The majority of congenital heart defects (CHDs) are thought to result from the interaction between multiple genetic, epigenetic, environmental, and lifestyle factors. Epigenetic mechanisms are attractive targets in the study of complex diseases because they may be altered by environmental factors and dietary interventions. We conducted a population based, case-control study of genome-wide maternal DNA methylation to determine if alterations in gene-specific methylation were associated with CHDs. Using the Illumina Infinium Human Methylation27 BeadChip, we assessed maternal gene-specific methylation in over 27,000 CpG sites from DNA isolated from peripheral blood lymphocytes. Our study sample included 180 mothers with non-syndromic CHD-affected pregnancies (cases) and 187 mothers with unaffected pregnancies (controls). Using a multi-factorial statistical model, we observed differential methylation between cases and controls at multiple CpG sites, although no CpG site reached the most stringent level of genome-wide statistical significance. The majority of differentially methylated CpG sites were hypermethylated in cases and located within CpG islands. Gene Set Enrichment Analysis (GSEA) revealed that the genes of interest were enriched in multiple biological processes involved in fetal development. Associations with canonical pathways previously shown to be involved in fetal organogenesis were also observed. We present preliminary evidence that alterations in maternal DNA methylation may be associated with CHDs. Our results suggest that further studies involving maternal epigenetic patterns and CHDs are warranted. Multiple candidate processes and pathways for future study have been identified. Birth defects are the leading cause of infant mortality, and congenital heart defects (CHDs) are among the most fatal of all birth defects Maternal folate supplementation has been shown to reduce the risk of CHDs To understand the mechanisms whereby gene-environment interactions lead to complex disease, epigenetic mechanisms must be considered Through the use of genome-wide DNA methylation array technology, we sought to determine if alterations in maternal gene-specific DNA methylation were associated with CHDs. We then further assessed candidate genes that were differentially methylated between mothers who had CHD-affected pregnancies and control mothers, and identified potential biological processes and pathways of biological relevance to CHDs.Genome-wide gene-specific DNA methylation was measured in 367 participants (180 cases and 187 controls). Various lifestyle characteristics were analyzed in our sample population to determine if these differed between cases and controls Table 1.To illustrate the representative distribution of methylation levels across more than 27,000 CpG sites, To determine if the associations between DNA methylation and cardiac defects were related to specific cardiac phenotypes, an analysis stratified by cardiac phenotypes was conducted (data not shown). Cardiac phenotypes included conotruncal, septal, and obstructive defects. Cases that had more than one cardiac defect type were included in all relevant strata. Among cases, 47.2% had atrial or ventricular septal defects (n = 85), 35.4% had right- and left-sided obstructive defects (n = 63), and 16.7% had conotruncal defects (n = 30). The 14 cases that did not fall within one of the three described main defect groups were excluded from the stratified analysis due to a small sample size. Analyses with sub-phenotype indicator variables included in the regression model were conducted and no statistically significant findings of sub-phenotype-specific methylation patterns were observed. Because gene-specific methylation patterns did not deviate significantly between cardiac phenotypes, cases were combined for subsequent analyses to maximize power to test our study hypotheses.10 for the 27,249 tests of association between gene-specific methylation and disease status. The observed quantiles are consistently higher than their expected values under the null hypothesis of no disease association, providing evidence of site-specific disease association for a large number of CpG sites.Table S1). The top 50 CpG sites ranked by statistical significance are listed in We identified 425 CpG sites that were differentially methylated between cases and controls with P<0.005, although an FDR threshold of 0.05 was not reached for any single CpG site  was performed to determine if the differentially methylated genes were significantly enriched in relevant biological processes and pathways of biological relevance to fetal heart development.Wnt signaling pathway. The GSEA results provided further evidence that the genes that were found to be differentially methylated appear to have biological relevance to CHDs. The results summaries generated from the GSEA, which includes the specific differentially methylated genes involved within each biological process and canonical pathway overlap, is included in Table S2 and Table S3 respectively.The 415 genes of interest were input into the GSEA program, and the top ten gene set overlaps were generated. To our knowledge, we have performed the first population based maternal case-control study to provide evidence of association between maternal gene-specific DNA methylation and CHDs. Further analysis of the differentially methylated genes revealed functionally relevant enrichment in biological processes and pathways previously shown to be involved in fetal development.No individual CpG site achieved genome-wide statistical significance. A combination of low variation in site-specific methylation between individuals and relatively modest sample size may account for the inability to reach statistical genome-wide significance. Thus, caution in interpreting the results of differentially methylated genes of interest must be noted. The advent of genome-wide association studies (GWAS) has led to an influx of studies structured to detect genetic variants associated with various human diseases, but limited studies have been conducted using genome-wide DNA methylation platforms. Issues regarding adjustments for multiple testing, statistically significant thresholds, and biologically relevant alteration levels, have not been established for high-throughput DNA methylation studies. The increased demand for genome wide epigenetic studies will lead to clearer definition of these issues in the future.In the current study, gene-specific methylation was measured in DNA isolated from peripheral blood lymphocytes. We observed slight alterations in methylation between cases and controls. Factors such as genetic variation, metabolites involved in one-carbon metabolism, and environmental exposures are possible mechanisms of differential methylation between cases and controls. The interpretation of results from DNA methylation studies must always be evaluated with caution as epigenetic marks may vary depending on cell type and can be influenced by various factors. Previous studies have used Illumina Infinium technology to assess DNA methylation in peripheral blood lymphocytes and have identified CpG sites associated with disease It has been established that various maternal factors may cause altered fetal growth and development EGFRGATA4Wnt5aGenes previously implicated in CHDs were identified within our differentially methylated genes. For example, multiple genes involved in the mitogen-activated protein kinases (MAPK) pathway are included in the list of differentially methylated genes. The MAPK pathway is involved in multiple cellular processes including transcription, proliferation, migration, survival, and differentiation GPX3, provide an example of a gene involved in the sustainment of a normal maternal environment. The GPX3 gene is involved in antioxidant potential in the body. Altered expression in GPX3 may result in a lower anti-oxidant potential, which may potentially lead to increased oxidative stress for the fetus.Alterations in maternal DNA methylation may also indirectly affect the fetus through changes in the maternal environment. Changes in the maternal environment can result in direct changes in gene expression in the developing fetus It is though that hypomethylation in the parental germline can influence genomic instability, and that this instability may subsequently cause genomic instability in the progeny Wnt signaling The importance of epigenetic factors in the development of complex disease is now unquestioned. Embryonic heart development requires a series of highly complex, coordinated and rapid morphogenesis processes. Specifically for CHDs, epigenetic factors may contribute to cardiomyocyte differentiation and chamber morphogenesis Certain methodological limitations of this study should be considered. The blood obtained to isolate genomic DNA was collected after pregnancy. We have previously described the rationale for the use of case-control study designs in human birth defects research studies Alterations in maternal DNA methylation converge with evidence from previous studies that indicate folate-dependent genetic and metabolic susceptibilities increase the risk of CHDs. The study has notable strengths including analyzing genome-wide gene specific DNA methylation in a large population of mothers. Extensive statistical modeling was used to adjust for any potential experimental confounders. We also adjusted for various lifestyle factors that have been shown to affect DNA methylation. The data presented here strongly suggest that maternal epigenetics may be a determinant of CHD risk. The non-invasive nature of the determination of DNA methylation in lymphocytes isolated from peripheral blood draw makes it an attractive candidate for disease risk assessment. Identification of genetic and epigenetic profiles associated with an increased risk of CHDs may add new dimensions to preconception risk assessment as well as further elucidate mechanisms involved in CHD development. Because DNA methylation may be influenced by diet or drugs, epigenetics may provide an avenue for future therapeutic intervention and prevention of CHDs.The National Birth Defects Research and Prevention Study (NBDPS) is an ongoing multi-site population-based case-control study investigating the etiology of 30 non-syndromic birth defects From eligible Arkansas NBDPS participants, we assessed DNA methylation in 180 cases and 187 controls for whom frozen blood samples were available. Blood samples were collected by research nurses during home visits in Arkansas that occurred between 2001 and 2008. Samples were collected by venipuncture after pregnancy. All participants signed informed consent approved by the UAMS Institutional Review Board.Information regarding selected lifestyle factors was obtained from in-home interviews conducted by research nurses using a Block Abbreviated Food-Frequency Questionnaire Gentra Systems, Minneapolis, MN).Fasting blood samples were collected in EDTA-Vacutainer tubes and immediately chilled on ice before they were centrifuged at 4000×g for 10 min at 4°C. DNA was isolated from the stored frozen blood samples according the manufacturer's protocol for the PureGene DNA isolation kit . In a total reaction volume of 25 µl, 2 µl of genomic DNA was used for absolute quantification for the RNase P assay on the ABI 7900HT Real Time PCR System, according to the manufacturer's protocol. After genomic DNA quantification, 500 ng of genomic DNA underwent bisulfite modification utilizing the EZ DNA Methylation-Direct Kit . The bisulfite-converted DNA was resuspended in 12 µl TE buffer and stored at -80°C until the samples were ready for analysis.Genomic DNA was quantified via the RNase P 20x assay  genes throughout the genome The standard protocol provided by Illumina was used for DNA methylation analysis. Briefly, 4 µl of bisulfite converted DNA was isothermally amplified at 37°C overnight. The amplified DNA product was fragmented by an endpoint enzymatic process. Fragmented DNA was precipitated, resuspended, and applied to an Infinium Human Methylation27 BeadChip and hybridized overnight. During hybridization, the amplified and fragmented DNA samples anneal to specific oligomers that are covalently linked to over 27,000 different bead types. Each bead type corresponds to the nucleotide identity and thus the methylation status at a bisulfite-converted cytosine in a specific CpG site. The bead chips were then subjected to a single-base extension reaction using the hybridized DNA as a template incorporating fluorescently labeled nucleotides of two different colors, each corresponding to the cytosine (methylated) or uracil (unmethylated) identity of the bisulfite-converted nucleotide at a specific CpG site. The fluorescently stained chip was imaged by the Illumina BeadArray Reader. Illumina's Genome Studio program was used to analyze BeadArray data to assign site-specific DNA methylation β-values to each CpG site. The proportion of methylation (β) for each subject at each CpG site was computed by first subtracting the background signal intensity of negative controls from both the methylated and unmethylated signals and then taking the ratio of the methylated signal intensity to the sum of both methylated and unmethylated signals. Thus, the β-value is a continuous variable ranging between 0 and 1.A total of one batch of two bead chips (24 samples) and eight batches of four bead chips were assayed. Cases and controls were randomly placed within the bead chips. Replicate samples were included in each batch for quality control and normalization measures. All replicate samples achieved a correlation coefficient of greater than 0.985 (data not shown). Using Illumina's Genome Studio, background adjusted β-values and assay control probe information was used for initial quality control. Subjects were required to pass three initial quality control criteria in order to be included in further analysis: 1) Subjects achieved a 95% CpG site call rate, which equals 26,250 sites called at an α<0.05 significance level; 2) Background signal for each subject was under 1000 units, determined from the controls dashboard in Genome Studio; and 3) Clear separation was observed in the non-polymorphic controls, also determined from the controls dashboard. Samples that did not meet these criteria were removed from the analysis. Individual CpG sites were examined using the detection p-value metric provided by Illumina in which the signal generated from each CpG site is compared to negative controls. A threshold of p<0.05 was used as a cutoff. CpG sites that did not reach this threshold were eliminated from the analysis. Using this criterion, 329 of 27,578 CpG sites (1.2%) had missing or invalid β-values in ten or more samples; these sites were dropped from subsequent analysis, leaving 27,249 CpG sites among 367 subjects.k nearest neighbor averaging Principal component analysis (PCA) was performed on the β-value data matrix of 367 subjects (rows) and 27,249 CpG sites (columns). Data were logit-transformed prior to PCA and subsequent association testing. As described above, 329 CpG sites with missing or invalid β-values in ten or more samples were removed from the data; before performing PCA, any remaining missing β-values were imputed using Figure S1). Regression analysis (not shown) revealed that these 20 PCs are variously associated with experimental factors that had been previously shown to be potential confounders under the Infinium Methylation platform The first 20 principal components (PCs) explain 62.8% of overall variance in the logit-transformed β-value matrix, and no single PC among the remaining 347 PCs explains more than 0.34% .After initial quality control and PCA of the β value data, a sample of 367 subjects (180 cases and 187 controls) was tested for association between disease status (i.e. case/control) and maternal gene-specific methylation. In this analysis, each CpG site was tested for association by regressing its logit-transformed β value on case/control status using multiple linear regression models. Additional covariates included these models were experimental batch (as a factor), bisulfite-conversion (BSC) efficiency, age, BMI, race (as a factor), and indicator variables of perinatal vitamin usage, alcohol drinking, and tobacco smoking. BSC efficiency for each subject has two channels, red and green. The red and green channels are highly correlated but are not sufficiently to be collinear, thus both channels were included in the models  p-values less than 0.005 was generated. The CpG sites of interest were to mapped to their corresponding genes and the list of genes were then tested for potential overlaps with biological processes and pathways using the Gene Set Enrichment Analysis (GSEA) Based on the results from our association testing, a list of differentially methylated CpG sites with Figure S1Variance explained by first 20 (of 367) principal components. The proportion of variance explained by each of the first 20 of 367 principal components, from a PCA of the 367-by-27,249 matrix of logit-transformed methylation β-values. Combined, the first 20 PCs explain 62.8% of overall variance, with no single PC among the remaining 347 explaining more than 0.34%.(PDF)Click here for additional data file.Figure S2Bisulfite conversion efficiency metrics scatterplot. Scatterplot of the red and green channels of bisulfite conversion efficiency for the 367 samples used in association testing, overlaid with a locally weighted scatterplot smoothing (LOWESS) curve.(PDF)Click here for additional data file.Table S1List of differentially methylated CpG sites at P<0.005.(DOC)Click here for additional data file.Table S2Gene Set Enrichment Analysis for Biological Processes.(XML)Click here for additional data file.Table S3Gene Set Enrichment Analysis for Canonical Pathways.(XML)Click here for additional data file.


Sensory hair cells in the zebrafish lateral line regenerate rapidly and completely after damage. Previous studies have used a variety of ototoxins to kill lateral line hair cells to study different phenomena including mechanisms of hair cell death and regeneration. We sought to directly compare these ototoxins to determine if they differentially affected the rate and amount of hair cell replacement. In addition, previous studies have found evidence of proliferative hair cell regeneration in zebrafish, but both proliferation and non-mitotic direct transdifferentiation have been observed during hair cell regeneration in the sensory epithelia of birds and amphibians. We sought to test whether a similar combination of regenerative mechanisms exist in the fish. We analyzed the time course of regeneration after treatment with different ototoxic compounds and also labeled dividing hair cell progenitors. Certain treatments, including cisplatin and higher concentrations of dissolved copper, significantly delayed regeneration by one or more days. However, cisplatin did not block all regeneration as observed previously in the chick basilar papilla. The particular ototoxin did not appear to affect the mechanism of regeneration, as we observed evidence of recent proliferation in the majority of new hair cells in all cases. Inhibiting proliferation with flubendazole blocked the production of new hair cells and prevented the accumulation of additional precursors, indicating that proliferation has a dominant role during regeneration of lateral line hair cells. Damage to auditory hair cells is associated with many factors, including exposure to various drugs, loud noise, and age-related degradation. Unfortunately, the majority of sensory hair cells do not regenerate in adult mammals Non-sensory supporting cells in the auditory and vestibular organs are the source of hair cell precursors, and two mechanisms of regeneration have been observed after hair cell damage. New hair cells are frequently derived from dividing precursors during the process of proliferative regeneration The zebrafish lateral line system provides an excellent model to study the process of hair cell regeneration. Zebrafish larvae are mostly transparent, and the lateral line is easily accessed on the body surface. Lateral line hair cells exhibit morphology and function similar to inner ear hair cells There are several lines of evidence that the type and extent of damage alter the course of hair cell regeneration. Differential exposure to copper affects the capacity for regeneration: treatment with low concentrations of copper elicits regeneration, while treatment with high concentrations prevents most hair cell replacement Taken together these data suggested that different types or levels of damage could elicit different responses with potentially distinct underlying molecular regulation. Here we revisit possible mechanisms of hair cell regeneration in the zebrafish lateral line system after exposure to several ototoxins. We compare the regenerative responses to copper, cisplatin, and the aminoglycosides neomycin and gentamicin, which we have previously suggested kill zebrafish lateral line hair cells by both distinct and overlapping mechanisms Danio rerio) embryos were obtained from pairings of *AB wild type or transgenic Et(krt4:EGFP)sqet4 adult fish . Larvae were fed live rotifers daily beginning at 4 days post-fertilization (dpf) with the exception of days when they were treated with an ototoxin. Experiments were conducted beginning at 3 or 5 dpf and were completed by 9 dpf. All animal procedures were approved by the University of Washington Institutional Animal Care and Use Committee, AWA number A3464-01.Zebrafish (Embryos at 3 or 5 dpf were incubated for between 30 min and 10 hours in EM (control treatment), neomycin , copper(II) sulfate , gentamicin , or cisplatin . All solutions were prepared by serial dilution when appropriate. Larvae were rinsed twice in fresh EM after treatment then returned to fresh EM or another solution as described.m-aminobenzoate methanesulfonate (MESAB), and viewed with an epifluorescent dissecting microscope. Ten neuromasts of the anterior lateral line  were scored using a semiquantitative scale where 0 = no signal, 1 = partial signal, and 2 = full signal for a total possible score of 20. Larvae were screened only once. In other experiments, individual hair cells from 7 neuromasts  were labeled with antibodies against parvalbumin as described below and counted using a Zeiss Axioplan 2 epifluorescent microscope. Eight larvae were assessed in each treatment group.Regeneration was measured in two ways. Initial experiments used a semi-quantitative scoring technique described previously To detect evidence of recent cell proliferation, ET4:GFP larvae were incubated in fresh EM containing 5 mM 5-bromo-2′-deoxyuridine (BrdU), a thymine analog, for 23 h immediately after a 1 hour (h) treatment with an ototoxin. BrdU-containing medium was replaced with fresh EM at 24 hpt and maintained for an additional 48 h. One reason for labeling DNA synthesis only during the initial stage of regeneration is that BrdU is a toxic mutagen and might affect hair cell survival To determine whether hair cell regeneration requires proliferation, larvae were incubated in fresh EM containing 5 μM flubendazole, which blocks microtubule assembly 2O. To label mature hair cells, larvae were blocked in 5% goat serum and incubated in mouse anti-parvalbumin primary antibody  followed by goat anti-mouse Alexa Fluor 488 or 568 secondary antibody . To label GFP in ET4:GFP larvae, mouse  or rabbit  anti-GFP primary antibody and goat anti-mouse or anti-rabbit Alexa Fluor 488 secondary antibody  were used. To label PHH3, rabbit anti-PHH3 primary antibody  and goat anti-rabbit Alexa Fluor 568 secondary antibody  were used. For the proliferation assay measuring incorporation of BrdU, larvae were subsequently rinsed in PBSDT (PBST with 1% DMSO) and 1 M HCl before being blocked in 10% goat serum again. Larvae were incubated in rat anti-BrdU primary antibody  followed by goat anti-rat Alexa Fluor 568 secondary antibody (1∶400). Larvae were viewed on a Zeiss LSM 5 confocal microscope using a 40X oil immersion objective. PASCAL image acquisition software was used to collect z-stacks of 3 neuromasts  per fish in 1 μm sections, which were later analyzed using ImageJ.Larvae were fixed in 4% paraformaldehyde and then rinsed in PBST  and ddHFigures were prepared using GraphPad Prism 5 and Adobe Illustrator CS3. Images were edited using Adobe Photoshop CS3. In all figures, error bars represent standard deviation. Differences between groups and time points were analyzed using Prism 5 with a one- or two-way ANOVA followed by a Tukey or Bonferroni posttest.We have previously shown that media composition alters hair cell toxicity We next compared hair cell regeneration after treatment with copper or neomycin on larvae at 5 dpf, when neuromast development is largely complete. As reported previously To examine whether treatment with neomycin or copper might bias regeneration toward proliferative or non-proliferative mechanisms, we used a pulse-chase paradigm to label new hair cells arising after damage with BrdU, a thymine analog that incorporates into DNA during S phase . Larvae p<0.0001). Approximately 40–50% of all hair cells in ototoxin-treated groups were BrdU-positive compared to ∼10% in control larvae, evidence of significant proliferative regeneration during the first 23 h of recovery , there was no such difference between 1 μM and 10 μM copper. Taken together, these data support the idea that a large number of hair cells are derived from dividing precursors after damage from multiple ototoxins.All experimental conditions resulted in significantly more proliferation compared to controls . One-wayp<0.001; . Althougp<0.001). These results support the idea that hair cells are derived from dividing precursors and do not support the idea that latent transdifferentiation can compensate in the absence of proliferation.Given that proliferative regeneration occurred after treatment with neomycin and with both low and high concentrations of copper, we sought to determine if preventing proliferation would uncover evidence of direct transdifferentiation. We treated 5 dpf larvae with neomycin or copper for 1 h and then incubated them in 5 μM flubendazole throughout the recovery period. Flubendazole blocks tubulin polymerization, preventing assembly of a mitotic spindle required for chromosome segregation p<0.001). This is consistent with the acquisition of a progenitor cell fate before subsequent hair cell differentiation.However, there was no significant difference between GFP- and parvalbumin-positive cells in larvae treated with flubendazole . This result strongly suggests that flubendazole arrests hair cell progenitors during mitosis by interfering with spindle microtubules, not at a later stage of differentiation.Impairment of microtubule assembly with flubendazole might impair differentiation of hair cells instead of blocking progenitor cell division. We therefore assayed the extent of regeneration during pharmacological inhibition of proliferation using transgenic ET4:GFP fish that express GFP in mature hair cells as well as pre-mitotic progenitors endazole , indicat p<0.05;  and coppA previous report demonstrated non-proliferative addition of hair cells after copper treatment in younger, 3 dpf animals during late embryogenesis We next tested whether proliferative regeneration is the predominant mechanism of hair cell renewal after damage by other ototoxic compounds. Gentamicin can induce both rapid and delayed patterns of cell death, which appear to be regulated by intracellular pathways that may be distinct from those that follow neomycin treatment p<0.001; To determine whether gentamicin treatment altered the rate of lateral line hair cell regeneration, we treated 5 dpf larvae with acute  or chronic  gentamicin and measured regeneration until 96 hpt. Approximately half of the hair cells remained at 6 h after the beginning of treatment and continued to decrease until 24 hpt , consist and measured regeneration until 96 hpt. Initial loss of hair cells was comparable to neomycin- and gentamicin-treated larvae, but regeneration was significantly delayed and remained incomplete at 96 hpt p<0.001; . IncubatOur results support a model in which new lateral line hair cells are derived from dividing progenitors irrespective of the type of ototoxic damage initiating regeneration. By labeling proliferating cells with BrdU, we found extensive incorporation of this marker after a variety of treatment conditions. This is consistent with other studies that used time-lapse microscopy or BrdU incorporation to observe hair cell regeneration We find that the drug flubendazole, an inhibitor of microtubule assembly Several other systems have been used to model auditory hair cell regeneration, and many of these make use of proliferative as well as non-proliferative regeneration. Although proliferative regeneration has been observed in birds A few possible explanations exist for the absence of direct transdifferentiation during regeneration of lateral line hair cells. Rapid proliferation may be sufficient such that an alternate mechanism is unnecessary. Complete regeneration in the zebrafish lateral line occurs within days compared to one or two months before functional recovery in the chick auditory system We found that in addition to blocking hair cell production, flubendazole prevented the accumulation of additional ET4:GFP-positive progenitors. This suggests that a mechanism responsible for producing new progenitors is sensitive to the rate of hair cell differentiation. Notch signaling has been implicated in regulating interactions among progenitors and differentiating hair cells. A recent report found that progenitors acquire their cell fate without dividing and in regions of low Notch expression located in polar compartments on opposite ends of the neuromast A major question is what mechanisms would need to be restored in mammals to promote hair cell regeneration. Although direct transdifferentiation seems simpler, it is not necessarily clear if proliferation or transdifferentiation provides a better opportunity to replace the large numbers of sensory hair cells required to restore auditory function in humans. Support cells undergo terminal mitosis in the mammalian cochlea while those in the vestibular system continue to proliferate in limited amounts atoh1, which specifies hair cell fate during both development and regeneration atoh1 is upregulated prior to the division of progenitor cells during regeneration of the chick basilar papilla atoh1 also can serve as a hindrance during hair cell regeneration. For example, atoh1 is expressed in some support cells in the mature mouse utricle after damage, but these rarely become new hair cells. Inhibition of Notch signaling increases both the amount of atoh1 expression and the number of cells that successfully transdifferentiate atoh1, is conserved during both phenomena. Therefore, one of the challenges to regenerating human auditory hair cells appears to be re-initiating these early developmental steps without disrupting the fully formed and complex structure of the inner ear.With or without proliferation, differentiation of progenitors into sensory hair cells requires expression of the transcription factor


In a double-blinded randomized controlled trial, Anja Haase-Fielitz and colleagues find that an infusion of sodium bicarbonate during open heart surgery did not reduce the risk for acute kidney injury, compared with saline control. Preliminary evidence suggests a nephroprotective effect of urinary alkalinization in patients at risk of acute kidney injury. In this study, we tested whether prophylactic bicarbonate-based infusion reduces the incidence of acute kidney injury and tubular damage in patients undergoing open heart surgery.p = 0.009). Sodium bicarbonate increased urinary pH . More patients receiving bicarbonate (83/174 [47.7%]) developed acute kidney injury compared with control patients . After multivariable adjustment, a non-significant unfavorable group difference affecting patients receiving sodium bicarbonate was found for the primary endpoint . A greater postoperative increase in urinary NGAL in patients receiving bicarbonate infusion was observed compared to control patients (p = 0.011). The incidence of postoperative renal replacement therapy was similar but hospital mortality was increased in patients receiving sodium bicarbonate compared with control .In a multicenter, double-blinded , randomized controlled trial we enrolled 350 adult patients undergoing open heart surgery with the use of cardiopulmonary bypass. At induction of anesthesia, patients received either 24 hours of intravenous infusion of sodium bicarbonate (5.1 mmol/kg) or sodium chloride (5.1 mmol/kg). The primary endpoint was the proportion of patients developing acute kidney injury. Secondary endpoints included the magnitude of acute tubular damage as measured by urinary neutrophil gelatinase-associated lipocalin (NGAL), initiation of acute renal replacement therapy, and mortality. The study was stopped early under recommendation of the Data Safety and Monitoring Committee because interim analysis suggested likely lack of efficacy and possible harm. Groups were non-significantly different at baseline except that a greater proportion of patients in the sodium bicarbonate group (66/174 [38%]) presented with preoperative chronic kidney disease compared to control , and formerly known as acute renal failure. Symptoms of AKI can be variable, with diagnosis of AKI based on laboratory findings (such as elevated blood urea nitrogen and creatinine), or clinical signs such as inability of the kidneys to produce sufficient amounts of urine. Globally, more than 10 million people are affected by AKI each year. AKI occurs in about one quarter of patients undergoing cardiac surgery and is associated with longer stays in the hospital and an increased risk of death. Treatment of AKI includes administration of intravenous fluids, diuretics, and, in severe cases, patients may require kidney dialysis.The mechanism for why AKI occurs during cardiac surgery is complex and thought to involve multiple factors relating to blood circulation, the immune system, and toxins released by the kidneys. In addition to treating AKI after it occurs, it is important to identify patients who are at risk for developing AKI prior to cardiac surgery and then apply techniques to prevent AKI during cardiac surgery. A number of interventions have been tested for preventing AKI during cardiac surgery, but there is currently no strong evidence for a standard way to prevent AKI. One intervention that has potential for preventing AKI is the administration of sodium bicarbonate during cardiac surgery. Sodium bicarbonate causes alkalinization of the urine, and it is thought that this could reduce the effect of toxins in the kidneys. A previous pilot study showed promising effects for sodium bicarbonate to reduce the likelihood of AKI. In a follow-up to this pilot study, here the researchers have performed an international randomized controlled trial to test whether administration of sodium bicarbonate compared to sodium chloride  during cardiac surgery can prevent AKI.350 patients undergoing open heart surgery with at least one risk factor for developing AKI were recruited across four sites in different countries . These patients were randomly assigned to receive either sodium bicarbonate (treatment) or saline control solution, given as a continuous infusion into the blood stream for 24 hours during surgery. Neither the researchers nor the patients were aware of which patients were assigned to the treatment group. The researchers measured the occurrence of AKI within the first 5 days after surgery and they found that a greater proportion of those patients receiving sodium bicarbonate developed AKI, as compared to those patients receiving saline control. On the basis of these findings the study was terminated before planned recruitment was completed. A key issue with this study is that a greater proportion of the patients in the sodium bicarbonate group had chronic kidney disease prior to open heart surgery. After adjusting for this difference in the statistical analysis, the researchers observed that the difference between the groups was not significant—that is, it could have happened by chance. The authors also observed that a significantly greater proportion of patients receiving sodium bicarbonate died in the hospital after surgery compared to patients receiving saline control.These findings suggest that giving an infusion of sodium bicarbonate to induce alkalinization of the urine during open heart surgery is not a useful treatment for preventing AKI. Furthermore, this treatment may even increase the likelihood of death. The researchers do not recommend the use of sodium bicarbonate infusion to reduce the risk of AKI after open heart surgery and stress the need for discontinuation of this therapy. Key limitations of this research study are the early termination of the study and the greater proportion of patients with chronic kidney disease prior to surgery.http://dx.doi.org/10.1371/journal.pmed.1001426.Please access these Web sites via the online version of this summary at acute kidney injuryThe Renal Association, a professional association for kidney doctors and researchers, provides information about The International Society for Nephrology and the International Federation of Kidney Foundations provide information about preventing acute kidney injury around the world and jointly initiated World Kidney Dayopen heart surgeryMedlinePlus has information on  Acute kidney injury is a global health problem with more than 10 million people affected annually Although the pathophysiology of cardiac surgery-associated acute kidney injury is complex, poorly liganded iron appears to be linked to its development suggesting that acute kidney injury, may, in part, be a sideropathy Given its biological rationale, a positive pilot trial, the broad discussion of the nephroprotective effects of bicarbonate infusion with positive risk benefit ratio, the lack of effective therapies, and the low costs, perioperative use of bicarbonate infusion has already impacted clinical practice of acute kidney injury prevention worldwide.Accordingly, we conducted a multicenter double-blinded randomized controlled trial to confirm or refute whether prophylactic urinary alkalinization with perioperative sodium bicarbonate infusion reduces the incidence of acute kidney injury and the magnitude of acute tubular damage compared to volume- and solute-equivalent sodium chloride infusion in a larger patient population undergoing open heart surgery.http://clinicaltrials.gov/ct2/show/NCT00672334).This study was an international, double-blinded, randomized controlled trial conducted at four university-affiliated hospitals Valvular surgery or concomitant valvular and coronary artery bypass graft surgeryRedo cardiac surgeryInsulin-dependent Type 2 diabetes mellitusEnd stage renal disease (serum creatinine concentration >300 µmol/l)Emergency cardiac surgeryPlanned off-pump cardiac surgeryKnown blood-borne infectious diseaseChronic inflammatory disease on immunosuppressionChronic moderate to high dose corticosteroid therapy Enrolled in conflicting research studyAge <18 yAllocation concealment to patients, anesthesiologists, cardiac surgeons, intensive care specialists, bedside nurses, outcome assessors, and investigators was ensured by central randomization through a clinical trial pharmacist who was not a co-investigator (Department of Pharmacy) at each study center. At each study center, the hospital pharmacy clinical trials coordinator used a Microsoft Excel-based (Microsoft Corp.) random number generator to create the randomization list using a permuted block strategy with blocks of six. The randomization list was kept locked on a password-secured computer that was placed in the Hospital Pharmacy Trials Area. Infusion bags were each delivered in separate shrink-wrapped black plastic bags that were identical in appearance. At each study center, the responsible hospital trial pharmacist was provided with emptied study medication infusion bags to assess intactness of the covering bag (shrink-wrapped black plastic bag). In two cases, the covering bag was damaged. The inner bag (beneath the black bag) was a neutral infusion bag which was not labeled with direct information about the study treatment. Treatment allocation was only revealed after the study had been completed, the database locked, and statistical analysis completed. During the initiation period of the study we assessed whether blinding of study treatment was still in place by frequent discussion with clinical and research personnel and participants. These discussions happened the day after surgery. The general response was that there was sufficient uncertainty regarding doubtless treatment allocation precluding the need for elaborate activities on blinding status.We used the same dosing of bolus of sodium bicarbonate and sodium chloride recently published in the prevention of cardiac surgery-associated acute kidney injury Apart from the addition of the infusion of study drug, clinical practice was not changed or modified for the purpose of the study. For non-study-related interventions, please see ACE inhibitors/angiotensin receptor 1 blockers:withdrawn on hospital admission Surgical approach and cardiopulmonary bypass:conducted according to the standard technique of each institutionAnesthetic techniques, including perioperative medications and fluid management:at the discretion of the attending anesthetist (documented)Analgesia:acetaminophen and morphine or tramadol with avoidance of non-steroidal anti-inflammatory drugsVancomycin and gentamicin:not used as perioperative anti-infective agentsPost-operative care, including hemodynamic, fluid, and analgesic management:at the discretion of the intensivists and nursing staff at each institutionThe primary endpoint was the number of patients who developed acute kidney injury. This was defined a priori as an increase in serum creatinine concentration greater than 25% or 0.5 mg/dl (44 µmol/l) from baseline to peak value at any time within the first 5 d after cardiopulmonary bypass. This endpoint has been used for previous studies of both cardiac surgery-associated acute kidney injury Secondary renal outcomes:changes of urinary NGAL concentration within the first 24 h after commencement of cardiopulmonary bypass were used to assess postoperative acute tubular damage as was previously done Number of patients who developed acute kidney injury according to the RIFLE criteria Number of patients who received acute renal replacement therapy during their hospital stayOther secondary outcomes:Changes in acid-base statusDuration of mechanical ventilation and length of intensive care unit and hospital stayIn-hospital mortality90-d mortalitySpecific adverse events:+]>150 mmol/l), hypokalemia ([K+]<3.5 mmol/l), alkalemia (pH>7.50), acidemia (pH<7.30), atrial fibrillation, acute kidney injury, initiation of acute renal replacement therapy and mortality  were recorded using the hospital electronic medical filing system or systematic telephone call of patients or their physicians at day 90Postoperative incidence of hypernatremia  on the ARCHITECT platform (Abbott Diagnostics) or ELISA (Antibodyshop), urine was sampled prior to induction of anesthesia, at 6 h and at 24 h after commencement of cardiopulmonary bypass. The laboratory investigators were blinded to the sample sources and clinical outcomes until the end of the study.p<0.05).Using data from a recently published randomized controlled trial on the use of bicarbonate for acute kidney injury prevention in cardiac surgical patients p<0.10. These variables were (i) clinically relevant : age, cardiopulmonary bypass time, chronic obstructive pulmonary disease, chronic kidney disease (eGFR <60 ml/min) p<0.10: center, previous myocardial infarction, and red blood transfusion.All following analyses were set a priori. All data were analyzed according to the intention-to-treat principle. In addition, the primary endpoint was analyzed per protocol. Per-protocol analysis excluded patients who were not operated on cardiopulmonary bypass or who were not drug-adherent defined as not receiving study infusion bolus before commencement of cardiopulmonary bypass or receiving <80% of the total weight-adjusted dose of study medication within the study period. Whilst there were insufficient events to facilitate multivariable analysis for mortality, to ensure all other observed results were not due to center or baseline imbalances, intention-to-treat analysis was repeated in multivariable models including all baseline variables that were considered to be clinically relevant or different between groups with univariate We also performed post hoc subgroup analyses including exclusion of the German study center, patients with eGFR >60 ml/min, and those receiving non-study-related bicarbonate infusion see . Serum ct test  or the Mann-Whitney U test and for categorical data with the use of Fisher exact test or chi-square test where appropriate. All tests were two-tailed and we considered a p-value<0.05 to indicate statistical significance.Continuous data were examined for normal distribution using histograms. Between-group comparisons for continuous data were performed with the use of the Student's We report values as mean with standard deviation (SD), or median with 25th to 75th percentiles or as ORs with 95% CI estimate as appropriate. Analysis was performed using SPSS, version 18.0 (SPSS Inc.) and SAS version 9.2 .n = 200]; The Mazankowski Alberta Health Institute, University of Alberta, Edmonton, Canada [n = 98]; The Austin Hospital, Melbourne, Australia [n = 47] and, The University College Dublin, Ireland [n = 5]). Between May 2008 and June 2011, 350 patients were randomized to receive either intravenous sodium bicarbonate (n = 174) or sodium chloride (n = 176) infusion. Details of patients screened and participating in the study are shown in the CONSORT diagram . There was no significant difference between treatment groups in type and duration of operation, intraoperative hemodynamic variables, and interventions  mmol and 408 (349–474) mmol for sodium chloride, p = 0.652, both administered within the 24-h study period.Drug adherence was 95.4% (166/174) for patients receiving bicarbonate and 96.6% (170/176) for patients receiving chloride study infusion, p<0.001) whereas urinary pH level decreased in control patients  compared with the control group .After multivariable adjustment for group imbalances at baseline , a non-significant group difference was found for the primary endpoint (adjusted p = 0.135).Also, after adjustment of peak creatinine for the dilutional effect of corresponding fluid-balance, no significant group difference for the primary endpoint was found .Per-protocol analysis of the primary endpoint including 166 patients receiving bicarbonate infusion and 170 patients receiving sodium chloride infusion , showed p = 0.011.A greater median postoperative increase in urinary NGAL from preoperative to peak value within the first 24 h postoperatively in patients receiving bicarbonate infusion (24.3 ng/ml [6.0–149.9]) indicated more pronounced acute tubular damage after surgery compared to control patients (12.2 ng/ml [0.0–76.6]), p = 0.015), higher peak serum creatinine (p = 0.001), and higher peak serum urea (p = 0.020)  classification = 0.020) .p = 0.031  in the bicarbonate group and 2.8% (n = 5) in the control group; p = 0.056 (n = 13] versus control group: 6.3% [n = 11]); p = 0.651.Overall mortality within 90 d was 7.5%  patients with preoperative chronic kidney disease and (ii) patients from the Australian, Canadian, and Irish study centers . The latAnalysis of patients excluding those with non-study bicarbonate infusion showed essentially unchanged major endpoints .p<0.001). Thirty patients in the control group and 14 patients in the bicarbonate group had a pH level below 7.30 with a minimum value of 7.17 in both groups (p = 0.011). Non-significantly more patients receiving sodium bicarbonate  had a plasma potassium <3.5 mmol/l compared to control  with the lowest potassium value of 3.0 mmol/l in both groups, p = 0.280. There was no significant difference in the incidence of new-onset atrial fibrillation , p = 0.838.Four patients developed hypernatremia during the study period with peak sodium concentration of 151, 152, 154, 158 mmol/l, respectively. Thirteen patients in the control group and 37 patients in the bicarbonate group had a pH level greater than 7.50 with a peak value of 7.59 in both groups  Group identified acute kidney injury to be a potentially preventable complication and recommended definitive randomized controlled trials for evaluating promising interventions to prevent and mitigate acute kidney injury In the present study, using the same scoring system This study demonstrates that there is no reduction of acute kidney injury or tubular protection after open heart surgery in patients who are administered sodium bicarbonate despite achieving adequate plasma and urinary alkalinization. This finding challenges the theory of attenuation of urinary mis-liganded iron compounds to be achieved by systemic alkalinization with bicarbonate. Experimental and clinical trials testing the effect of local urinary alkalinization without blood alkalinization—usually achieved by carbonic anhydrase inhibitors—are needed. We cannot exclude that perioperative systemic application of sodium bicarbonate and alkalinization of the blood had negative effects on unadjusted hospital mortality. There were insufficient events to facilitate multivariable analysis for in-hospital mortality, to ensure observed results were not due to center or baseline imbalances specifically given the group difference for preoperative chronic kidney disease. Despite the fact that the majority of patients died later in the course and no relationship of study bicarbonate infusion with mortality in the pilot study was observed Given our findings, we do not recommend the routine prophylactic use of sodium bicarbonate infusion for the purpose of urinary alkalinization and acute kidney injury prevention in patients undergoing open heart surgery, nor can we recommend conduct of larger scale clinical research in this setting. Using a preoperative risk stratification scoring system Given the positive effect of bicarbonate in the pilot trial and the negative effect in this randomized controlled multicenter trial, for sample size estimation in future preventive trials, we recommend using a more severe acute kidney injury endpoint definition such as doubling of serum creatinine or commencement of postoperative renal replacement therapy or a combination thereof. Such endpoints may decrease the chance of type I errors and may increase the likelihood of successful phase IIb trials. Finally, mechanistic studies linking alkalotic blood pH with organ damage are needed.The timing and dose of bicarbonate bolus and the duration of bicarbonate maintenance infusion may have been insufficient. However, study medication was administered using exactly the same infusion protocol with a slightly increased dose of maintenance infusion compared to the previous successful pilot trial Most patients were enrolled in one study center and a greater proportion of patients receiving sodium bicarbonate presented with preoperative chronic kidney disease compared to control; however, (i) multivariable, (ii) center-specific, and (iii) subgroup analyses revealed no nephroprotective effect of sodium bicarbonate. The study was prematurely terminated because of lack of efficacy and suggestion of harm; however, excellent study protocol adherence, no significant loss to follow-up, the use of kidney functional and acute tubular damage markers, and the use of preset sensitivity analyses strengthen the findings and the observation of possible harm make the likelihood of a positive finding implausible. Also, the internal and external validity of the results was achieved by double-blinding and a multicenter international design. Given the above observations, it appears unlikely that the negative outcome of this trial is due to type II error. We were not able to control for intentionally unblinding of study treatment by directly measuring pH value of the study treatment solution. Clinicians may have been able to guess group assignment on the basis of urinary and plasma pH levels being more alkaline in the treatment group after surgery (significant differences at 6 h and 24 h after surgery). However, assessment of such an attempt during study initiation for the day after surgery showed sufficient doubt, since there was some overlap in these values between the groups.In conclusion, this study demonstrates that in patients at high risk of acute kidney injury following open heart surgery, bicarbonate infusion alkalinized both blood and urine but did not result in a decrease in the incidence of acute kidney injury or attenuation of acute tubular damage. Importantly, prophylactic bicarbonate might possibly have increased mortality. On this basis of our findings we do not recommend the prophylactic use of perioperative infusions of sodium bicarbonate to reduce the incidence or severity of acute kidney injury in this patient group. Accordingly, discontinuation of growing implementation of this intervention in this setting would appear justified.Table S1Center-specific management of cardiopulmonary bypass and perioperative hemodynamic management.(DOC)Click here for additional data file.Table S2Major endpoints in post hoc subgroup analysis.(DOC)Click here for additional data file.Table S3Outcomes separated by study center.(DOC)Click here for additional data file.Text S1Trial protocol.(DOC)Click here for additional data file.Text S2CONSORT checklist (DOC)Click here for additional data file.


In the avian sound localization circuit, nucleus magnocellularis (NM) projects bilaterally to nucleus laminaris (NL), with ipsilateral and contralateral NM axon branches directed to dorsal and ventral NL dendrites, respectively. We previously showed that the Eph receptor EphB2 is expressed in NL neuropil and NM axons during development. Here we tested whether EphB2 contributes to NM-NL circuit formation.We found that misexpression of EphB2 in embryonic NM precursors significantly increased the number of axon targeting errors from NM to contralateral NL in a cell-autonomous manner when forward signaling was impaired. We also tested the effects of inhibiting forward signaling of different Eph receptor subclasses by injecting soluble unclustered Fc-fusion proteins at stages when NM axons are approaching their NL target. Again we found an increase in axon targeting errors compared to controls when forward signaling was impaired, an effect that was significantly increased when both Eph receptor subclasses were inhibited together. In addition to axon targeting errors, we also observed morphological abnormalities of the auditory nuclei when EphB2 forward signaling was increased by E2 transfection, and when Eph-ephrin forward signaling was inhibited by E6-E8 injection of Eph receptor fusion proteins.These data suggest that EphB signaling has distinct functions in axon guidance and morphogenesis. The results provide evidence that multiple Eph receptors work synergistically in the formation of precise auditory circuitry. The sensory epithelium of the auditory system is unique in that it contains an orderly representation of stimulus frequency that lacks information about auditory space. Our ability to localize sound sources relies on interaural time differences (ITDs) and interaural level differences (ILDs) encoded in auditory brainstem circuitry. Circuitry that detects ITDs is well characterized in the avian auditory brainstem , in whicDuring development, contralaterally projecting NM axons cross the midline at E4  and reacEph receptors comprise the largest class of receptor tyrosine kinases, and along with their ephrin ligands, are categorized into A and B subclasses based on structural similarity and binding affinities ,30. In gAt E10, when NM-NL contacts form, EphA4 is highly expressed in the dorsal, but not ventral, neuropil of NL . In our in ovo electroporation at E2 leads to prolonged plasmid expression as shown by EGFP reporter expression and by immunolabeling of protein encoded by the transfected plasmid. Transfection was directed focally to the auditory brainstem precursors at E2 by placing the electrodes at the level of r5 [As we have previously shown ,45, tranel of r5 . At E2, P = 0.09; Student’s t-test). To test the effect of reduced EphB2 signaling, we examined embryos transfected with kiEphB2, which acts as a dominant negative to inhibit forward signaling by interfering with endogenous receptor function [P < 0.0001) or in EphB2-transfected embryos (P = 0.0028). The significant increase in errors associated with kiEphB2 compared to EphB2 suggests that EphB2 forward signaling selectively affects targeting of NM axons to appropriate dendritic targets in NL and that it is a cell-autonomous effect since NM (axons) rather than NL (target) cells were transfected. Axon targeting in the NM-NL pathway was analyzed in E10 embryos after transfection. Each embryo was considered a single data point, and targeting errors were quantified across the central region of each right and left NL. As expected, control embryos with EGFP transfection showed few contralateral NM axons in the dorsal region of the NL neuropil Figure A, A’, wifunction  but actsWe examined the morphology of auditory brainstem nuclei in electroporated embryos. During data collection, abnormal NL morphologies were observed in which NL was either not organized into a flattened cellular monolayer and/or was reduced in length. In some instances, ipsilateral NM and NL were positioned in closer proximity to each other than expected for the age examined. The percentages of embryos within each group meeting any of these criteria for atypical NM-NL morphology are shown in Figure The electroporation studies suggest that EphB2 forward signaling regulates both axon guidance and auditory brainstem morphogenesis. We further tested this possibility by inhibiting forward signaling during the time that NM axons approach their contralateral NL target. We used unclustered soluble Eph receptor fusion proteins to inhibit forward signaling by endogenous ligands. The fusion proteins only differ at their C-terminal region, either having or lacking an Eph receptor extracellular domain, with an identical IgG-Fc region at the N-terminus. We first demonstrated that EphB1-Fc has the expected binding properties based on known expression patterns of ephrin-B ligands in the chick auditory brainstem Figure . Adjacenex ovo preparation that provided access to the brainstem at later embryonic ages and permitted injections in a localized region over the course of several days. Fusion proteins solubilized in PBS were injected into the developing hindbrain and fourth ventricle for four consecutive days from E6 to E9, when contralateral-projecting NM axons have already crossed the midline and are approaching their NL target [Because soluble fusion proteins are subject to degradation, the competitive inhibition assays were all performed in an L target . Since iex ovo had a mean of 9.80 errors ± 1.48 per 400 μm NL . In contrast, we found there was a significant increase in axon targeting errors from NM to contralateral NL after treatment with EphB1-Fc  and with EphA4-Fc  as compared to IgG-Fc controls. The effect of EphA4-Fc was significantly greater than that of EphB1-Fc  had some level of disorganization at the NL layer  of transfected embryos showing abnormal NL morphology Figures E, 6D. Th of transex ovo control cases (n = 18) also displayed some aberrant lateral projections but to a much lesser extent than treated embryos. These aberrant lateral projections were never observed with any of the in ovo preparations. Thus, it is possible the impaired lateral targeting is permitted within the ex ovo preparation and is exacerbated by the inhibition of Eph signaling. Several anatomical variables were quantified, including rostrocaudal and mediolateral lengths of NL . For embryos treated with EphA4-Fc, the rostrocaudal extent of NL was significantly reduced compared to IgG-Fc controls (P = 0.0169). An example of a severely reduced NL extent without NL disorganization is shown in Figure in ovo versus ex ovo experiments separately to exclude any differences associated with the preparations.We found that 17% of the L Figure E, F as w P = 0.019. An exa P = 0.019. An exa P = 0.019. An exaIn this study, we tested the role of EphB signaling in the guidance of NM axons to dorsal versus ventral dendritic regions of NL and in the morphogenesis of these auditory nuclei. We found that reduction of EphB2 forward signaling, either by kiEphB2 transfection or by local inhibition with soluble fusion proteins, resulted in a significant increase in the number of mistargeted contralateral NM axons as compared to controls. In addition, there were significantly more targeting errors when forward signaling through both EphA and EphB receptor subclasses were inhibited compared to inhibition of EphB receptors alone. While EphB2 electroporation, performed at E2, did not alter axon targeting, we found that it resulted in abnormal NL morphology in which NL was significantly shorter along both rostrocaudal and mediolateral brain axes than in controls. Morphological defects were also seen with fusion protein treatment, performed from E6 to E9. EphB1-Fc injections resulted in markedly aberrant NM projections at E10 that exceeded the contralateral NL boundary and projected laterally. EphA4-Fc injections resulted in malformed NL lamina, and aberrant laterally projecting axons, though less prominent than in EphB1-Fc treated embryos. NL was significantly shorter along the rostrocaudal axis with no change in mediolateral length compared to IgG-Fc injected and untreated controls. Together, these studies demonstrate several distinct functions for Eph proteins in the formation and connections of NM and NL.We previously showed that misexpression of EphA4 resulted in a significant number of targeting errors in which contralateral NM axons extended into the dorsal NL neuropil . The expThe electroporation studies carried out here examined the role of EphB2 in NM axons. We found that kiEphB2, but not EphB2, increased targeting errors by NM axon branches into contralateral dorsal NL. Because kiEphB2 decreases forward signaling while EphB2 increases it, our results suggest that forward signaling through EphB2 in NM axons is needed for the restriction of NM axons. The observation that EphB2 overexpression in NM axons did not have an opposite effect likely reflects the normally high precision in NM axon segregation. Together with our EphA4 study, these results suggest that bidirectional Eph signaling regulates the binaural segregation of NM axons in NL.Based on our previous expression analysis , forwardcis, whereby ephrins can bind to Eph receptors within the same cell [trans[The expression of EphB receptors and ephrin-B ligands in both axon and target suggests several possible roles for EphB signaling. In addition to axon guidance, Eph-ephrin signaling, and in particular, EphB signaling, has a well-documented role in synaptogenesis and synaptic plasticity -61. Becaame cell ,63 or seame cell ,65. Thesll [trans,63,66,67In our fusion protein studies, we found that infusion of EphA4-Fc resulted in significantly more targeting errors in the NM-NL pathway than infusion of EphB1-Fc, suggesting that inhibition of EphA and EphB signaling is more effective than inhibition of EphB signaling alone. These results are consistent with the observation of extensive targeting errors with EphA4 electroporation  and suggWhile our study focused on targeting of NM axons to distinct dorsal versus ventral NL regions, selective inhibition of the EphB class of receptors revealed an additional dimension of axon guidance for contralaterally projecting NM axons along the mediolateral axis. In particular, EphB fusion proteins resulted in pronounced lateral overgrowth by NM axons. Unlike the electroporation studies where misexpression was generally limited to NM axons and led to aberrant dorsoventral targeting, injection of fusion proteins into the hindbrain produced a broader inhibition that likely included EphB receptors in the NL neuropil. The observation of lateral overgrowth of NM axons using this approach may thus indicate that EphB signals arising in or near NL normally provide chemorepulsive cues that limit lateral growth.In addition to axon targeting errors, manipulations of Eph signaling also resulted in stereotyped morphological abnormalities. When EphB2 forward signaling was increased using plasmid electroporation at E2, prior to cellular migration and NL flattening into a monolayer, we observed a significant reduction in the size of NL. This effect may be a result of impaired migration of NL cells from the auditory anlage and/or from a reduction in total NL cells either by changes in cell fate specification or increased cell death. During normal development, NL undergoes extensive (84%) cell death as the monolayer forms . FurtherXenopus neural crest [Our observations are consistent with previous reports that EphB/ephrin-B signaling has been shown to guide normal cellular migration in mammalian neocortex, hippocampus and cerebellum , avian aal crest -76, and al crest  and hindal crest , wherebyal crest ,80. EphBal crest . Though al crest , the abnal crest  further ex ovo preparations from E6 to E9, though NL lamination appeared normal, we observed a tendency for NM axons to overshoot the lateral boundary of their contralateral NL target, suggesting that during normal development ephrin-B2 expressing axons may be limited to ventral NL neuropil by EphB2 forward signaling in NL cells. However, when forward signaling through both EphA and EphB was inhibited by EphA4-Fc injection, we observed more significant morphological defects. Similar to results for EphB2 electroporation, there was a reduced rostrocaudal extent of NL compared to controls. In contrast, the mediolateral extent was unchanged, suggesting that this axis is set earlier in development, while rostrocaudal extension may be more protracted.When EphB forward signaling was inhibited using fusion proteins in The differences in morphological defects between treatment groups suggest that Eph proteins have distinct roles during different developmental phases. Effects seen with electroporation at E2 could result from early morphogenetic events, such as cell proliferation and formation of the auditory anlage from precursors in distinct regions , as wellOur experimental results together with known developmental events seen during NM-NL development are summarized in Figure per se, or both. While it is not yet possible to determine how these proteins act at each stage, or which other molecules contribute to the assembly of this circuit, our results, together with previous studies, suggest that coordinated Eph signaling at multiple steps is needed for the formation, connectivity and boundary specification of the NM-NL circuit.Together with previous expression and misexpression studies, our results highlight the complex interactions between Eph molecules in NM-NL development. In our model, we propose that EphA4 signaling in dorsal but not ventral NL neuropil is an attractive cue to ipsilateral NM axons, whereas the repulsive nature of EphB signaling acts at the NL cell body layer disallowing axons on either side from crossing the NL monolayer. The segregation of ipsilateral and contralateral NM inputs to NL could thus arise from the distinct expression patterns of EphA4 and EphB2 along with the opposing effects of these proteins on axon growth. This view is consistent with data on dorsoventral targeting (present study and ), midlinGallus domesticus) were obtained from AA Laboratories  and stored at room temperature for up to two days prior to use. To initiate embryonic development, the eggs were placed in a rotating incubator at 39°C with a relative humidity above 70%. Chick embryos were either electroporated in ovo at E2 or transferred at E3 to a culture dish for ex ovo experimentation at E6 to E9. Ex ovo culture dishes were maintained in a non-rotating incubation chamber of similar temperature and humidity, allowing access to the developing embryo at later ages than the in ovo preparation. Each ex ovo culture preparation consisted of a disinfected polystyrene weigh dish (3.5 X 3.5 X 1 in.) into which the egg was opened, and a sterile square disposable petri dish bottom (100 X 100 X 15 mm) used as a lid. All embryos were sacrificed at E10 and the brainstem carefully dissected for in vitro whole mount axon tracing.Fertilized brown Leghorn chicken eggs ) was injected directly beneath the embryo to permit visualization. The vitelline membrane was removed with a fine tungsten needle and a small incision was made into the embryonic roof plate at the level of rhombomere 5 to provide access to the neural tube. Sterile PBS was placed over the area of interest to enhance electrical current and protect the embryo and underlying membranes from electrical burns. Paired needle-style gold-plated electrodes  were positioned straddling the area of interest, approximately 2 to 3 mm apart. Plasmid DNA (1 to 3 μg/μL in Tris/EDTA), colored with a small amount of Fast Green dye, was injected into the neural tube opening using a 1.2 mm pulled glass pipette attached to a Picospritzer . No more than 100 to 200 nL of plasmid DNA was delivered using a series of  injections of 20 to 50 ms duration and 20 psi. Voltage was delivered using a BTX electroporator, with amplitude of 9 to 12 Volts and 50-ms duration, in trains of four to six pulses of 100 ms intervals. A total of 20 to 30 pulse trains were delivered for each embryo, with polarity switched between pulse trains in order to achieve bilateral transfection. All openings in the eggshell were sealed with tape, and the eggs were placed in a humid, non-rotating 39°C incubation chamber for an additional eight days.in ovo electroporation. Full-length EphB2 was cloned into pCAX at the EcoR1 site and co-transfected with pCAX-EGFP. The pCAX construct encodes chicken β-actin promoter, a CMV-IE enhancer, polycloning sites and poly-A signal. Full-length EphB2 or kinase inactive EphB2  containing a point mutation in the intracellular kinase domain was cloned into the pMES vector at the EcoR1 site. The pMES construct contains a chicken β-actin promoter, a CMV-IE enhancer and also encodes an EGFP reporter with an internal ribosomal entry site. As a negative control, embryos were transfected with pCAX-EGFP alone. Transfection was assessed by examination of EGFP fluorescence in dissected brainstems and in sectioned material using an epifluorescent microscope. In addition, we performed immunostaining on transfected embryos using methods described previously for EphA4 and EphB2 detection following electroporation [Plasmid vectors were used to introduce DNA via poration ,17,44.A soluble recombinant protein containing the EphB1 receptor extracellular domain fused to the Fc region of Human IgG  was used to inhibit forward signaling through EphB receptors. Additionally, mouse EphA4-Fc (R&D systems) was used to inhibit forward signaling through EphA and EphB receptors. Recombinant proteins were diluted to 10 μg/mL in sterile 1X PBS and a small amount of methylene blue powder  was dissolved into the solution at 37°C immediately prior to use to aid with injection visibility. Final solution was injected into the developing hindbrain at the floor of the fourth ventricle with a 1.2 mm pulled glass pipette attached to a Picospritzer using pulses of 20 to 200 ms duration and intensity of 20 psi. Injections were made for four consecutive days, E6 to E9, when NM axons have already crossed the midline and are approaching their contralateral NL target. The total volume injected varied from approximately 1.5 to 5.5 μL per embryo for respective E6 to E9 ages. Human IgG1-Fc (RD Systems) was used as a negative control. Fusion protein binding specificity was confirmed by incubation of fixed 30 μm cryosections with EphB1-Fc or IgG-Fc overnight, followed by secondary (anti-Fc) fluorescent antibody incubation for two hours.ex ovo culture dish and the brainstem quickly dissected in oxygenated artificial cerebrospinal fluid . A pulled glass micropipette with an approximately 10 μm opening at the tip, was filled with rhodamine dextran amine  made at 6.25% with a 0.4% Triton X-100 in PBS, and attached by fine tubing to a Picospritzer. Using several pulses ranging from 10 to 50 ms at 20 psi, RDA was pressure injected into the dorsal midline of the medulla at the floor of the fourth ventricle to label only contralateral-projecting NM axons, as described previously [2/5% CO2 for 15 minutes. The tissue was then prepared for histologic sectioning as described in the next section and the axon tracings were later visualized by fluorescent microscopy.Axon tracing was used to identify and quantify targeting errors. E10 chick embryos were removed from the egg or the eviously . The tisTissue was fixed in 4% paraformaldehyde (PFA) in PBS for at least 2 hours, and then washed in 1X PBS for 10 minutes. For Vibratome sectioning, tissue was embedded in 2% low-melting agar  and mounted to the stage, fully immersed in 1X PBS during sectioning. For cryostat sectioning, tissue was cryoprotected in 30% sucrose overnight prior to embedding in OCT medium. For all tissues, sequential 50 μm sections in the coronal plane were collected on a subbed glass slide and dried on a slide warmer at 37°C. Most sections were counterstained with the nuclear stain bisbenzimide  to facilitate identification of NM and NL. Slides were coverslipped with Glycergel mounting medium  and stored in the dark at 4°C until analyzed. A separate group of samples underwent immunofluorescence staining to confirm plasmid expression or fusion protein location. These samples were sectioned on the cryostat as described above into 25 μm sections.Immunostaining was performed on a subset of samples to confirm plasmid expression with anti-EphB2 primary antibody or to confirm fusion protein localization with anti-Fc secondary antibody. Briefly, a hydrophobic pap-pen border was drawn around the sections and the slides were rinsed then incubated in blocking solution  for one hour at room temperature in a humid chamber. The slides were quickly rinsed with PBS and then incubated with the primary antibody for confirmation of plasmid expression, or with secondary antibody for visualization of fusion protein localization. For slides undergoing primary antibody labeling, slides were rinsed after one day then incubated with the secondary antibody for labeling. All secondary antibody labeling was performed with Alexa Fluor anti-Fc antibodies  used at a 1:1,000 dilution in blocking solution and incubated for two hours at room temperature in the dark.ex ovo cultured embryos, and successful midline axon tracing. Samples were coded and analyzed blind to experimental conditions. The slides were viewed on a Zeiss AxioSkop-2 epifluorescence microscope  using 10X, 20X or 40X objective lenses. To quantify NM-NL axon targeting errors, NL was viewed at 20 and 40X across several focal planes in order to allow for systematic analysis of the entire width of every section. Targeting errors were defined as axons arising from contralateral NM that crossed the NL cell body layer to terminate in the dorsal neuropil, visible as a cell-free layer with bisbenzimide staining and dorsal to NL somata. At this age in development, NL was typically visible across at least eight sections, or 400 μm of coronally sectioned brain. Since the central region of this extent is where NL presents robustly as a single layer of cells , this is the area where targeting errors can be most accurately quantified. Thus, for each embryo, n = 1 and a total of 400 μm of NL extent was analyzed, typically 200 μm (four adjacent sections X 50 μm each) from each left and right sides of the brain, at the central region along the NL axes. Mediolateral lengths were measured with Openlab software  by recording the distance between the medial-most and lateral-most borders of identifiable NL soma in equivalent sections. Rostrocaudal lengths were measured by counting the number of adjacent sections containing NL soma and multiplying by 50 (the thickness of each section). Left and right sides were recorded separately for anatomical measures. Once data collection was complete, the samples were decoded and grouped for comparison of means. Representative images were taken with Zeiss Axiocam digital camera and Openlab software. Any additional image analysis, tiling and color rendering or color merging was performed with Adobe Photoshop  and figures were prepared with Adobe Illustrator. All statistical analyses were done with JMP software  using a Student’s t-test and a significance value of P < 0.05.Experimental embryos with no gross abnormalities were used at E10 for data collection. Criteria for inclusion in further analysis were EGFP expression in the auditory brainstem of transfected embryos, four days of successful hindbrain injections for ED: Embryonic day; ILDs: Interaural level differences; ITDs: Interaural time differences; NL: Nucleus laminaris; NM: Nucleus magnocellularis; PBS: Phosphate buffered saline; PFA: Paraformaldehyde.The authors declare that they have no competing interests.MRA carried out the experiments and analysis for the electroporation and fusion protein studies. MRA and KSC contributed to the experimental design and manuscript preparation. Both authors have read and approved the final manuscript.


Plant-frugivore networks play a key role in the regeneration of sub-tropical forest ecosystems. However, information about the impact of habitat characteristics on plant-frugivore networks in fragmented forests is scarce. We investigated the importance of fruit abundance, fruiting plant species richness and canopy cover within habitat fragments for the structure and robustness of plant-frugivore networks in a mosaic forest landscape of South Africa. In total, 53 avian species were involved in fruit removal of 31 fleshy-fruiting plant species. Species specialisation was always higher for plants than for frugivores. Both species and network-level specialisation increased with increasing fruit abundance and decreased with increasing fruiting plant species richness and canopy cover within fragments. Interaction diversity was unaffected by fruit abundance and canopy cover, but increased slightly with increasing fruiting plant species richness. These findings suggest that especially the availability of resources is an important determinant of the structure of plant-frugivore networks in a fragmented forest landscape. Increasing impact of human land-use changes the structure and composition of ecosystems across the globe The interaction among fruiting plants and frugivorous birds can be both antagonistic and mutualistic. The fruit handling behavior of the animals determines if these act as predators or as legitimate seed dispersers A number of studies have shown that forest fragmentation and degradation lead to changes in community composition of birds and in plant-frugivore interactions Prunus africana were positively affected by human disturbance in a Kenyan forest Most of the studies investigating the impact of human activities on plant-frugivore relations studied interactions with single model plant species and yielded contrasting results Recently, the analyses of complex interaction networks have gained in importance Here, we present a study on the effects of fruit resource availability and canopy cover within forest fragments on plant-frugivore interaction networks in a fragmented sub-tropical forest landscape of South Africa. We assessed (1) overall levels of specialisation within networks in different forest fragments (2) the influence of resource availability, i.e. fruit availability and the diversity of fruiting tree species, as well as of canopy cover on the specialisation, interaction diversity and robustness of these networks. We expected to find (1) overall low levels of specialisation especially of frugivore species and (2) that high resource availability and a dense canopy cover of forest fragments lead to a higher interaction diversity and robustness of plant-frugivore networks.We conducted this study over two successive years, 2009/10 and 2010/11 in a heterogeneous scarp forest landscape in and around Vernon Crookes Nature Reserve (VCNR) situated on the south coast of South Africa’s KwaZulu-Natal Province . The necessary research permits for VCNR were obtained from Ezemvelo KZN Wildlife. All study sites outside VCNR were on private property owned by G. Archibald, who granted us access to his land.Monthly rainfall in the area ranges from 1–148 mm and annual temperature from 6–31°C We worked in forest fragments that are representative for the area, i.e. large natural forest, forest fragments occurring naturally and being completely surrounded by natural grassland as well as remnant forest fragments embedded in a matrix of sugar cane fields at the border of VCNR. All forest fragments harbour similar communities of plants and birds and occur within the same natural geographic range of scarp forests We established nine (200 m×200 m) study sites within forest fragments. The distance between study sites ranged from 0.53 to 1.06 km (mean = 0.80±0.3 km SD). Given the likelihood of strong edge effects in smaller forest fragments compared to the large fragments We measured habitat characteristics of local forest fragments by assessing, fruit abundance and fruiting plant species richness as well as canopy cover in each study site. We calculated fruit abundance (at the onset of fruit ripening) by estimating the number of fruits for each plant monitored and any other fleshy-fruiting plants within a radius of 50 m. We then calculated the mean fruit abundance for each study site, which ranged from 10,759 to 66,198 fruits  over the two years. To determine fruiting plant species richness, we identified all fleshy fruiting woody plants in each site to species level. Fruiting plant species richness ranged from 9 to 15 (mean = 12±3 SD) across study sites. We visually estimated canopy cover standing in the center of four (50×50 m) quadrates in each site and calculated the mean per site. Canopy cover ranged from 64 to 92% (mean = 84±14% SD) across study sites.We observed all fleshy fruiting plant species in each study site to assess interactions with frugivorous birds. Observations were undertaken during the main fruiting seasons in 2009/10 and 2010/11. Within this timeframe, plant species were observed at their peak of fruit ripening. Due to the low abundance of plant species as well as the low abundance of fruiting individuals, only one individual of each plant species was monitored in each site. We observed each plant in each study site for a total of 18 h, ideally split into 9 h per year. In cases where we could not achieve 9 h in the first fruiting year, we increased the number of observation hours in the second fruiting year to attain the standard total of 18 h per plant. We split the observations into three monitoring sessions, namely early morning (06∶00 am –09∶00 am), mid-morning (09∶00 am to 12∶00 am) and afternoon (2∶00 pm –5∶00 pm), conducted at three different days during the main fruiting period of each plant species in each year. Observations of species were evenly spread across the three sessions. Using binoculars  observations were carried out from a camouflaged hide at ca. 20 m distance to the plant individual. All plant visiting birds and their fruit handling behaviour on the plant were recorded. If more than one feeding bird was present on the plant (<0.5% of observations), one randomly chosen individual was selected for which fruit consumption was observed.p) with each frugivore species (f) in a quantitative interaction matrix, whereby interaction frequency was defined as the number of fruit-eating visitors per plant species d') of each plant (pd') and each frugivore (fd') species as the deviation of the actual interaction frequencies from a null model assuming that all partners are used in proportion to their availability. We calculated a weighted mean of the index per site for both plants and frugivores 2',H as the standardized two-dimensional Shannon entropy at the network level d' and 2'H range between 0 and 1, for complete generalisation and complete specialisation, respectively We compiled interaction frequencies of each plant species (vs. frugivores) on species specialization (d'). Trophic level was nested within plot for this analysis. Moreover, we used fruit abundance, fruiting plant species richness and canopy cover as explanatory variables to examine their effect on complementary specialisation (2'H), interaction diversity and network robustness in separate linear models. All statistical analyses were performed in R using packages base and nlme. We further tested for a correlation of interaction diversity and network robustness.All habitat characteristics, i.e. fruit abundance, fruiting plant species richness and canopy cover were uncorrelated . We used a linear mixed effects model to test the effect of the above-mentioned characteristics as well as of trophic level (plants Ficus burkei), Forest Knobwood (Zanthoxylum davyi) and Red-beech (Protorhus longifolia). Frugivores with most interactions on plants included the Dark-capped Bulbul (Pycnonotus tricolor), Cape White-eye (Zosterops virens) and Knysna Turaco (Tauraco corythaix)  .d') of the trophic levels within our networks was significantly higher for plants (0.39±0.02 SE) than for frugivores  was 0.44±0.02  and dispersal distances The increase in interaction diversity particularly with increasing fruiting plant species richness denotes that high diversity of food resources may promote network stability. In fact, we found a positive relationship between interaction diversity and robustness of our networks. Under such conditions, plants would profit from a broad spectrum of seed dispersing frugivores as this may reduce the clumping of seeds in the landscape. This may potentially aid the recruitment of their offspring in diverse communities. Nonetheless, neither fruit abundance nor fruiting plant species richness affected robustness at the network level. Changes of specialisation and interaction diversity along gradients of habitat characteristics are not reflected in varying robustness of the networks. This could be caused by the comparably high overall generalisation and redundancy within our networks, which makes the networks insensitive towards the loss of single frugivore species.Specialisation at both species and network level decreased slightly with increasing canopy cover, while interaction diversity and network robustness where unaffected. Decreasing specialisation with increasing canopy cover is congruent with results from a study in a Kenyan rainforest Our study shows that networks across local forest fragments were all characterized by redundancy in the associations between plants and frugivores, suggesting a stable coexistence of species in the plant-frugivore communities. Thus, secondary extinctions of mutualists, due to resource losses are unlikely in these networks Figure S1Plant-frugivore interaction networks in nine scarp forest fragments in Kwazulu-Natal, South Africa. Grey shades represent the frequency of interactions among frugivores and plants within a network, with black representing most frequent interactions.(EPS)Click here for additional data file.Table S1Effects of habitat characteristics and season  on the structure of plant-frugivore networks. Species specialisation (d'), network specialisation (2'H), interaction diversity and robustness of plant-frugivore networks (n = 9) in relation to fruit abundance, fruiting plant species richness, canopy cover (%) and season (2009/10 and 2010/11). A subset of the complete data set was used for these analyses. For each species where more than 6 h of observation in each year were available, we equally sampled a 6 h-subset of the dataset from the three observation sessions (see main text for details). Effects on all dependent variables were analysed using linear mixed effect models, with year nested within plot. To investigate effects on species specialization, trophic level was nested within year and plot. Given are t and P values. Note: all significant or marginally significant P values are highlighted in bold.(DOCX)Click here for additional data file.Table S2Common and scientific names of fleshy-fruiting plants in nine plant-frugivore networks.(DOCX)Click here for additional data file.Table S3Species codes, common and scientific names of frugivores (birds) in nine plant-frugivore networks.(DOCX)Click here for additional data file.Table S4Measures of plant-frugivore networks and habitat characteristics of nine scarp forest fragments in South Africa.(DOCX)Click here for additional data file.