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In vivo proximity labeling identifies cardiomyocyte protein networks during zebrafish heart regeneration
Strategies have not been available until recently to uncover interacting protein networks specific to key cell types, their subcellular compartments, and their major regulators during complex in vivo events. Here, we apply BioID2 proximity labeling to capture protein networks acting within cardiomyocytes during a key model of innate heart regeneration in zebrafish. Transgenic zebrafish expressing a promiscuous BirA2 localized to the entire myocardial cell or membrane compartment were generated, each identifying distinct proteomes in adult cardiomyocytes that became altered during regeneration. BioID2 profiling for interactors with ErbB2, a co-receptor for the cardiomyocyte mitogen Nrg1, implicated Rho A as a target of ErbB2 signaling in cardiomyocytes. Blockade of Rho A during heart regeneration, or during cardiogenic stimulation by the mitogenic influences Nrg1, Vegfaa, or vitamin D, disrupted muscle creation. Our findings reveal proximity labeling as a useful resource to interrogate cell proteomes and signaling networks during tissue regeneration in zebrafish.
Understanding how and why tissue regeneration occurs is a central objective of developmental biology. Genome-wide transcriptome or chromatin profiling experiments using RNA-seq, scRNA-seq, ATAC-seq, or ChIP-seq approaches have helped to identify novel factors, mechanisms, and concepts that have informed our understanding of regeneration in many tissues, injury contexts, and species (Goldman et al., 2017; Hoang et al., 2020; Johnson et al., 2020). Although key for detecting and inferring changes in gene transcription, chromatin organization, or DNA–protein associations, these strategies are limited in their abilities to identify protein and signaling complexes that carry out changes at the cell and tissue level. Co-immunoprecipitations and affinity-purification mass spectrometry (AP/MS/MS) are well-established techniques to probe protein interactions and networks. Yet, they require tissue dissociation and flow cytometry to acquire cell type-specific interactions, and weak interactions between proteins often escape detection (Perkins et al., 2010).
Proximity labeling is a relatively recent technology established to probe protein interactions in specific sub-tissue and subcellular compartments, represented by multiple versions including BioID2, TurboID, and APEX2 (Kim et al., 2016; Branon et al., 2018; Hung et al., 2016). Here, engineered ligases that attach biotin to lysine residues are fused with a protein of interest such that, when expressed in cells, proteins in close proximity are indiscriminately biotinylated. The biotinylated proteome is isolated by streptavidin affinity, and proteins are identified by quantitative mass spectrometry. Most studies have applied proximity labeling to in vitro tissue culture systems, whereas many fewer have explored in vivo networks in mice or Drosophila (Spence et al., 2019; Feng et al., 2020; Rudolph et al., 2020; Mannix et al., 2019; Cho et al., 2020). In the latter studies, proximity labeling captured the proteome within interneuron–neuron interactions, within muscle sarcomeres, and in the ring canal of Drosophila oocytes, producing new insights into protein network assemblies and signaling. The principles of proximity labeling should be capable of identifying key protein networks during dynamic cellular processes like embryonic development and tissue regeneration without the need for tissue disruption. Notably, proximity labeling has yet to be applied in central animal models of regeneration such as zebrafish and axolotl salamanders.
Unlike mammals, teleost zebrafish regenerate cardiac muscle after injury with minimal scar formation, based on division of pre-existing cardiomyocytes (Poss et al., 2002; Kikuchi et al., 2010; Jopling et al., 2010). Understanding mechanisms of innate cardiac repair in animals like zebrafish has the potential to define and overcome barriers to cardiac regeneration in humans. Evidence to date indicates that heart regeneration in zebrafish is a relentless process that outcompetes scarring to recover the heart function, rather than a linear series of molecular steps and synchronized cellular progressions. Intrinsic programs in cardiomyocytes and the ability to receive extrinsic proliferative cues are prime cardiomyocytes for cell division. Many ligands, receptors, and transcription factors have been shown to be required for heart regeneration (Xiao et al., 2016; Zhao et al., 2019; Dogra et al., 2017; Chablais and Jazwinska, 2012; Wu et al., 2016; Pronobis and Poss, 2020). Three secreted factors, Nrg1, Vegfaa, and vitamin D, have been demonstrated to have instructive mitogenic effects, characterized by the ability to induce cardiomyocyte proliferation in zebrafish in the absence of injury (Gemberling et al., 2015; Karra et al., 2018; Han et al., 2019). Nrg1/ErbB2 signaling can stimulate cardiomyocyte division in adult mice, most notably when ErbB2 is experimentally expressed in the form of a ligand-independent activator (Bersell et al., 2009; D'Uva et al., 2015; Gordon et al., 2009). ErbB2 signaling was recently reported to work in part through regulation of YAP, a transcriptional activator that can impact cytoskeletal remodeling and cell division in cardiomyocytes during cardiac growth or repair in mice (Leach et al., 2017; Aharonov et al., 2020; Heallen et al., 2013; Heallen et al., 2011). In zebrafish, Nrg1/ErbB2 signals in part by promoting glycolysis versus mitochondrial oxidative phosphorylation and reducing Tp53 levels through upregulation of its inhibitor Mdm2 (Shoffner et al., 2020; Honkoop et al., 2019; Fukuda et al., 2019).
Signal transduction mechanisms by which ErbB2 and other mitogenic factors support cardiomyocyte proliferation versus other outcomes could be illuminated by customized proteomic profiling. Proteomic studies of heart regeneration to date have faced challenges, including lack of cell-type specificity and limited detection of changes in poorly represented members of the proteome due to the abundance of sarcomeric proteins (Wang et al., 2013; Ma et al., 2018; Garcia-Puig et al., 2019). Here, we applied a transgenic BioID2 strategy to capture cell-specific proteome changes in cardiomyocytes during heart regeneration in adult zebrafish. We show that BioID2 can be used to track changes in protein levels in whole cardiomyocytes or their membrane compartments during regeneration. We also investigate the proximal proteome of ErbB2 in cardiomyocytes, finding an increase in association with the small GTPase Rho A during regeneration, a protein with activity we find to be essential for injury-induced regeneration and the proliferative response to mitogens. Our findings demonstrate the utility of proximity labeling as a resource to interrogate cell proteomes and signaling networks during tissue regeneration in zebrafish, and they identify a key role of small GTPases in injury-induced cardiogenesis.
Results and discussion
BioID2 detects cardiomyocyte proteomes in adult zebrafish
BioID2 employs the promiscuous BirA2, which attaches biotin molecules to the lysines of proteins within an approximate distance of 10 nm (Kim et al., 2016). We reasoned that the lysines of sarcomeric proteins might be less accessible when packed in supermolecular complexes, and therefore less likely to undergo biotinylation. To determine whether BioID2 is functional in cardiomyocytes of adult zebrafish, we generated a transgenic line with a cassette encoding BirA2 fused to GFP and placed downstream of the myosin light chain 7 (cmlc2, or mlc7) promoter (Figure 1A, B). BirA2-GFP was readily detected in adult zebrafish cardiac chambers, where it was distributed throughout the cytoplasm and nuclei of adult cardiomyocytes (Figure 1B). To test whether BirA2 expressed in this manner biotinylates the cardiomyocyte proteome, we supplied biotin either in the aquarium water or via intraperitoneal injections over three consecutive days. We found that effective protein biotinylation occurred only when biotin was delivered by injection (Figure 1D, E, Figure 1—figure supplement 1A–E). Thus, transgenic BioID2 systems can biotinylate proteins in cardiomyocytes of adult zebrafish.
To test whether BioID2 can detect proteins that are compartmentalized in cardiomyocytes, we generated transgenic zebrafish with BirA2-GFP expected to be localized to membranes via a CAAX motif (Figure 1A, C). We found that the majority of BirA2-GFP-CAAX signal associated with the cardiomyocyte plasma membrane in cmlc2:birA2-GFP-CAAX hearts (Figure 1C, Figure 1—figure supplement 2A, B). Although BirA2-GFP-CAAX was enriched at membranes, direct staining for biotin revealed signals at membranes and cytosol, suggesting release of labeled proteins from the membrane over the 3-day incubation period (Figure 1D–F, Figure 1—figure supplement 2C–G). To capture the membrane proteome of cardiomyocytes, we conducted the BioID2 assay on adult cmlc2:bira2-GFP or cmlc2:bira2-GFP-CAAX hearts (Figure 1G). Quantitative BioID2 analysis captured 1113 distinct proteins, of which 343 proteins showed a 2.5-fold enrichment in BirA2-CAAX samples when compared to BirA2-GFP samples (Supplementary file 1). The threshold of 2.5-fold has previously been used in in vivo BioID2 studies in mice (Uezu et al., 2016). Gene ontology analysis for cellular components of the 343 proteins revealed enrichment for proteins associated with cellular junctions, plasma membrane, and membrane-bound organelles (Figure 1H). Taken together, our results indicate that in vivo proximity labeling using BioID2 is functional in zebrafish and can profile proteomes of highly specialized cells like cardiomyocytes.
BioID2 detects proteome changes in cardiomyocytes during heart regeneration
To capture the changes in the protein network of cardiomyocytes during regeneration, we combined our BirA2 system with a transgenic system to ablate ~60% of cardiomyocytes through the tamoxifen (4-HT)-inducible expression of a Cre-induced diphtheria toxin A gene (Wang et al., 2011). We injected biotin for 3 days and collected hearts at 14 days post incubation (dpi), followed by purification of the biotinylated proteome (Figure 2A, B). Proliferation of cardiomyocytes in this injury model is most prominent around 7 dpi, with regeneration of the myocardium essentially complete by 28 dpi (Figure 2—figure supplement 1A–C). We assessed cmlc2:bira2-GFP hearts in two different conditions, uninjured (treated with 4-HT but no CreER transgene) and 14 dpi. Quantitative mass spectrometry analysis of pooled samples revealed that 208 proteins showed a 1.5-fold or greater change during regeneration when compared to uninjured hearts (Figure 2D, Supplementary file 2).
It is common in proximity labeling assays to normalize levels of the protein of interest to those of a diffuse BirA2-labeled protein. However, this requires a high threshold for protein changes to avoid false positives, diminishing the gating of proteins with subtle fold changes. Normalizing the regenerating heart data set of a transgenic strain to the uninjured control of the same strain allowed a low threshold. Gene ontology analysis (over-representation test) for biological processes characterized the proteins with increased levels during regeneration in the whole-cell BioID2 set as involved in wound healing, cardiac development, muscle cell differentiation, and response to wounding, whereas decreased proteins were enriched for metabolites and energy precursors (Figure 2D). The latter category includes the generation of ADP to ATP and oxidative phosphorylation, implying that these processes are dampened during dedifferentiation and proliferation of cardiomyocytes, as suggested by recent publications (Honkoop et al., 2019; Fukuda et al., 2019). The top 10% of increased proteins included ErbB2 and the mTOR signaling protein Rictora (Figure 2—figure supplement 1D), each previously implicated in heart regeneration (Gemberling et al., 2015; Sciarretta et al., 2015). We compared the BioID2 data set to published RNA-seq or ChIP-seq data sets, which each identified a much greater number of differential products than BioID2 (Goldman et al., 2017; Rudolph et al., 2020; Kang et al., 2016; Ben-Yair et al., 2019). Correlations were generally weak, with the highest being an RNA-seq data set from 14 dpi hearts at r = 0.39 (Figure 2—figure supplement 2A–D; Kang et al., 2016). We suspect that any post-transcriptional regulation incorporated in BioID2 data contributes to these weak correlations. Beyond this, there are several possible technical explanations, including differences in assessed timepoints, injury models, and cell types, and limitations of BioID2 in capturing targets. Regardless, our results indicate that proximity labeling with BirA2 can identify biological processes that are involved in tissue regeneration and proteins that have been reported to regulate cardiac repair.
To investigate how the membrane proteome changes after heart injury, we examined cmlc2:bira2-GFP-CAAX samples at 14 dpi (Figure 2A, B, Figure 2—figure supplement 3A–C). We identified 173 proteins with a 1.5-fold or greater change in membrane-associated proteins during regeneration when normalized to uninjured hearts (Figure 2C, E, Supplementary file 3), with the majority of these proteins reducing presence during regeneration. This contrasts with BirA2-GFP samples, which showed more proteins increasing levels during regeneration than those decreasing (Figure 2D vs. Figure 2E). Proteins with reduced membrane levels during regeneration showed over-representation of involvement in transmembrane transport, muscle development, and energy production (electron transport and glycolysis), while proteins increased at the membrane during regeneration were involved in protein complex assembly and organelle organization (Figure 2E). Intracellular restructuring is known to occur in dividing cardiomyocytes (Uribe et al., 2018). Upon comparison of the top and bottom 10% of the membrane profiling data with BirA2-GFP data (Figure 2—figure supplement 3D vs. Figure 2—figure supplement 1D), very few proteins were found in both data sets, suggesting specificity of the membrane BioID2 data.
Comparison of proteins found in BirA2-GFP and BirA2-GFP-CAAX data sets identified 44 shared proteins (Figure 2C). Of these, 16 proteins showed opposing changes in levels at the membrane or in the whole cell (Figure 2F). To visualize dynamism of proteins for which there were available candidate antibodies during regeneration, we assessed some of these proteins by immunofluorescence in a model of regeneration in which ~20% of the ventricle is resected (Poss et al., 2002). Erythrocyte membrane protein band 4.1 like 5 (Epb41l5) is involved in animal organ development and has been implicated as a positive regulator of Notch signaling, a pathway involved in heart regeneration (Matsuda et al., 2016; Zhao et al., 2014, Figure 2F, G). Epb41l5 was predominantly localized at the plasma membrane (Figure 2G, arrows) and increased presence throughout cardiomyocytes during regeneration, consistent with the BioID2 data. Talin 2, an adapter protein that couples integrin focal adhesions to the actin cytoskeleton (Figure 2—figure supplement 4A, B), displayed low levels in uninjured hearts (Figure 2—figure supplement 4B). Upon injury, Talin 2 levels increased near the wound site as well as in ventricular muscle away from the injury, with a punctate pattern appearing that resembled focal adhesions (Figure 2—figure supplement 4B). These results are consistent with a remodeling of focal adhesions during myocardial stress induction that has been reported for Talin 1 (Manso et al., 2013). To assess BioID2 results implicating proteins that increase levels at the cell membrane during regeneration, we identified antibodies against SET nuclear proto-oncogene a (Seta), which is involved in nucleosome assembly (Figure 2F, H, Figure 2—figure supplement 4A, C). Seta levels were undetectable in uninjured hearts, yet became prominent in compact muscle and epicardial tissue in injured ventricles (Figure 2H, Figure 2—figure supplement 4C). The Seta signal was perinuclear and colocalized with wheat germ agglutinin (WGA), which indicates that Seta associates in part with the nuclear membrane and endomembranes after heart injury (Figure 2H, arrows, Figure 2—figure supplement 4C). As Seta has been proposed to silence histone acetyltransferase-dependent transcription, its recruitment to nuclear and endomembranes could impact this function (Seo et al., 2001). Taken together, our data indicate that BioID2 can capture changes in the proteomes of cardiomyocytes during heart regeneration, providing a resource for candidate effectors.
Rho A interacts with ErbB2 during heart regeneration
Nrg1 ligand has been reported to bind its receptor ErbB4 on cardiomyocytes, causing heterodimerization of Erbb4 and its co-receptor ErbB2 (Bersell et al., 2009; Harskamp et al., 2016). To probe the network of ErbB2 signaling during heart regeneration in zebrafish, we generated the transgenic line cmlc2:erbb2-birA2-HA-P2A-GFP (Figure 3A), with BirA2 fused to the C-terminus of ErbB2. We detected ErbB2-BirA2-HA via western blotting analysis, although we could not visualize it by immunofluorescence (Figure 3—figure supplement 1A). We also detected biotinylation of cardiac proteins in this line in the presence of injected biotin (Figure 3—figure supplement 1A). To assess dynamic ErbB2 networks during heart regeneration, we crossed cmlc2:erbb2-birA2-HA-P2A-GFP with Z-CAT animals to ablate cardiomyocytes as described above and collected ventricles at 14 dpi (Figure 3B, Figure 3—figure supplement 1B–D). Quantitative mass spectrometry analysis identified 667 proteins, of which 108 showed a >1.5-fold change during regeneration when normalized to uninjured control hearts (Supplementary file 4). Using STRING and BioGRID databases, we identified nine known ErbB2 interactors in our data set. Three of these proteins showed increased association with ErbB2 during regeneration, whereas two had decreased association (Figure 3C, yellow and blue circles, respectively). Next, we examined the levels of these candidates as reported in published transcriptome and proteome data sets (Goldman et al., 2017; Wang et al., 2013; Ma et al., 2018; Kang et al., 2016; Ben-Yair et al., 2019), and found that overall expression levels were not predictive of ErbB2 association (Supplementary file 5). ErbB4, encoded by erbb4a and b paralogs, was not detected in any of the BioID2 data sets, which might either indicate that ErbB4 does not associate with ErbB2 in the zebrafish heart, or that their C-terminal regions are not in close proximity, or a limitation in sensitivity of the assays. The strongest detected increase was an association with the Rho GTPase Rho A-b, and pathway analyses indicated enrichment for proteins involved in cytoskeletal regulation by Rho GTPases (Figure 3C, D). Rho A activation by ErbB2 has been previously reported to promote pro-metastatic and invasive behavior of human breast cancer cells and contribute to mammary adenocarcinoma tumorigenesis (Brantley-Sieders et al., 2008; Worzfeld et al., 2012). Moreover, a recent study implicated Rho A mislocalization with aberrant spindle orientation leading to binucleation, a common feature of mammalian CMs that is thought to inhibit cardiomyocytes (CM) proliferation (Leone et al., 2018). To test whether ErbB2-Rho A association is increased during regeneration, we conducted co-immunoprecipitations (Figure 3E). Interestingly, Rho A was pulled down with ErbB2-BirA2-HA in regenerating heart samples, but not from uninjured myocardium, suggesting a context-dependent association (Figure 3E). Due to their high conservation, we were unable to identify which of the Rho A proteins, Rho A-a, Rho A-b, or Rho A-c, increased its association with ErbB2 (Figure 3—figure supplement 2).
As many direct interactors of Rho A have been identified, we searched our data set of whole cardiomyocyte changes at 14 dpi for these proteins. Levels of myocardial Rho A proteins detected by BioID2 were similar in uninjured and regenerating hearts. Yet, all known Rho A interactors detected in our cmlc2:birA2-GFP data set showed elevated levels after injury, including ErbB2 (59.2-fold; Figure 3F). Most of the detected Rho A interactors are involved in cytoskeletal organization such as moesin a, WASP-like actin nucleation promoting factor a, profilin 1, catenin δ1, and ROCK2a, or cell adhesion such as scribble and integrin-linked kinase (Figure 3F). The signal transducer Rho GTPase activating protein 21b, the guanine nucleotide binding protein (G protein) alpha 13a, which is involved in the formation of heart fields, and reticulon 4a, which plays a role in epithelial mesenchymal transition (EMT) transition, were also found to be increased after injury in the whole-cell BioID2 data set (Figure 3F, Ye et al., 2015; Zhao et al., 2015). Rho A signaling is known to facilitate cytoskeletal changes via Rho-associated protein kinases (ROCK), which have been reported to regulate the DNA binding activity of the cardiogenic transcription factor Gata4 and nuclear localization of SRF in cell culture (Kaarbo et al., 2013; Liu et al., 2003; Yanazume et al., 2002). ROCK2a levels were elevated in our whole-cell BioID2 data set, further confirmed by western blotting (Figure 3G), consistent with the idea that ErbB2–Rho A association could impact ROCK2 levels in cardiomyocytes after injury. Thus, BioID2 interrogation of the co-receptor ErbB2 captured the network of ErbB2 during heart regeneration, a potential resource to uncover novel ErbB2-interacting proteins, and revealed increased association of Rho A and ErbB2 in regenerating cardiomyocytes.
Rho A activity is essential for injury- and mitogen-stimulated cardiomyocyte proliferation
To evaluate a potential role for Rho A signaling during cardiac repair, we treated zebrafish with the Rho A inhibitor Rhosin for three consecutive days after partial ventricular resection and assessed indicators of cardiomyocyte proliferation (Figure 4A, B; Miranda-Rodríguez et al., 2017). In these experiments, Rhosin reduced the proliferation index by ~68% (Figure 4C). To test whether regeneration is inhibited or delayed when Rho A signaling is blocked, we generated a transgenic line that enables tamoxifen-inducible expression of a dominant-negative Rho A cassette in cardiomyocytes. (Figure 4F, top panel). In animals expressing a dominant-negative Rho A for 30 days after resection (Figure 4F), injury sites remained deficient in cardiac myofibers, whereas in control hearts had re-established a contiguous wall of muscle (Figure 4F). Proteomic analyses of our whole-cell BioID2 data set also implicated the small GTPases Rac1 and Cdc42 in heart regeneration; although protein levels remained unchanged, pathway analyses showed enrichment for signaling by Rho GTPases and its effectors (Figure 4D, E). Similar to Rho A, the interactome maps of Rac1b and Cdc42 revealed many known interactors increased in the whole-cell data set after injury (Figure 4—figure supplement 1A, B). Using inducible dominant-negative gene cassettes in transgenic animals, we found that Rac1 and Cdc42 are similarly required for normal heart regeneration (Figure 4F, middle and bottom panels). We also observed tendencies for lowered cardiomyocyte proliferation in each transgenic background at 7 days after resection of the ventricular apex (Figure 4G). Thus, our data implicate each member of the small GTPase trio – Rho A, Rac1, and Cdc42 – in zebrafish heart regeneration.
To further test whether Rho A function is required for the effects of ErbB2 signaling, we used transgenic fish enabling tamoxifen-inducible overexpression of Nrg1 ligand in cardiomyocytes (Figure 5A; Gemberling et al., 2015). Depending on the duration of Nrg1 overexpression, these animals display overt cardiomyocyte hyperplasia and thickening of the ventricular wall (Figure 5C; Gemberling et al., 2015). We examined Rho A levels from hearts that ectopically expressed Nrg1 for 14 days, and unlike during ablation-induced heart regeneration where Rho A levels are unchanged, we found an ~85% increase in total Rho A protein (Figure 5—figure supplement 1A,mB). Consistent with a mitogenic role, three daily injections of zebrafish with Rhosin reduced the cardiomyocyte proliferation index by ~71% in the presence of ectopic Nrg1 expression (Figure 5B–D). To examine whether Rho A is key for the activity of other cardiomyocyte mitogens, we tested Rho A levels and the effects of its inhibition during tamoxifen-induced myocardial vegfaa overexpression or cardiomyocyte proliferation induced by the vitamin D analog alfacalcidol (Figure 5A, B). Western blot analysis revealed that vegfaa-expressing hearts had elevated cardiac Rho A levels, whereas alfacalcidol-treated hearts displayed no significant change (Figure 5—figure supplement 1A, B). Inhibition of Rho A by Rhosin reduced cardiomyocyte proliferation by ~68% and ~65% during vegfaa overexpression and alfacalcidol treatment, respectively (Figure 5E–H). Thus, our data indicate that Rho A signaling is a major target during heart regeneration, required for normal cardiomyocyte proliferation in response to injury or presence of Nrg1, Vegfaa, or vitamin D.
Assessment of published single-cell RNA-seq data revealed detectable rhoaa expression in cardiomyocytes, endothelial cells, fibroblasts, and immune cells, and rhoab expression in cardiomyocytes and endothelial cells during zebrafish heart regeneration (Supplementary file 6; Honkoop et al., 2019). Additionally, published profiles of cardiomyocyte H3.3 occupancy (Goldman et al., 2017), an indicator of active gene expression, indicate an approximately twofold increase in H3.3 enrichment in rhoaa and rhoab gene bodies during heart regeneration (Supplementary file 6). Generation of RNA-seq data from ventricles of transgenic animals after 7 days of induced myocardial Nrg1 overexpression (see Materials and methods) revealed that rhoaa levels were weakly elevated (1.6-fold), whereas no changes in rhoab or rhoac were detectable (Supplementary file 6). Thus, rhoaa and rhoab are top candidates for Rho A function in cardiomyocytes, although conditional disruption of the rhoa genes in zebrafish is needed to distinguish which member(s) is key.
Here, we have applied the proximity labeling technique BioID2 to investigate cell-type-specific proteome changes during a key model for innate tissue regeneration, the renewal of cardiomyocytes upon massive injury to the zebrafish heart. We identified changes specific to cardiomyocytes at the levels of whole-cell, membrane compartment, and the molecular interface of a key pro-regenerative factor ErbB2. Our profiling and functional experiments implicate the GTPase Rho A as mediating the effects of Nrg1 and other mitogens in cardiomyocytes during regeneration and provide a resource for which other candidate regenerative effectors can be assessed. We expect that BioID2 and evolved iterations can be effectively applied in myriad ways to monitor proteome changes during dynamic developmental events like regeneration in many species, tissues, and injury contexts to provide high-resolution insights into essential networks of regeneration.
Materials and methods
Wild-type or transgenic zebrafish of the hybrid EK/AB strain at 3–8 months of age were used for all experiments. All transgenic strains were analyzed as hemizygotes. Published transgenic strains used in this study were cmlc2:CreER (Tg(cmlc2:CreER)pd10) (Kikuchi et al., 2010), used with β-act2:BSnrg1 (Gemberling et al., 2015), β-act2:BSvegfaa (Karra et al., 2018), or β-actin2:loxp-mCherry-STOP-loxp-DTA (Wang et al., 2011). Tamoxifen treatment to deplete cardiomyocytes was performed as described previously (Wang et al., 2011) by incubating cmlc2:CreER; βactin2:loxp-mCherry-STOP-loxp-DTA (Z-CAT) animals in 0.7 μM tamoxifen (Sigma-Aldrich, St. Louis, MO) for 16 hr. Nrg1 and Vegfaa expression were induced as described by incubating adult cmlc2:CreER; β-act2:BSNrg1 or cmlc2:CreER; β-act2:BSVegfaa animals, respectively, in 5 μM tamoxifen for 24 hr. For expression of DN-Rho A, DN-Rac1 and DN-Cdc42, and Cre-negative animals were bathed in 5 μM 4-HT for 24 hr. For treatment with Rho A inhibitor, 5 μM of a 250 μm Rhosin (Miranda-Rodríguez et al., 2017) solution were injected on three consecutive days prior to harvest. Clutch mates were treated with vehicle. EdU injections were performed as described (Shoffner et al., 2020). Procedures involving animals were approved by the Institutional Animal Care and Use Committee at Duke University, Protocol number A005-21-01.
Generation of transgenic zebrafish birA2 (a gift from Scott Soderling) was subcloned into the cmlc2 vector (Kikuchi et al., 2010) into BamHI/NheI sites to generate cmlc2:birA2. To generate cmlc2:birA2-GFP, an EGFP cassette was subcloned downstream of BirA2 using NheI/EcoRI sites. A CAAX-tag was inserted into the EcoRI site to generate cmlc2:birA2-GFP-CAAX. erbb2 cDNA was amplified with the following primers (forward 5′-GCCACCATGGAGGCGGACAGAAGTTT-3′; reverse 5′-TCAGGTGTACTCCTTGTGGCCG-3′) and then subcloned into cmlc2-birA2 vector using the BsiWI/NruI sites. HA-P2A-GFP was then inserted into cmlc2:erbb2-birA2 by using NheI/XhoI sites to generate cmlc2:erbb2-birA2-HA-P2A-GFP. Mouse GFP-DN-Rho A (forward 5′-ACCGCCATGGTGAGCAAGGGCGAAGAG-3′, reverse 5′- TCTGGTTGCCTTGTCTTGTGA-3′), GFP-DN-Rac1 (forward 5′-ACCGCCATGGTGAGCAAGGGCGAAGAG-3′; reverse 5′- GAAGAGAAAATGCCTGCTGTTGTAA-3′), and GFP-DN-Cdc42 (forward 5′-ACCGCCATGGTGAGCAAGGGCGAAGAG-3′; reverse 5′-CAACCCAAAAGGAAGTGCTGTATATTCTAA-3′) were a gift from Scott Soderling and were each cloned into βactin2:loxp-dsRed-STOP-loxp vector (Yanazume et al., 2002) by using the AgeI/NotI sites. Each of these constructs was co-injected with I-Sce-I enzyme mix into one-cell-stage embryos. One founder of each construct was isolated, although they were not maintained as stable lines. The full names of the transgenic lines are as follows: Tg(cmlc2:birA2-GFP)pd335, Tg(cmlc2:birA2-GFP-CAAX)pd336, Tg(erbb2-birA2-HA-P2A-GFP)pd337, Tg(βactin2:loxp-BFP-STOP-loxp-DN-Rho)pd68, Tg(βactin2:loxp-BFP-STOP-loxp-DN-Rac)1pd69, and Tg(βactin2:loxp-BFP-STOP-loxp-DN-Cdc42)pd70.
BioID2 assay and quantitative mass spectrometry analysis
Request a detailed protocolTwenty hearts from adult zebrafish 3–8 months old were pooled for each sample, and triplicates were used for each condition. Prior to isolation of hearts, 10 μl of a 500 μM biotin/PBS solution was intraperitoneally (IP) injected into adult zebrafish on three consecutive days. Ventricle and outflow tract but not atrium were extracted, rinsed in cold PBS, and collected in ice-cold RIPA buffer. Using an Eppendorf tube pestle, hearts were lysed by 50 strokes, then flash frozen in EtOH/dry ice, incubated on ice for 15 min, and then again homogenized with 50 strokes of an Eppendorf tube pestle. Lysates were then centrifuged at 21,000 rpm for 20 min at 4°C. The supernatant was transferred to a new tube. Meanwhile, NeutrAvidin beads were pre-washed in keratin-free conditions in a laminar flow hood. 25–50 μl of bead slurry were pipetted into a low-protein-binding tube and spun down for 30 s at 2000 rpm at room temperature (RT). Supernatant was removed with a 30 g needle attached to 1 ml syringe. Beads were washed five times with 500 ml RIPA buffer. Next, washed beads were resuspended in 100 μl RIPA buffer and added to each sample. Each tube was sealed with parafilm and incubated overnight with rotation in 4°C cold room. The next day, samples were centrifuged for 1 min at 3000 × g at 4°C to pellet beads, and the supernatant was removed using a 30 g needle. 500 μl 2% SDS in 50 mM ammonium bicarbonate in MS-grade water was added to the bead pellet, and the mixture was transferred into a low-protein-binding tube. Beads were then washed (Goldman et al., 2017) twice with 500 μl 0.5% SDS in 50 mM ammonium bicarbonate (Hoang et al., 2020), twice with 1% Triton X-100/1% deoxycholate/25 mM LiCl in 50 mM ammonium bicarbonate (Johnson et al., 2020), twice with 0.5 M NaCl in 50 mM ammonium bicarbonate, and two times with 50 mM ammonium bicarbonate solution. For each wash step, 500 μl wash solution was used and samples were rocked for 10 min at RT. Samples were then centrifuged at 2000 × g for 30 s at RT, and the supernatant was removed with a 30 g injection needle. To elute the biotinylated proteome, an elution buffer containing 2% SDS, 10% glycerol, 5% β-mercaptoethanol, and 62.5 mM Tris pH 6.8 (NO bromophenol blue) in MS-grade water was prepared and biotin was added freshly to a final concentration of 2.5 mM. 50 μl elution buffer was added to the washed beads. Samples were boiled for 5 min at 95°C on a heat block. During the elution, samples were slowly vortexed three times. Next, samples were centrifuged for 30 s at 2000 × g at RT, and the supernatant was carefully removed using a new 30 g needle attached to a 1 ml syringe and transferred to a new low-protein-binding tube. Samples were stored at −80°C until delivery to the Duke Proteomics and Metabolomics Shared Resource.
Samples were reduced with 10 mM dithiolthreitol for 30 min at 80°C and alkylated with 20 mM iodoacetamide for 30 min at RT. Next, they were supplemented with 15 μl of 20% SDS in 50 mM TEAB, a final concentration of 1.2% phosphoric acid, and 555 μl of S-Trap (Protifi) binding buffer (90% MeOH/100 mM TEAB). Proteins were trapped on the S-Trap, digested using 20 ng/μl sequencing grade trypsin (Promega) for 1 hr at 47C, and eluted using 50 mM TEAB, followed by 0.2% formic acid (FA), and lastly using 50% ACN/0.2% FA. All samples were then lyophilized and resuspended in 12 μl 1% Trifluoroacetic acid (TFA)/2% acetonitrile containing 12.5 fmol/μl yeast alcohol dehydrogenase (ADH_YEAST). From each sample, 3 μl was removed to create a QC Pool sample, which was run periodically throughout the acquisition period. Quantitative LC/MS/MS was performed on 3 μl of each sample using a nanoAcquity UPLC system (Waters Corp.) coupled to a Thermo Fusion Lumos high-resolution accurate mass tandem mass spectrometer (Thermo) via a nanoelectrospray ionization source. Briefly, the sample was first trapped on a Symmetry C18 20 mm × 180 μm trapping column (5 μl/min at 99.9/0.1 v/v water/acetonitrile), after which the analytical separation was performed using a 1.8 μm Acquity HSS T3 C18 75 μm × 250 mm column (Waters Corp.) with a 90 min linear gradient of 5–30% acetonitrile with 0.1% formic acid at a flow rate of 400 nl/min with a column temperature of 55°C. Data collection on the Fusion Lumos mass spectrometer was performed in a data-dependent acquisition mode of acquisition with an r = 120,000,000 (at m/z 200) full MS scan from m/z 375–1500 and a target Automatic Gain Control (AGC) value of 2e5 ions with a 2 s cycle time. Ion trap MS/MS scans were acquired with a Rapid scan rate, 100 ms max injection time, and a target AGC value of 5e3 ions. A 20 s dynamic exclusion was employed to increase depth of coverage. The total analysis cycle time for each sample injection was approximately 2 hr.
Following UPLC-MS/MS analyses, data were imported into Proteome Discoverer 2.2 (Thermo Scientific Inc), and analyses were aligned based on the accurate mass and retention time of detected ions ('features') using Minora Feature Detector algorithm in Proteome Discoverer. Relative peptide abundance was calculated based on area under the curve of the selected ion chromatograms of the aligned features across all runs. The MS/MS data was searched against the TrEMBL D. rerio database (downloaded in May 2018) with additional proteins, including yeast ADH1, bovine serum albumin (BSA), as well as an equal number of reversed-sequence ('decoys') false discovery rate determination. Mascot Distiller and Mascot Server (v 2.5, Matrix Sciences) were utilized to produce fragment ion spectra and perform the database searches. Database search parameters included fixed modification on Cys (carbamidomethyl) and variable modifications on Meth (oxidation) and Asn and Gln (deamidation). Peptide Validator and Protein FDR Validator nodes in Proteome Discoverer were used to annotate the data at a maximum 1% protein false discovery rate.
Relative peptide abundance of triplicates was averaged for each protein, and proteins were confirmed as positive with p<0.05. As biotinylation of proteins depends on lysine availability at the contact site, proteins with one unique peptide were included in the data if relative abundance fit the outlined criteria above. Injured hearts were normalized to uninjured hearts, and a fold change of 1.5-fold was used as a threshold for protein level change. For the membrane proteome, uninjured BirA2-GFP-CAAX-expressing hearts were normalized to BirA2-GFP hearts, and proteins with a 2.5-fold change were considered to be membrane-specific.
Cytoscape was used to create interactome maps. BioGRID and Scaffold were used to search for interactors of ErbB2, Rho A, Rac1b, and Cdc42. Panther gene list analysis was used for over-representation tests in cellular components, signaling pathways and reactome analysis of the BioID2 data sets.
BioID2 raw MS proteomics data sets have been deposited to MassIVE with the data identifier MSV000087028 and are also summarized in Supplementary files 7 and 8.
Request a detailed protocolHearts were removed and placed in Hanks Buffered Saline Solution supplemented with 20 U/ml heparin. Outflow tracts and atria were removed, and ventricles were pulled apart with tweezers to remove blood from the lumen. Washed ventricles were then placed in 1 ml Trizol and processed to recover RNA. Further enrichment for mRNA was performed by polyA isolation, and mRNA was processed into RNA-seq libraries for sequencing on an Illumina Hi-Seq 2000. RNA-seq data was deposited at GEO with the data identifier GSE168371.
Immunofluorescence and imaging
Request a detailed protocolPrimary and secondary antibody staining was performed as described (Kikuchi et al., 2011). Mef2/PCNA staining has been previously described (Kikuchi et al., 2010). EdU/Mef2 staining of unfixed sections of ventricles was performed as follows: fish were injected intraperitoneally with 10 mM EdU on three consecutive days prior to harvest. For tissue preparation, hearts were removed from animals and placed into ice-cold 30% sucrose/PBS, before mounting. Hearts were flash-frozen in EtOH/dry Ice and stored in −80°C. Cryosections were performed to generate 10-μm-thick sections, and slides were air-dried for 30 min and then stored at −20°C. For EdU/Mef2 staining, slides were incubated on a slide warmer for 10 min and then fixed in 3.7% formaldehyde/PBS at RT for 15 min. Next, slides were washed three times 5 min in PBS. EdU staining was performed by mixing 600 μl 1M Tris pH 8.5, 6 μl 1< CuSO4, 6 μl 10 mM azide, and 600 μl 0.5 M ascorbic acid (made fresh). Slides were incubated for 30 min at RT. Next, washes were performed in 2% Triton-X-100/PBS four times each 5 min at RT, and slides were blocked for 1 hr at RT in 2% Triton-X-100/PBS, 5% goat serum, and 1% BSA. Primary antibody anti-Mef2 (Abcam, ab197070) was incubated 1:100 in 2% Triton-X-100/PBS and 5% goat serum overnight at 4°C. The next day, slides were washed in 2% Triton-X-100/PBS four times each for 5 min at RT and stained with the secondary antibody for 2 hr at RT. Lastly. four 5 min washes in 2% Triton-X-100/PBS were performed, and slides were mounted in Vectashield hard set DAPI reagent.
A Zeiss 700 confocal microscope was used to image slides. For EdU/Mef2 and Mef2/PCNA staining, images were acquired of the three largest sections from each ventricle. Mef2+ and Mef2+/EdU+ or Mef2+/PCNA+ cells were counted manually using Fij (ImageJ) in a muscle strip 100 μm wide along the ventricular periphery or injury border zone. Values from the sections were averaged to compute a proliferative index for each animal. Four animals were sampled for each condition.
Biochemical analysis
Request a detailed protocolCo-immunoprecipitations of Erbb2-HA in whole zebrafish hearts were performed as follows. Hearts from 20 cmlc2:erbb2-birA2-HA-P2A-GFP; cmlc2:CreER; βactin2:loxp-mCherry-STOP-loxp-DTA animals treated with tamoxifen, or from 20 controls without cmlc2:CreER, were extracted 14 days after treatment. Ventricle and outflow tract was isolated, rinsed in cold PBS, and collected in ice-cold RIPA buffer. Using an Eppendorf tube pestle, hearts were lysed by 50 strokes, flash-frozen in EtOH/dry ice, incubated on ice for 15 min, and then again homogenized with 50 stokes of an Eppendorf tube pestle. Lysates were then centrifuged at 21,000 rpm for 20 min at 4°C, and the supernatant was transferred to a new tube. 50 μl were saved as an input control and kept on ice during the entire Co-IP protocol. Heart lysates were first pre-cleared with 20 μl of packed protein A beads (Sigma) per lysate on a rocker or tumbler at 4°C overnight. The next day, 1 μl of the anti-HA antibody (1 μl IgG rabbit for control) was incubated with 20 μl of protein A beads for 1 hr on a rocker or tumbler at 4°C. Next, antibody-bead mix was added to the pre-cleared heart lysates and incubated for 2 hr at on a rocker or tumbler at 4°C. Samples were three times washed with 500 μl lysis and spun down at 800 rpm for 1 min at 4°C. After the last wash, 50 μl of lysis buffer was added to the samples, and samples were boiled with Laemmli buffer and β-mercaptoethanol at 95°C for 10 min. Samples were stored at −20°C before western blot analysis.
For whole heart lysates, the first part of the Co-IP protocol was followed until lysates were centrifuged at 21,000 rpm for 20 min at 4°C. The supernatant was isolated, and a Bradford assay (BioRad) was performed to determine protein concentration. Then, samples were boiled with Laemmli buffer and β-mercaptoethanol at 95°C for 10 min. 25 μg protein from each whole heart sample was used in western blot analysis.
Antibodies and reagents
Request a detailed protocolPrimary antibodies used in this study: anti-Mef2 (Abcam, ab197070, 1:100), anti-Rho A (Santa Cruz, 26C4,1:1000), anti-epb41L5 (RPL39, Proteintech, 1:100), anti-Talin 2 (Boster Biological Technology, PB9961, 1:100), anti-Seta (Antibodies online, ABIN2786762, 1:100), anti-ROCK2 (Bioss Antibodies, BS-1205R, 1:500), anti-HA (Abcam, ab9110, 1:1000), anti-CT3 (DSHB, 1:200), anti-PCNA (1:200, Sigma, P8825), anti-Mef2 antibody used in PCNA/Mef2 staining (gift from Jingli Cao) and anti-MHC (DSHB, F59-S, 1:200). Secondary antibodies were Alexa Fluor 488 goat anti-rabbit IgG (H + L) (Thermo Fisher Scientific, 1:200), Alexa Fluor 547 goat anti-mouse IgG (H + L) (Thermo Fisher Scientific, 1:200), EdU probes (Thermo Fisher Scientific, E10187), HRP goat anti-mouse IgG (Thermo Fisher Scientific, 31430, 1:50,000), HRP goat anti-rabbit IgG (Thermo Fisher Scientific, 65–6120, 1:50,000), and HRP goat streptavidin IgG (Pierce, 21130, 1:5000). Reagents used in this study were biotin (Sigma-Aldrich, B4501, 0.5 mM), Rhosin (Calbiochem, 555460), alfacalcidol (Selleck Chemicals, S1466, 200 μM), EdU (Life Technology, A10044), protein A sepharose beads (Sigma-Aldrich, P3391), NeutrAvidin Resin (Piere, Thermo Fisher Scientific 29202), and WGA Alexa Fluor 633 conjugate (Invitrogen, W21404, 1:100).
Data availability
RNA-seq data were deposited at GEO with the data identifier GSE168371. BioID2 raw MS proteomics datasets have been deposited to MassIVE with the data identifier MSV000087028.
NCBI Gene Expression OmnibusID GSE168371. RNA-seq from Pronobis et al.
MassIVEID MSV000087028. MS proteomics from Pronobis et al.
ERBB2 drives YAP activation and EMT-like processes during cardiac regenerationNature Cell Biology 22:1346–1356.https://doi.org/10.1038/s41556-020-00588-4
Efficient proximity labeling in living cells and organisms with TurboIDNature Biotechnology 36:880–887.https://doi.org/10.1038/nbt.4201
Proteomics analysis of extracellular matrix remodeling during zebrafish heart regenerationMolecular & Cellular Proteomics 18:1745–1755.https://doi.org/10.1074/mcp.RA118.001193
Resolving heart regeneration by replacement histone profilingDevelopmental Cell 40:392–404.https://doi.org/10.1016/j.devcel.2017.01.013
Blockade of the erbB2 receptor induces cardiomyocyte death through mitochondrial and reactive oxygen species-dependent pathwaysJournal of Biological Chemistry 284:2080–2087.https://doi.org/10.1074/jbc.M804570200
The epidermal growth factor receptor pathway in chronic kidney diseasesNature Reviews Nephrology 12:496–506.https://doi.org/10.1038/nrneph.2016.91
Hippo signaling impedes adult heart regenerationDevelopment 140:4683–4690.https://doi.org/10.1242/dev.102798
RhoA regulation of cardiomyocyte differentiationTheScientificWorldJournal 2013:491546.https://doi.org/10.1155/2013/491546
An improved smaller biotin ligase for BioID proximity labelingMolecular Biology of the Cell 27:1188–1196.https://doi.org/10.1091/mbc.E15-12-0844
The RhoA/Rho kinase pathway regulates nuclear localization of serum response factorAmerican Journal of Respiratory Cell and Molecular Biology 29:39–47.https://doi.org/10.1165/rcmb.2002-0206OC
Dynamics of zebrafish heart regeneration using an HPLC-ESI-MS/MS approachJournal of Proteome Research 17:1300–1308.https://doi.org/10.1021/acs.jproteome.7b00915
Talin1 has unique expression versus talin 2 in the heart and modifies the hypertrophic response to pressure overloadJournal of Biological Chemistry 288:4252–4264.https://doi.org/10.1074/jbc.M112.427484
Signals for cardiomyocyte proliferation during zebrafish heart regenerationCurrent Opinion in Physiology 14:78–85.https://doi.org/10.1016/j.cophys.2020.02.002
In vivo analysis of cardiomyocyte proliferation during trabeculationDevelopment 145:dev164194.https://doi.org/10.1242/dev.164194
ErbB-2 signals through Plexin-B1 to promote breast Cancer metastasisJournal of Clinical Investigation 122:1296–1305.https://doi.org/10.1172/JCI60568
Rho/ROCK pathway contributes to the activation of extracellular signal-regulated kinase/GATA-4 during myocardial cell hypertrophyJournal of Biological Chemistry 277:8618–8625.https://doi.org/10.1074/jbc.M107924200
Decision letter
Lilianna Solnica-KrezelReviewing Editor; Washington University School of Medicine, United States
Didier YR StainierSenior Editor; Max Planck Institute for Heart and Lung Research, Germany
Lilianna Solnica-KrezelReviewer; Washington University School of Medicine, United States
Ryan S GrayReviewer; University of Texas at Austin - Dell Pediatrics Research Institute, United States
In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.
Acceptance summary:
The manuscript reports the application of proximity labeling approaches in zebrafish to a specific cell type or its membrane compartment to identify changes in proteome and protein interaction networks during heart regeneration. Using a transgenic approach to express the promiscuous Bir2 biotin ligase in whole cardiomyocytes, their membrane compartment, or fused to the regeneration regulator ErbB2 in zebrafish, the authors define cardiomyocyte-specific proteome changes and protein interactions during heart regeneration. They functionally validate a role of the identified ErbB2-RhoA interaction in regulation of cell proliferation during heart regeneration. The reported methods and data sets will be of interest to those interested in applying proximity labeling techniques to study proteome dynamics and protein interactions in vivo in specific cell types and tissues.
Decision letter after peer review:
Thank you for submitting your article "in vivo proximity labeling identifies cardiomyocyte protein networks during zebrafish heart regeneration" for consideration by eLife. Your article has been reviewed by 2 peer reviewers, including Lilianna Solnica-Krezel as the Reviewing Editor and Reviewer #1, and the evaluation has been overseen by Didier Stainier as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Ryan Gray (Reviewer #2).
The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.
The manuscript reports the application of proximity labeling approaches in zebrafish to a specific cell type or its membrane compartment to identify changes in proteome and protein interaction networks during heart regeneration. Using a transgenic approach to express the promiscuous Bir2 biotin ligase in whole cardiomyocytes, their membrane compartment, or fused to the regeneration regulator ErbB2 in zebrafish, the authors define cardiomyocyte-specific proteome changes and protein interactions during heart regeneration. They functionally validate a role of the identified ErbB2-RhoA interaction in regulation of cell proliferation during heart regeneration. Overall, the data are properly controlled and analysis of datasets are rigorously analyzed in comparison with previous progress for understanding the transcriptional responses during cardiac regeneration. The reported methods and data sets will be of interest to those interested in applying proximity labeling techniques to study proteome dynamics and protein interactions in vivo in specific cell types and tissues.
Essential Revisions:
1. Page 6 " Quantitative BioID2 analysis captured 1113 unique proteins, of which 343 proteins showed a 2.5-fold enrichment in BirA2-CAAX samples when compared to BirA2-GFP samples ". What do the authors mean as 1113 unique proteins, clearly they overlapped with those detected by BirA2-GFP. Please clarify.
2. Figure 2—figure supplement 1, D panel legend – do different lines show data from three separate experiments?
3. For the proteins that showed increased ErbB2 association during regeneration, it would be important to examine how their level of expression at RNA and protein level (if tools are available) changes during regeneration.
4. The authors conclude that the strongest association was with Rho Ab; can the authors comment on specificity of the antibodies used in IP experiments in Figure 3E? Can they distinguish between different RhoAs in zebrafish?
5. As the authors are using pretty blunt reagents to inhibit RhoAa function (Rhosin and DN-RhoA), which of the genes encoding RhoA is affected is unclear. It will be important to assess whether other rhoa genes (rhoaa, rhoac) are expressed in cardiomyocytes? Is their expression altered during regeneration and in transgenics expressing Nrg1. Given the ease of genome editing, this part of the manuscript would be significantly strengthened by experiments assessing heart regeneration in rhoAa mutant fish, or the authors have to have to include these caveats in their interpretations and conclusions.
6. How was cardiomyocyte proliferation during regeneration affected in the transgenic DN-RhoA fish?
https://doi.org/10.7554/eLife.66079.sa1Author response
Essential Revisions:
1. Page 6 " Quantitative BioID2 analysis captured 1113 unique proteins, of which 343 proteins showed a 2.5-fold enrichment in BirA2-CAAX samples when compared to BirA2-GFP samples ". What do the authors mean as 1113 unique proteins, clearly they overlapped with those detected by BirA2-GFP. Please clarify.
The 1113 unique proteins refer to individual proteins that have been identified in our BioID2 assays of BirA2-GFP and BirA2-GFP-CAAX samples. We now refer to them as follows on p. 6: "Quantitative BioID2 analysis captured 1113 distinct proteins, of which 343 proteins showed a 2.5-fold enrichment in BirA2-CAAX samples when compared to BirA2-GFP samples (Supplementary File 1)".
2. Figure 2—figure supplement 1, D panel legend – do different lines show data from three separate experiments?
We added to the figure legend of figure 2—figure supplement 1, and supplement 2 and 3: "Heat map summarizes fold changes measured in 3 separate pooled samples."
3. For the proteins that showed increased ErbB2 association during regeneration, it would be important to examine how their level of expression at RNA and protein level (if tools are available) changes during regeneration.
To evaluate the protein levels of ErbB2 associated proteins (Figure 3C) we have created a table (Supplementary File 5) listing the protein levels that are found in our whole cell BioID2 samples (BirA2-GFP), and as reported in the proteomic screens of Wang et al. 2013 and Ma et al. 2018. Supplementary File 5 also lists the mRNA levels of ErbB2 associated proteins found in previously published RNA-seq datasets (Kang et al. 2016 and Ben-Yair et al. 2019) and CM-specific ChIP-seq of H3.3 (Goldman et al. 2017). We added the following to the manuscript (p. 10): "Next, we examined the levels of these candidates as reported in published transcriptome and proteome datasets (1, 35, 36, 41, 42), and found that overall expression levels were not predictive of ErbB2 association (Supplementary File 5)."
4. The authors conclude that the strongest association was with Rho Ab; can the authors comment on specificity of the antibodies used in IP experiments in Figure 3E? Can they distinguish between different RhoAs in zebrafish?
Due to high conservation, we do not expect that the antibody we used can distinguish between RhoAa, RhoAb, or RhoAc. We have now included an alignment of the different Rho A proteins and the immunogen used to generate the antibody (Figure 3—figure supplement 2), and we also state in our manuscript on p. 11: "Due to their high conservation, we were unable to identify which of the Rho A proteins, Rho A-a, Rho A-b, or Rho A-c increased its association with ErbB2 (Figure 3—figure supplement 2)."
5. As the authors are using pretty blunt reagents to inhibit RhoAa function (Rhosin and DN-RhoA), which of the genes encoding RhoA is affected is unclear. It will be important to assess whether other rhoa genes (rhoaa, rhoac) are expressed in cardiomyocytes? Is their expression altered during regeneration and in transgenics expressing Nrg1. Given the ease of genome editing, this part of the manuscript would be significantly strengthened by experiments assessing heart regeneration in rhoAa mutant fish, or the authors have to have to include these caveats in their interpretations and conclusions.
We have expanded Supplementary File 6, and it now includes the mRNA levels of rhoaa, rhoab, and rhoac genes reported in single cell RNA-seq data (Honkoop et al., 2019), and new RNA-seq data from collected from zebrafish hearts with induced overexpression of Nrg1. These data indicate that mRNA levels of rhoaa increase after Nrg1 overexpression, whereas there is no evidence of changes in rhoab and rhoac. We summarize these results in our manuscript as follows (p. 13): "Assessment of published single cell RNA-seq data revealed detectable rhoaa expression in cardiomyocytes, endothelial cells, fibroblasts, and immune cells, and rhoab expression in cardiomyocytes and endothelial cells during zebrafish heart regeneration (33) (Supplementary File 6). Additionally, published profiles of cardiomyocyte H3.3 occupancy (1), an indicator of active gene expression, indicate a ~2-fold increase in H3.3 enrichment in rhoaa and rhoab gene bodies during heart regeneration (Supplementary File 6). Generation of RNA-seq data from ventricles of transgenic animals after 7 days of induced myocardial Nrg1 overexpression (see Methods) revealed that rhoaa levels were weakly elevated (1.6-fold), whereas no changes in rhoab or rhoac were detectable (Supplementary File 6). Thus, rhoaa and rhoab are top candidates for Rho A function in cardiomyocytes, although conditional disruption of the rhoa genes in zebrafish is needed to distinguish which member(s) is key."
6. How was cardiomyocyte proliferation during regeneration affected in the transgenic DN-RhoA fish?
We have now performed these analyses and added them to the manuscript. We conducted Mef2/PCNA staining on DN-Rho A, DN-Rac1 and DN-Cdc42 transgenic animals 8 days after myocardial induction of each transgene, and 7 days after resection of the ventricular apex. We found trends of reduced cardiomyocyte proliferation in each case and added the following to our manuscript (p. 12): "We also observed tendencies for lowered cardiomyocyte proliferation in each transgenic background at 7 days after resection of the ventricular apex (Figure 4G).
https://doi.org/10.7554/eLife.66079.sa2Article and author information
Author details
Life Sciences Research Foundation
- Mira I Pronobis
National Heart, Lung, and Blood Institute (R35 HL150713)
- Kenneth D Poss
American Heart Association
- Kenneth D Poss
Fondation Leducq
- Kenneth D Poss
Fonds De La Recherche Scientifique - FNRS (34772792 –SCHISM)
- Sumeet Pal Singh
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
We thank the Duke Zebrafish Shared Resource staff for zebrafish care; Erik Soderblum and the Duke University School of Medicine for the use of the Proteomics and Metabolomics Shared Resource, which provided mass spectrometry service; Scott Soderling for the BirA2 and Rho GTPase constructs; Jianhong Ou for help with comparisons of BioID2 and ChIP-seq data sets; Jingli Cao for reagents; and Frank Conlon, Scott Soderling, Valentina Cigliola, and Ruorong Yan for discussions. MIP was supported by the Life Science Research Foundation (LSRF) – Astellas Pharma Postdoctoral Fellowship. SPS was supported by MISU funding from the FNRS (34772792 – SCHISM). KDP acknowledges research support from NHLBI (R35 HL150713), American Heart Association, and Fondation Leducq.
Animal experimentation: Procedures involving animals were approved by the Institutional Animal Care and Use Committee at Duke University, Protocol number A005-21-01.
Senior Editor
- Didier YR Stainier, Max Planck Institute for Heart and Lung Research, Germany
Reviewing Editor
- Lilianna Solnica-Krezel, Washington University School of Medicine, United States
- Lilianna Solnica-Krezel, Washington University School of Medicine, United States
- Ryan S Gray, University of Texas at Austin - Dell Pediatrics Research Institute, United States
Version history
- Received: December 29, 2020
- Accepted: March 25, 2021
- Accepted Manuscript published: March 25, 2021 (version 1)
- Version of Record published: April 9, 2021 (version 2)
© 2021, Pronobis et al.
This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.
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I have a link that I use to get the most relevant posts that I feel comfortable answering. Fairly frequently I get back that 0 questions are tagged. A simple refresh fixes the problem, but it is becoming quite the thing to refresh every 3 or 4 visits to the link.
The link I am using is all or
tags so there really should never be any times that there are 0 questions tagged. In this example, there just happens to be a new question with activity, but most of the time it's completely empty page.
Is this a bug?
Edit: I have updated the link to this and but the issue still remains.
Edit 10/31/15: Just updating that the issue still remains. I am currently using this link which is really similar to the one before but with 2 less tags.

subset: commoncrawl


text:
Rajasthan is the largest state of the Republic of India in terms of area. It encompasses most of the area of the large, inhospitable Great Indian Desert (Thar Desert), which has an edge paralleling the Sutlej-Indus river valley along its border with Pakistan.
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Our BSc (Hons) Radiography (Diagnostic Imaging) course provides you with all the training and knowledge you need to become a fully qualified practitioner and you'll learn a variety of skills that will enable you to shape the future health and wellbeing of both individuals and society. You'll be taught how to work under pressure and to make decisions under significant time restraints.
This course will teach you to think critically and analytically and will encourage you to become self-aware, reflective and empathetic.
Once you've completed the course you'll have the ability to work within a multi-disciplinary team across the hospital, from A&E to surgery, and will be fully equipped to embark on a challenging and rewarding career in radiography.
*This course is accredited by the Health Care Professions Council (HCPC) for eligibility to apply for registration.*��
Key Information
- Course title:
- BSc (Hons) Radiography (Diagnostic Imaging)
- UCAS Code:
- Qualification:
- BSc (Hons)
- Duration:
- 3 years
- Study mode:
- Full-time
- Typical offer:
- UCAS Tariff points 112-120
- Accreditation:
- Accredited by the Health Care Professions Council (HCPC) for eligibility to apply for registration
Course leader
Shelley Blane
BSc, MSc - Senior Lecturer (Radiotherapy)
Shelley is the Course Lead for both BSc Radiography courses. She has a BSC (Hons) in Therapeutic Radiography and an MSc in Radiation Physics. She is a HCPC registered Therapeutic Radiographer and Clinical Scientist with over 27 years' experience in Radiotherapy.
Course details
During the BSc (Hons) Radiography (Diagnostic Imaging) course you will also benefit from a high-performance digital X-ray suite, including a direct radiography (DR) room and mock ward to simulate the hospital setting. With on-campus Ultrasound, MRI, X-Ray, and Fluoroscopy you can expected to be well prepared for your first clinical placements with 91���� University College hospital partners in the NHS.
Together with small group working, practical sessions, and lectures you will be able to take advantage of working on placement at local providers seeing the real world working without the pressures of a busy NHS department.
On the completion of topics, you will be supported through interactive quizzes and shown example assignments to focus your critical evaluation and knowledge retention.
Formal assessments take the form of practical exams undertaken in the clinical environment on placement, together with a competency record and placement logbook.
Academic understanding and knowledge are confirmed through written assignments and computer-based short question exams.
Case Studies
"The Reason I Became a Diagnostic Radiographer"
Matt Southam is Clinical Imaging lead for the department of radiology. We spoke to Matt about why he became a Diagnostic Radiographer and the opportunities radiography students have at 91���� UC.
"91���� UC is the right size campus for our cohort size. We are learning so much and having so many opportunities."
Gary Hicks is a First Year student on our BSc (Hons) Diagnostic Radiography course he tells us about his passion to work in a health-care setting, making a difference to the lives of his patients and how he is learning something new every day from his inspiring lecturers and use of 91���� University College's industry-leading facilities.
Unit breakdown
Diagnostic Radiography (Diagnostic Imaging)
- Professional Practice and Patient Care
- Radiation Physics
- Anatomy and Physiology for the Radiographer
- Research Skills and Principles
- Equipment of Diagnostic Radiography
- Pathophysiology for Diagnostic Radiographers part 1
- Introduction to Radiobiology����
- Professional Practice and Wellbeing
- Scientific principles of Medical Imaging part 1
- Radiographic Anatomy
- Research Methods
- Pathophysiology for Diagnostic Radiographers part 2
- Multidisciplinary Working
- Professional Practice and Clinical Confidence
- Advanced practice in Diagnostic Imaging
- Image Interpretation
- Scientific Principles of Medical Imaging part 2
Entry Requirements
If you're applying from the UK or Ireland
Essential for entry
Please note that applicants must hold 5 GCSEs A*-C including Maths and English, or equivalent (reformed GCSEs grade 4 or above) to be considered for the programme.
BBB-BBC or above to include one of; Biology/Human Biology/Mathematics/Chemistry or Physics. General Studies will not be considered.
BTEC Extended Diploma
DMM or above in a relevant Extended Diploma for example; Applied Science, Medical Science or Health and Social Care.
Access to HE Diploma
A total of 112-120 UCAS points in a relevant Access to HE Diploma for example; Science, Medicine, Human Science, Allied Health Professions.��
International Baccalaureate
Overall grade of 30-32 points or higher to include either Biology/Human Biology/Mathematics/Chemistry or Physics at Higher Level.
Scottish Highers/Advanced Highers
BBBBB or above in 5 Higher/Advanced Higher subjects to include either Biology/Human Biology/Mathematics/Chemistry or Physics.
Irish Leaving Certificate
H3,H3,H3,H3,H3 from 5 subjects at Higher Level to include either Biology/Human Biology/Mathematics/Chemistry or Physics.
T Level
Merit in any Health and Science pathway accepted.
If you have undertaken a qualification that's not listed, it may still be considered. Get in touch with our Admissions team by completing the form below or call us on 01202 436200 and we'll be happy to answer any questions you have.
Is there anything else that is required?
If you live in the UK, we'll need a satisfactory Disclosure and Barring Service (DBS) check. It's standard for all UK students and we'll be there to guide you through the process once you've applied.
To gain a place on the course applicants must also successfully undergo an interview process.�� More information about this process will be provided if/when applicants are invited to the interview stage.
*Please note: You are required to have an occupational health screening prior to commencement on the course*
Please note that an NHS placement is required as part of this course.����
For more information about our admissions policies and procedures, including our Admissions Complaints and Appeals Policy, see��our latest policies.��
International Students��
English language requirements
Test | Grade | Additional details |
IELTS (Academic) | 7 | with no less than 6.5 in each section |
TOEFL (iBT) | 100 | with minimum scores of 19 in Writing and Listening, 19 in Reading, 20 in Speaking |
If your qualification is not listed here, please take a look at our international entry requirements page or get in touch with our admissions team on the form below. Please note, to be accepted onto this course; you will also need to demonstrate that you meet the minimum standards of English. For further information on English language requirements, please see the dedicated English language requirements page. More information on permission to study in the UK, can be found on our Visas and Immigration page.
Course Fees
A new £5,000 annual maintenance grant will be available for Radiography students from September 2020. This is in addition to existing support available, including student loans. Students who study radiography (diagnostic and therapeutic) are also entitled to an additional £1,000, as the subject is seen as a 'shortage specialism' and important to delivering the NHS long term plan.
Tuition fees��
UK/ROI/Channel Islands and Isle of Man students – £9,250 per year��
International - £16,560 per year
Please note that these fees are for the 2024-25 academic year only and may be subject to increase in subsequent years of study. ��Any increase will be in line with our Tuition Fees Policy but will not be more than 5%.
Additional course costs
Your tuition fees will cover the cost of all mandatory elements of your course.�� There are additional course related costs are not included in the tuition fees. So you will need to budget for them when you plan your spending.����
UK-domiciled applicants are required to undergo an Enhanced Disclosure and Barring Service (DBS) check. Further information will be sent to those who accept our offer. Applicants must meet the costs of this check (£45 in 2023). Students from low-income backgrounds eligible for bursary support from the 91���� UC may be able to have this funded.
BSc (Hons) Radiography (Diagnostic Imaging) ADDITIONAL COSTS
For further fees guidance for all students at 91���� University College whether full time or part time please refer to our Tuition fees policy.
Visit our student finance pages for more information on:
Note: For students to be eligible for funding from Student Finance England, they must be studying on an eligible course at a provider registered with the Office for Students (OfS). 91���� University College is registered with the OfS.��and search for 91���� University College.
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The polymerase chain reaction (PCR) and RAPD fragments are potentially useful methods for identifying turfgrass cultivar breeding lines. RAPD markers were studied in 25 vegetatively propagated buffalograss lines using oligonucleotide random primers and agarose-gel electrophoresis to determine their potential for identifying cultivar breeding lines. The variation of RAPD markers was extensive. The RAPD markers produced by one random primer were sufficient to separate the 25 buffalograss lines. Cluster analysis baaed on' the RAPD markers produced by two random primers revealed that the 25 buffalograss lines generally fell into two groups: diploid and hexaploid. Three DNA extraction methods—sarcosyl lysis-chloroform extraction-isopropanol precipitation, sodium dodecyl sulfate (SDS) lysine-isopropanol precipitation, and boiling in the presence of Chelex-100 resin—and fresh or oven-dried tissues were tested for reproducibility of RAPD markers. The three DNA extraction methods, using dry or fresh plant tissues, produced highly comparable RAPD marker profiles. More than 80%1 of the RAPD markers was consistently detected in six replicate analyses. The above studies demonstrate that small quantities (5 mg) of oven-dried leaf tissue and several DNA extraction methods can be used for buffalograss fingerprint studies.
Poor and inconsistent germination is a problem in triploid watermelon. Nicking was shown effective in improving germination in triploid cultivars. In this experiment, we examined the effects of high and low medium moisture, and nicking on diploid and triploid seed germination. Germination for the diploid cultivar was unaffected by any treatment. At high moisture conditions, triploid seed germination was severely reduced to less than 15%, while nicking significantly improved germination up to 40%. However, this increase is still not commercially acceptable. When seed morphological components were measured for each cultivar, triploid seeds had a larger and highly variable air space as compared to the diploid seed. The data confirm that seed germination is not inhibited by the seedcoat alone, but appears to be highly sensitive to excessive water conditions.
Autotetraploid (4n) plants of Dieffenbachia maculata (Lodd.) G. Don 'Perfection' flowered poorly, compared to diploids (2n), following treatment with 250 or 500 mg foliar spray of gibberellic acid (GA3)/liter. GA3-treated 4n plants produced bracts that normally precede flowering but remained vegetative and produced additional distal shoots instead of flowers.
Floral characteristics, meiotic behavior, and sporad formation were compared in three Dendrobium tetraploids (amphidiploids) and their diploid counterparts. Amphidiploid flowers were larger than those of diploids. Diploid meiotic behavior varied; mean configurations at Metaphase I ranged from 14.3 bivalents and 9.4 univalents to 18.9 bivalents and 0.2 univalents. In amphidiploids, nearly all cells had 38 bivalents. Sporad formation also varied; diploids had 36% to 70% tetrads and amphidiploids had 97% to 100% tetrads. Preferential pairing and small chromosome size may promote bivalent formation in amphidiploids.
The breeding of hybrid rose cultivars is part of the ornamental rose industry, but there have been few studies concerned with the factors limiting reproduction in the genus Rosa L. In this study, 28 species of Rosa were examined under field conditions for self- and cross-compatibility relationships. Twenty-five specimens of 25 species were tested for self-compatibility. All of the 23 diploid plants tested exhibited more than 96%. Twelve of the 16 polyploid plants preserved the 96% self-incompatibility reaction. A distinct separation existed between self-compatible and self-incompatible polyploid plants. Four of the 5 diploid cross-fertilizations produced unexpected reciprocal differences. Significant reciprocal differences were not obtained from the polyploid level cross-fertilizations. Some parthenocarpy occurred in the R. stellata species. Pollen viability and abundance, daily maximum and minimum temperature, and maximum and minimum relative humidity were not correlated with the compatibility reactions.
Knowledge of the chromosome number in Rubus would be valuable when planning crosses and identifying plants, etc., however, preparation of tissue for microscopic evaluation and chromosome counting is difficult and time-consuming. Flow cytometry offers a more-efficient approach to this task. DNA flow cytometry was used to determine the nuclear DNA content in 22 Rubus genotypes. The genotypes represented a range of reported chromosome numbers from 2x to 12x. Six of the genotypes were representatives of Rubus ursinus, which is reported to have both 8x and 12x forms. Samples of nuclei were prepared from leaf discs of newly emerged and mature leaves following published protocols with some modifications. The DNA content was estimated by comparison of the fluorescence of Rubus nuclei with an internal DNA standard. There was an increase in nuclear DNA content concurrent with the increase in chromosome number. In these studies DNA flow cytometry could differentiate genotypes that differed by 2x, such as 6x and 8x, but could not reliably distinguish genotypes that differed by 1x, such as 7x vs. 8x or 6x. Aneuploids cannot be differentiated at this time.
A comparison of pretreatment, fixing, and staining methods for root tips of Fragaria × ananassa (2n=8x=56), a polyploid species with small chromosomes, was made to facilitate chromosome counting. Three pretreatments (8-hydroxyquinoline, α-bromonaphthalene, and p-dichlorobenzene), three fixatives (Farmer's, Carnoy's, and Newcomer's), and five stains (acetocarmine, lacto-propionic orcein, leucobasic fuchsin, altered carbol fuchsin, and alcoholic hydrochloric-acid carmine) were examined in a factorial design to determine which treatment combination produced the best chromosome preparation. Field propagated runners were grown in sand under greenhouse conditions with supplemental lighting to produce root tips for late morning collection. The treatment combinations of α-bromonaphthalene or 8-hydroxyquinoline, Farmer's fixative, and altered carbol fuchsin, or the combination of α-bromonaphthalene, Farmer's fixative, and alcoholic hydrochloric-acid carmine produced the most intensely-stained and well-defined preparations.
A single regeneration procedure using cotyledon explants effectively regenerated five commercially grown muskmelon cultivars. This regeneration scheme was used to facilitate gene transfers using either Agrobacterium tumefaciens (using `Burpee Hybrid' and `Hales Best Jumbo') or microprojectile bombardment (using `Topmark') methods. In both cases, the transferred genes were from the T-DNA region of the binary vector plasmid pGA482GG/cp cucumber mosaic virus-white leaf strain (CMV-WL), which contains genes that encode neomycin phosphotransferase II (NPT II), β-glucuronidase (GUS), and the CMV-WL coat protein (CP). Explants treated with pGA482GG/cpCMV-WL regenerated shoots on Murashige and Skoog medium containing 4.4 μm 6-benzylaminopurine (BA), kanamycin (Km) at 150 mg·liter-1 and carbenicillin (Cb) at 500 mg·liter-1. Our comparison of A. tumefaciens- and microprojectile-mediated gene transfer procedures shows that both methods effectively produce nearly the same percentage of transgenic plants. R0 plants were first tested for GUS or NPT II expression, then the polymerase chain reaction (PCR) and other tests were used to verify the transfer of the NPT II, GUS, and CMV-WL CP genes. This analysis showed that plants transformed by A. tumefaciens contained all three genes, although co-transferring the genes into bombarded plants was not always successful. R1 plants were challenge inoculated with CMV-FNY, a destructive strain of CMV found in New York. Resistance levels varied according to the different transformed genotypes. Somaclonal variation was observed in a significant number of R0 transgenic plants. Flow cytometry analysis of leaf tissue revealed that a significant number of transgenic plants were tetraploid or mixoploid, whereas the commercial nontransformed cultivars were diploid. In a study of young, germinated cotyledons, however, a mixture of diploid, tetraploid, and octoploid cells were found at the shoot regeneration sites.
Citrus fruit with sector chimeras were collected in commercial packinghouses and from the field. Chimeric fruit from eight cultivars of sweet oranges [Citrus sinensis (L.) Osbeck], grapefruit (C. paradisi Macf.), tangelo (C. paradisi × C. reticulate Blanco), and tangors (C. reticulate × c. sinensis) were found at a frequency of 0.009% to 0.271%. Tetraploid plants obtained from one type of sector mutant (termed gigas) and albino plants obtained from another type of sector mutant confirmed that some genetic mutations observed in fruit rind can be recovered in nucellar seedlings. The gigas chimeras were identified as a source of citrus tetraploids. Several types of potentially useful sector mutants with altered rind color were observed, and plants were produced from some mutant sectors by developed seed or culture of aborted ovules. HPLC analysis of rind tissues from sectors of one chimeric fruit revealed substantial quantitative and qualitative differences in pigment composition. Propagation of plants from mutant sectors may yield cultivars with improved fruit color, altered maturation date, and reduced disease or mite susceptibility and may eventually lead to breeding of seedless triploid hybrids.
Factors of crop management such as irrigation, cultivation, cultivar selection, and control of insect pests and plant diseases play important roles in watermelon production. To gain a better understanding of how intensity of crop management affects yield, we conducted a comparative study contrasting high and low intensity management in 1997, 1999, and 2000. High-intensity management (HM) included the use of trickle irrigation, black plastic mulch, insecticides, and fungicides, not used under low-intensity management (LM). We examined the effects of management intensity on watermelon productivity, the variation in such effects among watermelon cultivars, and the mediating effect of survival of watermelon plants, abundance of insect pests, and incidence of anthracnose (% leaves with anthracnose lesions). The results indicated that HM produced 100% greater marketable fruit yield per area and marketable fraction of total fruit than LM in 2 out of 3 years. The effect of management intensity on plant survival was related to this effect on yield in 1 out of 2 years, and contributed to the latter by increasing weight and number of marketable fruit per plant under HM. We detected no significant effect of abundance of insect pests and incidence of anthracnose on yield. There was variation in the effect of management intensity on yield among watermelon cultivars in 1 out of 3 years. The triploid `Gem Dandy' showed great differences in yield between HM and LM in 2 years, producing on average 28.9 Mg·ha-1 of marketable fruit yield under HM compared to 14.0 Mg·ha-1 under LM. `Gem Dandy' also produced 100% higher yield of marketable fruit per area, per plant, and marketable fraction of total fruit than the open-pollinated diploid `Allsweet' or the diploid hybrid `Sangria.' Each year during the 3-year study, all three cultivars had a similar density of insect pests, incidence of anthracnose, and plant survival after transplant and at harvest. This study provided information on the collective impact of multiple aspects of watermelon management on yield.


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Finding your suitable readers for star wars 6 movie poster is not easy. You may need consider between hundred or thousand products from many store. In this article, we make a short list of the best readers for star wars 6 movie poster including detail information and customer reviews. Let's find out which is your favorite one.
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1. Star Wars: A New Hope Movie Poster, 24-inch x 36-inch
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The first six Star Wars films are immortalized in this FilmCells presentation. This framed collectible features the film posters of the first six films, a certificate of authenticity, and six clips of real 35mm film from the Star Wars saga. Each officially licensed FilmCells presentation is made with careful attention to detail. It is double matted, placed in a quality frame, and contains an acrylic front and back allows natural light to enhance the film frames. This is all put together to bring you a collectible that looks great in any room. Each piece of film is hand-cut from reels to provide you with the best possible scenes to offer a unique and varied product. The film strip portrayed in the image above is only an example of the film cells you may receive. Please note, the cells may vary from piece to piece as they are hand selected from reels of film.
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Former Indiana Senator Dan Coats to be Trump pick for Director of National Intelligence
Coats has been critical of Russia and may soften tensions between Trump and the US intelligence community.
Donald Trump is set to tap former Republican senator Dan Coats as his pick for Director of National Intelligence, according to reports on Thursday (5 January).
The widely expected pick comes as sources told the Wall Street Journal (WSJ) that the President-elect is planning to overhaul the very office that Coats would be leading.
The 73-year-old former senator from Indiana served between 1989-99 and then from 2011 until 2017, before deciding not to run for re-election.
Coats is a vocal critic of Russia and Vladimir Putin, having been banned from the country in 2014 for supporting sanctions after the annexation of Crimea.
Trump and the intelligence community, which will be overseen by Coats in his role leading the Office of the Director of National Intelligence (ODNI), have not fostered a warm relationship in the days running up to his inauguration. Reports from the CIA that Russia attempted to interfere with the US election to help Trump win have been branded "ridiculous" by the incoming leader.
The ODNI was created after the 9/11 attacks and is tasked with coordinating the 16 US intelligence agencies.
The decision to choose someone openly critical of Russia may help Trump's standing with those worried about his relationship and stance toward the Kremlin - particularly after he picked former Exxon CEO Rex Tillerson to be his Secretary of State. Tillerson is said to have a close personal relationship with President Putin.
Coats has served on the senate intelligence committee and was the US Ambassador to Germany between 2001 to 2005, under then-President George W Bush. Coats will have to undergo a senate confirmation hearing before he takes office, the date of which has not yet been announced.
© Copyright IBTimes 2023. All rights reserved.


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News » Premier League news » Burnley news
Arsenal receive big injury boost ahead of Burnley clash
Arsenal receive big injury boost ahead of Burnley clash
Arsenal have provided a big injury boost ahead of the Premier League clash against Burnley on Saturday afternoon.
The Gunners recently returned to winning ways in the league with a 3-1 comeback win against Leicester City on the road.
During that game, Emile Smith Rowe was substituted before the half-time interval with a hip problem.
Initially, Mikel Arteta expressed his concern over the attacker, but it appears that he has avoided a significant injury.
In an update provided by the Gunners, it has been revealed that the 20-year-old could be available to face the Clarets.
"Assessments and scans this week have shown that it is not a significant injury," a statement on the club's website read.
"Emile will be assessed and checked regarding his availability ahead of Saturday's match."
Smith Rowe has been almost ever-present for the Gunners since the 3-1 win over Chelsea in the Premier League on Boxing Day.
The capital outfit are currently 10 points behind the final Champions League spot, but they have a game in hand.


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Street Seal Coat Schedule has been updated again. On Wednesday, July 27th, the Seal Coat Contractor will return to finish sealing various city streets. See attached map for locations. Seal Coat is spray applied in two coats with a dry time of 2 to 3 hours per pass, so the streets being sealed will be temporarily closed during this time. On the date scheduled for treatment, all cars need to be moved from the street by 8:00 am. Streets being sealed will be closed from approximately 8:00 am until 5:00 pm. Access can be achieved in cases of emergency. Parking on adjacent streets is recommended during the daytime. Parking in driveways is ok if you DO NOT plan on moving the vehicle between 8:00 am and 5:00 pm. The Seal Coat treatment should not be driven on until it has set up (dried). It will stain surfaces such as concrete, carpet, or flooring if tracked from construction areas. Please have all lawn watering devices turned off the evening before the scheduled date. Please help keep the streets clean and dry for a smooth and lasting project. Traffic control will be set in areas of work being completed and picked up by the end of the work day. Notification of adjacent properties will be made door to door 24 hours in advance of the work.
The City of Hays regrets any inconvenience this may cause to the public. If there are any questions, please call the Office of Project Management at 628-7350 or Circle C Paving at (316) 573-1650.


text: Getting your tyres up to temperature before you head out on track is vital if you're going to set your best time or get off to the perfect start off the line. If your tyres are too cold you could end up losing the front end on the first corner, but having your tyres at the optimum temperature can help you to keep the grip you need to fly off the line, through the first sector and off towards pole position or the race victory. Capit tyre warmers are designed to help both amateur and professional motorcycle racers to prepare their tyres for practice, qualifying and race sessions; fixing onto the front and rear tyres with ease and providing heat for the rubber to cling onto before you turn the throttle. Once away your tyres will be able to hold onto the heat (provided that you're going fast enough, of course), but without using Capit tyre warmers you could find yourself falling out of contention - or out of the race altogether. Having produced car and motorcycle tyre warmers in Italy since 1996, Capit are a name you can trust, so make sure you've got them in your garage before your next race meeting.