Division-dependent telomere shortening correlating with age triggers senescence on a cellular level and telomere dysfunction can facilitate oncogenesis. Therefore, the study of telomere biology is critical to the understanding of aging and cancer. The domestic chicken, a classic model for the study of developmental biology, possesses a telomere genome with highly conserved aspects and distinctive features which make it uniquely suited for the study of telomere maintenance mechanisms, their function and dysfunction. The purpose of this review is to highlight the chicken as a model for aging research, specifically as a model for telomere and telomerase research, and to increase its utility as such by describing developments in the study of chicken telomeres and telomerase in the context of related research in human and mouse.
MECP2, an X-linked gene encoding the epigenetic factor methyl-CpG-binding protein-2, is mutated in Rett syndrome (RTT) and aberrantly expressed in autism. Most children affected by RTT are heterozygous Mecp2+/- females whose brain function is impaired post- natally due to MeCP2 deficiency. While prior functional investigations of MeCP2 have focused exclusively on neurons and have concluded the absence of MeCP2 in astrocytes, here we report that astrocytes express MeCP2, and MeCP2 deficiency in astrocytes causes significant abnormalities in BDNF regulation, cytokine production, and neuronal dendritic induction, effects that may contribute to abnormal neurodevelopment. In addition, we show that the MeCP2 deficiency state can progressively spread at least in part via gap junction communications between mosaic Mecp2+/- astrocytes in a novel non-cell-autonomous mechanism. This mechanism may lead to the pronounced loss of MeCP2 observed selectively in astrocytes in mouse Mecp2+/- brain, which is coincident with phenotypic regression characteristic of RTT. Our results suggest that astrocytes are viable therapeutic targets for RTT and perhaps regressive forms of autism.
Mutations in MECP2, encoding methyl-CpG-binding protein 2 (MeCP2), cause the neurodevelopmental dis- order Rett syndrome (RTT). Although MECP2 mutations are rare in idiopathic autism, reduced MeCP2 levels are common in autism cortex. MeCP2 is critical for postnatal neuronal maturation and a modulator of activity-dependent genes such as Bdnf (brain-derived neurotropic factor) and JUNB. The activity- dependent early growth response gene 2 (EGR2), required for both early hindbrain development and mature neuronal function, has predicted binding sites in the promoters of several neurologically relevant genes including MECP2. Conversely, MeCP2 family members MBD1, MBD2 and MBD4 bind a methylated CpG island in an enhancer region located in EGR2 intron 1. This study was designed to test the hypothesis that MECP2 and EGR2 regulate each other’s expression during neuronal maturation in postnatal brain devel- opment. Chromatin immunoprecipitation analysis showed EGR2 binding to the MECP2 promoter and MeCP2 binding to the enhancer region in EGR2 intron 1. Reduction in EGR2 and MeCP2 levels in cultured human neuroblastoma cells by RNA interference reciprocally reduced expression of both EGR2 and MECP2 and their protein products. Consistent with a role of MeCP2 in enhancing EGR2, Mecp2-deficient mouse cortex samples showed significantly reduced EGR2 by quantitative immunofluorescence. Furthermore, MeCP2 and EGR2 show coordinately increased levels during postnatal development of both mouse and human cortex. In contrast to age-matched Controls, RTT and autism postmortem cortex samples showed significant reduction in EGR2. Together, these data support a role of dysregulation of an activity-dependent EGR2/ MeCP2 pathway in RTT and autism.
Epigenetic mechanisms have been proposed to play a role in the etiology of autism. This hypothesis is supported by the discovery of increased MECP2 promoter methylation associated with decreased MeCP2 protein expression in autism male brain. To further understand the influence of female X chromosome inactivation (XCI) and neighboring methylation patterns on aberrant MECP2 promoter methylation in autism, multiple methylation analyses were peformed on brain and blood samples from individuals with autism. Bisulfite sequencing analyses of a region 0.6 kb upstream of MECP2 in brain DNA samples revealed an abrupt transition from a highly methylated region in both sexes to a region unmethylated in males and subject to XCI in females. Chromatin immunoprecipitation analysis demonstrated that the CCTC-binding factor (CTCF) bound to this transition region in neuronal cells, consistent with a chromatin boundary at the methylation transition. Male autism brain DNA samples displayed a slight increase in methylation in this transition region, suggesting a possible aberrant spreading of methylation into the MECP2 promoter in autism males across this boundary element. In addition, autistic female brain DNA samples showed evidence for aberrant MECP2 promoter methylation as an increase in the number of bisulfite sequenced clones with undefined XCI status for MECP2 but not androgen receptor (AR). To further investigate the specificity of MECP2 methylation alterations in autism, blood DNA samples from females and mothers of males with autism were also examined for XCI skewing at AR, but no significant increase in XCI skewing was observed compared to controls. These results suggest that the aberrant MECP2 methylation in autism brain DNA samples is due to locus-specific rather than global X chromosome methylation changes.
Nagarajan et. al., Autism Research (2008)
Male and female embryonic stem (ES) cell lines were derived from the area pellucidae of Stage X (EG&K) chicken embryos. These ES cell lines were grown in culture for extended periods of time and the majority of the cells retained a diploid karyotype. When reintroduced into Stage VI–X (EG&K) recipient embryos, the cES cells were able to contribute to all somatic tissues. By combining irradiation of the recipient embryo with exposure of the cES cells to the embryonic environment in diapause, a high frequency and extent of chimerism was obtained. High-grade chimeras, indistinguishable from the donor phenotype by feather pigmentation, were produced. A transgene encoding GFP was incorporated into the genome of cES cells under control of the ubiquitous promoter CX and GFP was widely expressed in somatic tissues. Although cES cells made extensive contributions to the somatic tissues, contribution to the germline was not observed.
Primordial germ cells (PGCs) are the precursors of sperm and eggs1. In most animals, segregation of the germ line from the somatic lineages is one of the earliest events in development2; in avian embryos, PGCs are first identified in an extra-embryonic region, the germinal crescent, after approximately 18 h of incu- bation. After 50–55 h of development, PGCs migrate to the gonad and subsequently produce functional sperm and oocytes3,4. So far, cultures of PGCs that remain restricted to the germ line have not been reported in any species5,6. Here we show that chicken PGCs can be isolated, cultured and genetically modified while main- taining their commitment to the germ line. Furthermore, we show that chicken PGCs can be induced in vitro to differentiate into embryonic germ cells that contribute to somatic tissues.
The versatility and utility of the domestic chicken as a developmental model was recently celebrated in a special issue of the journal Developmental Dynamics [(2004) 229, 413-712.] The chicken is one of the primary models for vertebrate developmental biology and a model organism for the study of virology, immunology, cancer and gene regulation (Tickle, 2004; Antin and Konieczka, 2005). With a 6.6X draft sequence of its genome completed, the chicken is poised to become even more valuable in traditional fields of study and also in aging research.
The earliest recorded descriptions of the chicken as a model for biological processes are attributed to Hippocrates and Aristotle who both wrote about embryonic development in fertilized chicken eggs. Twentieth century embryologists authored numerous treatises describing, diagramming, and providing detailed photographs of the chicken during development (Hamburger and Hamilton, 1951; Romanoff, 1960; Eyal-Giladi and Kochev, 1976) which promoted use of the chicken embryo as a model for study of mechanisms including morphogenesis; neurogenesis; somatogenesis; limb, limb-digit and craniofacial development; left-right symmetry; axis development and others. The extensive use of the chicken as a model for early vertebrate development and its role in biomedical research has of necessity produced a detailed and comprehensive body of knowledge about basic chicken biology (Stern, 2005; Scanes et al., 2004). Add to all of this the accessibility of the chicken embryo, the relative economy of breeding and maintaining chickens and the ease of manipulation of embryonic and adult tissues and the chicken becomes an obvious choice as a model for the study of organismal and cellular senescence.
Telomere-binding proteins, their interaction partners and transcription factors play a prominent role in telomere maintenance and telomerase activation. We examined mRNA expression levels of tankyrase 1 and 2, TRF1 and 2, c-myc, TERT and TR in Gallus domesticus, the domestic chicken, by quantitative real-time PCR, establishing expression profiles for three contrasting cell systems: the pluripotent gastrula, differentiated embryo fibroblasts and transformed DT40 cells.
This study examined telomerase activity and gene expression profiles for three genes in Gallus gallus domesticus: telomerase reverse transcriptase (chTERT), telomerase RNA (chTR), and c-myc. Expression of these genes was studied in chicken embryonic stem (chES) cells, chicken embryo fibroblasts (CEFs), and DT40 cells using quantitative real-time polymerase chain reaction. Our results establish that, relative to transcription levels in telomerase-negative CEFs, chTERT and chTR are up-regulated in telomerase-positive chES cells. Transcription levels of chTERT, chTR, and c-myc are dramatically up-regulated in telomerase-positive DT40 cells, relative to CEFs and chES cells. These results are consistent with a model in which telomerase activity is up-regulated in proliferating embryonic stem cells requiring stable telomeres to endure multiple rounds of cell division; down-regulated in differentiated, lifespan-limited cells; and dramatically up-regulated in immortalized, transformed cells for which uncontrolled proliferation is correlated with c-myc dysregulation and telomerase activity.