Elevated glucocorticoid alters the developmental dynamics of hypothalamic neurogenesis in zebrafish.

Exposure to excess glucocorticoid (GC) during early development is implicated in adult dysfunctions. Reduced adult hippocampal neurogenesis is a well-known consequence of exposure to early life stress or elevated GC, however the effects on neurogenesis during development and effects on other brain regions are not well understood. Using an optogenetic zebrafish model, here we analyse the effects of GC exposure on neurogenesis during development in the whole brain. We identify that the hypothalamus is a highly GC-sensitive region where elevated GC causes precocious development. This is followed by failed maturation and early decline accompanied by impaired feeding, growth, and survival. In GC-exposed animals, the developmental trajectory of hypothalamic progenitor cells is strikingly altered, potentially mediated by direct regulation of transcription factors such as rx3 by GC. Our data provide cellular and molecular level insight into GC-induced alteration of the hypothalamic developmental trajectory, a process crucial for health across the life-course.

The Effects of Early Life Stress on the Brain and Behaviour: Insights From Zebrafish Models.

The early life period represents a window of increased vulnerability to stress, during which exposure can lead to long-lasting effects on brain structure and function. This stress-induced developmental programming may contribute to the behavioural changes observed in mental illness. In recent decades, rodent studies have significantly advanced our understanding of how early life stress (ELS) affects brain development and behaviour. These studies reveal that ELS has long-term consequences on the brain such as impairment of adult hippocampal neurogenesis, altering learning and memory. Despite such advances, several key questions remain inadequately answered, including a comprehensive overview of brain regions and molecular pathways that are altered by ELS and how ELS-induced molecular changes ultimately lead to behavioural changes in adulthood. The zebrafish represents a novel ELS model, with the potential to contribute to answering some of these questions. The zebrafish offers some important advantages such as the ability to non-invasively modulate stress hormone levels in a whole animal and to visualise whole brain activity in freely behaving animals. This review discusses the current status of the zebrafish ELS field and its potential as a new ELS model.

Transgenic Mice Overexpressing PG1 Display Corneal Opacity and Severe Inflammation in the Eye.

Antimicrobial peptides (AMPs) are of interest as alternatives to antibiotics or immunomodulators. We generated and characterized the phenotypes of transgenic mice overexpressing protegrin 1 (PG1), a potent porcine cathelicidin. No obvious differences were observed between PG1 transgenic and wild-type mice in terms of growth, development, general behaviour, and the major immune cell population. However, PG1 transgenic mice intranasally infected with Staphylococcus aureus resulted in a reduction in microscopic pulmonary injury, improved clearance of bacteria, and lower proinflammatory cytokine secretion, compared to those of wild-type mice. On the other hand, approximately 25% of PG1 transgenic mice (n = 54/215) showed corneal opacity and developed inflammation in the eye, resulting ultimately in phthisis bulbi. Immunohistochemical analyses revealed that PG1 and its activator, neutrophil elastase, localized to the basal cells of the cornea and glands in eyelids, respectively. In addition, apoptosis indicated by a Terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL)-positive signal was detected from flat cells of the cornea. Our study suggests that the expression regulation or localization of AMPs such as PG1 is important to prevent their adverse effects. However, our results also showed that the cytotoxic effects of PG1 on cells could be tolerated in animals, except for the eyes.

Analysis of allele-specific expression using RNA-seq of the Korean native pig and Landrace reciprocal cross.

OBJECTIVE: We tried to analyze allele-specific expression in the pig neocortex using bioinformatic analysis of high-throughput sequencing results from the parental genomes and offspring transcriptomes from reciprocal crosses between Korean Native and Landrace pigs. METHODS: We carried out sequencing of parental genomes and offspring transcriptomes using next generation sequencing. We subsequently carried out genome scale identification of SNPs in two different ways using either individual genome mapping or joint genome mapping of the same breed parents that were used for the reciprocal crosses. Using parent-specific SNPs, allele-specifically expressed genes were analyzed. RESULTS: Because of the low genome coverage (~4x) of the sequencing results, most SNPs were non-informative for parental lineage determination of the expressed alleles in the offspring and were thus excluded from our analysis. Consequently, 436 SNPs covering 336 genes were applicable to measure the imbalanced expression of paternal alleles in the offspring. By calculating the read ratios of parental alleles in the offspring, we identified seven genes showing allele-biased expression (P < 0.05) including three previously reported and four newly identified genes in this study. CONCLUSION: The newly identified allele-specifically expressing genes in the neocortex of pigs should contribute to improving our knowledge on genomic imprinting in pigs. To our knowledge, this is the first study of allelic imbalance using high throughput analysis of both parental genomes and offspring transcriptomes of the reciprocal cross in outbred animals. Our study also showed the effect of the number of informative animals on the genome level investigation of allele-specific expression using RNA-seq analysis in livestock species.

Determination of complete sequence information of the human ABO blood group orthologous gene in pigs and breed difference in blood type frequencies.

The sequence information of the genomic form of the human ABO blood group orthologous gene (erythrocyte antigen A, EAA) is not complete in pigs. Therefore, we cloned and characterized the nucleotide sequence of EAA intron 7, which is critical to understand genetic difference between A and 0 blood groups in pigs, covering complete genomic sequence information of EAA excluding a ~560bp unsequencible gap. We also analyzed genetic polymorphisms within EAA intron 7 and exon 8. We found difference in A0 blood group frequencies among pig breeds. In addition, we designed a new genomic DNA-based A0 blood group typing method and improved the accuracy and simplicity of the typing.

ß2-microglobulin gene duplication in cetartiodactyla remains intact only in pigs and possibly confers selective advantage to the species.

Several ß2-microglobulin (B2M) -bound protein complexes undertake key roles in various immune system pathways, including the neonatal Fc receptor (FcRn), cluster of differentiation 1 (CD1) protein, non-classical major histocompatibility complex (MHC), and well-known MHC class I molecules. Therefore, the duplication of B2M may lead to an increase in the biological competence of organisms to the environment. Based on the pig genome assembly SSC10.2, a segmental duplication of ~45.5 kb, encoding the entire B2M protein, was identified in pig chromosome 1. Through experimental validation, we confirmed the functional duplication of the B2M gene with a completely identical coding sequence between two copies in pigs. Considering the importance of B2M in the immune system, we performed the phylogenetic analysis of B2M duplication in ten mammalian species, confirming the presence of B2M duplication in cetartioldactyls, like cattle, sheep, goats, pigs and whales, but non-cetartiodactyl species, like mice, cats, dogs, horses, and humans. The density of long interspersed nuclear element (LINE) at the edges of duplicated blocks (39 to 66%) was found to be 2 to 3-fold higher than the average (20.12%) of the pig genome, suggesting its role in the duplication event. The B2M mRNA expression level in pigs was 12.71 and 7.57 times (2-ΔΔCt values) higher than humans and mice, respectively. However, we were unable to experimentally demonstrate the difference in the level of B2M protein because species specific anti-B2M antibodies are not available. We reported, for the first time, the functional duplication of the B2M gene in animals. The identification of partially remaining duplicated B2M sequences in the genomes of only cetartiodactyls indicates that the event was lineage specific. B2M duplication could be beneficial to the immune system of pigs by increasing the availability of MHC class I light chain protein, B2M, to complex with the proteins encoded by the relatively large number of MHC class I heavy chain genes in pigs. Further studies are necessary to address the biological meaning of increased expression of B2M.

Analysis of the vomeronasal receptor repertoire, expression and allelic diversity in swine.

Here we report a comprehensive analysis of the vomeronasal receptor repertoire in pigs. We identified a total of 25 V1R sequences consisting of 10 functional genes, 3 pseudogenes, and 12 partial genes, while functional V2R and FPR genes were not present in the pig genome. Pig V1Rs were classified into three subfamilies, D, F, and J. Using direct high resolution sequencing-based typing of all functional V1Rs from 10 individuals of 5 different breeds, a total of 24 SNPs were identified, indicating that the allelic diversity of V1Rs is much lower than that of the olfactory receptors. A high expression level of V1Rs was detected in the vomeronasal organ (VNO) and testes, while a low expression level of V1Rs was observed in all other tissues examined. Our results showed that pigs could serve as an interesting large animal model system to study pheromone-related neurobiology because of their genetic simplicity.

Development of a simultaneous high resolution typing method for three SLA class II genes, SLA-DQA, SLA-DQB1, and SLA-DRB1 and the analysis of SLA class II haplotypes.

The characterization of the genetic variations of major histocompatibility complex (MHC) is essential to understand the relationship between the genetic diversity of MHC molecules and disease resistance and susceptibility in adaptive immunity. We previously reported the development of high-resolution individual locus typing methods for three of the most polymorphic swine leukocyte antigens (SLA) class II loci, namely, SLA-DQA, SLA-DQB1, and SLA-DRB1. In this study, we extensively modified our previous protocols and developed a method for the simultaneous amplification of the three SLA class II genes and subsequent analysis of individual loci using direct sequencing. The unbiased and simultaneous amplification of alleles from the all three hyper-polymorphic and pseudogene containing genes such as MHC genes is extremely challenging. However, using this method, we demonstrated the successful typing of SLA-DQA, SLA-DQB1, and SLA-DRB1 for 31 selected individuals comprising 26 different SLA class II haplotypes which were identified from 700 animals using the single locus typing methods. The results were identical to the known genotypes from the individual locus typing. The new method has significant benefits over the individual locus typing, including lower typing cost, use of less biomaterial, less effort and fewer errors in handling large samples for multiple loci. We also extensively characterized the haplotypes of SLA class II genes and reported three new haplotypes. Our results should serve as a basis to investigate the possible association between polymorphisms of MHC class II and differences in immune responses to exogenous antigens.

Defining the genetic relationship of protegrin-related sequences and the in vivo expression of protegrins.

Protegrins (PGs) are potent antimicrobial peptides that act on a broad spectrum of microorganisms, including bacteria, fungi and some enveloped viruses. We analyzed the expression pattern of protegrins in 17 different pig tissues using RT-PCR, and developed an anti-(PG-1) polyclonal IgG. Western blot analysis using the antibody showed that protegrins are mainly present as prepropeptide forms in normal tissues, rather than as mature peptides. Immunohistochemical analysis showed that protegrin expression was specific to a few cell types, including neutrophils, pulmonary club, epithelial and Leydig cells. Genetic analyses of the five previously reported protegrin sequences showed that they are encoded at a single locus, rather than from multiple paralogous genes. By genotyping 28 animals across five breeds, we identified eight different alleles of the PGs.

The complete swine olfactory subgenome: expansion of the olfactory gene repertoire in the pig genome.

BACKGROUND: Insects and animals can recognize surrounding environments by detecting thousands of chemical odorants. Olfaction is a complicated process that begins in the olfactory epithelium with the specific binding of volatile odorant molecules to dedicated olfactory receptors (ORs). OR proteins are encoded by the largest gene superfamily in the mammalian genome. RESULTS: We report here the whole genome analysis of the olfactory receptor genes of S. scrofa using conserved OR gene specific motifs and known OR protein sequences from diverse species. We identified 1,301 OR related sequences from the S. scrofa genome assembly, Sscrofa10.2, including 1,113 functional OR genes and 188 pseudogenes. OR genes were located in 46 different regions on 16 pig chromosomes. We classified the ORs into 17 families, three Class I and 14 Class II families, and further grouped them into 349 subfamilies. We also identified inter- and intra-chromosomal duplications of OR genes residing on 11 chromosomes. A significant number of pig OR genes (n = 212) showed less than 60% amino acid sequence similarity to known OR genes of other species. CONCLUSION: As the genome assembly Sscrofa10.2 covers 99.9% of the pig genome, our analysis represents an almost complete OR gene repertoire from an individual pig genome. We show that S. scrofa has one of the largest OR repertoires, suggesting an expansion of OR genes in the swine genome. A significant number of unique OR genes in the pig genome may suggest the presence of swine specific olfactory stimulation.

Genome-level identification, gene expression, and comparative analysis of porcine ß-defensin genes.

BACKGROUND: Beta-defensins (ß-defensins) are innate immune peptides with evolutionary conservation across a wide range of species and has been suggested to play important roles in innate immune reactions against pathogens. However, the complete ß-defensin repertoire in the pig has not been fully addressed. RESULT: A BLAST analysis was performed against the available pig genomic sequence in the NCBI database to identify ß-defensin-related sequences using previously reported ß-defensin sequences of pigs, humans, and cattle. The porcine ß-defensin gene clusters were mapped to chromosomes 7, 14, 15 and 17. The gene expression analysis of 17 newly annotated porcine ß-defensin genes across 15 tissues using semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) showed differences in their tissue distribution, with the kidney and testis having the largest pBD expression repertoire. We also analyzed single nucleotide polymorphisms (SNPs) in the mature peptide region of pBD genes from 35 pigs of 7 breeds. We found 8 cSNPs in 7 pBDs. CONCLUSION: We identified 29 porcine ß-defensin (pBD) gene-like sequences, including 17 unreported pBDs in the porcine genome. Comparative analysis of ß-defensin genes in the pig genome with those in human and cattle genomes showed structural conservation of ß-defensin syntenic regions among these species.

Identification and classification of endogenous retroviruses in the canine genome using degenerative PCR and in-silico data analysis.

Pooled genomic DNA from 10 dogs was subjected to polymerase chain reaction with primers targeting the retroviral pro/pol region. Sequence analysis of 120 clones obtained by PCR revealed 81 of retroviral origin. Subsequent analysis of the dog genome (CanFam 2.0) by BLAST investigation using degenerate PCR products and previously identified retroviral sequences permitted the identification of additional retroviral γ and ß sequences. A phylogenetic analysis using the retroviral protease (PR) and reverse transcriptase (RT) sequences in the dog genome resulted in identification of 17 γ and 7 ß families. In addition, we also identified 167 spuma-like ERV elements from CanFam 2.0 based on sequence homology to murine (Mu)ERV-L and human (H)ERV-L. Our results could contribute to the understanding of the influence of retroviruses in shaping the genome structure and altering gene expression by providing quantitative and locational information of ERV loci and their diversity in the dog genome.