Gene expression atlas of the domestic goat and comparative analysis of immune signatures with sheep

Goats are an economically important livestock species providing a resource of meat and milk across the world. They are of particular importance in developing countries contributing to sustainable agriculture, alleviation of poverty and utilisation of marginal grazing. Recently, a highly contiguous refence genome (ARS1) of the domestic goat was released. However, gene expression information on the domestic goat is particularly limited when compared to other ruminants. Despite great genetic similarity, ruminants vary in their susceptibility to similar pathogens, but the underlying molecular mechanisms remain largely unknown. To elucidate the molecular basis of variation in disease response in small ruminants, a gene expression atlas of the domestic goat was generated from a subset of 22 tissue and cell types and compared to the previously developed sheep gene expression atlas. Fifty-four mRNA-Seq (poly-A selected) 75bp paired-end libraries spanning all major organ systems in the domestic goat were produced, generating a total of 8.7×108 paired end sequence reads. The tissues and cell-types sampled were all transcriptionally complex, with each expressing at least 50% of the total protein coding genes at detectable levels. 18,528 protein coding genes (out of a possible 21,343) had detectable expression in at least one tissue sampled, enabling the capture of 90% of the reference transcriptome. Additionally, of the 21,343 protein coding genes in the ARS1 reference transcriptome 7,036 (33%) had no informative gene name. Using the HISAT2 annotation pipeline, informative gene names were assigned to 1,114 (15%) of the previously un-annotated protein coding genes in ARS1, greatly expanding the previously available genetic and genomic resources available for goat. Using network cluster analysis, genes were assigned to specific biological pathways or cell populations based on expression profiles. Clusters of genes in the liver, gastro-intestinal tract and those involved in innate immunity are analysed and discussed in detail. Additionally, a protocol to isolate goat bone marrow derived macrophages (BMDM) and culture them in the presence of macrophage colony stimulating factor (CSF1) was developed and optimized. The goat BMDM were characterised using light microscopy to confirm morphology as well as flow cytometry to investigate the cell surface markers. Flow cytometry results revealed that goat BMDM express CD14, CD16 and CD172a on the surface similar to sheep macrophages. When exposed to bacterial lipopolysaccharide (LPS), goat BMDM responded by inducing inflammatory cytokines such as TNF, interferon-associated genes including IFI6, IFIT3 and IFNG and interleukins such as IL10RA, IL12B, IL16 and IL1RAP similar to sheep BMDM. However, unlike sheep, goat BMDM produced detectable levels of nitric oxide (NO) post-LPS stimulation. The goat BMDM post-LPS stimulation were also analysed with RNA-Seq to reveal hundreds of upregulated genes further expanding the transcriptional data available for goat. Finally, the data generated from the network cluster analysis of the goat was used to run a comparative analysis with the larger gene expression atlas of the domestic sheep, revealing transcriptional differences between the two species which may underlie the mechanisms controlling disease variation

Comprehensive transcriptional profiling of the gastrointestinal tract of ruminants from birth to adulthood reveals strong developmental stage specific gene expression

One of the most significant physiological challenges to neonatal and juvenile ruminants is the development and establishment of the rumen. Using a subset of RNA-Seq data from our high-resolution atlas of gene expression in sheep (Ovis aries) we have provided the first comprehensive characterization of transcription of the entire gastrointestinal (GI) tract during the transition from pre-ruminant to ruminant. The dataset comprises 164 tissue samples from sheep at four different time points (birth, one week, 8 weeks and adult). Using network cluster analysis we illustrate how the complexity of the GI tract is reflected in tissueand developmental stage-specific differences in gene expression. The most significant transcriptional differences between neonatal and adult sheep were observed in the rumen complex. Comparative analysis of gene expression in three GI tract tissues from age-matched sheep and goats revealed species-specific differences in genes involved in immunity and metabolism. This study improves our understanding of the transcriptomic mechanisms involved in the transition from pre-ruminant to ruminant by identifying key genes involved in immunity, microbe recognition and metabolism. The results form a basis for future studies linking gene expression with microbial colonization of the developing GI tract and provide a foundation to improve ruminant efficiency and productivity through identifying potential targets for novel therapeutics and gene editing

Species-specificity of transcriptional regulation and the response to lipopolysaccharide in mammalian macrophages.

Mammalian macrophages differ in their basal gene expression profiles and response to the toll-like receptor 4 (TLR4) agonist, lipopolysaccharide (LPS). In human macrophages, LPS elicits a temporal cascade of transient gene expression including feed forward activators and feedback regulators that limit the response. Here we present a transcriptional network analysis of the response of sheep bone marrow-derived macrophages (BMDM) to LPS based upon RNA-seq at 0, 2, 4, 7, and 24 h post-stimulation. The analysis reveals a conserved transcription factor network with humans, and rapid induction of feedback regulators that constrain the response at every level. The gene expression profiles of sheep BMDM at 0 and 7 h post LPS addition were compared to similar data obtained from goat, cow, water buffalo, horse, pig, mouse and rat BMDM. This comparison was based upon identification of 8,200 genes annotated in all species and detected at >10TPM in at least one sample. Analysis of expression of transcription factors revealed a conserved transcriptional millieu associated with macrophage differentiation and LPS response. The largest co-expression clusters, including genes encoding cell surface receptors, endosome–lysosome components and secretory activity, were also expressed in all species and the combined dataset defines a macrophage functional transcriptome. All of the large animals differed from rodents in lacking inducible expression of genes involved in arginine metabolism and nitric oxide production. Instead, they expressed inducible transporters and enzymes of tryptophan and kynurenine metabolism. BMDM from all species expressed high levels of transcripts encoding transporters and enzymes involved in glutamine metabolism suggesting that glutamine is a major metabolic fuel. We identify and discuss transcripts that were uniquely expressed or regulated in rodents compared to large animals including ACOD1, CXC and CC chemokines, CD163, CLEC4E, CPM, CSF1, CSF2, CTSK, MARCO, MMP9, SLC2A3, SLC7A7, and SUCNR1. Conversely, the data confirm the conserved regulation of multiple transcripts for which there is limited functional data from mouse models and knockouts. The data provide a resource for functional annotation and interpretation of loci involved in susceptibility to infectious and inflammatory disease in humans and large animal species

Functional annotation of the transcriptome of the pig, Sus scrofa, based upon network analysis of an RNAseq transcriptional atlas

The domestic pig (Sus scrofa) is both an economically important livestock species and a model for biomedical research. Two highly contiguous pig reference genomes have recently been released. To support functional annotation of the pig genomes and comparative analysis with large human transcriptomic data sets, we aimed to create a pig gene expression atlas. To achieve this objective, we extended a previous approach developed for the chicken. We downloaded RNAseq data sets from public repositories, down-sampled to a common depth, and quantified expression against a reference transcriptome using the mRNA quantitation tool, Kallisto. We then used the network analysis tool Graphia to identify clusters of transcripts that were coexpressed across the merged data set. Consistent with the principle of guilt-by-association, we identified coexpression clusters that were highly tissue or cell-type restricted and contained transcription factors that have previously been implicated in lineage determination. Other clusters were enriched for transcripts associated with biological processes, such as the cell cycle and oxidative phosphorylation. The same approach was used to identify coexpression clusters within RNAseq data from multiple individual liver and brain samples, highlighting cell type, process, and region-specific gene expression. Evidence of conserved expression can add confidence to assignment of orthology between pig and human genes. Many transcripts currently identified as novel genes with ENSSSCG or LOC IDs were found to be coexpressed with annotated neighbouring transcripts in the same orientation, indicating they may be products of the same transcriptional unit. The meta-analytic approach to utilising public RNAseq data is extendable to include new data sets and new species and provides a framework to support the Functional Annotation of Animals Genomes (FAANG) initiative

A mini-atlas of gene expression for the domestic goat (Capra hircus)

Goats (Capra hircus) are an economically important livestock species providing meat and milk across the globe. They are of particular importance in tropical agri-systems contributing to sustainable agriculture, alleviation of poverty, social cohesion, and utilisation of marginal grazing. There are excellent genetic and genomic resources available for goats, including a highly contiguous reference genome (ARS1). However, gene expression information is limited in comparison to other ruminants. To support functional annotation of the genome and comparative transcriptomics, we created a mini-atlas of gene expression for the domestic goat. RNA-Seq analysis of 17 transcriptionally rich tissues and 3 cell-types detected the majority (90%) of predicted protein-coding transcripts and assigned informative gene names to more than 1000 previously unannotated protein-coding genes in the current reference genome for goat (ARS1). Using network-based cluster analysis, we grouped genes according to their expression patterns and assigned those groups of coexpressed genes to specific cell populations or pathways. We describe clusters of genes expressed in the gastro-intestinal tract and provide the expression profiles across tissues of a subset of genes associated with functional traits. Comparative analysis of the goat atlas with the larger sheep gene expression atlas dataset revealed transcriptional similarities between macrophage associated signatures in the sheep and goats sampled in this study. The goat transcriptomic resource complements the large gene expression dataset we have generated for sheep and contributes to the available genomic resources for interpretation of the relationship between genotype and phenotype in small ruminants

Species-Specific Transcriptional Regulation of Genes Involved in Nitric Oxide Production and Arginine Metabolism in Macrophages

Activated mouse macrophages metabolize arginine via NO synthase (NOS2) to produce NO as an antimicrobial effector. Published gene expression datasets provide little support for the activation of this pathway in human macrophages. Generation of NO requires the coordinated regulation of multiple genes. We have generated RNA-sequencing data from bone marrow–derived macrophages from representative rodent (rat), monogastric (pig and horse), and ruminant (sheep, goat, cattle, and water buffalo) species, and analyzed the expression of genes involved in arginine metabolism in response to stimulation with LPS. In rats, as in mice, LPS strongly induced Nos2, the arginine transporter Slc7a2, arginase 1 (Arg1), GTP cyclohydrolase (Gch1), and argininosuccinate synthase (Ass1). None of these responses was conserved across species. Only cattle and water buffalo showed substantial NOS2 induction. The species studied also differed in expression and regulation of arginase (ARG2, rather than ARG1), and amino acid transporters. Variation between species was associated with rapid promoter evolution. Differential induction of NOS2 and ARG2 between the ruminant species was associated with insertions of the Bov-A2 retrotransposon in the promoter region. Bov-A2 was shown to possess LPS-inducible enhancer activity in transfected RAW264.7 macrophages. Consistent with a function in innate immunity, NO production and arginine metabolism vary greatly between species and differences may contribute to pathogen host restriction

Cross-species inference of long non-coding RNAs greatly expands the ruminant transcriptome

Additional file 3. This file contains all supplementary tables relating to lncRNA identification via the conservation of synteny. Table S3. lncRNAs inferred in one species by the genomic alignment of a transcript assembled with the RNA-seq libraries from a related spdecies. Table S12. Presence of intergenic lncRNAs both in sheep and cattle, in regions of conserved synteny. Table S13. Presence of intergenic lncRNAs both in sheep and goat, in regions of conserved synteny. Table S14. Presence of intergenic lncRNAs both in cattle and goat, in regions of conserved synteny. Table S15. Presence of intergenic lncRNAs both in sheep and humans, in regions of conserved synteny. Table S16. Presence of intergenic lncRNAs both in goat and humans, in regions of conserved synteny. Table S17. Presence of intergenic lncRNAs both in cattle and humans, in regions of conserved synteny. Table S18. High-confidence lncRNA pairs, those conserved across species both sequentially and positionally

Identification of Unique Family-Specific Markers for Molecular Detection of Arboviruses: Application to Bunyaviridae and Togaviridae Families

Recent incidences of emerging arbovirus (arthropod-borne) disease outbreaks have led to heavy losses globally. Molecular virology offers a range of methods, able to accelerate and improve the diagnosis of arbovirus diseases but these rely heavily on the availability of robust PCR-based markers of the causative agents. The great genetic diversity exhibited by viral genomes makes it difficult to design specific signatures or markers since most methods start with a multiple sequence alignment (MSA). This study aimed at exploring the use of recently developed software (PriMux) prototyped on viral pathogens, that designs multiplex compatible degenerate primers without need for an initial MSA. It was used to design family-specific molecular signatures for arboviruses and members from the bunyaviridae and togaviridae virus families were used as models. Keywords: Arboviruses, Bunyaviru