be of broad interest to embryologists, stem cell biologists, and signaling pathway enthusiasts. However, the evidence to support the study's most exciting conclusions are still quite preliminary (see below). Therefore, while this study has the potential to provide exciting new insight to signaling in blastocyst formation, the story does not yet rest on a concrete foundation, and it's really hard to know if the authors' model is accurate.• Replication unknown. For the majority of figures, the sample size is not reported (neither for number of embryos examined nor for the number of embryo cells examined). Therefore, it is not possible to evaluate whether the reported phenotypes are highly penetrant or even representative.Tead4 null embryos the same cell number? Could this explain the increase in CDX2 expression (as opposed to overexpression of NICD)• Ambiguous embryo staging. Throughout the manuscript, the authors refer to embryos at the morula stage. However, the morula stage encompasses several dynamic stages (8-cell to <32-cell stage). Therefore, it is not possible to know that the comparisons are apples-to-apples across different litter, treatments, and culture conditions, without a more granular view (precise embryo cell number counts). For example, in Figure 1—figure supplement 4, are the • In several places, the data seem overinterpreted. For example:- Figure 2: the claim is made that Verteporfin and RO4929097 inhibit YAP/TEAD and Notch signaling, respectively. However, the authors do not provide an independent assessment of the efficacy of these particular treatments. Does each treatment window allow RO inhibit expression of the N signaling reporter CBF1? Does Verteporfin inhibit the nuclear localization of YAP ? While the authors provided some of these data in their previous manuscript , the treatment windows differ between the two studies. Without knowledge of the degree of inhibition, it is not possible to rule out the possibility that a signaling pathway is active in a particular time or place.- Figure 4: the authors state that N signaling determines cell positioning. However, the authors have not confirmed whether the genetic manipulations . Increasing the sample size would be one way to address this. Finally, it is nice that wild type, unrecombined cells serve as an internal control in these experiments. However, I am slightly concerned that there is no control to examine whether the fluorescent proteins themselves or some other feature not related to Notch, could skew cellular contributions. This is a relatively minor concern. Finally, "Different N levels determines cell position" seems an overly strong conclusion, since many cells with altered N signaling do not change position.Rbpj null embryos, only two null embryos were evaluated . This probably does not provide adequate biological replication and could explain why one of the proposed RBPJ targets (Tbx3) was a false positive.– For the RNA-seq analysis of • In some places, the data do not support the conclusions stated.Cdx2 prior to blastocyst formation is based on analysis of qPCR data shown in Figure 2A, where Cdx2 levels are compared between treated and control embryos (left and right panels). However, if you compare the Cdx2 expression levels in RO-treated and Verteporfin-treated embryos, their levels are similar. By contrast, it is the variation within the control embryos that makes it seem like the effect of RO is statistically significant while the effect of the Verteporfin is not. Finally, do Rbpj null embryos phenocopy RO treatment at the early/morula stage?- The conclusion that Notch, but not YAP/TEAD4 are essential for regulating expression of - Subsection “Absence of crosstalk between the Notch and Hippo signalling pathways in the early mouse embryo”, last paragraph: if the maternally provided Cre recombines in oocytes, then no mosaicism should be observed in embryo cell recombination. Do the authors mean that the maternally provided Cre recombines during the embryonic stages? Probably this is just a semantic issue.Reviewer #2:Having previously shown that Notch signalling regulates the expression of CDX2 in conjunction with YAP/TEAD complexes in TE cells, the authors investigate the role of the pathway during early preimplantation development. Using a variety of approaches , the authors claim that Notch plays an unexpected role in regulating genes that act as repressors of early naïve pluripotency prior to compaction, thereby initiating differentiation events that drive early specification of the trophectoderm.The manuscript is clearly written and presents potentially interesting findings. However, in some cases, it was let down by either its experimental design, a lack of extensive controls or an inappropriate number of independent biological replicates. Please find below a list of major points that I think need to be addressed before the manuscript is ready for publication1) The importance of Notch signalling during early preimplantation development-/-Rbpj embryos develop into apparently morphologically normal blastocysts  and have also completely recovered a normal CDX2 expression pattern after the 16 cell-stage .If Notch signalling is crucial prior to compaction to initiate TE differentiation, it is not clear why 2) Lack of quantification of CDX2 nuclear levels in mutant embryos-/-Rbpj embryos and the impact on CDX2 expression in a separate figure to Figure 1 which makes comparisons between the various genotypes difficult. CDX2 nuclear levels seem lower in -/-; Tead4+/-Rbpj and +/-; Tead4-/-Rbpj embryos , whereas in -/-Rbpj embryos cells are either described as positive or negative for CDX2 . This discrepancy is unexplained in the manuscript but proper quantification of nuclear CDX2 would go some way in properly describing the results and allow comparisons between embryos of different genotypes.The authors present results obtained with -/-Tead4 embryos3) Absence of description of -/-Tead4 embryos is not presented in Figure 1A and elsewhere in the manuscript. Since a previous report suggests that TEAD4 accounts for most of CDX2 expression in the nucleus at E2.5 , it needs to be addressed whether it is truly the case to find what is the exact contribution of both Notch and YAP/TEAD4. In particular at the 8-cell stage, since the authors claim that "there is an earlier requirement for Notch than for Hippo in the regulation of Cdx2, and that both pathways exert non-redundant roles".It seems that the data for -/-Tead4 embryos on CDX2 expression4) Weak effect of N1ICD in -/-Tead4 embryos seems to have a marginal effect at best  as nuclear CDX2 intensity levels go up from around 2.25 to 2.50. It seems that this was quantified at the E3.5. Considering the authors' conclusions regarding the role of Notch signalling prior to the morula stage, it seems that it would be more relevant to investigate the effect of N1ICD in -/-Tead4 embryos at earlier stages and it may be that nuclear CDX2 expression is rescued more impressively at these stages. Additionally, to further asses the relative importance of Notch and Tead4 at early stages, the reverse experiment would be to try and rescue nuclear CDX2 expression in -/-Rbpj embryos using a constitutively active form of TEAD4 for instance.In Figure 1—figure supplement 4, expressing N1ICD in 5) Lack of clarity in the GOF and LOF regarding recombination eventsIn the GOF and LOF experiments, the authors resort to immunostaining to identify the different cell recombination events. However, the GOF cells were triple positive because of antibody cross-reactivity. In addition, the images presented are unclear and, the examples given, fail to alleviate concerns about the validity of the approach and why it was used. If fixation reduced the fluorescence signal, could the embryos have been imaged live prior to that? Alternatively, have the authors attempted to validate their method by other means ? The fact that the less likely recombination event was observed the most often using this method adds to the concern that it may not accurately identify the right type of cell.6) Number of independent biological replicates in RNA-seq experiments-/-Rbpj morulae were used in RNA-seq experiments. It seems unreasonable to justify this by the decision of wanting to strictly compare litter mates.Only 2 7) Quantification of transient transgenic experimentFigure 5E-F – It is not clear why the authors quantified positive and negative embryos. Unless I misunderstood the experiment, it seems that embryos injected with the mutated transcriptional enhancer should either drive H2B-mRFP weakly or not at all in comparison to the wild type construct. As a result, it seems that signal intensity should have been quantified instead.8) CRISPR experimentFigure 5G – Not enough information is provided to understand what mutations were obtained in the CRISPR experiment. Was a repair construct lacking the enhancer sequence introduced to generate a knockin in the endogenous locus? If not, were the different mutated embryos harbouring the same mutations?Reviewer #3:Cdx2 expression and control TE fate. Importantly, by studying null mutants for the Notch effector Rbpj as well as using mosaic Notch gain and loss of function mutations, they also identify that Notch signalling appears to regulate not only TE fate, but have a more general role in promoting differentiation.The current manuscript by Menchero and colleagues describes an analysis of the role of Notch signalling during early pre-implantation development. The authors had previously shown that Notch and Yap/Tead signalling act together to specify trophectoderm (TE) identity. Here using a series of elegant single cell expression analyses combined with genetic and inhibitor experiments they demonstrate that these are parallel pathways, with Notch signalling acting earlier than Hippo signalling to affect Rbpj/Notch signalling is required for this process or if the regulation of Prdm14 and Dppa3 is reflecting other roles of Notch signalling. Therefore, it would be interesting to have a broader picture of the role of Notch in exit of pluripotency and if Tle4 is an important target during this process. Using naïve pluripotent ES cells (2i+Lif) and their differentiation will most likely be the most straightforward way to address this question.Together, these are very interesting findings that provide important new insight into pre-implantation development. The role of FGF and Hippo signalling have been extensively analysed, therefore insight into the roles of other pathways is welcome. The analysis of the relative roles of Notch and Hippo on the regulation of TE fate is conclusive. In contrast to this, the manuscript would benefit from some clarification regarding the role of Notch signalling in the exit of naive pluripotency. Specifically, it would be important to understand if  Essential revisions:In revising the manuscript we would ask you in particular to address the four main concerns raised during the review process, namely:1) To improve the data to substantiate your claim that Notch signalling regulates cell positioning.We have further substantiated our claim that Notch signalling regulates cell position in the preimplantation embryo and the validity of the Notch mosaic mouse model by:- increasing the number of cells examined at the morula stage in the iChr-Notch-Mosaic model.- using non-recombined blastomeres in morula and blastocyst mosaic as a second internal control.- checking the specificity of the antibody panel used to distinguish different recombination events in embryos by testing them in fluorescent-based sortied ES cells pf the same genotype as the iChr-Notch-Mosaic mice.- including a new control using a mouse line carrying the same construct as the iChrNotch-Mosaic, but that does only carry the fluorescent proteins with no fusions to DNMaml1 or NICD (iChr-Control-Mosaic).These results are now shown in revised Figure 4, Figure 4—figure supplement 1, and Figure 4—figure supplement 3.-/- embryos subjected to single cell sequencing.2) To increase the number of RbpjRbpj-/- embryo in the RNA-seq and re-done the analysis, confirming our previous results. These results are now shown in revised Figure 5, Figure 5—figure supplement 1, Figure 5—source data 1, and Figure 5—source data 2.We have included a further 3) Strengthening of the data that shows that Notch and Hippo operate in parallel.We have considerably strengthened the data that shows that Notch and Hippo act in parallel by:- separating the data of the correlation analysis of CDX2, Yap and VENUS for morulas in two different windows based on cell number (8-16 and 17-32 cells).- increasing the number of Verteporfin treated embryos analysed by RT-qPCR in different time windows.- including new controls that show that drug treatments work as expected in these time windows by decreasing fluorescence from the CBF:VENUS reporter or downregulating known YAP-TEAD4 targets in preimplantation embryos.These results are now shown in revised Figure 1, Figure 1—figure supplement 2, Figure 2, and Figure 2—figure supplement 1.4) A more in-depth analysis of the differentiation potential of iChr-Notch mosaic ES cell.We have carried out novel differentiation experiments of ES cells with different Notch levels that were grown and maintained in 2i+LIF conditions, in order to examine the their potential starting from the naïve state. These results are now shown in revised Figure 6—figure supplement 4.The issues raised with regard to these points are clearly articulated in the reviewers’ comments below.See response to each of the issues raised by the reviewers below, along with the description of the corresponding changes in the main text and in the figures.Reviewer #1:[…] All of these findings would be of broad interest to embryologists, stem cell biologists, and signaling pathway enthusiasts. However, the evidence to support the study's most exciting conclusions are still quite preliminary (see below). Therefore, while this study has the potential to provide exciting new insight to signaling in blastocyst formation, the story does not yet rest on a concrete foundation, and it's really hard to know if the authors' model is accurate.• Replication unknown. For the majority of figures, the sample size is not reported (neither for number of embryos examined nor for the number of embryo cells examined). Therefore, it is not possible to evaluate whether the reported phenotypes are highly penetrant or even representative.Rbpj and Tead4 mutants, we have included the number of embryos analysed on top of the corresponding panels . In other cases  we have included this information in the figure legends. In all other cases, samples sizes (number of embryos and/or cells) are indicated in the figure legends.We are sorry for not including this basic information and thanks the reviewer for pointing it out. We have carefully revised all of the data presented in the manuscript and included sample size where needed. For example, in the case of CDX2 staining in the different allelic combinations of the • Ambiguous embryo staging. Throughout the manuscript, the authors refer to embryos at the morula stage. However, the morula stage encompasses several dynamic stages (8-cell to <32-cell stage). Therefore, it is not possible to know that the comparisons are apples-to-apples across different litter, treatments, and culture conditions, without a more granular view (precise embryo cell number counts). For example, in Figure 1—figure supplement 4, are the Tead4 null embryos the same cell number? Could this explain the increase in CDX2 expression (as opposed to overexpression of NICD)Rbpj and Notch1 mutant embryos . We have also changed the image of the double heterozygous +/-;Tead4+/-Rbpj shown in Figure 1A so it is at the same 8-cell stage as other embryos shown in this panel (see response to reviewer #2 below).We agree with the reviewer that cell number counts is a more accurate description of embryos stages in these time windows, and where possible we have separated the data from embryos in early (8-16 cells) and late (17-32) stages. We have done so for the correlations analysis of levels of CDX2, YAP and VENUS shown in Figure 1C and Figure 1—figure supplement 2, allowing us to observe nicely the gradual increase in correlation along development of CDX2 with VENUS (as a readout of Notch activity) and YAP, while correlation of Venus with YAP remains low until the blastocyst. We also show this distinction when we quantify nuclear levels of CDX2 in In the case of the pharmacological treatments , we could not accurately count cell numbers per embryo as these where live and directly placed in lysis buffer for RNA extraction. Manipulation such as fixing and staining nuclei would have compromised the quality of the RNA, thus not allowing to perform RT-qPCR on this limited amount of material.Tead4 mutant phenotype by overexpression of N1ICD , we followed the reviewer’s advice and carefully counted the number of cells of the embryos analysed. While on average embryos had 40-65 cells, one of the /-Tead4 embryos was considerably earlier with only 25 cells. We therefore excluded this embryo from the analysis and included two new -/-Tead4 embryos with a cell number within the range of the others previously analysed. When we repeated the comparison of nuclear CDX2 intensity levels between -/-Tead4 and -/-N1ICD-GFP;Tead4 embryos, we found no significant differences .As for the experiment of the rescue of the ferences . In ligh• In several places, the data seem overinterpreted. For example:- Figure 2: the claim is made that Verteporfin and RO4929097 inhibit YAP/TEAD and Notch signaling, respectively. However, the authors do not provide an independent assessment of the efficacy of these particular treatments. Does each treatment window allow RO inhibit expression of the N signaling reporter CBF1? Does Verteporfin inhibit the nuclear localization of YAP ? While the authors provided some of these data in their previous manuscript , the treatment windows differ between the two studies. Without knowledge of the degree of inhibition, it is not possible to rule out the possibility that a signaling pathway is active in a particular time or place.We agree with the reviewer that we do not formally show that drug treatments in the time windows used result in inhibition of the signalling pathways analysed. We have carried out new experiments to address this issue, treating embryos from the CBF1-VENUS line with the Notch pathway inhibitor RO4929097 from 2-cell to morula and from morula to blastocysts. In both cases, we observe a robust downregulation of VENUS expression, proving that we are effectively inhibition Notch signalling as expected. These results are shown in the new Figure 2—figure supplement 1, and nicely complement our previous findings .As for the effect of Verteporfin on Hippo signalling, it has been described that this drug acts by disrupting the association of the YAP/TEAD complex . It is true, as the reviewer points out, that the report by Wang et al., 2016 suggests that treatment with Verteporfin causes YAP translocation to the cytoplasm. We therefore examined in detail subcellular localization of YAP in Verteporfin and DMSO (control) treated embryos, but we did not observe changes in nuclear or cytoplasmic localization .Cdx2  or Gata3 . Sure enough, we observe a reduction in Cdx2 expression in Verteporfin treated embryos from morula to blastocyst window , and of Gata3  in embryos treated from 2-cell to morula .Nevertheless, we believe this does not mean that the drug is not acting as expected. The above-mentioned paper was carried out in tissue cultured cells, and from the images provided it is difficult to quantitatively assess the extent of nuclear-cytoplasmic shuffling of YAP. An independent way to support our claim that Verteporfin is interfering with YAP/TEAD4 in the time windows used, is to check the expression of known YAP/TEAD4 targets such as As for the degree of inhibition we obtain, we do not suggest that we are completely blocking the pathways. Pharmacological treatments at these stages are tricky, as high doses results in embryo lethality , so at best we obtain a partial reduction. Nevertheless, these experiments also offer unique opportunities to treat the embryos in very precise time windows, what is not possible using genetic approaches.- Figure 4: the authors state that N signaling determines cell positioning. However, the authors have not confirmed whether the genetic manipulations , we would not be able to distinguish green fluorescence from the VENUS reporter or from the EGFP fusion to dominant negative MAML1.Hes1 or Hey genes. However, data from our RNA-seq and that of others show that the levels of expression of these genes in mouse preimplantation development is zero or extremely low. Therefore, we decided to measure nuclear levels of CDX2 as a readout of Notch signalling, as this is the only bona-fide target of Notch identified and validated so far in mouse preimplantation stages. We find a perfect correlation between CDX2 and Notch activity determined by the loss- and gain-of-function systems used. We have included these new results in the revised version of the manuscript and show them in Figure 4—figure supplement 1B.The other possibility is to examine the expression of known Notch targets in other contexts, such as Another concern with this figure is whether an increase in ICM contribution is meaningful, when there were so few N loss of function cells surveyed to begin with (n = 6 cells). Increasing the sample size would be one way to address this.We agree with the reviewer this number was somewhat low, so we have setup and analysed more embryos from this cross in order to increase the sample size. We have now analysed 10 morula and 11 blastocysts. We used embryos containing cells derived from all three possible recombination events plus unrecombined cells, and also embryos that at least had recombined cells for both the Notch gain- and loss-of-function events (see response to reviewer #2 below). We have now analysed 29 Notch loss-of-function cells in the morula instead of 6 as before. Alongside, we have also increased the sample size for all other events  at both morula  and blastocyst  stages. Details are provided in the revised legend for Figure 4. In the new analysis, we still observe differential contribution of cells to inner or out positions dependant on Notch activity, although in a more gradual manner. For example, Notch loss-of-function cells are more frequently located in inner positions in the blastocyst  and also in the morula .Finally, it is nice that wild type, unrecombined cells serve as an internal control in these experiments.We thank the reviewer for this nice and very useful suggestion to use unrecombined cells as a second internal control in addition to the Cherry cells. We have counted these cell in both morula and blastocyst stages finding that their distribution in inner or outer positions is identical to recombined Cherry wild type cells . As noted above, in this re-analysis we have included not only embryos in which we observe all three possible recombination events but also those in which we observed at least recombination leading to Notch loss- and gain-of-function cells . We have updated Figure 4C-E; Figure 4—figure supplement 2A, B; and Figure 4—figure supplement 3B, C (see below) with this data.However, I am slightly concerned that there is no control to examine whether the fluorescent proteins themselves or some other feature not related to Notch, could skew cellular contributions. This is a relatively minor concern.CreERT2Polr2a driver as before . When we examined the contribution of the different recombination events, we find that the majority correspond to Cherry+ cells , the first and most probable event, in contrast to what we observed with the iChrNotch-Mosaic where Cerulean+ Notch gain-of-function cells were more abundant . This control also shows that the type of recombination event that has taken place does not influence the in-out distribution of cells in the blastocyst . Overall, this new set of control experiments confirms and further supports our previous claim that forced activation of the Notch pathway offer a competitive advantage to Cerulean+ cells that therefore are present in a higher than expected proportion in the resulting embryos.The reviewer is right in noting that skews in distribution of cells harbouring different recombination events could be due to factors other than different activation state of the Notch pathway. in order to address this valid and important concern, we have carried out a new set of experiments in which we use a control mice line that carries the same construct as the Notch mosaic, but including only the fluorescent reporters not fused to dominant-negative MAML1 or constitutively active NICD , crossed with the Finally, "Different N levels determines cell position" seems an overly strong conclusion, since many cells with altered N signaling do not change position.influences cell position". We have also changed a sentence in the last section of the Introduction from “differences in Notch levels determines cell fate acquisition in the blastocyst” to “differences in Notch levels contribute to cell fate acquisition in the blastocyst” along the same line.We do agree with the reviewer that the title of this section was an over-interpretation. We have now changed it to "Different N levels - For the RNA-seq analysis of Rbpj null embryos, only two null embryos were evaluated . This probably does not provide adequate biological replication and could explain why one of the proposed RBPJ targets (Tbx3) was a false positive.Rbpj-/- embryos. In our original analysis this was due to the technical hurdles involved in obtaining adequate material, as embryos where processed in batch and genotypes were only inferred later from sequence data. On the other hand, we have used this approach as an initial discovery tool to identify other putative Notch pathway targets, such as Tle4, that we then have confirmed by independent means. Nevertheless, we have carried out new RNA-seq experiments and been able to obtain data from another Rbpj null embryo. We have included this data and repeated the analysis, confirming and replicating our previous results and observations. We have updated Figure 5, Figure 5—figure supplement 1 and Figure 5—source data 1 and 2 correspondingly. We have also updated the GEO database entry with the newly generated data.We certainly have to agree with the reviewer that n=2 is a low number for analysis of Tbx3, we still identify it as a differentially expressed gene containing a putative RBPJ site in an open chromatin region in its vicinity. We therefore do not think it is a false positive, as its expression is significantly reduced in RO treated embryos . The fact that deletion of the putative RBPJ site does not affect enhancer activity in transient transgenic assays, and that the deletion of the corresponding genomic region does not reduce endogenous Tbx3 expression suggests that this is not the only transcriptional input required. Further unidentified sites could be acting together with this unique RBPJ site, as is the case for the TEE enhancer we previously characterized for Cdx2 . A more detailed analysis of the regulation of Tbx3 in the preimplantation embryo is of clear interest and will sure merit further work, but we believe it is out of the scope of the present manuscript.Regarding • In some places, the data do not support the conclusions stated.- The conclusion that Notch, but not YAP/TEAD4 are essential for regulating expression of Cdx2 prior to blastocyst formation is based on analysis of qPCR data shown in Figure 2A, where Cdx2 levels are compared between treated and control embryos (left and right panels). However, if you compare the Cdx2 expression levels in RO-treated and Verteporfin-treated embryos, their levels are similar. By contrast, it is the variation within the control embryos that makes it seem like the effect of RO is statistically significant while the effect of the Verteporfin is not.Cdx2 expression , thus confirming our previous interpretation. Interestingly, what we do observe now is a significant downregulation of Gata3 upon Verteporfin treatment in this early time window. As discussed above, Gata3 has been established as a TEAD4 target independent of Cdx2 , so this might be reflecting this early input of Hippo signalling on Gata3. As for the differential early and late effects of Notch inhibition on Cdx2 expression, we have confirmed the RTqPCR results independently by adding the quantification of nuclear CDX2 expression levels in early morula (8-16 cells) from Rbpj  and Notch1  null embryos .We have followed the reviewer’s advice and increased the sample size for Verteporfin treated embryos in the 2-cell to morula time window. In this way, we have reduce the variability in DMSO treated embryos and still find no significant differences in Finally, do Rbpj null embryos phenocopy RO treatment at the early/morula stage?Rbpj and Notch1 null embryos , showing a reduction of expression in early 8-16 cell morulas but not in later embryos. This data complements our previous estimate of the number of CDX2 positive cells in these embryos , providing a more fine-grained analysis.Yes, as mentioned above we have now included detailed quantification of the nuclear levels of CDX2 in - Subsection “Absence of crosstalk between the Notch and Hippo signalling pathways in the early mouse embryo”, last paragraph: if the maternally provided Cre recombines in oocytes, then no mosaicism should be observed in embryo cell recombination. Do the authors mean that the maternally provided Cre recombines during the embryonic stages? Probably this is just a semantic issue.Sox2-CRE recombines in the oocyte, we should not expect mosaicism. However, as we noted in our previous work , this Cre driver does result in mosaic recombination. Therefore, even if CRE is expressed in the oocyte as previously described , it must be acting at later stages. Nevertheless, we have now removed this data from the revised version of the manuscript (see above).The reviewer is right that if the maternal Reviewer #2:[…] The manuscript is clearly written and presents potentially interesting findings. However, in some cases, it was let down by either its experimental design, a lack of extensive controls or an inappropriate number of independent biological replicates. Please find below a list of major points that I think need to be addressed before the manuscript is ready for publicationWe thank the reviewer for the comments, which we have addressed in this revised version. Among other new experimental data, we have added controls as requested and also increased the number of biological replicates in key experiments.1) The importance of Notch signalling during early preimplantation development-/- embryos develop into apparently morphologically normal blastocysts  and have also completely recovered a normal CDX2 expression pattern after the 16 cell-stage .If Notch signalling is crucial prior to compaction to initiate TE differentiation, it is not clear why RbpjCdx2 and to regulate cell position in the embryo. At no point do we conclude that Notch signalling, or Rbpj, is absolutely necessary to specify the TE, but that in acts in concert and along time with other signalling pathways such as Hippo in this process. The lack of an overt morphological phenotype affecting the TE indicates that this pathway is required but that it can be compensated by others triggered independently by cell polarisation. This fits with the hypothesis that the early mammalian embryo acts as a self-organizing system , integrating genetic, morphological and physical forces to achieve its final form at the blastocyst stage. However subtle, the fact that double -/-;Tead4-/-Rbpjembryos fail to reach the blastocyst stage  further supports a role for Notch, together with Hippo, in these early stages.As we discuss extensively in this manuscript, as well as in our previous work , we contend that the input of Notch is necessary in these early stages for the proper expression of TE markers such as 2) Lack of quantification of CDX2 nuclear levels in mutant embryos-/- embryos and the impact on CDX2 expression in a separate figure to Figure 1 which makes comparisons between the various genotypes difficult. CDX2 nuclear levels seem lower in Rbpj-/-; Tead4+/- and Rbpj+/-; Tead4-/- embryos , whereas in Rbpj-/- embryos cells are either described as positive or negative for CDX2 . This discrepancy is unexplained in the manuscript but proper quantification of nuclear CDX2 would go some way in properly describing the results and allow comparisons between embryos of different genotypes.The authors present results obtained with RbpjRbpj-/- and Notch1-/- embryos, we could carry out correct quantification and compare between embryos. This data is now shown in revised Figure 2D, G and Figure 2—figure supplement 2B, E.We do agree with the reviewer that data at some points is distributed among multiple figures and panels, what can make direct comparisons not easy. We have tried different ways to correct this, but after multiple attempts we still believe that the way the data is presented is the best to follow the story. Regarding quantification of nuclear expression levels shown in Figure 1 for different genotypes, and as discussed the response to reviewer #1 above, we were not able to quantify nuclear expression levels for CDX2 in this set of experiments due to inconsistencies in microscopy settings across different days and litters (see above). However, in an independent set of experiments where we examined nuclear CDX2 expression in morula stage -/- embryos3) Absence of description of Tead4-/- embryos is not presented in Figure 1A and elsewhere in the manuscript. Since a previous report suggests that TEAD4 accounts for most of CDX2 expression in the nucleus at E2.5 , it needs to be addressed whether it is truly the case to find what is the exact contribution of both Notch and YAP/TEAD4. In particular at the 8-cell stage, since the authors claim that "there is an earlier requirement for Notch than for Hippo in the regulation of Cdx2, and that both pathways exert non-redundant roles".It seems that the data for Tead4+/-;Tead4-/-Rbpj embryo that we thought could be representative of the effect of lack of Tead4 on CDX2 expression. Nevertheless, we have now completed this description by including the immunostaining for CDX2 in all genotypes in Figure 1—figure supplement 1B. Unfortunately, and as described above and in the response to reviewer #1, we could not quantify accurately expression in this experiment, so we can only reach conclusions based on qualitative assessment of antibody stainings. In any case, we do feel sure enough to state that CDX2 is expressed in Tead4-/- 8-cell embryos, although at lower levels than what can be observed in wildtypes, and not completely absent as we see in double KO embryos. This is in line with the cited work of Yagi et al.  and that of other authors , that we have included in the revised text of the manuscript.We originally did not include this information due to the high number of different genotypes obtained from the cross for the double KO embryos , that would have resulted in a too complex figure. However, we did show CDX2 expression in a 8cell stage -/- embryos on CDX2 expression4) Weak effect of N1ICD in Tead4-/- embryos seems to have a marginal effect at best  as nuclear CDX2 intensity levels go up from around 2.25 to 2.50. It seems that this was quantified at the E3.5. Considering the authors' conclusions regarding the role of Notch signalling prior to the morula stage, it seems that it would be more relevant to investigate the effect of N1ICD in Tead4-/- embryos at earlier stages and it may be that nuclear CDX2 expression is rescued more impressively at these stages. Additionally, to further asses the relative importance of Notch and Tead4 at early stages, the reverse experiment would be to try and rescue nuclear CDX2 expression in Rbpj-/- embryos using a constitutively active form of TEAD4 for instance.In Figure 1—figure supplement 4, expressing N1ICD in Tead4Tead4-/- embryos. As a matter of fact, and following the advice of reviewer #1, we carefully examined the cell number of the embryos analysed in this experiments. After removing a too early Tead4-/- embryo, and despite including two other embryos with a comparable cell number (40-65), the reanalysis showed no significant difference in CDX2 expression  that we would need to generate from scratch. The time, space in the mouse facility, and effort this would mean made this experiment not feasible in a reasonable time frame in order to revise the manuscript. Therefore, and in light of the results described above and in the response to reviewer #1, we have decided to remove this experiment from the revised version of the manuscript, We believe this does not affect significantly our work or change the conclusions reached.The reviewer rightly points out that there is only a weak effect of N1ICD in rescuing CDX2 expression in Rbpj-/- embryos by forced expression of an active form of TEAD4, this would certainly be of great interest. However, this is an extremely complex experiment from a logistics point of view. We would need to generate and maintain a colony of Rbpj+/- mice big enough to generate KO embryos in sufficient number. Due to the lethality of homozygotes, this would require separate heterozygous male (to use as studs) and female (to use for superovulation and the production of embryos) colonies. On top of that, embryos resulting from the het cross would need to be individually microinjected with the TEAD4 construct, immunostained, and imaged. Only after this process could embryos be genotyped, and only one quarter of them would be homozygous KOs. Taking into account the variability and mosaicism inherent to microinjection experiments, the number of mutant embryos that we would need to analyse in order to obtain a robust result would be large. We simply do not have the capacity, in terms of mouse space, funding and time, to carry out this experiment.As for the reviewer’s suggestion to perform the reverse experiment, that is to rescue the expression of CDX2 in 5) Lack of clarity in the GOF and LOF regarding recombination eventsIn the GOF and LOF experiments, the authors resort to immunostaining to identify the different cell recombination events. However, the GOF cells were triple positive because of antibody cross-reactivity. In addition, the images presented are unclear and, the examples given, fail to alleviate concerns about the validity of the approach and why it was used. If fixation reduced the fluorescence signal, could the embryos have been imaged live prior to that? Alternatively, have the authors attempted to validate their method by other means ? The fact that the less likely recombination event was observed the most often using this method adds to the concern that it may not accurately identify the right type of cell.We agree with the reviewer that ideally using endogenous fluorescence single to follow the recombination events would have been best. Unfortunately, in this mouse line signal is too weak to observe directly, either in live embryos or fixed specimens. That is why we used immunostaining to distinguish different recombined cells. As for the reviewer’s suggestion to genotype individual cells after immunostaining, although possible in principle, in our opinion it would result extremely challenging. First, dissociation of fixed cells from the embryos (as required for immunostaining) is difficult if not impossible without cross-contamination of neighbouring cells. And second, purification of DNA from this material would again be not that easy.Nevertheless, the concern raised by the reviewer is valid, so we devised a way to independently test the panel of antibodies used to identify each different recombination event. For this, we used ES cells carrying the same construct, that were separated by FACS according to their fluorescence . This is the same strategy used in the experiments described in Figure 6. We then tested all three antibodies  in each of the three sorted populations, finding the expected cross-reactivities: anti-CHERRY recognized the wildtype and gain-of-function recombined cassettes, anti-GFP the loss-of-function and the gain-of-function cassette, and anti-HA the gain-of-function cassette. We have included these results in the revised version in Figure 4—figure supplement 1A. As for the concern of the reviewer that the less likely recombination event was observed the most often, we have addressed this issue by including a new control experiment using an iChrControl-Mosaic line .6) Number of independent biological replicates in RNA-seq experiments-/- morulae were used in RNA-seq experiments. It seems unreasonable to justify this by the decision of wanting to strictly compare litter mates.Only 2 RbpjRbpj-/- embryos analysed and repeated the analysis, reaching essentially the same conclusions as before (see response to reviewer #1 above). Updated results are shown in Figure 5, Figure 5—figure supplement 1 and Figure 5—source data 1 and 2, as well as in the GEO database.We have increased the sample size of 7) Quantification of transient transgenic experimentFigure 5E-F – It is not clear why the authors quantified positive and negative embryos. Unless I misunderstood the experiment, it seems that embryos injected with the mutated transcriptional enhancer should either drive H2B-mRFP weakly or not at all in comparison to the wild type construct. As a result, it seems that signal intensity should have been quantified instead.We deeply apologize for the lack of clarity in the way we explain this set of experiments in the text, as the reviewer rightly points out. Reading again the original version, we can see how we took for granted certain methodological aspect that are far from clear and cannot be readily followed in the description we provide. We will try doing so in the following paragraph, as well as correcting the manuscript in order to provide a better explanation of the experiment.Oct4 distal enhancer, or the Cdx2 TEE), this proportion increases to 2-80%. Examples of this approach can be found in previous publications from our group . In the case of the Tle4-up genomic fragment, the wildtype construct drives expression in approximately 30% of microinjected embryos, and thus we consider it as a positive enhancer. However, when we mutate the putative RBPJ binding site only 13% embryos showed reporter activity. We therefore concluded that mutation of this sequence resulted in diminished enhancer activity.  On the other hand, the Tbx3-i7 fragment drove reporter activity in 56% of embryos, what did not change significantly upon mutation of the putative RBPJ site . In this case, we conclude that this genomic fragment does contain enhancer activity, but this is not dependant exclusively on the RBPJ site.In these transient transgenic experiments, each embryo is individually injected with the construct at the one-cell stage, allowed to develop to the desired stage  and then scored for activity of the reporter. Because of the technique itself, embryos will be mosaic for the transgene, and we do not have control of the amount of construct received by an individual embryo, or even by different cells of the same embryo. Therefore, we cannot use signal intensity or number of positive cells as a reliable readout of construct activity. We thus score as positive any embryo that shows activity even in a single cell. When we use an empty vector , we obtain up to 10-15% of positive embryos. However, when testing fragments showing activity (such as the 8) CRISPR experimentFigure 5G – Not enough information is provided to understand what mutations were obtained in the CRISPR experiment. Was a repair construct lacking the enhancer sequence introduced to generate a knockin in the endogenous locus? If not, were the different mutated embryos harbouring the same mutations?Tle4-up element, we recover two different deletions, and in the case of the Tbx3-i7 element three, all within the expected range. This data is now shown in Figure 5—figure supplement 2E, F, and we have corrected the text where necessary.We apologize once more for the lack of experimental detail provided in the original version of the manuscript. For these experiments, we used two gRNA flanking the putative RBPJ binding site, with the aim of generating deletion of approximately 150 bp in both cases. Due to the fact that each individual embryos needs to be processed for genotyping by PCR and expression analysis by RT-qPCR, we cannot obtain enough material to check in the same embryos the exact deletion generated. In order to provide more information on this point, we have carried out new deletion experiments, and in this case used the embryos for genomic DNA extraction, followed by PCR and cloning of the fragments of the expected size for the deletions. Sanger sequencing of individual clones shows that in the case of the deletion of the Reviewer #3:[…] The manuscript would benefit from some clarification regarding the role of Notch signalling in the exit of naive pluripotency. Specifically, it would be important to understand if Rbpj/Notch signalling is required for this process or if the regulation of Prdm14 and Dppa3 is reflecting other roles of Notch signalling. Therefore, it would be interesting to have a broader picture of the role of Notch in exit of pluripotency and if Tle4 is an important target during this process. Using naïve pluripotent ES cells (2i+Lif) and their differentiation will most likely be the most straightforward way to address this question.Tle4 and Tbx3 following the same trend as established early epiblast markers . Interestingly, when we examined the expression of later differentiation markers such as Eomes or Gata6, we did not see any differences related to varying Notch activity. This suggests that Notch signalling is regulating early transition from naïve pluripotency, but not directly specific lineage differentiation events. We have now included these results in the text and present them in Figure 6—figure supplement 4.This is a very interesting suggestion by the reviewer, so we repeated this experiment with ES cells grown in 2i+LIF. We observe the same trend as when using LIF+serum, in that Notch gain-of-function cells upregulated its putative targets


At 24 h titanium treated with SLA showed a higher percentage of cell viability (47.3 ± 8.1%) compared to titanium IV treated with argon plasma, which presented a percentage of 79.1 ± 1.1%. Grade V titanium treated with argon plasma presented a higher viability percentage 91.3 ± 3.0% whereas nontreated Grade V titanium presented 53.3 ± 4.0%. Cells cultivated on the surfaces with an argon-plasma treatment were enlarged in comparison to non-treated discs. The cells with smaller circularity with a greater spread and spindle shape were the ones cultivated on the Grade V titanium surface. Cells seeded on treated titanium IV and titanium V, treated or not, showed higher mitochondrial activity over nontreated titanium IV. (4) Conclusions. Cells cultivated on those Grade V titanium discs that were decontaminated with argon plasma presented higher levels of cell adhesion and proliferation, lower mitochondrial damage and a higher mean cell area compared to those not decontaminated with argon plasma.(1) Background. Titanium is characterized by its biocompatibility and resistance to stress and fatigue. Treatment with argon plasma may favour growth of human osteoblasts with respect to cell adhesion and proliferation. The aim of this study was to analyse the behaviour of human osteoblasts (MG-63) on Grade IV and V titanium possessing a sand-blasted, acid-etched (SLA) surface. SLA is a widely used surface treatment to create micro- and macroretentions to enhance osteoconductive properties on the surface. (2) Methods. One group of each grade of titanium was decontaminated with argon plasma and compared. On each disc, 20 × 10 There are different degrees of titanium in which there is an increase in the elements Fe, O and N as the degree increases. Grade IV titanium is the most commonly used in manufacturing of implants, but also different titanium alloys with other elements  have been made to achieve Grade V titanium  with non-thermal argon plasma. The null hypothesis was that there is no difference at the level of cell morphology, spreading, viability or mitochondrial activity between the surfaces.This in vitro study was carried out at the Centre for Scientific Instrumentation (CIC) of the University of Granada to study osteoblast cell growth on different commercially modified Grade IV and V titanium discs . Four different types of discs were used: Grade IV titanium discs with SLA treatment and no argon-plasma treatment (GR4NT); Grade IV titanium discs treated with SLA and argon plasma (GR4TR); Grade V titanium discs treated with SLA, but not treated with argon plasma (GR5NT) and Grade V discs with SLA and argon plasma (GR5TR). Five discs were used for each group.a) of 1.5 ± 0.2 µm [Treatment with SLA  was based on coarse grain (250 to 500 mm) silicon particles blasted following an acid etching treatment using a mixture of hydrochloric and sulphuric acids on the titanium surfaces, obtaining a surface roughness , placed into an ISO 7 clean room . The machine is equipped with a radio-frequency generator. The gas flow rate is controlled by a caudalimeter MKS Vortex , flow meters and Ar (>99.9990% purity). After reaching 10-Pa base pressure, Ar was introduced in the reactor chamber and the treatment was performed using 590 J/mL power-to-flow-rate ratio. The operating pressure was 20 Pa and the treatment time was 15 min.4 cells. Three discs were employed for each type of surface and the experiments were triplicated. Microwells without discs were used as negative controls. After 4 h of incubation, the cell culture was controlled by an Olympus CKX41SF2 phase contrast microscope . Then, the microwells were completely filled with Dulbecco’s Modified Eagle Medium and incubated for a period of 24 h.The MG-63 cell line was used in this study because it can retain the differentiated phenotype over consecutive subcultures and shows a faster growth than the primary bone-forming lines, which makes it a good in vitro model. MG-63 cells  were cultivated in T75 culture flasks with Dulbecco’s Modified Eagle Medium  enriched with a 1% antibiotic  and 10% foetal bovine serum (Biowest). After performing two cell subcultures and achieving an 80% confluence, the cells were seeded on the disc surfaces in a 24-microwells plate at a previously calculated concentration of 2 × 10Cell viability was assessed after 6 h, 24 h and 48 h, on the 4 types of surfaces , in triplicate, to thereby determine cell proliferation. The WST-1 assay  was used both in the sample and control wells. As a positive control of cell damage, xantine oxidase  was used. Xantine oxidase is an enzyme that upon contact with cells, releases free radicals and therefore reduces the fluorescence intensity issued by the cells on spectrophotometry. A value of 100% of viability was given to the negative control (cells seeded on wells without discs), and viability values of the samples were obtained as a percentage of this value.For morphological cell analysis and determination of mitochondrial energy balance of the cells that adhered to the discs, osteoblasts were cultured on the discs on the four types of surfaces analysed , in triplicate.After an incubation period of 24 h, cell staining was performed using Phalloidine-TRITC , which stains the F-actin of the cell cytoskeleton. DAPI was used  to stain the nuclei. The images were taken with an Axio Imager Z1 fluorescence microscope  with in 40× magnification.Likewise, cell size measurements were taken with an ImageJ v1.50e . The segmentation tool was employed to detect cell shapes automatically and to measure the cell area and circularity. This latter parameter is dimensionless and based on the formula  circle) .Finally, to evaluate the mitochondrial energy balance of the MG-63 cells, microphotographs were taken after culture staining with JC-1 . At low concentrations, this dye forms monomers with green fluorescence; at higher concentrations, it forms aggregates with a red emission. This red/green ratio reveals the membrane potential. Images were taken with the Axio Imager Z1 fluorescence microscope  and the red/green ratio was measured with the FC500MPL cytometer .The comparison of the groups for each of the analysed variables was made using IBM SPSS Statistics 24.0 software . The Kolmogorov–Smirnov test was performed to verify normal distribution. Homogeneity equality of variance was checked using Levene’s test. One-way analysis of variance (ANOVA) was calculated. The Bonferroni test was applied for comparing the groups. A 5% level of statistical significance was established (p < 0.05).Cell viability percentage was calculated regarding control viability (100%) after 6 h, 24 h and 48 h on the four types of surfaces .The data obtained in this study indicate that cell viability measured after 6 and 24 h was higher on both titanium Groups IV and V treated with argon plasma . On the other hand, viability at 48 h was greater on both titanium V groups (treated or not) over titanium Grade IV groups. Nevertheless, no statistically significant differences were observed.Fluorescence microphotographs were used for the morphological analysis after 24 h of incubation. MG-63 showed philopodia-like cell processes that indicate the cell adhesion to the surface .Both argon plasma groups showed a greater average cell area in comparison with those without cold plasma treatment.Statistically significant differences were found between the GR4NT-GR5TR and GR5NT-GR5TR groups. Cells cultivated on the surfaces with an argon-plasma treatment were enlarged in comparison to cells on non-treated discs. Regarding circularity values, the values on the GR4NT surface were larger compared to the other groups. This was interpreted as more round cells and, therefore, less extended or adapted on the surface without treatment with argon plasma. No statistically significant difference was found in the group that did not receive decontamination treatment .Regarding cell area, statistically significant differences were found between groups GR4NT-GR5TR, GR4TR-GR5TR and GR5NT-GR5TR. The cells with smaller circularity values (and therefore with a greater spread and spindle shape) were the ones cultivated on the Grade V titanium surface.Mitochondrial energy balance was analysed on the four surfaces using green and red filters  and confThe ratio was calculated using the results obtained with the cytometer. Higher mitochondrial activity was observed in the MG-63 cells in the GR4TR than in the GR4NT group; nevertheless, lower mitochondrial activity was observed in the GR5TR than in the GR5NT group, with the GR5TR group’s energy balance similar to the negative control.Environmental contamination of the surface of dental implants, prior to their placement, by particles such as carbon, sodium or calcium is an inevitable fact, which involves, according to the literature, a decrease in osteoconductivity or protein adsorption and, consequently, a decrease in the maximum contact of the implant surface with the bone . FurtherThe objective of our research was to study whether decontamination with argon plasma led to greater cell viability compared with the non-decontamination of Grade IV and V titanium implant surfaces with SLA treatment. Furthermore, other cell parameters were assessed, such as cell area and circularity, which could indicate the degree of cell adaptation on the surfaces we studied. Finally, the mitochondrial energy balance was analysed as a measure of cell stress and apoptosis.There are several studies in the literature that have analysed the effect of argon-plasma treatment on titanium surfaces, which has been widely used in recent years, mainly due to its versatility .In this study, it was observed that cell viability after 6, 24 and 48 h was higher on titanium surfaces that were decontaminated with argon plasma. No statistically significant differences were found between the groups after 48 h and this could be due to cell confluence. The viability values found on the titanium IV argon-treated group are similar to the ones found by Rizo-Gorrita et al., who conducted a study on Grade IV titanium implants with SLA surfaces in which treatment with argon plasma was employed and compared with machined surfaces . MG-63 cIn that study, XPS (X-ray photoelectron spectroscopy) analysis  was done and it showed a lower carbon content after argon-plasma treatment. This study completes the latter from a morphological and cellular activity point of view. From both studies it could be interpreted that argon treatment leads to a lower content of contamination particles on the titanium surface and it improves cell viability, leading to better cell spreading on the surface and higher mitochondrial activity.Other authors have also compared surface treatment with argon plasma with techniques such as oxygen plasma or UV radiation. Henningsen et al. compared surface decontamination techniques using UV radiation, oxygen or argon plasma with respect to carbon deposition, viability and cell growth after 48 h. It was found that none of the techniques changed the surface of the discs; however, they did improve their wettability, showing a significant difference of the group compared to the control, but no significant difference between argon plasma and oxygen plasma was found. Furthermore, a reduction in the carbon deposition levels was observed with the three treatments. At the same time, an increase in the thickness of the layer of titanium oxide was observed, which was greater after decontamination with argon plasma. Nevertheless, better anchorage and cell growth was observed at 48 h on the discs treated with argon plasma compared to those treated with oxygen plasma, UV radiation and control. Overall, the authors reported that cells on discs treated with argon plasma showed slightly better behaviour than cells on discs treated with oxygen plasma concerning cell attachment and cell viability . Choi etOn the other hand, Smeets et al. obtained higher cell proliferation and viability after 24 and 48 h with cold atmospheric oxygen plasma compared to cold atmospheric argon plasma when osteoblasts were seeded on a zirconium surface. According to the authors, this phenomenon could be explained by the fact that the wavelength used in oxygen plasma (240 nm) is closer to the wavelength needed for expressing the photocatalytic activity of the zirconia surface (213 nm). UV light and argon plasma generated further wavelengths (about 250 nm) . In anotCanullo et al. and Coelho et al. studied the effect of argon-plasma treatment on early biological response using different surface modification techniques on dental implants. Non-thermal plasma of argon (NTP) showed a positive response in terms of osteoblast adhesion and protein adsorption. Within its limitations, the study highlighted the potential benefits of implant surface treatment with argon plasma (12 min) or UV (3 h) ,24. In oDe Queiroz et al. analysed the effect of argon-plasma treatment on grade II titanium surfaces regarding the quantification of cell viability by comparing it with untreated surfaces. A viability of 72 h in excess of 80% was obtained on the treated surfaces, as well as better cell adhesion on increasing the hydrophility of the surface with argon-plasma treatment . Our stuIn the study by Tavares et al., which was conducted on titanium discs using polished commercially pure titanium  .2) was obtained in titanium V with argon plasma than in the rest of the groups; those cells seeded on SLA surfaces treated with argon plasma were larger. From this result, it may be interpreted that this treatment improves the surface by favouring greater cell spreading.Concerning quantification of the measurement of cell size, a greater average cell area  groups, with a greater spread and spindle shape. Cells seeded on titanium IV were rounder and therefore less spread over the surface, meaning a better cell behaviour on this surface. Similar results were obtained by other authors decontaminating the surface with argon plasma applied using microwaves. It was observed that preosteoblastic cells cultivated on SLA-treated titanium were greater in number and area, showing more spread within cells than those cultivated on untreated surfaces on which cells were seen to be rounder and smaller .Pistilli et al. assessed protein adsorption and cell adhesion on four types of titanium discs in which argon plasma was compared with a control. An increase in levels of protein adsorption and cell adhesion was observed in all groups treated with argon plasma, being those discs with a rougher surface in which higher values were observed .Naujokat et al. conducted an in vivo study on which implant surfaces were prepared with argon plasma for 4 min prior to insertion into pig bone. Neither macroscopic nor microscopic changes were observed after treatment. The primary stability of the implants was not affected. Bone-to-implant contact (BIC) values were investigated. Higher BIC values and bone density were found in those implants that had been prepared in advance. The authors claimed that this technique could be performed routinely by the specialist using a pen-type plasma device before inserting the implant but the effect could be lower than an argon chamber method .In a study performed by Annunziata et al. , the resIn the study performed by Duske et al., the potential of cold argon plasma complemented by 1% oxygen was investigated for treating rough titanium surfaces contaminated with plaque biofilms. To classify the results, treatment with mechanical cleaning (brushing) was included. Neither a single treatment with CAP (cold atmospheric plasma) nor a single brushing provided a completely decontaminated surface. Consequently, osteoblast cells could not develop normally in the presence of bacteria. A combined treatment of brushing followed by CAP treatment led to a clean surface, thus enabling the cells to grow in a comparable fashion to those cells in the sterile control. Therefore, the synergetic antimicrobial effect of the combined application with brushing and CAP seems to be a promising strategy for decontaminating implants with a rough titanium surface .Once again, there is evidence in a study performed by Teixeira et al. that treatment of dental implants with argon plasma at room temperature can produce substantial improvements in biomechanical fixation at the early stages of implantation, improving osseointegration .In a study by Garcia et al., several cleaning processes were carried out that produced different levels of cell adhesion to the surfaces of the implant abutments. From a descriptive perspective, implant abutments cleaned with argon plasma showed greater cell adhesion than the control group . The effect of argon-plasma treatment was also assessed as influential on the distribution of soft tissues. The area occupied by the cells was higher in the plasma group than in the untreated group, with a statistically significant difference. Furthermore, there was no bacterial contamination in 100% of the abutments of the decontaminated group, while in the control group, 40% presented surface contamination. Besides, a biopsy of the gum around the abutments was taken to study the orientation and density of the collagen fibres, those in the plasma group having greater density with a predominance of oblique fibres .In another study, Canullo et al. suggested that the removal of contaminating organic particles from titanium dental implants using argon plasma may enable better maintenance of the bone level compared to steam cleaning for 5 s. The importance of using clean and sterile dental implants in clinical practice became clear .We can say that after comparing titanium surfaces treated or not with argon plasma, significant changes were observed in cellular behaviour and therefore it is necessary to reject the null hypothesis of equal cellular response. Cell viability was increased in cells cultured on treated surfaces at 6 and 24 h, reaching stability in the viability between all groups at 48 h due to the cell confluence. On the other hand, cell size was larger in those cells seeded on treated surfaces and generally with low circularity values with respect to the untreated groups, being interpreted as better cell spreading on the surface. In this study, an assay was performed to determine mitochondrial energy balance using JC-1 staining after 24 h of incubation. This type of analysis has been carried out by other authors on different types of titanium surfaces . The higThe comparison between both types of titanium treated with argon plasma should be cautious. The materials are different. According to our study, GR5TR presented a better overall cellular behaviour with respect to GR4TR. As both materials are suitable to use within the field of oral implantology, there are studies that show a best long-term performance on Grade IV than Grade V titanium, respectively . Neverth2) compared to non-treated Grade V titanium and Grade IV titanium (treated or not). Grade IV titanium is the most commonly used type of titanium, and it showed an improvement in cellular behaviour and greater cell size on surfaces treated with argon plasma. One of the limitations of this study was the number of analysed samples and cell line used, but the data of this study indicate that the use of argon plasma as an intervention for decontaminating the surfaces of titanium implants may lead to an improvement in the growth, cell size, spreading and mitochondrial activity of the MG-63 cells that cover them. Future in vitro and clinical studies should corroborate these experimental results.Considering the results that we obtained in our study, argon-plasma- treated titanium V discs showed higher levels of cell adhesion and proliferation of MG-63 cells, lower mitochondrial damage and a larger cell area (µm


In case of a cardiac arrest, start of cardiopulmonary resuscitation by a bystander before the arrival of the emergency personnel increases the probability of survival. However, the steps of high-quality resuscitation are not known by every bystander or might be forgotten in this complex and time-critical situation. Mobile phone apps offering real-time step-by-step instructions might be a valuable source of information.The aim of this study was to examine mobile phone apps offering real-time instructions in German or English in case of a cardiac arrest, to evaluate their adherence to current resuscitation guidelines, and to test their usability.Our 3-step approach combines a systematic review of currently available apps guiding a medical layperson through a resuscitation situation, an adherence testing to medical guidelines, and a usability evaluation of the determined apps. The systematic review followed an adapted preferred reporting items for systematic reviews and meta-analyses flow diagram, the guideline adherence was tested by applying a conformity checklist, and the usability was evaluated by a group of mobile phone frequent users and emergency physicians with the system usability scale (SUS) tool.P<.01, n=19) and “Mein DRK” .The structured search in Google Play Store and Apple App Store resulted in 3890 hits. After removing redundant ones, 2640 hits were checked for fulfilling the inclusion criteria. As a result, 34 apps meeting all inclusion criteria were identified. These included apps were analyzed to determine medical accuracy as defined by the European Resuscitation Council’s guidelines. Only 5 out of 34 apps  fulfilled all criteria chosen to determine guideline adherence. All other apps provided no or wrong information on at least one relevant topic. The usability of 3 apps was evaluated by 10 mobile phone frequent users and 9 emergency physicians. Of these 3 apps, solely the app “HELP Notfall” (median=87.5) was ranked with an SUS score above the published average of 68. This app was rated significantly superior to “HAMBURG SCHOCKT” (median=55; asymptotic Wilcoxon test: z=−3.63, Implementing a systematic quality control for health-related apps should be enforced to ensure that all products provide medically accurate content and sufficient usability in complex situations. This is of exceptional importance for apps dealing with the treatment of life-threatening events such as cardiac arrest. In case of a cardiac arrest, cardiopulmonary resuscitation (CPR) has to start as soon as possible . HoweverA possible solution to allow immediate assistance might be a mobile phone app offering real-time step-by-step instructions. Mobile electronic devices such as mobile phones are ubiquitously available and a bystander is likely to have them available at site . Mobile The aim of this study was to systematically detect apps giving German or English step-by-step instructions to perform CPR by an adult bystander and determine both their adherence to current medical guidelines and their usability.Our 3-step approach combined (1) a systematic review of currently available apps guiding a medical layperson through a resuscitation situation, (2) an adherence testing to medical guidelines, and (3) a usability evaluation of the determined apps. The study was approved by the institutional review board of Universitätsmedizin Greifswald with the case number BB 055/17.To date, there is no standardized search method for identifying mobile health apps. We used an approach similar to other studies -27. The We focused on the 2 largest and most popular stores for mobile apps—Apple App Store (for Apple iOS apps) and the Google Play Store (for Android apps). Smaller stores such as Amazon App store, Windows Store, Samsung Apps, or Blackberry World were not included in this study -31. The All identified apps were screened. Apps that were found under different keywords but had the same name and were developed by the same company were considered redundant.For assessment of eligibility, the remaining apps were examined regarding the study’s inclusion criteria. Apps not coherent with 1 or more of these criteria were excluded from further evaluation. The following inclusion criteria were used in a descending rank order: availability on both Google Play Store and Apple App Store, language of the app either German or English, free of charge, covered the topic resuscitation, resuscitation of human beings, provides real-time step-by-step instructions, no duplicate under different names, and no technical problems. The inclusion criterion “availability in both stores” was chosen to find an app that can be recommended in, for example, basic life support trainings and is usable by the majority of mobile phone users without being restricted to a subgroup. Apps were classified as duplicates if they were developed by the same company and had the same interface but had different names. All remaining apps were included for evaluation of guideline adherence.We analyzed the quality of content based on the adherence to the European Resuscitation Council Guidelines for Resuscitation 2015 , and theRemaining apps were evaluated using the system usability scale (SUS) developed by John Brooke . This toSauro and Lewis detected a rate of 11% of coding errors when working with a conventional SUS score with positive and negative statements . TherefoBarnum recommends asking multiple groups to evaluate with SUS to emphasize different aspects of a given system . RegardiIn addition, the emergency physicians were asked to rank the apps according to the quality of teaching different aspects of high-quality CPR: 7 aspects were developed in 2 successive brainstorming sessions of 8 emergency physicians and paramedics based on the German translation of the European Resuscitation Council guidelines. Aspects, all researchers involved associated with high quality CPR were collected and evaluated. The criteria were as follows: “The app comprehensively explains the opening of the airway,” “The app points out the problem of agonal breathing,” “The app emphasizes the importance of complete recoil of the chest after each compression,” “The app indicates that pauses in chest compressions should be minimized,” “The app helps the medical layperson to find the correct frequency for chest compression,” “The design and user-interface supports an optimal execution of cardiopulmonary resuscitation,” and “The app requests the medical layperson to continue with cardiopulmonary resuscitation until the arrival of emergency service.”t test was used to assess significance levels. To assess significance level in the absence of normal distribution, Mann-Whitney U test was used between the 2 groups testing the same app and Wilcoxon test between the same people testing different apps.Statistical processing of the data was carried out using IBM SPSS Statistics, version 26.0 , and Microsoft Excel 2010 . We assessed normal distribution by the Shapiro-Wilk test; median and interquartile range were calculated. In case of normal distribution, The results of the systematic review are depicted in The results of the analysis of guideline adherence are depicted in The usability evaluation with the SUS tool was conducted in October 2017. Of the 5 apps that met the criteria, 2 had to be excluded before starting the usability evaluation: 1 app was no longer available in both app stores  and the other app  was excluded because it showed fundamental differences between the version of the Google Play Store and Apple App Store. The version of the Google Play Store contained pictures and movies explaining all steps, and the text was read aloud, if desired. None of these features were available in the Apple App Store version. This gap would profoundly influence the results of the SUS, leading to the decision to exclude this app in the usability evaluation.The group of mobile phone frequent users consisted of 10 participants  with a median age of 23 years (minimum=20 years and maximum=25 years) and the group of emergency physicians of 9 participants  with a median age of 37 years (minimum=32 years and maximum=56 years). The SUS participants assessed the apps on their own mobile phone. iPhone 6Plus, provided by the researchers, was used by 2 emergency physicians.P<.01, n=19) and also significantly higher than “Mein DRK” . The median SUS of “HAMBURG SCHOCKT” was significantly higher than “Mein DRK” . The median SUS scores did not differ significantly between the group of mobile phone frequent users and emergency physicians. The SUS results are shown in The median SUS of “HELP Notfall” was significantly higher than “HAMBURG SCHOCKT”  fulfilled all defined criteria of adherence to the guidelines of the European Resuscitation Council and the American Heart Association. All other apps gave no or incorrect information on at least one relevant topic. Regarding the usability, only 1 out of the 3 apps was evaluated with an SUS score above the published average of 68 .The systematic review of apps available on Google Play Store and Apple App Store took place between May 26, 2017, and June 23, 2017. After excluding redundant hits; duplicates apps; and apps that were not ubiquitously available, free of charge, or did not provide step-by-step instructions in English or German for resuscitation of human beings, 34 apps remained.Similar to other studies, the search in Google Play Store yielded far more results than the Apple App Store . One reaVarious aspects increase the difficulties for a medical layperson to find a suitable app: the sheer volume of apps to choose from can overwhelm the user . Which aOf the 34 examined apps, only 5  apps fit all of our criteria of guideline-adherent resuscitation. This alarming result is concordant with that of other studies examining the content of mHealth apps -54. The It has been shown in multiple studies -60 that Determining a global quality management for apps is complicated by different legislations in different countries and multiple concerned governmental agencies  ,68. AlthOf the 3 apps examined with regard to usability, the app “HELP Notfall” had the highest median SUS score (87.5), followed by “HAMBURG SCHOCKT” (55) and “Mein DRK” (32.5). This difference was seen in both evaluating groups (mobile phone frequent users and emergency physicians). There was no significant difference between the evaluating groups.On the basis of a work by Sauro et al, an SUS score above 68 is rated as a value above average . Such a The systematic review of apps was conducted in the 2 main app stores Apple App Store and Google Play Store, whereas smaller stores such as Amazon App Store, Windows Store, Samsung Apps, or Blackberry World were not included. The review was completed by only 1 researcher (LS), leaving a possibility of misjudging inclusion or exclusion criteria. However, the criteria were phrased distinctly and clearly to diminish this risk, and 2 other researchers (BM and CM) made spot checks. If an app was available either in Google Play Store or in Apple App Store but not in both stores, it was excluded because a recommendable CPR app should be usable by the majority of the population. This criterion led to the exclusion of 2218 search results, which was the majority of all search hits. Furthermore, we decided to choose “free of charge” as an inclusion criterion to enlarge the group of possible future users. A study by Lim et al conducted in different countries worldwide showed that the most important factor influencing people in the process of downloading an app was the price of the app and that 57% of users will not download apps they have to pay for . We do nThe SUS was evaluated by mobile phone frequent users and emergency physicians. As described in the Methods section, groups at high risk of witnessing a cardiac arrest were not included. This might bias the results.To date, it is not known whether the use of an app providing step-by-step instructions in a CPR situation increases the rate or quality of bystander resuscitation and leads to higher survival rates among the victims. To broadly recommend the use of such apps, further studies are needed to evaluate positive and negative effects.Only few studies systematically evaluating CPR apps exist -54,77-79Contrary to all other studies, we did not select a reduced number of apps for the SUS evaluation but did a comprehensive evaluation of all apps that fit the inclusion criteria.This work combined a systematic review of currently available resuscitation apps with an assessment of guideline adherence and an evaluation of usability. The search resulted in 3890 hits. Of 34 apps that met the inclusion criteria, only 5  fulfilled all of the criteria applied to determine guideline adherence. All other apps gave no or incorrect information on at least one relevant topic. Furthermore, our evaluation of usability revealed that only 1 of the 3 apps tested had an above average usability rate according to SUS. Implementing a systematic quality control for health-related apps should be enforced to ensure medical accuracy and sufficient usability. This is of superior importance for apps focusing on the treatment of life-threatening events such as cardiac arrest.