Syncope, defined as a transient loss of consciousness caused by transient global cerebral hypoperfusion, affects 30–40% of humans during their lifetime. Vasovagal syncope (VVS) is the most common cause of syncope, the etiology of which is still unclear. This review summarizes data on the genetics of VVS, describing the inheritance pattern of the disorder, candidate gene association studies and genome-wide studies. According to this evidence, VVS is a complex disorder, which can be caused by the interplay between genetic factors, whose contribution varies from monogenic Mendelian inheritance to polygenic inherited predisposition, and external factors affecting the monogenic (resulting in incomplete penetrance) and polygenic syncope types. Syncope, or fainting, is characterized by global cerebral hypoperfusion, transient loss of consciousness with disturbed postural tone, disturbance of the cardiovascular and respiratory systems, and spontaneous recovery back to the normal state .A reduction in systemic blood pressure (BP) causing a decrease in cerebral blood flow plays a major role in the pathogenesis of syncope . In turnApproximately 40% of humans during their lifetime have transient loss of consciousness . VasovagA surrogate marker, the head-up tilt test (or prolonged passive head up tilt testing) has been used to diagnose VVS since the late 1980s. In this test, the patient is moved from the horizontal to vertical position using a special tilting table in order to simulate the neurally mediated reflex and induce syncope. The probability that VVS is induced during the head-up tilt test is 40–60%. The positive head-up tilt test response is associated with frequent VVS episodes and may be indicative of significant severity of orthostatic disorders [p < 0.01). Among individuals with VVS unrelated to medical triggers, 94% of subjects had a family history of syncope [The first step in studying hereditary diseases is often to perform a familial aggregation analysis. If familial aggregation is revealed, the patient’s relatives have a higher risk of developing the disease compared to the average risk within a population, which is inversely proportional to the genetic distance from the proband. The earliest studies focused on the inheritance of VVS have identified familial aggregation of syncope. Kleinknecht and Lenz  reported syncope .p < 0.01) had at least one first degree relative also suffering from VVS. Furthermore, 37% of children with VVS had both a sibling and a parent with syncope vs. 4% of controls (p < 0.05). It was inferred that VVS is a complex disorder with both inherited genetic and external factors involved in its development.In an early study, the family history of 30 children with VVS was examined . For 24 In a study  involvinStudying family history records identified families in which several generations suffered from VVS ,15. ThusTherefore, family aggregation studies of VVS inevitably demonstrated that probands with VVS were more likely to have a positive family history compared to the control group. A number of researchers have put forward a skeptical hypothesis that family aggregation might be observed randomly due to the high syncope rate among the population ,17; othep = 0.018) and for syncope related to typical vasovagal triggers  (p < 0.001). The results of this study are also consistent with the assumption that VVS is a complex disorder, with both genetic and environmental factors contributing to its development. According to [Twin studies allow one to examine the contribution of genetics to the development of a disease/phenotype by comparing the concordance in monozygotic and dizygotic twins. The first twin study focusing on syncope was conducted in 659 twin pairs from the Australian Twin Registry with respect to syncope related to blood/injury/injection fear . A signirding to , the numA total of 2,694,442 subjects from several Swedish nationwide registries were enrolled in a recent large-scale study , includiThe reported data are indicative of the role played by the genetic component in the development of VVS. However, as mentioned in the review by Sheldon and Sandhu , a signiThe effect of sex and age on the risk of VVS can be another reason for erroneous results. Indeed, a study enrolling 62 medical students and their families reported that females were more likely to experience syncope than males . By the Hence, the individual susceptibility to VVS largely depends on one’s sex and age, as well as on the sex of the parent who suffers from VVS. These data are consistent with the theory of a potential contribution of epigenetic factors to the development of VVS. Epigenetic regulation is not related to nucleotide sequence changes in the genome, but affects the transcriptional level of the genes important for phenotype development via covalent modification of DNA or histone proteins. Since sex hormones can modulate gene expression, the differences in prevalence of VVS in males and females can be attributed to the sex-hormone-dependent epigenetic mechanisms.Overall, the observed complex inheritance pattern demonstrates that VVS has complex  origins: its development can be regulated by interplay between the genetic and epigenetic, as well as environmental factors. Importantly, environmental factors can modulate epigenetic processes , and theWhat is the ratio between the different inheritance patterns of VVS? The findings reported in  provide The conventional approach to searching for genes involved in disease/phenotype development still remains relevant and is based on analyzing the association of individual candidate genes with the phenotype. An assumption that a gene is possibly associated with the phenotype is made based on the function of the gene product (the “phenotype-to-gene” approach). According to views on pathogenesis of VVS, one can expect that the genes whose products regulate the functioning of the autonomic nervous system and the cardiovascular system are mainly involved in syncope development. It is worth mentioning that genes are ascribed to either of these categories rather tentatively, since the autonomic nervous system regulates the function of all internal organs, including the heart and blood vessels, while variations in functioning of the cardiovascular system trigger a response from the nervous system. In most cases, researchers analyze polymorphic variants of genes, represented by single nucleotide polymorphisms (SNPs).Data obtained from case-control studies on the association between carriership of polymorphic variants of a certain candidate gene and susceptibility to VVS are summarized in Before we discuss the results obtained by assessing the involvement of individual genes in the development of VVS, we would like to point out the methodological flaws that some of the cited studies have. In a conventional association study, the groups being compared need to be characterized according to the ESC Guidelines for the diagnosis and management of syncope . HoweverSince such neurotransmitters as norepinephrine, epinephrine, and serotonin are believed to play a major role in the development of syncope, genetic factors of susceptibility to VVS are primarily searched for among genes encoding receptors, carrier proteins, and enzymes partaking in the synthesis of these mediators.ADRA1A gene encoding the alpha-1A adrenergic receptor, the SNP rs1048101 (1039T > C) is responsible for a Cys347Arg substitution at the C-terminal end of alpha-1 adrenergic receptor; this substitution can affect receptor–protein interactions and, therefore, signal transduction from the receptor to the cell. Hernández-Pacheco et al. compared groups consisting of 89 tilt-positive patients and 40 healthy tilt-negative subjects without a history of VVS, heart or lung disease, and revealed a positive association between VVS and carriership of the C allele and the CC genotype  (p < 0.001) [ADRA1A gene were compared in tilt-positive and tilt-negative patients with VVS. Thus, Sorrentino et al. [ADRA1A rs1048101 and head-up tilt response in 129 patients suffering from VVS without a history of cardiovascular disease or carotid sinus syndrome who were not taking medications affecting the cardiovascular system. A recent study observed differences in allele/genotype frequencies for neither rs1048101 nor other SNPs of the ADRA1A gene  when comparing 88 tilt-positive and 46 tilt-negative patients with VVS [The genes encoding adrenergic receptors are the most interesting candidate genes for VVS; the contribution of polymorphic variants of these genes was studied both to the development of VVS and head-up tilt test response. In the < 0.001) . The aut< 0.001) . The reso et al.  revealedwith VVS .ADRB1 to the development of VVS. This gene mediates positive chronotropic and inotropic effects in the cardiac muscle tissue and acts as a target for beta blockers. Researchers focused on two SNPs in the coding region of the ADRB1 gene, rs1801253 (Arg389Gly) and rs1801252 (Ser49Gly), which affect the receptor function and its response to adrenoblockers. The Arg389Gly polymorphism resides in the C-terminal region of the beta 1 adrenergic receptor, affects its binding to G protein and, thereby, activation of adenylate cyclase. The Arg389 variant was shown to stimulate adenylate cyclase more efficiently and enhances signal transduction from adrenergic receptors compared to Gly389 [p = 0.012) [p < 0.001) were revealed [p = 0.02) [p = 0.012); these patients also showed a better response to beta blocker therapy compared to those with the CG genotype (Arg389Gly) (p < 0.001).Most of the studies listed in 01252 SerGly, whico Gly389 . Compario Gly389 ,39. Howe= 0.012)  and, sho = 0.02) . Sorrent = 0.02)  found no = 0.02) . PatientADRB2, a comparison was performed only between tilt-positive and tilt-negative patients with VVS. No association of ADRB2 rs1042713 or ADRB2 rs1042714 with head-up tilt test response was found [p = 0.04). They also found an association between rs4994 in the ADRB3 gene encoding beta 3 adrenergic receptor and the head-up tilt test response (p = 0.03).For variants of the beta 2 adrenergic receptor gene, as found . Márquezas found  also fouADRA1A rs1048101 and VVS was observed [Therefore, data on contribution of polymorphic variants of genes encoding alpha and beta adrenergic receptors to genetic susceptibility to VVS are rather controversial. When patients with VVS were compared to healthy controls, only an association between observed , whereasobserved ). When aDBH gene encoding dopamine beta-hydroxylase, which catalyzes conversion of dopamine to norepinephrine. Comparison of tilt-positive and tilt-negative patients with VVS found no association between this SNP and head-up tilt test response  within the promoter of this gene for patients with VVS compared to healthy controls [There currently is no agreement regarding the role of serotonin in VVS development. An assumption was made that since serotonin is related to BP regulation and is found in the brain regions involved in the development of VVS, this neurotransmitter may contribute due to the antisympathetic effects mediated by the central nervous system . Table 1 rs25531  and rs47 rs25531  in the scontrols .HTR1A) with VVS was revealed only in males (p = 0.005). This polymorphic variant resides in the promoter region of the gene directly within the binding site of the transcription factor NUDR, which can affect the receptor expression level [COMT gene encoding catechol-O-methyl transferase related to a reduction in the enzyme level was associated with a lower risk of VVS in males and higher risk in females (p = 0.017). The COMT gene is known to have different effects on cerebral function and dysfunction in males and females, and is involved in sex-specific dimorphism of susceptibility to mental disorders [SLC6A4 gene; however, the differences did not reach statistical significance (p = 0.059). A conclusion has been drawn that males carrying any of the aforementioned three allelic variants of the genes involved in serotoninergic regulation are protected against VVS compared to females and to other males not carrying these allelic variants. Undoubtedly, these results need to be reproduced using independent samples.A study  conducteon level . As repoon level , the A aisorders . It shouisorders  observedA large group of candidate genes refers to the genes whose products are involved in functioning of the cardiovascular system . Special focus is placed on the genes whose products participate in purinergic signal transduction (adenosine receptors), nitric oxide metabolism, functioning of potassium channels, and of the renin–angiotensin–aldosterone system.ADORA2A gene encoding adenosine A2A receptor with susceptibility to VVS, as well as to induction of VSS during the head-up tilt test, was investigated in three publications. The study [p < 0.0001), as well as with a high frequency of syncopal episodes (p = 0.004), however, no association with susceptibility to VVS has been found. The SNP rs5751876 is responsible for the synonymous substitution Tyr361Tyr. The researchers suggested that this polymorphism may affect gene expression level and protein folding. In a more recent study, analyzing a much greater number of patients (n = 347) with a history of at least one syncopal episode of unknown etiology and 85 controls without a history of syncopal episodes, no association of this SNP with head-up tilt test response or VVS was detected [The association of the SNP rs5751876 in the he study , which idetected . Sheldondetected  also obsGNAS1 gene encoding protein G subunit alpha, the SNP rs5443 of the GNB3 gene encoding protein G subunit beta 3, and the SNP rs4606 of the RGS2 gene encoding the G protein signaling regulator 2; these SNPs have also been analyzed by other researchers. A comparison of VVS patients with healthy controls revealed that none of these genes are associated with VVS [GNAS1 [GNB3 [RGS2 genes [GNB1 gene encoding G protein subunit beta 1 and c.87 + 34G > A of the GNG2 gene encoding G protein subunit gamma 2 with VVS was detected [GNAS1 and GNB3 genes and syncope severity was revealed in patients with a history of more than three syncopal episodes over the preceding two years [GNB3 gene were found to be associated with a history of VVS  (p < 0.001) [RGS2 gene were found to be associated with the number of syncopal episodes (p = 0.04) [GNAS1, GNB3 or RGS2) and head-up tilt test response, such an association was found only in study [GNAS1 gene (p < 0.001). Many receptors involved in signal transduction in patients suffering from VVS belong to the group of G protein-coupled receptors. The potential role of genes encoding G proteins in the formation of genetic predisposition to VVS has mainly been investigated by Lelonek et al. In the series of studies, they analyzed the SNP rs7121 of the with VVS ,46,47. OS [GNAS1 , GNB3 [2S1 [GNB3 , and RGSS2 genes ; the samS2 genes  was rathdetected . No asso< 0.001) ; polymorHence, the best reproducibility of findings has been achieved by analyzing the association between G protein genes and VVS. These data allow one to infer that the analyzed polymorphic variants of the G protein genes have no significant contribution to VVS susceptibility.KCNJ5 (a gene encoding one of the potassium channel proteins) and VVS was identified when compared 74 tilt-positive patients without cardiac, endocrine, or neurological disorders and 208 healthy blood donors (p = 0.001) [KCNJ3 gene. Negative results were also obtained in this study for the SNP rs138806839 of the CHRM2 gene encoding acetylcholine receptors M2 (muscarinic receptors) capable of modulating muscarinic potassium channels  plays a crucial role in the regulation of vascular tone, blood flow, and BP. However, there were no association between the polymorphic regions of the  and VVS .ACE gene encoding angiotensin-converting enzyme [AGT, rs5186 of the AGTR1 gene encoding angiotensin II [EDN1 [EDNRA [EDN1 gene (rs10478694) was shown to be associated with a positive head-up tilt test response (p = 0.048). Carriers of this allele have increased endothelin-1 production. Since endothelin-1 has a vasoconstrictive effect and is expected to prevent VVS, researchers suggest that its involvement in the development of VVS is not confined to affecting vascular tone and is possibly related to a different mechanism [Association of VVS with polymorphic variants of other candidate genes whose products are involved in regulation of vascular tone and BP  have ale p = 0.0. CarrierTherefore, studies focusing on the role of individual candidate genes in VVS patients are rather controversial, which can be explained by small sample size and ethnic differences in the groups being compared, as well as the fact that it is rather labor-intensive to diagnose this condition. Furthermore, patients with VVS subdivided into tilt-positive or tilt-negative were used as controls in a number of studies, or patients with typical and atypical syncope were compared. A plausible reason for non-reproducibility of the results is that the control groups were formed from conditionally healthy individuals without any prior diagnostics, which is especially important because of the high frequency of VVS (up to 25%) in the general population. Meanwhile, the association data for a number of genes  have been obtained in a single study and need to be reproduced. Taking into account the complex pathogenesis and heterogeneous clinical course of VVS, it is fair to assume that this disorder is polygenic. However, none of the studies analyzed the overall contribution of candidate genes to its development.Unlike the “candidate gene” approach when a hypothesis regarding potential involvement of a gene in phenotype is put forward according to its nature and function of the gene product (the “phenotype-to-gene” approach), the genome-wide searching for genes involved in disease/phenotype development employs panels of genetic markers with known chromosomal localization. These panels can be used to identify the genomic regions where these genes localize, and then search for the genes directly associated with the phenotype within these genomic loci is performed. This approach can be defined as the “genome-to-gene” approach. Polymorphic variants distributed over the entire genome more or less uniformly serve as genetic markers. Most typically, those are SNPs. Insertions/deletions (indels) and mini- or microsatellite repeats are also analyzed rather commonly. Microarrays and other modern technologies allow one to simultaneously identify as many as several dozen thousand to several million polymorphisms within a single sample.Genome-wide studies were originally applied to analyze the linkage between a disease and chromosomal loci in families where several members had this disease. Later, genome-wide association studies (GWAS) were used as a more powerful tool for studying human genetic architecture.SLCO3A1, ST8SIA2 and NR2F2 candidate genes residing within the linkage interval detected no mutations. Linkage to the chromosome 15q26 region was excluded in two additional large families, suggesting that different genes may be relevant in the development of VVS in these families. The first genome-wide study of VVS was conducted by linkage analysis in a large family presumably with autosomal dominant inheritance  using microarray-based SNP genotyping data . In thisp < 0.05) was revealed. In patients with VVS, the CNV segments were longer than those in healthy controls. However, the copy number presumably is not directly related to the pathophysiology of syncope, since no identical CNVs specific for individuals diagnosed with VVS have been identified.Demir et al.  conducteThe accuracy of assessments made in association studies directly depends on the number of DNA samples being studied. It is noteworthy that both patients and controls need to belong to the same ethnic group. A large-scale GWAS for syncope and collapse was conducted in 2020 . This stp value for 1,000,000 comparisons needs to be <5 × 10−8). The p value for the lead SNP rs12465214 was 5.8 × 10−15; for the other four SNPs in this locus , the p value ranged from 1.0 × 10−9 to 3.3 × 10−8. Carriership of the rs12465214*C risk allele was characterized by a hazard ratio of 1.13  [A new locus on chromosome 2q32.1 associated with VVS was identified at a significance level complying with modern requirements of GWAS results (the Bonferroni-adjusted 10–1.17) .p = 8.82 × 10−6).Results of this study were validated using the Danish Neonatal Screenings Biobank database . SamplesZNF804A gene encoding zinc finger protein 804A is the most plausible candidate gene associated with the risk of developing VVS. The SNP rs12465214 resides at a distance of approximately 250 kb from this gene and affects its expression as shown by the transcriptome analysis also conducted in [ZNF804A gene was preferentially expressed in the brain, cerebral arteries, and endocrine tissue rather than in the cardiac muscle tissue [Functional annotation identified 26 genes in the locus associated with a risk of syncope ; the resucted in . Using qe tissue .ZNF804A gene is known to be associated with schizophrenia and bipolar disorder [The disorder ,59. Its disorder . Based odisorder  using thdisorder . Howeverdisorder .Enhanced expression in patients with VVS was shown for other genes encoding zinc finger proteins  in a study involving a Chinese cohort of children with VVS- and age-matched controls .ZNF804A gene encoding zinc finger protein 804A have been obtained and validated using GWASs on two large ethnically homogeneous cohorts [Therefore, convincing findings on the association of the rs12465214*C risk allele residing on chromosome 2 in close proximity to the  cohorts . This pr cohorts . Such do cohorts .However, this study has serious limitations related to the fact that the definition of the disease was given using the ICD-10 code “syncope and collapse,” without differentiating between syncope subtypes. Since VVS is the most common type of syncope , there iThe question regarding the genetic nature of VVS was raised in the late 1980s, and is still far from being fully answered.Data on the inheritance of VVS accumulated thus far are indicative of its familial aggregation; the genetic component can vary from a monogenic Mendelian inheritance pattern to polygenic inherited predisposition. The observed genetic heterogeneity can be correlated with both the diversity of VVS triggers and its clinical heterogeneity.Both genetic and external factors contribute to the development of VVS; they can play a role in monogenic (resulting in incomplete penetrance) and polygenic syncope. The effect of external and epigenetic factors makes polygenic VVS a conventional complex  disorder. Interplay between these factors is responsible for the complex inheritance pattern. Since the effect of external factors can modulate epigenetic processes, these mechanisms cannot be fully delineated.While the familial analysis data are rather convincing, the conventional approach to searching for the genes involved in syncope development by analyzing the association with individual candidate genes (selected according to VVS pathophysiology and function of the gene product) has not yielded unambiguous results yet. Although the range of candidate genes is rather broad, genes whose association with VVS that can be confirmed using independent samples or at least obtained by comparing large cohorts of patients and controls sufficient to perform robust statistical analysis are yet to be discovered.The “genome-to-gene” approach has been used to analyze genetic architecture in a few studies and has yielded fundamentally important results. The genome-wide linkage analysis of VVS in several multiplex families with autosomal dominant inheritance patterns revealed the mutant locus 15q26 in only one family . These dZNF804A gene in locus 2q32.1. The C2H2 transcription factor containing zinc finger protein 804A (ZNF804A) encoded by the ZNF804A gene can play a pivotal role in the protein–protein interaction network by participating in various regulatory and signaling pathways involved in syncope development.Hadji-Turdeghal et al.  believe LOC105373776 and LOC102724340, in the positive and negative DNA strands, respectively. Although the functions of these genes are unknown, we presume that these very genes might be responsible for the role of the 2q32.1 region as a locus associated with the risk of syncope development. As reported in the NCBI database [We believe, however, that it is not the only possible interpretation of the results. When discussing localization of the lead SNP associated with syncope, rs12465214, on chromosome 2, Hadji-Turdeghal et al.  classifidatabase , these gdatabase . It is qIdentifying genetic and epigenetic factors involved in VVS has proven to be a promising field of research, not only to improve knowledge of risk factors for VVS, which could be of help for prevention, but also to improve the understanding of the pathophysiology of syncope subtypes, and to optimize and personalize the treatment of patients with syncope in the future.


Ambulatory blood pressure monitoring (ABPM) is important in evaluating average 24-hour blood pressure (BP) levels, circadian rhythm, sleeping BP and BP variability but many patients are reluctant to use standard ABPM devices.We compared two validated ABPM devices, the BPro tonometric wrist monitor and the A&D TM-2430 oscillometric upper arm monitor, for agreement of recordings and acceptability in 37 hypertensive patients (aged 55±9 years).P <0.01) and awake periods . Comparable paired readings showed no significant difference in systolic BP but diastolic BP (DBP) values were higher with the wrist compared to the arm monitor  with similar differences awake and sleeping. Bland-Altman analysis showed some large discrepancies between individual arm and wrist monitor measurements. More patients found the wrist monitor more comfortable to use than the arm monitor.Successful BP measurements were less frequent with the wrist-type than the arm-type device during the sleeping (66.3% vs. 92.9%, Despite the difference in individual BP measurements and the systematic overestimation of DBP values with the BPro device, wrist monitors with good patient acceptability may be useful to facilitate ABPM in some patients to provide additional information about cardiovascular risk and response to antihypertensive therapies. Ambulatory blood pressure monitoring (ABPM) is recommended in recent hypertension guidelines to confirm the diagnosis of hypertension, to identify white-coat and masked hypertension, and to monitor blood pressure (BP) control –3. It alHome BP monitoring (HBPM) using validated automatic devices is also recommended and has the particular advantage of improving adherence with treatment regimens , 2, 7. EThe arm-cuff ABPM was recommended as a standard ABPM device and it has been used widely since the noninvasive ABPM was introduced . HoweverSome studies have compared this device with other conventional arm-cuff ABPM devices including the A&D TM-2430 in complicated hypertensive patients , the A&DIn the present study, we compared the BPro wrist type ABPM with the A&D TM 2430 upper arm ABPM , which was validated according to the Association for the Advancement of Medical Instrumentation (AAMI) and British Hypertension Society protocols (grade A/A) and recommended for clinical use in 1998 . We evalFrom January 2013 to June 2014, consecutive suitable Chinese patients with mild to moderate essential hypertension who attended the outpatient clinics in the Prince of Wales Hospital, Hong Kong were invited to enter the study. Mild to moderate essential hypertension was defined as sitting clinic systolic BP (SBP) from 140 to 169 mmHg or sitting clinic diastolic BP (DBP) from 90 to 109 mmHg (130–169 mmHg or 80–109 mmHg for patients with diabetes mellitus). According to the European Society of Hypertension (ESH) BP device validation protocol (12), ≥33 subjects were required to gain a statistical power of 90% . A total of 37 hypertensive patients with mean (±SD) age 55±9 years (range 18–79 years) completed this comparison study and the statistical power was 99% with a two-sided alpha of 0.05. Informed consent was obtained from each patient before undertaking any study related activity. This study was a sub-study of the main study which was approved by the Joint Chinese University of Hong Kong-New Teritories East Cluster Clinical Research Ethics Committee with a reference number of CRE-2011.616-T.Before starting the wrist and arm ABPM recordings, the clinic seated BP was measured in both arms using an automatic arm monitor  to ensure there was no difference in BP between arms. The average BP was calculated from three clinic seated BP measurements  taken at 1 minute intervals. This average BP was entered into the wrist ABPM device before starting the 24-hour ABPM recordings. The tonometer in the wrist strap of the BPro was placed over the left radial artery. The arm monitor cuff was applied to the right upper arm.The wrist monitor was set to measure BP every 15 minutes during the 24-hour period and the arm monitor was set to measure BP at 15 minute intervals during 07:00~22:00 and every 1 hour during 22:00~07:00. Subjects were requested to keep the arm still and parallel to the trunk when the arm-cuff was inflated and to keep the left wrist at heart level when the wrist-type measurements were being performed. The patients were encouraged to continue their usual daily activities but not to engage in vigorous physical exercise such as running, climbing, or playing sports. A daily activity record form was given to each patient. At the end of the 24-hour ABPM, each patient was asked to choose which device they would prefer if they were having ABPM in the future. ABPM recordings were excluded from the analyses if the percentage of valid readings was below 70% or if readings were lacking for an interval more than 2 hours or if there were less than 14 readings during the daytime period, or less than 7 readings during the nighttime period.ABPM profiles obtained by wrist and arm devices were edited for measurement errors and outliers according to conventional criteria; SBP readings >250 or <70 mmHg, DBP readings >150 or <40 mmHg, pulse pressure  values >150 or <20 mmHg, and readings with a change of ≥50 mmHg in DBP between consecutive readings were eliminated. The valid paired readings measured within ≤5 minutes of each other and having a difference in heart rate ≤5 beats/minute by the wrist and arm devices were compared. The 24-hour BP was calculated for each device as the average of all paired readings obtained during the 24-hour ABPM. Awake and asleep BP values were calculated as the average of all paired readings measured during the hours of daytime activity or nighttime sleep, respectively. The daytime activity and nighttime sleep period were based on the information obtained from the patient’s daily activity record form.x2-test. Bland–Altman plots were used for the comparison of the readings from the two monitors. The correlations between the values from the two monitors were evaluated by Pearson’s correlation coefficients and intra-class correlation (ICC) agreements.All statistical analyses were carried out using SPSS software, version 18.0 . The individual BP values obtained using the two monitors were evaluated as mean ± standard deviation (SD). The group values and proportions were compatible with a normal distribution. The group means were compared using paired Student’s t-tests. The group proportions were compared using the χ2. There were 14 male (38%) patients in this study. There were 14 patients taking amlodipine as antihypertensive treatment and the other patients were on no antihypertensive treatment. Eight patients were on anti-hyperglycemic treatment for type 2 diabetes mellitus.Thirty-seven participants completed the study. The wrist monitor was preferred by 27 patients as the patients reported that it caused fewer disturbances than the arm monitor, especially during sleep, and overall it was more comfortable to use, while the other 10 patients preferred the arm monitor as they had discomfort in the wrist area when using the wrist monitor. Another reason for preferring the wrist monitor was that it is placed on the radial artery and can measure BP without removing clothing for a cuff application. As shown in P <0.01). There were a greater (P <0.01) number of successful readings for the sleeping period using the wrist-type device (21±8 readings) compared with the arm-type device (10.5±4.8 readings). The frequency of attempted readings was greater with the wrist-type than the arm-type device during 22:00~07:00 so the success rate was lower with the wrist-type device during the sleeping period  and the awake period .The success rates of the BP measurements according to the wrist-type and the arm-type devices are shown in P <0.01; 88±14 vs. 91±13 mmHg, P <0.01; 79±13 vs 83±12 mmHg, P <0.01). There was no significant difference in the SBP measurements between the two devices. Pearson’s correlations and intra-class correlations were significant for all comparisons. A regression analysis was performed to identify if any baseline factors such as age, sex, body weight, BMI, 24-hour SBP and DBP measured by the two devices were related to the discordance and it was found there were only the 24-hour SBP values measured by the two devices which were related to the differences in SBP between the two devices. None of the factors tested were significantly related to the differences in DBP between the two devices.The average BP levels are shown in The Bland–Altman plot was used to graphically present the difference between each of the wrist and arm measurements plotted against the average of the two measurements. Ambulatory blood pressure monitoring is important in evaluating average 24-hour BP levels, circadian rhythm, sleeping BP and BP variability but many patients are reluctant to use standard upper arm ABPM devices , 19–22. A previous study which compared the BPro device with the A&D TM 2431 arm monitor examined BP measurements at different arm positions and found that the BP values provided by the wrist monitor were almost identical in three arm positions and the BP was significantly higher for the wrist monitor than for the arm monitor in the arm-raised position .One of the problems in the use of devices that measure BP at the upper arm is that arm circumference varies and physicians or patients may use cuffs which do not match the arm circumference. The measured BP values will be lower than the true value when a large cuff is used in a slim patient and BP will be higher than the true value when a narrow cuff is used in obese patients so it is important to use the correct cuff size for the patient with arm-type monitors –25. HoweOne disadvantage of the wrist monitor is that when the position sensor moves to an unsuitable position, the BP measurement will fail. In our study, the successful recoding rate with the wrist monitor was considerably lower than with the arm-type device. The success rate of BP measurement with the BPro was as low as 58.9% for 24 hours, 56.2% for the waking period and 66.3% for the sleeping period. However, because the frequency of measurements was greater with the wrist monitor than with the arm monitor during the sleeping period there were more successful readings with the wrist monitor than with the arm monitor at nighttime.A significant number of patients prefer devices that measure BP at the wrist because of their convenience and they are usually more comfortable to use compared with the arm-cuff type device. The wrist monitor measures BP without compression of the wrist but with slight pressure on the radial artery. In the present study, 10 patients had some discomfort using the wrist monitor. We found this could usually be relieved by adjusting the position or tightness of the wrist monitor and patients could be taught how to do this themselves. Another advantage of the wrist monitor is that there is less noise from the device during the night, which is an important issue in measuring the true BP during the sleeping period.There was a wide range of differences between readings with the arm monitor and the wrist monitor with a 2SD difference of -37.7 to 39.1 mmHg for 24 hour SBP and -30.9 to 23.1 mmHg for 24 hour DBP . This maSome studies have shown that mean asleep BP evaluated by ABPM was the most significant predictor of event-free survival in hypertensive patients . High leThe Hygia Chronotherapy Trial reported that routine ingestion of ≥ 1 antihypertensive medication at bedtime, as opposed to upon waking, was associated with better ABPM control, especially the asleep BP, and with reduced CVD events . HoweverAnother potential advantage of tonometric devices is that they can be used to estimate central aortic systolic pressure using the N-point moving average method. The BPro device is not only capable of 24-hour ABPM but also for capturing radial arterial waveforms. Results for central aortic systolic pressure from the BPro and SphygmoCor devices in patients with type 1 diabetes showed strong correlations .Central BP changes are thought to have a major influence on the reduction of CVD events with different antihypertensive therapies –36. The The present study has several limitations: firstly, the BP values compared for the two devices were not taken at exactly the same time but the time difference was limited to be ≤5 minutes and the heart rate difference between recordings was required to be ≤5 beats/minute to avoid situations when there was a sudden change in activity. Secondly, the number of readings that could be compared between the two devices was limited. Thirdly, although there was a wide range in age of the patients, there were not sufficient very elderly patients to determine if age-related changes in arterial stiffness affected the results.The mean SBP values both awake and sleeping showed reasonable agreement for the wrist monitor and the arm monitor but mean DBP were systematically overestimated by about 4 mmHg with the wrist monitor over 24 hours. There were considerable discrepancies for individual readings between the two devices as shown by the Bland–Altman plots. More patients found the wrist monitor more comfortable to use than the arm monitor. The successful recoding rate was considerably lower with the wrist monitor but this could be overcome by having more frequent attempted recordings. Wrist monitors, such as the BPro, may be useful for repeated monitoring to assess changes in 24-hour and sleeping BP which could facilitate adjustment of medications.S1 Data(XLS)Click here for additional data file.


Ciona embryo, to investigate the essential gene regulatory network that orchestrates morphogenesis of the notochord, a structure necessary for the proper development of all chordate embryos. Although numerous transcription factors expressed in the notochord have been identified in different chordates, several of them remain to be positioned within a regulatory framework. Here, we focus on Xbp1, a transcription factor expressed during notochord formation in Ciona and other chordates. Through the identification of Xbp1-downstream notochord genes in Ciona, we found evidence of the early co-option of genes involved in the unfolded protein response to the notochord developmental program. We report the regulatory interplay between Xbp1 and Brachyury, and by extending these results to Xenopus, we show that Brachyury and Xbp1 form a cross-regulatory subcircuit of the notochord gene regulatory network that has been consolidated during chordate evolution.Gene regulatory networks coordinate the formation of organs and structures that compose the evolving body plans of different organisms. We are using a simple chordate model, the  Ciona, an invertebrate chordate whose larvae are characterized by a fast-developing and tractable notochord, a compact genome, and unrivaled ease of transgenesis (Ciona notochord develops within approximately 1 day after fertilization (Ciona) orthologs, other transcription factors are expressed in the Ciona notochord (Ciona counterpart of X-box binding protein 1 (Xbp1)  , a basic1 (Xbp1) . The UPR1 (Xbp1) . Three E1 (Xbp1) . In turn1 (Xbp1) . Physiol1 (Xbp1) . Loss ofc lethal , and Xbpm anemia .Xenopus  and showed that this gene is predominantly expressed in notochord and epidermis  on embryos carrying a null mutation in the Ci-Bra coding region . Compared to stage-matched control embryos  cDNA downstream of the r region . Transgethe tail . Lastly,the tail  , expressing either the Xbp1ublished , and theublished . Five geH.C8.749 , were id as well . We alsoPitx, a well-characterized homeobox gene mainly expressed in the anterior sensory vesicle, epidermis, and oral siphon primordium  include notochord cells, their precursors, and/or broader territories encompassing the notochord . The remimordium , and Node embryo . These fe embryo  and in te embryo . In addie embryo , in circe embryo ; accordirve cord  is exprerve cord .Ciona-/ascidian-specific’ genes, which appear to be either specific to Ciona  or to be present in Ciona and other ascidians, but currently lack identifiable counterparts in the genomes of organisms from other divisions; nevertheless, five of these ascidian-specific genes  contain recognizable protein domains  relies upon a single T-box binding site that is likely targeted by Ci-Bra and/or Tbx2/3  of the Xenopus putative orthologs of Cr-Xbp1-downstream notochord genes; however, 6 of the remaining genes are reportedly expressed in the notochord, while for the remaining 19 genes expression in this structure has not been reported . WMISH of Xenopus embryos at NF stage 35/36 with a Shh probe indicates that, compared to sibling controls, Shh expression is discontinuous in the notochord of Xbra and Xbp1 morphant embryos, and often confined to its posterior regions  (Ci-Bra coding region (DBD::VP16) show numerous round, poorly differentiated transgenic cells dispersed throughout their tails.In f Ci-Bra , expressra (DBD)  or shRNAg region . On the DBD::VP16 or Xbp1DBD,using embryos expressing GFP as controls. This approach identified 109 genes whose expression is influenced either directly or indirectly by Cr-Xbp1, 71 of which were found expressed in the notochord, either through this study or through the analysis of previously published in situ hybridization data. Eighteen of these genes currently lack clear orthologs in organisms other than Ciona and other ascidians. The presence of recognizable protein domains and sequence homology with proteins identified in different organisms allowed the tentative classification of the remaining 53 genes into 15 GO categories. The most ample of these categories include genes whose predicted products either participate in signaling pathways or in the formation of the abundant ECM secreted by the developing notochord cells. 36 of the 71 Cr-Xbp1-downstream notochord genes (50.7%) had been previously reported as targets of Ci-Bra by microarray screens , and was cloned downstream of the DBD::VP16::GFPBra>Xbp1 construct was generated by fusing the DBD of Xbp1 to the VP16 transactivation domain, as previously described .C. robusta embryos were electroporated with 50 µg of either DBD::GFP,Bra>Xbp1 or DBD::VP16::GFPBra>Xbp1, or Bra>GFP plasmid. Fluorescent transgenic embryos from the same clutch were manually selected in comparable amounts from all three experimental samples using a Zeiss SteReo Discovery V12 epifluorescence microscope at ~6.25 hpf at 21°C, corresponding to initial tailbud I/II according to the standardized developmental table at 18°C  cut-off of 2.0 were further considered. Changed mRNA levels are expressed as ‘up’ or ‘down’ regulated in Tables S1 and S2. The complete dataset has been deposited into the NCBI Gene Expression Omnibus (GEO) under accession number GSE46751.Xenopus Xbp1.L was amplified by PCR from stage 12 cDNA using the primers:ATGGTGGTCGTGGGAGCC-3′F: 5′-TTAAAAATGTACATCAAACT-3′R: 5′-based on the published sequence . A 1190 X. laevis embryos were staged according to GCGCAGCTCTCGGTCGCACTCATTC-3′) targets both the short (Xbra.S) and the long (Xbra.L) forms of Xbra. For Xbp1 (Xbp1MO), we used a mix (1:1) of two MOs targeting the Xbp1-S (5′-GACATCTGGGCCTGCTCCTGCTGCA-3′) and Xbp1-L (5′-GCCCAACAAGAGATCAGACTCAGAG-3′). All three translation-blocking MOs have been previously validated (Xbra and Xbp1 pre-mRNA splicing. Xbp1MOS (5′-TCTGGAAGAGATCAAACACATGACA-3′) targeting the intron 2/exon 3 junction of both forms of Xbp1, and a mix (1:1) of two MOs:The procedures were performed in accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health, approved by New York University Institutional Animal Care and Use Committee, under animal protocol #150201. alidated . To furtAGTACCTACTGAAGAGAAAGCACAA-3′5′-ACCTACTGAAGGGAAAGCACAAAGA-3′5′-Xbra, respectively (XbraMOS)  in the equatorial region of both dorsal blastomeres at the four-cell stage (NF stage 3), and the embryos were analyzed by in situ hybridization at NF stage 12 or stage 35/36. Each injection was performed on at least three independent batches of embryos.targeting the intron 5/exon 6 junction of the short and long forms of XbraMOS) . In eachXenopus embryos at the appropriate developmental stages were fixed in MEMFA , stained for Red-Gal  to visualize the lineage tracer (LacZ mRNA), and processed for in situ hybridization. Antisense digoxygenin-labeled probes  were synthesized using as templates cDNAs encoding Xbra/Tbxt (Xbp1 (pGEMT-Xbp1), and Shh  and reverse-transcribed using SuperScript IV VILO Master Mix (Thermo Fisher Scientific) according to the manufacturer’s instructions, and used for PCR with Illustra PuReTaq Ready-To-Go PCR beads . The following primer sets spanning the entire coding sequence were used: 5′-ATGAGTGCGACCGAGAGCTG-3′Xbra_fwd:5′-TTAGATTGATGGTGGTGCAA-3′Xbra_rev: 5′-ATGGTGGTCGTGGGAGCC-3′Xbp1_fwd: 5′-TTAAAAATGTACATCAAACT-3′.Xbp1_rev:  Ciona X-box binding protein (Xbp1), a basic leucine zipper transcription factor, in notochord morphology, in downstream gene regulation providing novel targets and as an evolutionarily conserved feedback interactor with Bra as shown in Xenopus. The manuscript is well written and suggests a conserved regulatory subcircuit between Xbp1 and Bra in Ciona and Xenopus.Wu et al. establish the role of  In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Decision letter after peer review:eLife. Your article has been reviewed by 2 peer reviewers, and the evaluation has been overseen by Marianne Bronner as the Senior and Reviewing Editor. The reviewers have opted to remain anonymous.Thank you for submitting your article "Xbp1 and Brachyury establish an evolutionarily conserved subcircuit of the notochord gene regulatory network" for consideration by The reviewers have discussed their reviews with one another. Both reviewers are enthusiastic about your manuscript but also suggest some additional experiments and changes to the text for clarity. We refer you to their detailed comments, attached below, and ask that you revise the paper accordingly to the best of your ability. We look forward to receiving a revised version of the manuscript.Reviewer #2:Xenopus, microarray screening of Xbp1 targets in Ciona upon overexpression of Xbp1-variants and by an individual validation of the endogenous expression in tailbud embryos notably in notochord cells.Wu et al. provide evidence that Xbp1 and Bra regulate each other's expression using gene interference in Ciona and Xenopus. This is convincingly reported in each of the model organism by at least two independent approaches of loss of function. In Ciona, a Bra null mutant affects Xbp1 expression while Xbp1 shRNA or overexpression of truncated Xbp1 variants under the Bra promoter affect notochord cell shape and intercalation while Xbp1 full length under Foxa.a overactivates the Bra promoter. In Xenopus, translation blocking and splice blocking MOs reciprocally affect Bra and Xbp1 as well as gastrulation and dorsal blastopore closure. A second Xbp1 target gene is affected in each system, Shh in Xenopus and Fibrillin in Ciona.The strength of this manuscript is a well written and presented support of a conserved regulatory subcircuit between Xbp1 and Bra in both, Ciona and Furthermore, 109 Xbp1 targets (including Fibrillin) were identified in microarray screening from comparing embryos overexpressing GFP under the Bra promoter or GFP-tagged Xbp1 truncations that contained either the DNA binding domain (DBD) only or its fusion with the VP16 transactivator. Upon individual validation, 80 of these target genes (presented as ISH patterns on tailbud stages) included notochord expression and encompassed several ascidian specific genes. A thorough annotation is provided in two Supplement tables for notochord or non-notochord genes, respectively, and includes knowledge from previous studies notably about regulation by Bra, Foxa.a and Tbx2/3. Overall, three functional groups within Xbp1 targets are proposed that include the UPR (unfolded protein response), TGFß signaling and morphogenetic effectors (such as Fibrillin). This is well discussed in the notochord functional context.A weakness is that only limited data is presented on endogenous or WT Xbp1 (in Ciona) affecting target gene expression or notochord morphology. Only one type of experiment with full length Xbp1 is presented as a graph of Bra promoter overactivation, but without pictures. In the same line, for the screening, only truncated Xbp1 versions were used, supposing a dominant negative effect of the DBD but of contradictory/unclear effects of VP16. Here, too, no WT Xbp1 was used, neither to analyse notochord morphology.Concerning the original data sets from microarray screening these are shown as Supplement tables only but not summarized as a Table in the body of the paper. Furthermore the screening logic and expected output are not well explained to the reader causing confusion in particular about the elimination of positive or negative fold change (FC) of expression normally obtained as output of such screens – here changed to absolute values (and deposited as such at NCBI).In addition, while a potentially valuable interpretation is put forward about different subcircuits regulating notochord formation it is not documented or explained how coregulation by Bra, Tbx2/3 and/or FoxA compares to the proposed independent circuit involving Bhlh-tun1.Comments for the authors:To be more convincing and aid the readers in their own judgement of data the following suggestions to eliminate the raised criticisms may help.1) Lack of Xbp1-WT GOF data:- Please provide pictures for the Bra promoter activation in Foxa.a driven Xbp1-WT overexpression.- Please provide a comparative experiment for effects of Xbp1-WT, Xbp1-DBD and Xbp1-VP16.2) Lack of explanation for partially overlapping effects of DBD or DBD-VP16 (such as on Fibrillin):- Please more explicitely explain the logic notably seemingly repressive function of Xbp1-VP16 on Fibrillin; is this an indirect activation of a repressor ? please provide a possible interpretation.3) Lack of explanation for the substractive screening approach and the 'absolute FC' output, also in the source data deposited at NCBI:- Please more explicitely explain the 'absolute' FC; original (if so) negative or positive FC should probably be kept.4) Lack of evidence provided for a suggested different Bhlh-tun1 subcircuit:- Precise examples should be named, listed or a table provided for common and/or different target genes (including corresponding citations).5) Please add a note in proof on the recent Reeves et al. study from 2021.Reviewer #3:Xenopus is known. In both Xenopus and Ciona they show that Xbp1 is acting downstream of Bra in notochord development. It is unclear if Xbp1 is directly or indirectly regulated by Bra.The paper nicely demonstrates that Xbp1 gene in is involved in notochord development in Ciona. As noted in the manuscript, the role of Xbp1 in notochord development in Xenopus and Ciona and thereby finding another gene linked to Bra that may be involved in chordate notochord development.The impact of this work is to link Bra to Xbp1 in The manuscript focuses on describing lots of genes identified in a subtractive microarray study, there is not enough information to explain what they did. As I am unclear how this was performed it is hard to evaluate the data presented.Xenopus and shows that this gene is regulated either directly or indirectly by Bra. It adds to the list of conserved genes involved in notochord development in vertebrate and the non-vertebrate chordate Ciona.In summary, the paper identifies a new gene in Ciona, already known in Strengths:Manipulation of Xbp1 in two different organisms to investigate the role of these gene in notochord development demonstrates that Xbp1 is indeed either directly or indirectly downstream of Bra in notochord development.This work adds another gene to the network of transcription factors conserved between vertebrate and ciona notochord development.Weakness:The data does not address whether or not there is a direct interaction between Bra and Xbp1.Xenopus in this study so their statements regarding co-option of genes involved in the unfolded protein response in notochord development is limited to Ciona.There is no analysis of genes downstream of Xbp1 in The authors use subtractive microarrays to identify genes downstream of Xbp1. There is hardly any information about this approach and so it is hard to review the resulting data.There are several places stating data not shown which makes it hard to evaluate.I found the section on transcriptional targets of Xbp1 hard to follow, simply setting out what the authors are doing would help improve this section so readers can follow. The current description is as follows – "two subtractive microarray screens were carried out in triplicate using the constructs described above".Please explain what subtractive microarray screens are. What was the purpose of these screens? How were the different constructs shown in the section above used in these microarray screens and in what concentration. How was the data from the two-time points used? What was subtracted from what? How did this approach identify 109 genes? In the methods section it seems there are two-time points at which embryos were collected and three constructs, but I'd like to understand what the analysis was.There is an assumption that most of the 109 genes are transcriptional targets of Xbp1, but I wonder if the changes in expression could be due to other factors such as loss of notochord cell identity or response to electroporation of DNA into the embryos. It is hard to evaluate the claims without understanding what was done.The authors suggest through identification of Xbp1-downstream notochord genes they find evidence of the early co-option of genes involved in the unfolded protein response to the notochord development program. In the text it lists all the genes that are differentially regulated in their analysis -with relation to the UPR – "Other gene ontology categories include genes presumably involved in the UPR, such as protein folding…" This is shown in figure 3. What % of the 109 genes are UPR genes. Could differential expression of UPR genes be a result of the way the experiment was done and the use of over expression constructs? Are these genes up or down regulated by the Bra>Xbp1dbd::GFP or Bra>Xbp1::VP16::GFP. I am aware that the control has GFP, but what is the size of the coding regions of the other constructs, how much was electroporated, I could not find the ug of constructs used for these experiments. I would like to know more about how the experiment was done to rule out the possibility that the UPR genes were differentially expressed as a result of electroporation of these constructs.Xenopus is Shh. In the discussion they mention Bmp being known to be perturbed by Xbp1 knock down in Xenopus and that similar results were seen in Ciona.The text should clearly point out that their observations regarding the co-option of genes involved in the unfolded protein response in notochord development relates to Ciona only. In the manuscript the only data I see on genes downstream of Xbp1 studied in Xenopus notochord. Could they have a figure showing what fraction of the 109 genes identified are also found in the Xenopus notochord?It would be helpful to know if the genes identified as downstream of Xbp1 in the Ciona are also expressed in the Xbp1 knock out mice have been studied. In the publications about these mice are there any notochord defects mentioned? If in the knockout mice there are notochord defects this should be stated. If there are notochord defects this provides evidence in another vertebrate of the role of Xbp1 in notochord development.The authors state that Xbp1 in the mammalian notochord has not been studied. There is ample single cell RNA-seq datasets from embryos in mammalian systems in which the authors could look for notochord expression of Xbp1. It would help the manuscript if this data was utilized to identify if expression of this gene is conserved in other vertebrates.Xenopus notochord, it would be helpful to state what has been learned from this study that was not appreciated in the previous analysis of Xbp1 in Xenopus to clearly show the knowledge added by this study.Since Xbp1 has already been studied in the There are several statements regarding data followed by data not shown. The data should be shown. Reviewer #2:[…] (1) Lack of Xbp1-WT GOF data:- Please provide pictures for the Bra promoter activation in Foxa.a driven Xbp1-WT overexpression.Ci-Bra promoter, and we have mutagenized a few of them in an effort to determine whether there is a direct interaction between Xbp1 and the Ci-Bra promoter. Since we have not obtained conclusive evidence of such interaction, and there are still numerous putative binding sites that would have to be mutagenized in order to have a definitive answer, we have removed this result from the revised manuscript. However, we did insert a microphotograph of an embryo displaying the phenotype induced by the Foxa.a-driven full-length Xbp1 overexpression .Over the past months we have identified putative Xbp1 binding sites in the 3.5-kb - Please provide a comparative experiment for effects of Xbp1-WT, Xbp1-DBD and Xbp1-VP16.FL) also induces an overexpression of Xbp1 in the notochord. In sum, the revised Figure 2 now displays a comparison of the effects of the overexpression of Xbp1-WT, Xbp1-DBD and Xbp1-VP16 in the developing notochord.In revised Figure 2, we have added a microphotograph of an embryo expressing Xbp1 shRNA (panel C). We have also added two panels  to show the results of the ectopic expression of the full-length (FL) wild-type Xbp1 protein in CNS and endoderm, through the Foxa.a promoter region; this construct (Foxa.a>Xbp12) Lack of explanation for partially overlapping effects of DBD or DBD-VP16 (such as on Fibrillin):- Please more explicitely explain the logic notably seemingly repressive function of Xbp1-VP16 on Fibrillin; is this an indirect activation of a repressor ? please provide a possible interpretation.We have added the interpretation of the repressive function of Xbp1::VP16 on pages 8 and 13 of the revised manuscript.3) Lack of explanation for the substractive screening approach and the 'absolute FC' output, also in the source data deposited at NCBI:- Please more explicitely explain the 'absolute' FC; original (if so) negative or positive FC should probably be kept.We have added a detailed explanation of the goals and details of the microarray screens to the text. As per the fold-change, we wish to clarify that the results were provided in the ‘absolute FC’ format after being analyzed and statistically validated, therefore there is no ‘original’ positive or negative FC. The data that we report in the manuscript have been kept consistent with the source data that we have deposited in the NCBI GEO database. For these reasons, we have maintained this format in the manuscript as well.4) Lack of evidence provided for a suggested different Bhlh-tun1 subcircuit:- Precise examples should be named, listed or a table provided for common and/or different target genes (including corresponding citations).Ciona notochord GRN, the ascidian-specific transcription factor Bhlh-tun1 .”We have clarified this concept by rephrasing the sentence as follows: “No overlap was found between the notochord genes downstream of Cr-Xbp1 and those controlled by another node of the All the information currently available on Bhlh1 and its target genes in the notochord and corresponding citations is included in our published work .5) Please add a note in proof on the recent Reeves et al. study from 2021.We have added a citation of this paper and how it might relate to our findings in the Discussion section, on page 22 of the revised text.Reviewer #3:[…] I found the section on transcriptional targets of Xbp1 hard to follow, simply setting out what the authors are doing would help improve this section so readers can follow. The current description is as follows – "two subtractive microarray screens were carried out in triplicate using the constructs described above".Please explain what subtractive microarray screens are. What was the purpose of these screens? How were the different constructs shown in the section above used in these microarray screens and in what concentration. What was subtracted from what? How did this approach identify 109 genes?We have removed “subtractive” from the text and we have clarified the purpose and the methodology of these screens, and how this approach identified 109 genes (page 8). We have added the concentration of the constructs (50 micrograms) and further explanation in the Methods section (page 23-24) and in the legend to figure 1.How was the data from the two-time points used? What was subtracted from what? How did this approach identify 109 genes? In the methods section it seems there are two-time points at which embryos were collected and three constructs, but I'd like to understand what the analysis was.We are thankful to this Reviewer for spotting our mistake in the Methods section. Differently from our previously published work on Tbx2/3, the Xbp1 microarray screen did not involve two experimental time points but only one. We have corrected this mistake in the revised manuscript.There is an assumption that most of the 109 genes are transcriptional targets of Xbp1, but I wonder if the changes in expression could be due to other factors such as loss of notochord cell identity or response to electroporation of DNA into the embryos. It is hard to evaluate the claims without understanding what was done.We have added a detailed explanation of the goals and details of the microarray screens to the revised text and we explain below the reasons why we believe that the results of this screen are specific (please see below).The authors suggest through identification of Xbp1-downstream notochord genes they find evidence of the early co-option of genes involved in the unfolded protein response to the notochord development program. In the text it lists all the genes that are differentially regulated in their analysis -with relation to the UPR – "Other gene ontology categories include genes presumably involved in the UPR, such as protein folding…" This is shown in figure 3. What % of the 109 genes are UPR genes.Ciona notochord have been reported to participate in the UPR in other model organisms.On page 17 we answer this question through the following sentence: At least 50% of the Cr-Xbp1-downstream genes expressed in the Could differential expression of UPR genes be a result of the way the experiment was done and the use of over expression constructs? Are these genes up or down regulated by the Bra>Xbp1dbd::GFP or Bra>Xbp1::VP16::GFP. I am aware that the control has GFP, but what is the size of the coding regions of the other constructs, how much was electroporated, I could not find the ug of constructs used for these experiments. I would like to know more about how the experiment was done to rule out the possibility that the UPR genes were differentially expressed as a result of electroporation of these constructs.We have added the amount of constructs used for each experiment in the Methods section and figure legends. The coding region of GFP is ~720 bp (240 aa), the coding region of Xbp1DBD::GFP is approximately 1284 bp, for a total of ~428 amino acid residues. The coding region of Xbp1::VP16::GFP is ~1442 bp. We have used GFP as a control also in another published microarray screen, for Ciona Tbx2/3 . There is limited overlap between the targets of Tbx2/3 and those of Xbp1, and this overlap mostly includes Ciona/ascidian-specific genes that do not appear to be involved in UPR. This suggests that the results of these microarray screens are specific to each transcription factor and that the activation of UPR genes is not due to the electroporation procedure, but is rather due to the specific effect of Xbp1 on its transcriptional targets.Xenopus is Shh. In the discussion they mention Bmp being known to be perturbed by Xbp1 knock down in Xenopus and that similar results were seen in Ciona.The text should clearly point out that their observations regarding the co-option of genes involved in the unfolded protein response in notochord development relates to Ciona only. In the manuscript the only data I see on genes downstream of Xbp1 studied in Ciona, we found evidence of the co-option of genes involved in the unfolded protein response to the notochord developmental program.”The co-option of UPR genes in notochord development is suggested by the work of Tanegashima et al. (2009). In the Introduction (page 2) we have clarified that “Through the identification of Xbp1-downstream notochord genes in Xenopus notochord. Could they have a figure showing what fraction of the 109 genes identified are also found in the Xenopus notochord?It would be helpful to know if the genes identified as downstream of Xbp1 in the Ciona are also expressed in the Xenopus orthologs of Ciona Xbp1-downstream notochord genes.To answer this question, we have added a supplemental table (Table S3) that contains the information currently available on Xbp1 knock out mice have been studied. In the publications about these mice are there any notochord defects mentioned? If in the knockout mice there are notochord defects this should be stated. If there are notochord defects this provides evidence in another vertebrate of the role of Xbp1 in notochord development.The authors state that Xbp1 in the mammalian notochord has not been studied. There is ample single cell RNA-seq datasets from embryos in mammalian systems in which the authors could look for notochord expression of Xbp1. It would help the manuscript if this data was utilized to identify if expression of this gene is conserved in other vertebrates.We appreciate this helpful suggestion, and indeed we had been trying to find some information on the role of Xbp1 in the mouse notochord. We had contacted a few of the scientists involved in this published research to address this important point, but none of those who responded had a specific answer to our questions. Nevertheless, we have expanded the references on the mouse Xbp1 KO experiments and we now discuss the mouse single-cell RNA-Seq results and other data that tentatively suggest that Xbp1 might be involved in notochord formation in mouse embryos as well.Xenopus notochord, it would be helpful to state what has been learned from this study that was not appreciated in the previous analysis of Xbp1 in Xenopus to clearly show the knowledge added by this study.Since Xbp1 has already been studied in the Xenopus and is required for the proper development of the notochord in this vertebrate. The information gathered using Ciona on the genes influenced by Xbp1 will guide future studies on the notochord genes controlled by Xenopus Xbp1.”In the Discussion section we clarify as follows: “the results of this study have uncovered a regulatory connection between Brachyury and Xbp1 that is maintained in There are several statements regarding data followed by data not shown. The data should be shown.To address this point we have added two supplemental figures to this revised manuscript. Figure S2 shows microphotographs of ten additional novel notochord genes identified through this work (previously listed as ‘data not shown’ in Table S1). Figure S4 shows the expression of eight Xbp1 target genes with no published data, which we have found to be expressed in tissues other than the notochord (from Table S2). In addition, we have reorganized the figure showing Ciona/ascidian-specific notochord genes  and we have added a pie graph that summarizes the results of GO and InterPro searches. New or additional insets have been added to existing figures to provide additional information.All three mentions of ‘data not shown’ that were present in the original text have been removed by filling in the gaps in information to the best of our abilities.We have removed the expression patterns of genes for which we did not have convincing images or for which we were not able to repeat the in situ hybridizations, and therefore these genes are now listed as ‘not analyzed’ in Table S2 and in the text. This explains the change in the number of notochord genes in Table S1 and throughout the text, from 80 to 71.


Bari transposons. Bari is a “normal” transposon family that has colonized the genomes of several Drosophila species and introduced genomic novelties in the melanogaster species. We discuss how these results have contributed to advance the field of TE research and what future studies can still add to the current knowledge.Transposable elements (TEs) have been historically depicted as detrimental genetic entities that selfishly aim at perpetuating themselves, invading genomes, and destroying genes. Scientists often co-opt “special” TEs to develop new and powerful genetic tools, that will hopefully aid in changing the future of the human being. However, many TEs are gentle, rarely unleash themselves to harm the genome, and bashfully contribute to generating diversity and novelty in the genomes they have colonized, yet they offer the opportunity to develop new molecular tools. In this review we summarize 30 years of research focused on the  Transposable elements are fundamental genetic units in the genomes of virtually all living organisms. TEs could be depicted as the characters of a happy ending fairytale. Initially regarded as “junk” and “useless”, TEs turned out to be considered as evolutionarily flagships after reconsidering the role they have had and still have in shaping genomes and their functioning. Moreover, the characterization of many transposition systems has led to the development of efficient DNA integration tools  as well It is a matter of fact that the hallmark of all TEs, i.e., the ability to integrate into chromosomes, is the most interesting aspect of TEs for many biologists, due to their many possible applications in a large group of fields in Life Sciences.Efficient genome integration tools are indeed desirable to disrupt genes, either in a random or targeted way, and to introduce exogenous DNA into the preferred cellular or animal model. A special application of the latter practice is gene therapy, which consists in the introduction of the non-pathological allele in the affected cell type of a patient that suffers from a genetic-based disease, with the aim to rescue the illness phenotype.In the past 40 years many transposition systems have been studied in detail with the aim to set up new and efficient DNA integration tools.P-element was the first transposon-based transposition tool to be employed in functional genomics [Historically, the Drosophila genomics ,6. Unforgenomics , which mTc1/mariner are more tractable for this kind of application. Tc1/mariner elements belong to the Class II of the eukaryotic transposons and are widely distributed, from bacteria [IS630/Tc1/mariner superfamily.Elements of the bacteria  to highebacteria , with feSleeping Beauty (SB) and the piggyBac (PB) elements [https://www.caramba-cart.eu; accessed 15 December 2021) currently uses an advanced SB-based transposon technology for therapeutic gene delivery [More in general, the so-called “cut and paste” DNA transposons are the best candidates to develop molecular tools for transgenesis because of their simple mechanism of transposition and their poor requirement of host factor . Two of elements . SB is udelivery .However, not all the known transposition systems support this kind of application. Most known TEs have limited transposition performances  or they have low flexibility  to allow the development of efficient genome integration systems. Nevertheless, many TEs are studied for their role in shaping genome structure  and geneTc1/mariner superfamily of TEs the Bari family was discovered 30 years ago in the former Institute of Genetics at the University of Bari . Such discovery led to the foundation of a new research line, which is still currently under investigation, in a laboratory up to that time devoted to the study of glutamine synthetase [Among the nthetase ,17,18.Bari elements, and frame these results in a comprehensive view in the field of TE research. We also provide more extensive hypotheses on the role of Bari transposons in the genome of Drosophilidae species.In this review, we comprehensively summarize the results of 30 years of research that concern the Drosophila Bari transposons occurred during the characterization of the h39 region of the mitotic chromosomes. This is a complex repetitive locus in Drosophila melanogaster, adjacent to the second chromosome’s centromere [The discovery of the ntromere . It was ntromere . The phentromere , the maiins16 (R16) deletion [Tc1/mariner superfamily. It was named Bari1 after its discovery in the Italian city of Bari, where the laboratory was based, and making the wish (which would later come true!) that other Bari elements might be discovered to complete the series.The team headed by Prof. Caizzi and Prof. Pimpinelli hypothesized that additional genetic and molecular entities could map in the h39 region, which could also account for the phenotype associated with the region deletion. In the main effort of characterizing the h39 region at the molecular level, they identified a novel repetitive sequence, uniquely mapping to this region. Originally, differential hybridization technique was used to identify, isolate, and subsequently clone h39-specific sequences. The existence of extraordinary genetic toolkits, such as precisely mapped chromosome rearrangements  undoBari1 is a DNA transposon belonging to the IS639/Tc1/mariner superfamily with 26 bp long inverted repeats (IRs) and three direct repeated sequences (DRs) [Paris [S [minos [SB [es (DRs) , servinges (DRs) , since tParis [S , minos [S [minos , and SB inos [SB .per se a great advancement in the field of genome structure and evolution, the characterization of the peculiar arrangement of Bari1 in a heterochromatic locus was breathtaking. The precise head-to-tail organization of roughly 80 Bari1 copies in the h39 region of the mitotic chromosomes of D. melanogaster was featured by the systematic deletion of the very first two nucleotides in each copy. To our knowledge, this enigmatic organization still has no comparable described examples in the field of TEs. While other Drosophila species contain Bari-like transposons (see the Section “The Bari Family Grows Up”), the heterochromatic Bari1 cluster seems to be specific to the melanogaster species. This organization suggests a recent-and species-specific evolutionary origin since it is specific to a single species. It also allows the discrimination of two sibling species  at the molecular level. Moreover, a second minor Bari1 cluster has been recently described, which maps on the X chromosome of D. melanogaster [Bari1. Surprisingly, both the main and the small clusters share the same di-nucleotide deletion at the 5′ end of each element, and the head-to-tail organization. In addition, both clusters share a heterochromatic localization, although on different chromosomes. In a retrospective view, it is worth noting that the presence of an additional Bari1 tandem repeat was also evident since the 1993 paper. Indeed, in one of the Southern blot hybridization analyses presented in the paper, it was evident that the Rspins16 background still contains an additional Bari1 sequence block arranged in a tandem repeat configuration  from D. erecta and D. diplacantha demonstrated the presence of another type of Bari element, which was named Bari2, in the genome of many species in which the Bari1 element was also found. Bari2 differs from Bari1 in structure, since it is characterized by long TIRs  and no coding potential, due to numerous invalidating indels and frameshift mutations that disrupt its ability to encode a transposase  form of Bari2 or a recent elimination of the euchromatic copies.Former studies, mainly performed using Southern blot hybridization techniques, demonstrated the presence of homologous nogaster . Weak ornsposase A. Not a Bari1 and the evolutionary knocked out Bari2 elements was also difficult to understand. While the comparison of the ORF (and its protein product) of Bari1 and the reconstructed consensus sequence of Bari2 clearly suggested their evolutionary relationship, their TIRs are very dissimilar both in sequence and structure. Bari2 is indeed featured by long TIRs, identical to each other. Only at the DRs level the two families share evident homology, a sign that both derived from an ancestral Bari transposon [Bari2 have been preserved in extant Drosophila species is not clear. An intriguing hypothesis, that parallelizes the one proposed for the human Hsmar1 element, is that Bari2 TIRs have been co-opted to titrate some endogenous nuclear protein [Bari elements.The evolutionary link between the potentially active ansposon . Why the protein ,41 involBari1, possessing short TIRs and intact ORF, and Bari2, possessing long TIRs and disrupted ORF, was subsequently identified with the discovery of the third Bari family in D. mojavensis [Bari3 was next identified in species of the obscura and the willistoni groups [Bari3 insertions and the polymorphism observed in geographically distinct populations suggested that it is an active transposon [Bari3 transposases share 80% similarity with Bari1, and it is featured by long TIRs with an IR/DR structure  are frequently found in eukaryotic genomes [Bari-derived MITEs have been identified in D. sechellia [Bari-derived MITEs can be categorized either as short- or long-MITEs, depending on their sequence length. While both forms share the same terminal sequences, the internal sequence can be highly variable in length, with the long form exhibiting sequences unrelated to Bari transposons [Bari-derived MITEs are maintained for this regulatory purpose.The evolutionary scenario observed in the extant Drosophila species is complicated by the presence of an additional group of non-autonomous sequences called MITEs B. MITEs  genomes ,44,45,46echellia  and in oechellia . Bari-densposons . It has nsposons . TherefoHorizontal transfer (HT) is one of the most obscure yet fascinating aspects underlying the evolutionary dynamics of the genomes ,49. TEs Bari1 moved horizontally several times during the evolution among Drosophilidae species. Several works provide evidence that Bari1 HT occurred between D. melanogaster and D. simulans [D. melanogaster and D. yakuba [D. melanogaster and D. sechellia [Bari1 elements also occurred between D. biarmipes and D bipectinata [Bari1 elements in the two species are nearly identical in sequence, despite that the host species divergence dates back to 27 million years ago [Several studies suggest that simulans , between. yakuba , and betechellia . Moreoveectinata . Bari1 eears ago .Bari1 transposon was made in Drosophila [P-element system: a helper plasmid (the transposase source) and a donor plasmid (the transposon source marked with a white reporter gene), were injected into genotypically suitable fly embryos with the goal of genetically transforming the recipient strain. After the initial excitement due to the identification of few transposition events , their molecular characterization turned out to be puzzling and frustrating, since the observed phenotype was due to transposition of the NOF-FB transposon rather than the transposition of Bari1 from the donor plasmid. To make a long story short, the expression of the Bari1 transposase from the helper plasmid has possibly deregulated the NOF-FB transposon [Bari1 transposase and the transposition of NOF-FB in vivo.An initial effort to develop a new chromosomal integration tool based on the osophila . A binaransposon , whose tansposon ,58,59,60Bari elements through a transposition assay were made using cultured cells as experimental systems. Setting up a binary system, in which the donor transposon was marked with a reporter cassette and the transposase was expressed by a helper plasmid, did not result in a significant integration over the background in Drosophila and human cultured cells [Tc1-like transposons, does not significantly improve the transposition efficiency of Bari1 in heterologous transposition assays (RMM unpublished observations).Similar attempts to observe the transposition of ed cells ,61. FinaBari elements led to question whether Bari transposons are transposition-competent. Excluding the Bari2 sub-family, which is entirely composed of non-autonomous elements [Bari1 and Bari3 sub-families have what it takes to be regarded as functional.The reiterated and unsuccessful attempts to develop an efficient transposition system from elements , membersIS630/Tc1/mariner transposase, the fundamental domains of this enzyme are the DNA binding domain, the GRPR-like domain (which mediates protein–protein interactions), the nuclear localization signal, the homeo-like domain, and the catalytic domain [Bari1 and Bari3. Furthermore, the functionality of the DNA binding domain has been tested in vitro for both transposons [Bari transposons coming from population genetics studies.Standing to the general architecture of the c domain . All thensposons ,61. In aBari1 is an active transposon in natural populations of D. melanogaster. Early studies were performed in 46 populations of D. melanogaster, which suggested both Bari1 inter- and intra-stock polymorphisms [Bari1. The strongest evidence that Bari1 is a functional transposon comes from the observation of an excision event in a population established from field-collected flies. The excision event, involving one of the two adaptive Bari1 insertions in the genome of D. melanogaster, was characterized at the molecular level, demonstrating that it was due to genuine transposition  [A series of population genetics analyses strongly suggest that orphisms . Junakovorphisms  and the orphisms  of Bari1otprint) .Bari1 transposon with the outcome of the experimental transposition assays, we argue that Bari1 could be tightly regulated, or alternatively we speculate that Bari1 could be subjected to some unknown types of activation to become transposition-competent. Further and extensive in vitro-directed molecular evolution studies would clarify whether hyperactive variants can be obtained to develop Bari-based transposition systems.In conclusion, combining the indirect genetic evidence of the mobility of The transposition activity of TEs is tightly regulated at various levels. The copy number per haploid genome is a critical factor for the resulting fitness of the whole organism since an excessive TE load can be deleterious for various reasons, ranging from the gene inactivation to the disturbance of the physiological expression networks .Tc1/mariner elements, two possible types of regulation can be predicted. One is the transposon self-regulation while the other one is the epigenetic regulation.Standing to the current knowledge on Bari elements are abundant, it is conceivable to hypothesize this mode of autoregulation.One mode of self-regulation is exerted through the dissemination throughout the genome of inactive TE copies that still retain the transposase binding ability. Defective copies can act as buffer to titrate endogenous levels of the transposase . Since dBari elements through post-transcriptional processing of the transposase mRNA. Few reported data come from overexpression of the transposase in S2R+ cultured cells and in D. melanogaster embryos [Bari1 and Bari3 transcripts undergo alternative splicing that is potentially translated into a truncated transposase protein. It can be predicted that the transcriptional de-repression of Bari elements can inhibit the transposition through the expression of a dominant-negative transposase form. The presence of alternatively spliced transcripts of Bari1 has also been reported in a D. melanogaster HSP83 mutant that deregulates many transposons, including Bari1 [Bari1 and Bari3 transposase genes in which all the conventional, unconventional and cryptic splicing sites have been eliminated, could hopefully enhance the transposition efficiency of both systems.There is some experimental evidence in favor of the self-regulation of  embryos ,61  are the most abundant non-coding RNAs in the germline, aiming at the genome safeguard against TE movement . Their aBari1 and Bari2 are described [Bari transposons are among the plethora of Drosophila TEs that are regulated by the piRNA pathway in the germline  that in turn regulate the transposition frequency.Despite the lack of experimental evidence, both Bari1 as a query. Roughly 3800 of them recognize the Bari1 transposase gene. Notably, a small proportion of piRNAs matching the sequence across the monomer-to-monomer junctions—typical of the Bari1 heterochromatic clusters—can also be found  gene cluster (Bari-Jheh insertion), while the 91F insertions are associated to the cyp12a4 gene (Bari1-Cyp12a4 insertion). Further studies in which ChIP-seq data were integrated with gene expression analyses and phenotypic assays demonstrated that the Bari-Jheh insertion introduced extra antioxidant response elements upstream of Jheh1 and Jheh2 genes [Bari-Jheh is differentially associated to H3K27me3 in stress vs. non-stress oxidative conditions, suggesting the addition of histone marks to the intergenic region between Jheh2 and Jheh3 genes, and its association with histone marks enrichment in the promoter of Jheh1 gene [The early distribution studies  suggeste project , it appeeh1 gene .Bari1-Cyp12a4 insertion overlaps with the 3′ end of the Cyp12a4 gene and it contributes to the stability of the mature transcript. It has been demonstrated that Bari1-Cyp12a4 supplies the polyadenylation sequence to the Cyp12a4 transcript [The anscript .Bari1-Cyp12a4 showed an enrichment of H3K27me3 under oxidative stress conditions [In addition, the nditions , suggestTEs can also act as potent epigenetic modifiers that could change both gene expression and chromatin structure  with impBari1 elements in the h39 region of the 2nd chromosome are targets of HP1, a chromatin protein mainly involved in heterochromatin formation [Bari1 also contains Polycomb responsive elements that could collaborate in recruiting additional heterochromatin components [Bari1 are exploited in the genome. It is therefore possible that both Bari1 clusters could have acquired a structural role in the heterochromatin of D. melanogaster, such as to ensure proper chromosome conformation and stability over the cell cycle.Large-scale mapping of in vivo binding sites of chromatin proteins using tethered dam methyltransferase ,97 showeormation . It has mponents . This coBari1 transposition system, the strength of the Bari1 promoter was tested. Luciferase promoter assays [+) revealed that Bari1 has a weak promoter if compared to the strong constitutive copia promoter [Tc1/mariner transposons, which suffer from overproduction inhibition and therefore tend to limit the amount of transposase [Bari1 promoter drives the reporter transcription also in human, bacteria, and yeast cells [Bari3 also showed a similar promiscuous activity  important to deepen our understanding of TE biology.In this review, we summarized the current scientific literature concerning the transposable elements belonging to the Bari transposons’ biology are still not completely known and should be clarified, such as the low transposition rate in vitro. Studies on wild-collected fly populations will likely be the key to obtaining insights into the regulation of the Bari elements, as suggested by studies of Junakovic et al. [Many aspects of the c et al. .Bari transposase could be intrinsically error-prone, and only occasionally it gives rise to productive transposition events. Fixing the transposase could possibly allow to obtain a robust transposition system. We still have to understand how the blurry promoter has evolved, and how it can be used to develop new tools for the genetic manipulation.It is possible that, as suggested by different studies, the Finally, the determination of the structural and functional role of the heterochromatic clusters is a task that deserves further investigations, and will be accomplished with the aid of the genome editing technologies.We think that more secrets associated to TEs remain to be discovered, and the continuous effort in dissecting the most diverse TEs in nature will contribute to clarifying the role of these genetic elements in evolution, hopefully attracting more interest in the field of TE biology, especially from emerging scientists.