Tuning the cell fate of neurons and glia by microRNAs

Abstract

The proper function of the nervous system depends on precise production and connection of distinct neurons and glia. Cell fate determination of neurons and glia is tightly controlled by complex gene expression regulation in the developing and adult nervous system. Emerging evidence has demonstrated the importance of noncoding microRNAs (miRNAs) in neural development and function. This review highlights current discoveries of miRNA functions in specifying neuronal and glial cell fate. We summarize the roles of miRNAs in expansion and differentiation of neural stem cells, specification of neuronal subtypes and glial cells, reprogramming of functional neurons from embryonic stem cells and fibroblasts, and left-right asymmetric organization of neuronal subtypes. Investigating the network of interactions between miRNAs and target genes will reveal new gene regulation machinery involved in tuning the cell fate decisions of neurons and glia.

Introduction

One fascinating phenomenon in the nervous system of invertebrates and vertebrates is the precise regulation of cell fate determination. Distinct neurons and glia are derived from neural stem cells (NSCs) or specific neural progenitors (NPs) and glial progenitors, respectively, in different regions of the developing central nervous system (CNS) by complex temporospatial gene regulation [1–5]. Even in adult brains, tightly controlled and diverse neurogenesis is critical for proper brain functions [6–8]. Resulting from extensive investigations of molecular mechanisms of cell fate determination, NSCs, specific progenitors, embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs) and even fibroblasts have been programmed or reprogrammed into specific neuronal and glial types for treatments of neurological disorders [9–12].

Emerging studies have shown that like protein coding genes, microRNAs (miRNAs) play essential roles in cell fate determination. miRNAs, found in almost all eukaryotic cells, are a group of 18–22 nucleotide (nt) highly conserved small noncoding RNAs, which normally negatively regulate target gene expression by binding to messenger RNAs (mRNAs), typically in the 3’ untranslated region (3’UTR) [13,14]. Exciting studies have demonstrated important roles of miRNAs in neural development and neurological diseases [15–18]. In this review, we will highlight miRNA-mediated neuronal and glial specification from NSCs, specific progenitors, ESCs, iPSCs and fibroblasts, and left-right organization of specific neuronal subtypes in the nervous system.

miRNAs regulate expansion and differentiation of NSCs and NPs

A feature of NSCs is their ability to self-renew to expand the NSC pool. Some miRNAs have been identified that promote self-renewal and proliferation of NSCs and NPs, and inhibit differentiation in both the developing and adult nervous system (Figure 1 and Table 1). In the embryonic mouse cerebral cortex, miR-19 in the miR-17-92 cluster has been found to promote NSC proliferation and radial glial cell (RGC) expansion by targeting Pten [19●]. Interestingly, miR-92, another miRNA in the miR-17-92 cluster, has been shown to inhibit transition of intermediate progenitors (IPs) from RGCs by targeting Tbr2 [19,20]. Dual regulation by members of the miR-17-92 cluster on numbers of RGCs and IPs is critical for controlling the proper progenitor pool and brain sizes [19]. miR-134 has been shown to be essential for the maintenance of cortical NPs by targeting doublecortin (Dcx) and/or Chordin-like 1 (Chrdl-1) [21].

Figure 1.

A scheme of the roles of miRNAs in cell fate determination. miRNAs that regulate neural stem cell (NSC) self-renewal and proliferation, neuronal differentiation, astrogliogensis, and oligodendrocyte differentiation are listed.

Table 1.

A list of miRNAs that regulate neuronal and glial cell fate determination.

Cell fate determination		miRNAs	Species and tissues	Targets	Reference
NSC proliferation		miR-17-92 cluster	Mouse embryonic cortex	Pten, Tbr2	[19, 20]
		miR-134	Cultured embryonic mouse cortical NSCs	Chrdl-1, Dcx	[21]
		miR-184	Cultured adult mouse forebrain NSCs	Numbl	[22]
		miR-195	Cultured adult mouse forebrain NSCs	MBD1	[23]
		miR-25	Cultured adult mouse forebrain NSCs	?	[24]
		miR-137	Cultured adult mouse forebrain NSCs	Ezh2	[25]
		miR-129, miR-155, miR-214, miR-222	Xenopus retina progenitor	Oxt2, Vsx1	[26]
NSC differentiation		Let-7b	Adult and embryonic mouse forebrain NSCs	TLX, Cyclin D1, lin-28	[28, 29]
		miR-9	Adult and embryonic mouse forebrain NSCs NPs in Zebrafish NPs in Xenopus	TLX Fgf8-1, FgfR1 Hairy1	[30] [32] [33]
		miR-137	Embryonic mouse forebrain NSCs	LSD1	[31]
		miR-26b	Zebrafish neural tube	Ctdsp2	[34,35]
		miR-125b	Cultured mouse forebrain NSCs	Nestin	[36]
		miR-124	Mouse neuroblastoma cell linesAdult SVZ NSCs	PTBP1 Sox9	[37] [38,39]
Neuronal subtype specification	Dopaminergic neurons (DN)	miR-133b	Embryonic mouse midbrain	Pitx3	[41]
		miR-132	Embryonic stem cells	Nurr1	[42]
		miR-7a	Suppressing DN differentiation	Pax6	[43]
	Projection neurons	miR-9-2/3	Embryonic mouse cortex	Foxg1, Pax6, Gsh2	[44]
	Olfactory neurons	miR-200 family	Mouse and zebrafish	Foxg1, Zfhx1, Lfng	[45]
	Spinal motor neurons	miR-17-3p	Mouse spinal motor neuron progenitors	Olig2	[46]
		miR-9	Chick spinal motor neurons	FoxP1	[47,48]
Gliogenesis	Astrogliogenesis	miR-125b	Neural cell line	?	[49]
	Oligodendrocyte differentiation	miR-219 and miR-338	Postnatal rat and mouse CNS	PDGFRα, Sox6, FoxJ3, ZFP238, Hes5	[50,51]
		miR-17-92 cluster	Neonatal mouse brain	?	[52]
		miR-7	Embryonic mouse brain	Pax6, NeuroD4	[53]
		miR-23	Mouse fibroblast and neural cell lines	Lamin B1	[54]

NSCs: Neural stem cells; NPs: neural progenitors; SVZ: subventricular zone; CNS: central nervous system.

In adult NSCs, Liu et al. have shown that miR-184 promotes adult NSC proliferation by repressing Numb-like (Numbl) [22]. Meanwhile, miR-184 is suppressed by methyl-CpG binding protein 1 (MBD1), suggesting that a regulatory network of miRNAs controls adult NSC expansion [22]. A similar regulatory loop has been identified between MBD1 and miR-195, which also positively regulates adult NSC proliferation [23●]. Moreover, miR-25, a member of the miR-106-25 cluster, has been shown to promote adult NSC proliferation, potentially through regulation of genes in the insulin/insulin-like growth factor-1 pathway [24]. miR-137, which is regulated by DNA methyl-CpG-binding protein (MeCP2) and transcription factor (TF) Sox2, promotes adult NSC proliferation and inhibits differentiation by targeting Ezh2, a histone methyltransferase and Polycomb group protein [25]. In the Xenopus retina, miR-129, miR-155, miR-214 and miR-222 have been found to promote progenitor proliferation by targeting Oxt2 and Vsx1 [26].

An interesting observation of miRNA regulation is that it often forms a feedback loop with its target genes in the process of controlling cell fate. Schwamborn et al. have shown that Let-7 is a target of TRIM32 and suppresses NSC proliferation [27]. Let-7b enhances differentiation by targeting the nuclear receptor TLX and the cell cycle regulator cyclin D1 [28]. Interestingly, further investigation has shown that let-7 normally suppresses lin-28 protein expression, and lin-28 also blocks let-7 expression by binding to the let-7 precursor and inhibiting its biogenesis [29]. In addition, miR-9, a CNS-enriched miRNA, has been shown to suppress mouse NSC expansion and induce differentiation through a feedback regulation of TLX [30]. TLX further recruits histone lysine-specific demethylase 1 (LSD1), which is a target of miR-137, and modulates proper expression of miR-137, which normally suppresses NSC proliferation [31]. These studies suggest that miRNAs play a critical role in ensuring proper numbers of NSCs and NPs by either directly silencing target genes, or forming a regulatory loop with targets.

miRNAs that inhibit NSC self-renewal and enhance differentiation have also been identified. In addition to targeting TLX, miR-9 has been shown to inhibit NP proliferation and elevate differentiation by suppressing several genes in the fibroblast growth factor signaling pathway such as Fgf8-1 and FgfR1 in zebrafish, and by targeting hairy1 in Xenopus [32,33]. miR-26b has been reported to induce neuronal differentiation by suppressing its host gene ctdsp2 in the zebrafish neural tube [34●,35]. In NSC cultures, miR-125b has been found to inhibit NSC proliferation by repressing the neural precursor marker Nestin [36]. miR-124 is another well-studied CNS-enriched miRNA that has been shown to induce differentiation of embryonic NSCs and NPs by targeting the global splicing repressor PTBP1 and to promote nervous system-specific alternative splicing [37]. In the adult brain, miR-124 plays a positive role in regulating neuronal differentiation of adult NSCs in the subventricular zone (SVZ) by suppressing Sox9 expression [38,39].

As studies that examine the function of miRNAs in NSC/NP proliferation and differentiation are accumulating, it is becoming clear that most miRNAs can be characterized into two groups based on their general roles: they promote either proliferation such as the miR-17-92 cluster, or differentiation such as miR-9 and miR-124 (Figure 1). However, the diversity and complexity of individual miRNAs in cell fate determination appear to rely on different species, specific regions in the nervous system, distinct cell context, and mostly the availability and direct physical interaction of their target genes.

Cell fate determination of neuronal subtypes by miRNAs

The complex functions of the nervous system depend on circuit formation built upon specification and connection of distinct neuronal subtypes. miRNAs have also been shown to play important roles in specifying neuronal subtypes. A profiling study has shown specific expression of miRNAs in glutamatergic and GABAergic neurons, and subtypes of GABAergic neurons [40●]. Due to their role in dopamine production, dopaminergic neurons (DNs) are essential for normal cognitive functions and voluntary movement. A negative feedback loop between paired-like homeodomain TF Pitx3 and miR-133b has been identified in the midbrain during DN differentiation and maturation: Pitx3 induces the expression of miR-133b, which also represses Pitx3 expression and suppresses DN maturation and function [41]. miR-132 has been shown to be highly expressed in tyrosine hydroxylase (TH)-positive DNs and to inhibit DN differentiation from ESCs by targeting Nurr1, one of the key TFs in DN differentiation [42]. miR-7a has been found to suppress DN differentiation by targeting Pax6 in the SVZ of postnatal mouse brains [43].

Genetic deletion of miR-9-2 and miR-9-3 results in malformation of the cerebral cortex in mice, suggesting that miR-9 plays a role in regulating projection neuron development [44●]. Members of the miR-200 family have been shown to be critical for neurogenesis of olfactory neurons by targeting Foxg1, Zfhx1 and Lfng [45]. In the spinal cord, miR-17-3p is required for patterning of motor neuron progenitors by targeting Olig2 [46]. miR-9 has been shown to modify spinal motor neuron subtype specification by balancing FoxP1 expression levels [47,48]. Because a miRNA can have multiple targets, neuronal subtype specification is likely achieved through cell type-specific miRNA expression and a balanced outcome of overall target gene expression, which eventually favors generation of a specific cell type.

miRNAs and gliogenesis

Many miRNAs are also involved in gliogenesis, including astrogliogenesis and oligodendrocyte differentiation. An in vitro study has shown that miR-125b positively regulates astrogliogenesis and promotes astrocyte proliferation [49]. Deletion of Dicer in the oligodendrocyte linage causes impaired oligodendrocyte differentiation, which can be partially rescued by ectopic expression of oligodendrocyte lineage-specific miR-219 [50]. miR-219 and miR-338 promote oligodendrocyte differentiation by suppressing PDGFRα, Sox6, FoxJ3, ZFP238 and Hes5 [50,51]. Moreover, studies have shown that miR-19b in the miR-17-92 cluster promotes oligodendrocyte precursor proliferation by regulating Akt signaling, and miR-7 enhances the generation of oligodendrocyte lineage cells by targeting proneuronal differentiation factors such as Pax6 and NeuroD4 [52,53]. Furthermore, miR-23 has been shown to suppress Lamin B1 expression and promote oligodendrocyte differentiation [54]. Compared to studies of miRNAs in neuronal specification, reports of miRNAs in glial development are still sparse (Table 1). Whether miRNAs are directly involved in the cell fate switch between neurons and glia is unclear. Identifying more glial-specific miRNAs will further advance our understanding of glial cell fate determination [55].

miRNAs promote the neuronal fate from ESCs, iPSCs and fibroblasts

Because of the therapeutic potential, induction of different types of neuronal cells from ESCs, iPSCs and fibroblasts has become a hot topic. Excitingly, miRNAs have been found to play roles in the reprogramming process. A miRNA profiling study has shown that miR-9 and miR-124a are highly expressed in ESC-derived culture during neuronal differentiation, which is consistent with their roles in promoting differentiation of NSCs and NPs [56], although one study has shown that miR-9 elevates proliferation of NPs that are derived from human ESCs [57]. Let-7 and miR-125 have been found to be strongly induced during neuronal differentiation from ESCs [29,58]. Further investigation has shown that miR-125 promotes neural conversion of human ESCs into SOX1-positive NPs by repressing SMAD4, suggesting that miR-125 is involved in the Bone Morphogenetic Protein (BMP)-mediated classic signaling transduction of neural lineage commitment from ESCs [59]. A study that analyzes 13 human ESC lines and 26 human iPSC lines has found that an increased miR-371-3 expression level favors the neurogenic differentiation propensity of human ESC and iPSC lines [60●].

Together with other TFs, some neural-specific miRNAs have been used to directly induce reprogramming of fibroblasts into neuronal lineages. miR-9/9* and miR-124, along with three neurogenic TFs NeuroD2, Ascl1 and Mytl1, have been shown to efficiently convert human fibroblasts into functional neurons [61●]. Another investigation has shown that miR-124, together with two TFs MYT1L and BRN2, is able to reprogram postnatal and adult human fibroblasts into functional neurons [62●]. These studies suggest that although miRNAs alone are not sufficient for neuronal reprogramming of ESCs, iPSCs and fibroblasts, neural-enriched miRNAs elevate the efficiency of reprogramming.

miRNAs specify neuronal left-right asymmetry

The nervous system is mostly bilaterally symmetric at the anatomical level, but also displays morphological and functional left-right asymmetry to some extent. miRNAs have been shown to play a role in neuronal left-right asymmetry in the nervous system of C. elegans. lsy-6 was the first identified miRNA that controls the left-right asymmetry of ASE left (ASEL) and ASE right (ASER) gustatory neurons by targeting cog-1, a negative regulator of ASEL neuronal cell fate [63]. Further investigation has shown that a C2H2 zinc finger TF lsy-2 regulates ASEL/R asymmetry by modulating transcription of lsy-6 in ASEL neurons [64]. Another zinc-finger TF die-1 has been shown to activate the expression of lsy-6 only in ASEL, but not in ASER. miR-273, an ASER-specific miRNA, has been found to repress die-1 expression to determine ASEL/R asymmetry [65]. Furthermore, miR-71 has been observed to play a role in regulating left/right identification of Amphid Wing Cell C (AWC) olfactory neurons by repressing TIR-1/SARM1 adaptor proteins in the calcium signaling pathway [66]. A most recent study has further identified a mechanism by which the lsy-6 locus is primed in the precursor for the left neuron by chromatin decompaction [67●]. However, the role of miRNAs in asymmetric neuronal organization in the vertebrate nervous system is still unknown.

Conclusions

It is becoming evident that cell fate determination of neurons and glia is tightly controlled by both protein coding genes and noncoding miRNAs. Because one miRNA has multiple target genes, cell fate specification by miRNAs is likely an overall outcome of balanced protein outputs, even though one or a few targets are probably major players. miRNA-mediated gene expression regulation shares similarities with that of TFs, which normally have binding motifs on promoters of multiple genes, including miRNAs, even though TFs act as both activators and repressors, while miRNAs largely negatively control target gene expression. For example, genome-wide quantification has revealed a range of genes that are directly or indirectly regulated by TFs such as Pax6 and Tbr1 in the mouse cortex [68–70]. Similarly, perturbing miRNA expression also affects many target genes, including TFs, in neurons [71–73].

Therefore, miRNAs, in parallel with TFs, form networks with target genes in gene expression regulation. Using genome-wide approaches in combination with functional analyses should advance mechanistic knowledge of miRNA actions in cell fate determination. Moreover, identifying more tissue- and cell type-specific miRNAs and uncovering how their expression is regulated by TFs will further accelerate research in miRNA-mediated cell fate determination.

Highlights.

miRNAs are small noncoding RNAs that normally silence target gene expression.

Many miRNAs have enriched expression in the central nervous system.

miRNAs are required for proliferation and differentiation of neural stem cells.

Neuronal subtypes are specified by miRNAs by suppressing specific target genes.

Networks of miRNA-target are critical in cell fate determination of neurons and glia.