Drosophila, the MZT is preceded by the transcription of a small number of genes that initiate sex determination, patterning, and other early developmental processes; and the zinc-finger protein Zelda (ZLD) plays a key role in their transcriptional activation. To better understand the mechanisms of ZLD activation and the range of its targets, we used chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-Seq) to map regions bound by ZLD before (mitotic cycle 8), during (mitotic cycle 13), and after (late mitotic cycle 14) the MZT. Although only a handful of genes are transcribed prior to mitotic cycle 10, we identified thousands of regions bound by ZLD in cycle 8 embryos, most of which remain bound through mitotic cycle 14. As expected, early ZLD-bound regions include the promoters and enhancers of genes transcribed at this early stage. However, we also observed ZLD bound at cycle 8 to the promoters of roughly a thousand genes whose first transcription does not occur until the MZT and to virtually all of the thousands of known and presumed enhancers bound at cycle 14 by transcription factors that regulate patterned gene activation during the MZT. The association between early ZLD binding and MZT activity is so strong that ZLD binding alone can be used to identify active promoters and regulatory sequences with high specificity and selectivity. This strong early association of ZLD with regions not active until the MZT suggests that ZLD is not only required for the earliest wave of transcription but also plays a major role in activating the genome at the MZT.The earliest stages of development in most metazoans are driven by maternally deposited proteins and mRNAs, with widespread transcriptional activation of the zygotic genome occurring hours after fertilization, at a period known as the maternal-to-zygotic transition (MZT). In  Drosophila melanogaster. ZLD had been previously shown to control the activation of a small number of genes expressed prior to the MZT. Here, using an experimental technique (ChIP-Seq) that allowed us to visualize where on the genome a protein is bound, we show that, approximately an hour prior to the MZT, ZLD is bound to most of the genomic regions active at the MZT. This suggests that ZLD may act as a kind of an “on switch” for the zygotic genome, poising regions where it binds for activation at the MZT, and this raises the possibility that similar master regulators of the MZT exist in other species.The newly fertilized eggs of most animal species begin development with a series of rapid cell divisions. During this time of rapid DNA replication, there is little or no transcription of the embryo's genome, with the synthesis of new proteins being directed by a store of maternally deposited mRNAs. Several hours after fertilization, at a period known as the maternal-to-zygotic transition (MZT), transcription of the embryo's genome begins in earnest, but little is known about how this process is initiated. In this paper we investigate the role of a protein known as Zelda (or ZLD) at the MZT in the laboratory model insect  Delayed activation of the zygotic genome during the early phases of embryogenesis is a nearly universal phenomenon in metazoans. Immediately following egg activation, the zygotic genome is largely transcriptionally quiescent, with development controlled by maternally contributed mRNAs and proteins Drosophila melanogaster, the fertilized egg undergoes a series of replication cycles without cytoplasmic divisions to generate a syncytial blastoderm CAGGTAG and related “TAGteam” elements, in their regulatory regions, the removal of which abolishes early activation In zld lead to defects in early embryonic mitosis and severe cellularization defects by mitotic cycle 14 Several factors present in the early embryo that bind to TAGteam elements have been identified CAGGTAGWe have, for several years, been investigating the genome-wide binding of the transcription factors that regulate the anterior-posterior and dorsal-ventral patterning of transcription during and immediately following the MZT. We used chromatin immunoprecipitation coupled with DNA microarray hybridization (ChIP-chip) to identify the regions bound during mitotic cycle 14 by 21 of these patterning transcription factors. While the regions bound by any particular factor are, predictably, enriched for its target sequence, in virtually every case the most strongly enriched sequence was not the specific target, but Although we were particularly interested in the possible role of ZLD at the MZT (mitotic cycle 14), we felt it was essential that we investigate ZLD binding when it is known to activate early zygotic transcription, as well as at the onset of and during the MZT. We therefore collected embryos from population cages and fixed them for chromatin extraction at three timepoints following egg-laying: 60–90 minutes, targeting mitotic cycles 8 and 9 when ZLD levels increase D. melanogaster females do not always lay eggs immediately following fertilization, meaning that while these bulk embryo collections were timed to target a particular stage, they invariably contained a small number of older embryos. Since, at this stage of development, even moderately older embryos contain substantially more DNA, even a small fraction of contaminating older embryos can represent a substantial fraction of purified chromatin. We therefore hand sorted each pool by individually examining every embryo under a light microscope and removing those that did not have the distinguishing morphological characteristics of the stage that sample was targeting.In a typical ChIP experiment, chromatin would be prepared directly from these timed embryo collections. However, Through this laborious procedure we obtained pure pools containing approximately 1, 0.2, and 0.1 g of embryos respectively for cycles 8–9, late cycle 13 and early cycle 14, and late cycle 14 . For simD. melanogaster reference sequence using Bowtie We performed immunoprecipitations using previously described affinity-purified anti-ZLD antibodies Although we used a relatively small amount of input chromatin for each sample, the ChIP-Seq data were of high quality, with well-resolved peaks and high signal-to-noise ratio . We idenCAGGTAGCAGGTAG motif at least once, as opposed to the random expectation of 1.8%. Similarly, CAGGTA and AGGTAG, two shorter versions of the motif, appear, respectively, in 94% and 75% of the most highly bound regions. There is also a strong correlation between the number of occurrences of CAGGTAG in a region and the magnitude of ZLD binding  We analyzed these regions for enriched occurrences of sequence motifs using a variety of computational algorithms  binding . Surprissc, zerknüllt (zen) and even-skipped (eve)  . ZLD wased (eve) . The obse genome . Indeed,CAGGTAG binding protein, Liang et al. In their paper describing ZLD as a Given this strong relationship between ZLD promoter binding in cycle 8 embryos and changes in transcription upon ZLD depletion, we next examined the relationship between ZLD binding and the onset of zygotic transcription in wildtype embryos. We took advantage of a recently published high-resolution time course of zygotic gene expression in the early embryo odd-paired (opa) and leak (lea) are not expressed until mitotic cycle 14, but were highly bound by ZLD at cycle 8  and dorsal-ventral (D-V) patterning in the early embryo are significantly enriched for To examine whether ZLD binding affects subsequent transcription factor binding or is simply associated with it, we examined the relationship between the presence of transcription factor target sequences, ZLD binding and transcription factor binding for the subset of factors whose binding specificity is known. As expected, the presence of a target sequence alone is a poor predictor of binding of the corresponding factor  are found in all three stages . For exacycle 14 . This dyCAGGTAG and ZLD binding (CAGGTAG site (compared to 1.8% expected at random), while an astonishing (relative to other studied factors) 64% of genomic CAGGTAG sites are indeed bound by ZLD. The unusually high fraction of CAGGTAG sites bound by ZLD at cycle 8 suggests that chromatin at this stage is in a fairly accessible state. By cycle 13, only 50% of CAGGTAG sites are bound  have been experimentally characterized D. melanogaster blastoderm, the state of chromatin shapes—and does not simply reflect—transcription factor binding CAGGTAG sites and ZLD.There is increasingly good evidence that difference in chromatin state across the genome at the MZT play a major role in determining which regions are active. We and others have recently assayed the state of chromatin in cycle 14 embryos and shown that regions of concentrated transcription factor binding are strongly associated with regions of “open” chromatin CAGGTAG sites were enriched in active promoters and regions of transcription factor binding at the MZT CAGGTAG sites is a major driver of changes in transcription factor binding at the MZT between different Drosophila species CAGGTAG sites in vivo; that there is a tight connection between ZLD binding, chromatin state and MZT activity; and, crucially, that ZLD binding precedes, by at least several mitotic cycles, transcription factor binding and transcription at regions active at the MZT. Thus it is in precisely the right places at the right time to act as a generic activator of the MZT.We already knew that CAGGTAG sites) to the mosaic of open and closed domains known to exist in cycle 14 CAGGTAG sites). If this is correct, we suggest ZLD likely plays a role in managing this transition, recruiting or repelling chromatin remodeling proteins to the regions where it is bound in uniformly open chromatin at cycle 8 and thereby ensuring they remain open at cycle 14. It is, however, also possible that early ZLD binding to its MZT targets may represent opportunistic binding of the protein to accessible regions containing CAGGTAG sites, with its MZT-specific activity arising from binding closer in time to the MZT.Although little is known about the chromatin state in the early embryo, our data support a model in which the genome transitions from a fairly uniform open state  before Alb1 is expressed Our proposed function for ZLD is reminiscent of the so-called “pioneer” transcription factors. This concept was introduced to describe the role of FoxA1 in regulating gene regulation in the developing mammalian liver. In the undifferentiated endoderm, FoxA1 is bound to the enhancer of the hepatocyte-expressed albumin gene . Embryos were collected for 30 minutes, and then allowed to develop for 60, 120 or 180 additional minutes before being harvested and fixed with formaldehyde. The fixed embryos were staged and hand sorted in small batches using an inverted microscope  for a total of processing time of 140 min , with power setting at “H”. We used 3.7 µg chromatin from cycle 8, 6.6 µg from cycle 13 and 6 µg from cycle 14 in the chromatin immunoprecipitation reaction, using the affinity purified anti-ZLD antibody, following the procedure described previously D. melanogaster genome,  using Bowtie Sequenced reads were mapped to the April 2006 assembly of the http://eisenlab.org/software/grizzly.We developed a model-based multi-peak algorithm—Grizzly Peak—to accurately identify significant ZLD bound loci across the genome. Grizzly Peak is an iterative model-based peak fitting method, which we modified from Capaldi et al. Identified peaks were expanded to 300 bp around each binding event (peak center), and were analyzed for enriched motifs. We used three de novo motif discovery tools. First we used MEME (version 4.5.0) Each called peak was assigned a genomic functional annotation based on FlyBase gene annotations , including the position of exons and transcript start and end points. According to the position of the peak center position, we categorized each peak into one of six genomic categories: (1) Promoter peaks – from 500 bp upstream to 150 bp downstream to an annotated start site; (2) Coding sequence (CDS) peaks – overlapping any exon; (3) 5′-UTR peaks – overlapping a transcript, but not CDS or promoter; (4) 3′-UTR peaks; (5) Intron peaks; and (6) Intergenic peaks – downstream of genes or more than 500 bp upstream. Each peak was then assigned to the nearest gene.To estimate the random expected distribution of ZLD peaks relative to genome annotations, we devised a simple strategy to assign every peak to a new randomized position that maintained the number of peaks, their sizes, their distribution over chromosomes and their relative distances from each other. First we randomly reordered the peaks in each chromosome, practically mixing between strong and weak peaks. Second, we randomly shuffled the linker distances between every pair of adjacent peaks. Finally, we repositioning each peak at a new randomized position and repeated the analyses at hand.We used single-embryo zygotic expression data from Lott et al. Raw gene expression data from Liang et al. http://bdtnp.lbl.gov/Fly-Net. We applied Grizzly Peak to identify the exact binding position for each factor within the 1% FDR  enriched regions. We then analyzed the co-occurrence of ZLD peaks vs. the top 300 peaks of each factor. As a control, we repeated this analyses after randomly repositioning all the peaks per TF using the random shuffling approach described above. We then compared the coverage of these randomized positions with ZLD, and calculated the percent of recognition elements that are bound in the presence or absence of ZLD. We focused on eight well-studied factors with simple recognition motifs . We identified all genomic occurrences of these motifs, and calculated the fraction of bound motifs  were obtained from MacArthur et al. http://www.ncbi.nlm.nih.gov/geo/) under accession number GSE30757. A browser with ZLD binding and other related data discussed in the manuscript can be accessed at http://eisenlab.org/data/ZLD.Raw and mapped sequencing reads are available from the National Center for Biotechnology Information's GEO database  Hand-sorted embryos used for ZLD chromatin-immunoprecipitation during cycle 8 (left), cycle 13 (center), late cycle 14 (right). (B) Classification of ZLD peaks based on relative binding strength at three timepoints. We identified 12,135 peaks in pooled reads from all timepoints, and then used reads from individual timepoints to compute occupancy at each of these sites and classified peaks based on change from cycle 8 to cycle 13, and from cycle 13 to late cycle 14.(EPS)Click here for additional data file.Figure S2CAGGTAG (top) and CAGGTA (bottom) motifs within 150 bp of ZLD peaks at cycle 8 (peaks grouped in to bins of 100).Strength of ZLD binding correlates with number of ZLD target sites. Average number of (EPS)Click here for additional data file.Figure S3opa and leak) bound by ZLD but not expressed until MZT.Binding and expression of two genes ((EPS)Click here for additional data file.Table S1ZLD peaks at cycle 8.(XLS)Click here for additional data file.Table S2ZLD peaks at cycle 13.(XLS)Click here for additional data file.Table S3ZLD peaks at late cycle 14.(XLS)Click here for additional data file.Table S4ZLD peaks in combined data.(XLS)Click here for additional data file.Table S5Master data table with ZLD binding, transcription factor and polymerase ChIP, and expression data.(XLS)Click here for additional data file.


Chloroplasts are semiautonomous organelles which possess their own genome and gene expression system. However, extant chloroplasts contain only limited coding information, and are dependent on a large number of nucleus-encoded proteins. During plant evolution, chloroplasts have lost most of the prokaryotic DNA-binding proteins and transcription regulators that were present in the original endosymbiont. Thus, chloroplasts have a unique hybrid transcription system composed of the remaining prokaryotic components, such as a prokaryotic RNA polymerase as well as nucleus-encoded eukaryotic components. Recent proteomic and transcriptomic analyses have provided insights into chloroplast transcription systems and their evolution. Here, we review chloroplast-specific transcription systems, focusing on the multiple RNA polymerases, eukaryotic transcription regulators in chloroplasts, chloroplast promoters, and the dynamics of chloroplast nucleoids. Chloroplasts are believed to have arisen from an endosymbiotic event between a photosynthetic cyanobacterium and the ancestral eukaryotic cell. Although chloroplasts of modern plants and algae have retained the genome of the symbiont, that genome has markedly shrunk over endosymbiotic evolution. Many chloroplast-encoded genes were lost or transferred to the nucleus soon after endosymbiosis. Thus, chloroplast genomes of extant land plants have only 50 protein-coding genes involved in photosynthesis, gene expression, lipid metabolism and other processes, 30 tRNA genes and full sets of rRNA genes. In spite of their small genomes (0.15 Mbp in land plant chloroplasts versus 3 Mbp in cyanobacteria), chloroplast gene expression is regulated by more complex systems compared to the simple prokaryotic regulatory system. Chloroplast gene expression is mediated by two distinct types of RNA polymerase (RNAP) and is highly dependent on post-transcriptional regulation, such as the processing of polycistronic transcripts, intron splicing and RNA editing. Moreover, recent RNA-seq analyses of chloroplast transcripts identified unexpected diversifications of RNA molecules, such as non-coding and antisense RNAs . HoweverArabidopsis thaliana have suggested that 60% of the chloroplast proteome may have been newly acquired from the nuclear genome of host cells after the endosymbiotic event  and nucleoid proteins at an early stage of their evolution. Genomics and proteomics analyses of chloroplast proteins in ic event . Indeed,Figure 1A) However, all genes for chloroplast sigma factors have been transferred to the nuclear genome, whereas genes for core subunits are typically retained in the chloroplast genome as rpoA, rpoB, rpoC1, and rpoC2.Chloroplast gene expression is largely dependent on prokaryotic machineries derived from the ancestral cyanobacterium. The bacterial multi-subunit RNAP is composed of a core Rpo complex, which has the catalytic enzyme activity, and a sigma factor, which recognizes promoter sequences . ChloropHordeum vulgare), iojap mutants of maize and tobacco mutants with disrupted rpo genes generated by gene targeting using chloroplast transformation . RpoTmp and RpoTp likely represent nuclear encoded RNAP (NEP) enzyme in chloroplasts . Among  merolae , and theydomonas . The HLPvolution . Howeverns of HU . Recentlns of HU . SWIB4 tnucleoid . These fFigure 1B). Human mitochondrial nucleoids have been shown to form layered structures, the central core involved in replication and transcription, and the peripheral region where translation and complex assembly may occur . In fact, land plants have a number of novel host cell-derived transcription regulators and DNA-binding proteins that are involved in the regulation of chloroplast transcription. Thus, it seems likely that chloroplast transcription is mediated by a hybrid system of prokaryotic and eukaryotic origin. Further molecular characterization of pTACs and plastid nucleoid proteins would provide novel insights into the unique plastid gene expression system and as yet known mechanisms of plastid differentiation.Recent proteomic and transcriptomic researches and the development of novel ChIP and imaging technologies have advanced the understanding of the molecular basis of RNAP complexes and nucleoid architecture. In land plants, neither the nuclear nor chloroplast genome encodes prokaryotic transcription factors and nucleoid proteins, whereas chloroplasts retain prokaryotic-type RNAP (The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


DUX4 is expressed in the testis and epigenetically repressed in somatic tissues. Facioscapulohumeral muscular dystrophy (FSHD) is caused by mutations that decrease the epigenetic repression of DUX4 in somatic tissues and result in mis-expression of this transcription factor in skeletal muscle. DUX4 binds sites in the human genome that contain a double-homeobox sequence motif, including sites in unique regions of the genome as well as many sites in repetitive elements. Using ChIP-seq and RNA-seq on myoblasts transduced with DUX4 we show that DUX4 binds and activates transcription of mammalian apparent LTR-retrotransposons (MaLRs), endogenous retrovirus (ERVL and ERVK) elements, and pericentromeric satellite HSATII sequences. Some DUX4-activated MaLR and ERV elements create novel promoters for genes, long non-coding RNAs, and antisense transcripts. Many of these novel transcripts are expressed in FSHD muscle cells but not control cells, and thus might contribute to FSHD pathology. For example, HEY1, a repressor of myogenesis, is activated by DUX4 through a MaLR promoter. DUX4-bound motifs, including those in repetitive elements, show evolutionary conservation and some repeat-initiated transcripts are expressed in healthy testis, the normal expression site of DUX4, but more rarely in other somatic tissues. Testis expression patterns are known to have evolved rapidly in mammals, but the mechanisms behind this rapid change have not yet been identified: our results suggest that mobilization of MaLR and ERV elements during mammalian evolution altered germline gene expression patterns through transcriptional activation by DUX4. Our findings demonstrate a role for DUX4 and repetitive elements in mammalian germline evolution and in FSHD muscular dystrophy.The human double-homeodomain retrogene  DUX4 that is normally expressed in testis and repressed in muscle. Sometimes muscle repression fails, causing the disease facioscapulohumeral muscular dystrophy (FSHD). We find that DUX4 binds many TE types and can activate their transcription. Some activated TEs have been co-opted as novel promoters for human genes. DUX4's activation of these genes via TEs might be important in the biology of normal testis and may contribute to the FSHD disease process. Our findings raise the possibility that DUX4 and TEs co-evolved, as TEs may have hijacked DUX4 to aid their retrotransposition while DUX4 may have utilized TEs to modify its transcriptional network in the evolving germline.Transposable elements (TEs) are found in most genomes, and many TEs create extra copies of themselves in new genomic locations by a process called retrotransposition. TEs are often thought of as genomic parasites that must be suppressed, because retrotransposition can cause great harm to their host organism. However, during evolution, the functions encoded by TEs have sometimes been co-opted to the advantage of the host genome as novel genes or as gene regulatory regions. We studied a human transcription factor called  DUX4 is a member of a small family of double-homeodomain genes that also includes paralogs DUXA, DUXB, DUXBL, DUXC and murine DuxDUX4 gene likely arose from retrotransposition of the DUXC mRNA, with subsequent deletion of the DUXC gene from the primate genome DUX4 exists in the primate genome as part of a ∼3.3 kb repeat unit called D4Z4, found in macrosatellite arrays in the subtelomeric regions of human chromosomes 4 and 10. Poorly studied arrays are also found in other genomic regions and appear to contain interrupted versions of the DUX4 ORF The transcription factor DUX4 is expressed in germ-line cells of the testis and is epigenetically repressed in somatic tissues SMCHD1, a gene necessary for repeat-mediated epigenetic repression, result in decreased epigenetic repression of DUX4 in skeletal muscle, causing a human muscle disease, facioscapulohumeral muscular dystrophy  DUX4 expression in a subset of nuclei in FSHD muscle cells DUX4 in human skeletal muscle activates the expression of many genes normally expressed in the germline and that DUX4 binds a double-homeobox motif at ∼60,000 sites in the mappable genome MaLRs, or mammalian apparent LTR-retrotransposons, are distantly related to endogenous retroviruses (ERVs), and comprise ∼3.7% of the human genome with ∼350,000 copies of various subclasses of MaLR elements Retrotransposition provides a way to spread regulatory sequences to large numbers of new genomic locations in short evolutionary time. Some families of repetitive elements appear to have been involved in large-scale rewiring of transcriptional networks DUX4, as well as RNA-seq data from FSHD patient and control muscle cells. We find that repetitive elements are bound and activated by DUX4, including primate-specific MaLR and ERV LTRs, and that some full-length repeat elements are transcribed in response to DUX4. In addition, some DUX4-bound LTRs form novel first exons of annotated genes, long-noncoding (lnc) RNAs, and antisense RNAs. Together, our findings demonstrate that DUX4's transcriptional network is mediated in part through binding to repetitive elements, including some primate-specific retroelements that could contribute to lineage-specific patterns of gene expression To determine whether DUX4 binding to repetitive elements can affect transcriptional networks, we analyzed ChIP-seq and RNA-seq datasets in skeletal muscle cells that ectopically express DUX4 in human myoblasts overlap MaLR LTR elements In previously published work, we observed that many of the ∼60,000 DUX4 binding sites identified by ChIP-seq following expression of An analysis of repetitive element content among DUX4 ChIP-seq peaks shows that enrichment is heavily biased towards LTR elements  and S3, It remains challenging to accurately map sequence reads to repetitive elements with high sequence identity To examine whether DUX4 binding sites are functionally important, especially those in repetitive elements, we determined whether they are evolutionarily conserved. We find that DUX4-bound motifs have tolerated fewer sequence changes than flanking sequences during the evolution of placental mammals , primateDUX4-transduced and control myoblasts. In a conservative analysis, we identified 738 DUX4-activated transcripts within 1 kb on either side of a DUX4 ChIP-seq peak and six DUX4-repressed transcripts . Peaks with more than one motif also show greater ChIP-seq peak height, a proxy measurement for DUX4 occupancy  from ccupancy .DUX4-bound repetitive elements are just as likely to initiate a transcript as DUX4 binding sites in unique sequence (∼1.2% of both classes show activation), with 454/738 (∼62%) of DUX4-initiated transcripts arising in or near a repetitive element. The DUX4-bound repeat types most likely to be transcribed are HSATII elements (23% of bound HSATII repeats show activation) and MLT2A1 ERVL-LTRs (5.4% of bound MLT2A1 LTRs are activated)  and S3. DUX4-transduced myoblasts shows that 184 of these regions are activated in the presence of DUX4 , of which 120 are MaLRs and the rest a mix of other ERV classes. These activated internal MaLR/ERV regions tend to be flanked on one or both sides by a DUX4-bound LTR, where transcription seems to initiate. Repeats whose internal regions are activated tend to be younger than repeats that do not show obvious activation, considering only repeats flanked by at least one DUX4-bound LTR  and encode fragments of various THE1 internal ORFs as well as fragments of some ERV gag and pol ORFs, although none encode full-length proteins.DUX4 retrogene and the spread of repetitive elements around the genome had the potential to alter germ cell promoter usage and the germ cell transcriptional network during primate evolution. Analysis of spliced RNA-seq reads that join a DUX4-bound region with a gene sequence identified 238 previously unannotated DUX4-activated promoters for human genes  for validation  inhibits myogenesis by repressing myogenin and Mef2C DUX4-expressing myoblasts suggests the existence of transcripts that initiate ∼40 kb upstream of HEY1's first annotated exon in a THE1B-MaLR retrotransposon and splice via two additional exons to the second exon of HEY1  that splice to exon 2 of PPCS, as suggested by RNA-seq data and verified by RT-PCR, 5′-RACE and Sanger sequencing (PPCS isoforms (RefSeq NP_001070915). This MLT1B element is found in the syntenic location in diverse mammalian genomes including those of primates, rodents, carnivores and bats, indicating that it inserted in our genome at least 90 million years ago.A DUX4-bound MLT1B-MaLR element initiates quencing . The preNT5C1B  gene and uses either of two donor sites to splice to exon 2 of NT5C1B . Comparison of DUX4 binding and activated transcripts to a recently published dataset of lncRNAs DDX10 gene in the antisense orientation . We identified many of the same repeat classes already highlighted by our analyses of uniquely mappable reads, indicating that many DUX4-bound and activated repeats have diverged enough that standard methods are effective  and S3.  samples , a basel samples .Our ChIP-seq data also demonstrated that HSATIIs are bound by DUX4, with ∼1.9-fold enrichment of HSATII sequences among individual reads and 30 DUX4 peaks in mappable HSATII regions (∼5-fold enrichment). Furthermore, each HSATII ChIP-seq peak appears to derive from multiple tandemly-arranged DUX4 binding sites. The 30 HSATII peaks span bigger genomic regions (median peak width 1.2 kb) than other ChIP-seq peaks (median width 0.4 kb) and contain multiple matches to DUX4's consensus motif - the 299 annotated HSATII regions in the human genome assembly contain a total of 820 DUX4 motifs.DUX4. To determine whether these transcripts are expressed in normal germ cell biology and in FSHD muscle, we used RT-PCR to assay for their presence in FSHD patient cells and various tissues from healthy individuals, using Sanger sequencing to confirm that each amplified product derives from the expected locus . RNA-seq profiles show that most of the genes, lncRNAs and internal repeat regions we tested using RT-PCR are expressed in FSHD myotubes but not controls (DUX4 transduced myoblasts (−15). Expression ratios of internal MaLR/ERV regions in FSHD myotubes versus controls are also well-correlated with activation levels in lenti-DUX4-transduced myoblasts  were detected in the testis RNA-seq despite our ability to detect all tested transcripts by RT-PCR  DUX4 over-expression is pathogenic HEY1 gene (induced by DUX4-mediated activation of an upstream THE1B element) can inhibit myogenesis by repressing myogenin and Mef2C DUX4 over-expression in myoblasts Brca1 aberrantly transcribe satellite sequences leading to genome instability CSF1R proto-oncogene and de-repression of THE1B elements is widespread DUX4-containing D4Z4 repeat is hypomethylated in certain tumors DUX4 de-repression in cancers might mediate the activation of HSATII, CTAs and/or THE1B promoters.Our findings may also have clinical implications for cancer biology. The DUX4 target HSATII is expressed in a number of cancers DUX4 is a primate-specific retrogene and a member of a small gene family that has experienced substantial change during mammalian evolution DUX4 orthologs and of DUX paralogs are still unknown, we note that an alignment of DUX family homeodomain sequences DUX4 and the parental DUXC gene. Determining whether other members of the DUX gene family also bind and regulate retrotransposons will illuminate the importance of these largely unstudied genes and retroelements in the biology and evolution of mammalian germ cells and in muscle disease.All experiments were performed with approval of the Institutional Review Board of the Fred Hutchinson Cancer Research Center.http://great.stanford.edu).We wrote a number of custom scripts using R DUX4, or from negative control myoblasts that do not express DUX4. The human genome contains multiple near-identical copies of DUX4: in our experiments, we used the full-length splice form of the most distal DUX4 copy on chromosome 4q35, because this is the copy that appears to be expressed and pathogenic in FSHD patient muscle DUX4 isoform is also expressed in testis, along with other copies containing minor sequence variants whose functional consequences are currently unknown Our ChIP-seq data were previously published For further analysis of DUX4 binding sites we refined peak locations by identifying the single 17-mer subsequence with highest score to the DUX4 PWM, rather than using the entire peak region (ChIP-seq resolution is limited by fragment size of ∼200 bp), making the simplifying assumption that each peak contains a single DUX4-binding site. We then determined whether each peak's best-scoring subsequence overlaps a repetitive element. To estimate peak-level enrichment for each repeat type, we divided the proportion of all peaks that overlap each repeat type (observed) by the proportion of base-pairs in the sequenced genome within that repeat type (expected). We previously found that DUX4 binding sites are distributed relatively uniformly across different types of genomic regions  The DUX4 binding motif includes an average of 5.03 G or C residues among its 17 bases. In order to create an AT-matched set of genomic locations, we randomly selected 17-bp regions from the human genome, eliminating any that overlap assembly gaps, retaining those whose sequence contains 4–6 G or C residues, and downsampling the final set to contain 63,795 sites to match the dataset of DUX4 binding sites. We then determined whether these randomly selected 17-mers overlapped repetitive elements, and counted the types of repeats found among overlapping elements. We performed this sampling 10 times, and use the mean fraction of sites overlapping repeats to calculate enrichment measures shown in the columns of In order to determine whether DUX4-bound repeats also tend to be bound by other regulatory factors, we obtained ENCODE ChIP-seq peak locations for 161 regulatory factors via the UCSC Genome Bioinformatics “wgEncodeAwgTfbsUniform” tables. For each factor, we chose a single representative ENCODE dataset, and determined the number of peaks that overlap DUX4 ChIP-seq peaks assigned as bound repeats (see above). Using the entire peak regions  for both DUX4 and the other TFs (rather than binding sites defined at higher resolution) allows us to ask the biologically relevant question of whether TFs bind in the vicinity of DUX4, rather than asking whether binding sites are exactly overlapping.et al.In our standard ChIP-seq analysis (above), we ignored sequencing reads that map to multiple genomic locations to ensure that called peaks likely represent true binding sites. However, this method is blind to binding in very recently duplicated regions, so we used an alternative bioinformatic method very similar to that of Day DUX4 constructs, the packaging constructs used during lentivirus preparation, or Illumina adapter sequences. We constructed an alternative repeat-based “reference genome”, where each repeat type is represented by a “chromosome” comprising every genomic instance of that repeat, with an amount of flanking sequence on each side equal to half the length of a sequence read, concatenated together with a intervening stretches of Ns that are each longer than a sequencing read. We then used BWA to map filtered reads to the repeat-based reference genomes, without filtering results for uniquely mapping sequences. We used the samtools idxstats program In more detail, we first filtered ChIP-seq read datasets to remove low quality sequences, and reads that match our lentivirus-These enrichment estimates are “dampened” because ChIP samples contain background DNA derived from unbound genomic regions (50–90% of reads); although immunoprecipitation depletes unbound sequences it cannot completely eliminate them. Background proportions differ between experimental and control samples, and background fraction undoubtedly contains many repetitive sequences. These read-based estimates are therefore likely a very conservative measure of true enrichment in the bound DNA fraction.Our RNA-seq data are available from GEO with accessions GSE45883 and GSE51041.DUX4. After 48 hours (54-1 cells) or 24 hours (MB135 cells), RNA was extracted, poly(A) selected, and subjected to Illumina sequencing using standard protocols to generate 100 bp single-end reads. As negative controls, we also sequenced RNA from untransduced 54-1 cells, and from MB135 cells transduced with lentivirus carrying GFP.Two human myoblast cell lines (54-1 and MB135) were each transduced with lentivirus carrying http://www.urmc.rochester.edu/fields-center) and were cultured in DMEM/F-10 media (Gibco) in the presence of 20% heat-inactivated fetal bovine serum (Gibco), 1% penicillin/streptomycin (Gibco). Media was supplemented with 10 ng/ml rhFGF (Promega) and 1 µM dexamethasone (SIGMA). Myoblasts were fused at 80% confluence in DMEM/F-12 Glutamax media containing 2% KnockOut serum replacement formulation (Gibco) for 36 hours. RNA was extracted, poly(A) selected, and subjected to Illumina sequencing using standard protocols to generate 100 bp single-end reads.In addition, we sequenced RNA from two FSHD1 and three control myotube samples. Primary myoblast cell lines were received from the University of Rochester biorepository . In addition, we note that we are only examining polyadenylated transcripts and may be ignoring others; however, full-length transcripts of many repetitive elements are polyadenylated, including those of ERVs, L1s and Alu elements Our analysis of RNA-seq reads falls into two general categories, both described in detail below.First, we performed a read-based analysis as we had done for ChIP-seq reads, combining read counts across all instances of a particular repeat type; this method does not allow us to determine which instance of a repeat type is activated, merely that one or more elements of that class shows activation, but unlike standard methods, it does allow examination of recently duplicated sequences. We used the same read-based method as we did for the ChIP-seq reads (see above) on our RNA-seq reads. Although this method uses the BWA read-mapping tool and will therefore fail to map spliced reads, it does not suffer from a limitation of tophat that it suppresses mappings for reads mapping to many genomic locations.etc.) using tophat and DESeq, a method that limits our ability to examine highly similar multicopy sequences. We mapped reads to the genome using tophat etc.) using the bedtools coverageBed function Second, we considered individual genomic locations  that are activated to very high levels in DUX4-expressing cells but are “off” in cells that do not express DUX4, very consistently at about 1/1000 of the number of reads found in 54-1 cells over-expressing DUX4. The most likely explanation is that a small amount of RNA from another sample contaminated the untreated 54-1 cell RNA sample before sequencing. Although this issue only affects genes expressed to very high levels, it causes a technical problem for the DESeq statistical analysis method we used, because contaminating reads for genes expressed to high levels make dispersal estimation difficult (data not shown). This issue contributes to the conservative nature of our conclusions.We encountered a minor technical issue: the RNA-seq read dataset for one sample, the untreated 54-1 negative control, has very low levels of contaminating reads from a lentivirus-In order to examine the age of activated internal repeat regions relative to internal repeat regions that do not show obvious activation , we firsTo analyze diversity of transcribed HSATII repeat units among RNA-seq reads , we firsDUX4-overexpressing myoblast cell lines (or in both of the FSHD patient myotube samples). For each peak-gene (or peak-lncRNA) pair, we estimated a DUX4 activation ratio, by comparing the proportion of reads linking that peak and gene in the two DUX4-expressing myoblasts (or two FSHD patient myotubes) with the proportion of reads in the two control myoblast samples (or three control myotubes). Again, we added 0.5 to each read count to avoid problems with division by zero. We then filtered the peak-gene (or peak-lncRNA) list to only include pairs with a DUX4 activation ratio of ≥2.In order to identify DUX4-bound regions that are used as previously unannotated promoters for genes or lncRNAs, we first used bedtools' intersectBed function Human tissue RNAs were purchased from BioChain  and had been DNase-treated by the supplier.DUX4 or GFP (as in our RNA-seq experiments). 24 hours after transduction, RNA was harvested for RT-PCR or 5′ RACE. Non-transduced 54-1 and MB200 cells were differentiated into myotubes by growing to 100% confluency and adding differentiation media for 48 hours .Primary human myoblasts  were collected and cultured as previously described . 54-1 primary myoblasts were transduced with a lentiviral vector expressing either Total RNA was isolated from cultured cells using the RNeasy Mini Kit (Qiagen) followed by Invitrogen's protocol for DNase I (Amplification Grade) treatment with the addition of RNaseOUT (Invitrogen) to the reaction. DNase I reaction components were removed using the RNeasy Mini Kit (Qiagen). RNA was eluted using 50 µl of RNase-free water, and the volume was reduced using a SpeedVac.°C 30 min and then 55°C 30 min). Reactions were cleaned using the QIAquick (Qiagen) PCR purification system and eluted with 50 µl of water. Negative control samples corresponding to each cDNA sample were prepared by omitting reverse transcriptase.cDNA synthesis was performed using 1 µg of RNA, SuperScript III reverse transcriptase (Invitrogen) and random hexamers (Roche) according to the manufacturer's instructions  agarose gels in TBE, cloned and sequenced using BigDye Terminators (Applied Biosystems).PCR reactions were performed with 10% PCRx Enhancer solution (Invitrogen) and Platinum Taq polymerase (Invitrogen) using 10% of the cDNA reaction as template in a total reaction volume of 20 µl in thin-walled MicroAmp reaction tubes (Applied Biosystems). Primers are listed in HEY1, MLT1B-PPCS, and MLT1E1A-NT5C1B transcripts was performed on total RNA using the GeneRacer kit (Invitrogen). Prior to PCR with gene-specific primers and GeneRacer 5′ primers, the RT reaction was cleaned using QIAquick spin columns (Qiagen) as described above. Gene-specific reverse primers are listed in 5′ RACE for the THE1B-Dataset S1HEY1, PPCS and NT5C1B, we provide the sequence of the 5′ RACE clone that extends furthest upstream, in fasta format.5′ RACE sequences. For (TXT)Click here for additional data file.Figure S1Consensus sequences for most bound repeat types contain a DUX4 binding motif. We scanned repeat consensus sequences with a PWM representing DUX4's binding preferences. In all panels, the dashed gray vertical line represents an ad hoc PWM score threshold of 9.75: most ChIP-seq peaks exceed this threshold. (A) For each repeat type, we plot read-based ChIP-seq enrichment estimate (y-axis) against best PWM score in the consensus (x-axis). We show only repeats with ≥1000 mapped reads, because enrichment estimates derived from fewer reads are less biologically significant and more error-prone. Orange and red datapoints show repeats enriched (≥2-fold) among DUX4 binding sites by either the peak-based (≥100 peaks) or the read-based method (≥1000 reads); most contain a good DUX4 motif in their consensus sequence. To explore exceptions, we investigated three repeat types: MLT1A ; THE1A ;and HAL1 .  Distribution of best PWM scores for every genomic instance of each repeat type, demonstrating that consensus motif scores may not represent genomic instances well. Blue vertical lines show best consensus motif scores. Text below each title shows how many genomic instances of the repeat contain ≥1 motif scoring ≥9.75. (B) MLT1A repeats are enriched among DUX4 binding sites. Although their consensus has no good motif, a reasonable proportion of genomic repeat instances do, explaining DUX4 binding. (C) Some repeats, like THE1As, have a good motif in their consensus and are enriched for DUX4 binding; many genomic instances contain a motif identical to that of the consensus sequence. (D) Other repeats, like HAL1s, have a good motif in their consensus but only in a few genomic instances, explaining the lack of DUX4 binding. Such discrepancies between consensus PWM scores and repeat instances can occur because repeat instances acquire post-insertion mutations, and/or because of inaccuracy in the consensus.(PDF)Click here for additional data file.Figure S2Activated internal repeats are younger than those that do not show activation. Repeat age is estimated using divergence per site from consensus sequence as a proxy (as reported by RepeatMasker). We show divergence for a filtered dataset of the internal regions of LTR-type elements that are close to DUX4-bound LTRs, showing those that are transcriptionally activated to a statistically significant level (“activated”) separately from the remaining elements (“not activated”). The activated elements show lower divergence from consensus sequences (i.e. are younger) than the remaining elements.(PDF)Click here for additional data file.Figure S3UCSC browser screenshot showing details of THE1C genomic region, including RNA-seq data. We use the UCSC Genome Browser (PDF)Click here for additional data file.Figure S4UCSC browser screenshot showing details of ERVL genomic region on chromosome 14, including RNA-seq data. We use the UCSC Genome Browser (PDF)Click here for additional data file.Figure S5UCSC browser screenshot showing details of ERVL genomic region on chromosome 11, including RNA-seq data. We use the UCSC Genome Browser (PDF)Click here for additional data file.Figure S6UCSC browser screenshot showing details of HEY1 genomic region, including RNA-seq data. We use the UCSC Genome Browser (PDF)Click here for additional data file.Figure S7UCSC browser screenshot showing details of PPCS genomic region, including RNA-seq data. We use the UCSC Genome Browser (PDF)Click here for additional data file.Figure S8UCSC browser screenshot showing details of NT5C1B genomic region, including RNA-seq data. We use the UCSC Genome Browser (PDF)Click here for additional data file.Figure S9UCSC browser screenshot showing details of MLT1C-lncRNA genomic region, including RNA-seq data. We use the UCSC Genome Browser (PDF)Click here for additional data file.Figure S10UCSC browser screenshot showing details of THE1C-lncRNA genomic region, including RNA-seq data. We use the UCSC Genome Browser (PDF)Click here for additional data file.Figure S11UCSC browser screenshot showing details of DDX10-antisense genomic region, including RNA-seq data. We use the UCSC Genome Browser (PDF)Click here for additional data file.Figure S12DUX4-transduced myoblast cell line . In the alignments, each sequence read is shown as a very thin gray line, stacked densely on top of one another. In many regions of the alignment where coverage is very deep, IGV displays only a subset of reads for enhanced visibility. IGV shows positions in each sequence read that do not match the consensus sequence as small colored tick marks, color coded . It is clear from the number and diversity of non-reference bases among the aligned reads that multiple HSATII variants are transcribed.Multiple variants of the HSATII repeat are expressed. We aligned HSATII RNA-seq reads to the HSATII consensus sequence (see Methods), and used the IGV browser (PDF)Click here for additional data file.Table S1DUX4 to control myoblasts.DUX4-binding site locations in the hg19 version of the human genome assembly. The “repetitive element” column shows whether the peak's best DUX4 motif is in unique sequence, or if not what kind of repetitive element it is in. The last two columns show results from differential expression analysis of the peak plus 1 kb of flanking sequence on each side, comparing expression levels in myoblasts transduced with lentivirus carrying (XLSX)Click here for additional data file.Table S2Enrichment estimates for each repeat type among ChIP-seq peaks, ChIP-seq reads and RNA-seq reads. Repeat types are shown sorted by peak-based enrichment estimate. See legend to (XLSX)Click here for additional data file.Table S3Enrichment estimates for each repeat family among ChIP-seq peaks, ChIP-seq reads and RNA-seq reads. Repeat families are shown sorted by peak-based enrichment estimate.(XLSX)Click here for additional data file.Table S4HEY1 does not appear in this list for technical reasons, because several intervening exons are spliced between the DUX4-bound repeat and the annotated gene, so that no 100 bp sequencing read directly joins the repeat and HEY1.DUX4-bound regions used as previously undescribed alternative promoters for annotated genes (see Methods). We note that (XLSX)Click here for additional data file.Table S5DUX4-bound regions used as previously undescribed alternative promoters for annotated lncRNAs (see Methods).(XLSX)Click here for additional data file.Table S6Primers used for RT-PCR and 5′-RACE experiments.(XLSX)Click here for additional data file.


The origins of our current understanding of control of transcription elongation lie in pioneering experiments that mapped RNA polymerase II on viral and cellular genes. These studies first uncovered the surprising excess of polymerase molecules that we now know to be situated at the at the 5′ ends of most genes in multicellular organisms. The pileup of pol II near transcription start sites reflects a ubiquitous bottle-neck that limits elongation right at the start of the transcription elongation. Subsequent seminal work identified conserved protein factors that positively and negatively control the flux of polymerase through this bottle-neck, and make a major contribution to control of gene expression. The initiation phase of the RNA polymerase II (pol II) transcription cycle involves multiple events, including recruitment of general transcription factors and pol II to the promoter, melting of the DNA template, initiation of RNA synthesis, and pol II promoter clearance, which marks entry into the elongation phase. The stochastic nature of all of these steps poses a potential problem if it becomes necessary to mount a rapid activation of transcription. Following initiation pol II often encounters a rate-limiting barrier that appears to lie between early elongation and productive elongation. The transition between these two phases of the transcription cycle has now been characterized as a powerful regulatory switch used to increase or decrease gene expression in a signal-responsive fashion. Here we review the early discoveries that laid the foundation for a detailed understanding of transcriptional regulation at this transition.in vivo and purified from infected cells revealed two additional unusual features of transcription from the late promoter. First, two pause sites were mapped around positions +15 and +40 relative to the start site by identifying the junctions between unlabelled RNA made in vivo and labeled RNA extended in vitro [Nearly 30 years ago it was reported by the late Yosef Aloni and colleagues that run-on transcripts made in nuclei from SV40 infected cells were strongly biased toward the 5′ end of the late transcription unit suggesting that pol II accumulated in the promoter-proximal region . Analysiin vitro . Second,hsp70 [c-myc genes even though the genes were not actively expressed [It was not long before the first evidence emerged that pol II also piles up near the transcription start sites of cellular genes. High levels of pol II were found to accumulate at the 5′ ends of the Drosophila heat shock gene hsp70 , 7, and xpressed , 9. Thesxpressed . Subsequxpressed  and adenxpressed –15. As axpressed , 16. Thexpressed . Recentlxpressed –20. Indexpressed –23. Whilin vitro pulse chase experiments of Coppola and colleagues showed that pol II can pause close to the start site and then resume elongation [in vivo polymerase mapping studies has been that they result from a similar “promoter-proximal pausing” phenomenon. That at least some promoter-proximal polymerase can resume elongation is demonstrated by nuclear runon experiments; indeed, these polymerases would not be detectable by this method if they could not elongate and incorporate labeled nucleotides. However, the possibility that some fraction of the promoter-proximal polymerases terminate prematurely and never enter the productive elongation phase cannot be eliminated. The evidence for premature termination is quite clear for the SV40 late and HIV viral genes [c-myc transcription in microinjected Xenopus oocytes, but the physiological relevance of this phenomenon remains unproven [What is the root cause for why pol II is so unevenly distributed across so many genes? The original ongation . Since tal genes , 25, butunproven . Recentlunproven . WhetherHsp70 and c-myc genes [Hsp70, the amount of paused pol II prior to heat shock correlated with the amount of mRNA made after heat shock [An important question to emerge from the early studies of pol II localization on viral and cellular genes was: “What is the purpose of pol II piling up at the start sites of genes even before they are activated?” One answer to this question quickly emerged from studies of three genes with regulated transcriptional output: the cellular yc genes , 8, 9 anyc genes . In eachat shock . Therefoat shock . A thirdat shock , 29, andHow is the flux of pol II from the promoter-proximal region into the body of a gene controlled? The first important clue was again provided by a virus; in this case HIV1. Groundbreaking work of Kao and colleagues showed that the viral transactivator protein Tat had the novel ability stimulate elongation by pol II . Withoutc-myc [HIV Tat is an unusual transactivator because it binds to the nascent RNA transcript. Therefore, the question remained open as to whether conventional DNA-bound activators can influence transcriptional elongation. Part of the answer to this question came with the demonstration that Tat could activate transcription when tethered to a DNA-binding site in the promoter . Subsequc-myc , 33, 34.c-myc , 36.tour de force of classical biochemistry, the Handa and Price labs took advantage of this inhibitor to identify the core negative and positive factors that control the “yin and yang” of transcriptional elongation. Handa's lab identified the DRB-sensitivity-inducing factor (DSIF) as Spt4/5 a conserved pol II binding complex that is required for inhibition of elongation near 5′ ends [How do activators like HIV Tat and cellular transcription factors stimulate pol II transit away from the promoter-proximal region and into the downstream region of the gene for productive mRNA synthesis? The solution to this problem was provided by landmark studies that uncovered novel inhibitors of elongation and the factors that antagonize them. This story started with an early insight into how the ATP analogue 5, 6-dichloro-1-ß-D-ribofuranosylbenzimidazole (DRB) inhibits pol II transcription. Pulse labeling of RNA in adenovirus-infected cells revealed that DRB inhibited chain elongation but not initiation . In a to 5′ ends . Soon af 5′ ends . The cou 5′ ends . PTEFb w 5′ ends , 42 that 5′ ends  which it 5′ ends , 45.c-fos and NFkB targets [Elucidation of the interplay between positive- and negative-elongation factors provided a basis for understanding how transcription factors can regulate elongation. The vital missing piece of the puzzle was filled in with the discovery that Tat when bound to TAR in the nascent HIV1 transcript contacts PTEFb through Cyclin T1 and this interaction is required for stimulation of transcriptional elongation , 42, 46. targets , 47. PTE targets , 49 and  targets  and chro targets .Tremendous advances have been made in understanding control of gene expression at the level of transcriptional elongation since the early days when it was identified on a few viral and cellular genes. Now this mechanism is recognized to be at least as important as control of the initiation step in pol II transcription. Still, important questions remain unresolved about the nature of promoter-proximally accumulated pol II. It is still not clear how many of these paused polymerases have backtracked and are destined ultimately to resume elongation and how many are destined for premature termination. These scenarios suggest the possibility of distinct targets for regulation by controlled polymerase release into the body of the gene. It will be interesting to see how these targets might be used in various developmental and signal-responsive contexts.


Odocoileus virginianus) post-mortem and long-term in live, captive Rocky Mountain elk (Cervus elaphus). The prototype dart with a 12.7 mm (0.5 inch) needle length and no powder charge resulted in the shallowest mean (± SD) penetration depth into gelatin blocks of 27.0 mm (±5.6 mm) with 2.0 psi setting on the Dan-Inject CO2-pressured rifle. Eighty percent of PIT were successfully injected in the muscle mass of white-tailed deer post-mortem with a mean (± SD) penetration depth of 22.2 mm . We injected PIT successfully into 13 live, captive elk by remote delivery at about 20 m that remained functional for 7 months. We successfully demonstrated that PIT could be remotely delivered in darts into muscle mass of large mammals and remain functional for >6 months. Although further research is warranted to fully develop the technique, remote delivery of PIT technology to large mammals is possible using prototype implant darts.Methods to individually mark and identify free-ranging wildlife without trapping and handling would be useful for a variety of research and management purposes. The use of Passive Integrated Transponder technology could be an efficient method for collecting data for mark-recapture analysis and other strategies for assessing characteristics about populations of various wildlife species. Passive Integrated Transponder tags (PIT) have unique numbered frequencies and have been used to successfully mark and identify mammals. We tested for successful injection of PIT and subsequent functioning of PIT into gelatin blocks using 4 variations of a prototype dart. We then selected the prototype dart that resulted in the least depth of penetration in the gelatin block to assess the ability of PIT to be successfully implanted into muscle tissue of white-tailed deer ( Lepus timidus), desert tortoise (Gopherus agassizii), Wryneck (Jynx torquilla), and Adélie penguins (Pygoscelis adeliae) at PIT readers comparable to a recapture Researchers are often challenged with identifying methods for humane and efficient marking and identification of free-ranging wildlife for a variety of research and management purposes. The use of Passive Integrated Transponder technology could be a more efficient method for mark-recapture analysis of various wildlife species. Passive Integrated Transponder tags (PIT) have unique numbered frequencies and have been used to successfully mark and subsequently identify mountain hare , deer (Odocoileus spp.), and black bear (Ursus virginianus), remote delivery of PIT could be followed by scanning of injection site at check stations after being harvested by hunters. Marking remotely with PIT would be a random sample of the population, similar to capturing or trapping large mammals for marking with ear-tags or radiocollars provided that sampling designs were set-up and followed. More importantly, remote delivery of PIT would be less invasive to study animals, safer for researchers, and potentially more practical and economical. Use of PIT may provide more detail on harvest rates, population estimates, and movements not previously possible due to the expense and logistics of radiotelemetry technology and monitoring of individuals.Most mark-recapture studies are limited by the number of animals monitored, which is limited by the expense of capture for marking, radiotelemetry equipment, and monitoring protocols and methods  and Kemp's ridley (Lepidochelys kempii) seaturtles and American eels (Anguilla rostrata) was considered superior to subcutaneous injection because quick encapsulation rendered PIT more stable with less migration than in subcutaneous injections As with any invasive technology that can be used to permanently mark large mammals, humane and behavioral concerns for the study animal should be considered Antilocapra americana), African elephants (Loxodonta africana), and a variety of primates Remote delivery of PIT would likely be comparable to remote darting and injection of chemical immobilization drugs that is routine in wildlife research and no detrimental effects from infections caused by remote darting have been documented This was a preliminary study with the overall goal of evaluating whether PIT tags can be delivered remotely. Our study was designed in phases to address animal welfare and to prevent use of technology on a live animal should it fail in controlled tests. To this end, we had 3 primary objectives: 1) to test prototype darts, 2) inject PIT into muscle tissue, and 3) to assess longevity of PIT functionality in a mammal. Specifically, our first objective was to select the prototype dart design that would inject PIT upon impact the shallowest depth to determine if the idea was feasible prior to animal testing. To test that darts were able to inject PIT into muscle tissue as it did in gelatin, our second objective was to use the selected prototype dart from objective 1 to assess the ability of PIT to be successfully implanted into muscle tissue using remote delivery into hind quarters of white-tailed deer post-mortem. Our third objective was to determine the potential for use of PIT in free-ranging wildlife by assessing remote delivery and longevity of functioning PIT in live, captive elk.We prepared a mixture of 5% gelatin powder and 1200 ml of water on a hot plate heated to 230°C and stirred with a metal spatula until the powder was completely dissolved in solution. We poured the mixture into 2-ply plastic bags confined by a box to create a square mold with dimensions of 15×10×15 cm. We refrigerated the entire mold to allow gel solution to solidify prior to removing bag from the mold (hereafter referred to as gelatin block). We set up a wooden case to contain the gelatin block equipped with a digital video camera to record impact of dart and injection of PIT into the gelatin block. We used 2.1 mm×12.5 mm glass PIT read at 134.2 kHz . We used a Dan-Inject rifle model JM Standard  to project the loaded prototype implant darts  and to inject PIT into the gelatin block upon impact. The prototype implant dart was 1 cc  with a 12-gauge needle and contained a rod within the needle to assist in injection of PIT into the gelatin block . Gelatin2 (psi) setting. We did not vary psi throughout the trials for consistency of each dart type and because number of darts available limited the number of trials for each objective. The 4 prototype darts were:To maximize the efficiency of the reader to detect PIT, we wanted to implant PIT as shallow in the muscle as possible because remote delivery of PIT subcutaneously would not be possible. To address this issue, we used 10 replicates of 4 prototype darts and measured the depth PIT penetrated the gelatin block fired using a 2.0 pounds/inch1 cc, 12 gauge needle, 25.4 mm (1 inch) needle length with powder charge (Dart 1).1 cc, 12 gauge needle, 25.4 mm (1 inch) needle length without powder charge (Dart 2).1 cc, 12 gauge needle, 12.7 mm (0.5 inch) needle length with powder charge (Dart 3).1 cc, 12 gauge needle, 12.7 mm (0.5 inch) needle length without powder charge (Dart 4).P≤0.05.We selected darts similar to a study on wound characteristics from remote darting of immobilization drugs that showed longer needle length and rapid injection  resulted in more contamination of wound cavities compared to shorter needle length and slow injection To determine depth and ability of PIT to implant in animal tissue, we darted hind quarters of 4 white-tailed deer that were collected post-mortem (cadavers) with 5 PIT per hind quarter at 20 meters. We used darting of cadavers instead of gelatin blocks covered in hide because covering with a hide is difficult to mimic the taunt nature of hide and muscle and could result in only partial penetration of the dart body through hide We remotely darted 14 captive elk, individually identified with numbered eartags, in the hind quarter using the rifle at about 20 meters at 2.5 psi with the prototype implant dart used in muscle mass of deer cadavers. The captive elk are maintained under USDA standard operating procedures for captive wildlife . The captive elk were all males that were 3 years-of-age during the study and were free-ranging behind a 2.0-m tall fence covering 7 ha. The dart needle was sealed with wide-spectrum antibiotic ointment prior to injection to help prevent infection post-darting and to hold PIT inside dart needle during remote delivery. Sealing the dart needle with antibiotic ointment was necessary because PIT occasionally fell out of the dart during flight in preliminary evaluations of remote delivery. The 3 failed first attempts had the dart eject from the elk hind quarter prior to successfully implanting the PIT while darts from the 11 successful deliveries remained in the elk for >5 minutes; we darted elk with PIT a second time for the 3 failed attempts. Twenty-four hours after remote injection, elk were run through a squeeze chute handling system and the reader was scanned over the injection site to determine successful injection and functioning of PIT. The squeeze chute was only used during handling of captive elk and was not accessible to captive elk or free-ranging wildlife when not in use for this research. Data on injection site condition , location of PIT, and frequency of PIT was recorded for each elk. If we were unable to read the PIT, the elk was remotely darted with an additional PIT with subsequent reading of PIT about 30 days later. We visually monitored elk within 20 m daily for 21 days to determine if any change in gate or condition of elk occurred in response to darting with PIT. We then determined proper PIT functioning in elk monthly by a combination of squeeze chutes or scanning with reader by hand during feeding operations. Use of all elk for the purposes of our study was approved by the National Wildlife Research Center Animal Care and Use Committee Quality Assurance protocol 1802.Darts 1 and 3 that included a powder charge penetrated the gel block the deepest with mean (± SD) penetration depths of 40.7 mm (±8.1 mm) and 42.0 mm (±8.5 mm), respectively . Dart 2 n = 6); PIT were difficult to accurately locate for depth measurement in the hind quarters. Two PIT remained in the dart's needle upon impact but were successfully implanted on the second remote delivery attempt. We were able to read 95% of PIT in the cadavers after darting and frequency was able to be read after excision from muscle. One PIT could not be read in cadavers and was found to be broken upon recovery from muscle tissue.Using Dart 4, we had a 90% success rate of PIT being injected from dart into muscle mass of cadavers at 2.5 psi setting for dart rifle although 2 PIT were implanted only in skin. Eighty percent of PIT actually injected in the muscle mass of cadavers with a mean (± SD) penetration of 22.2 mm (±3.8 mm) for those that could be accurately measured  to detect individual PIT up to 50 mm in gelatin blocks and 26 mm in muscle tissue. Individual desert tortoises were monitored near culvert crossings under highways with an automated system that recorded unique frequency, date, and time of crossing up to 75 mm Only one of 20 PIT injected into the muscle tissue of cadavers broke upon impact and could not be read. We feel this may be related to dart manufacture causing a tight fit of the PIT in the dart needle. We had to bore out a few dart needles because PIT was not easily inserted into the dart. This is a simple problem to solve by the manufacturer or can be easily fixed in the field by testing for complete submersion of PIT into dart needle with no friction. Conversely, too loose a fit caused a few PIT to fall out of the dart during flight and were recovered on the ground within a meter of the target. Sealing the dart needle with an antiobiotic ointment solved this problem along with providing protection from potential infection. Further evaluation on remote delivery of PIT from greater distances from the target and higher psi settings would be needed to increase use of remote delivery unless an automated delivery system was designed The impetus for this study was the idea of marking large numbers of individuals in the field without the cost and hazards of capture typically required for marking large mammals with unique identifiers . No long-term methods currently exist for marking large mammals with unique identifiers that does not require capture but see . MarkingDarting rifles are quite common among researchers to capture large mammals and range from $500 to $2,000. The PIT readers vary in cost with the reader being the least expensive ($475) and a 6-antennae multiplexing transceiver system around $25,000 with each antennae capable of being placed out to 10 m from a central location. The PIT range in price depending on the quantity ordered was from $5 to $8 per PIT. The darts were prototypes at a cost of $6 per dart that would likely decrease if a market for PIT-delivery darts was established and mass production were initiated. Depending on study objectives, costs of marking with PIT seems relatively inexpensive, considering cost of capture for equipping with VHF/GPS collars, tracking equipment, personnel, and software often exceeds $50,000 to initiate a study of about 20 animals.The impetus for the research was the perceived need of state-agencies to mark large numbers of free-ranging cervids for mark-recapture analysis that can be used to more accurately estimate population size. Although PIT has been used in a variety of research designs, delivery of PIT remotely also requires additional considerations to address practicality and animal welfare concerns. Further research is needed to determine PIT tag loss and retention, potential for post-harvest consumption of PIT, multiple injections per animal with PIT, potential tissue damage, and behavior of study animals beyond the time-scale of this study. Furthermore, readers of PIT would need to be explored to determine if study objectives could be achieved such as can study animals be funneled into an area to read injected PIT because of the limitations of PIT readers.In conclusion, research on PIT has been occurring for decades but has not reached its potential for use in large mammals. Although several components of remote delivery of PIT still needs to be explored, remote delivery is possible, and proper function of PIT occurred for at least 7 months. If manufacturers of PIT could increase strength and durability of PIT for remote darting at greater distances and velocities, utility of PIT in wildlife research could increase. Furthermore, if manufacturers could increase the distance that readers can detect PIT beyond several meters, it could potentially expand the use of marking with PIT by researchers. Similar to remote darting for chemical immobilization and biobullet delivery, animal welfare is comparable while providing a valuable tool to mark large numbers of animals with minimal cost and hazards to biologists and study animals.


CYP19A1, but it is unclear which are direct targets and whether the C134W mutation alters their regulation. Recently, it has been reported that FOXL2 forms a complex with steroidogenic factor 1 (SF-1) which is a known regulator of aromatase in granulosa cells.Granulosa cell tumors (GCT) of the ovary often express aromatase and synthesize estrogen, which in turn may influence their progression. Recently a specific point mutation (C134W) in the FOXL2 protein was identified in >94% of adult-type GCT and it is likely to contribute to their development. A number of genes are known to be regulated by FOXL2, including aromatase/In this work, the human GCT-derived cell lines KGN and COV434 were heterozygous and wildtype for the FOXL2:C134W mutation, respectively. KGN had abundant FOXL2 mRNA expression but it was not expressed in COV434. Expression of exogenous FOXL2:C134W in COV434 cells induced higher expression of a luciferase reporter for the ovarian specific aromatase promoter, promoter II (PII) (−516bp) than expression of wildtype FOXL2, but did not alter induction of a similar reporter for the steroidogenic acute regulatory protein (StAR) promoter (−1300bp). Co-immunoprecipitation confirmed that FOXL2 bound SF-1 and that it also bound its homologue, liver receptor homologue 1 (LRH-1), however, the C134W mutation did not alter these interactions or induce a selective binding of the proteins. A highly conserved putative binding site for FOXL2 was identified in PII. FOXL2 was demonstrated to bind the site by electrophoretic mobility shift assays (EMSA) and site-directed mutagenesis of this element blocked its differential induction by wildtype FOXL2 and FOXL2:C134W.These findings suggest that aromatase is a direct target of FOXL2:C134W in adult-type GCT via a single distinctive and highly conserved binding site in PII and therefore provide insight into the pathogenic mechanism of this mutation. The forkhead transcription factor FOXL2 has recently emerged as a critical regulator of ovarian function. It is one of the first molecular markers of ovarian development STAR, CYP17 and aromatase), inflammation (e.g. NFAT and PTGS2/COX2) and apoptosis or detoxification (e.g. MNSOD) STAR gene Despite the biological importance of FOXL2, many details of its action remain unknown or are the subject of conflicting reports. A number of putative target genes for FOXL2 have been identified including genes involved in steroidogenesis . However, reverse transcribed-quantitative PCR (RT-qPCR) analysis of the FOXL2 transcript showed that where the KGN had robust FOXL2 mRNA expression, the COV434 cells had little or no FOXL2 expression .In normal granulosa cells, aromatase is known to be up-regulated by follicle stimulating hormone (FSH) signaling via cAMP and in cooperation with the protein kinase C (PKC) pathway. Aromatase and SF-1 expression were measured by RT-qPCR in KGN and COV434 cells, following separate and combined treatment with forskolin (FSK) and phorbol myristate acetate (PMA) . StrikinThe finding that COV434 cells do not express FOXL2 provided the opportunity to examine the influence of ectopic FOXL2 expression on the regulation of a firefly luciferase reporter for the ovarian specific promoter of aromatase, PII (PII −516bp-luciferase) Given that the SF-1 and LRH-1 proteins are key regulators of aromatase The nature and context of DNA binding sites of transcription factors give insight into the action of those transcription factors. We sought to determine where in PII, FOXL2 bound to regulate the promoter. FOXL2 is a highly conserved protein To determine whether the identified putative forkhead binding element in PII was bound by FOXL2 we performed an EMSA using 38bp probes centered on the site . We geneTo test whether the identified forkhead element was a functional regulator of the promoter and whether it could explain the increased stimulation of PII −516bp-luciferase by the FOXL2:C134W  we generThe potentially oncogenic FOXL2:C134W mutation is remarkable for its molecular specificity and therefore it provides an opportunity to define a new pathogenic mechanism. Here we confirmed that the KGN cell line is heterozygous for the FOXL2:C134W mutation and found that it has robust FOXL2 expression. The COV434 cell line was wildtype for FOXL2 but had low or no expression. FSK/PMA treatment induced aromatase robustly in KGN cells but not in COV434 cells. These findings combined with a dramatic difference in cellular morphology The FOXL2:C134W mutation was found to increase FOXL2 induction of an aromatase reporter but not one for StAR, indicating that the mechanism of FOXL2 action on the two promoters is different and that aromatase may belong to an undefined subset of genes that are influenced by the mutant protein. Notably, the mutation appeared to circumvent the requirement for FOXL2 stimulation of aromatase on concurrent PKA and PKC signaling . The StAFew co-regulators have been described for FOXL2. It has been reported to form complexes with SMAD3 The sequence and context of DNA elements bound by transcription factors give insight into the action of those factors. Here we identified a highly conserved region in PII containing a forkhead element that conferred its sensitivity to increased stimulation by FOXL2:C134W  and 5. OFSHB)   [44]. InFSHB)  and impoA second observation we have made of our identified element is that it is also a putative insulin response sequence (IRS), which as a group have the consensus T(G/A)TTT(T/G)(G/T) Drosophila gene that controls sex determination in that species A third observation is that the identified forkhead element is immediately flanked by a potential binding site for members of the doublesex and mab-3 related transcription factor (DMRT) family so that the core sequence of this binding site  in silico modeling based on crystal structures of other forkhead protein family members Finally, a fourth and key observation made of the identified sequence is that it is actually a forkhead sequence and not a binding site for members of a different protein family. FOXL2 forms complexes with other transcription factors (e.g. SF-1) and it is likely to bind DNA both directly and indirectly  using primers previously described The RNA was prepared using the Trizol reagent  according to the manufacture's instructions and cDNA was generated using AMV reverse transcriptase  with poly-dT primers. RT-qPCR was performed on a Rota-Gene RG-3000  using previously described primer sets for SF-1 Renilla luciferase signals generated by co-transfection of a Renilla luciferase expression construct Cells were plated at 15% confluence and transiently co-transfected with constructs of interest and either PII −516bp-luciferase For Western analysis of expression from the FLAG-tagged FOXL2 expression constructs, lysates were generated by the addition of lysis buffer containing 1% Igepal CA-630 (Sigma-Aldrich), 24hr following transfection. The proteins were separated on a SDS-PAGE gel and visualized by standard methods using anti-FLAG antibody . For co-immunoprecipitation, lysates were generated in the same manner and then mixed with Anti-FLAG M2 Affinity Gel (Sigma-Aldrich). The beads were washed three times with 20 volumes of lysis buffer and then boiled in denaturing sample loading buffer. The remaining proteins were separated on a SDS-PAGE gel and visualized using commercially available antibodies for LRH-1  and FLAG , and with anti-sera for SF-1 that was kindly provided by Dr. Ken-ichirou Morohashi. Densitometry was performed with EZQuantGel .DNA homology analysis was performed via the University of California Santa Cruz (UCSC) Genome Browser 5′-tttcttgggcttccttgttttgacttgtaaccataaat-3′ and 5′- tttcttgggcttccttggggtgacttgtaaccataaat-3′ (mutant) were prepared by end labeling annealed oligos with γ32P-ATP using T4 Polynucleotide Kinase (Promega) and purification with MicroSpin™ G-50 Sephadex columns (Amersham Biosciences). EMSA were performed by incubating the probes with in vitro translation products generated with the TNT Quick transcription/translation kit (Promega) either with or without appropriate antibodies. The complexes were separated on non-denaturing polyacrylamide gels and visualized by drying and subsequent overnight exposure to autoradiography Hyperfilm MP at −80C . Densitometry was performed with EZQuantGel .Probes of the sequences Figure S1FOXL2:wt and FOXL2:C134W constructs were expressed equally following transfection. The FOXL2:wt (wt) and FOXL2:C134W (m) constructs were transiently transfected into COV434 cells and lysates were prepared 24hr later. The lysates of three experiments were visualized together on a single Western blot using anti-FLAG antibody. (A.) Western blot. (B.) Densitometry of bands detected in B. Mean of three experiments, error bars are ±SEM.(0.85 MB TIF)Click here for additional data file.Figure S2FOXL2:wt and FOXL2:C134W bound to the promoter probe and bound less to the mutated probe in a similar manner. Densitometry of three EMSA blots including the example shown in (0.50 MB TIF)Click here for additional data file.


X, which is considered a primary determinant of sodium sensing in the brain. Only partial data exist describing the NaX distribution pattern and the cell types that express NaX in both the rat and mouse brain. To generate a global view of the sodium detection mechanisms in the two rodent brains, we combined NaX immunofluorescence with fluorescent cell markers to map and identify the NaX-expressing cell populations throughout the network involved in hydromineral homeostasis. Here, we designed an anti-NaX antibody targeting the interdomain 2–3 region of the NaX channel’s α-subunit. In both the rat and mouse, NaX immunostaining was colocalized with vimentin positive cells in the median eminence and with magnocellular neurons immunopositive for neurophysin associated with oxytocin or vasopressin in both the supraoptic and paraventricular nuclei. NaX immunostaining was also detected in neurons of the area postrema. In addition to this common NaX expression pattern, several differences in NaX immunostaining for certain structures and cell types were found between the rat and mouse. NaX was present in both NeuN and vimentin positive cells in the subfornical organ and the vascular organ of the lamina terminalis of the rat whereas NaX was only colocalized with vimentin positive cells in the mouse circumventricular organs. In addition, NaX immunostaining was specifically observed in NeuN immunopositive cells in the median preoptic nucleus of the rat. Overall, this study characterized the NaX-expressing cell types in the network controlling hydromineral homeostasis of the rat and mouse. NaX expression pattern was clearly different in the nuclei of the lamina terminalis of the rat and mouse, indicating that the mechanisms involved in systemic and central Na+ sensing are specific to each rodent species.The Scn7a gene encodes for the specific sodium channel Na Indeed, the NaX channel shares <50% sequence identity with this Na+ channel family and the structural specificity of the NaX channel lies in the marked discrepancies within the consensus amino acid sequences characterizing the voltage sensor domain (S4 segment), the pore region (P domain), and the inactivation gate  with several other atypical Na+ channels that are strongly homologous to each other . These Na+ channels were previously cloned  from cultured astrocytes  provided the first indication of both the brain localization and the functional properties of this atypical Na+ channel in vivo  demonstrated a restricted pattern of Nav2.3 (NaX) throughout the central nervous system with its highest expression in the four following circumventricular organs (CVOs): the subfornical organ (SFO), the vascular organ of the lamina terminalis (OVLT), the median eminence (ME), and the neurohypophysis. The presence of NaV2.3 in brain regions that are critical for hydromineral balance is in agreement with the putative function of this atypical Na+ channel, which has been related to Na+ homeostasis in mice. Indeed, behavioral testing of the Nav2.3 null mutant mice (Nav2.3−/−) indicates that these animals do not show salt aversion under dehydrated conditions and that site-directed transfer of the NaX gene into the SFO restores salt aversion  in the SFO and OVLT of the mouse  in the SFO and OVLT, thereby indicating that NaX was exclusively expressed in ependymal cells and astrocytes in these organs  was synthesized in the CHUL research centre . Briefly, the 495 bp corresponding cDNA sequence (accession number NM_031686) was amplified with the following PCR primers: forward, 5′-GGGGGATCCGCTTCATACGATGCTACCACAGAA-3′ and reverse, 5′-GGGCTCGAGAAGCTTCGACTTTTCATGTTCATAG-3′, included the restriction enzyme site BamHI and XhoI, respectively (underlined sequences).The amplified PCR fragment was cloned in plasmid expression pGEX-4T vector in frame with the glutathione S-transferase (GSH-T) tag, and transformed in E. coli BL21-Gold (D3) strain. The resulting DNA construction was purified and sequenced on the both strand to check unwanted mutation. Rat NaX fusion protein was induced with 0.1 mM IPTG, 2 h at 37°C and purified on glutathione sepharose 4B column. The purified fusion protein was injected into rabbit for immunization and the antibodies were affinity purified by GenScript Company .The polypeptide from the cytoplasmic interdomain 2–3  and fifteen C57BL/6 male mice (20–25 g) were purchased from Charles River  1 week before experimentation. The animals were housed in plastic cages and acclimated to standard laboratory conditions . The animals had free access to tap water and regular rat chow . The experimental procedures were performed according to the guidelines of the Canadian Council on Animal Care and approved by the animal care committee of our institution .The rats and mice were deeply anesthetized  and euthanized by transcardiac perfusion with cold saline (4°C), immediately followed by the perfusion of a fixative solution . The brains were quickly removed from the skull, post-fixed 6 h in the fixative solution and then immersed for 40 h at 4°C in a sterile phosphate buffer salt (PBS) solution containing 20% sucrose. The brains were frozen on dry ice and mounted on a microtome stage. Coronal sections (30 μm for rats and 20 μm for mice) were collected in a cryoprotectant solution and stored at −20°C. Before use, the brain sections were rinsed in sterile PBS solution.l-lysine-coated slides, air-dried and coverslipped with Vectashield® mounting medium for fluorescence .The primary and secondary antibodies used in this study are listed in Table 2–5% CO2) and containing 2 mM KCl, 1 mM CaCl2, 3 mM MgCl2, 26 mM NaHCO3, 1.2 mM NaHPO4, 10 mM d-glucose, 200 mM sucrose, pH 7.4. Osmolality was adjusted to 298–300 mOsm/l with mannitol. Coronal brain slices (350 μm thick) were cut with a vibratome  then transferred in aCSF continuously bubbled with a gas mixture (95% O2–5% CO2). Brain regions of interest were micropunched, transferred in 1.5 ml microtube and freeze in dry ice. Tissue was kept at −80°C until to use.Rats and mice were prepared as for tissue preparation for immunofluorescence without tissue fixation step. Briefly Brains were quickly removed from the skull and submerged in ice-cold (2°C) artificial CSF (aCSF) continuously bubbled with a gas mixture  and primers: MOUD66: 5′-ACTGTGTTCCGAATCCTCTG-3′/MOUD59: 5′-TCATGTCTCCATACTCCAGG-3′ for NaX, and MOUD82: 5′-GTCTTCACTACCATGGAGAAGG-3′/MOUD83: 5′-TCATGGATGACCTTGGCCAG-3′ for GAPDH  agarose gel.Total RNA from brain micropunches were extracted and DNase-treated with the PureLink™ RNA Micro kit according to the manufacturer’s instructions . The concentration was determined by measurement of absorbance at 260 nm with an absorbance reader . Reverse transcription (RT) PCR experiments were performed in two steps. First, cDNA synthesis was carried out with the Transcriptor first-strand cDNA synthesis kit . RT reaction used a mix of random hexamer and oligo-dT primers of the kit with 400 ng of total RNA in a final volume of 20 μl. Three microliters of the first-strand cDNA synthesis reaction was used as template for PCR amplification. PCR fragments were amplified with MaximaHigh-resolution images were collected on a i90 Nikon fluorescence microscope  coupled to a Hamamatsu 1394 ORCA-285 monochrome camera and exploited by Simple PCI software version 5.3.0.1102 . Finally, all images were adjusted for brightness and contrast using Adobe Photoshop, and the figures were assembled in Adobe Illustrator.X channel in the rat and mouse brain. Accordingly, we developed a specific antibody directed against the cytoplasmic loop linking domains 2 and 3 (ID2–3) of the NaX channel. This region was targeted because protein sequence analysis revealed that ID2–3 is unique among the voltage-gated Na+ channel family members. Indeed, the targeted 165 amino acids sequence . Note that the antibody titer was defined as the maximal dilution giving an OD450 nm signal greater than 0.1 and was calculated at a 1/512,000 dilution for the antibody batch used in the present study. Subsequently, the specificity of the anti-NaX antibody was verified in situ with immunohistochemical staining. Using thin (30 μm) brain slices, we evaluated NaX immunostaining in the rat MnPO (positive control) and BST , were incubated with the anti-NaX antibody. In addition, fluorescent NaX immunostaining was combined with various cell markers to identify the cellular phenotype that expressed the NaX channel. Thus, NaX immunostaining was performed in combination with anti-NeuN, anti-GFAP, or anti-vimentin immunohistochemistry to identify neurons, glial cells or ependymal cells and tanycytes, respectively. Immunostaining was also performed with oxytocin and vasopressin-associated neurophysin to distinguish between oxytocin and vasopressin-expressing magnocellular neurons.To examine the specificity in the NaX.Although rats and mice are phylogenetically close animals, our anatomical analysis revealed noticeable differences in terms of patterns and cell types in the brain that expressed NaX immunostaining in both the rat and the mouse was detected in the CVOs, the OVLT, the SFO, the ME, and the area postrema (AP). The pattern and cellular phenotype for NaX expression in both rats and mice showed similarities only in two CVOs, which were the ME and the AP. In the ME, NaX immunostaining was essentially observed in the ventral portion of the wall of the third ventricle and in the radial processes penetrating the neuropil. NaX immunostaining colocalized with vimentin immunostaining only , of vimentin staining  and of GFAP staining  ruled out possible colocalization of NaX immunostaining with tanycytes or glial cells in the rat and mouse AP. In the other CVOs, NaX cellular localization was strongly divergent  and oxytocin (OT–NP) immunofluorescence . These results show that NaX immunostaining colocalizes with the magnocellular vasopressinergic and oxytocinergic neurons in the SON  and antinatriuretic hormone (oxytocin) in rodents. Nans Table . NaX immN Figure A. To fur Figures B,C and i Figures A,B. MoreP Figure .X antibody specifically designed against the ID2–3 region of the channel’s α-subunit in order to compare the mouse and rat’s expression patterns among the brain areas involved in the control of hydromineral balance. In addition, double immunohistochemical staining was performed to characterize the phenotype of the cells that expressed NaX in both rodent brains. Our results indicate that the expression pattern of NaX and the cell type distribution show similarities between the rat and mouse, as well as noticeable differences. In these two rodents, NaX was clearly present in the primary regions involved in hydromineral homeostasis, which are the SFO, OVLT, and the ME. Interestingly, NaX was also found in the AP, the SON, and the PVN of these two species, thereby supporting the claim that this channel is involved in the osmoregulatory process and control of salt appetite. NaX was strongly expressed in the rat but not in the mouse MnPO. However, the most noticeable differences observed between the rat and mouse concerned the phenotype of the cells expressing NaX in the forebrain CVOs. Indeed, in the rat, NaX is present specifically in neurons of the SFO, OVLT, and MnPO, as well as in the ependymocytes lining the two CVOs. In the mouse, NaX is only expressed in the ependymocyte layers boarding the SFO and the OVLT. This comparative anatomical study was carried out with a highly sensitive anti-NaX antibody, which identified the pattern of NaX expression and clarified the specificity of the NaX-expressing cell types in the brain regions that are involved in sodium detection in the rat and mouse.The present study used a NaX in the rat and mouse brain, we produced an antibody against the ID2–3 region of the rat NaX protein. This region is highly conserved between the rat and mouse but shows weak homology with other voltage-gated sodium channels. A mandatory step in validating this anti-NaX antibody was to assess the purification level and the specificity toward the NaX channel. We showed that the polyclonal anti-NaX antibody specifically bound the immunogenic peptide without interference from the GST protein. The anti-NaX antibody labeled the rat MnPO nucleus, which has been previously described as being a NaX-positive brain nucleus , like previously observed  or the FoxP2-immunoreactive neurons in specific divisions of the parabrachial nucleus  of the SON and PVN in the rat and mouse represents a major finding of this anatomical study. The expression of the NaX channel by both the vasopressinergic and oxytocinergic MNCs was unexpected according to a previous mapping study of the NaX gene in the mouse brain , but not abolished, by the application of gadolinium, which is a specific TRPV blocker (Voisin et al., X channel. Indeed, such a current amplitude is well in the range of the sodium inward current flowing through NaX in the MnPO neurons recorded in situ (Grob et al., The presence of Nae et al.  used a NX expression in all the brain areas involved in the monitoring of plasma and CSF Na+ concentration in the rat and mouse. Beside the expected location of NaX in the forebrain CVOs, NaX was found in the AP, the ME and in the magnocellular neuroendocrine cells, which synthesize and release vasopressin and oxytocin to regulate systemic water and Na+ balance. The noticeable discrepancy concerns the NaX-expressing cell types in the network controlling Na+ sensing in the rat and mouse. The pattern of NaX expression was clearly different in the nuclei of the lamina terminalis, because NaX was expressed in neurons, ependymocytes, and tanycytes of the rat, whereas NaX was restricted to ependymocytes and tanycytes in the mouse. In addition, NaX was not expressed in the mouse MnPO. The difference in NaX expression pattern and in NaX-expressing cell types strongly suggests that the mechanisms involved in systemic and central Na+ sensing are specific to each rodent species.The present study reveals the common and strategic NaThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.