Project description:Primordial germ cells (PGCs), major cell resource used in the production of germline chimeras in birds, have been used in conservation of avian genetic resources and production of transgenic animals. Numerous bird species have been put on the brink of extinction due to habitat loss and degradation caused by environmental destruction and climate change, but research on PGCs is limited to specific poultry, such as chickens. Although it has recently been expanding to various bird species, it is still difficult to utilize PGCs due to biological differences and difficulties in in vitro long-term culture. Here, we constructed a single-cell landscape of chicken gonadal PGCs with established long-term culture systems of PGCs and compared them with those of the vocal learning wild bird, the zebra finches. Our results identified the interspecific differences in signaling pathways in gonadal PGCs and somatic cells, respectively. In particular, the NODAL and insulin signaling pathways were more active in zebra finch than in chickens, whereas the FGF downstream signaling pathway known to be important for the proliferation of chicken PGCs, was more active in chickens. These differences may contribute to optimizing the in vitro culture conditions of zebra finch PGCs. This study is the first cross-species single-cell transcriptomic analysis targeting birds, and laid an essential groundwork to contribute to the restoration of endangered birds and the production of transgenic birds by securing sufficient PGCs from various bird species in the future.
Project description:To investigate the cellular basis of parental species bias at birdsong, we performed single nuclei RNA-seq for six zebra finch and owl finch F1 hybrid juvenile birds.
Project description:To investigate the cellular basis of parental species bias at birdsong, we performed single nuclei RNA-seq for six zebra finch and owl finch F1 hybrid juvenile birds.
Project description:DNA methylation is tightly linked with gene expression regulation and has long been regarded a stable epigenetic mark in postmitotic cells. However, it recently became clear that postnatal brains appear to show stimulus-induced de novo CpG methylation or active demethylation related to neuronal plasticity. Due to striking homologies between the brains of birds and mammals, songbirds, especially the zebra finch, propose an attractive model for investigating the genome-wide DNA methylation profile and DNA methylation reconfiguration during brain development. In order to obtain a first genome-wide compendium of genes under putative DNA methylation control, we performed MethyCap-seq experiments on two recently cultured zebra finch cell lines, G266 and ZFTMA, also upon AZA-induced demethylation. First, the MethylCap-seq methodology in zebra finch was validated by comparison with RRBS generated data. Subsequently, quantitative analysis identified 30,700 significantly demethylated loci upon AZA-treatment. Further examination revealed enrichment of these regions in exons and promoters. To assess the influence of methylation on gene expression, RNA-seq experiments were performed. Comparison of the RNA-seq and MethylCap-seq results showed that at least 357 of the 3,457 AZA-upregulated genes are putatively regulated by methylation in the promoter region, for which a pathway analysis showed obvious enrichment for neurological networks. A subset of genes was validated using qPCR and CpG pyrosequencing. To our knowledge, this study provides the first genome-wide DNA methylation map of the zebra finch genome as well as a comprehensive set of genes of which transcription is under putative methylation control. MethylCap-seq and RNA-seq experiments were performed on DMSO- and AZA-treated zebra finch cell lines, i.e. G266 and ZFTMA. As a quality control, also an untreated ZFTMA sample was analyzed with MethylCap-seq and RRBS.
Project description:We queried a songbird brain to discover behaviorally regulated transcriptional mechanisms relevant for speech behavior. About 10% of zebra finch genes showed regulation during singing, and most were brain-region specific. We propose that the brain-regional diversity of the singing-regulated gene networks is derived both from differential combinatorial binding of transcription factors and the epigenetic state of these genes before singing begins. To test this hypothesis, we measured H3K27ac two brain regions that participate in song production. The examination of H3K27ac in two brain regions of zebra finch in singing and silent conditions
Project description:The zebra finch is one of the most commonly studied songbirds in biology, particularly in genomics, neuroscience and vocal communication. However, this species lacks a robust cell line for molecular biology research and reagent optimization. We generated a cell line from zebra finch embryonic fibroblasts using the SV40 large and small T antigens, designated CFS414. This cell line demonstrates an improvement over previous songbird cell lines through continuous and density-independent growth, allowing for indefinite culture and monoclonal line derivation. Cytogenetic, genomic, and transcriptomic profiling established the provenance of this cell line and identified the expression of genes relevant to ongoing songbird research. This single-cell RNA sequencing experiment provided information on the gene expression landscape of the cell line, informing on its cell type, transcriptomic stability, and value to researchers utilizing the zebra finch as a model organism.
Project description:We have conducted a Cross-Species Microarray analysis by hybridizing genomic DNA from the common whitethroat (Sylivia communis) on a newly developed Affymetrix costum array designed for the zebra finch (Taeniopygia guttata), the Lund-zf array. We have also quality tested the Lund-zf array by hybridizing zebra finch genomic DNA to the platform.
Project description:The unraveling of heterogenous cell populations, — previously masked in bulk transcriptome measurements — are made possible through analysis of single-cell transcriptomics. The goals of this study are to examine single-cell NGS-derived gonadal cell transcriptome profiling and to evaluate heterogeneity of gonadal germ cells in avian species. We performed single-cell RNA sequencing using embryonic gonadal cells of HH stage 28 from male and female zebra finches. We identified 12,489 male transcripts and 10,046 female transcripts. We annotated 9 different cell types from a total of 20 clustered single cells and identified that primordial germ cells, which have been considered as single population, can be distinguishable into three subtypes. The three PGC subtypes were specifically enriched for genes with expression patterns related to germness or pluripotency, suggesting functional differences in PGCs according to the three subtypes. Our study represents the first single-cell RNA seq analysis of gonadal transcriptomes in zebra finch. Our results revealed the first heterogeneity and intrinsic changes of gonadal primordial germ cells, which have hitherto been considered as a single population in avian species. We conclude that single-cell RNA seq based transcriptome analysis would offers new perspectives on avian primordial germ cells.