Drosophila heart, focusing on the two major cardioblast subpopulations: generic working myocardial cells and inflow valve-forming ostial cardioblasts. By screening a large collection of randomly induced mutants, we identified several genes involved in cardiac patterning. Further analysis revealed an unexpected, specific requirement of EGF signaling for the specification of generic cardioblasts and a subset of pericardial cells. We demonstrate that the Tbx20 ortholog Midline acts as a direct target of the EGFR effector Pointed to repress ostial fates. Furthermore, we identified Edl/Mae, an antagonist of the ETS factor Pointed, as a novel cardiac regulator crucial for ostial cardioblast specification. Combining these findings, we propose a regulatory model in which the balance between activation of Pointed and its inhibition by Edl controls cardioblast subtype-specific gene expression.For coordinated circulation, vertebrate and invertebrate hearts require stereotyped arrangements of diverse cell populations. This study explores the process of cardiac cell diversification in the  Organs contain many different kinds of cells, each specialised to perform a particular role. The fruit fly heart, for example, has two types of muscle cells: generic heart muscle cells and ostial heart muscle cells. The generic cells contract to force blood around the body, whilst the ostial cells form openings that allow blood to enter the heart. Though both types of cells carry the same genetic information, each uses a different combination of active genes to perform their role.During development, the cells must decide whether to become generic or ostial. They obtain signals from other cells in and near the developing heart, and respond by turning genes on or off. The response uses proteins called transcription factors, which bind to regulatory portions of specific genes.The sequence of signals and transcription factors that control the fate of developing heart muscle cells was not known. So Schwarz et al. examined the process using a technique called a mutagenesis screen. This involved triggering random genetic mutations and looking for flies with defects in their heart muscle cells. Matching the defects to the mutations revealed genes responsible for heart development.Schwarz et al. found that for cells to develop into generic heart muscle cells, a signal called epidermal growth factor (EGF) switches on a transcription factor called Pointed in the cells. Pointed then turns on another transcription factor that switches off the genes for ostial cells. Conversely, ostial heart muscle cells develop when a protein called ‘ETS-domain lacking’ (Edl) interferes with Pointed, allowing the ostial genes to remain on. The balance between Pointed and Edl controls which type of heart cell each cell will become.Many cells in other tissues in fruit flies also produce the Pointed and Edl proteins and respond to EGF signals. This means that this system may help to decide the fate of cells in other organs. The EGF signaling system is also present in other animals, including humans. Future work could reveal whether the same molecular decision making happens in our own hearts. Drosophila heart see for example Tbx20 promotes working myocardial fate by restricting Tbx2 expression and enabling the expression of chamber myocardium-specific genes  comprises several types of cardiomyocytes  and non-contractile pericardial cells (PCs) (Nkx2-5 ortholog tinman (tin), the Gata4 ortholog pannier (pnr) and the Dorsocross1-3 T-box genes  (The ls (PCs) . The prols (PCs) . Subsequled Doc) .+/Even-skipped[Eve]+ EPCs, Tin+ TPCs, and Odd-skipped[Odd]+ OPCs; tin, oCBs (a.k.a. Svp-CBs) specifically express the COUP-TFII ortholog seven-up (svp) and Doc (Doc is repressed in gCBs in a tin-dependent manner (tin expression in turn depends on the Tbx20 ortholog midline (mid/nmr2). The mid gene is first activated in gCB progenitors, but later, like its paralog H15/nmr1, becomes expressed in all cardioblasts , which build the main portion of the contractile tube (‘working myocardium’), and ostial cardioblasts (oCBs), which form bi-cellular valves (ostia) for hemolymph inflow. Due to Hox gene inputs, ostial progenitor specification is limited to the abdominal region . Current and Doc . Previout manner . Robust ioblasts . In oCBsse cells . In the se cells .+ lineage as the most prominent example for the latter  and the chaperon Star (S) for its conversion from a membrane-bound into its active form  and Yan/Aop (encoded by anterior open/aop). While PntP2 becomes an active transcriptional activator upon phosphorylation by MAPK, the transcriptional repressor Yan is negatively regulated by MAPK , relies on the protease Rhomboid  as a crucial regulator of the specification of inflow valve-forming oCBs. Edl possesses a SAM domain, which mediates binding to the SAM domain-containing ETS factors PntP2 and Yan, thereby inhibiting their activity as a transcriptional activator or repressor, respectively  and four generic CBs  per hemisegment are to be expected. To analyze the cardiac pattern of mutants in more detail, we performed immunofluorescent double stainings for Doc and H15  to label oCBs and all CBs, respectively. We then genetically and in part also molecularly mapped the mutations responsible for CB pattern anomalies  or Lbe- (posterior pair) subtypes. Since the above-mentioned spitz group mutants often feature a single pair of gCBs in each abdominal hemisegment, we tested whether these cells are preferentially Lbe+ or Lbe-, which would indicate that one of the two gCB progenitor types may be more sensitive to impaired EGF signaling. However, our finding that both types are about equally represented in rho mutants  has no significant effect on CB number or pattern  the larger the total number of CBs  are siblings of oCBs, we also analyzed PCs in EGF-related mutants by Odd/Eve as well as Odd/Zfh1 double-stainings  is generated by rho overexpression with tinD +tinCΔ4-GAL4  is not significantly reduced in Star mutants if compared to the wild type  but is largely dispensable or even detrimental for the specification of the numb gene  as part of a multi-gene deletion and named this mutant Df(2R)edl-S0520 edl-S0520 and Df(2R)edl-L19 . By contrast, overexpression of edl in the entire mesoderm via twist-GAL4 results in an increase of CB numbers . In agreement with the proposed function of Edl as a negative regulator of PntP2 , all Notch pathway mutants for which CB patterning data are available feature a significantly smaller fraction of oCBs than pnt mutants  regarding to the total number of CBs is also apparent in  mutants . The maxe system , is not svp. In svp mutants all oCBs are converted into Tin+/Doc- CBs due to de-repression of tin  as well as genetic disruption of pntP2 resulted in a near-complete loss of cardiac PntP1 , like PntP1, shows expression in early gCB progenitors  by Mid. Consistent with the latter, we observe a Doc-like expansion of svp expression in mid loss-of-function mutants  observed in the same genetic background argues for the involvement of a Svp-dependent positive feedback loop in ectopic cardiac svp activation in gCBs, as has been predicted previously based on svp overexpression studies  is strongly increased as compared to edl mutants, but reduced in comparison with mid mutants, with total CB numbers being similar to those of edl mutants. In some cases, a near wild-type pattern is observed  was recently shown to drive mid expression specifically in gCBs  devoted to the reception of early gCB-specific inputs. Consistent with our assumption that this enhancer is also a target of Pnt, very little midE19-GFP activity is detectable in pnt mutants (edl overexpression (VP16 (not shown). An observed reduction of midE19-driven GFP levels in many of the retained Tin+ gCBs of rho mutants  over myocardial fate , a phenotype different from that of mutants defective in EGF pathway components upstream of Pnt . By contrast, EGF signals are dispensable (in high doses even unfavorable) for the development of progenitors of oCBs and their sibling OPCs. Thus, EGF signaling clearly has a lineage-specific function, which is most easily explained by a requirement for progenitor selection and cell fate specification. This interpretation does not preclude contributions to cell survival (which might depend on differentiation) or lineage-specific divisions . Notably, in most hemisegments of the analyzed EGF pathway mutants, the number of gCBs is reduced by even numbers and remaining gCB pairs are usually of the same subtype regarding Lbe expression, arguing for a requirement prior to completion of the final mitotic division at the progenitor stage. Since we have only minor evidence for apoptosis and fate conversions into other cell types in EGF-related mutants  we propose that many of the missing gCBs are not selected as highly Delta-expressing CB progenitors upon reduced MAPK signaling activity . Insteadtwi-GAL4 driver reduces the numbers of all cardiac cells except the Eve+ progenitors (tinD-GAL4-driven rho). We propose that the specification of these progenitors requires the context of the definitive cardiogenic mesoderm, whereas premature MAPK activation in all mesoderm cells negates any pro-cardiogenic effects due to the massive expansion of Eve+ clusters  at the expense of the cardiac progenitors in the neighboring C14/C16 clusters  as well as the restriction of PntP2-dependent PntP1 expression in cardiac progenitors. Molecularly, Edl can modulate the activities of PntP2 as well as Yan , Alvarez et al. proposed that PntP2 acts independent of MAPK signaling to limit the number of CBs  does not cause the cardiac phenotypes observed with early pan-mesodermal drivers. Furthermore, depending on enhancer structure, target genes may be either quickly activated by PntP2 alone or require higher levels only achieved upon additional PntP1 buildup (particularly for sustained expression). In case of the mid gene, our model includes both possibilities  motif’ (with the consensus ATT[TG]CC or GG[CA]AAT in antisense orientation)  or Yan . Therefore, a combination of closely spaced Tin and ETS binding sites might be a key signature in enhancers of working myocardial genes, although additional features must be present in their architecture to distinguish them from Tin+ETS binding site-containing enhancers active in pericardial cells or their progenitors  . Mutatiogenitors . The difgenitors . Hence, rho and/or edl. The Rhomboid protease is a key determinant in the decision of which cells will activate the more broadly expressed EGF Spitz and thus emanate signaling activity. A prime candidate for an instructive cue to anterior-posterior positioning within each segment could be Hh , in which we observe a strong increase in the gCBs:oCBs ratio . On the other hand, mutants with diminished Hh pathway activity, including some that were recovered by our EMS screen because of their partial CB losses (i.e. smoothened mutants), do not display a biased reduction of either oCBs or gCBs . Hence, the regulation of rho and the role of hh during CB diversification await more detailed analysis.Our work clearly identifies Pnt and Edl as crucial transducers of spatio-temporal inputs during cardiac cell diversification, but open questions remain regarding the initial source for the differential activities. Our model proposes that factors which tilt the balance between PntP2 activity and Edl will have a major impact on CB subtype choice see . Thus, a studies . In thespression . Furtheron of hh . This woedl expression levels might also determine the outcome of the competition between Edl and Pnt. The edl gene was found to be positively regulated by EGF signaling, and to be a target of Pnt and Yan, and thus was proposed to provide a negative feedback system for EGF inputs edl-S0520, S0167Egfr, S2145Egfr, S2307Egfr, S2561Egfr, S3330kuz, S3832kuz, S0669mam, S4648mam, S0021mid, S2961mid, S1342numb, S3992numb, S4439numb, S3547pyr , UAS-svp.I, 2xPE-twi-GAL4, twi-SG24-GAL4, tinD-GAL4, UAS-tin#2, EC40{tin-ABD}T003-1B1; tin, UAS-p35 were as described previously (1 = aopIPaop (UAS-aop.ACT-IIa (1bib (L19 = Df(2R)edl-L19edl (edl and some neighboring genes deleted) and UAS-edl-X , 8mam  (1spi = IIA14spiIIA (B0453Star (tinCΔ4-GAL4 (Df(2R)Exel7157, and about 180 additional deficiencies spanning chromosome 2 .The mutants pyrS3547 , spiS338eviously . In addi = aopIP , UAS-aop.ACT-IIa , bib1 , mam8 , mid1  , spi1 = IIAIIA14 , StarB04+edl from a transgene were generated anew by standard P-element transgenesis using the previously described edl[+t18] rescue construct edl-S0520 was mapped by non-complementation of lethality with Df(2R)Exel7157, Df(2R)edl-L19 and Df(2R)ED3636, but the cardiac phenotype was only reproduced in trans with Df(2R)Exel7157, Df(2R)edl-L19 and k06602edl. Novel alleles of Egfr and Star were mapped using a candidate gene approach.Novel EMS-induced mutants were obtained from our screen for embryonic heart and muscle defects and mapped to a particular gene through extensive complementation testing analogous to the previously described procedure . Many alS-18a-13b-16c.1 control were analyzed by sequencing of overlapping PCR products covering the coding sequence and splicing sites of the candidate gene as described (Df(2R)edl-S0520 and its approximate break points were determined by iterative PCR amplification tests. The insertion of k06602P{lacW}edl near the edl transcription start site was confirmed by PCR using primers binding to the 5' P end and adjacent genomic DNA. Although the integrity of the both P element ends could be confirmed by PCR, no genomic edl sequences expected next to the 3' P end could be amplified using several primer pairs shown to amplify control DNA. This indicates that k06602P{lacW}edl is associated with a deletion in edl. Details of the deletion mapping are listed in Several EMS alleles and the unmutagenized escribed . Detailsmid180-GFP reporter constructs were generated according to a similar lacZ construct published by EcoRI-CGTGCCTCCCACTTCAGGGCGG-3' and the backward primer 5'-BamHI-TTAATTTCATTTTTCACTCTGCTCACTTGAGATTCCCCTGCTTTGTCTGCGGCTCCGCATTTTCT-3' were used to amply DNA from y w flies. The predicted ETS binding site matching the antisense sequence of published ETS binding motifs (mid180-mETS-GFP by replacing the invariable TCC core (bold) with AAA in the backward primer. Amplicons were cloned into EcoRI/BamHI of pH-Stinger-attB  templates for in vitro transcription were generated by PCR  from DNA of wild-type flies or flies carrying UAS-rho(ve.dC) or UAS-pntP2, respectively.Digoxigenin-labeled antisense riboprobes against eviously . T7 promEmbryos were mounted in Vectashield (Vector Laboratories). Images were acquired on a Leica SP5 II confocal laser scanning microscope and projected using Leica LAS-AF and ImageJ. 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 favorably evaluated by K VijayRaghavan (Senior Editor) and three reviewers, one of whom, Utpal Banerjee, is a member of our Board of Reviewing Editors. The following individual involved in review of your submission has agreed to reveal his identity: Rolf Bodmer (Reviewer #3).Thank you for sending your article entitled "Diversification of heart progenitor cells by EGF signaling and differential modulation of ETS protein activity" for peer review at eLife publication. Nevertheless, the reviewing editor finds considerable merit and novelty in the manuscript given that this is an area of Drosophila developmental biology that needs attention provided by the manuscript.The revisions asked for by the reviewers are rather extensive and in the post-review discussion, the concern was raised that they are not achievable in a two-month period, and even that some of the concerns may be difficult to meet to the satisfaction of an Summary:In this paper, the authors show that EGFR signaling has a specific and defined role in the specification of cardioblast cell fates. Their work shows that active EGFR signaling through the Spitz signaling involving Rhomboid and Star activate the EGFR signaling in the gCB cells and is required for the activation and maintenance of Tinman expression. This process requires the participation of both isoforms of Pointed and also requires the function of Tbx20 ortholog, Midline. In contrast, the specification of the oCB requires an active attenuation of EGFR signaling and this is mediated by the expression of the gene ETS domain lacking (Edl) which has been shown to bind to ETS domain protein and block their function.Drosophila.Using loss of function and gain of function studies the authors convincingly support their model for the role of EGFR signaling in the specification of cardioblast fate during heart development in Essential revisions:1) In the paper the authors refer to the fact that EGFR signaling functions at multiple stages, one being its role in the specification of cardioblast fate. They use loss of function mutants and overexpression and dominant negative version driven by broadly expressed GAL4 lines to come of the conclusion. The results will be strengthened if EGFR signaling is blocked at specific time points to rule out the possibility that a prior EGFR step might contribute to the phenotype.2) The authors should show the activity status of EGFR during these fate specification events by using the activated MAPK antibody to define how this correlates with the phenotype.3) Even though this paper is directed towards defining the role of EGFR in cardioblast fate, the role of Notch signaling during these events have been totally ignored. Is it possible that Notch signaling is required for the activation of Edl, which then blocks the activation of Pnt mediated transcription to promote oCB fate?4) Although the authors suggest that the decrease in gCBs is due to the adoption of other cell fates, they do not identify which fates these cells take on. A better understanding of this through staining with markers for other cell types, clonal analysis or lineage tracing would be helpful, especially to distinguish this model from one in which mutant cells undergo apoptosis or fail to divide.5) While the final figure shows a brief enhancer analysis, this aspect of the paper is under-developed. This point is specifically germane to Figure 7, for which there are a number of concerns:pointed can bind specifically to the ETS sites in the enhancer, and that its binding is abrogated by the mutations.A) There is no indication that B) Since the role for midline in cardiac diversification is expected to occur during stage 12, it is unclear why the enhancer analyses (panels D and E) are presented at stage 16.midE19 enhancer is clearly not reporting the entire midline expression pattern, so how generally applicable are the findings from the analysis of just this single enhancer?C) Related to this, the seven-up at later stages ?D) Since midline is expressed at later stages throughout the cardiac tube, how does it fail to suppress 6) How does hedgehog impact EGF expression and receptor activity? This is a concern because, as the work stands, we are not left with a comprehensive understanding of how cardioblast diversification is triggered that incorporates existing published data.wg and in particular hh signaling, upstream of rho needs better elaboration. Also, the Discussion should be more explicit on the fact that the current studies mainly address a permissive/transducing role for pnt/edl rather than a spatially instructive role, and/or discuss what the scenarios could be that relate to spatially instructive signals.7) The question of spatial integration , attributing the spatial cues to +/Lbe+ and Tin+ only; their distinction should be included in the Discussion.8) Also, the gCBs come in two flavors Tin Essential revisions:1) In the paper the authors refer to the fact that EGFR signaling functions at multiple stages, one being its role in the specification of cardioblast fate. They use loss of function mutants and overexpression and dominant negative version driven by broadly expressed GAL4 lines to come of the conclusion. The results will be strengthened if EGFR signaling is blocked at specific time points to rule out the possibility that a prior EGFR step might contribute to the phenotype.DN driven by 24B-GAL42xPE-twi+how  was merely intended to demonstrate that EGFR signaling activity is required in the mesoderm and exclude indirect influences from potential disruptions in the ectoderm in mutants. The discussed mesodermal EGF signaling activities might not be fully separable by blocking EGFR at specific time points with conventional methods due to potential temporal overlap as well as technical reasons discussed below.First, we want to clarify that the manuscript proposes multiple functions of EGF signaling in the cardiogenic mesoderm only for gCB progenitor selection as such (Pnt-independent function) as well as for a potential boost in MAPK activity that feeds into CB subtype diversification (Pnt-dependent function). Our experiment that uses dominant negative EGFR+ C2 cluster, slightly before MAPK activity in the DA1-generating C15 cluster and the nearly simultaneously appearing cardiogenic clusters C14/C16 . While gCBs and subsets of PCs are strongly affected in amorphic Star and rhomboid mutants as well as in 24B-GAL4>>EGFRDN2xPE-twi+how embryos, oCBs, oCB-related OPCs and Eve-positive pericardial cells are not , demonstrating that mesodermal EGFR activities are clearly specific to particular cardiac lineages. This and the normal early patterning of the rho expression clusters in the cardiogenic mesoderm themselves  also argue against an earlier broad function. The specificity is analogous to published data in certain lineages in the somatic mesoderm, where EGF signaling, via loss-of-function approaches similar to ours, was shown to affect only certain types of muscles  and adult muscle precursors . These functional aspects of EGF signaling in the mesoderm are now mentioned more specifically in the Introduction.Secondly, we would like to point out that there are no indications for an early pan-mesodermal function of EGF signaling. In the early mesoderm, MAPK activity is strictly dependent on FGF signaling , and in the fully migrated mesoderm, earliest MAPK activity is observed in the FGF-dependent EveDN at levels capable of out-competing endogenous EGFR. We have tried to block EGF signaling using EGFRDN driven by the later active tinD+tinCΔ4-GAL4 driver, but this did not result in any abnormalities in CB formation. Since formation of the EGF-dependent DA1 muscle was also entirely normal in this background, we conclude that this driver does not produce sufficient amounts of dominant-negative EGFRDN for EGF signaling attenuation at the time of cardioblast specification. However, a mild but significant reduction in gCBs was observed using 24B-GAL4 without 2xPE-twi-GAL4 , even though this driver was still too weak to generate any DA1 defects (n=3). Aiming at a more efficient, controllable knock-down of EGFR activity we have tried to knock-down Rhomboid, the most restricted factor of EGFR signaling, via inducible RNAi. Unfortunately, this turned out to be unsuccessful, presumably because tissue-specific RNAi is generally very inefficient during embryonic development of Drosophila . Note that effective expression of positively acting factors in the pathway  is inherently easier to achieve, since there is no competition with the endogenous counterpart. This  explains the differences in the efficiency of the different drivers in loss- and gain-of-function experiments.Furthermore, we think that the experiment shown in Figure 1J actually represents a partial and rather late attenuation of EGF signaling due to delays in the GAL4 system in combination with the need to accumulate EGFR2) The authors should show the activity status of EGFR during these fate specification events by using the activated MAPK antibody to define how this correlates with the phenotype.Figure 2. The data are consistent with the assertion that EGF signaling is the major source of MAPK activity in CB progenitors. . This is in agreement with a CB fate maintaining function of FGF signaling proposed by Grigorian et al. To clarify the EGFR status in the cardiogenic mesoderm, we have performed stainings with anti-diphospho-MAPK antibodies in combination with cardiogenic mesoderm markers in wild-type embryos and compared them to stainings of various EGF-related genotypes. The most important results of this analysis are shown in Concerning activated MAPK level differences between CB subtypes: These are technically difficult to prove by standard dpMAPK antibody stainings. Furthermore, differential input to the different progenitor subtypes may not solely rely on level differences, but could also be achieved via the timing/duration of activity. Thus to get a full understanding, activity would have to be monitored for each cell type over the entire time course of early cardiac development. However, since MAPK activation is expected to be upstream of cardioblast diversification events, there is no marker available that will unambiguously identify oCB vs. gCB progenitors in the early cardiogenic mesoderm, making it impossible to assign dpMAPK antibody signals with 100% certainty to particular CB progenitor subtypes at critical stages.3) Even though this paper is directed towards defining the role of EGFR in cardioblast fate, the role of Notch signaling during these events have been totally ignored. Is it possible that Notch signaling is required for the activation of Edl, which then blocks the activation of Pnt mediated transcription to promote oCB fate?pnt phenotypes). Several observations argue against a positive oCB-subtype determining function of Notch signaling.The revised manuscript now more extensively deals with Notch-related aspects  Genetic data show that Notch signaling activity generally antagonizes CB fate.mastermind (mam), big brain (bib) and kuzbanian (kuz). We did not include these mutants in the first version of the manuscript, because Notch signaling components, particularly mam and kuz, have been previously reported to cause supernumerary CBs irrespective of their CB subtype . In response to your request, we added a new figure supplement showing cardioblast patterning data for various Notch signaling-impaired genotypes including a novel molecularly characterized bib allele . Consistent with the previous reports, our data show that Notch signaling mutants feature a strong increase in both oCB and gCB numbers. Some variations in the oCB:gCB ratio are expected to occur between different Notch-related gene mutations or alleles due to variable impact  on the multiple functions of Notch, which involve lateral inhibition and fate decisions linked to asymmetric division, the latter being relevant specifically for the oCB lineage. In case of the novel mam allele, S0669mam, the oCB:gCB ratio was elevated relative to the wild type, but not as much as in pnt mutants. By contrast, in mutants with a strong maternal component such as kuz, the increase in oCBs lags a bit behind that of gCBs presumably due to partially functional lateral inhibition during selection of the oCB progenitors . Many Notch-related mutations may cause oCB:gCB ratios that lie somewhere in between . Of note, hyperactivation of the Notch pathway via constitutive active Notch driven by tinCΔ4-GAL4 or 24B-GAL4how causes a complete or near complete loss of cardioblasts. In those cases where some cardioblasts are formed, the fraction of oCBs was not increased compared to the wild type .; a normal oCB:gCB ratio was also observed upon mesoderm-specific overexpression of dominant-negative MamN driven by edl expression is not abolished in Notch pathway mutants.2) Cardiac edl as a Notch target in a Drosophila cell culture system , it made sense to test whether this is the case in our system as well. Thus, we analyzed edl expression  in wild type embryos  and compared it to embryos with impaired Notch activity . These mutants do not display any obvious reductions of edl expression in the cardiogenic mesoderm, further arguing against a substantial role of Notch in determining CB subtypes.Nevertheless, because a previous report identified 4) Although the authors suggest that the decrease in gCBs is due to the adoption of other cell fates, they do not identify which fates these cells take on. A better understanding of this through staining with markers for other cell types, clonal analysis or lineage tracing would be helpful, especially to distinguish this model from one in which mutant cells undergo apoptosis or fail to divide.+ and Tin-(Odd+) PCs are not expanded in compensation for gCBs, but are rather reduced  as are many somatic muscles . We discuss that a minor fraction of cells may undergo apoptosis or adopt oCB fates. But these effects are small and they do not fully explain the fate of all lost progenitors.The sum of our data suggests that missing gCBs do not simply differentiate into one particular alternate cell type in mutants with diminished EGF signaling. At the end of embryogenesis, TinStar mutants carrying a pnr-lacZ reporter that marks the definitive cardiogenic mesoderm (excluding EPC progenitors) and early CBs and OPCs . Although these experiments tended to favor the idea that the presumptive gCBs cells become part of the pool of uncommitted Mef2+/Lmd+ dorsal mesoderm cells , results were not fully conclusive due to insufficient specificity of the marker. Even in the wild type, low level expression was also detected in a few Mef2+/Lmd+ cells in the vicinity of developing abdominal CBs/OPCs and in a broader dorsal mesoderm region in thoracic segments; later stages also displayed expression in passing hemocytes. The somewhat broader expression of the marker in the wild type is actually consistent with the idea that some more cells obtain the competence to develop into cardiac cells than will be selected by opposing MAPK and Notch activities. Thus, if some pro-cardiogenic cells fail to get properly selected upon attenuation of EGF signaling they will not be discriminable from other cells of the region. A potential increase in the number of unselected pro-cardiogenic cells is technically difficult to prove since this pool is poorly defined and the number of missing progenitors is relatively small in comparison to the remaining dorsal mesoderm cells.To follow up on the fate of the unaccounted cells, we analyzed We modified wording in the respective paragraphs of the Results and Discussion sections to acknowledge the uncertainties regarding the fate of presumptive gCBs.5) While the final figure shows a brief enhancer analysis, this aspect of the paper is under-developed. This point is specifically germane to Figure 7, for which there are a number of concerns:A) There is no indication that pointed can bind specifically to the ETS sites in the enhancer, and that its binding is abrogated by the mutations.midE19 enhancer, we have provided several strong indications for this:Although we agree that there is currently no evidence for direct binding of Pnt to the ETS sites in the midE19 enhancer responds to the absence of Pnt with a very severe drop in activity.First, the Second, upon ectopic Pnt expression the enhancer gains activity even in Tin-negative cardioblasts.mid180, loses activity upon mutation of a single motif that matches the well-characterized ETS binding signature .Third, the minimal enhancer version, mid180 and never observed for the eve mesoderm enhancer. Since the latter is considered a well-established Pnt target , we attribute this to technical difficulties that are likely to be caused by the following obstacles: (1) gCB progenitors only represent a very small number of cells in the context of whole embryos, (2) Pnt is likely to bind many target genes in more abundant cell types, and (3) active Pnt may occupy targets only for a short time.Due to the sequence match with the well-established ETS binding signature, we think that additional in vitro binding studies would yield results that are entirely predictable and thus would not be helpful in generating new insights. Therefore we tried to detect binding in vivo by chromatin immunoprecipitation (ChIP) using anti-PntP1  or anti-GFP with chromatin from C-terminally tagged Pnt::GFP embryos. However, enrichment was inconsistent for the ETS site-containing region of midE19 interaction on the basis of our genetic data. We would like to emphasize that even in the unlikely case that Pnt only indirectly regulates mid expression or via a different enhancer, the regulatory logic in our model (now more in the focus in the simplified model version) would still be maintained. We acknowledge the fact that direct binding has not been validated in the Discussion.Nevertheless, we are still confident about the Pnt-B) Since the role for midline in cardiac diversification is expected to occur during stage 12, it is unclear why the enhancer analyses (panels D and E) are presented at stage 16.Figure 8 and Figure 9). Simultaneously the former Figure 7—figure supplement 1 has been embedded into the main figure.There is a delay in the detectability of the GFP reporter and we find the current representation with the straight alignment of bilateral cardioblast rows more instructive. We have now expanded the previous Figure 7 by including reporter analysis in embryos at earlier stages . For a more suitable presentation we decided to split this expanded figure into two separate main figures .A notable observation is that the less robust C) Related to this, the midE19 enhancer is clearly not reporting the entire midline expression pattern, so how generally applicable are the findings from the analysis of just this single enhancer?midE19-driven reporter gene reflects the alternating pattern of early gCB-specific mid expression and it is responsive to Pnt activity just like the endogenous mid gene during germ band retraction stages . The observation that this enhancer does not drive gene expression in oCBs after germ band retraction as detected for mid in the genomic context , suggests that additional enhancers or interaction with other cis-regulatory regions are required to reproduce all aspects of cardiac mid expression. This is now stated more clearly in the manuscript. What causes the later uniform expression of mid in all cardioblasts is outside of the topic of this work as it is regulated by pnt-independent, yet unknown mechanisms .The pattern displayed by the D) Since midline is expressed at later stages throughout the cardiac tube, how does it fail to suppress seven-up at later stages ?svp gene activity becomes fixed prior to appearance of Mid protein in oCBs. This is consistent with the observation that svp activation requires Tin , which is present in oCB progenitors but absent from maturing oCBs. We added this proposed explanation to the Discussion.One possible explanation is that the chromatin structure determining 6) How does hedgehog impact EGF expression and receptor activity? This is a concern because, as the work stands, we are not left with a comprehensive understanding of how cardioblast diversification is triggered that incorporates existing published data.See joint response to comments 6+7 below.7) The question of spatial integration , attributing the spatial cues to wg and in particular hh signaling, upstream of rho needs better elaboration. Also, the Discussion should be more explicit on the fact that the current studies mainly address a permissive/transducing role for pnt/edl rather than a spatially instructive role, and/or discuss what the scenarios could be that relate to spatially instructive signals.svp expression . Mutants with diminished Hh pathway activity, including some that were recovered by our EMS screen because of their partial CB losses (i.e. smo mutants), do not display a biased reduction of oCBs . On the other hand, loss of patched, which encodes a negative regulator of Hh signaling activity and therefore would be expected to favor oCBs, leads to a relative and in some alleles absolute reduction of oCBs in comparison gCBs, although absolute CB numbers are highly variable between embryos and alleles . In the presumed amorphic mutant 9ptc we found an increase in CB numbers that was solely based on supernumerary gCBs. Although this may also be is caused indirectly via changes in the ectoderm, this observation is in agreement with the previously reported positive input of Hh signaling on rho expression  and the positive impact of rho on gCBs described in our manuscript. Therefore, we added inputs from Hh to the current working model (in the extended version), but labeled it with a question mark for unknown details. Since potentially specific effects of Hh signaling may be masked by interaction with ectodermal Wg signaling or other feedback mechanisms, this is not a matter of a few-months investigation, but a full-scale project. We are currently setting up such a project, because we agree with the critique that differences in Pnt activities, whether promoted by MAPK signaling or attenuated by Edl ultimately must be connected to segmental inputs.To make the issue of spatial inputs more apparent and also to address the second part of this comment we have made modifications to the discussion, referring more extensively to published data  and mentioning some of our own unpublished observations regarding components of the Hh pathway in the final paragraph of the Discussion. Our current data are not in agreement with the view that the Hh pathway directly promotes oCB fate/+/Lbe+ and Tin+ only; their distinction should be included in the Discussion.8) Also, the gCBs come in two flavors Tin+/Lbe+ and Tin+/Lbe- gCB subtypes and analyzed the frequency of their retention in mutants with impaired EGF signaling. Although we did not detect a significant bias in the retention/loss of a particular  gCB subtype, this analysis provided some valuable additional information, which we added to the manuscript . We found that residual gCBs usually occur either as Lbe+ or Lbe- pairs, but rarely as mixed Lbe+/Lbe- pairs or single cells. This further supports our notion that EGF function is required prior to completion of the final mitotic division at the gCB progenitor stage.In response to the reviewers request, we paid more attention to the distinction of Tin


Primary cilia detect extracellular cues and transduce these signals into cells to regulate proliferation, migration, and differentiation. Here, the function of primary cilia as signaling hubs of growth factors and morphogens is in focus. First, the molecular mechanisms regulating the assembly and disassembly of primary cilia are described. Then, the role of primary cilia in mediating growth factor and morphogen signaling to maintain human health and the potential mechanisms by which defects in these pathways contribute to human diseases, such as ciliopathy, obesity, and cancer are described. Furthermore, a novel signaling pathway by which certain growth factors stimulate cell proliferation through suppression of ciliogenesis is also described, suggesting novel therapeutic targets in cancer. Primary cilia detect extracellular cues, such as mechanical flow and chemical stimulation, and transduce these signals into the cell.Figuretrans‐autophosphorylation of multiple tyrosine residues. Trans‐autophosphorylation releases cis‐autoinhibition, which activates various downstream signaling proteins containing Src homology‐2 (SH2) or phosphotyrosine‐binding (PTB) domains. These proteins bind to the autophosphorylated RTKs through their SH2 or PTB domains and propagate downstream signaling pathways, such as the mitogen‐activated protein kinase (MAPK) cascade, phosphatidylinositol 3‐kinase (PI3K)‐Akt kinase (AKT) cascade, and Janus kinase (JAK)‐signal transducer and activator of transcription (STAT) cascade.The signaling of growth factors and morphogens is also mediated by primary ciliaVarious negative‐feedback mechanisms have important roles in the regulation of growth factor signaling.2Primary cilia have three compartments, the basal body, the transition zone, and the axoneme.3Two pathways to generate primary cilia, namely, the extracellular and intracellular pathways, have been demonstrated in the assembly of primary cilia responding to serum withdrawal.The current understanding of the intracellular pathway of ciliogenesis can be summarized as follows. First, small cytoplasmic vesicles are transported from the Golgi apparatus to the mother centriole, along using microtubule and actin networks in kinesin/dynein and myosin‐dependent manners, respectively, resulting in the formation of a ciliary vesicle and the conversion from the mother centriole to the basal body.FBXW7. The ubiquitinated NDE1 is then degraded in proteasome, which inhibits the formation of the ciliary disassembly complex, resulting in ciliogenesis.The assembly of primary cilia responding to serum withdrawal is also induced by inhibition of Aurora A (AurA), one of the most important mitotic kinases for cell cycle control, which is also involved in the disassembly of primary cilia (described in the following sections). AurA associates with nudE neurodevelopment protein 1 (NDE1), oral‐facial‐digital syndrome 1 (OFD1), and centrosomal P4.1‐associated protein (CPAP) as a ciliary disassembly complex where CPAP works as a scaffold protein.42+/calmodulin signaling, the noncanonical wingless (WNT) pathway, and phosphatidyl inositol signaling.2+ binds to multiple motifs on AurA and activates it.2).When quiescent mouse 3T3 fibroblasts or human RPE1 cells are stimulated with serum, primary cilia disassemble after serum stimulation.5FigureWe have recently found that knockdown of trichoplein, a centriolar protein originally identified as a keratin‐binding protein (and now as an activator protein of AurA),KCTD17, was the E3 ligase that could polyubiquitinate trichoplein.KCTD17 was constant regardless of the presence or absence of serum, we hypothesized the existence of deubiquitinases (DUBs) that could deubiquitinate trichoplein dependent on the serum. Using global screenings of DUBs and their subsequent characterization, we revealed that ubiquitin‐specific peptidase 8 (USP8) could deubiquitinate and stabilize trichoplein and that the DUB activity of USP8 was regulated by EGFR tyrosine kinase through phosphorylation of tyrosine residues at 717 and 810 of USP8.Given that trichoplein was stabilized by proteasome inhibitors in serum‐starved RPE cells, we performed global screenings of E3 ligases and revealed that potassium channel tetramerization domain‐containing 17 (KCTD17), associated with the scaffold protein Cullin 3 (Cul3) and RBX1 as an E3 ligase complex called CRL3KCTD17 and suppresses ciliogenesis in the presence of serum, whereas NDEL1 is degraded via the ubiquitin–proteasome system (UPS) in the absence of serum, resulting in the disappearance of trichoplein at the mother centriole and ciliogenesis.The balance between ubiquitination and deubiquitination of trichoplein is also modulated by NDE1‐like 1 (NDEL1),6RSK)‐Na+/H+ exchanger (NHE1) and PI3K‐AKT‐ NHE1 pathways. If primary cilia are defective, these signaling modules are not activated, suggesting that localization of PDGFRα in primary cilia is critical in response to PDGF‐AA to regulate cell migration. The activated NHE1 translocates to the leading and mobile edge of the cell  and organizes the actin cytoskeleton with Ezrin/Radixin/Moesin proteins, which enable directional cell migration. The localization of PDGFRα is regulated by Cbl E3 ubiquitin ligases.Primary cilia and RTKs, especially PDGFRα, play critical roles in cell migration.72+, currents.2) partly through the phosphorylation of PIP2 into phosphatidylinositol ‐triphosphate P3) by PI3K and the hydrolysis of PIP2 by PLCγFigure2. When EGF signaling is activated, the local concentration of PtdInsP2 is decreased, which causes activation of PKD2, resulting in increased Ca2+ influx.EGF signaling regulates not only cell proliferation but also the development of various tissues and the maintenance of body homeostasis. In the kidney, EGFR is localized in the primary cilia of epithelial cells of the thick ascending loop of Henle and the distal convoluted tube within the vicinity of polycystin 2 (PKD2).8FigureAlthough the human body is externally symmetrical, the visceral organs are arranged asymmetrically in a stereotyped manner.9Figureβγ. The free Gβγ triggers the release of Tctex‐1 from the dynein complex, enabling subsequent phosphorylation and recruiting of the phosphorylated Tctex‐1 to the transition zone of the primary cilia of radial glial cells at the ventricular zone.The formation of the cerebral cortex begins with the transition from neuroepithelial to radial glial cells, the proliferating progenitors of the developing neocortex, in the ventricular zone10FigureCiliopathies, such as Bardet‐Biedl syndrome (BBS) and Alström syndrome (ALMS), often accompany obesity, suggesting that the impairment of primary cilia may be involved in the pathogenesis of obesity.POMC/αMSH and AgRP‐producing neurons in the arcuate nucleus send their axonal projections to second‐order neurons in the paraventricular nucleus (PVN). Melanocortin 4 receptor (MC4R), a common receptor for αMSH and AgRP, is strongly expressed in the ciliated PVN neurons expressing single‐minded 1 (SIM1), where αMSH and AgRP activate and inhibit MC4R, respectively.11FigureAlthough primary cilia are lost in a wide range of cancer types, as described in the section below, primary cilia can also promote tumor progression in different types of cancer, including medulloblastoma, basal cell skin cancer, and basal‐like breast cancer.The context‐dependent functions of primary cilia are also related to the resistance mechanism of SMO inhibitors.2+nS degron in most of its substrates, including GLI2, GLI3, WW domain‐containing transcription regulator 1 (WWTR1), β‐catenin, programmed cell death 4 (PDCD4), and NFκB inhibitor (IκB).2+nS degron in GLI2 and GLI3 are phosphorylated by protein kinase A, glycogen synthase kinase 3, and casein kinase 1. β‐TRCP recognizes the phosphorylated degron and processes GLI2/3 to GLI2/3‐R, resulting in the suppression of hedgehog signaling.Hedgehog signaling in cancer can be regulated by the UPS.Primary cilia disappear from growing cells, even in the context of tumors where progression is positively correlated with the presence of primary cilia.12Primary cilia are lost in a wide range of cancer types, including clear‐cell renal‐cell carcinoma,Figure3, serving as a docking site for proteins that have phospholipid‐binding domains, including AKT. If AKT binds to PIP3, it is activated through autophosphorylation on two stimulatory serine/threonine residues. The activation of the Ras‐Raf‐MEK‐ERK and PI3K‐AKT pathways promotes cell proliferation. The recent findings that activation of RTKs, such as EGFR, deciliates primary cilia through activation of a USP8‐trichoplein‐AurA pathway may provide a novel explanation for the loss of primary cilia frequently observed in cancer cells with aberrant RTK signaling.RTK signaling is also frequently activated in various cancer cells13Growth factor and morphogen signaling through primary cilia are undoubtedly important for both health and disease. Although many mechanisms underlying these signaling events have been elucidated, many questions still exist. The clinical phenotypes observed in ciliopathies are highly heterogeneous, even in the same syndrome, depending on the organ system in which primary cilia are impairedOmics approaches, such as transcriptome analysis and mass spectrometry, may be useful tools to reveal the complexity of growth factor and morphogen signaling through primary cilia. For example, transcriptome analysis uncovered that TGFβ signaling that is activated at the ciliary pocket, the proximal part of the primary cilium residing in the cytoplasm within an invagination of the plasma membrane, is associated with differentiation into cardiomyocytes.The molecular mechanisms underlying the regulation of cell proliferation by growth factors and morphogens through primary cilia remain to be completely elucidated. The recent finding that activation of EGFR induces deciliation through a USP8‐trichoplein‐AurA pathway suggests that inhibition of this pathway is able to block the proliferation of some cells. DUBs  can suppress ciliogenesis.The authors declare no conflict of interest.


It is estimated that up to 20% of all types of human cancers worldwide are attributed to viruses. The genome of oncogenic viruses carries genes that have protein products that act as oncoproteins in cell proliferation and transformation. The modulation of cell cycle control mechanisms, cellular regulatory and signaling pathways by oncogenic viruses, plays an important role in viral carcinogenesis. Different signaling pathways play a part in the carcinogenesis that occurs in a cell. Among these pathways, the Wnt signaling pathway plays a predominant role in carcinogenesis and is known as a central cellular pathway in the development of tumors. There are three Wnt signaling pathways that are well identified, including the canonical or Wnt/β-catenin dependent pathway, the noncanonical or β-catenin-independent planar cell polarity (PCP) pathway, and the noncanonical Wnt/Ca2+ pathway. Most of the oncogenic viruses modulate the canonical Wnt signaling pathway. This review discusses the interaction between proteins of several human oncogenic viruses with the Wnt signaling pathway. Viruses are known as one of the major agents of human cancers . Risk faReviewOncogenic viruses and cellular pathwaysViral Carcinogenesis is a time-consuming process in which various cellular pathways are involved . Many ofWnt signaling pathways2+ pathway  family receptor that transduces the corresponding signal to the phosphoprotein Dishevelled (Dsh) protein inside the cell cytosol , 9. Dsh Epstein bar virus (EBV) and Human herpesvirus-8(HHV-8) Herpesviridae family. These viruses have a coding-genomic double-stranded DNA for enzymes involved in replication, repair, and biosynthesis of viral nucleic acid. EBV and HHV-8 can establish latent infections within the lymphoid cells and tissues that can be activated when the host immune system is suppressed , compared to EBV-negative lines, it was shown that in contrast to epithelial cells, β-catenin accumulates in the cytoplasm but not in the nucleus . In. InHerpe nucleus , 64, 65.Hepatitis B virus (HBV) and Hepatitis C virus (HCV)Hepadnaviridae family, containing a partially double-stranded DNA genome and several structural and soluble proteins. One of the most important proteins of HBV is HBx, considered as an oncoprotein because of its carcinogenic characteristics  -68. HBV eristics , 70. Severistics . It has eristics , 71, 72.eristics . Researc pathway , 73, 74.in vitro , 25, 26.in vitro , 76. Resin vitro .Flaviviridae family. HCV contains several structural proteins  and non-structural proteins , associated with the pathogenesis of HCV  have suggested that the HCV core protein increased the expression of Wnt signaling proteins, via hypermethylation of the SFRP1 promoter, resulting in epigenetic silencing of SFRP1 expression  and Human Immunodeficiency Virus (HIV)et al. (2013) showed that interactions between the nef virus and β-catenin proteins resulted in T-cell transmigration  suggested that HIV infection and HIV-1 gp120 cause accumulation of Wnt3a and β-catenin and s activation of the Wnt/β-catenin signaling pathway Papillomaviridae (wart viruses) family. These viruses are equipped with several oncoproteins and are associated with some malignancies in humans and other species  is a member of the fections , 107.Although a direct association between CMV and carcinogenicity has not been found, the evidence suggests that the virus can interfere with cellular pathways such as the Wnt signaling pathway , 108. ThIn addition, dysregulated Wnt signaling results in impurities in the fetus and its growth . StudiesAdenoviridae family), containing a double strand DNA genome, have different viral groups. Adenoviruses infect both humans and animals , prevention of β-catenin proteasomal degradation via interaction with the destruction complex proteins such as GSK3β and APC.Taken together, according to the mentioned mechanisms and analysis of the results it seems that oncogenic viruses have common evolutionary mechanisms for modulation and interaction with the Wnt signaling pathway especially canonical pathway, which results in carcinogenesis of the tissue involved. Therefore, it is seen that interactions between viral oncoproteins and the Wnt signaling pathway potentially have an intense role in carcinogenesis, which can provide new insights into the pathogenesis of viral cancers and possible therapeutic strategies. However, more precise molecular mechanisms of these interactions and their role in viral carcinogenesis need to be further studied.


Importantly, pre-treatment of cells with 2-deoxy-D-glucose reduced TBT-mediated lipid accumulation, thereby suggesting a role for active glycolysis during the process of lipid accumulation. As other RXRA binding ligands can promote RXRA protein turnover via the 26S proteasome, TBT was tested for such an effect in the two liver cell lines. We found that TBT, in a time- and dose-dependent manner, significantly reduced steady-state RXRA levels in a proteasome-dependent manner. While TBT promotes both RXRA protein turnover and lipid accumulation, we found no correlation between these two events at the single cell level, thereby suggesting an additional mechanism may be involved in TBT promotion of lipid accumulation, such as glycolysis.A subset of environmental chemicals acts as “obesogens” as they increase adipose mass and lipid content in livers of treated rodents. One of the most studied class of obesogens are the tin-containing chemicals that have as a central moiety tributyltin (TBT), which bind and activate two nuclear hormone receptors, Peroxisome Proliferator Activated Receptor Gamma (PPARG) and Retinoid X Receptor Alpha (RXRA), at nanomolar concentrations. Here, we have tested whether TBT chloride at such concentrations may affect the neutral lipid level in two cell line models of human liver. Indeed, using high content image analysis (HCA), TBT significantly increased neutral lipid content in a time- and concentration-dependent manner. Consistent with the observed increased lipid accumulation, RNA fluorescence  Endocrine disrupting chemicals (EDCs) are a growing class of compounds with highly varied structures and mechanisms of action that interfere with endocrine and metabolic functions by posing as either agonists or antagonists of physiological hormones ,2. A subbona fide obesogen and an established reference compound. Here, we analyzed, by imaging and high content analysis (HCA), the effects of TBT in human liver cell lines and determined that even picomolar concentrations of TBT, lower than levels found in human samples (~20 nM), cause a glycolysis-dependent increase in lipid content. We delved into TBT mechanism of action and showed that TBT can quickly activate lipogenic target genes, as determined by single molecule RNA FISH and single cell analysis. Interestingly, TBT also affected the levels of its two main target NRs, PPARG and RXRA, but in opposite directions, with PPARG being increased, while RXRA was decreased in a 26S proteasome-dependent manner. Moreover, at the single cell level, RXRA levels did not correlate with lipid content, similar to our previous results in adipocytes where coregulator proteins did not correlate with NR levels and lipid content  of HepaRG cells treated with vehicle (left column) or TBT 50 nM (right column) for 72 h. D) quantification of PPARG nuclear intensity at the single cell level represented as a box plot. E-F) RXRA/PPARG correlation analysis (Spearman r) in vehicle vs. TBT treated cells, respectively.(TIF)Click here for additional data file.S1 File(XLSX)Click here for additional data file.


Mechanotransduction, the process by which physical forces are converted into chemical signaling, has been observed in several cell types –3. This Interestingly, there is evidence that the forces exerted by the cytoskeleton itself can influence the phosphorylation and conformation of signaling molecules, and this mechanotransduction subsequently controls protein function, and ultimately has an impact at the cellular level. For example, Babich et al. showed that perturbing F-actin dynamics decreases Phospho Lipase C Gamma 1(PLCγ−1) phosphorylation, leading to a reduction of calcium flux in T-cells . AdditioIn this compelling report, Jankowska and colleagues show that in primary T-cells, engagement of VLA-4 or LFA-1 with their ligands significantly decelerates centripetal actin flow at the IS. This study also compliments earlier reports describing inward movement of signaling microclusters (MCs) , 16. FurHowever, several questions remain. It is not obvious how inhibiting actomyosin retrograde flow affects signaling downstream of the TCR. Specifically, is rapid ARF necessary for tyrosine phosphorylation of signaling clusters, or is there another mechanism by which ARF characterized by a slower lamellipodial velocity dampens signaling molecule function? We recently discovered a molecular mechanism that may explain some of the recent findings by Babich et al. and Jankowska et al. on the mechanistic level . We showHence, due to the fact that SHP-1 plays an inhibitory role in T-cell signaling , it is pSuch a model suggests the possibility of a much larger system controlled by the cytoskeleton, which precisely tunes lymphocyte signaling. It may also explain how lymphocytes such as T-cells and NK cells rapidly survey their surroundings, and process various signals in a coordinated and organized manner in a complex immune environment. It would be interesting to study whether additional signaling molecules are influenced by the forces exerted through cytoskeletal flow, and how such force transduction ultimately tunes lymphocyte function. Additionally, more work is required to understand how ARF is regulated in the early and later stages of immune synapse formation, and how it is differentially regulated in various compartments of the IS. Finally, these novel results raise the exciting possibility of manipulating immune cell cytoskeletal networks to influence cellular function.All authors listed have made substantial, direct, and intellectual contribution to the work and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Evidence for nation-states suggests that the long-standing negative relationship between fertility and economic development might turn positive at high levels of development. The robustness of the reversal continues to be debated. We add to this discussion from a novel angle by considering whether such a reversal could also occur at the sub-national level within highly developed countries. Our contributions are both theoretical and empirical. We first discuss important trends which might foster the emergence of a positive fertility–development relationship across regions of highly developed countries. These include shifts in family policies, changes in the spatial organisation of the economic sphere, and selective international and internal migration processes. In order to explore whether we observe tendencies towards a reversal, we investigate data covering 20 European countries subdivided in 256 regions between 1990 and 2012. We document a weakening of the negative relationship between fertility and economic development within many countries, and among some countries the emergence of a positive relationship. These findings do not seem to be driven by postponement effects alone. However, there is substantial variation in the fertility and the economic development levels at which such tendencies towards a reversal are observed.The online version of this article (10.1007/s10680-018-9485-1) contains supplementary material, which is available to authorized users. Dominant demographic theories such as the demographic transition and second demographic transition frameworks postulate a negative relationship between development and fertility. As societies progress, fertility tends to decrease Kirk . ProposeConcurrent with these theoretical shifts are shifts in the empirical evidence. While a global national-level comparison still tends to demonstrate a negative relationship between fertility and development levels, recent evidence suggests that among high-income countries this relationship may transition from negative to positive. Myrskylä et al.  found thHowever, whether such a reversal is occurring remains contested. Ryabov  applied The goals of this paper are to add the sub-national regional dimension to this discussion both in terms of theoretical considerations and empirical findings, and to explore to what extent changes in postponement contribute to the observed patterns in European countries. Within European countries, sub-national regions vary in their levels of income/development and fertility, which can be adequately captured with cross-country comparative data. Relative to cross-country differences, regions within countries tend to be generally more similar in their cultural and policy frameworks , it is important to consider these aspects in our analyses of changes in the fertility–income relationship. To explore how postponement affects our analysis, we compare models using the total fertility rate (TFR) as a fertility measure with models in which the TFR is replaced by a tempo-adjusted TFR. To account for the potential distortion effects due to the post-communist transition, we separate the sample between Western European countries and the former communist Eastern European countries. As a final comment, the 2007 economic recession may also represent a short-term distortion. We explore this further in the online appendix of consistency checks.In this section, we briefly outline the data and variables used. For a detailed discussion of the data, including summary statistics and our decisions on corrections, we refer readers to the “To calculate the TFR and an estimate of a tempo-adjusted TFR, age-specific birth count and population data were drawn from a combination of sources. Most of the data were obtained through Eurostat. However, there are some large gaps in the time series for a number of countries during the period of observation. We thus made substantial efforts to fill in these gaps using data directly derived from national statistical offices. This also allowed us to correct some inconsistencies in the Eurostat data see “”. For soEconomic data were obtained from Cambridge Econometrics (CE). These data primarily originate from Eurostat and were adjusted by CE to 2005 Euros using purchasing power parity deflators. Employee compensation per capita is chosen as our measure of per capita income, as it accounts for household income levels better than the more commonly used Gross Domestic Product (GDP). GDP, which is calculated in the dataset as Total Gross Value Added, plus taxes and less subsidies, captures total income generated by both firms and households. This provides a good measure to compare the overall economic development of different areas. However, as we are interested in variation in private household incomes across regions, it is potentially a challenge that GDP comprises both labour and capital income. Capital owners may be less likely to reside in the same region relative to labourers, so information on capital income may be less relevant for fertility decisions in the region. For that reason we focus on the aforementioned employee compensation, which consists of wages and salaries, and of employers’ social contributions. This measure of income should more closely approximate the labour income earned by individuals in the different regions. Employment participation rates and the demographic structure of the population can vary across our regions. We focus, however, on employee compensation per capita instead of compensation per worker or per “active population” member school children, students, and pensioners) to gauge the experienced economic well-being of the population.3We acknowledge that employee compensation per capita may miss income generated by households in other ways. If regions differ significantly in how that income is generated, employee compensation may reflect a distorted measure of actual household income levels. Sub-national regions might, for example, vary in the amount of remittances received. In this case, employee compensation per capita will overestimate household incomes in sending regions and underestimate household incomes in receiving regions. If remittances are important for the relationship, then using GDP per capita as a measure of economic development that includes received remittances spent locally should result in significantly different model estimates. That we do not observe substantial differences between models using GDP per capita and employee compensation per capita indicates that these distortions do not likely represent a significant source of bias.As a first step of analysis, we look at trends in the cross-sectional relationship between income and fertility levels across NUTS 2 regions for each of the sample countries between 1990 and 2012. We do this by performing cross-sectional regressions on a by-country annual basis. Thus, for each year and for each country, TFRs are regressed on the lag of adjusted employee compensation per capita. Figures From Fig. If we turn to the Western European countries in the centre and the south of Europe Fig. b, one trFigure The negative relationship observed in all Eastern European countries in the early 1990s weakened throughout the 1990s and 2000s, so that we witness for most of these countries fertility–income correlations of around zero in 2012. Only Romania experienced something of a reversion in the final years of our study period. However, this seems to be an artefact of the shortcomings in the Romanian population estimates for the 2000s that created a downward bias in fertility rates in less developed regions and an upward bias in the more developed ones see “”. As to Overall, the trends of these cross-sectional associations provide support that over the last two decades most European countries have experienced a lessening of the negative association between economic development and fertility across their regions. In many countries, this association has even turned positive. However, the fertility and economic levels at which countries report such tendencies vary substantially across countries. To illustrate this, we show in Fig. Figure The preceding analysis looked at the evolution of the cross-sectional associations between income and fertility levels over time within different European countries. In this second part of our empirical section, we investigate how changes in income are related to changes in fertility by applying country-by-year fixed effects models. We emphasise these models are not specified to identify causal effects of income on fertility, but to provide a broad perspective on the association between these two variables across European regions. With that goal in mind, the models below include the TFR or the tempo-adjusted TFR as dependent variable, and as explanatory variables, the prior year log of regional income and its square (as measured by employee compensation per capita). The square term is included to test for the convexity of the fertility–income relationship. For unobserved factors affecting income and fertility jointly , we attempt to control for with the fixed effects.The country-by-year fixed effects model is based on the assumption that regions within a country are identical in those factors related to both income and fertility. We justify this assumption with the observation that, for Europe, the national level is most relevant for aspects that might affect the general levels of income and fertility in a country  at time t (measured in years), in country c, and in region i. The first term, χc * λt, controls for the country-by-year fixed effects. The inclusion of this interaction term accounts for a country-specific effect that varies over time. This is in contrast to the inclusion of country and year fixed effects, which would control for the time-constant country-level factors and the time-varying factors common to all of the countries in the panel. Income per capita is represented by inc and appears in both the linear and the squared terms. The error term ɛi,c,t is assumed with conditional mean zero. We estimate this model separately for the full sample, for regions part of Western European countries (including former West Germany), and for regions part of the Eastern European countries (including former East Germany).In notation form, our main model is specified as follows:Estimates from the model using TFR are presented in Table To examine the extent to which fertility postponement may be contributing to the above results, we attempt to control for tempo effects within the different countries. The arguably most sophisticated approach suggested in the literature requires parity-specific fertility by age for the different regions . A recent example of such an approach is Wood et al. . The patOne additional restriction is that we only have access to income data which was measured at the place of work, while a measurement at the place of residence would be preferable. This might create biases when big cities or metropolitan areas constitute their own regions into which people commute into for work. However, this data limitation would bias against the identification of a convex relationship shown in Tables Overall, our paper provides theoretical support for the view that a turnaround in the relationship between fertility and economic development might occur as well at the sub-national level in highly developed countries. Our empirical analysis indicates that such tendencies are already visible, particularly in Western Europe. However, it is too early to draw definite conclusions whether we are indeed witnessing a turnaround to a genuine positive relationship between income and fertility, or simply the emergence of a less or non-negative relationship. As sub-national parity-specific register or register-like data become increasingly available across Europe, future research might be able to investigate shifts in the sub-national fertility–income association with more detailed data in the years to come.Supplementary material 1 (DOCX 121 kb)Below is the link to the electronic supplementary material.


Naphthoquinones may cause oxidative stress in exposed cells and, therefore, affect redox signaling. Here, contributions of redox cycling and alkylating properties of quinones  to cellular and inter-cellular signaling processes are discussed: (i) naphthoquinone-induced Nrf2-dependent modulation of gene expression and its potentially beneficial outcome; (ii) the modulation of receptor tyrosine kinases, such as the epidermal growth factor receptor by naphthoquinones, resulting in altered gap junctional intercellular communication. Generation of reactive oxygen species and modulation of redox signaling are properties of naphthoquinones that render them interesting leads for the development of novel compounds of potential use in various therapeutic settings. All vitamin K forms (vitamers) are 2-methyl-1,4-naphthoquinone  to O2 and hydrogen peroxide—both with and without facilitation by superoxide dismutases. Alternatively, 1,4-naphthoquinones may undergo a two-electron reduction to the corresponding hydroquinone, which is catalyzed by NAD(P)H:quinone oxidoreductase-1 -oxidation of the reaction product under concomitant generation of ROS . It requaphorase ). Althouaphorase   and “electrophile response element” (EpRE) . AREs weIn line with the weak effects of menadione on Nrf2 signaling in these models, menadione did not stimulate ARE-dependent signaling in cultured neuronal cells—whereas its 5-hydroxylated derivative, plumbagin, strongly activated Nrf2/ARE signaling . PlumbagCaenorhabditis elegans, serves as a convenient in vivo model for research on metabolic and stress signaling because central signaling pathways are highly conserved from worm to mammalia. Plumbagin elicited an enhanced expression of genes known to be controlled by ARE activation [C. elegans ortholog of Nrf2, SKN1. Exposure of C. elegans worms to non-toxic concentrations of naphthoquinones, such as juglone, plumbagin and naphthazarin, not only not affected C. elegans life span, but, rather, resulted in life span extension (by some 10%) at low quinone concentrations [The roundworm, tivation . Like pltrations ,33, a cltrations . Depletitrations .In addition to interaction with Keap1, posttranslational modification was described as a means of regulating Nrf2 activity. For example, ERK, JNK and p38 mitogen-activated protein kinases (MAPK) all phosphorylate Nrf2, albeit with only minor contribution to Nrf2 nuclear accumulation and Nrf2-dependent target gene expression . As seveIn summary, some naphthoquinones stimulate Nrf2-dependent activation of ARE-dependent gene expression, followed by enhanced formation of protective proteins, such as enzymes involved in glutathione biosynthesis—and NQO1. This naphthoquinone-induced Nrf2-dependent protective response appears to be highly conserved, as the same signaling cascade is stimulated in roundworms and mammalia.i.e., through mechanisms independent of growth factor stimulation of the respective receptor. The extent of EGFR and ErbB2 activation was shown to correlate with the capability of the respective naphthoquinones to cause the generation of superoxide and depletion of glutathione (GSH): both were demonstrated in HaCaT human keratinocytes exposed to 1,4-naphthoquinone, menadione, juglone or plumbagin, and all quinones strongly stimulated EGFR and ErbB2 activation and downstream signaling [2O2, whose reduction to water would be catalyzed by glutathione peroxidases and would occur at the expense of GSH (which would be oxidized to GSSG)  family, and of RTK-dependent signaling. For example, exposure to naphthoquinones may elicit tyrosine phosphorylation of the ErbB-family RTKs, the epidermal growth factor (EGF) receptor (EGFR) and the related ErbB2 ,35,36, oSSG) see . InteresSSG) see .Interestingly, RTK signaling may also be impaired by suitable naphthoquinones: PDGFR inhibitors on a naphthoquinone basis were synthesized and described recently, targeting PDGFR but not EGFR signaling in vascular smooth muscle cells. These quinones are 5,8-dimethoxy-1, 4-naphthoquinone derivatives with intermediate-chain alkyl moieties , as well as the phosphoinositide 3'-kinase (PI3K)/Akt cascade are stimulated in various cells exposed to naphthoquinones, including menadione [RTK stimulation results in activation of several signaling cascades emanating from these receptors, including activation of MAPKs. Specifically, ERK-1, ERK-2, and ERK-5 MAPKs . Such diffusible compounds include nutrients, such as glucose, signaling molecules and ions, such as cAMP or Ca2+, and even oligonucleotides [MAPK were among the first kinases identified as Cx43 kinases [MAPK was demonstrated to result in lowered conductivity of Cx43 gap junctional channels [i.e., by microinjection of a non-membrane-permeant fluorescent dye into single cells and assessing the extent of its diffusion into neighboring cells. Numbers of cells showing up as fluorescent after a set period of time are compared and taken as a measure of intercellular communication via gap junctions . MAPK aleotides ,48. The leotides , and gapleotides ,50. Next kinases ,52. Cx43see MAPK ,35. Inhi of GJIC ,12. Otheing DMNQ  or NSC 9ing DMNQ .i.e., the quinones with lowest activities in terms of inducing oxidative stress and stimulating EGFR—see i.e., the presence or absence of a methoxy-moiety), which will alter the quinone also with respect to its alkylation and redox cycling properties, changes the cellular response to the quinone in terms of GJIC. While DMNQ is a mere redox-cycling naphthoquinone, MeONQ, like menadione, potently depletes GSH from red blood cells, indicating that, in addition to redox cycling, it may also alkylate thiols [vs. DMNQ did not significantly alter the quinone’s impact on GJIC. In line with previous data [Some of the quinones listed in e thiols . In linee thiols . Neitherous data  it appeaMAPK and Cx43 phosphorylation, affects inter-cellular communication in addition to intracellular signaling responses .Whether naphthoquinones affect GJIC in ways other than through connexin phosphorylation, remains to be determined—it is conceivable that stress-responsive mechanisms of posttranscriptional regulation of connexin levels  in cellsMenadione was demonstrated to inhibit protein tyrosine phosphatase(s) dephosphorylating activated EGFR and ErbB2 ,36—suggep-benzoquinone) and an exclusive redox cycler, DMNQ, also stimulated EGFR and EGFR-dependent signaling, yet only for menadione was an inhibition of a PTPase controlling the EGFR detectable [Menadione is capable of alkylating nucleophilic moieties in cells, including thiols ,11, and tectable .To test for the inhibitory effects of naphthoquinones in the absence of redox cycling-fostering conditions, isolated PTPase PTP-1B , a PTPasi.e., under non-redox cycling conditions—suggesting that non-alkylating, non-oxidative inhibition of PTPases by naphthoquinones is feasible as well. In line with this, NSC 95397 , however, simple naphthoquinones as described in this article are mostly “potentials”—gin, see . Here, ti.e., towards cell death, by forcing additional ROS upon these cells. For example, one approach successfully tested in cell culture and in mice was the overexpression of manganese superoxide dismutase to catalyze the dismutation of superoxide to hydrogen peroxide—combined with an impairment of peroxide removal, e.g., through drugs interfering with glutathione metabolism [et al., synthesized a set of multifunctional selenium- and tellurium-containing naphthoquinones [i.e., to facilitate oxidative damage. The chalcogen added in position 3 would be oxidized by ROS, but the resulting selenoxide or telluroxide would subsequently be reduced easily by cellular components, such as thiols, resulting in the recycling of the chalcogen and the widespread oxidation of cellular thiols and proteins of the “cellular thiolstat” [MAPK signaling in rat liver epithelial cells. Both seleno- and telluro-menadiones strongly stimulated ERKMAPK in rat liver epithelial cells, and activation of ERKMAPK was via stimulation of EGFR signaling. These compounds elicited EGFR tyrosine phosphorylation, and inhibition of EGFR, as well as MEK1 and MEK2 abrogated ERKMAPK activation [MAPK signaling by multifunctional naphthoquinones will result in a loss of GJIC, this has not been tested so far.The reasoning frequently put forward for research on the use of quinones in cancer therapy is that cancer cells are more susceptible to oxidative damage than their non-cancerous counterparts are: their already elevated levels of ROS  would matabolism ,64. Naphquinones . These miolstat” . Exposurtivation . AlthougAs described above, naphthoquinones may inhibit PTPases in many ways, and depending on the identity of the PTPase targeted this may affect cellular proliferation. For example, a ligand-independent stimulation of EGFR would be regarded pro-proliferative, owing to the crucial role of this receptor in triggering cell division processes. If, however, the PTPase were to be required for a crucial phase in proliferation to proceed, such as during cell cycle phase transitions, the inhibition of this PTPase may be anti-proliferative. The naphthoquinone NSC 95397 was identified as a selective and potent inhibitor of Cdc25 phosphatases . Cdc25 dMutations leading to an overactivity of EGFR (for example through overexpression or generation of a dominant-active mutant) as well as other ErbB family members frequently occur in the genomes of cancerous tissues  [As described above, EGFR activation by naphthoquinones may occur independently of a ligand (such as EGF), and indirectly through targeting a PTPase (protein) . From thprotein) . Clinica