Ciliate protozoa are an integral part of the rumen microbiome and were found to exert a large effect on the rumen ecosystem itself as well as their host animal physiology. Part of these effects have been attributed to their ability to harbor a diverse ecto- and endo-symbiotic community of prokaryotic cells. Studies on the relationship between the protozoa population and their associated prokaryotic community in the rumen mainly focused on the methanogens, revealing that protozoa play a major role in enhancing methanogenesis potential. In contrast, little is known about the composition and function of the bacteria associated with rumen protozoa and the extent of this association. In this study, we characterize the prokaryotic communities associated with different protozoa populations and compare their structure to the free-living prokaryotic population residing in the cow rumen. We show that the overall protozoa associated prokaryotic community structure differs significantly compared to the free-living community in terms of richness and composition. The methanogens proportion was significantly higher in all protozoa populations compared to the free-living fraction, while the Lachnospiraceae was the most prevalent bacterial family in the protozoa associated bacterial communities. Several taxa not detected or detected in extremely low abundance in the free-living community were enriched in the protozoa associated bacterial community. These include members of the Endomicrobia class, previously identified as protozoa symbionts in the termite gut. Our results show that rumen protozoa harbor prokaryotic communities that are compositionally different from their surroundings, which may be the result of specific tropism between the prokaryotic community and protozoa. Ciliate protozoa are a diverse group of eukaryotic microorganisms, ubiquitous across many environments and are known to live in close association with their surrounding prokaryotic community . The proCiliate protozoa are an integral part of the rumen microbiome and form a large proportion of its microbial biomass . In the in vitro studies demonstrate a close metabolic interaction between methanogens and ciliates, in which mixed or individual species of ciliates incubated with the rumen prokaryotic community lead to an increase in methane production  and 806R (5′-GGACTACNVGGGTWTCTAAT-3′). Sequencing was performed on a MiSeq platform using the paired end protocol (2 × 250 bp). For the 18S rDNA sequencing, primers specifically designed for ciliates were used from SRP156491.The Research Laboratory Hylab  performed amplicon sequencing of the ruminal DNA samples as follows: 20 ng of DNA was used in a 25 μl PCR reaction with primers, using PrimeStar Max DNA Polymerase (Takara) for 20 cycles. The PCR reaction was purified using AmpureXP beads, and then a second PCR was performed using the Fluidigm Access Array primers for Illumina to add the adaptor and index sequences. For this reaction 2 μl of the first PCR were amplified in a 10 μl reaction for 10 cycles. The PCR reaction was purified using AmpureXP beads and the concentrations were measured by Qubit. The samples were pooled, run on a DNA D1000 screentape (Agilent) to check for correct size and for the absence of primer-dimers product. The pool was then sequenced on the Illumina MiSeq, using the MiSeq V2-500 cycles sequencing kit. The primers sequences used for the 16S rDNA bacteria and archaea were taken from the updated sequences (2015–present) of the earth microbiome project2).Downstream processing of the 16S rDNA data, up to the generation of OTU tables was performed in QIIME v.1.9.1 . After rAfter taxonomic assignment, reads belonging to Chloroflexi and Cyanobacteria phyla were further removed from the dataset, as these sequences may be the product of chloroplast amplification commonly found in protozoa and cannot confidently be attributed to the bacterial domain . For taxt-test procedure was used to assess the differences in the mean variability between samples from different fractions across animals and time points. The heatmap and hierarchical clustering of the 16S rDNA relative abundance data was generated with the heatplus package in R  using the Bray–Curtis dissimilarity metric based on OTU composition  was used and plotted using the PAleontological STatistics software (PAST) . Bonferrage in R . For therocedure  were conP < 0.05; Figure P < 0.01; Figure P < 0.01; Figure We quantified the abundance of the bacteria and methanogens and their relative prevalence in association with the different protozoa populations and the free-living community using real time PCR. This analysis revealed that the methanogens/bacteria ratio within the protozoa fractions is significantly higher compared to the ratio found in the free-living fraction . The smaller filter P-10 had a mixed population of Dasytricha, Entodinium and Isotricha, and its filtrate, P-<10 almost exclusively contained members of Dasytricha , but no such discrimination could be observed in the protozoa associated prokaryotic communities  with averages ranging between 50% and 80%, followed by the Methanomassiliicoccaceae with averages ranging between 13% and 39% and Methanomicrobium ranging between 0.94% and 7.4% across the fractions.In the free-living community, the Methanomassiliicoccaceae family was the dominant taxon, accounting for an average of 50% of all methanogens reads, followed by the Mbb. gottschalkii, Mbb. wolinii, and Mbb. ruminantium represented the vast majority of OTUs from the Methanobrevibacter genus, and exhibited significant differences both between the free-living and protozoa associated population and between the different protozoa fractions . Mbb. ruminantium was in a significantly higher proportion in all the protozoa fractions  compared to the free-living population (0.83%) .Using RIM-DB, a database specifically oriented toward identification and annotation of rumen methanogenic archaea, we assessed the distribution within the taxonomic groups observed across the different fractions. OTUs that had a high similarity (OTU similarity > 97%) with sequences from this database where thus categorized to several different clades within each of the families or genera observed . Mbb. gos Figure . FractioMethanobrevibacter, had different proportions between the fractions. The proportion of OTUs associated with Group_9 was significantly higher in the free-living fraction, accounting for an average of 21.13% of the total methanogenic OTUs compared to the protozoa associated fractions . In addition, different proportions were observed within this group between the protozoa fractions, with P-100 and P-60 exhibited the lowest proportion with an average of 1.07% and 3.3%, respectively, compared to a significantly higher proportion in fractions P-40, P-10, and P-<10, with 11%, 10.5% and 5.9 %, respectively , and exhibited a non-significant lower proportion in P-40 , P-10 (3.8%: P = 0.07), and P-<10  fractions . Other phyla that had significantly different proportion between the free-living and protozoa fractions include the Actinobacteria, Proteobacteria, and Elusimicrobia which were higher in all of the protozoa associated communities . Both Actinobacteria and Fibrobacteres exhibited a significant differential proportion between the protozoa fractions. Fibrobacteres was uniquely enriched in the P-100 fraction .The Bacteroidetes and Firmicutes were the most prevalent phyla within all the samples, which is in agreement with previous rumen microbiome studies . Howevers Figure . In contWe further depicted the 35 most abundant families out of a total of 315 families that had a mean relative abundance of at least 0.5% in at least one of the fractions Figure .P < 0.01, Figure The higher proportions observed within the Firmicutes phylum in the protozoa associated fractions was mostly driven by the presence of the Lachnospiraceae family. This family appeared in a significantly higher proportion in all of the protozoa associated communities with an average of 12.7%, 25.4%, 32%, 28.9%, and 24% for the P-100, P-60, P-40, P-10, and P-<10 fractions, respectively, compared to an 3.75% on average in the free-living community  revealed significant differences in the overall structure of the prokaryotic communities associated with protozoa compared to the free-living community. These results are in agreement with the previously shown differences in methanogenic population structure associated with protozoa compared to the free-living population in the rumen, and suggest that these compositional differences extend to the whole prokaryotic community . In addiMethanobrevibacter was the dominant genus in most of the protozoa fractions  methanogenic group in the rumen  and Blautia (OTU similarity > 95%). S. sucromutans, the only cultivated representative of the Syntrophococcus genus, is a known rumen acetogen while Blautia has several members known to reside in the rumen identified as acetogenic (The identity and role of most members of Lachnospiraceae and Ruminococcaceae in the rumen remains unclear, however, several recent studies assessing reductive acetogenesis in the rumen uncovered that both Lachnospiraceae and Ruminococcaceae were prominent in culture media enriching for homoacetogenic functions . Similarhanogens . Howeverhanogens . Strengtetogenic . Our resetogenic .Euplotes aediculatus. Other bacterial taxa exclusively seen in the protozoa community include members Sphingobacteria, and Flavobacterium. Interestingly, members of these taxa have been previously shown to be engaged in a symbiotic relationship with the ecto-parasitic ciliate Ichthyophthirius multifiliis (In addition to the differential abundance of shared taxa between the free-living and protozoa associated fraction. We observed several bacterial taxa not detected or appearing in low abundance in the free-living samples and enriched in the protozoa associated fractions. These included Proteobacterial lineages such as Rickettsiales and other undefined Alpha-proteobacteria. tifiliis .Our analyses revealed the high prevalence of OTUs belonging to the Endomicrobia class, which could reach up to 10% of the protozoa associated bacterial communities. Several studies identified members of this class engaged in an obligate endosymbiotic relationship with flagellate protozoa species in the wood feeding termites . NotablyThe identity and role of prokaryotic microorganisms associated with protozoa remains largely unclear. To our knowledge this is the first comprehensive study of the overall prokaryotic population found associated with different ciliate protozoa populations, aimed at characterizing the structure of both bacterial and archaeal taxa. The protozoa feed on the prokaryotic members of the microbiome and therefore it is possible that a part of the prokaryotic populations observed serve this purpose . The difThe specificity of each prokaryotic community across different protozoa fraction isolated, the proportional dominance of several taxa, and the observed enrichment of bacteria previously linked to protozoa symbiosis in other environments, including the cellulose degrading termite gut system, suggest that the prokaryotic community may have a wide variety of functional interactions, conserved across different environments, with their neighboring ciliate population.EJ and BL conceived and performed the experiments and analyzed the data. EJ wrote the manuscript.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.


Venous blood was collected immediately before and after saturation. Following gene expression profiling, post-saturation gene activity changes were analyzed. Protein biomarkers for inflammation, endothelial function, and fibrinolysis: Il-6, CRP, ICAM-1, fibrinogen, and PAI-1, were measured in plasma. Post-saturation gene expression changes indicated acclimatization to elevated ppO2 by extensive downregulation of factors involved in oxygen transport, including heme, hemoglobin, and erythrocytes. Primary endogenous antioxidants; superoxide dismutase 1, catalase, and glutathione synthetase, were upregulated, and there was increased expression of genes involved in immune activity and inflammatory signaling pathways. The antioxidant vitamin supplements had no effect on post-saturation gene expression profiles or vascular function biomarkers, implying that the divers preserved their homeostasis through endogenous antioxidant defenses.Saturation diving is an established way to conduct subsea operations with human intervention. While working, the divers must acclimatize to the hyperbaric environments. In this study, genome-wide gene expression and selected plasma biomarkers for vascular function were investigated. We also examined whether antioxidant vitamin supplements affected the outcome. The study included 20 male professional divers, 13 of whom took vitamin C and E supplements in doses of 1,000 and 30 mg daily during saturation periods that lasted 7–14 days. The dives were done in a heliox atmosphere with 40 kPa oxygen partial pressure (ppO Health risks in saturation diving are managed through procedures initially established in the 1960s, which have since developed with accumulated research and empirical knowledge.Professional saturation divers live onboard Diving Support Vessels (DSVs) in hyperbaric pressure chambers during work assignments. Their assignments may last up to 3 weeks in Norwegian waters, and even longer in international waters. When commuting from the hyperbaric living chambers onboard the DSV to work on the seabed, the divers are transported in pressurized diving bells. They leave the bell through a door in the bell floor to perform physically demanding underwater work, comprising handling of tools and equipment, rigging and welding of pipelines. In order to remain fit, the divers must acclimatize to the hyperbaric environment, including elevated partial pressures of oxygen  during work assignments on the Norwegian Continental Shelf in 2015. The protocol was approved by the Norwegian Regional Committee for Medical and Health Research Ethics (REK), approval reference number 2015/351, and by the Norwegian Petroleum Safety Authority. The study subjects were individually informed and provided written consents before inclusion, and all procedures were conducted according to the Declaration of Helsinki principles for ethical human experimentation.The study subjects were 20 healthy male divers, who held valid health certificates for working as saturation divers. All subjects underwent the diving contractor’s standard medical examinations before and after saturation diving, including measurements of weight, height, pulse, and blood pressure. Information regarding allergies, smoking, previous/current injuries or illnesses and current medication were also registered.2max) to determine if they fulfill requirements for aerobic fitness by use of direct oxygen uptake measurement. In this study, the diving contractor used a Woodway PPS Med treadmill ; combined with a METALYZER® 3B analyzer and MetaSoft® Studio software . The oxygen uptake tests were supervised by the hyperbaric nurse on duty, following a standardized protocol  and the control group (n = 7). Table 1 describes the participating divers’ subgroups demographics, experience, VO2max and body-mass index (BMI).Prior to saturation diving, the subjects were randomly divided into two groups: the antioxidant vitamin group , were supplied to the vitamin group during the pre-saturation medical examination. The divers in the vitamin group consumed two tablets of vitamin C (500 mg/tablet) and one capsule of vitamin E (30 mg/capsule) with dinner for the duration of saturation and decompression; i.e., every day from entry to exit from the pressure chambers. All the divers in the vitamin group reported that they took the vitamins as instructed. Apart from the vitamin intervention, no constraints were put on the divers’ diet or life-style choices as part of this study.2 was kept at 40 kPa in the pressure chambers for the duration of the saturation period. The divers were organized in teams of 3, with each team working daily 12 h shifts. A bell dive takes up to 8 h; with the diver locked out and working in the water for maximum 6 h, including a mandatory 30 min restitution and rehydration break in the bell between the third and fourth hour of work. The maximal diving depth was 125 msw.Diving operations took place in June–July 2015, approximately 200 km west of the Stavanger coast in Norway, conducted in accordance with the NORSOK U-100 requirements . The div2 was increased to 60 kPa. In order to protect the divers from hypothermia from work in 4°C water, seawater was heated onboard the DSV and pumped down, through the diving bell and out into the divers’ wet suits. After a rotation of one (3 divers) or two (17 divers) weeks in saturation, the divers were decompressed following table 12 in NORSOK U-100 requirements  and kept refrigerated at 4°C.Blood samples were collected by standard phlebotomy of the median cubital vein at two time-points: the first before the divers entered the hyperbaric living chambers (pre-saturation), and the second shortly after completed decompression back to surface (post-saturation). For plasma, 3.5 ml venous blood was collected on citrated and heparinized tubes . After filling, the tubes were gently inverted 8–10×. Hematocrits were measured with the citrated blood samples, using capillary tubes and a Haematokrit 200 centrifuge . To prepare plasma, the blood tubes were centrifuged (Hettich GmbH and Co. KG) for 10 min at 1,800 × For RNA, 2.5 ml blood was collected in PAXgene blood RNA tubes . A single batch of PAXgene tubes was used in order to limit technical variation. After filling, the tubes were kept upright at room temperature for 4 h before they were refrigerated at 4°C. All samples were transported in temperature-controlled cubes  at 4°C to NTNU in Trondheim, where they were frozen at -80°C until all material was collected for analysis.For reasons of logistics on the DSV, and uniformity in the analyses, all samples were kept refrigerated at 4°C for exactly 14 days prior to freezing at -80°C. In order to determine whether this affected the quality of the subsequent analyses, two control samples from healthy males were added to each plasma set-up: one that had been frozen at -80°C on the day of collection, and another from the same donor that was kept at 4°C for 14 days and then frozen at -80°C. These controls gave similar results for all biomarkers, indicating that the plasma collected on the DSV was unlikely to be deteriorated. The PAXgene tubes were expected to sufficiently stabilize the RNA profiles during the cold storage.Total RNA was extracted from thawed blood using the PAXgene Blood RNA kit version 2 . RNA quantity and quality were measured on an Agilent 2100 Bioanalyzer , giving RNA concentrations of 52–357 ng/l, and RNA integrity numbers  ranging 1 for gene expression analysis using lumi Bioconductor package version 1.1.0 . An absolute threshold for transcription change was set to 0.5. The pathways were ranked according to the probability of a chance occurrence on the background of all probes on the Illumina humanHT-12 v4 Expression BeadChips microarray. In all analyses, the significance level was set at P < 0.05, using false discovery-adjusted P-values.The filtered microarray data was transferred to the R statistical computing softwareon 1.1.0 . Inter-son 1.1.0 . Genes tPrior to plasma analyses, the expected power was assessed on the SISA online statistical power calculator , using bP < 0.05 considered significant.In order to determine appropriate statistical tests for group-wise comparison of the plasma data, the normality and homogeneity of variances in the data were examined using Kolmogorov–Smirnov and Levene’s tests. When biomarker data were normally distributed or became so after transformations, a one-way ANCOVA test was applied. In cases where data were not normally distributed, a non-parametric Mann–Whitney test was applied. All analyses were performed in IBM SPSS Statistics software , with Heparinized plasma was used for analysis of high-sensitivity CRP , total cTM  were used to quantify interleukin 6 (IL-6) . All controls were within the target ranges set by the manufacturer, and plasma protein concentrations were calculated from the standard curves using the sample mean OD values with blank correction.Commercial ELISA immunoassay kits  from R&D systems6 (IL-6) , interceThe normal ranges indicated by the producer for CRP, ICAM-1, IL-6, fibrinogen, and PAI-1 were, respectively: <5 mg/L, 0.435–9.57 pg/ml, 106–337 ng/ml, 1.9–4.2 g/l, and 2.66–69.3 ng/ml.3 in accordance with MIAME standards. The access code is E-MTAB-4491 (The microarray data are openly available in the EMBL-EBI ArrayExpress repositoryTAB-4491 .Table 2). Hematocrit levels were within the normal range (Table 2).The saturation diving operations were conducted according to plan, and blood samples were collected from all study subjects as per the protocol. All data for total cholesterol, HDL, LDL, triglycerides, and creatinine were within normal range , and unaal range  pre-satuFigure 2, sample relations for the primary (PC1) and secondary (PC2) PLS components revealed two major traits. First, the samples were almost completely separated along PC1 according to whether they had been collected before or after saturation. This implied that saturation diving was the predominant cause of variation in the data. Second, intake of antioxidant vitamins had no effect on the two primary PLS components. In further gene expression analysis, data from both groups of divers were therefore merged.In order to examine the relations between the samples collected from saturation divers with and without antioxidant vitamins, and before and after diving, we performed an exploratory multilevel PLSs regression analysis on the microarray data. As illustrated in Supplementary Table S1.Genome-wide effects of saturation diving on transcription were determined by comparing the pre- and post-saturation microarray data. A total of 12,201 transcripts were identified as differentially expressed: 5,452 were downregulated, and 6,749 were upregulated after saturation diving. Complete lists of differentially expressed transcripts and their corresponding genes are presented as Figure 3, log fold expression changes for genes involved in central aspects of blood oxygen transport are plotted according to their placement in the process; from the synthesis of oxygen-carrying heme molecules, the synthesis and activity of different hemoglobin types into which heme is built, through the final step of erythropoiesis in which reticulocytes released from the bone marrow are differentiated into erythrocytes, and finally the activity of mature circulating erythrocytes in the blood stream.For biological interpretation of the gene expression data, we first considered the genes that displayed the largest fold change post-saturation. Among those with established function, the data comprised a disproportionally large number of genes involved in oxygen transport, all of which were downregulated. Further inspection revealed more downregulated genes with similar function. In Supplementary Table S1). Genes involved in glutathione turnover, GSR and GPX, showed variable results.Several gene coding for antioxidant factors were differentially expressed after saturation. Among them, all three primary endogenous cytosolic antioxidants; superoxide dismutase 1 (SOD1), catalase (CAT), and glutathione synthetase (GSS), were upregulated, whereas the mitochondrial superoxide dismutase, SOD2, was downregulated (Supplementary Table S1).In order to identify biological pathways that may be affected by saturation diving, we performed functional clustering analysis of genes that were differentially expressed. As predictable from the inspection of genes with the largest fold-change in expression, the downregulated genes were largely associated with anemic conditions. The upregulated genes were primarily involved in immune responses and inflammatory signaling, indicating that there was increased inflammatory activity in the divers’ blood at the time they completed their hyperbaric saturation (P= 0.279), IL-6 (P = 0.968), ICAM-1 (P= 0.588), fibrinogen (P = 0.464), and PAI-1 (P = 0.536) (Table 3).There was no difference between the two diving groups, with and without antioxidant vitamin supplementation, for CRP (This study had two objectives. The first was to identify and characterize indicators of acclimatization to the hyperbaric environments in professional saturation diving, on genetic activity level and plasma biomarkers of vascular function. The second objective was to assess whether daily intake of antioxidant vitamins C and E affected the outcome of either of the above.2 for 14–21 days; from 21 kPa in the normal ambient air to a range of 40–60 kPa in the heliox gas mixture they were breathing in saturation and on work excursions. It is reasonable to conclude that a reduction in blood oxygen transport represents acclimatization against the hyperoxia. Our results are in agreement with reports of decreased hemoglobin levels and erythrocyte counts in experimental saturation diving (For the first objective, peripheral blood gene expression profiling revealed extensive downregulation of genes involved in oxygen transport, including the production and activity of heme, hemoglobin and erythrocytes, at the time when the divers had completed their saturation diving assignments. At that time, they had been exposed to elevated ppOn diving . Hyperoxn diving .During work excursions, saturation divers experience dehydration due to physical activity, the use of seawater-heated diving suites, and in some cases failure to rehydrate . A loss For the second study objective; the assessment of antioxidant vitamin supplement effects, we found no changes in the blood collected after saturation diving for either gene expression or plasma biomarker levels.Effective management of oxidative stress is important for health preservation. While this is normally provided by the body’s endogenous redox systems, diving challenges the physiological balance by inducing ROS production in excess of normal doses. In saturation diving, hyperoxia along with absolute and partial pressure changes and inert gas exchange during decompression are likely sources of excess ROS . In the Sparse data are available showing the effect of antioxidant vitamins on vascular function in relation to diving, but many experiments were conducted on the general population. However, reports on the latter are conflicting. A 2015 systematic review of vitamin C and E supplementation concluded that while either vitamin C or E alone appeared to improve endothelial function in healthy individuals, their combination might have little effect . There iSimultaneous measurement of a panel of biomarkers can provide a comprehensive assessment of oxidative stress effects on vascular function. In this study, protein biomarkers were chosen in order to cover key features of vascular function; i.e., inflammation, endothelial function, and fibrinolysis. CRP is a biomarker of inflammation and endothelial function ; its incElevated fibrinogen is a predictor of cardiovascular disease risk , while PElevated circulating levels of LDL and triglycerides are risk factors for atherosclerosis and cardiovascular disease. Their effects are countered by HDL, which acts as an antioxidant and anti-inflammatory molecule promoting endothelial repair . ElevateTaken together, the unchanged levels of the biomarkers chosen to assess inflammation, endothelial function, and fibrinolysis suggest that any potential effects of saturation diving on vascular function were resorbed; the diver’s bodies had returned to their pre-saturation state by the end of decompression. The absence of antioxidant vitamin effects also indicates that the divers retained their vascular homeostasis through their own endogenous antioxidant defenses, as is also supported by the observed upregulation of SOD1, CAT, and GSS. Endogenous antioxidants that primarily or exclusively act in the mitochondria, e.g., SOD2, were downregulated or showed variable results. However, both the copy number and function of mitochondria may be affected by hyperoxia , which iThere are limitations to this study. First, the collection of blood took place during real-life diving operations, and was conducted so as not to disturb the work. As the divers were not available for tests from their time of entry until they exited the pressure chambers, sample collections were only performed pre- and post-saturation. At that time acclimatization to the hyperbaric heliox atmosphere appear to have restored their redox balances, thus curbing any potential effects of the antioxidant supplements. Considering that antioxidant supplements have been found to reverse liver dysfunction during the initial days of experimental saturation diving , we cannIn conclusion, acclimatization to professional saturation diving was associated with extensive downregulation of genes involved in oxygen transport, upregulation of endogenous antioxidants, immune activity and inflammatory signaling. Daily antioxidant vitamin C and E intake in recommended doses had no effect on the outcome of either genetic activity or vascular function biomarkers. However, this study did not address responses that might occur during the saturation phase. Future research to determine whether antioxidant supplements might protect professional divers’ redox balances during underwater work would require sample collections to be performed in saturation.FK, IE, and AM designed the study. AH and AF conducted the statistical analyses. ØL obtained the necessary consents. FK managed the blood and data sampling, initiated the manuscript, and all the co-authors contributed to the writing and approval of the final manuscript.FK and ØL were employed by TechnipFMC in Norway. The remaining 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.


However, the ability of this compound to function as a nucleating agent to improve other macromolecule properties has not been explored. This report scrutinizes the influence of the cationic complex as a novel nucleating agent on the spherulitic morphology, crystal structure, and isothermal and non-isothermal crystallization behavior of the Poly(3-hydroxybutyrate) (PHB) bacterial origin. The incorporation of the CpFe+ into the PHB materials caused a significant increase in its spherulitic numbers with a remarkable reduction in the spherulitic sizes. Unlike other nucleating agents, the SEM imageries exhibited a good dispersion without forming agglomerates of the CpFe+ moieties in the PHB matrix. Moreover, according to the FTIR analysis, the cationic organoiron complex has a strong interaction with the PHB polymeric chains via the coordination with its ester carbonyl. Yet, the XRD results revealed that this incorporation had no significant effect on the PHB crystalline structure. Though the CpFe+ had no effect on the polymer’s crystal structure, it accelerated outstandingly the melt crystallization of the PHB. Meanwhile, the crystallization half-times (t0.5) of the PHB decreased dramatically with the addition of the CpFe+. The isothermal and non-isothermal crystallization processes were successfully described using the Avrami model and a modified Avrami model, as well as a combination of the Avrami and Ozawa methods. Finally, the effective activation energy of the PHB/CpFe+ nanocomposites was much lower than those of their pure counterparts, which supported the heterogeneous nucleation mechanism with the organometallic moieties, indicating that the CpFe+ is a superior nucleating agent for this class of polymer.Cationic cyclopentadienyliron (CpFe The development of biopolymers has attracted great interest over the years as a replacement for petroleum polymers. The key feature of this interest arises from the detrimental impact of traditional polymers on the world’s ecosystems. As a result, many researchers seek to exploit the synthesis of polymeric materials from renewable resources that will not persist in the environment after disposal ,2,3,4. SAmong the polys, polyester of 3-hydroxybutyrate (PHB) is an attractive example of a biodegradable and biocompatible polymer ,8, whichA general comparison of biodegradable polymers to traditional classes revealed certain drawbacks that pose challenges to their commercial applications. For example, in the case of PHB, its manufacturability has been limited due to its stiff and brittle character, narrow processing window, and high cost ,14,15. A4Cl [2, and ZnO nanoparticles [2 inorganic nanotubes [Several strategies have been reported to overcome some of the aforementioned disadvantages ,17. One 4Cl , melamin4Cl , boron n4Cl ,21, talc4Cl ,23, cyan4Cl , cyclode4Cl , lignin 4Cl , and the4Cl . Recent 4Cl ,29, TiO2articles , graphitarticles , and WS2anotubes .Despite the prevalence of previous studies that have explored a tremendous number of nucleating agents, this topic continues to be researched in order to overcome the drawbacks that have been associated with such nucleating agents ,41,42,43Over the past two decades, our work has been focused on utilizing the cationic cyclopentadienyl iron moieties in designing novel classes of monomeric and polymeric materials ,47,48,49+ complex as a novel nucleating agent on the isothermal and non-isothermal crystallization behavior of PHB using Differential Scanning Calorimetry (DSC) and polarized optical microscopy (POM). The crystallization kinetics and the activation energy have also been analyzed using several models, such as Avrami, Ozawa, a combination Avrami and Ozawa model, and the differential isoconversional method of Friedman. This work presents the first example of exploring the effect of the CpFe+ nanocomposites at the isothermal crystallization condition, 100 °C. The obtained results revealed that the size of the PHB’s spherulite was found to be as large as 1 mm, while the spherulite number was extremely small. This behavior could be attributed to the exceptional purity of the PHB that was prepared via bacterial origin [+ nanocomposites displayed a Maltese cross section with regular extinction rings. The presence of the cationic cyclopentadienyliron (CpFe+) moieties led to the production of smaller spherulites than that existing in the pure PHB, where the CpFe+ nanoparticles were trapped in the center of the spherulites in the form of brown colored dots. Also, the CpFe+ particles were well distributed in the PHB matrix, which is clearly seen in the SEM images in + correspondingly increased the number of small spherulites. These results categorized the CpFe+ as one of the most highly efficient nucleating agents of PHB.l origin . It is a+ nanocomposites were examined using scanning electron microscope (SEM). + species without forming agglomerates in the polymer matrix. This behavior remained incessant while increasing the nucleating agent ratio. Additionally, the incorporation of the iron moieties into the polymer matrix triggered the formation of the helical structures of the polymer chains during the crystallization process, which can be observed in some of the SEM images [The morphologies of the PHB and PHB/CpFeM images ,55,56.+ moiety, pure PHB, and PHB/CpFe+ nanocomposites with a 99:1%, respectively, are illustrated in −1, while their stretching bands were observed at 469 and 553 cm−1. These peaks were shifted to a higher frequency, 1055 and 1410 cm−1, after the decoordination process and the blending with the PHB polymer; in addition, the spectrum exhibited a disappearance of the complexed arene C–H band at 3097 cm−1. On the other hand, the cyclopentadienyliron moieties became more susceptible to the coordination with the ester carbonyl of the polymer, as seen in −1 in the pure PHB spectrum was assigned to the ester carbonyl group within the polymer matrix. Upon blending the polymeric materials with the CpFe+ complex, a decrease in the carbonyl stretching frequency was observed while maintaining its value, which confirmed the formation of a chelating complexation to the cationic iron moieties. Furthermore, the frequencies of the complexed cyclopentadienyl C–H stretching band and the CH3 bands of the PHB chains, in the range of 2901–2987 cm−1, were increased after the blending process with the preservation of their values, which supported the complexation behavior of the iron centers towards the PHB matrixThe FTIR spectra of the CpFe+, and the PHB/CpFe+ nanocomposites. The characteristic Bragg reflection peaks of the CpFe+ complex can be observed at 2θ = 19.77, 20.08, and 21.48, which is indexed on the bases of the face centered cubic (FCC) structure of iron and found to be identical to those reported for the standard iron metal. The average crystal size of the CpFe+ was calculated, according to the Scherrer equation, and found to be around 42 nm. However, blending this organometallic complex with the PHB, followed by the thermal processing of the prepared sheets, enhanced the tendency of the iron center to coordinate with the ester carbonyl of the polymer as described previously and illustrated in + was one of major factors that facilitated the excellent distribution of the metallic centers within the polymer matrix. Furthermore, the coordination process led to the disappearance of the diffraction peaks of the CpFe+ moiety at the (021) area. On the other hand, the metallic moieties exhibited a crystal matching with the polymeric materials that was evidenced by the perfect alignment of the diffraction peaks, which can be observed through the growth of its peaks at the (101) and (110) areas. It is also noticeable that the diffraction peaks of the PHB became sharper and narrower than the pure PHB ones with the increasing of the percentage of the iron complex, which is an indication of the increasing crystallinity of the modified polymer. Utilizing the Scherrer equation, the pure PHB exhibited a crystal size of 32.7 nm, while the PHB/CpFe+ nanocomposites displayed crystal sizes of 35.5, 36.8, and 37.2 nm for 0.5%, 1%, and 3% of the CpFe+ nanocomposites. In spite of the fact that the diffraction peaks of the modified polymeric materials retained their values, which suggested that the presence of the metallic moieties did not alter the structure of the polymer, increasing the ratio of the iron moieties reduced the intermolecular interactions between the C=O group and the CH3 group of the PHB due to the formation of a coordination interaction with the CpFe+ complex. This behavior resulted in the increase of the intensity and the narrowing of the (110) peak area with a value of 2θ (16.88°) (marked with asterisk). In order to verify the consistency of this performance, an extra ratio of the CpFe+ (10%) was blended with the PHB. c, is an indication of a faster crystallization rate. The values of the crystallization temperatures (Tc) and the crystallization enthalpy (∆Hc) are reported in The Differential Scanning Calorimetry (DSC) cooling curves of the PHB and its nanocomposites are presented in +) complexes in the PHB matrix resulted in a shift of the crystallization peaks to a higher temperature, + complexes. These results could be attributed to the presence of the strong interaction between the organoiron centers (CpFe+) and the PHB matrix. As can be seen in + acts as an efficient nucleating agent and suggested that the PHB crystallization occurs through a heterogeneous nucleating mechanism [c for all the PHB nanocomposites were higher than those of the pure PHB. g), cold crystallization temperature (TCC), and melting temperature (Tm) are listed in + nanocomposites were around 2 °C, which was independent of the PHB composition.The composition of the PHB nanocomposites manipulates the position and shape of the crystallization peak. The presence of the cationic iron  to the selected crystallization temperature, ranging from 90° to 128 °C. The exothermic crystallization peaks were recorded as a function of the crystallization time. It is crucial to note that the crystallization peaks were broader at higher crystallization temperatures (Tc), indicating a lower crystallization rate. The numerator represents the integrated heat flow generated until time t, and the denominator is the total heat flow produced in the isothermal crystallization until t = ∞. It can be detected that in the X(t) range, between 0.2 and 0.8, the curves are straight. The isothermal crystallization kinetics of the PHB and its nanocomposites were analyzed based on the Avrami Equation, Equation (3):The plot of k, n, and t0.5, are all reported in t0.5 is defined as the time at which the relative crystallinity is 50%. As seen in t0.5 decreased with the decrease of the crystallization temperature and increased the percentage of the CpFe+ in the PHB/nanocomposites, which is a required behavior for processing the polymer materials. These results indicated that the rate of the PHB crystallization process is faster when the super cooling temperature is higher within the selected crystallization temperature range. At the given crystallization temperature, 120 °C, the t0.5 of the PHB crystallization process in the PHB nanocomposites was much lower than that of the pure analogues, while all the curves of the isothermal crystallization kinetics were almost parallel with each other, as shown in n values were almost constant and approximately equal to 2. The value of n can be attributed to the presence of thermal crystallization during the crystallization process [k, which is related to both the nucleation and the growth processes, increased with the decrease in the crystallization temperature and the increase of the ratio of the CpFe+ complex. For instance, at 120 °C, the crystallization rate of the PHB with the incorporation of 3 % of the CpFe+ moieties was found to be 87% faster than that of its pure analogue. These results verify the hypothesis that CpFe+ is a superior nucleating agent for this type of polymer.The crystallization parameters,  process . Another process  is that In the polymer processing techniques, the temperature changes continuously. Therefore, investigating the crystallization kinetics under non-isothermal conditions was essential. In general, the exothermic crystallization peak becomes broader and shifts to lower temperatures with the increasing of the cooling rate of all the PHB samples. The dependence of the crystallization temperature (Tc) on the cooling rate for the PHB/nanocomposites is shown in + moieties. The relative crystallinity X(T) was evaluated from the DSC experimental data in The attained data disclosed that the crystallization temperature of the PHB in its nanocomposites was higher than those of the pure counterparts for all the studied cooling rates. These results indicated that the enhancement of the crystallization temperature arises from the presence of the CpFe0 and T∞ are the onset and the end crystallization temperature, respectively, and the ∂ H is the enthalpy of the process while the temperature is converted into the time scale, using Equation (5) as follows:The TThe T is the temperature at the crystallization time, and the t should be effectively corrected [The Avrami model , the Ozaorrected , and it t are two adjustable parameters [t values, at any given composition, increase by increasing the cooling rate and, at any given cooling rate, increase with the increase in the content of the CpFe+. A similar trend was detected for an inverse half-life time of crystallization, as shown in It is well known that the Avrami equation assumes that during the isothermal crystallization process, the nucleation rate remains constant. However, the nucleation rate is not constant when the PHB samples are crystallized under non-isothermal crystallization conditions . Thereforameters ,63,64. ACombining both the Avrami and Ozawa models was proposed by Liu  at a givAfter rearrangement, it became Equation (8):b is the ratio of the Avrami exponent (n) to the Ozawa exponent (m):The + nanocomposites. A good linearity was achieved for all the PHB samples, as shown in + nanocomposites decreased with increase of the CpFe+ content, indicating a pronounced effect of CpFe+.Several models in the literature have been proposed to determine the effective activation energy . The isoThe subscript Vyazovkin  stated te PHB in .+ ratio of 97:3. The reduction of the effective activation energy values in the nanocomposites has been attributed to the occurrence of the heterogeneous nucleation mechanism with the organometallic moieties, which can be distinguished in the polarized optical microscopy images, In general, the effective activation energy of the pure PHB in its nanocomposites is much lower than that of the pure form. The lowest activation energy value was obtained at the PHB/CpFe+ moieties with the PHB prompted the formation of more nuclei at high crystallization temperatures, which, consequently, has the lowest effective activation energy.The activation energy is closely related to the transport of the macromolecular chains to the growing surface and nucleation steps . Figure + complex was degraded by two steps. The initial degradation step corresponded to the cleavage of the arene ring, while the second step was comprised of the decomposition of the organic moieties. The maxima degradation temperatures of the CpFe+ for the first and second steps were 243 °C and 410 °C, respectively. On the other hand, the PHB/CpFe+ nanocomposites decomposed through a one-step degradation mechanism, which was found to be higher than that of the pure PHB by 10 °C. Furthermore, the thermal degradation temperatures of the PHB/ CpFe+ nanocomposites were almost constant and were independent of their ratios, as shown in + complex improved the thermal stability of the PHB due to the strong chemical interaction, as described previously in the text.The thermal stability of the PHB in the nanocomposites was determined using TGA and DTGA, as shown in 6-arene-η5-cyclopentadienyliron complex (CpFe+) was prepared in accordance with the previously reported methodology [1H NMR spectrum exhibited two singlet resonances at 5.6 and 6.9 ppm, which corresponded to the equivalent cyclopentadienyl protons and the complexed aromatic arene protons, respectively. In the meantime, the FTIR spectrum displayed an out of plan bending peak, corresponding to the complexed arene C–H at 816 cm−1, while their stretching bands were observed at 3097, 469, and 553 cm−1. Furthermore, the frequencies of the complexed cyclopentadienyl C–H stretching band resonated in the range of 2901–2987 cm−1.The pure bacterial Poly(3-hydroxybutyrate) (PHB), in the form of white powder, was kindly provided by Dr. Haenggi, Biomer Company, Germany. The PHB was more than 98%, the content of the polyhydroxyvalerate (PHV) was less than 1%, and the remaining cell membrane and membrane lipids were ca. 1%. The weight average molecular weight was about 600,000 g/mol. The melt flow rate (MFR) at 180 °C with 2.16 kg was 10 g/10 min; methylene chloride (99.9%) was supplied from Sigma Aldrich, Germany. No further purification was performed on the materials. The ηhodology . The str+ composites with different weight ratios were prepared through a casting film technique using methylene chloride as a solvent. For each film, approximately 2.00 g of the total sample were mixed and dissolved in 50 mL of methylene chloride with stirring at 40 °C for 45 min to ensure the complete dissolution of both components. After that, the solution was kept at room temperature for 120 min and then casted onto a glass Petri dish at room temperature to slowly evaporate the solvent and to form homogenously a film. The cast film samples were molten between hot melt press at 200 °C for 2 min. The thickness of the specimen was measured using a digital micrometer and was found to be 0.67 ± 0.01 mm.PHB/CpFeThe spherulitic morphologies of the samples were examined on an Olympus CX31 polarizing optical microscope (POM) equipped with a digital camera system, E330. Samples weighing 3–5 mg were melted on glass slips with cover slips to form 20–50 mm thick films. Each specimen was melted at 200 °C for 2 min on a hot stage and then was cooled to the selected crystallization temperature of 100 °C. The polarized optical micrographs were recorded after 1 h annealing at the crystallization temperature of 100 °C.Wide angle X-ray diffraction, XRD, measurements were conducted by a Shimadzu XRD-6100 X-ray diffractometer with Cu-Kα radiation . The X-ray diffraction patterns were carried out at ambient temperature in the 2θ range of 10–80° with a scanning step and rate of 0.02° and 2°/min, respectively.The transition temperatures of the samples were detected using a differential scanning colorimeter DSC-Q2000 from TA instruments Co. with a Universal Analysis 2000. The DSC temperature and heat flow were calibrated with indium according to the methodology mentioned previously . All the 146 J/g .For the study of the non-isothermal crystallization, the samples were heated to 200 °C rapidly and held for 2 min, and then, the DSC curves were recorded during cooling at various cooling rates . The crystallization temperature during cooling  complex as a nucleating agent for the enhancement of the properties of the PHB biodegradable polymer. The influence of this metallic complex on the crystallization behavior of the bacterial PHB has been studied profoundly. The new nucleating agent displayed an excellent distribution into the polymer matrix without any sign of agglomeration. Meanwhile, the blending process was assisted by the coordination of the CpFe+ with the ester carbonyl of the polymer chains. Furthermore, the incorporation of the CpFe+ had a multitude of outcomes. For instance, merging the cationic organoiron complex into the PHB materials shifted the melt crystallization temperature 26 °C higher than that of its pure analogue. It also promptly increased the crystallization rate of the bacterial PHB by 87% with no influence on the polymeric crystal lattice. The isothermal and non-isothermal crystallization behaviors of the polymer have been elucidated using the Avrami model, the modified Avrami model, and the combination Avrami and Ozawa model. In addition, the presence of the cationic nucleating agent lowered the effective activation energy of the PHB, owing to its heterogeneous nucleation mechanism with the polymer matrix, which is an indication of a faster crystallization rate that leads to a shortened production cycle time for the polymer processing techniques. All these results signify that the CpFe+ is a superior novel nucleating agent and that the complex has been deemed to be a good candidate for high-temperature polymer processing techniques and commodity applications.This study represents the first example of employing the cationic cyclopentadienyliron (CpFe


Tiparp is highly expressed in the brain; however, its function in this organ is unknown. Here, we used  –/–Tiparp mice to determine Tiparp’s role in the development of the prefrontal cortex. Loss of Tiparp resulted in an aberrant organization of the mouse cortex, where the upper layers presented increased cell density in the knock-out mice compared with wild type. Tiparp loss predominantly affected the correct distribution and number of GABAergic neurons. Furthermore, neural progenitor cell proliferation was significantly reduced. Neural stem cells (NSCs) derived from –/–Tiparp mice showed a slower rate of migration. Cytoskeletal components, such as α-tubulin are key regulators of neuronal differentiation and cortical development. α-tubulin mono-ADP ribosylation (MAR) levels were reduced in –/–Tiparp cells, suggesting that Tiparp plays a role in the MAR of α-tubulin. Despite the mild phenotype presented by –/–Tiparp mice, our findings reveal an important function for Tiparp and MAR in the correct development of the cortex. Unravelling Tiparp’s role in the cortex, could pave the way to a better understanding of a wide spectrum of neurological diseases which are known to have increased expression of TIPARP.2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-inducible poly-ADP-ribose polymerase (TIPARP) is an enzyme that adds a single ADP-ribose moiety to itself or other proteins. Tiparp is highly expressed in the brain; however, its function in this organ is unknown. Here, we used  Tiparp knock-out mouse our findings reveal an important function of MAR by this enzyme in the correct development of the cortex.2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-inducible poly-ADP-ribose polymerase (TIPARP) is an enzyme which adds a single ADP-ribose moiety to itself or other proteins and is highly expressed in the brain. However, its function in this organ remains unknown. Here, we show that Tiparp affects neural progenitor cell proliferation and migration, and its loss leads to aberrant organization of the mouse cortex, predominantly by disrupting the correct distribution and number of GABAergic neurons. Cytoskeletal components, such as α-tubulin, are key regulators of neuronal differentiation and cortical development, here, we show that Tiparp is part of a complex that interacts with α-tubulin and plays a role in its mono-ADP ribosylation (MAR). Despite the mild phenotype presented by the +) as a substrate to transfer one or more molecules of ADP-ribose to specific amino acid residues on target proteins. TIPARP mono-ADP ribosylates itself and other proteins including, TANK-binding protein 1 (TBK1) and the aryl hydrocarbon receptor -inducible poly-ADP-ribose polymerase  is expressed in many different tissues, including liver, heart, spleen, brain, and reproductive organs . TIPARP tor AHR; . TIPARP tor AHR; . It conttor AHR; . In HeLator AHR; . Moreovetor AHR; .–/–Tiparp mice exhibit an increased sensitivity to dioxin-induced toxicities primarily due to increased AHR activity  in adults . This suad libitum. The –/–Tiparp mice where exon 3 of the Tiparp gene was removed were generated in C57BL/6N strain. A full description of the generation of these mice has been previously described (Tiparp+/+) control mice or –/–Tiparp mice were used in all experiments. Mice of either sex and wherever possible same sex littermates were used in these studies.All experiments were approved by the Norwegian Animal Research authority (FOTS ID 10420). Mice were housed in a 12/12 h light/dark cycle and had access to food and water escribed . The aniTbp). Primer sequences are provided in Mice were euthanized by open-drop exposure to isoflurane and cervical dislocation. The brains were extracted in ice-cold PBS, various brain regions were dissected and flash frozen in liquid nitrogen. The tissue was then homogenized in TRIzol reagent (Thermo Fisher Scientific) and total RNA and RNA from mouse neural stem cells (mNSCs) were isolated with Aurum Total RNA Mini kit (Bio-Rad) according to the manufacturer’s instructions. All RNA was reverse transcribed using High-Capacity cDNA Reverse Transcription kit (Thermo Fisher Scientific). Transcript levels were quantified by qPCR using KAPA SYBR Fast Universal kit (Sigma-Aldrich). The amplified genes were normalized to tata-binding protein . Fixed brains were washed with PBS and incubated overnight in 30% (w/v) sucrose before embedding in Optimal Cutting Temperature compound (OCT). All mouse brain tissue was cryo-sectioned into 15-μm-thick sections, which were air dried before use. Unless noted otherwise, sagittal section from comparable lateral regions within a third from the midline were used.For Nissl staining, sections were placed in 0.1% cresyl violet solution for 10 min at 37° before rinsing them in distilled water and then differentiated in 95% ethyl alcohol for 15 min. The sections were then dehydrated in 100% alcohol twice for 5 min. The sections were cleared in xylene twice for 5 min before mounting.mNSCs were grown on poly-D-lysine coated glass cover slips, whereas COS-1 cells were grown on non-coated cover slips. Both cell types were fixed for 15 min in 4% PFA before proceeding with immunofluorescence staining.www.cellprofiler.org; Broad Institute; www.knime.com; For immunofluorescence sections or cells were rinsed in PBS then blocked for 45 min in in 0.1% Triton X-100, 10% BSA in PBS at room temperature. Sections were then incubated overnight at 4°C with primary antibodies. They were washed in PBS and incubated at room temperature for 1 h with species-appropriate Alexa Fluor or cyanine dye conjugated secondary antibodies and Hoechst 33258 (Sigma) and mounted with Vectashield anti-fading mounting medium (Vectorlabs). The antibodies used are provided in 2/5%CO2) artificial CSF . Coronal brain slices of 350 μm containing the cerebral cortex at the emergence of the dorsal hippocampus were cut using a vibrating microtome . Slices were stored and allowed to recover at room temperature in a submerged chamber in oxygenated standard ACSF  for at least 1 h before experimentation. The slices were then transferred to a submersion chamber that was perfused with the oxygenated ACSF at 31°C for whole-cell recording. The cortex primary neurons were visually identified by their pyramidal shape and regular spikes with an up-right infrared-differential interference contrast (IR-DIC) microscope (Olympus BX51WI) and captured with a CoolSNAP EZ with SSD ICX285 (Photometrics) video camera. Spontaneous IPSCs and EPSCs were recorded by using patch clamp technique in a whole-cell configuration. Patch electrodes were fabricated from borosilicate glass capillaries with an outer diameter of 1.5 mm (Sutter Instruments) using an automated puller . The internal pipette solution for studying excitatory synapses contained 130 mM K-methylsulfate, 5 mM NaCl, 1 mM CaCl2, 20 mM HEPES, 0.5 mM EGTA, 3 mM Mg-ATP, and 0.4 mM Na3GTP-GTP , osmolarity was 295–305 mOsmol, and pipette resistance was 5–7 MΩ.For whole-cell patch-clamp experiments, mice (P14–P21) were euthanized as described above, and the brain rapidly removed to ice-cold oxygenated , different holding potentials were applied . Recordings and pre-processing of data were made with WinWCP (University of Strathclyde). The signals were typically low-pass filtered with a corner frequency (−3 dB) of 3 kHz and sampled at 6 kHz by DigiData 1322A (Molecular Devices).0 is the maximum amplitude of the event (determined as the maximum current of the selected event/response), τ is the time constant, τRISE for event’s rise phase and τDECAY for decay phaseData analysis was performed with p-CLAMP 10 (Molecular Devices) and OriginLab 8 (OriginLab Corp.). sPSCs were detected with a threshold of 5–7 pA, depending on the noise level via template-based algorithm and by visual verification. Analyses of the kinetic properties of sPSCs included only well separated , monophasic PSCs which appeared to rise in a monotonic fashion without visible deviation of the rising phase and which decayed exponentially. The rise and decay time courses of individual PSCs were estimated by curve fitting with the exponential function:Tiparp+/+ and –/–Tiparpmice were established and cultured as described previously  and the meninges were removed. Each forebrain was kept separately in 1ml of Accutase and put at 37°C for 30 min and triturated with a pipette half way through the incubation. The cells were then triturated again to obtain an almost a single cell suspension in 1 ml of culture medium  and centrifuged 100 × g for 4 min. The pellet was then resuspended in 1ml of culture medium and the non-digested tissue was left to settle by gravity for about 1 min. Cell suspension was then plated in a well of a 12-well plate. The cells were cultured as a monolayer with laminin. Differentiation of the mNSCs in the three lineages was done as previously described  of α-tubulin was verified by immunoprecipitation as previously described . In brieH532A plasmids . U values for the Mann–Whitney test are reported in the Extended Data All data were presented as means and SEM. A two-tailed Mann–Whitney 10.1523/ENEURO.0239-19.2019.f1-1Extended Data Figure 1-1Figure 1-1, DOC file.U values for the Mann–Whitney tests in 10.1523/ENEURO.0239-19.2019.f4-1Extended Data Figure 4-1Figure 4-1, DOC file.U values for the Mann–Whitney tests in 10.1523/ENEURO.0239-19.2019.f5-1Extended Data Figure 5-1Figure 5-1, DOC file.U values for the Mann–Whitney tests in –/–Tiparp and +/+Tiparp mice at two to four months of age. The distribution of cells along the thickness of the cortex was analyzed by determining the y-position of each Nissl-stained cell in comparable sagittal brain sections followed by data binning. –/–Tiparp mice had a significantly higher percentage of cells in the upper layers of the cortex compared with the lower ones  as well as in the brainstem, which included the diencephalon, and the cerebellum, albeit at lower levels , but more in the lower third of the cortex compared with +/+Tiparp ones  calretinin and lower (40%) parvalbumin-positive neurons, but higher calbindin (148%) expressing cells compared with +/+Tiparp littermates (–/–Tiparp mice had lower number of cells (49%) surrounded by these structures  curves from action potentials to assess the firing rate of pyramidal neurons and assessed the minimal current injection required to trigger enough membrane depolarization for the cells to generate an action potential. Both EPSCs and IPSCs were recorded. However, no significant differences were observed in any of these parameters between neurons isolated from +/+ mice A–F. The +/+ mice F, sugges+/+Tiparp and –/–Tiparp littermates. Three independent lines were established from two litters. There were no significant differences in the expression of the basic NSC markers at either mRNA level  between genotypes. mNSCs derived from both genotypes expressed comparable protein levels of SOX2 and Nestin . Histone H3 is specifically phosphorylated during mitosis where metaphase chromosomes are phosphorylated and on exit of the cell cycle a global dephosphorylation of H3 occurs  through regulation of the cell cycle and migration. Furthermore, the –/–Tiparp mice showed differences of GABAergic neuron distribution with lower amounts of calretinin and parvalbumin-positive interneurons, but higher numbers of calbindin-expressing neurons. The molecular mechanisms underlining this observation remain to be determined.Furthermore, adult Kip1 (Ahr influences the migration and differentiation of neurons and specifically of GABAergic neurons . Other sKip1 . MoreoveKip1 . Tiparp Kip1 . TherefoKip1 .in utero exposure to synthetic glucocorticoids results in aberrant distribution of neurons throughout the cortex, similar to what we report in this study (Tuba1a mutant mice have defects associated with cortical Layers II/III and IV and aberrant neuronal migration (Tuba1a mutant mice (However, Tiparp could also function independently of Ahr. Its expression can be induced by other nuclear receptors important in neuronal development such as glucocorticoid receptor and platelet-derived growth factor receptor . Indeed,is study . The comis study . Cytoskeis study . Tuba1a igration . A recenigration . Here, want mice . Collect


Drosophila larvae. A re-analysis of the literature reveals that through paired presentations of an odor A and a sugar reward (A+) the animals learn that the reward can be found where the odor is, and therefore show an above-baseline preference for the odor. In contrast, through unpaired training (A/+) the animals learn that the reward can be found precisely where the odor is not, and accordingly these larvae show a below-baseline preference for it . In addition, we present previously unpublished data demonstrating that also during a two-odor, differential conditioning protocol (A+/B) both these learning processes take place in larvae, i.e., learning about both the rewarded stimulus A and the non-rewarded stimulus B . Secondly, after briefly discussing published evidence from adult Drosophila, honeybees, and rats, we report an unpublished data set showing that relative to baseline behavior after truly random presentations of a visual stimulus A and punishment, rats exhibit memories of opposite valence upon paired and unpaired training. Collectively, the evidence conforms to classical findings in experimental psychology and suggests that across species animals associatively learn both through paired and through unpaired presentations of stimuli with reinforcement – with opposite valence. While the brain mechanisms of unpaired learning for the most part still need to be uncovered, the immediate implication is that using unpaired procedures as a mnemonically neutral control for associative reinforcement learning may be leading analyses astray.Finding rewards and avoiding punishments are powerful goals of behavior. To maximize reward and minimize punishment, it is beneficial to learn about the stimuli that predict their occurrence, and decades of research have provided insight into the brain processes underlying such associative reinforcement learning. In addition, it is well known in experimental psychology, yet often unacknowledged in neighboring scientific disciplines, that subjects also learn about the stimuli that predict the absence of reinforcement. Here we evaluate evidence for both these learning processes. We focus on two study cases that both provide a baseline level of behavior against which the effects of associative learning can be assessed. Firstly, we report pertinent evidence from  However, it can be equally important to learn where and when a reward will not be found . Essentially, this rule holds that the more we remember, the less we learn. In other words, the amount of reward learning about A depends on the difference between the reward received in the presence of A minus the reward predicted by A, the so-called ‘prediction error’. Considering multiple training trials , the prediction error is large and positive for the first A+ trial. This is because much more reward is received than is predicted (‘pleasant surprise’). As training progresses, the reward will eventually be fully predicted such that the prediction error is zero and no further learning accrues to A.In Pavlovian conditioning, a stimulus A (in Pavlovian terminology: the conditioned stimulus or CS) is presented along with a reinforcer + (in Pavlovian terminology: the unconditioned stimulus or US). By such paired A+ training, an association is formed between A and the reinforcer . In the Drosophila that provides evidence for A+ and A/+ learning relative to a control condition that prevents the behavioral expression of associative memories. Then we report on so far unpublished experiments regarding these learning processes in differential conditioning in this paradigm, and briefly evaluate pertinent literature on adult flies, honey bees and rats. Finally, we present unpublished data demonstrating associative learning through paired and unpaired training in a fear-conditioning paradigm in rats, relative to a control condition that prevents the formation of associative memories.Already in early studies in the field, unpaired training was introduced as a control procedure for reinforcement learning e.g., . In suchDrosophila larva is an ecologically plausible study case for Pavlovian conditioning . An odor A is presented together with a sugar-containing substrate; a tasteless substrate is then presented without an odor . These animals can learn that the reward can be found where the odor is . Importantly, a second group of larvae is trained unpaired, i.e., the odor and the reward are present on different dishes . These animals can learn that the reward can be found where the odor is not . After typically three such training cycles, the animals are transferred to a test Petri dish where their preference for A is assessed. This usually reveals a higher preference for A after paired than after unpaired training . This difference in preference between paired-trained and unpaired-trained animals indicates how much the contingency between odor and reward matters for the larvae’s odor preference, and can thus serve as a measure of associative memory. However, is this due to associative memory in the paired-trained group, associative memory in the unpaired-trained group, or both? The observation that the larvae approach or avoid the odor after a given training procedure is not in itself an argument in this respect, because odors are not neutral to experimentally naive larvae, but support moderate levels of attraction   . This beigure 2) . The samigure 2) , 1968. Iigure 2) . Still, igure 2) , and notFigure 1, green box plots). This is an effect specific to learned behavior, as olfactory behavior in experimentally naive larvae is not likewise affected . The equal level of odor preference in paired-trained and unpaired-trained animals in the presence of the reward can thus be used as a baseline, reflecting olfactory behavior specifically cleared of associative memories. The following Section “Evidence for Unpaired-Memory in Larval Drosophila” discusses what a re-analysis of previously published experiments using such a baseline approach can reveal about the memories formed though paired and unpaired training of odor and taste reinforcement.A second strategy is not to try to prevent the formation of associative memory, but to prevent its behavioral expression. How can this be done? Fortunately, the behavioral expression of associative memory in larvae has been found to depend on the circumstances of testing . Specifin-amyl acetate as the odor and fructose as the reward. In this and the following analyses, we pooled the data for paired-trained and unpaired-trained animals tested under baseline conditions , and compared them to animals that were paired-trained or unpaired-trained and tested under non-baseline conditions , using pairwise statistical tests . Associative memory after paired or unpaired training would manifest itself as a difference between the respective group and the baseline. Indeed, paired odor-reward training increased odor preference compared to baseline, whereas unpaired training decreased odor preference compared to baseline . This result has been reproduced four times in three follow-up studies      and confgure 1B) . Interesigure 3) . TogetheDrosophila larvae can be conditioned to associate odors with taste punishment such as highly concentrated salt, or quinine , this makes it possible to measure odor preference after paired or unpaired punishment training, and to compare the levels of preference against baseline – which in this case is determined by testing the animals in the absence of the punishment. It turned out that after paired odor-punishment training, larvae prefer the odor less than at baseline, whereas after unpaired punishment training they prefer the odor more than at baseline   ( quinine . Importa quinine . This ca quinine . Given tgure 1C) . In othegure 1C) . Specifiigure 3) .Figure 3). This is adaptive because in both cases it keeps the larvae on target . Secondly, the way in which the presence of the reinforcer during the test affects the behavioral expression of memory is determined in turn by the used reinforcer, not by memory valence : for example, the presence of the reward suppresses the behavioral expression of reward-memory both after reward-paired and after reward-unpaired training, although these two types of training establish memory of opposite valence. This is adaptive because in both cases learned behavior is about obtaining the desired outcome .These results show two points of conceptual relevance. Firstly, the way in which microbehavior is affected is determined by memory valence, not by the used reinforcer: for example, when heading toward the odor source turns are suppressed both by reward-paired and by punishment-unpaired memory . We conclude that during differential conditioning, too, larvae learn both about the reward-paired and about the reward-unpaired odor. The same principle, with reversed sign, applies in the aversive domain as well .To address this problem, we modified the differential, two-odor conditioning paradigm , levels of PER toward B are initially elevated, arguably because of generalization from the first A+ training trial. As training progresses, however, the response levels to B decrease  is repeatedly paired with a punishing foot-shock reinforcement (+) (serving as US) . Intendea.k.a. safety learning). However, as discussed in Section “How to Determine Baseline Odor Preferences in Larval Drosophila,” a firm conclusion would require a proper baseline measure of freezing to disentangle whether the difference in freezing between paired-trained and unpaired-trained conditions results from either one of these two types of training, or from both.One suggested approach to measuring positively valenced memory after unpaired training takes advantage of the contextual learning capabilities of rodents . In thesa.k.a. the startle probe). This response consists of a short-latency contraction of all body muscles and can be measured by motion-sensitive devices . However, a firm conclusion would again require a proper baseline against which to measure startle after unpaired training.A second approach takes advantage of the startle response, which can be elicited by a sudden, loud noise  . Rats were submitted to 15 presentations of a light stimulus A (5 s duration) and foot-shock punishment  with intertrial-intervals ranging between 90 and 150 s. Different groups of rats underwent one of three training conditions: (1) for one group stimulus A preceded the shock (Paired group); (2) one group received unpaired presentations of A and shock, such that the inter-stimulus-interval was never shorter than 12 s (Unpaired group); and (3) one group underwent the truly random procedure . Confirming Using the truly random procedure, startle has turned out to be the same in the presence and in the absence of A, i.e., the Startle Difference Scores are zero . After uTo summarize, from experiments using startle modulation as a bivalent behavioral read-out and the truly random procedure to determine baseline behavior, we conclude that paired and unpaired training establish oppositely valenced associative memories in rats.Drosophila, honeybees, and rats confirms a general principle of classical experimental psychology: that animals learn through both paired and unpaired presentations of a stimulus A with reinforcement, and that the resulting associative memories are opposite in valence. This warns against using the unpairing of A with a reward or punishment as a control for the effects of associative learning. Indeed, unpaired presentations of A and reinforcement are not a safe procedure in controlling for associative learning effects – because such a procedure can in itself establish associative memory for A as a signal that a reward or punishment will not occur.The evidence presented from larval and adult Drosophila associatively learn about both stimuli – with opposite valence. Arguably, the behavior after any differential conditioning experiment might thus be a result of either or both of two types of learning process that need to be disentangled in order to fully understand the results.Importantly, as we show here, this applies not only to ‘absolute,’ non-differential conditioning, but to differential conditioning as well: when stimulus A is presented paired with reinforcement and, in the same experimental subjects, another stimulus B is presented unpaired from reinforcement, larval Drosophila larva in particular has demonstrated its potential for in-depth analyses of the genetic and neuronal mechanisms of reinforcement learning . All experiments with rats were approved by the local ethical committee .Supplementary Data Sheet S1.The data for all presented behavioral experiments can be found in the Figure 4.MS, MF, and BG wrote the manuscript. SS and MS performed and analyzed the experiments displayed in 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.


At early somite stages, nkx2.3+ cells located at lateral region of pharyngeal endoderm give rise to the pouch epithelium except a subpopulation expressing pdgfαa rather than nkx2.3. A small-scale screen of chemical inhibitors reveals that BMP signaling is necessary to specify these progenitors. Loss-of-function analyses show that BMP2b, expressed in the pharyngeal ectoderm, actives Smad effectors in endodermal cells to induce nkx2.3+ progenitors. Collectively, our study provides in vivo evidence for the existence of pouch progenitors and highlights the importance of BMP2b signaling in progenitor specification.Pharyngeal pouches, a series of outpocketings that bud from the foregut endoderm, are essential to the formation of craniofacial skeleton as well as several important structures like parathyroid and thymus. However, whether pharyngeal pouch progenitors exist in the developing gut tube remains unknown. Here, taking advantage of cell lineage tracing and transgenic ablation technologies, we identified a population of  in vivo evidence that, in the early somite stages, nkx2.3+ cells are present in the lateral pharyngeal endoderm and give rise to the pouch epithelium. We further reveal that ectodermal BMP2b is essential for the activation of Smad effectors in endodermal cells, thereby facilitating pouch progenitor specification. Collectively, our discoveries shed new light on the cellular and molecular mechanisms of pharyngeal pouch development.Pharyngeal pouches are essential to the formation of craniofacial skeleton as well as several important structures like parathyroid and thymus, but whether their progenitors exist in the developing gut tube remains unknown. Our study provide  A prominent feature of vertebrate embryos is the presence of a series of bulges on the lateral surface of the head, which are known as pharyngeal arches. Past work has established that a key event in pharynx development is the formation of a series of outpocketings-known as pharyngeal pouches-from the foregut endoderm ,2. Althopdx1 can be detected in the most medial cells of the bilateral sheets at the 10-somite stage (14 hpf), a very early time point relative to pancreas morphogenesis . Co. Conkx2.Tg(sox17:GFP) transgenic embryos stably expressed GFP fluorescence in unaltered expression domains at 6- and 10-somite stages when compared with control embryos, suggesting that the pan-endoderm was properly developed (Tg(nkx2.3:mCherry) transgenic embryos were treated with DMH1. Treatment started at the bud stage and lasted until the mCherry-labeled pouch progenitors were examined at the 17-somite stage. When compared with control embryos, the number of pericardial progenitors in mCherry-expressing clusters of DMH1-treated embryos was reduced  embryos. These embryos overexpressed the dominant negative BMP receptor 1a (dnBmpr1a) [nkx2.3 expression in the lateral pharyngeal endoderm at the 17-somite stage  , excludite stage . Interesithelium .nkx2.3+ pharyngeal pouch progenitors at the early somite stages.Together, these data suggest that BMP signaling is necessary but not sufficient for the formation of the lateral pharyngeal endoderm that contains nkx2.3 expression and pouch structures in embryos deficient for BMP activity revealed a direct role of BMP signaling in the generation of nkx2.3+ pouch progenitors within the lateral pharyngeal endoderm at the early somite stages. However, a previous study showed that while BMP signaling was crucial for endodermal pouch morphogenesis, it was not necessary for lateral pharyngeal endoderm identity [pdgfαb, while no obvious effect on pdgfαa expression [As we had described above, the loss of identity . In thispression . Given tfoxa1 is expressed not only in the medial pharyngeal endoderm but also in the first pouch [foxa1 was not notably changed in the medial pharyngeal endoderm, but obviously decreased in the first pouch at 26 hpf in embryos treated with 10 μM Dorsomorphin  embryos were ablated after early-stage MTZ treatment, the nkx2.3+ pouch epithelium disappeared  fish to Tg(nkx2.3:mCherry). Using the resulting dynamic mCherry expression, the pouch progenitor live reporter was examined by time-lapse confocal imaging.The loss of al cells , we spec+/GFP+ cells initially appeared from the 9-somite stage  expression in zebrafish. This work revealed that the pharyngeal endoderm was subject to BMP signaling as early as the 10-somite stage [+/GFP+ cells gradually emerged. To address this issue, immunostaining was used to determine the status of phosphorylated Smad1/5/8 (p-Smad1/5/8) in Tg(sox17:GFP) embryos, which served as intracellular effectors for BMP signaling. Confocal optical sections revealed that, by the 6-somite stage (12 hpf), endogenous Smad1/5/8 was robustly activated in the lateral-most region of the pharyngeal endoderm, where pouch progenitors were presumed to be specified  transgenic embryos were treated with DMH1 from the bud stage. At the 10-somite stage, approximately 7 or 8 mCherry+/GFP+ cells were found in the lateral pharyngeal endoderm of control embryos. However, these cells were nearly absent in the DMH1-treated group  to drive a destabilized form of GFP  embryos revealed that bmp2b was expressed in the pharyngeal ectoderm overlying the nkx2.3+ progenitors during the early somite stages  embryos injected with bmp2b MO  embryos incubated with MTZ showed that the lateral pharyngeal endoderm contains both BMP-sensitive and non-sensitive subpopulations. These findings lead us to hypothesize that bmp2b is required only for the generation of BMP-sensitive pouch endoderm. To directly test this hypothesis, we examined the expression of multiple pharyngeal endodermal markers in bmp2b morphants at 26 hpf. When compared with control embryos, we found that bmp2b morphants had normal development of the medial pharyngeal endoderm with foxa1 expression  transgenic line to explore whether the defective development of pharyngeal pouches in bmp2b-deficient embryos was due to the loss of pouch progenitors. Consistent with the defects of pouch formation in bmp2b morphants  or wild-type embryos resulted in the near absence of the pouch and pericardial progenitors, a phenotype similar to that of swrta72 mutants (bmp2b-depletion in heat-shocked Tg(hsp70l:caBmpr1b-GFP) embryos  embryos. When compared with control embryos, the p-Smad1/5/8 level was decreased in the ventral-most pharyngeal endodermal cells in bmp2b morphants  [nkx2.3+ pericardial progenitors were depleted at the early somite stages in zebrafish embryos using the MTZ/NTR system, severe pericardial edema was observed at later stages. Given these findings, it is possible that pericardial deficiencies might also result from the failure of pericardial progenitors to generate on either one or both sides.An unanticipated finding is that a non-endodermal population of absence) ,27. Congabsence) ,45. In fTg transgenic embryos, pharyngeal pouch progenitors expressed both mCherry and GFP, while pericardial progenitors only expressed mCherry. Although these two different progenitor types were distinguished by their different locations after developmentally-regulated cell migration, the future identification of endogenous biomolecular markers for each type of progenitors will aid the study of cell fate determination.In the somite-stage wnt2bb is expressed in restricted, bilateral domains of the lateral plate mesoderm and directly induces the adjacent endoderm to form liver anlage [in vivo evidence that the liver and pancreas originate from common progenitors; moreover, that proximity to the source of mesodermal bmp2b influences cell fate [During the early somite stages of vertebrate development, signals from surrounding tissues induce the specification of lineage-specific progenitors for several foregut organs . For exar anlage . Single-ell fate . Previouell fate . Howeverbmp2b-but not bmp4 or bmp7a-played an essential role in pouch progenitor specification. bmp2b is dynamically expressed in multiple tissues during the gastrulation and segmentation stages and has been shown to be important for multiple stages of endoderm development [bmp2b expression in a spatio-temporal manner during zebrafish embryo development, several independent lines of evidence converge in support of pharyngeal ectoderm-derived BMP2b as an important regulator of the induction of nkx2.3+ pouch progenitors. More specifically: (1) Endogenous Smad1/5/8 proteins, the intracellular effectors of BMP signaling, are activated in the lateral-most region of the head endoderm as early as the 6-somite stage. (2) Inhibition of BMP signaling during the early somite stages by either applying BMP inhibitors or overexpressing a dominant-negative BMP receptor significantly decrease the number of pouch progenitors. This suggests that BMP signaling is required for cell fate determination. (3) bmp2b begins to be expressed from the 6-somite stage in the pharyngeal ectoderm overlying the nkx2.3+ pouch progenitors. (4) Inactivation of bmp2b using an antisense morpholino does not notably impact the formation of the medial pharyngeal endoderm, but does impair the phosphorylation of Smad1/5/8 and the specification of pouch progenitors. Interestingly, ectopic induction of BMP signaling cannot induce more pouch progenitors during the early somite stages and cannot increase pouch epithelium at later stages. Collectively, these findings imply that BMP signaling is necessary but not sufficient for pouch progenitor specification.Using results from a small-scale screen of chemical inhibitors, we identified BMP signaling was required to specify pouch progenitors from the head endoderm during the early somite stages. Subsequent to this finding, we determined that the BMP ligand gene elopment ,39. Althnkx2.3+ pouch progenitors in the lateral pharyngeal endoderm. Disrupting BMP signaling through the application of chemical inhibitors such as Dorsomorphin and DMH1 during early somite stages disturbs the generation of the lateral pharyngeal endoderm. Ultimately, this leads to severe loss of the pouch epithelium. These observations are different from previous findings, which indicated that BMP signaling was necessary for proper pouch morphogenesis, but not crucial to the genesis of the pouch epithelium [pdgfαa [nkx2.3 and nkx2.7 expression were almost completely abolished in the pharyngeal region. Comparatively, pdgfαa expression in the pharyngeal region was retained. Based on these observations, we speculate that, excepting the nkx2.3+ cells, the lateral pharyngeal endoderm might contain another distinct cell population expressing pdgfαa, but not nkx2.3. Moreover, its development dose not dependent on BMP signaling. In support of this hypothesis, our studies of MTZ-induced depletion of nkx2.3+ pouch progenitors showed that the nkx2.3+ pouch epithelium was disrupted, while the nkx2.3-/pdgfaa+ lateral pharyngeal endoderm persisted. This finding also suggests that the nkx2.3-/pdgfaa+ pouch epithelium should not be derived from nkx2.3+ pouch progenitors. In the future, it will be interesting to discern whether a subset of pouch progenitors exist and contribute to nkx2.3-/pdgfaa+ lateral pharyngeal endoderm.Our results unambiguously demonstrate the existence of ithelium . This di [pdgfαa . Howevernkx2.3 and pdgfαa display nested expression patterns along the dorsal-ventral (DV) axis of the pouch epithelium. In particular, nkx2.3+ cells are located in the ventral regions of each pouch. When nkx2.3+ pouch epithelium development was eliminated, it seemed that the residual pdgfαa+ cells were incapable of migrating laterally. This was evidenced by their nearly straight-line, dorsal location. Time-lapse microscopy studies revealed a collective cell migration during the pouch epithelium outgrowth [nkx2.3+ pouch epithelium functions as the leading edge of cells that direct the migratory behavior for the collective cell migration [Another interesting observation is that utgrowth . It is tigration . Similarigration –51. Previgration ,52. The igration ,53,54. TOur zebrafish experiments were all approved and carried out in accordance with the Animal Care Committee at the Institute of Zoology, Chinese Academy of Sciences (Permission Number: IOZ-13048).Tg(fli1:EGFP) [Tg(sox17:GFP) [Tg(cmlc2:EGFP) [Tg(nkx2.5:ZsYellow) [Tg(hsp70:dnBmpr1-GFP) [Tg(hsp70:caBmpr1b-GFP) [swrta72 [Tg(UAS:NTR-mCherry), Tg(nkx2.3:mCherry), Tg(nkx2.3:EosFP), Tg(nkx2.3:EGFP-CAAX) and Tg. To generate the Tg(nkx2.3:EosFP), Tg(nkx2.3:EGFP-CAAX), and Tg transgenic lines, the 5.5 kb of the nkx2.3 upstream regulatory sequence was cloned into a Tol2 transposon-based vector [Zebrafish strains were maintained in stand laboratory conditions. Embryos were obtained from natural zebrafish matings, raised in Holtfreter’s solution at 28.5°C, and staged by morphology as previously described . We usedi1:EGFP) , Tg(sox1x17:GFP) , Tg(cmlcc2:EGFP) , Tg(nkx2sYellow) , Tg(hsp7pr1-GFP) , Tg(hsp7r1b-GFP) , swrta72[swrta72 , Tg(UAS:d vector .sox32 MO (5’-CAGGGAGCATCCGGTCGAGATACAT-3’), bmp2b MO (5’-CGCGGACCACGGCGACCATGATC-3’), bmp4 MO (5’-GTCTCGACAGAAAATAAAGCATGGG-3’) and bmp7a MO (5’-GCACTGGAAACATTTTTAGAGTCAT-3’) were used as previously described [Morpholinos were purchased from Gene Tools . The standard control morpholino (5’-CCTCTTACCTCAGTTACAATTTATA-3’), escribed –61. Micrescribed –64.in vitro from linearized plasmids using the MEGAscript Kit (Ambion) according to the manufacturer’s instructions. Whole-mount in situ hybridizations and frozen sections were performed according to previously published methods [in situ hybridizations, anti-digoxigenin-POD  and anti-fluorescein-POD  were used as primary antibodies to detect digoxigenin-labled pdgfαa probes and fluorescein-labeled nkx2.3 probes, respectively. The signals were detected using a TSA system, including Cyanine 3 amplification reagent , Fluorescein amplification reagent , and 1x plus amplification diluents .Digoxigenin-UTP-labeled RNA probes were synthesized  methods –64. For Immunofluorescent staining was performed as previously described . Embryosegfp  and bmp2b RNAscope probes . For labeling, Amp4 Alt A-FL  was used. Preamplifier hybridization of Amp1 and amplifier hybridization of Amp3 were both extended to 3 hours. Signal enhancement of Amp2 and labelling with Amp4 were both extended to 1.5 hours. All images were taken using a Nikon A1R+ confocal microscope.Whole-mount RNAscope assay was conducted using the RNAscope Fluorescent Multiplex Reagent Kit  according to published protocols with some modifications . WithoutFor the EosFP photoconversion and lineage tracing experiments, all embryos were anesthetized and embedded in 0.8% low-melt agarose  in glass-bottom dishes . EosFP fluorescent proteins were converted upon irradiations with violet-blue light (405 nm) using a Nikon A1R+ confocal microscope (20× objective) until the green fluorescence in the regions of interest disappeared. For lineage tracing, converted embryos were immediately imaged, removed from the agarose, and raised in dark conditions until later evaluation. All confocal stack pictures were processed using Nikon NIS-Elements AR 4.13.00 software.Pharmacological treatment was performed by incubating live embryos in the dark in Holtfreter’s solution supplemented with inhibitors as follows: Dorsomorphin , DMH1 , U0126 , SP600125 , SB203580 , CCT036477 , and Metronidazole (MTZ) . Control embryos were treated with equivalent amounts of DMSO . MTZ was added as early as the 32-cell stage and washed out until 17 somite stage. For heat shock, embryos were subjected to 39.5°C heat shocks for 20 min at 9 hpf and then incubated at 28.5°C until harvested.S1 FigA) Tg embryos at 28 hpf with mCherry fluorescence (red) in the pericardium and EGFP fluorescence (green) in the heart. Single plane images showed that the GFP-positive myocardial cells were surrounded by the mCherry-positive cells. Scale bar, 50 μm. (B) In situ hybridization of nkx2.3 in whole mount embryos at 36 hpf . Sagittal section of the same embryo was shown in the right panel.((TIF)Click here for additional data file.S2 FigA-D) mCherry and ZsYellow fuorescence in Tg embryos at indicated developmental stages. Single plane images showed that no cells within the paired cords (A-C) and the pericardium (D) were double-positive for mCherry and ZsYellow. Dorsal views with anterior to the top. Scale bars, 50 μm.((TIF)Click here for additional data file.S3 FigTg embryos injected ng cMO or ng sox32 MO at 36 hpf. Ventral views, anterior to the top. Scale bar, 50 μm.mCherry fluorescence in the pericardium of (TIF)Click here for additional data file.S4 FigA) Tg(nkx2.3:EosFP) embryos were photoconverted in the right-side nkx2.3+ cluster at the 17-somite stage. At 36 hpf, embryos were imaged in the green and red channels in the pericardium. Ventral views, anterior to the top. Scale bar, 50 μm. (B) Tg(nkx2.3:EosFP) embryos were photoconverted in the right-side nkx2.3+ cluster at the 10-somite stage, and then these embryos were imaged in the pericardium at 28 hpf. Scale bar, 50 μm.((TIF)Click here for additional data file.S5 FigA-B) Tg embryos were treated with 50 mM MTZ during different time intervals and then harvested at the indicated developmental stages for in vivo confocal imaging (A) and in situ hybridization (B). Scale bars, 50 μm. (C-D) Depletion of nkx2.3+ progenitors has no obvious effect on endoderm formation. Tg or Tg embryos were treated with 50 mM MTZ from the 32-cell stage to the 17-somite stage. Then these embryos were subjected to in vivo confocal imaging (C) and in situ hybridizations (D) at the 17-somite stage. In panel D, embryos are viewed from the dorsal aspect, and the white dotted lines indicate the region of the pericardium. Scale bars, 50 μm. (E-F) Depletion of nkx2.3+ progenitors leads to obvious pericardial edema. Tg embryos were treated with 50 mM MTZ from the 32-cell stage to the 17-somite stage, and then these embryos were harvested at 28 hpf for in vivo confocal imaging . Their morphological defects were shown in . Red Arrowheads indicate the pericardium.((TIF)Click here for additional data file.S6 FigTg(sox17:GFP) embryos were treated with 10 μM DMH1 from bud stages until harvested for confocal imaging. Dorsal views with anterior to the top. Scale bars, 50 μm.(TIF)Click here for additional data file.S7 FigA-B) Knockdown of bmp4 perturbed asymmetrical left-right patterning. Tg(cmlc2:EGFP) embryos was injected with ng bmp4 MO at one-cell stage. Defects in cardiac jogging was visualized by EGFP expression at 30 hpf. Different types of EGFP expression fluorescence in the heart were shown in ventral views (A). The ratios were shown in (B). Scale bars, 50 μm. (C-D) Knockdown of bmp7a resulted in a range of dorsalized phenotypes. Wild-type embryos were injected with ng bmp7a MO at the one-cell stage and imaged at 36 hpf. Representative dorsalized morphologies (C1-C3) are shown in (C) and their ratios are shown in (D). Scale bar, 100 μm.((TIF)Click here for additional data file.S8 Figsox17 expression in swrta72 embryos at the bud stage. The swrta72 mutant embryos can be easily recognized owing to their elongated shape. Note that the swrta72 mutants showed nearly normal endoderm specification but delayed convergence of endodermal cells.The (TIF)Click here for additional data file.S9 FigDuring the early somite stages, the ectodermal cells (orange) express and secret BMP2b proteins (yellow), which play an essential role in the specification of pouch progenitors (pink) from adjacent pharyngeal endoderm (green). PPP, pharyngeal pouch progenitor.(TIF)Click here for additional data file.