Improvements in poultry production within the past 50 years have led to increased muscle yield and growth rate, which may be contributing to an increased rate and development of new muscle disorders in chickens. Previously reported muscle disorders and conditions are generally associated with poor meat quality traits and have a significant negative economic impact on the poultry industry. Recently, a novel myopathy phenotype has emerged which is characterized by palpably “hard” or tough breast muscle. The objective of this study is to identify the underlying biological mechanisms that contribute to this emerging muscle disorder colloquially referred to as “Wooden Breast”, through the use of RNA-sequencing technology.We constructed cDNA libraries from five affected and six unaffected breast muscle samples from a line of commercial broiler chickens. After paired-end sequencing of samples using the Illumina Hiseq platform, we used Tophat to align the resulting sequence reads to the chicken reference genome and then used Cufflinks to find significant changes in gene transcript expression between each group. By comparing our gene list to previously published histology findings on this disorder and using Ingenuity Pathways Analysis (IPA®), we aim to develop a characteristic gene expression profile for this novel disorder through analyzing genes, gene families, and predicted biological pathways.Over 1500 genes were differentially expressed between affected and unaffected birds. There was an average of approximately 98 million reads per sample, across all samples. Results from the IPA analysis suggested “Diseases and Disorders” such as connective tissue disorders, “Molecular and Cellular Functions” such as cellular assembly and organization, cellular function and maintenance, and cellular movement, “Physiological System Development and Function” such as tissue development, and embryonic development, and “Top Canonical Pathways” such as, coagulation system, axonal guidance signaling, and acute phase response signaling, are associated with the Wooden Breast disease.There is convincing evidence by RNA-seq analysis to support localized hypoxia, oxidative stress, increased intracellular calcium, as well as the possible presence of muscle fiber-type switching, as key features of Wooden Breast Disease, which are supported by reported microscopic lesions of the disease.The online version of this article (doi:10.1186/s12864-015-1623-0) contains supplementary material, which is available to authorized users. In terms of consumption, poultry meat is highly regarded as one of the most efficient food sources; it contains high quality nutrients such as protein, and is relatively inexpensive for purchase. However, muscle disorders of commercial chickens such as deep pectoral myopathy (DPM) -3 and whSihvo et al. 2014  specificPreviously described muscle disorders in chickens, such as white striping and nutritional myopathy, have been shown to affect the integrity of the pectoralis major muscle in chickens. White striping is identified through the observation of white fatty lines on the breast muscle running in the same direction as the muscle fibers of the breast muscle . It is sNutritional myopathy also affects the breast muscle of broiler chickens and is generally associated with low levels of vitamin E and selenium in diet . The maiThe overall objective of this paper is to use RNA-sequencing to identify biological and molecular pathways that are involved in the underlying etiology and pathogenesis of this emerging muscle disorder of the pectoralis muscle in commercial broiler chickens. We used RNA-sequencing as this powerful technology provides comprehensive and accurate gene expression data, performing better than previous technologies such as microarrays . Also, uChickens used in this study were all males from a high breast meat yield purebred line of broiler breeder chickens raised on the floor, in pens of 300, by Heritage Breeders in the Delmarva region of the United States. Birds were given free access to both feed and water, and were housed according to optimal industry growing standards. Birds were euthanized at 47 days of age by cervical dislocation. After euthanasia, pectoral muscle samples were collected at necropsy. Based upon clinical examination and manual palpation of pectoral muscle for evidence of palpable “hardening", eight affected and eight unaffected birds were identified; affected birds were those deemed to have “severe” to “moderately-severe” lesions, while unaffected birds showed no apparent gross lesions. Samples from the pectoralis major were taken from the caudal aspect of the right superficial pectoral muscle, perpendicular to myofibers and to the keel bone. Samples were taken with the Keyes biopsy punch (8 mm) manufactured by HNM. Roughly one to two grams of tissue was extracted from each sample location and immediately flash frozen in liquid nitrogen. The protocols were submitted to, and the use of the collected samples for research was approved by, the University of Delaware Agricultural Animal Care and Use Committee (protocol number: 44 12-15-13R).mirVana™ miRNA Isolation Kit according to the manufacturer’s protocol (Life Technologies®). Multiple quality checks were performed to assess the RNA quality and integrity using NanoDrop 1000 and Angilent Bioanalyzer 2100 to measure RNA concentrations following the established manufacturer protocol. The RNA integrity number of samples was larger than 8, which is considered acceptable for cDNA library preparation.Total RNA was extracted from the breast muscle tissue samples using the Eleven samples, five affected and six unaffected, were prepared to create cDNA libraries for RNA-sequencing. All samples used for RNA-sequencing were also strictly verified for correct identity  and was then calculated as 2 log2(fold change) for genes with a positive log2(fold change) and −1/(2 log2(fold change)) for genes with a negative log2(fold change) before submission to IPA.Cufflinks v2.1.1  was used2(fold change) for gene expression between affected and unaffected birds were calculated from the NanoString results to be compared with the log2(fold change) of gene expression from the RNA-sequencing results.To verify the gene expression data we used NanoString nCounter® technology previously described by Geiss et al. 2008 . We seleThe total number of reads per sample provided from RNA-sequencing can be found in the Additional file Expression data for each gene within each sample was used to create a heatmap for cluster classification. The heatmap was generated using the R package “plots” Fig. . This cl2(fold change) for normalized NanoString count data against our RNA-sequencing data using the Pearson pair-wise correlation coefficient were very high, with a value of 0.861 across all overlapped genes tested in RNA-seq.We verified our RNA-sequencing data using the Nanostring nCounter gene expression assay. Comparison of the logEnsembl ID of differentially-expressed (DE) genes, i.e., genes with a q-value < 0.05 and a fold change higher than 1.3, were submitted to IPA® for functional annotation as well as mapping to canonical pathways and identifying upstream regulators. The differentially expressed genes with positive or negative fold change values are considered to be, respectively, upregulated or downregulated genes in affected birds relative to their expression in unaffected birds. Among the 1637 significant DE genes identified, IPA recognized 1390 of these genes for analysis. The Ingenuity Knowledge Base®, which supports the mapping of genes, relies on ortholog information for only human, mouse, and rat. Some genes went unmapped because there is no known ortholog with either of these species . A summary of the top ten up-regulated and top ten down-regulated genes in the affected birds, along with the fold change for each gene is presented in Table A summary of the genes identified in the IPA® category of “Top Biological Functions” is presented in Table The resultant RNA-seq profile for Wooden Breast indicated by these analyses includes evidence of altered intracellular calcium, hypoxia, oxidative stress, potential fiber-type switching, and cellular repair within affected muscle. Additionally, there is direct correlation between RNA-sequencing data and the histologic lesions observed in the Wooden Breast myopathy.ATP2A2) gene, which is upregulated in birds affected with Wooden Breast, encodes for sarco(endo)-plasmic reticulum Ca2+ -ATPase isoform 2a and 2b (SERCA2a/SERCA2b) [SERCA2a and SERCA2b differ in the location where they are generally expressed with SERCA2a showing high levels of expression in the heart and in slow-twitch skeletal and smooth muscle, whereas SERCA2b is expressed in all tissues [2+[SERCA2 leads to quicker calcium uptake and larger amounts of calcium loading within the ER [SERCA expression could be influenced by extrinsic factors such as disease [ATP2A2 in Wooden Breast birds may be occurring in response to increased amounts of intracellular calcium within muscle cells, potentially leading to up-take and loading calcium within the sarcoplasmic reticulum at faster than normal rates in affected birds.Through analysis of DE genes in this study, there is ample evidence to suggest that intracellular calcium overload with the potential to impact membrane integrity is occurring in the pectoralis major muscle of broilers affected with Wooden Breast. The ATPase, Ca++ transporting, cardiac muscle, slow twitch 2 (SERCA2b) . SERCA2a tissues . SERCA2  tissues  and transsues [2+. SERCA pssues [2+, 20. Incn the ER . Previou disease . The uprPVALB), another upregulated gene in Wooden Breast birds, is essential to calcium buffering and regulating calcium concentrations within the cell [PVALB in birds affected within the current study may be compensatory to avoid a significant rise in intracellular calcium levels. This type of compensatory change has also been hypothesized in other muscle disorders such as Duchenne dystrophy in humans, in which excess calcium negatively impacts the muscle [PVALB expression may act to stop hypercontraction of the muscle by inducing relaxation through calcium buffering and binding.Parvalbumin (the cell . PVALB athe cell . The upre muscle . Therefo2 (PLA2), increase the overall production of ROS, and may also lead to excess mitochondrial calcium [2 disrupts mitochondrial function causing the formation of lysophospholipids [PLA2G4A gene, encoding cytosolic phospholipase A2 (PLA2), and phospholipase B1 (PLB1) were upregulated in birds affected with Wooden Breast. PLA2G4A has previously been implicated in broad spectrum skeletal muscle myopathies in broiler chickens [PLA2G4A and PLB1, and increased intercellular calcium may be causing changes in membrane integrity in Wooden Breast chickens.Abnormal accumulation of intracellular calcium can occur because of damaged cellular membranes of muscle cells or during episodes of metabolic imbalance between calcium and other ions . The res calcium . Damage holipids  that in holipids . In the chickens . OverallPLOD2), transforming growth factor beta 3 (TGFB3), asparagine synthetase (ASNS), matrix metallopeptidase 2 (MMP2), transient receptor potential cation channel, subfamily A, member 1 (TRPA1), muscle-specific carbonic anhydrase III (CA3), and 6-phosphofructo-2-kinase (PFKFB3). Hypoxia-inducible factor 1 (HIF-1) is an important transcription factor to consider when discussing hypoxia. HIF-1 regulates many genes involved in angiogenesis, energy metabolism, vasomotor regulation, and cell proliferation as well as cell survival, and it is critical in maintaining oxygen homeostasis within the cell [HIF-1A) and aryl hydrocarbon receptor nuclear translocator (ARNT), are not differentially expressed between normal birds and those affected by Wooden Breast, our significant gene list suggests the activation of this transcription factor in affected birds. It has been previously reported that in skeletal muscle HIF-1A is highly expressed in both hypoxic and normoxic conditions [HIF-1A is not differentially expressed between affected and unaffected birds in the present study, though there is specific evidence for hypoxic conditions in the Wooden Breast profile.A number of significant differentially expressed genes in the present study have been found to be regulated by HIF-1 (Hypoxia-inducible factor- 1) (HIF-dependent expression), suggesting a hypoxic state in Wooden Breast. These genes include procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 , characterized as a HIF-dependent gene, is also upregulated in birds affected with Wooden Breast. MMP2 is a member of a large family of matrix metalloproteases and is typically associated with the degradation of cellular components such as the basement membrane and type IV collagen [HIF-1A and MMP2 increased. More specifically, they detected increased gene expression of MMP2 in capillary endothelium of muscle cells. Although the likely activation of HIF-1 within the current study may be the primary cause for the upregulation of HIF-dependent MMP2, it must also be considered that MMP2 expression may be increased due to vascular smooth muscle stretch and remodeling secondary to venous damage or inflammation in birds affected with Wooden Breast.Matrix Metallopeptidase-2 , asparagine synthetase (ASNS), and muscle specific carbonic anhydrase III (CA3) were previously reported as regulated by HIF-1. TGFB3, which is upregulated in affected birds in our study, has been previously found to be upregulated in traumatized muscle during instances of chronic inflammation and regenerating muscle tissue, which is evident microscopically in this study (data not shown), and which stimulates a hypoxic microenvironment [ASNS is upregulated in affected chickens. Cui et al. 2007 [ASNS in relation to tumor growth. They reported a significant increase in expression of ASNS in response to inadequate glucose levels. The upregulation of ASNS, possibly due to low glucose levels, also correlates with previous observations, such as the downregulation of PFKFB3, suggesting deficient glucose metabolism. CA3 is also differentially expressed between the two groups of chickens in the present study, and is upregulated in affected birds. CA3 has previously been found to increase within the muscle tissue of human hypoxia-trained athletes [+ ions delivery to the interstitial fluid of the cell in order to combat muscle fatigue [CA3 in affected muscle may be primarily associated with the antioxidant role of its protein product [Other differentially expressed genes within our study such as transforming growth factor, beta 3 , may be a major contributor to the Wooden Breast phenotype. Many DE genes between the two groups of broilers being studied suggest an increase in ROS in birds affected with Wooden Breast. Increases in ROS within the skeletal muscle are typically generated as a response to increased contractile activity or after periods of major muscle disuse . IncreasHSF2), heat shock 27 kDa protein family, member 7 (HSPB7), heat shock 70 kDa protein 4-like protein (HSPA4L), and heat shock 105 kDa/110 kDa protein 1 (HSPH1). Another gene within this family that is also differentially expressed but found to be downregulated in affected chickens is FK506 binding protein (FKBP51). Heat shock proteins are broadly regulated, and are a fairly large gene family, meaning that members respond to different stimuli [CRH), a major factor important to stress response pathways, was identified as a top upregulated gene in affected birds (fold change = 14.31) in the current study. CRH expression has been previously found to aid in the protection of neurons against oxidative stress [Oxidative stress can elicit high amounts of stress within the body and to specific tissues that undergo direct increases in ROS. Because of oxidative stress, the activation of stress response-related pathways and genes are appropriate reactions to prevent further damage. In the current study, the differential expression of many genes within the heat shock family and other stress response-related genes suggests the occurrence of oxidative stress within the breast muscle of affected chickens. The genes within the heat shock family that are upregulated in affected chickens include: heat shock transcription factor 2  myopathy, render skeletal muscle to be more susceptible to oxidative damages by depleting or overwhelming the antioxidant defense system , 62. SucSELO) is found to be downregulated in Wooden Breast affected broilers. SELO was recently characterized by Dudkiewicz et al. 2012 [SELO, along with other selenoprotein-related genes [SELO, another family of antioxidative enzymes glutathione peroxidase 7 and 8  are observed to be upregulated in birds affected with Wooden Breast, potentially because of elevated ROS levels and also to compensate for changes to SELO. In past studies, GPX7 has been shown to provide antioxidant functions against high levels of ROS in order to combat oxidative damages in humans [CA3). Within the current study, CA3 is one of the top differentially expressed genes with a fold change of 28.47. It has been proposed that CA3 has the ability to serve as an anti-oxidizing agent against damaging molecules, has both regulatory and reparative functions, and is a critical player in the antioxidant defense system within skeletal muscle [Nevertheless, selenoprotein-related deficiencies are strongly associated with oxidative stress damage, as selenoproteins are essential for counteracting oxidative stress ; the selal. 2012 , and it al. 2012 . Interesed genes . The down humans . It was n humans . Anotherl muscle .MB). Myoglobin is mainly distributed within Type I or aerobic muscle (slow twitch) fibers and serves to store oxygen. High myoglobin content is typically correlated with “red” meat, which has a high density of Type I fibers [In the current study, one of the most differentially expressed genes in affected samples was myoglobin  in the present study may support the hypothesis of fast-to-slow switch in Wooden Breast myopathy. TNNI1 expression likewise differs by muscle type and, similar to MB, is also limited to expression in only slow skeletal fibers [TNNI subfamily has further been found to play a role in the mediation of skeletal muscle relaxation and contraction [MYOZ2) and myosin binding protein C, slow type (MYBPC1). MYOZ2 expression has been reported in only cardiac and slow-twitch skeletal muscle, and acts to control for calcineurin, which is a positive regulator of slow-type fibers [TNNI1, MYOZ2, and MyBPC1 are upregulated in affected birds, potentially indicating fiber-type switching to type I fibers following myofiber damage in the Wooden Breast myopathy.Fiber type switching from fast-to-slow twitch fibers has also been suggested in turkeys exhibiting PSE muscle conditions . Upregull fibers . The TNNtraction . Other se fibers , 78. Sime fibers . In thisRyR1 gene in pigs has been previously correlated with the occurrence of malignant hyperthermia, a skeletal muscle myopathy, which leads to defects in Ca2+ channel functioning [Jordan et al. 2004  suggest ctioning , PSE-typctioning , and rigctioning . It has ctioning , proteinctioning  and muscctioning , possessctioning . Callipyctioning . Thus, tMany genes involved in cellular repair and transport are differentially expressed within our dataset. It is highly probable that these genes are linked to the Wooden Breast myopathy in an effort to compensate for muscle damage in this degenerative muscle disease.CSRP3) (fold change = 23.17), which is also referred to as muscle LIM  protein within the literature [CSRP3 expression is present only in striated muscle, including skeletal muscle, and it plays a large role in myogenic differentiation [CSRP3 also correlates with myotube growth and formation [CSRP3 knockout mice and found that deficient mice displayed an array of skeletal muscle defects such as shorter sarcomere length and muscle atrophy. They suggested that CSRP3 has a role in the maintenance of skeletal muscle and also supports skeletal muscle regeneration following injury through structural repair and gene regulation [CSRP3 may significantly increase in expression after periods of exercise in humans [CSRP3 is mainly expressed in slow skeletal muscle, compared to its low expression in fast skeletal muscle [CSRP3 expression was upregulated during fast-to-slow fiber type switch in rats [CSRP3 could impact meat quality since CSRP3 is involved in both myofiber generation and fiber-type switching.One of the top genes upregulated in affected chickens is cysteine and glycine-rich protein 3  also referred to as CCN3 [CCN gene family takes part in critical roles during cell development, wound healing, tissue homeostasis, and varying conditions including cancer and fibrosis [NOV is necessary and required for normal muscle maintenance and use, showing that the over-expression of NOV leads to increased cell proliferation and promotes the survival of cells expressing myogenin. Myogenin is an essential regulator of myogenesis, and crucial to muscle maintenance, regeneration, and repair [NOV expression has also been previously linked with multiple components of cellular repair, as well as wound healing and endothelial adhesion and migration [Along a similar line, another highly upregulated gene in this study is nephroblastoma overexpressed  also contribute to cellular repair and regeneration in skeletal muscle. In the current study MUSTN1, which is considered essential for skeletal muscle growth and regeneration [MUSTN1 has also been reported in domestic chickens; its expression is higher in broiler chickens than it is in layer chickens. This observation supports the notion that MUSTN1 may be involved in the regulation of muscle hypertrophy [MUSTN1 expression was also upregulated in correlation to exercise-induced muscle damage, supporting its possible involvement in muscle damage repair [MUSTN1 in affected birds likely indicates compensatory hypertrophy or muscle repair secondary to muscle damage rather than physiologic hypertrophy during bird growth.Other differentially expressed genes within the current study, such as musculoskeletal embryonic nuclear protein 1 , Rho GTPase activating protein 20 (ARHGAP20), and Rho GTPase activating protein 40 (ARHGAP40). Another significant member of this family that is downregulated is Rho-related BTB domain containing 3 (RHOBTB3). The Rho GTPase family are important regulators of the organization of the actin cytoskeleton [ARHGAP10 is essential to cell differentiation within the cytoskeleton [Other key contributors to the actin cytoskeleton system, such as members of the Rho GTPase family, could also indicate cell differentiation or movement in the reparative phase of the disease. Within our dataset there are four members of this family that are upregulated. These include: Rho GTPase activating protein 10  and fibroblast growth factor (FGF), and also include many cytoskeleton related genes such as, actin related protein complex (ARPC) and many member of the myosin heavy and light chain families (MYH/MYL). As shown in Fig. EGF) and fibroblast growth factor 10 (FGF10) which are upregulated, and fibroblast growth factor 1 (FGF1) which is downregulated. It is more likely that growth factors are expressed differently in affected birds due to repair and regeneration occurring in the diseased muscle. The upregulation of EGF is important since EGF is previously known to promote myoblast proliferation to aid in muscle repair [FGF1 is unexpected, since it is known that FGF1 contributes greatly to skeletal muscle proliferation [FGF1 when studying the response to muscle stretch, or stretch hypertrophy, in chickens. Mitchell et al. 1999 [MYH2, MYH6, MYH10), and four myosin light chain genes . Myosin is essential for muscle contraction and comprises most of the myofibrillar proteins within muscle cells [Results from IPA show that the canonical pathways “Actin Cytoskeleton Signaling”  and “Axonal Guidance Signaling”  have probable functions in tissue repair during Wooden Breast muscle disease. The “Actin Cytoskeleton Signaling” pathway contains 36 significant differentially expressed genes from our gene list. Most of the genes within this pathway relate to specific growth factors such as, epidermal growth factor , fibrinogen alpha chain (FGA), fibrinogen beta chain (FGB), and fibrinogen gamma chain (FGG), kininogen 1 (KNG1), plasminogen (PLG), serpin peptidase inhibitor, clade A, member 1 (SERPINC1), and von Willebrand factor (VWF), which are all found to be downregulated , coagulation factor X (F10), coagulation factor II (F2R), and protein S  (PROS1). The reasons for the involvement of the coagulation pathway and its overall downregulation in Wooden Breast remain to be elucidated.The inflammation observed microscopically in birds affected with Wooden Breast suggests that genes involved in the coagulation system pathway would likely be upregulated due to vascular (venous) damage and inflammation. It is highly regarded that inflammation promotes the activation of coagulation . Howeverted Fig. . Other DFeatures of the gene expression profile  brought to light by this study have strong correlation to the histologic lesions reported previously by Sihvo et al. 2014  and obse2+ within the cell leads to hypercontraction either as a result of poor Ca2+ -ATPase performance [A major characteristic of increased intracellular calcium levels is myofiber hypercontraction. Hypercontraction associated with skeletal muscle degeneration has been previously identified across species , 113. Itformance  or becauformance . Ultimatformance . Histoloformance , 117 andformance . The “hyformance  would alBirds affected with Wooden Breast also show characteristics of histologic changes and clinical symptoms associated with hypoxia , 8, poteCOL3A1, COL4A3, COL4A4, COL4A5, COL5A1, COL5A2, COL6A1, COL6A2, COL6A3, COL8A1, COL8A2, COL11A1, COL12A1, COL14A1, COL16A1, COL21A1, COL24A1, COL25A1, and COL27A1. Furthermore, there is additional microscopic evidence to corroborate a hypoxic state; beyond fibrosis, the accumulation of lipid within affected muscle is also apparent histologically as variably-sized discrete vacuoles within the sarcoplasm of affected muscles (data not shown). Hypoxia has been previously linked with increases in lipid storage in cultured cells [To study the impact of hypoxic environment in mice, Lopes-Ferreira et al. 2001  investiged cells , cardiaced cells , and alsed cells . In suppPrevious muscle disorders described in chickens, such as deep pectoral myopathy (DPM), have been associated with hypoxia resulting from restricted blood flow to the muscle as a predominant cause of muscle degeneration , 121. ThThe gene expression profile brought to light within this study and previously reported histology of Wooden Breast suggests a pro-inflammatory response is taking place in affected birds, which involves the influx of immune cells. In the human muscular disease, Duchenne muscular dystrophy (DMD), cellular membrane damage caused by high intracellular calcium levels and inflammation leads to immune responses observed by a dramatic rise in inflammatory cell concentration within the affected area , 123. ImHistologically, it is seen that tissue repair is occurring through mechanisms such as inflammation, myofiber regeneration, and fibrosis , as wellUsing RNA-sequencing and pathway analysis tools such as IPA, we were able to define possible primary and secondary factors affecting the breast muscle of affected chickens. We were further able to link RNA-sequencing data to histologic findings in our lab and previously published reports of Wooden Breast myopathy  to show The present work should be considered a first step towards elucidating the underlying genetic fingerprint of the Wooden Breast myopathy in commercial broilers. With the RNA-sequencing profile identified by this work, there is a potential to find specific genetic markers characteristic for this disease for use in diagnosis and detection of subclinical or carrier birds. Future work with RNA-sequencing across the time course of disease development would be beneficial. Since birds in the present study were all collected at market age, performing a time-series study using RNA-sequencing could help determine the specific age of onset of this disease and may help distinguish primary from secondary mechanisms contributing to Wooden Breast and associated gross and histologic lesions. Histochemical staining for specific fiber-type could further be implemented to confirm if a fiber-type switch is occurring in affected birds. Since it is also possible that a single gene or a few candidate genes are principle causal factors in this disorder, conducting a genome-wide association study (GWAS) could also be greatly beneficial.To our knowledge, this is the first study on the Wooden Breast disease in commercial broiler chickens to focus on RNA-sequencing data and gene expression profiling. Although the underlying cause of this disease still remains unknown, this work has brought forward many factors associated with the pathogenesis of this muscle disease. This work further shows the importance of using RNA-sequencing in biologic correlation with gross and microscopic disease lesions to study and compare muscle disorders across species. While more research must be performed to gain insight into the specific origin of Wooden Breast myopathy in chickens, RNA-sequence profiling has the potential to directly contribute to disease diagnosis and research for myopathies across animal species, including humans.Samples used in this study were obtained from Heritage Breeders, LLC. The data supporting the results are included within the article and its additional files. Readers may contact the corresponding author for additional information.


Glutamatergic synapse size remodeling is governed not only by specific activity forms but also by apparently stochastic processes with well-defined statistics. These spontaneous remodeling processes can give rise to skewed and stable synaptic size distributions, underlie scaling of these distributions and drive changes in glutamatergic synapse size “configurations”. Where inhibitory synapses are concerned, however, little is known on spontaneous remodeling dynamics, their statistics, their activity dependence or their long-term consequences. Here we followed individual inhibitory synapses for days, and analyzed their size remodeling dynamics within the statistical framework previously developed for glutamatergic synapses. Similar to glutamatergic synapses, size distributions of inhibitory synapses were skewed and stable; at the same time, however, sizes of individual synapses changed considerably, leading to gradual changes in synaptic size configurations. The suppression of network activity only transiently affected spontaneous remodeling dynamics, did not affect synaptic size configuration change rates and was not followed by the scaling of inhibitory synapse size distributions. Comparisons with glutamatergic synapses within the same dendrites revealed a degree of coupling between nearby inhibitory and excitatory synapse remodeling, but also revealed that inhibitory synapse size configurations changed at considerably slower rates than those of their glutamatergic neighbors. These findings point to quantitative differences in spontaneous remodeling dynamics of inhibitory and excitatory synapses but also reveal deep qualitative similarities in the processes that control their sizes and govern their remodeling dynamics. Synaptic plasticity is widely believed to constitute a fundamental mechanism for altering network function. An (implicit) extension of this belief is an assumption that spontaneous changes in synaptic function should not occur to any significant degree. Where excitatory synapses are concerned, recent studies have questioned the validity of this assumption. Where inhibitory synapses are concerned, however, much less is known. Here we followed the spontaneous remodeling dynamics of inhibitory synapses for days, and analyzed these dynamics within a statistical framework previously developed for glutamatergic synapses. Like their excitatory counterparts, sizes of individual synapses fluctuated considerably. Similarly, these spontaneous fluctuations were governed by a well-defined statistical process which assures that synaptic size distributions remain constant. Contrary to the aforementioned assumption, these spontaneous fluctuations drove changes in synaptic size configurations; interestingly, however, change rates were slower for inhibitory synapses. Unlike excitatory synapses, suppressing network activity barely affected inhibitory synapse remodeling dynamics, synaptic configuration change rates or synaptic size distributions. Our findings thus point to quantitative differences in spontaneous remodeling dynamics of inhibitory and excitatory synapses, but also indicate that the processes that control their sizes and govern their remodeling dynamics are fundamentally similar. Otherwise, physiologically relevant modifications would be gradually lost, or drowned in a sea of spurious changes.Activity-driven changes in synaptic properties are widely believed to constitute a fundamental mechanism for altering network function. This belief also (implicitly) implies that synapses, when synaptic tenacity). Indeed, when molecular contents of individual synapses are followed over many hours and days, these exhibit very considerable fluctuations, even in the absence of specific activity patterns, or, for that matter, any activity at all )ng e.g. –61). We . We AR lng e.g. ,62) and  and AR lng . Ind. IndAR lntiation  .in vivo as well [in-vivo . It was shown that key functional properties of such networks are much more sensitive to spontaneous changes in inhibitory connections as compared to spontaneous remodeling of excitatory ones, and it was concluded that inhibitory plasticity has a far larger potential for changing network functionality than the extensively-studied excitatory plasticity. Our findings that inhibitory synapse configurations are less labile than those of excitatory ones would seem to be in line with this idea, as they would imply that spontaneous changes in network function might be slower that what might be expected from observations of glutamatergic synapse “volatility”. Conversely, this finding might be interpreted to suggest that inhibitory synapses represent an effectively stable foundation that minimizes the impact of excitatory synapse size fluctuations, an interpretation that would be consistent with the relative insensitivity of inhibitory synapse remodeling to dramatic changes in network activity levels . How susee also ).Although configurations of inhibitory synapses were less labile that those of their glutamatergic counterparts, such configurations changed nonetheless. Thus, quantitative differences notwithstanding, the emerging realization that excitatory synapse properties can change spontaneously seems to apply to inhibitory synapses as well. It is of particular note in this respect that major features of excitatory and inhibitory remodeling dynamics—the volatility of individual synapse sizes, the stability of their size distributions, the skewed shape of these distributions and the evolution of synaptic configurations—are all captured remarkably well by a statistical process that is essentially stochastic . It thus6 cells were then plated on thin-glass multielectrode array (MEA) dishes  whose surface had been pre-treated with polyethylenimine (Sigma) to facilitate cell adherence. The preparations were then transferred to a humidified tissue culture incubator and maintained at 37°C in a gas mixture of 5% CO2, 95% air, and grown in medium containing minimal essential medium , 25 mg/l insulin (Sigma), 20 mM glucose (Sigma), 2 mM L-glutamine (Sigma), 5 mg/ml gentamycin sulfate (Sigma) and 10% NuSerum (Becton Dickinson Labware). Half the volume of the culture medium was replaced three times a week with feeding medium similar to the medium described above but devoid of NuSerum, containing a lower L-glutamine concentration (0.5 mM) and 2% B-27 supplement (Invitrogen).Primary cultures of rat cortical neurons were prepared as described previously  using a Primary cultures of rat hippocampal neurons for antibody labeling experiments were prepared as described previously . BrieflyExogenous DNA was introduced into neurons by means of a 3rd generation lentiviral expression system. These vectors code for the fluorescently tagged postsynaptic protein PSD-95:EGFP  and the expression vector. HEK cell transfection was performed using Lipofectamine 2000 (Invitrogen) in 10cm plates when the cells had reached 80% confluence. Supernatant was collected after 48 hours, filtered through 0.45μm filters, aliquoted, and stored at -80°C.in-vitro (DIV) by adding predetermined amounts of the filtered supernatant to each MEA dish.Transduction of cortical cultures was performed at 5 days A MEA system was used to continuously monitor the electrical activity of the network through 59, 30 μm diameter recording electrodes, arranged in an 8x8 array, spaced 200 μm apart. The dishes contain 59 rather than 64 electrodes because the corner electrodes are missing, and one of the remaining leads is connected to a large substrate embedded electrode designed to be used as a reference (ground) electrode. The flat, round electrodes are made of titanium nitride, whereas the tracks and contact pads are made of transparent Indium Tin Oxide.Network activity was recorded through a commercial 60-channel headstage/amplifier  with a gain of 1024x. The amplified signal was multiplexed into 16 channels, amplified by a factor of 10 by a 16 channel amplifier  and then digitized by an A/D board  at 12 KSamples/sec per channel. Data acquisition was performed using AlphaMap . All data was stored as threshold crossing events with the threshold set to -40μV. Electrophysiological data were imported to Matlab  and analyzed using custom written scripts.All imaging was performed on neurons grown on thin glass MEA dishes as described above. These particular MEA dishes are fabricated of very thin glass (180 μm), which allows for the use of high numerical aperture, oil immersion objectives and are thus ideally suited for high-resolution imaging . ScanninmTurq2:Gephryin and PSD-95:EGFP were excited using 457 nm (Cobolt) and 488 nm (Coherent) solid state lasers, respectively. Fluorescence emissions were read through 467–493 nm and 500–550 nm bandpass filter . Time-lapse recordings were usually performed by averaging six frames collected at each of 8 focal planes spaced 0.8 μm apart. All data were collected at a resolution of 640 x 480 pixels, at 12 bits/pixel, with the confocal aperture fully open. Data was collected sequentially from up to 12 predefined sites, using the confocal microscope robotic XYZ stage to cycle automatically through these sites at 1 hour (or 24 hour) time intervals. Focal drift during the experiment was corrected automatically by using the microscopes' "autofocus" feature.2 sterile mixture was continuously streamed into the dish at very low rates through a third port with flow rates regulated by a high precision flow meter . The base of the headstage/amplifier and the objective were heated to 37°C and 36°C respectively using resistive elements, separate temperature sensors and controllers, resulting in temperatures of 36–37°C in the culture media.MEA dishes were covered with a custom designed cap containing inlet and outlet ports for perfusion media and air mixtures, a reference ground electrode and a removable transparent glass window. The MEA dish was continuously perfused with feeding media (described above) at a rate of 2.5 ml/day by means of a custom built perfusion system based on an ultra-slow flow peristaltic pump  and silicone tubing. The tubes were connected to the dish through the appropriate ports in the custom designed cap. A 95% air / 5% COAll imaging data analysis was performed using custom written software (“OpenView”). Special features of this software allow for automated / manual tracking of individual synaptic puncta and measurements of fluorescent intensities of these over time (described in detail in ). 9 × 9 For measuring distributions of puncta intensities, areas were placed programmatically on fluorescent puncta at each time step using identical parameters but no tracking of individual puncta was performed (see Because mTurq2:Geph (and PSD-95:EGFP) expression levels varied slightly from one neuron to another, mTurq2:Geph (and PSD-95:EGFP) puncta fluorescence data from each neuron were first normalized to mean mTurq2:Geph (or PSD-95:EGFP) puncta fluorescence of that neuron, allowing us to pool data from different neurons and experiments, correcting for differences in expression levels and potential variations in optical parameters between experiments. Consequently, all fluorescence values throughout the study are expressed as fractions of mean neuronal mTurq2:Geph (or PSD-95:EGFP) fluorescence at initial time points.Tetrodotoxin  was diluted in 100 μl of medium drawn from the culture dish while on the microscope. The mixture was subsequently returned to the dish and mixed gently. Applications to the dish were complemented by simultaneous addition to the perfusion media. Final concentrations in the dish and perfusion media were 1 μM. In one experiment, bicuculline (Sigma) was added as well.AR γ2 or β2,3 subunits  were prepared in phosphate buffered saline (PBS) at the predetermined concentration. The primary antibodies were labeled with fluorescent Fab’ fragments  according to the manufactures instructions . At 14–18 DIV, primary hippocampal cultures were incubated for 1 hour at 37°C with the primary/Fab’ mixtures  or a primary antibody against Vesicular GABA transporter (VGAT) lumenal domain fluorescence-labeled with Oyster 488  and imaged immediately.Primary antibodies against extracellular epitopes of the GABA Systems ). After S1 FigA) A two minute raster plot of action potentials recorded from the MEA 59 electrodes. Each dot represents a single action potential. Recording is from the same experiment shown in B) An enlarged, 2-second portion of the trace shown in A.(TIF)Click here for additional data file.S2 FigA) Changes in the number of mTurq2:Geph puncta in 6 neurons in the same experiment as in B) Mean fluorescence intensities of mTurq2:Geph puncta in the same 6 neurons as in A) (~266 synapses). Fluorescence values were normalized to mean fluorescence at t = 0. C) The slight decline in mTurq2:Geph puncta fluorescence is not due to photobleaching. Changes in mTurq2:Geph puncta fluorescence intensities pooled from 16 neurons in 2 experiments in which images were obtained at 24 hour instead of one hours intervals. All values in represent means ± standard deviations.(TIF)Click here for additional data file.S3 FigActivity was normalized to baseline activity levels at t = -24 hours. Average of 4 experiments.(TIF)Click here for additional data file.S4 FigA) Changes in the number of mTurq2:Geph puncta in a single experiment in which bicuculline (6 μM) was added together with TTX. Counts for each neuron were normalized to initial puncta counts at t = 0. ~210 synapses from 8 neurons. B) Mean fluorescence intensities of mTurq2:Geph puncta for the same 8 neurons as in A). Note the similarity with (TIF)Click here for additional data file.S5 FigA) Before exposure to TTX. B-D) After exposure to TTX. Solid lines are linear regression fits. Unity lines are shown as dashed red lines. Note the transient change in regression line trends during the 12 hour window following exposure to TTX (B), and the recovery of these trends in subsequent time windows . Same data as in (TIF)Click here for additional data file.S1 DatasetThe figure to which data pertains to is indicated in the tab of each worksheet. Data are provided as fractions of mean fluorescence (i.e. as a fraction of the mean fluorescence measured for the particular neuron each synapse belongs to). All times are in hours. For the data of Figs (XLSX)Click here for additional data file.


Sepsis is a global burden and the primary cause of death in intensive care units worldwide. The pathophysiological changes induced by the host’s systemic inflammatory response to infection are not yet fully understood. During sepsis, the immune system is confronted with a variety of factors, which are integrated within the individual cells and result in changes of their basal state of responsiveness. Epigenetic mechanisms like histone modifications are known to participate in the control of immune reactions, but so far the situation during sepsis is unknown.In a pilot approach, we performed combined chromatin immunoprecipitation followed by high-throughput sequencing to assess the genome-wide distribution of the chromatin modifications histone 3 lysine 4 and 27 trimethylation and lysine 9 acetylation in monocytes isolated from healthy donors (n = 4) and patients with sepsis (n = 2). Despite different underlying causes for sepsis, a comparison over promoter regions shows a high correlation between the patients for all chromatin marks. These findings hold true also when comparing patients to healthy controls. Despite the global similarity, differential analysis reveals a set of distinct promoters with significant enrichment or depletion of histone marks. Further analysis of overrepresented GO terms show an enrichment of genes involved in immune function. To the most prominent ones belong different members of the HLA family located within the MHC cluster together with the gene coding for the major regulator of this locus—CIITA.We are able to show for the first time that sepsis in humans induces selective and precise changes of chromatin modifications in distinct promoter regions of immunologically relevant genes, shedding light on basal regulatory mechanisms that might be contributing to the functional changes occurring in monocytes. Sepsis is a global burden and the primary cause of death on ICUs all over the world –3. DurinHistones can be posttranslationally modified by the enzyme-catalyzed addition of chemical groups to their N-terminal tails, e.g. acetylation, phosphorylation or methylation. The specific presence or absence of these histone modifications in promoter regions is functionally correlated with the expression of the associated genes in defined genomic regions . TrimethBy influencing gene expression, histone modifications seem to be indirectly associated with the regulation of different kinds of cell functions. Therefore, also the regulation of histone modifying enzymes like histone deacetylases (HDACs) seems to play a key role in inflammatory gene expression. It has been demonstrated that HDAC3-deficient murine macrophages lack the ability to express inflammatory genes after LPS (lipopolysaccharide) stimulation, which is attributable to a secondary effect by the loss of LPS-induced IFN-β (interferone-β) expression . MoreoveBesides manipulation of acetylation through histone deacetylases, also changes in methylation have been found to be important during immune reactions. The histone demethylase Jmjd3 (jumonji domain containing 3) has been identified to regulate immune response in murine macrophages after induction by the transcription factor NF-kB . It remoThis is in line with the finding, that continuous interleukin-4 stimulation induces an increase in Jmjd3 expression and decreases H3K27 methylation at the promoter regions of alternative macrophage marker genes followed by an increased transcriptional activation of these genes .Animal experiments using the cecal ligation and puncture (CLP) model, mimicking the clinical condition of polymicrobial peritonitis, showed both a decreased H4ac and H3K4me3 with reduced recruitment of RNA polymerase II and associated with decreased cytokine gene expression in lung macrophages. These findings correlated with the induction of Interleukin-1 receptor-associated kinase-M, a negatively regulating protein of the TLR signaling pathways. Interestingly, only few genes showed a transiently reduced expression in response to LPS during sepsis .Despite all evidence from animal experiments, the global pathophysiological mechanisms are still unknown and the mounting evidence that genomic responses in mouse models only poorly mimic the situation during human inflammatory diseases raises the ultimate need for primary cell analysis from patients suffering from sepsis .++ CD16−) seems to play an important part in the initial pro-inflammatory defence mechanism [Monocytes are key players in the innate immune system and their cellular functions are of high importance during early immune reactions. During sepsis, the classical monocyte subpopulation CD14++ CD16− echanism . Studiesechanism .Despite all efforts in immune and sepsis research the exact mechanisms leading to the pathological monocyte dysfunction are still unknown. An analysis of epigenetic modifications of human immune cells may help to understand the genomic alterations occurring and therefore unravel novel pathophysiological mechanisms in patients suffering from sepsis.In order to set the stage for epigenomic studies in the future, we conducted genome-wide histone modification analysis of human CD14++ CD16- monocytes from two patients suffering from sepsis. Our study demonstrates for the first time that sepsis in humans does not result in an uncontrolled remodeling of the chromatin landscape, but in selective and precise changes of chromatin modifications in distinct promoter regions of immunologically relevant genes.To assess the genome-wide distribution of the chromatin modifications H3K4me3, H3K27me3 and H3K9ac in CD14++ CD16- monocytes from patients suffering from sepsis  of their promoter regions for at least one of the examined posttranslational modifications . EmployiMonocytes belong to the group of antigen-presenting cells. Antigen-presentation is a crucial process during early immune responses required for full activation of the host response. Upon uptake, microorganisms are digested within the phagolysosomes and peptides are embedded into MHC class II complexes and presented on the surface of the cell for recognition by CD4+ T helper cells. During late sepsis a diminished expression of monocytic HLA-DR, which is a member of the MHC class II molecules, is a well known characteristic and closely correlates to the global loss of monocyte function, ultimately leading into an immuneparalytic state of the host . The genOverall, our results indicate changes in chromatin modifications, which seem to appear in a very specialized manner. As an example of high interest in sepsis, we found alterations in the MHC class II locus and its corresponding master regulator CIITA. In line with results of earlier studies in monocytes from septic patients, which show a decreased expression of MHC II genes as well as the gene encoding CIITA , our finAnother finding with potential implications in the late pathophysiology of the disease is the alterations of the promoter regions of IL-1β as well as the IL1 Receptor 2 (IL1R2). While the promoter of IL-1β is depleted of active marks , both marks are accumulating at the IL1R2 promoter . RemarkaSepsis might lead to changes in the expression of certain important transcriptional regulatory factors, resulting in changes in the modification pattern of downstream promoters. Interfering with these factors might be a promising therapeutic approach, for which already experimental evidence exists. By applying the synthetic histone I-BET, which mimics acetylated histones, pro-inflammatory gene expression is abolished during systemic inflammation . In addiOur first results might be promising, but it is a long way to step out of the dark of applying treatment in a blackbox scenario towards the light of guided, surgical precise modulation of the epigenome. Before this might happen, we need to conduct further studies with larger sample sizes, and eventually including more histone modifications and especially more cell types—both hematopoetic and somatic. Moreover, as sepsis is a dynamic syndrome, we need to understand the spatiotemporal kinetic of epigenomic patterns and their functional consequences as well as differences depending on the underlying cause or focus of infection. Nevertheless, we believe that this bottom-up approach “from promoter to target” will raise new therapeutic options for the treatment of sepsis and other inflammatory diseases.All together this study provides the first comprehensive ressource of the epigenomic changes occurring in CD14 ++ CD16- monocytes of patients suffering from sepsis. The genome-wide maps of three histone modifications indicate that a defined set of immunologically relevant genes undergo specific alterations of their associated histone modification patterns in human sepsis.Ethics Statement: All experiments were conducted according to the principles expressed in the Declaration of Helsinki, with approval of the ethics committee of the medical faculty of the Justus-Liebig-University of Giessen, Klinikstrasse 32, D-35385 Giessen, Germany . Subjects provided written, informed consent on forms approved by the Institutional Review Board.Cell separation: For monocyte separation 30 ml blood sample was taken and immediately processed from 2 patients suffering from sepsis and 4 healthy donors. Ficoll-based density gradient centrifugation was used to isolate peripheral blood mononuclear cells. To isolate CD14++ CD16- monocytes, magnetic cell sorting  was conducted using CD16 MicroBeads (Miltenyi Biotec) to deplete CD16++ monocytes and CD14 MicroBeads (Miltenyi Biotec) to finally isolate CD14++ CD16- monocytes.Purity of CD14++ CD16- monocytes was measured using flow cytometry after incubation with FITC anti-human CD14 Antibody (BioLegend).6 cells per 200 μl lysis buffer) sonication was performed using Branson sonifier (fragment size 150–600 base pairs) under constant cooling. Preparation of immunoprecipiation was conducted after seperation of 10% input using agarose A/G-beads . Chromatin was mixed with antibodies for H3K4me3, H3K27me3 and H3K9ac at 4°C in a rotator for 3 hours. Different concentrations of salt wash buffer were to clear chromatin bead complex, before reverse Cross-linking (RNase A followed by 10% SDS/Proteinase K mix). Illustra GFX PCR DNA and Gel Band Purification kit  was used to purify DNA. DNA was eluted in pure H2O and quantitation of ChIP-DNA determined by Qubit Assay .ChIP protocol: ChIP was performed as described before . CD14++ Library amplification: We used MicroPlex Library Preparation kit (Diagenode) for library amplification with the presence of SYBR Green in a real-time PCR machine as previously described . AMPure Sequencing: Illumina HiSeq was used for sequencing of 4 barcoded libraries  for each patient and healthy donor on a single lane. Cluster generation was performed using the Illumina cluster station, sequencing on the Illumina HiSeq 2500 followed a standard protocol. The fluorescent images were processed to sequences using the Genome Analyzer Pipeline Analysis software 1.8 (Illumina). All sequencing data was deposited at NCBI’s gene expression omnibus (GEO) under the accession number GSE62908.http://www.bioconductor.org).Processing of ChIP-seq reads: ChIP-Seq reads were converted to fastq format and aligned to a precompiled hg19 reference index with BOWTIE . Sequenchclust function using the "complete" method.Differential promoter binding analysis: Read counts were determined for the promoters of all annotated hg19 RefSeq transcripts. The RefSeq transcriptional start sites (TSSs) were used to identify promoter intervals ranging from −1 to +1 kb around the TSS. For each promoter the number of reads mapping to the respective interval were extracted for patient and control samples. DESeq  was usedVisualization of binding profiles: After extension of reads, continuous coverage vectors were calculated and normalized per million reads to account for differential library sizes. Additonally the coverage vectors were smoothed in a 1000 bp window, quantile-normalized and subsequently converted into bigwig format for visualization in genome browsers. The coverage vectors were used to collect data in windows of different sizes spanning transcriptional start sites using the bigWigSummary tool  applicatIdentification of enriched GO terms: Clusters identified by hierarchical clustering as described above were used to identifiy over-reprasented GO-terms using DAVID .Statistics were calculated by the Mann-Whitney signed Rank test or paired t-test using R.S1 FigBoxplots shows z-scaled read counts of MHC genes for individual patients (#P1 and #P2: red boxes) and controls (#C1-#C4: blue boxes) for three modifications.(TIF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 TableSummary statistics describing differential association of histone modifications comparing patients and controls over 743 gene promoters with significant changes for at least one histone modification.(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.


Salvelinus alpinus) is a polymorphic fish species, and, in Lake Thingvallavatn in Iceland, four sympatric morphs have evolved distinct craniofacial structures. We conducted a gene expression study on candidates from a conserved gene coexpression network, focusing on the development of craniofacial elements in embryos of two contrasting Arctic charr morphotypes (benthic and limnetic).Understanding the molecular basis of craniofacial variation can provide insights into key developmental mechanisms of adaptive changes and their role in trophic divergence and speciation. Arctic charr . A literature review and Gene Ontology analysis indicated that a significant proportion of the network genes play a role in extracellular matrix organization and skeletogenesis, and motif enrichment analysis of conserved noncoding regions of network candidates predicted a handful of transcription factors, including Ap1 and Ets2, as potential regulators of the gene network. The expression of Ets2 itself was also found to associate with network gene expression. Genes linked to glucocorticoid signalling were also studied, as both Mmp2 and Sparc are responsive to this pathway. Among those, several transcriptional targets and upstream regulators showed differential expression between the contrasting morphotypes. Interestingly, although selected network genes showed overlapping expression patterns in situ and no morph differences, Timp2 expression patterns differed between morphs.Four Arctic charr morphs were studied: one limnetic and two benthic morphs from Lake Thingvallavatn and a limnetic reference aquaculture morph. The presence of morphological differences at developmental stages before the onset of feeding was verified by morphometric analysis. Following up on our previous findings that Our comparative study of transcriptional dynamics in divergent craniofacial morphologies of Arctic charr revealed a conserved network of coexpressed genes sharing functional roles in structural morphogenesis. We also implicate transcriptional regulators of the network as targets for future functional studies.The online version of this article (doi:10.1186/2041-9139-5-40) contains supplementary material, which is available to authorized users. Unravelling the developmental and genetic basis of morphological and functional diversity is a fundamental step in understanding the evolutionary processes involved at both intra- and interspecific levels. The extensive diversity of teleost fish has been a fertile model system for studying such variation–5. CloseSalvelinus alpinus) is a highly suitable species for such studies. It shows extensive morphological variation throughout its geographic distribution and sports many cases of distinct polymorphisms which have evolved rapidly and repeatedly[Arctic charr  analysis[Mmp2 and Sparc expression levels has been reported[Adaptations can arise through structural or regulatory changes in individual genes, several loci or coordinated deployment of coregulated genes, 30. In analysis. Severalanalysis–37, and reported–40, suggMmp2 and Sparc might be part of a larger network of genes with correlated expression during craniofacial morphogenesis and test whether such a network would show differential expression in developing heads of contrasting Arctic charr morphotypes. To accomplish this goal, we identified genes with strong expressional correlation to Mmp2 and Sparc in other species and selected those which also showed differential expression in developmental transcriptome profiles in contrasting Arctic charr morphotypes. Here we report that a network of functionally related genes shows coexpression in the developing head of Arctic charr embryos and is differentially expressed between benthic and limnetic morphotypes. The network genes share conserved binding motifs for a set of transcription factor (TFs), including Ap1 and Ets2. Interestingly, Ets2 itself is differentially expressed between the benthic and limnetic Arctic charr morphs during craniofacial development and shows strong expressional correlation with the network as well as spatiotemporal overlap in expression pattern.In view of this information, we decided to further investigate the expression dynamics and potential regulators of these genes. We wanted to find out whether s) units, defined as the time it takes for one somite pair to form at a given temperature[Ripe parent fish from three of the Lake Thingvallavatn Arctic charr morphs—PL  morph, SB morph and LB morph—were sampled in 2010 during their respective spawning periods. For each morph, eggs from several females were pooled and fertilized using milt from several males. We also set up pooled crosses from a limnetic aquaculture stock (AC) from the Hólar College breeding programme. Eggs were reared at approximately 4°C to 5°C in hatching trays  under constant water flow and in complete darkness at Hólar College experimental facilities in Verið, Sauðárkrókur, Iceland. The water temperature was recorded twice daily, and the average was used to estimate the relative age of the embryos using tau-somite (τperature.s). Samples were fixed in 4% paraformaldehyde. A total of 53 individuals  were stained for cartilage (Alcian blue) and bone (Alizarin red) using a modified protocol for zebrafish[For morphometric comparisons of PL, LB, SB and AC morphs, we selected newly hatched embryos  version 5.0 using orthologs of Mmp2 and Sparc[Mmp2 and Sparc were retrieved for the three vertebrates with the largest available data sets  using the mutual rank . Genes with reliability scores less than three were discarded[et al., unpublished data) that also show craniofacial expression in zebrafish (according to the Zebrafish Model Organism Database (ZFIN) up to August 2013)[To gauge a potential network of coexpressed craniofacial genes, we searched COXPRESdb .The coexpression relationships between genes from the resulting list, along with selected glucocorticoid (GC) effectors (see reasoning below), were visualized across divergent vertebrate species using FunCoup version 2.0. FunCoupMmp2 and Sparc in humans and mice  using the Database for Annotation, Visualization and Integrated Discovery (DAVID) v6.7[Mmp2 and Sparc in humans and mice, as well as the WEB-based GEne SeT AnaLysis Toolkit  v2[To characterize the coexpression module, we performed Gene Ontology (GO) overrepresentation analysis on top-ranked genes coexpressed with both ID) v6.7. The GO talt) v2.s), and, for simplicity, these time points will be referred to as stages 1, 2, 3, 4, 5, 6 and 7, respectively. Extraembryonic membranes were punctured, and the embryos were stored in RNAlater solution  at -20°C. For RNA extraction, embryos were dechorionated and decapitated in front of the pectoral fin under a light microscope . Two separate extractions were made for each morph and time point. For each replicate, six heads were placed in TRI Reagent  and homogenized with a disposable Kontes Pellet Pestle cordless motor tissue grinder . RNA was prepared according to the manufacturer’s instructions and dissolved in 50 μl of RNase-free water. To remove DNA contamination, the RNA was treated with DNase I . The quantity of the resulting RNA was measured using a NanoDrop ND-1000 UV/VIS spectrophotometer . The quality of the RNA was evaluated by agarose gel electrophoresis by determining the integrity of the 18S and 28S RNA bands. cDNA was prepared from 1 μg of RNA in a total reaction volume of 20 μl using the High Capacity cDNA Reverse Transcription kit  according to the manufacturer’s protocol. The absence of genomic DNA was confirmed by preparing several samples without addition of reverse transcriptase. cDNA was diluted threefold in nuclease-free water for further use in qPCR experiments.We studied embryos of four morphs, collected at seven time points spanning early craniofacial cartilage formation to a prehatching time point . We used the high conservation of exon–intron boundaries between orthologous genes and aligned charr contigs to the genomic sequences of zebrafish and salmon orthologs from the Ensembl database (http://useast.ensembl.org/Danio_rerio/Info/Index) and salmonids species database  using the National Center for Biotechnology Information Spidey software (http://www.ncbi.nlm.nih.gov/spidey). Primers were designed using Primer Express 3.0 software (Applied Biosystems) and checked for self-annealing, heterodimers and hairpin structures by using OligoAnalyzer 3.1  [t (target) as follows: ΔCqtarget = Cqtarget – Cqreference. The geometric mean of Cq values of two validated craniofacial reference genes, If5a1 and Actb, was used for ΔCq calculations[target – ΔCqcalibrator). A biological replicate from AC at 155 (τs) was chosen as the calibrator sample to calculate the differential mRNA expression of each target gene. Relative expression quantities (RQ) were calculated based on the expression level of the calibrator sample (E-ΔΔCq)[post hoc tests were used to contrast benthic and limnetic morphotypes. To assess expression similarity of the RT-qPCR-amplified genes, Pearson correlation coefficients (r) were calculated for all gene pairs using the data from all morphs and time points. R (http://www.r-project.org) was used for all statistical analyses[RT-qPCR was performed in 96-well PCR plates on an 7500 Real-Time PCR System (Applied Biosystems) using 2× Fermentas Maxima SYBR Green qPCR Master Mix  with a 10-μl final reaction volume. Each biological replicate was run in duplicate together with a no-template control in each run for each gene. RT-qPCR was performed as described previously. FluoresgPCR.nl) to analyulations. All sam(E-ΔΔCq). The RQ analyses.http://genome.ucsc.edu)[http://genie.dartmouth.edu/scope)[Danio rerio, Tetraodon nigroviridis and Oryzias latipes—with annotated genome and available background sequences in SCOPE. We retained motifs that were present in noncoding regions near at least 12 (70%) of the 17 genes. We screened for potential TF binding sites using STAMP[Gasterosteus aculeatus, which has high-quality genome sequences available for the conserved noncoding sequences of the genes under study.Tests for the enrichment of motifs, aimed at uncovering potential transcriptional regulators, were conducted for 17 genes in the coexpression module showing expression differences between limnetic and benthic charr. The conserved noncoding regions of each gene (including sequences in promoter and 5′ untranslated regions conserved across fish species) were retrieved using the UCSC Genome Browser (csc.edu). The conu/scope) and BioPu/scope). SCOPE ung STAMP, with thng STAMP. Similarng STAMP by settiin situ hybridization was performed following a standard procedure adapted for Atlantic salmon[Ctsk, Ets2, Mmp2, Ogn, Sfrp1, Sparc and Timp2) generated PCR products of around 400 to 700 bp  for 20 to 60 minutes, depending on the developmental stage. The hybridization was performed with 1 μg/ml DIG-labelled RNA probes at 70°C for 12 hours. The hybridized embryos were incubated with alkaline phosphatase-conjugated anti-DIG antibody (Roche Diagnostics) at 4°C overnight, and the hybridization signals were visualized using nitro blue tetrazolium chloride/5-bromo-4-chloro-3-indolyl-phosphate, toluidine salt . The specificity of antisense probes was also verified by running control experiments with sense probes. Samples were imaged on a Leica MZ10 F binocular microscope (Leica Microsystems).Whole-mount c salmon. EmbryosP <0.001 by ANOVA) morph effect and W2 (16.59%) also differed between morphs (P <0.05), whereas W3 (10.27%) did not. Pairwise comparisons using Mahalanobis distances showed significant differences between morphs . Of 347 genes coexpressed with both genes in at least one of these species, 150 were expressed during Arctic charr embryonic development, and 31 of those genes were among the genes suggested to be differentially expressed between benthic and limnetic morphotypes, based on preliminary transcriptome profiles from the SB and AC morphs . Twenty-two of these genes were selected for further analysis, based on craniofacial expression pattern in zebrafish . FigureMmp2 and Sparc are known to be directly responsive to GC[Mmp2 and Sparc, which also showed benthic–limnetic differential expression, are known to be responsive to GC  and a few known modulators of GC activity, that is, the TFs P300, Cebpa, Ets1 and Ets2, and Star, an upstream component of steroidogenesis[Srebp1, which regulates the expression of Star[Potential regulators of the developmental differences between Arctic charr morphotypes are of particular interest. Three observations led us to include GC signalling in the analysis. First, both ve to GC, 62. Secnd Pmp22–68). Thind Pmp22, 69, 70.database, 71–77. ogenesis–84. In a of Star and showAngptl4, Anxa1, Mmp9 and Sfrp1) . Four of the GC target genes showed conserved coexpression with the network . qPCR was performed on samples extracted from dissected heads of SB, LB, PL and AC embryos, and the relative expression of the genes was normalized to the expression of two stable craniofacial reference genes, Actb and If5a1[post hoc tests) . The expression over time also varied by morph for all genes . A closer look at the clusters revealed that gene expression in the PL and benthic morphs was more similar at the first two time points (stages 1 and 2), whereas AC showed expression similar to benthic morphs at the last time point (stage 7)  in the conserved coexpression network   showed consistently higher expression in developing heads of the benthic morphs  differed slightly but significantly between the limnetic and benthic morphs, but the receptor did not cluster with genes from the coexpression network. As shown in Figure Nr3c1 did not overlap completely with differences in network gene expression between morphs, but two effectors of the pathway—the activator Hsd11b1l, and Ets2, which can function as a coactivator for Nr3c1 transcriptional activity[Two of the eight selected effectors of the GC pathway , had strong positive correlation with almost all of the genes in the network. Interestingly, a few genes showed significant negative correlation with the network genes (whereby the increased expression of one gene is associated with decreased expression of another), most notably P300 and Sgk1. Lum, which was originally predicted to show coexpression with Mmp2 and Sparc, does not appear to form a part of this coexpression module in developing Arctic charr heads. To conclude, the analysis confirms the existence of a coexpression network conserved between vertebrates  over all morphs and time points Figure . A stron8 genes oD. rerio, T. nigroviridis and O. latipes) were retrieved, and SCOPE was used to screen for enriched motifs. From a list of 40 to 60 significantly enriched motifs in each fish species  were initially selected for whole-mount in situ hybridization studies to investigate their expression patterns, using AC as a reference morph. The expression pattern of the genes was profiled at the earliest, intermediate and latest time points under study   and Ets2, a potential upstream regulator, showed similar overlapping craniofacial expression in anterior and ventral facial elements and pharyngeal arches in all four charr morphs. Only Timp2 showed spatiotemporal differences between the morphs.No major spatial expression pattern differences were observed between the morphs (data not shown), indicating that the differential expression observed in qPCR experiments was caused mainly by differences in expression levels within tissues and not due to spatiotemporal differences in expression patterns. However, when the expression of Figures B and5C.Mmp2 and Sparc, are expressed at higher levels in benthic than in limnetic morphs[Mmp2 and Sparc in different vertebrate species. Coexpressed genes showing craniofacial expression in zebrafish and differential expression in transcriptional profiles of two contrasting Arctic charr morphs  were then selected for further study. This allowed the identification of a conserved network of genes which is differentially expressed between benthic and limnetic Arctic charr morphs, as well as the identification of potential upstream transcriptional regulators.In this study, we have taken steps towards uncovering molecular mechanisms associated with the rapid and extensive phenotypic divergence of Arctic charr in Icelandic lakes. Following up on our previous finding that two ECM-associated factors related to bone development, c morphs, we askeMmp2 and Sparc is differentially regulated in the morphotypes. Distinct coexpression modules have been found to correlate with specific developmental stages or structures, such as in the zebrafish blastula and segmentation[The fact that all the genes from the initial list of coexpression candidates that we examined in this study showed correlated expression dynamics and significantly higher expression in benthic than limnetic morphs supports the idea that a coexpression network including entation. Althougentation, 88. Forentation.Lum is one of these genes  were selected for further study. We found that five of the eight additional GC transcriptional targets were differentially expressed between benthic and limnetic morphs and showed strong expressional correlation with the network genes [Hsd11b1 and Hsd11b2 modulate GC metabolism within the cell at the prereceptor level by catalysing the interconversion of hormonally active cortisol and inactive cortisone[11β-hsd3 (Hsd11b1l) is present instead of Hsd11b1[Hsd11b1l expression correlates well with the network genes and that Nr3c1 is expressed at higher levels in benthic morphs at certain timepoints. It is therefore possible that higher levels of GCs are present in the benthic morphs as well as a more robust response (higher GR levels).During cranial skeletal development of the mouse, GC signalling has been suggested to regulate Wnt signalling in early osteoblastogenesis. Wnt sigSfrp1, 94. In Sfrp1. Moreoveormation–97. GCs 3c1 (GR). In addiortisone. In fish Hsd11b1. Our finAp1 and Nfkb[Together, the data imply a possible interplay of GC signalling with the coexpression network which would be either direct, by driving network gene expression, or indirect, as a consequence of affecting effectors of GC activity. Analysis of conserved noncoding regions of the genes under study did not show significant enrichment for motifs matching GC-responsive elements. However, GC transcriptional activation can be mediated through interaction of the monomeric GC receptor with other DNA-bound TFs, such as and Nfkb. In addiet al. and KHK unpublished data). At the molecular level, such divergences in the orofacial compartment can be directly associated with the network we have identified. During the morphogenetic process, the shape and size of craniofacial elements is rapidly changing, and these shape changes are likely to involve substantial matrix remodelling. Our results show that genes known to affect craniofacial morphogenesis that also play important roles in ECM deposition and remodelling display differential expression in the developing head of benthic and limnetic morphotypes. Mmp2, Mmp9, Timp2, Ctsk and Sparc all play roles in ECM remodelling[Col1a1 and the proteoglycans Lum, Ogn and Dcn are components of the ECM[Col1a1, Ctsk and Mmp2, which play roles in intramembranous ossification[Common garden experiments focused on posthatching development have shown how head shape diverges among the Lake Thingvallavatn morphs. The contrasts are especially clear between the benthic and limnetic morphotypes and are reflected in functional elements, such as the relative length of the lower jaw. This maodelling, 100–103 the ECM, 105. Fufication.Because of this tight association of the network with osteogenic and ECM sculpting processes, we postulate that the network could play an important role in the development of subtle differences in the trophic apparatus between morphotypes via spatial and/or temporal differences in ECM deposition and ossification. This is further supported by the observed overlap in expression patterns of selected genes from the network in the perichondrial region of the forming lower jaw Figure , where oTimp2 differed between the AC morph and the Lake Thingvallavatn morphs and showed visible differences in expression levels between the limnetic (PL) and benthic (SB and LB) morphs. Timp2 has a highly conserved expressional correlation with the other network genes , ETS family, Nfkb and Smad3/4 particularly interesting because of their characterized genetic interaction with genes of the network in other vertebrate species  TFs are heterodimers of Jun, Fos or Atf that bind to a common DNA site[Ap1 factors can regulate different target genes in distinct biological processes, including normal development, depending on the relative abundance of Ap1 subunits[Tgfβ) that play a crucial role in regulating the specification of cranial neural crest cells[Ap1, ETS family, Nfkb and SMADs have been reported by analysis of several responsive gene promoters in other vertebrates  had already been selected for the present study as examples of less specific upstream effectors of GC transcriptional activity and because Ets2 overexpression in mice has been reported to cause craniofacial abnormalities[Ets2 fits completely to the network described here is therefore of great interest. Ets2 expression is higher in developing heads of benthic morphs and shows both positive correlation and spatiotemporal overlap with other network genes, which suggests a functional relationship. However, more detailed analysis is needed to evaluate whether Ets2 (or some of the other predicted TFs) holds a potential master regulatory status in the coexpression network and morph-related differences and whether genetic separation in those or other regulators/binding sites have played a role in morph formation.Having identified a network of differentially expressed genes with a potential role in crucial events which may underlie some of the differences between Arctic charr morphotypes, we were interested in discovering how this network might be differentially regulated between the morphs. A search for conserved motifs in three distant teleost fish species revealed potential binding sites for nine known TFs in the majority of the coexpressed genes Table . Among tDNA site. Differesubunits. In addist cells. Interesmalities. The facGene expression studies can provide novel and valuable insights into the molecular circuitry involved in the fine-tuning of variable morphology. Nevertheless, they can only imply, not directly identify, the triggers of the molecular cascades underlying variation and divergence. In general, causative genetic or environmental differences must underlie observed differences such as those seen in the tropic apparatus of Arctic charr morphs. The identification of causal genetic changes is not trivial. Such chet al.[The upstream regulators of the gene network shown here to be differentially expressed in charr morphotypes are obviously of focal interest, the causalities of later developmental events are also of importance when studying morph-related differences. Using a common garden setup, Parsons et al., 113 stuEts2, a differentially expressed member of the ETS TF family shows a strong expression correlation and overlapping expression pattern in craniofacial structures with network genes. Identification of genetic differences, such as in Ets2 or other regulators, influencing this conserved expression network can cast light on the agents used by evolution to shape trophic apparatus diversity in salmonids and possibly other fishes.Our present study addresses the molecular mechanisms underlying the craniofacial divergence in the highly polymorphic Arctic charr. We have identified a network of coexpressed genes which are differentially expressed in benthic and limnetic morphotypes during developmental stages, spanning the emergence of craniofacial skeletal elements to hatching. Members of the gene network share biological functions mainly involved in ECM organization and skeletal formation. Moreover, coexpression connection between members of the network is conserved across distant vertebrate species, which suggests a crucial role of the genes in different developmental and physiological processes. The results of this study also predict a set of potential upstream TFs regulating the network. In particular, Arctic charr–specific primers for cDNA cloning and qPCR.(XLSX 13 KB)Additional file 1:Relative expression of candidate genes in the developing head of four Arctic charr morphs. Relative expression of 12 candidate genes (with strong coexpression relationship in vertebrates) in the developing head of AC, PL, SB and LB at seven developmental stages. Gene expression was measured by qPCR, and expression levels were normalized with respect to the geometric means of two craniofacial reference genes (Actb and If5a1). The relative expression level for each gene is depicted by setting a replicate of the AC morph at stage 1 to an arbitrary unit of 1. Error bars represent standard deviation calculated from two biological replicates. The letters A, L, P and S above the bars indicate significantly higher expression than for AC, LB, PL and SB, respectively Additional file 2:Ranking of genes coexpressed with bothMmp2andSparc,Gene Ontology (GO) and TF enrichment analyses. Genes coexpressed with both Mmp2 and Sparc in humans, mice and zebrafish were retrieved using COXPRESdb. The genes were filtered by setting the mutual rank (MR) to the top-ranked 500 and the reliability score of 3. In the list of Mmp2 and Sparc coexpressed genes, the column titled ‘Transcriptome Presence’ indicates that an orthologous gene was present in Arctic charr transcriptome profiles. ‘Differential Expression’ indicates differences in expression levels between the AC and SB morphs in the same data. ‘Craniofacial Expression’ indicates craniofacial expression in zebrafish. The last two columns were used to select genes for further analysis in Arctic charr. The top-ranked (MR scores) of genes coexpressed with both Mmp2 and Sparc for humans and mice were used as input for GO enrichment analysis using DAVID v6.7. GO terms with direct link to skeletal formation are highlighted in blue. Similar input lists of genes were used for transcription factor enrichment analysis in humans and mice through WebGestalt v2. Enriched TFs that were later predicted to be the potential regulators of the network are highlighted in orange. (XLSX 163 KB)Additional file 3:Analyses of candidate gene expression using two-way ANOVA for effects of morph and time. Results show significant effects of morph, time and morph × time interaction on the expression of genes coexpressed with both Mmp2 and Sparc, as well as selected GC downstream genes and upstream effectors. (XLSX 15 KB)Additional file 4:Analyses of candidate gene expression using two-way ANOVA for effects of morphotypes and time. Results show significant effects of benthic and limnetic morphotypes, time and, to lesser extent, morphotype × time interaction on the expression of genes coexpressed with Mmp2 and Sparc, as well as selected GC downstream genes and upstream effectors. (XLSX 15 KB)Additional file 5:Relative expression of eight downstream transcriptional targets of GC signalling. Relative expression of candidate genes in the developing head of AC, PL, SB and LB at seven developmental stages, as measured by qPCR. Gene expression levels were normalized with respect to the geometric means of two craniofacial reference genes (Actb and If5a1). The relative expression level for each gene is depicted by setting a replicate of the AC morph at stage 1 to an arbitrary unit of 1. Error bars represent standard deviation calculated from two biological replicates. The letters A, L, P and S above the bars indicate significantly higher expression than in AC, LB, PL and SB, respectively (P <0.05) as calculated by Tukey’s HSD post hoc tests. (TIFF 358 KB)Additional file 6:Relative expression of eight upstream effectors of GC signalling. Relative expression of candidate genes in the developing head of AC, PL, SB and LB at seven developmental stages, as measured by qPCR. Gene expression levels were normalized with respect to the geometric means of two craniofacial reference genes (Actb and If5a1). The relative expression level for each gene is depicted by setting a replicate of the AC morph at stage 1 to an arbitrary unit of 1. Error bars represent standard deviation calculated from two biological replicates. The letters A, L, P and S above the bars indicate significantly higher expression than in AC, LB, PL and SB, respectively (P <0.05) as calculated by Tukey’s HSD post hoc tests. (TIFF 339 KB)Additional file 7:Motif sequence enrichment analysis of candidate genes. Two lists of motif sequences identified by SCOPE and transcription factor binding sites predicted by BioProspector in conserved noncoding regions of 17 coexpressed genes in three and four teleost fish species, respectively. The motifs identified by BioProspector were run against known PWMs in the TRANSFAC database using the STAMP tool. (XLSX 36 KB)Additional file 8:Craniofacial expression pattern of selected members of the coexpression network.In situ hybridization revealing the anterior and ventral craniofacial expression pattern of Ctsk, Mmp2, Ogn, Sfrp1, Sparc and Ets2 at stage 1 (A) and stage 7 (B), ventral and lateral views showing the overlapping expression of the genes in the facial area anterior to and surrounding the mouth, as well as in the pharyngeal arches. Sparc displays ubiquitous and relatively less specific expression pattern during head development, and the expression of Sfrp1 is hardly detectable at the last time point. Dashed red boxes emphasize on important expression patterns. Scale bar = 1 mM. (TIFF 6 MB)Additional file 9:A summary of known interactions between the predicted transcription factors in different vertebrate species.(PDF 448 KB)Additional file 10:Regulatory interactions between predicted transcription factors and candidate genes in different vertebrate species.(PDF 476 KB)Additional file 11: