In this study, we utilized a custom-designed microarray platform to probe the transcription of both protein-coding and noncoding transcripts during ventral forebrain-derived NSC lineage restriction, neuronal and oligodendrocyte (OL) lineage specification, neuronal-glial fate transitions, and progressive stages of OL lineage elaboration including myelination. Expression profiles for mRNAs in our study were similar to previously reported findings [62
] showing modulation of key signaling pathways, transcription factors, and epigenetic regulators that help to define the molecular codes governing neural lineage elaboration. Additionally, we also observed expression of components of the myelin sheath.
Although we examined protein-coding genes, we focused primarily on characterizing the expression profiles of long ncRNAs, which are being increasingly appreciated in the regulation of developmental gene expression programs [3
]. Previous studies of neural differentiation have reported expression profiles for only a small number of long ncRNAs, such as Nkx2.2AS
and the imprinted H19
]. In contrast, we examined a large spectrum of long ncRNAs and found dramatic changes in expression that are unique to specific stages of cellular differentiation. Many of these ncRNAs are transcribed from complex genomic loci that encompass protein-coding genes with defined roles in neuronal and glial lineage elaboration, and these pairs of ncRNAs and associated protein-coding genes often exhibited coordinated expression during developmental transitions. Therefore, we suggest that these ncRNAs may influence the expression of the associated protein-coding genes similar to previously characterized examples such as Nkx2.2AS
] and Evf2
]. For example, many ncRNAs were associated with transcription factors that were themselves differentially regulated during OL differentiation [62
]. Long ncRNAs, such as those identified in this study, may therefore contribute to the combinatorial transcriptional codes involved in lineage specification and terminal differentiation including myelination.
The coordinated expression of long ncRNAs with critical neural developmental protein coding genes suggests that both are subject to shared regulatory mechanisms and that long ncRNAs are integrated into complex environmentally-mediated neural and glial developmental gene expression programs. We, therefore, considered whether the well-documented ability of epigenetic mechanisms to promote decisive changes in neuronal and glial gene expression programs also includes the capacity to regulate long ncRNA expression. Indeed, treatment with the HDAC inhibitor, TSA, effectively arrests the developmental progression of OLPs into PMOs [23
] and also affects the expression of long ncRNAs, suggesting that these are not only regulated by epigenetic modifications but also these transcripts are incorporated into a broader lineage specific developmental gene expression program. Furthermore, HDAC inhibition also has differential effects on certain ncRNAs in an environmentally responsive manner. For example, treatment of OLPs with TSA in the presence (inductive) or absence (stochastic) of CNTF results in opposing profiles of Neat1
expression, implying that long ncRNAs might be deployed in a context-specific manner to dynamically modulate seminal fate decisions within the OL lineage. Although the use of HDAC inhibitors has been essential for interrogating the epigenetic mechanisms that govern OL lineage maturation, the cellular effects of TSA administration are complex and our understanding of the mechanisms mediating ncRNA expression are still evolving. Nonetheless, long ncRNAs may represent important targets for regenerative therapies in the CNS because they exhibit both exquisite site-selective as well as genome wide effects [5
] associated with dynamic OL developmental transition states characterized by considerable epigenetic flux [22