Ontogeny of telomerase in chicken: impact of downregulation on pre- and postnatal telomere length in vivo

Telomeres are the termini of linear chromosomes composed of tandem repeats of a conserved DNA sequence. Telomerase provides a mechanism for proliferating cells to offset telomeric sequence erosion by synthesizing new repeats onto the end of each parental DNA strand. Reduced or absent telomerase activity can lead to telomere shortening and genome instability. Telomeres and telomerase have not previously been characterized during ontogeny of any avian species. In the present study, telomerase activity in the chicken model was examined from early differentiation embryos through to adulthood. Telomerase activity was detected in all early embryos (preblastula through neurula) and in tissues throughout organogenesis. Subsequently, telomerase was downregulated in the majority of somatic tissues, either pre- or postnatally. A subset of tissues, such as intestine, immune and reproductive organs, exhibited constitutive activity. The impact of telomerase downregulation on telomere length was investigated and a telomere reduction of 3.2 kb in somatic tissues compared with germ line was observed in 5-year-old adults. The present results suggest that the telomere clock function is a conserved feature of avians as well as mammals. Knowledge regarding the relationships among telomerase regulation, proliferation/senescence profiles and differentiation status will be useful for numerous applications of chicken cells.     

Gene duplication and fragmentation in the zebra finch major histocompatibility complex

Background

Due to its high polymorphism and importance for disease resistance, the major histocompatibility complex (MHC) has been an important focus of many vertebrate genome projects. Avian MHC organization is of particular interest because the chicken Gallus gallus, the avian species with the best characterized MHC, possesses a highly streamlined minimal essential MHC, which is linked to resistance against specific pathogens. It remains unclear the extent to which this organization describes the situation in other birds and whether it represents a derived or ancestral condition. The sequencing of the zebra finch Taeniopygia guttata genome, in combination with targeted bacterial artificial chromosome (BAC) sequencing, has allowed us to characterize an MHC from a highly divergent and diverse avian lineage, the passerines.

Results

The zebra finch MHC exhibits a complex structure and history involving gene duplication and fragmentation. The zebra finch MHC includes multiple Class I and Class II genes, some of which appear to be pseudogenes, and spans a much more extensive genomic region than the chicken MHC, as evidenced by the presence of MHC genes on each of seven BACs spanning 739 kb. Cytogenetic (FISH) evidence and the genome assembly itself place core MHC genes on as many as four chromosomes with TAP and Class I genes mapping to different chromosomes. MHC Class II regions are further characterized by high endogenous retroviral content. Lastly, we find strong evidence of selection acting on sites within passerine MHC Class I and Class II genes.

Conclusion

The zebra finch MHC differs markedly from that of the chicken, the only other bird species with a complete genome sequence. The apparent lack of synteny between TAP and the expressed MHC Class I locus is in fact reminiscent of a pattern seen in some mammalian lineages and may represent convergent evolution. Our analyses of the zebra finch MHC suggest a complex history involving chromosomal fission, gene duplication and translocation in the history of the MHC in birds, and highlight striking differences in MHC structure and organization among avian lineages.     

Dynamics of telomere erosion in transformed and non-transformed avian cells in vitro

Although vertebrate telomeres are highly conserved, telomere dynamics and telomerase profiles vary among species. The objective of the present study was to examine telomerase activity and telomere length profiles of transformed and non-transformed avian cells in vitro. Non-transformed chicken embryo fibroblasts (CEFs) showed little or no telomerase activity from the earliest passages through senescence. Unexpectedly, a single culture of particularly long-lived senescent CEFs showed telomerase activity after over 250 days in culture. Transformed avian lines (six chicken, two quail and one turkey) and tumor samples (two chicken) exhibited telomerase activity. Telomere length profiles of non-transformed CEF cultures derived from individual embryos of an inbred line (UCD 003) exhibited cycles of shortening and lengthening with a substantial net loss of telomeric DNA by senescence. The telomere length profiles of several transformed cell lines resembled telomere length profiles of senescent CEFs in that they exhibited little of the typical smear of terminal restriction fragments (TRFs) suggesting that these transformed cells may possess a reduced amount of telomeric DNA. These results show that avian telomerase activity profiles are consistent with the telomerase activity profiles of human primary and transformed cells. Further, monitoring of telomere lengths of primary cells provides evidence for a dynamic series of changes over the lifespan of any specific cell culture ultimately resulting in net telomeric DNA loss by senescence.     

DNA repeat arrays in chicken and human genomes and the adaptive evolution of avian genome size

Background

Birds have smaller average genome sizes than other tetrapod classes, and it has been proposed that a relatively low frequency of repeating DNA is one factor in reduction of avian genome sizes.

Results

DNA repeat arrays in the sequenced portion of the chicken (Gallus gallus) autosomes were quantified and compared with those in human autosomes. In the chicken 10.3% of the genome was occupied by DNA repeats, in contrast to 44.9% in human. In the chicken, the percentage of a chromosome occupied by repeats was positively correlated with chromosome length, but even the largest chicken chromosomes had repeat densities much lower than those in human, indicating that avoidance of repeats in the chicken is not confined to minichromosomes. When 294 simple sequence repeat types shared between chicken and human genomes were compared, mean repeat array length and maximum repeat array length were significantly lower in the chicken than in human.

Conclusions

The fact that the chicken simple sequence repeat arrays were consistently smaller than arrays of the same type in human is evidence that the reduction in repeat array length in the chicken has involved numerous independent evolutionary events. This implies that reduction of DNA repeats in birds is the result of adaptive evolution. Reduction of DNA repeats on minichromosomes may be an adaptation to permit chiasma formation and alignment of small chromosomes. However, the fact that repeat array lengths are consistently reduced on the largest chicken chromosomes supports the hypothesis that other selective factors are at work, presumably related to the reduction of cell size and consequent advantages for the energetic demands of flight.     

Interspecific germline transmission of cultured primordial germ cells

In birds, the primordial germ cell (PGC) lineage separates from the soma within 24 h following fertilization. Here we show that the endogenous population of about 200 PGCs from a single chicken embryo can be expanded one million fold in culture. When cultured PGCs are injected into a xenogeneic embryo at an equivalent stage of development, they colonize the testis. At sexual maturity, these donor PGCs undergo spermatogenesis in the xenogeneic host and become functional sperm. Insemination of semen from the xenogeneic host into females from the donor species produces normal offspring from the donor species. In our model system, the donor species is chicken (Gallus domesticus) and the recipient species is guinea fowl (Numida meleagris), a member of a different avian family, suggesting that the mechanisms controlling proliferation of the germline are highly conserved within birds. From a pragmatic perspective, these data are the basis of a novel strategy to produce endangered species of birds using domesticated hosts that are both tractable and fecund.     

Effects of sexual maturation and Salmonella infection on the expression of Toll-like receptors in the chicken vagina

Toll-like receptors (TLRs) are a critical component of the innate immune response in many vertebrates, including avian species. The recent findings of chicken TLRs (cTLRs) expression in ovarian follicles and in the chicken ovary in vivo, as well as the changes in their expression in response to lipopolysaccharide or Salmonella enteritidis (SE) infection, have broad implications for reproductive physiology and for the prevention of transmission of zoonotic diseases to humans through the consumption of contaminated poultry eggs. Because the main route of egg contamination is from infection of the oviduct and mainly from the vagina, the aim of this study was to investigate the expression of the ten cTLRs identified to date in the chicken oviduct in vivo, to determine whether sexual maturation affects their mRNA abundance and to investigate whether SE infection alters the expression of TLRs in the chicken vagina. RNA was extracted from the vagina of healthy prepubertal, sexually mature and aged birds, and from sexually mature and aged SE infected birds. RT-PCR analysis revealed that all types of cTLRs apart from TLR1-1 were expressed in the vagina of sexually mature birds. Quantitative real-time PCR analysis revealed that the mRNA abundance of TLR2-1, 2-2 and 4 differ with respect to sexual maturation in the chicken vagina. SE infection resulted in a significant induction of TLR5 and 15 in the vagina of sexually mature birds, and in a significant induction of TLR2-1, 4 and 15 in the vagina of aged birds, while a significant down-regulation was observed for TLR7 in the vagina of sexually mature birds. These findings suggest that a TLR mediated immune response mechanism exists in the chicken vagina, playing a crucial role in preventing microbial pathogens from being incorporated into newly forming eggs.     

Telomere biology in mammalian germ cells and during development

The development of an organism is a strictly regulated program in which controlled gene expression guarantees the establishment of a specific phenotype. The chromosome termini or so-called telomeres preserve the integrity of the genome within developing cells. In the germline, during early development, and in highly proliferative organs, human telomeres are balanced between shortening processes with each cell division and elongation by telomerase, but once terminally differentiated or mature the equilibrium is shifted to gradual shortening by repression of the telomerase enzyme. Telomere length is to a large extent genetically determined and the neonatal telomere length equilibrium is, in fact, a matter of evolution. Gradual telomere shortening in normal human somatic cells during consecutive rounds of replication eventually leads to critically short telomeres that induce replicative senescence in vitro and probably in vivo. Hence, a molecular clock is set during development, which determines the replicative potential of cells during extrauterine life. Telomeres might be directly or indirectly implicated in longevity determination in vivo, and information on telomere length setting in utero and beyond should help elucidate presumed causal connections between early growth and aging disorders later in life. Only limited information exists concerning the mechanisms underlying overall telomere length regulation in the germline and during early development, especially in humans. The intent of this review is to focus on recent advances in our understanding of telomere biology in germline cells as well as during development (pre- and postimplantation periods) in an attempt to summarize our knowledge about telomere length determination and its importance for normal development in utero and the occurrence of the aging and abnormal phenotype later on.     

Application of chicken microarrays for gene expression analysis in other avian species

Background

With the threat of emerging infectious diseases such as avian influenza, whose natural hosts are thought to be a variety of wild water birds including duck, we are armed with very few genomic resources to investigate large scale immunological gene expression studies in avian species. Multiple options exist for conducting large gene expression studies in chickens and in this study we explore the feasibility of using one of these tools to investigate gene expression in other avian species.

Results

In this study we utilised a whole genome long oligonucleotide chicken microarray to assess the utility of cross species hybridisation (CSH). We successfully hybridised a number of different avian species to this array, obtaining reliable signals. We were able to distinguish ducks that were infected with avian influenza from uninfected ducks using this microarray platform. In addition, we were able to detect known chicken immunological genes in all of the hybridised avian species.

Conclusion

Cross species hybridisation using long oligonucleotide microarrays is a powerful tool to study the immune response in avian species with little available genomic information. The present study validated the use of the whole genome long oligonucleotide chicken microarray to investigate gene expression in a range of avian species.

     

Ultralong telomeres shorten with age in nestling great tits but are static in adults and mask attrition of short telomeres

Telomere length (TL) is increasingly being used as a biomarker of senescence, but measuring telomeres remains a challenge. Within tissue samples, TL varies between cells and chromosomes. Class I telomeres are (presumably static) interstitial telomeric sequences, while terminal telomeres have been divided in shorter (Class II) telomeres and ultralong (Class III) telomeres, and the presence of the latter varies strongly between species. Class II telomeres typically shorten with age, but little is known of Class III telomere dynamics. Using multiple experimental approaches, we show great tits to have ultralong telomeres, and we investigated age effects on Class II and III telomeres using a longitudinal approach (our method excludes Class I telomeres). In adults, TL averaged over the whole distribution did not significantly change with age. However, more detailed analyses showed that Class II TL did shorten with age, and, as in other species, the longest Class II telomeres within individuals shortened more quickly with age. In contrast, Class III TL did not shorten with age within individual adults. Surprisingly, we found the opposite pattern in nestlings: Class III TL shortened significantly with age, while the age effect on Class II TL was close to zero. Thus, Class III TL may provide information on developmental history, while Class II TL provides information on telomere dynamics in adulthood. These findings have practical implications for telomere studies and raise the interesting question of what causes variation in TL dynamics between chromosomes within individuals and how this is related to development.     

江西老虎脑省级自然保护区鸟类资源调查

2017年11—12月、2018年7—8月、2018年11—12月,在江西老虎脑省级自然保护区开展鸟类资源调查,调查发现该保护区有鸟类17目53科240种。其中,非雀形目16目22科94种,雀形目31科146种。本区鸟类中有留鸟115种,冬候鸟57种,夏候鸟42种,旅鸟26种;古北界87种,广布种22种,东洋界131种。其中,国家重点保护鸟类31种,列入中国红色名录濒危等级(EN)物种有1种,黄胸鹀Emberiza aureola;易危等级(VU)物种有2种,白颈长尾雉Syrmaticus ellioti、白眉山鹧鸪Arborophila gingica;近危等级(NT)物种有19种。中国特有种4种,灰胸竹鸡Bambusicola thoracica、白颈长尾雉、白眉山鹧鸪、黄腹山雀Parus venustulus。多样性分析结果显示,该区鸟类群落Shannon-Wiener指数、Pielou指数分别为4.135、0.778,通过与周围包括武夷山、井冈山、齐云山、九连山4个保护区比较,显示研究区域物种种数较高。     

Measuring vertebrate telomeres: applications and limitations

Telomeres are short tandem repeated sequences of DNA found at the ends of eukaryotic chromosomes that function in stabilizing chromosomal end integrity. In vivo studies of somatic tissue of mammals and birds have shown a correlation between telomere length and organismal age within species, and correlations between telomere shortening rate and lifespan among species. This result presents the tantalizing possibility that telomere length could be used to provide much needed information on age, ageing and survival in natural populations where longitudinal studies are lacking. Here we review methods available for measuring telomere length and discuss the potential uses and limitations of telomeres as age and ageing estimators in the fields of vertebrate ecology, evolution and conservation.     

Chromosomal mapping of chicken mega-telomere arrays to GGA9, 16, 28 and W using a cytogenomic approach

Four mega-telomere loci were mapped to chicken chromosomes 9, 16, 28, and the W sex chromosome by dual-color fluorescence in situ hybridization using a telomeric sequence probe and BAC clones previously assigned to chicken chromosomes. The in-common features of the mega-telomere chromosomes are that microchromosomes are involved rather than macrochromosomes; in three cases (9, 16, 28) acrocentrics are involved with the mega-telomeres mapping to the p arms. Three of the four chromosomes (9, 16, W) encode tandem repeats which in two cases (9 and 16) involve the ribosomal DNA arrays (the 5S and 18S-5.8S-28S gene repeats, respectively). All involved chromosomes have a typical-sized telomere on the opposite terminus. Intra- and interindividual variation for mega-telomere distribution are discussed in terms of karyotype abnormalities and the potential for mitotic instability of some telomeres. The diversity and distribution of telomere array quantity in the chicken genome should be useful in contributing to research related to telomere length regulation - how and by what mechanism genomes and individual chromosomes establish and maintain distinct sets of telomere array sizes, as well as for future studies related to stability of the chicken genome affecting development, growth, cellular lifespan and disease. An additional impact of this study includes the listing of BAC clones (26 autosomal and six W BACs tested) that were cytogenetically verified; this set of BACs provide a useful tool for future cytogenetic analyses of the microchromosomes.     

Accumulation of short telomeres in human fibroblasts prior to replicative senescence

The loss of telomere repeats has been causally linked to in vitro replicative senescence of human diploid fibroblasts (HDFs). In order to study the mechanism(s) by which telomere shortening signals cell senescence, we analyzed the telomere length at specific chromosome ends at cumulative population doublings in polyclonal and clonal HDFs by quantitative fluorescence in situ hybridization. The rate of telomere shortening at individual telomeres varied between 50 and 150 bp per population doubling and short telomeres with an estimated 1-2 kb of telomere repeats accumulated prior to senescence. The average telomere length in specific chromosome ends was remarkably similar between clones. However, some exceptions with individual telomeres measuring 0.5-1 kb were observed. In the fibroblast clones, the onset of replicative senescence was significantly correlated with the mean telomere fluorescence but, strikingly, not with chromosomes with the shortest telomere length. The accumulation of short telomeres in late passages of cultured HDFs is compatible with selection of cells on the basis of telomere length and limited recombination between telomeres prior to senescence.     

The role of humoral factors in the regression of leukemia in chickens as measured by in vitro colony formation

Leukemic myeloblasts induced by avian myeloblastosis virus in the chicken formed small compact (type II) colonies in semi-solid agar medium. Normal yolk sac cells from 12-day old embryos formed large diffuse (type I) colonies under the same conditions. Type I colony formation (but not type II) was strictly dependent upon the presence in the medium of a colony stimulating factor (CSF) present in fresh chicken serum or conditioned medium. Serum CSF levels were determined for normal, leukemic, and birds which had spontaneously regressed from myeloblastic leukemia. When type I colony formation was used as the assay, serum CSF levels of leukemic birds were found to be significantly lower than levels in either normal or regressed birds. When the same sera were tested for their ability to induce type II colonies, leukemic birds demonstrated a significantly higher CSF level than either normal or regressed sera. Regressed chickens had serum CSF levels similar to normal birds.     

Differentiation of female chicken primordial germ cells into spermatozoa in male gonads

In avian species, the developmental fate of different-sex germ cells in the gonads is unclear. The present study attempted to confirm whether genetically female germ cells can differentiate into spermatozoa in male gonads using male germline chimeric chickens produced by the transfer of primordial germ cells (PGC), and employing molecular biological methods. As a result of Southern hybridization, specific sequences of the W chromosome (the female specific sex chromosome in birds) were detected in the genomic DNA extracted from one out of four male germline chimeric chickens. When two-color in situ hybridization was conducted on the spermatozoa of this germline chimera, 0.33% (average) of the nuclei of each semen sample showed the fluorescent signal indicating the presence of the W chromosome. The present study shows that female PGC can differentiate into spermatozoa in male gonads in the chicken. However, the ratio of produced W chromosome-bearing (W-bearing) spermatozoa fell substantially below expectations. It is therefore concluded that most of the W-bearing PGC could not differentiate into spermatozoa because of restricted spermatogenesis.     

Mhc diversity in two passerine birds: no evidence for a minimal essential Mhc

Humans express an array of Mhc genes, while the chicken has an Mhc that is relatively small and compact with fewer expressed genes. Here we ask whether the "minimal essential Mhc" of the chicken is representative for birds. We investigated the RFLP genotypes in 55 great reed warblers Acrocephalus arundinaceus and 10 willow warblers Phylloscopus trochilus to obtain an overview of the number of class II B genes. There were 13-17 bands per individual in the great reed warblers and 25-30 in the willow warblers, and every individual had a unique RFLP genotype. The high number of RFLP bands indicates that both species have a large number of class II B genes although some may be pseudogenes. Seven different class II B sequences were detected in a great reed warbler cDNA library. There was considerable sequence divergence between the cDNA sequences in exon 2 (peptide-binding region, PBR), whereas they were very similar in exon 3. The cDNA sequences were easily alignable to a classical chicken class II B sequence, and balancing selection was acting in the PBR. One of the cDNA sequences had two deletions and is likely nonfunctional. Finally, the polymorphic class I and class II B RFLP fragments seemed to be linked in the five studied great reed warbler families. These and previous results suggest that birds of the order Passeriformes in general have more Mhc class I and II B genes than birds of the order Galliformes. This difference could be caused by their phylogenetic past, and/or by variance in the selection pressure for maintaining a high number of Mhc genes.     

Robust and ubiquitous GFP expression in a single generation of chicken embryos using the avian retroviral vector,RCASBP

Functional genomics in avian models has lagged behind that of mammals, and the production of transgenic birds has proven to be challenging and time-consuming. All current methods rely upon breeding chimeric birds through at least one generation. Here, we report a rapid method for the ubiquitous expression of GFP in chicken embryos in a single generation (G-0), using the avian retroviral vector, Replication-Competent Avian sarcoma-leukosis virus, with a Splice acceptor, Bryan RSV Pol (RCASBP). High-titre RCASBP retrovirus carrying eGFP was injected into unincubated (stage X) blastoderms in ovo. This resulted in stable and widespread expression of eGFP throughout development in a very high proportion of embryos. Transgenic tissues were identified by fluorescence and immunohistochemistry. These results indicate that chicken blastodermal cells are permissive for infection by the RCASBP virus. This system represents a rapid and efficient method of producing global gene expression in the chicken embryo. The method can be used to generate avian cells with a stable genetic marker, or to induce global expression of a gene of choice. Interestingly, in day 8.5 embryos, somatic cells the embryonic gonads were predominantly GFP positive but primordial germ cells were GFP negative, indicating viral silencing in the embryonic germline. This dichotomy in the gonads allows the isolation or enrichment of the germ cells through negative selection during embryonic stages. This transgenic chicken model is of value in developmental studies, and for the isolation and study of avian primordial germ cells.     

A smörgåsbord of markers for avian ecology and evolution

The polymerase chain reaction has been a boon to the study of molecular ecology and population genetics of birds. But the nagging truth is that for many bird species, the number of polymerase chain reaction (PCR) primer pairs that one can pick off the shelf and expect to amplify their target loci with ease is frustratingly small. Now, studying DNA sequence variation in natural populations of birds just got a whole lot easier. This issue of Molecular Ecology reports a large-scale bioinformatics search for exonic sequences conserved between the chicken and zebra finch genomes and flanking polymorphic introns that has generated a staggering 242 PCR primer pairs that readily amplify their single-copy target loci in five avian species spanning ~100 million years of avian evolution ( Backström et al . 2008 ). As proof of principle, these primers have also been used to survey the genomic landscape in over 110 kb of intronic sequence in the collared flycatcher, a model species in ecology and evolution. These resources pave the way for easy multilocus study of evolving populations and lineages of birds, and bring the goal of quickly turning nonmodel species in to ecological genomic models tantalizingly close.     

Cis- and trans-acting influences on telomeric position effect in Drosophila melanogaster detected with a subterminal transgene

One model of telomeric position effect (TPE) in Drosophila melanogaster proposes that reporter genes in the vicinity of telomeres are repressed by subterminal telomere-associated sequences (TAS) and that variegation of these genes is the result of competition between the repressive effects of TAS and the stimulating effects of promoters in the terminal HeT-A transposon array. The data presented here support this model, but also suggest that TPE is more complex. Activity of a telomeric white reporter gene increases in response to deletion of some or all of the TAS on the homolog. Only transgenes next to fairly long HeT-A arrays respond to this trans-interaction. HeT-A arrays of 6-18 kb respond by increasing the number of dark spots on the eye, while longer arrays increase the background eye color or increase the number of spots sufficiently to cause them to merge. Thus, expression of a subtelomeric reporter gene is influenced by the telomere structure in cis and trans. We propose that the forces involved in telomere length regulation in Drosophila are the underlying forces that manifest themselves as TPE. In the wild-type telomere TAS may play an important role in controlling telomere elongation by repressing HeT-A promoter activity. Modulation of this repression by the homolog may thus regulate telomere elongation.     

Analysis of cDNA and genomic clones coding for the pro alpha 1 chain of calf type II collagen.

A bovine cDNA library constructed from fetal cartilage RNA was screened with a pro alpha 1(II) collagen specific chicken cDNA. A recombinant clone (Bc 7), with an insert of 1 kb, was identified and shown to contain sequences exhibiting 85% homology with the chicken pro alpha 1(II) collagen C-propeptide. Interspecies comparison strongly suggested that one potential glycosylation site present in the avian C-propeptide is not utilized, since this site is absent in the bovine chain. In addition, two overlapping genomic clones (Pal 3 and Pal 4) were isolated and partially characterized. These clones span 23 kb of DNA and contain approximately 17 kb of the pro alpha 1(II) calf gene. Sequencing of exon 1 has determined the length of the 3' untranslated region and the exact location of the polyadenylation attachment site.