Abstract
Genomic imprinting is an epigenetic mechanism causing monoallelic expression in a parent-of-origin-specific manner. Disruption of imprinted genes causes various neurological and psychiatric disorders. However, the role of imprinted genes in the brain is largely unknown. Different cell types within distinct brain regions can influence the genomic imprinting status, but imprinted genes in single cell types within distinct brain regions have not been characterized on a genome-wide scale. To address this critical question, we used a multi-stage approach, which combined genetically engineered mice with fluorescence-based laser capture microdissection (LCM) to capture excitatory neurons, inhibitory neurons and astrocytes as single cells in layer 2/3 of mouse visual cortex. RNA sequencing determined parental expression patterns on a genome-wide scale in the captured cells within specific brain regions. The expression level of cell-type-specific genes for excitatory neurons (13 genes), inhibitory neurons (16 genes) and astrocytes (20 genes) confirmed the LCM-captured cells maintained their cellular identities. The parent-of-origin-specific expression pattern of imprinted genes, including maternally expressed Meg3 and paternally expressed Peg3, provided evidence that the status of known imprinted genes was also maintained. Although our platform remains to be improved, our findings demonstrate the parental expression pattern can be analysed not only at the level of a single cell type but also at the level of specific cortical layers. Our approach has the potential to reveal novel regulatory modules associated with plasticity through genomic imprinting mechanisms in different cell types, not only in the visual cortex but also in other brain regions.
Highlights
The genomes inherited from each parent are not functionally equivalent due to genomic imprinting, which is established during gametogenesis and maintained throughout life [1,2,3]
Single cells with red fluorescent signals from layers 2/3 of the visual cortex were isolated with laser capture microdissection (LCM) (Fig 1a, middle)
To distinguish parent-of-origin-specific monoallelic expression, we took advantage of single nucleotide polymorphisms (SNPs), which were identified from the genomic DNA of C57BL/6J (B6) and CAST/EiJ (CAST) strain mice
Summary
The genomes inherited from each parent are not functionally equivalent due to genomic imprinting, which is established during gametogenesis and maintained throughout life [1,2,3]. Genomic imprinting is an epigenetic process through which monoallelic gene expression occurs in a parent-of-origin-specific manner. Understanding genomic imprinting is limited because it is spatiotemporally dynamic, and due to challenges associated with identifying environmental-, gender-, species-, cell-type- or gene isoform-specific parental expression effects [3,4,5]. There is an unmet need to identify imprinted genes in the brain and to understand their underlying physiological function. This is complicated by the heterogeneous nature of brain tissues and the multifaceted regulation of genomic imprinting. It is critical to comprehensively determine parent-of-origin-specific gene expression in the brain, at least at a cellular resolution within a distinct brain region. Despite the importance of genomic imprinting in brain function, the numbers and identities of imprinted genes that have been proposed are still debated due to the heterogeneity of the brain and the complexity of screening techniques and statistical analysis [14,15,16]
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