Identification and characterisation of novel transcripts involved in the proliferation, differentiation and developmental networks of the mouse cerebral cortex.

Abstract

Cerebral corticogenesis involves specific influence of intrinsic and extrinsic mechanisms, which are triggered spatiotemporally. During mouse embryogenesis, the mouse cerebral cortex develops from a relatively homogenous band of mitotic multipotent progenitor cells into a complex laminated structure between embryonic day 11.5 (E11.5) and 18.5 (E18.5). Identification of molecular targets and regulatory networks involved in cerebral corticogenesis is crucial for the understanding of the development and function of the cortex. Global transcriptome profiling of the mouse cerebral cortex at various developmental stages using Serial Analysis of Gene Expression (SAGE) technique identified 561 differentially expressed tags/transcripts (DETs). Hierarchical and genomic clustering of DETs showed common functional ontologies and molecular networks that are associated with neurological disorders in human. In addition, 4 genomic loci at Sox4, Sox11, Nrgn and Camk2n1 were significantly represented by embryonic and adult-specific DETs when compared to other genomic loci. These genomic loci have multiple over-lapping sense and natural antisense transcripts (NATs) featuring different polyadenylation signal sites and spatiotemporally regulated expression profiles. The study suggests that these antisense transcripts have an important role in cerebral corticogenesis and neuronal/glial cell differentiation or function. These NATs were further characterized using Fluorescence In situ Hybridization (FISH) probes specific to the sense and antisense transcripts of Sox4, Nrgn and Camk2n1 RNA in trypsinized adult brain cells. The analysis showed colocalization of sense and antisense transcripts and confirmed the formation of sense-antisense double stranded RNA (dsRNA) in the cytoplasm. Overexpression of Sox4 antisense transcripts did not regulate Sox4 transcription or translation processes. Instead, Sox4 dsRNAs serve as templates for the generation of a small RNA, namely Sox4_sir3, which is an endogenous small interfering RNA (siRNA). Its biogenesis is dependent on Dicer1 activity as well as the formation of dsRNA between Sox4 sense and antisense transcripts. Sox4-sir3 is expressed specifically in the germinative zones and in specialized neurons throughout brain development. This is the first demonstration in the mammalian system that cytoplasmic sense-NAT dsRNAs serve as templates for the production of novel endogenous siRNAs adding a new dimension to the long-debated controversial role of NATs in the genome. Small RNAs such as microRNAs (miRNAs) can repress translation of proteincoding mRNA or direct mRNA decay by recruiting RNA-induced silencing complex (RISC) machinery. Massively parallel sequencing of an E15.5 developing mouse brain further identified 4 putative miRNAs and one of them was confirmed as a novel miRNA, M1181. M1181 was spatiotemporally expressed throughout mouse embryo development including mouse embryonic stem cells, E3.5 blastocysts, and embryos aged between E7.5 and E15.5. Between E13.5 and E17.5, M1181 expression was confined to the cortical plate of the cerebral cortex and the ventricular zone of midbrain. In adult mice, M1181 was strongly expressed in the brain, particularly the olfactory bulb, cerebrum, thalamus and midbrain. Taken together, the expression pattern of M1181 implicates its role as a potential novel regulator in early embryonic development involving ES cell pluripotency, neural tube formation and adult central nervous system function. In a nutshell, novel transcripts involved in the developmental networks of the brain particular the cerebral cortex were identified using a variety of genomic and in silico approaches. A new role and related mechanism for novel Sox4 NATs especially in the biogenesis of small RNA were described and this landmark discovery add to our understanding of the versatility of NAT function in mammalian biology.