Convergent microRNA actions coordinate neocortical development

1 3 DOI 10.1007/s00018-014-1576-5 Cellular and Molecular Life Sciences Cell. Mol. Life Sci. (2014) 71:2975–2995 RevIew Convergent microRNA actions coordinate neocortical development Olga Barca‑Mayo · Davide De Pietri Tonelli Received: 17 September 2013 / Revised: 11 January 2014 / Accepted: 27 January 2014 / Published online: 12 February 2014 © The Author(s) 2014. This article is published with open access at Springerlink.com Introduction The structure of the mammalian neocortex, the site where higher cognitive behaviors are generated, is organized in six layers and is composed of different neuronal subtypes and glia. Pyramidal-projection glutamatergic neurons are the predominant type of cortical neurons, accounting for 75–85 % of the total neuronal population, depending on the species. The remaining 15–25 % of cortical neurons are GABAergic interneurons [1–3]. The neocortex is generated by the development of the foremost region of the neural tube: the telencephalon. This region comprises the dorsal telencephalon (generating almost exclusively excitatory glutamatergic projection neurons) and the ventral telencephalon (producing inhibitory GABAergic interneurons). Pyramidal neurons of the dorsaltelencephalon are born locally in the germinal zones [i.e., the ventricular (vZ) and subventricular zone (SvZ)], migrate toward the cortical plate by somal translocation along radial glia (RG) (Fig. 1a), and assemble in an inside-out manner to establish the six layers that characterize the laminar structure of the neocortex [4]. In contrast, GABAergic interneurons arise from the germinal zones of the ventral telencephalon and make long journeys, following tangential migratory routes, to their final destinations in the neocortex [4]. Finally, but at a later developmental time compared to cortical neurons, germinal zones give rise to the two major glial cell types of the neocortex, namely, astrocytes and oligodendrocytes [5]. Thus, neocortical development (or corticogenesis) is a complex neurodevelopmental process that requires the precise coordination of cell proliferation, differentiation, as well as subtype specification and migration. How are the pyramidal-projection glutamatergic neurons generated during neocortical development? Several years Abstract Neocortical development is a complex process that, at the cellular level, involves tight control of selfrenewal, cell fate commitment, survival, differentiation and delamination/migration. These processes require, at the molecular level, the precise regulation of intrinsic signaling pathways and extrinsic factors with coordinated action in a spatially and temporally specific manner. Transcriptional regulation plays an important role during corticogenesis; however, microRNAs (miRNAs) are emerging as important post-transcriptional regulators of various aspects of central nervous system development. miRNAs are a class of small, single-stranded noncoding RNA molecules that control the expression of the majority of protein coding genes (i.e., targets). How do different miRNAs achieve precise control of gene networks during neocortical development? Here, we critically review all the miRNA–target interactions validated in vivo, with relevance to the generation and migration of pyramidal-projection glutamatergic neurons, and for the initial formation of cortical layers in the embryonic development of rodent neocortex. In particular, we focus on convergent miRNA actions, which are still a poorly understood layer of complexity in miRNA signaling, but potentially one of the keys to disclosing how miRNAs achieve the precise coordination of complex biological processes such as neocortical development. Keywords Rodents · Cerebral cortex · Development · MicroRNAs O. Barca-Mayo · D. De Pietri Tonelli (*) Department of Neuroscience and Brain Technologies, Istituto Italiano di Tecnologia, via Morego 30, 16163 Genoa, Italy e-mail: da[email protected] 2976 O. Barca-Mayo, D. De Pietri Tonelli 1 3 of study have contributed to clarifying the transcriptional mechanisms that control the generation of cortical neurons during development [6, 7], but in recent years, novel important mechanisms of gene regulation have been discovered. examples of these mechanisms include epigenetics [8] and noncoding RNAs [9]. microRNAs (miRNAs) are a class of small, single-stranded noncoding RNA molecules that control the expression of the majority of protein coding genes (i.e., targets), mainly at the post-transcriptional level [10]. miRNAs and their binding sites in target messenger RNAs (mRNAs) are often evolutionarily conserved among distant organisms [11, 12]. Moreover, miRNAs that are expressed in the developing central nervous system (CNS) exert a prominent role as regulatory molecules that coordinate gene networks during neocortical development, as well as in brain function and dysfunction [13–18]. How do the different miRNAs achieve precise control of gene networks during neocortical development? Here, we critically review all the miRNA-target interactions validated in vivo, with relevance to the generation and migration of pyramidal-projection glutamatergic neurons and initial formation of cortical layers in the embryonic development of the neocortex in mice and rats (hereinafter collectively referred to as rodents). In particular, we first describe the miRNA biogenesis pathway, and then the diversity of the neural stem and progenitor cells (NSCs/NPCs) that give rise to pyramidal-projection glutamatergic neurons. Next, we describe the pattern of miRNA expression, and review the studies that have used the genetic inhibition of miRNA biogenesis as a strategy to investigate the global role of miRNAs in the context of neocortical development. Finally, we review the studies in which manipulation of specific miRNAs have been used as a strategy to identify targets and mechanisms that control neocortical development in vivo. Moreover, here we focus on convergent miRNA action on targets, which is still a poorly understood layer of complexity in miRNA signaling, but potentially one of the keys to disclosing how miRNAs achieve the precise control of molecular programs, pathways and biological functions that coordinate complex biological processes such as neocortical development. Fig. 1 Cell biology and miRNA expression in developing rodent neocortex. a In the early phase of cortical development, the telencephalon is composed of single layer neuroepithelial (Ne) cells that divide at the ventricular (apical) surface of the ventricular zone (vZ). In this phase, Ne cells undergo a massive expansion, mostly by symmetric self-amplifying divisions (curved arrow). At the onset of neurogenesis, Ne cells progressively start to divide asymmetrically. Ne cells can generate the Cajal-Retzius neurons, which migrate out from the vZ toward the basal lamina (basal) and form the preplate (PP). During development, the PP is split into the marginal zone (MZ) and subplate (SP) by incoming neurons, giving rise to the cortical plate (CP). During the mid phase of cortical development, Ne cells give rise to additional and fate-restricted subtypes of neural progenitors cells (NPCs). NPCs deriving from Ne cells include radial glia (RG) and short neural precursors (not shown) collectively referred to as apical NPCs, and progenitors that delaminate from the vZ and divide in the subventricular zone (SvZ), basal progenitors (BP), and outer radial glia (oRG) (collectively referred to as basal NPCs). Apical and basal NPCs can generate progenitors, neurons, or both, and undergo a limited number of symmetric self-amplifying divisions (dashed curved arrows). During mid and late phases of cortical development, several types of glutamatergic cortical projection neurons are generated by apical or basal NPCs. Newborn glutamatergic cortical neurons migrate radially from the vZ/SvZ, and by somatic translocation along the basal processes of RG cells, cross the intermediate zone (IZ) and accumulate into the CP in an inside-out way. The earlyborn neurons form the postnatal cortical layers vI–v (L vI–v) (deep layers), while later-born neurons accumulate above the deep layers, forming the cortical layers Iv–II (superficial layers, L III–II). Layer I (L I) originates from the MZ. b microRNAs (miRNAs) show dynamic patterns of expression in developing rat cortex. The expression pattern of miRNAs in developing rat cortex can be roughly classified into four main trends: (1) miRNAs that are continuously expressed throughout development (pink dashed line); (2) miRNAs that start to be expressed during early development and decline in their expression during development, or perinatally (purple and green dashed lines); (3) miRNAs that start to be expressed in mid or late development and then also remain expressed in postnatal brain (orange and blue dashed lines); and (4) miRNAs that are specifically expressed during a short period during development (curved lines). Co-expressed miRNAs might cooperate to modulate the activities of transcription factors and signaling networks, which are required during specific phases of cortical development ▸ 2977 Convergent microRNA actions coordinate neocortical development 1 3 miRNA biogenesis microRNAs (miRNAs) are short 18–22 nucleotide (nt), single-stranded noncoding RNAs that bind primarily to the 3′ untranslated region (UTR) of target mRNAs to repress their translation and stability [19–22]. Biogenesis of miRNA requires sequential steps; miRNA are generally transcribed by RNA polymerase II as immature nuclear precursors called pri-miRNAs, which are stem-loop containing transcripts. In mammals, pri-miRNAs can also be organized in “clusters,” which are transcripts containing multiple stemloop structures that often give rise to highly similar miRNAs. An example of such a transcript is the miR-17–92 cluster, encoding for some of the best-characterized miRNAs expressed in developing neocortex (see below). PrimiRNAs are typically processed by a nuclear complex of proteins named the “microprocessor”, which is formed by a type III-like ribonuclease protein, Drosha, and by the RNA binding protein Dgcr8 encoded by the DiGeorge syndrome critical region gene 8. After cleavage, the microprocessor releases 60–100 nt-long hairpin-containing intermediate precursors called pre-miRNAs. Pre-miRNAs are then exported to the cytoplasm by the export 5-Ran GTPase shuttle system. In the cytoplasm, pre-miRNAs are again processed into mature miRNAs by another RNase IIIlike ribonuclease protein dicer; they are processed into an 18–22 nt duplex. Alternative miRNA biogenesis pathways have also been found in mammals, and currently it has been shown that miRNA biogenesis can occur following noncanonical pathways, which do not require the microprocessor or dicer [23, 24]. After these steps, one strand (or in a few cases both strands) of the dicer-cleaved pre-miRNA is loaded into RNA-induced silencing complex (RISC), which contains RNA binding proteins of the Argonaute family [25]. RISC-loaded miRNA (i.e., mature miRNA) can interact with target mRNAs. This interaction is typically guided by the binding between the 6–8 nt at 5′ end of the miRNA (i.e., the seed region) and the miRNA binding site(s), which can be located in any region of the target mRNA (i.e., 5′, 3′ UTRs or coding region). Recently, seedless miRNA interactions with mRNA targets have also been described [26– 28]. Moreover, despite the interaction of a miRNA with its target mRNAs typically inducing the post-transcriptional silencing of the target through inhibition of its translation and/or mRNA destabilization [10], a few miRNAs that can activate translation of targets have also been found [29, 30]. This evidence adds further complexity to the mechanism of miRNA-mediated control of gene expression. In recent years, it has become apparent that miRNA biogenesis and decay are subject to sophisticated control in the central nervous system (CNS) [23]. For example, it has been shown that brain-derived neurotrophic factor (BDNF), a secreted protein member of the Neurotrophin family of growth factors, can coordinate the translation of specific subsets of mRNAs by potentiating miRNA biogenesis in neurons. Briefly, BDNF can stimulate the maturation of dicer-dependent miRNAs by increasing the levels of dicer protein, and thereby cause a general decrease in the translation of transcripts that are targeted by these miRNAs. In parallel, BDNF also rapidly induces the expression of Lin28 protein, an RNA-binding protein that by preventing the processing of a subset of anti-proliferative pre-miRNA induces pluripotency (see below), and thus causes the selective decrease/loss of mature miRNAs that depend on Lin28. These parallel roles of BDNF are therefore thought to cause a selective upregulation in translation of a certain set of transcripts that are normally repressed by Lin28-dependent miRNAs [31]. On the other hand, it has been shown that synaptic stimulation accelerates miRNA decay in neurons [32]. This evidence therefore suggests that miRNA turnover might play an important role during neocortical development. The cell biology of cortical neurogenesis One of the first steps towards the development the neocortex is the subdivision of the embryonic telencephalon into two halves along the dorso-ventral (Dv) axis. The ventral half the subpallium will develop into the basal ganglia, and the dorsal half, also known as pallium, will form the cortex. At the molecular level, the patterning of telencephalon relies on the restricted expression of transcription factors that define the specific regions inside the telencephalon itself, as well as rates of cell proliferation, differentiation and programmed cell death, leading to distinct cell subtypes and morphologies [33]. Initial studies reported only a marginal role of miRNAs in the early patterning of vertebrate forebrain [34–36]. Recent evidence, however, reveals that persistence of domain boundaries between different areas might be tuned by miRNAs. Indeed, a dorsal shift of pallial–subpallial boundary was observed in the developing telencephalon of mice knockouts for two of the three genes encoding miR-9 [37], one of the most abundant miRNAs of the developing cortex. Despite this evidence, the role of miRNAs in the control of the early patterning of the telencephalon still remains a poorly understood aspect of forebrain development, and will require further studies. Following the initial patterning of telencephalon, cortical development can be roughly subdivided into three main phases, notably, early, mid and late phase (Fig. 1a). These subsequent phases of cortical development are partially overlapping among each other, and some of the subtypes of NSCs/NPCs and cortical neurons that are generated coexist in space and time during neocortical development (Fig. 1a). In the early phase of cortical development, the 2978 O. Barca-Mayo, D. De Pietri Tonelli 1 3 telencephalonis is composed of a single layer of epitheliallike cells, the neuroepithelial (Ne) cells, which are the primary NSCs of the developing cortex and divide at the ventricular (apical) surface of the vZ (Fig. 1a). In this phase, Ne cells undergo a massive expansion, mostly by symmetric self-amplifying divisions (i.e., one Ne cell generates two daughter Ne cells). At the onset of cortical neurogenesis (which in the dorsal telencephalon of the mouse initiates around e10, and about 2 days later in the rat), Ne cells progressively start to divide asymmetrically, giving rise to one Ne cell plus either a postmitotic neuron or a more differentiated (i.e., fate-restricted) subtype of NPCs that successively replace Ne cells (Fig. 1a). In the developing cortex, the earliest postmitotic neurons that are generated by Ne cells are mainly the Cajal-Retzius, which migrate out from the vZ toward the basal lamina and accumulate in the preplate (PP, Fig. 1a). The PP is a transient cell structure that is later split into subplate (SP, a developmental zone located immediately below the forming cortical plate, Fig. 1a) and marginal zone (MZ, the most superficial cortical layer that becomes layer I in the postnatal cortex, Fig. 1a) by incoming neurons that form the cortical plate (CP, Fig. 1a). The CP is a cell dense structure of the developing cortex that contains postmigratory neurons and that is expanded in layers II–vI in the postnatal cortex (Fig. 1a). In addition to postmitotic neurons, asymmetrically dividing Ne cells also generate fate-restricted subtypes of NPCs (Fig. 1a). The fate-restricted subtypes of NPCs belong to two main categories, those that remain epithelial and divide at the apical surface of the vZ (RG—and short neural precursors, here collectively referred to as apical progenitors); and the progenitors that delaminate from the vZ and divide in the SvZ, or above it [basal progenitors (BP)—also called intermediate, or non-surface, or SvZ progenitors; and outer radial glia (oRG)—also known as intermediate RG, here collectively referred to as BP] [38, 39] (Fig. 1a). The different subtypes of NPCs can be identified by specific morphological characteristics, as well as by expression of a distinct set of proteins [38, 39]. Similarly to Ne cells, fate-restricted NPCs can generate progenitors, neurons, or both. However, in contrast to Ne cells, fate-restricted NPCs undergo a reduced number of symmetric self-amplifying divisions, and most of them undergo divisions that give rise to differentiated progeny [40] (Fig. 1a). During mid and late phases of cortical development, several waves of different glutamatergic cortical projection neurons are generated by fate-restricted NPCs, either by apical or basal division. Newborn glutamatergic cortical neurons migrate radially from the vZ/SvZ, and by somatic translocation along the basal processes of RG cells, they cross the intermediate zone (IZ), and accumulate in the CP in an inside-out way (Fig. 1a). In particular, the early-born neurons form the deep cortical layers vI–v, while later-born neurons accumulate above the deep layers, forming the upper cortical layers Iv–II [6, 41] (Fig. 1a). Finally, later aspects of cortical development include, but are not limited to, neurite outgrowth and the dendritic elaboration of cortical neurons, which lead to the formation of short and long projections, and synaptogenesis with their target sites. In rodents, these aspects are mostly concluded in the first 2–3 postnatal weeks [42] (Fig. 1a). In addition to cortical neurons, several types of glial cells, such as astrocytes and oligodendrocytes, are present in the adult mammalian cortex. In rodents, despite recent evidence indicating that astrocytes precursor cells are generated during embryonic corticogenesis [43], most of the astrocyte and oligodendrocyte cells can be detected after the disappearance of cortical NPCs, in the early postnatal cortex [5]. Given that the role of miRNAs in these later aspects of corticogenesis has been extensively reviewed elsewhere [13–17], here we will not discuss these aspects in detail. In short, neocortical development is a complex process that results from the execution of a precise developmental program that is controlled in a spatially and temporally specific manner. extracellular signals and networks of intrinsic/intracellular factors that govern this developmental program require a precise and coordinated orchestration of their expression, and miRNAs are certainly part of this mechanism. Dynamic expression of miRNAs during neocortical development It is now clear that a tightly regulated event, such as neocortical development, needs a coordinated expression of genes and miRNAs that converge to maintain the proper developmental program. Several studies have profiled miRNA expression in embryonic neocortex of rodents, and provided a crucial first step toward the identification of the possible function(s) of miRNAs in the control of cortical development [44–48]. These studies identified a cohort of miRNAs whose expression dynamically changes during cortical development (Fig. 1b), and provided evidence that in developing cortex some miRNAs share similar dynamics of expression (Fig. 1b), suggesting that co-expressed miRNAs might act cooperatively to modulate the activities of signaling networks that are required during specific phases of cortical development (Fig. 1a). According to these data, the expression pattern of miRNAs in developing cortex can be roughly classified into four main trends (Fig. 1b): (1) miRNAs that are continuously expressed throughout development (e.g., some miRNA members of the let-7 family and miR-9/9*); (2) miRNAs that start to be expressed during early development and decline their expression during development, or around perinatal stage (e.g., miR-125b, miR-181 family, and miRNAs encoded by the miR-17-92 2979 Convergent microRNA actions coordinate neocortical development 1 3 cluster, and by its paralogous genes miR-106a-363, miR106b-25, collectively referred to as miR-17-92 subfamily); (3) miRNAs that start to be expressed in mid or late development and then remain expressed also in postnatal brain (e.g., miR-124, miR-128, etc. ); and (4) miRNAs that are specifically expressed during a short period during development. The latter category is particularly interesting, because it might control specific stages of cortical development such as cell proliferation, viability, neuronal differentiation, migration, and neuronal network formation. MiRNAs that are continuously expressed throughout corticogenesis The let-7 family of miRNAs is among the miRNAs that show a continuous expression throughout cortical development (Fig. 1b). let-7 is encoded by the Lethal-7 (let-7) gene, one of the founding miRNAs discovered in the nematode worm Caenorhabditis elegans (C. elegans), and is evolutionarily conserved in vertebrates [49]. In mammals, the let-7 family of miRNAs comprises several mature miRNA sequences that differ only in a few nucleotides. The small differences in their seed regions are thought to discriminate let-7 family members for their specific target genes, thus leading to distinctive biological consequences [50]. let-7 family members play significant roles in proliferation of NSCs/NPCs and in the control of neurogenesis [50] (see below). Mature miR-9 and -9* also show high expression levels throughout cortical development (Fig. 1b). Interestingly, miR-9 and -9*share the same pre-miRNA precursor transcript. As introduced above, typically only one mature miRNA can be detected as a stable product cleaved from a double strand pre-miRNA. However, in mammals the precursor for these miRNAs is transcribed by three loci (i.e., miR-9-1, miR-9-2 and miR-9-3), which are all coexpressed in the developing mouse cortex [37]. Therefore, it is possible that miR-9 and -9* derive from the cleavage of independent pre-miRNA precursors during brain development. Despite this hypothesis, the exact mechanism of miR-9 and -9* biogenesis in developing cortex remains obscure. Some sets of miRNAs share dynamic expression patterns during corticogenesis As introduced above, the early phase of cortical development is characterized by the fast proliferation and expansion of NSCs/NPCs. In the rat cortex, this phase occurs roughly between embryonic day 10 (e10) and e11 (Fig. 1a). In recent study, Yao et al. [48] performed miRNA profiling in developing and postnatal rat cortical tissues [from e10 to post-natal day 28 (P28)], and found that nearly 40 % of all miRNAs expressed during cortical development had the highest abundance at e10, while decreasing at later stages (Fig. 1b). Among those miRNAs, miR-34c, -152, -219-5p, -301b, -449a, -451 and -532-5p were tenfold more abundant at e10 than at any other stage, providing a hint that those miRNAs may play important roles in the regulation of NSC/NPC proliferation and viability [48]. In another study from Nielsen et al. [46], it has been reported that only 7. 2 % of miRNAs underwent significant changes between e11 and e13, whereas the majority of them did not change expression during this developmental time window. It should be noted, however, that some of the miRNAs found to be highly expressed between e10 and e11 in Yao’s study, such as miR-20b*, -126, -143, -183, -199a, -200b/c, -214, -222 and -292-3p, are not overlapping with those of Nielsen’s study [46, 48]. This discrepancy might be due to the different sensitivity of the methods used, to the different cell sources, or to the existence of strain-specific miRNAs. Further detailed discussion about the systematic comparison of commercially available miRNA profiling platforms, as well as RNA extraction and quality control methods, can be found in a recent paper from Git and colleagues [51]. Around e12–e13 (i.e., at the onset of cortical neurogenesis in rats), several changes in the expression of miRNA are observed (Fig. 1b) [48]. At this stage, some miRNAs that are enriched in the early phase of cortical development (e.g., the miR-181 family and miR-199a) gradually start to decrease, and eventually their expression stops before birth, suggesting a possible role of these miRNAs in control of proliferation and possibly other aspects of early cortical development (Fig. 1a) [48]. At the same stage, other miRNAs are at the peak of their expression (e.g., miR-7, -7a and miR-191, Fig. 1b), suggesting a possible role for these miRNAs at the onset of neurogenesis (Fig. 1a) [48]. Conversely other miRNAs, such as those encoded by the miR-17-92 subfamilies of miRNAs (not shown), and others belonging to a well-characterized category of CNSenriched miRNAs, namely miR-99a -124a and miR-266, start to be upregulated (Fig. 1b) [46]. In addition to these miRNAs, between e13 and birth, other miRNAs start being expressed (Fig. 1b), followed by their downregulation after birth [44]. The miRNAs encoded by the miR-17-92 cluster are amongst the best-characterized miRNAs in mammals, and are expressed in many tissues. At the functional level, knockout mice for the miR-17-92 cluster (and its paralogous genes) display early embryonic lethality, and miRNAs encoded by this cluster play essential roles in the control of NSC/NPC self-renewal, and subtype specification [52]. In the developing rat cortex, between e21 and birth most of the glutamatergic cortical neurons reach their final laminar destination and start projecting axons and dendrites toward their final targets (Fig. 1a). At this stage, the expression of miR-19b decreases rapidly; miR-137 shows a peak in its expression; whereas other miRNAs such as let-7b, 2980 O. Barca-Mayo, D. De Pietri Tonelli 1 3 miR-128 and miR-185 start to be expressed (Fig. 1b) and their expression increases in the postnatal days (especially between P14 and P28) [48]. In rodents, major sensory inputs are established and most glial cells are generated during the first 2–3 postnatal weeks [5]. For example, eye opening occurs around P13 and is thought to result in activity-dependent neuronal remodeling. Consistently, some of the miRNAs that are enriched during late embryogenesis, or at the perinatal stage, such as miR-128 (Fig. 1b) and miR-29a (not shown), tend to increase over time [48], suggesting a role for these miRNAs in late aspects of brain development, such as fine control of cortical connectivity, or gliogenesis. Detection of miRNA expression in corticogenesis: what’s next? miRNA profiling is very informative and has allowed us to gain important information about temporal expression of miRNAs during corticogenesis. However, this approach also has limitations. For example, conventional miRNA profiling does not provide information with respect to spatial expression of miRNAs. Moreover, the high heterogeneity of the cells types present in the developing neocortex, or during the early postnatal weeks (when major sensory inputs are established and most glial cells are generated [5]), might contribute to the changes observed in miRNA profiles. In order to have a complete picture of the pattern(s) of miRNA expression in the various cortical cell types, some recent studies have started to perform cell-type-based analysis of miRNA profiles in the mouse brain [53, 54]. In particular, these studies profiled the expression of miRNAs in various neuronal subtypes, such as glutamatergic and GABAergic neurons [53], or in neurons and glia [54]. It would also be interesting to apply this approach to perform profiling of miRNA expression in specific subpopulations of NSCs/NPCs in developing cortex. For example, given that oRG has been implicated in the evolutionary expansion of the neocortex in primates and human, in particular in the generation of the outer SvZ (a transient germinative layer that is typical of the development of gyrencephalic cortex [38, 39]), future comparative studies on miRNA expressed in oRG might shed new light on the molecular mechanisms at the base of expansion and evolution of the neocortex. On the other hand, techniques such as negative sensors for miRNAs [55, 56], in situ hybridization [57, 58], or the use of transgenic organisms expressing green fluorescent protein (GFP) under the control of promoters of miRNAs genes [59] allow us to gain insight into spatial and temporal dynamics of the expression of a given miRNA. By using these techniques, it was recently uncovered that the expression pattern of miR-124, one of the most abundant miRNAs of the mammalian brain and generally regarded as being specifically restricted to post-mitotic neurons in the developing cortex [60, 61], starts its expression in NPCs [56, 62, 63]. Thereby, the use of cell-type based miRNA profiling in combination with techniques providing spatial information and dynamics of miRNA expression will be important to gain a better insight into possible functions of miRNAs in the control of specific aspects of cortical development. miRNA depletion approach to investigate the global requirement for miRNAs during neocortical development The correlation of some sets of miRNAs with distinct phases of cortical development (Fig. 1) suggests that coexpressed miRNAs might coordinate gene expression to regulate specific aspects of cortical development, such as NSC/NPC expansion and their differentiation. How is it possible to functionally investigate the role of co-expressed miRNAs during cortical development in vivo? For this purpose, several studies have used depletion of miRNAs, by means of genetic inactivation of the RNase III enzyme dicer or other essential proteins for miRNA biogenesis such as Dgcr8. Mice null for dicer, or Dgcr8, are not viable and die in utero before the onset of cortical neurogenesis, indicating that miRNAs are critical for mammalian development [64, 65]. To bypass the early embryonic lethality and investigate the global role of miRNAs in specific phases of cortical development (or cortical cell types), several mouse mutant lines carrying conditional deletions of dicer have been created. In these studies, conditional deletion of Dicer was obtained in vivo with Cre-recombinase driven by Foxg1, Emx1; Nes, Nex, human GFAP, and Camk2 promoters [36, 66–78]. Overall, conditional deletion of dicer in the embryonic neocortex resulted in gross anatomical abnormalities, and provided evidence that miRNAs regulate important aspects of cortical development, such as cell viability, self-renewal and commitment (i.e., the program engaged by stem or progenitor cells that leads to their differentiation) of NPCs, as well as differentiation, migration and maturation of glutamatergic cortical neurons, and in turn, the proper formation of cortical layers. Cell loss upon conditional deletion of dicer in embryonic neocortex In embryonic neocortex, conditional deletion of dicer in vivo (although with some noticeable differences depending on the onset of Cre-recombinase expression) generally causes loss of neurons, and in some cases also loss of 2981 Convergent microRNA actions coordinate neocortical development 1 3 NSC/NPC pools [36, 66–78]. Overall, a plausible explanation for cell loss upon conditional deletion of dicer in embryonic neocortex is increased cell death. However, which specific cell types are actually lost, as well as the mechanisms responsible for cell death upon dicer deletion, are still unresolved questions (see limits of the global miRNA-depletion approach, below). Cell loss could be triggered, for example, by impaired differentiation of dicer-deleted NSCs/NPCs (and newborn neurons), which might fail to exit the cell cycle upon differentiation and therefore undergo cell death. In some studies, the loss of neurons was due to increased apoptosis [36, 66– 72, 75]. However, in other studies and in agreement with in vitro evidence [79], neuronal loss observed upon dicer deletion in vivo is not always due to a dramatic increase in apoptosis [67, 73, 76, 78, 80, 81]. On the other hand, cell loss in dicer-deleted embryonic cortices might also be due to the progressive decline of proliferation of dicer-deleted NSCs/NPCs, leading to their proliferative arrest and death. Remarkably, despite selfrenewal of NSCs/NPCs seeming to be less affected by dicer deletion than cell fate transitions [68, 79, 82] (see also cell fate transitions and developmental maturation of NSCs/NPCs below), several studies reported progressive arrest in NSC/NPC proliferation upon dicer deletion in embryonic cortices [36, 66, 68, 69, 71, 73, 75]. Two recent studies characterized in detail the proliferation of dicer-deleted NPCs during late cortical and hippocampal development, and both studies found that miRNAs are essential for maintenance of NPC pools [71, 73]. In the first study, reduced proliferation of hippocampal NPCs was observed at e15. 5, upon dicer deletion obtained with Emx1 and Nestin promoters [71]. In the second study, dicer deletion was obtained with the human GFAP promoter, and this resulted in a significant impairment in RG cell proliferation at P15 and P40. This proliferation defect was mostly attributed to “High-temperature requirement A serine peptidase 1” (HtrA1) gene product, whose overexpression in the developing neocortex recapitulated some of the phenotypes observed in dicer deleted cortices. The results of these two studies indicate that at late developmental stages, miRNAs functions are essential for the maintenance of the NPC pool in both hippocampus and cortex. It remains to be seen whether the increased apoptosis observed at certain stages of development, or the limited sensitivity of self-amplifying NSCs/NPCs to the dicer deletion, reflect a real characteristic of the miRNA pathway (e.g., that might be required/dispensable for maintenance of the NPC pool at certain developmental stages), or is due to some technical limitation of the conditional dicer deletion approach (see limits of the global miRNA-depletion approach, below). Cell fate transitions and developmental maturation of NSCs/NPCs are impaired upon conditional deletion of dicer in embryonic neocortex Perhaps the most recurrent theme resulting upon selective deletion of dicer in cortical NSCs/NPCs is that miRNA depletion often impairs cell fate transitions (e.g., the transition from apical to BP, or the differentiation of NSCs/NPCs to cortical projection neurons) or developmental maturation toward more restricted progenitor cell types (e.g., progressive restriction of RG competence). In agreement with this statement, a wide spectrum of defects, such as premature, delayed, or reiterated generation of certain subtypes of neural stem and progenitors cells (both apical or BP), neurons, or glia, has been reported upon dicer deletion in developing neocortex [36, 66, 68, 69, 71, 73, 75, 76, 81]. This evidence strongly supports our initial observation that in developing mouse, neocortex miRNA functions are less required for self-amplification of NSCs/NPCs, but are particularly important during cell-fate transitions [68]. In particular, miRNA-depleted embryonic NSCs from dicer-deleted cortices can self-renew for several days in vivo [68] or in vitro [79, 82], but are unable to differentiate in neurons or astrocytes, and exhibited a marked dependency on exogenous mitogens for survival [68, 79, 82]. These data are also consistent with similar observations during the differentiation of dicer-deleted murine embryonic stem cells [65, 83, 84] in vitro. More recently, it has been observed that conditional deletion of dicer also impairs the developmental maturation of NSCs/NPCs. In particular, it has been shown that dicer deletion results in the progressive restriction of RG competence [76]. Another example is the transition between the pre-neurogenic Ne cells and the neurogenic RG [36]. Given that there are very few known mechanisms implicated in the regulation of either of these processes, it is therefore particularly interesting that miRNAs might also be involved in these important aspects of cortical development. In short, these common themes for miRNA function in developing cortex are reminiscent of those observed for the founding miRNAs lin-4 and let-7 in C. elegans; that clearing and repressing previously expressed transcripts facilitates progression to the following developmental stage [19–22]. Defective cortical lamination and migration upon dicer deletion in embryonic neocortex As introduced above, the cerebral cortex is a highly organized laminar structure that results from the precise positioning of subsequent waves of cortical glutamatergic neurons that are generated in the course of cortical development 2982 O. Barca-Mayo, D. De Pietri Tonelli 1 3 (Fig. 1a). The first study addressing this aspect in detail reported laminar defects in the postnatal neocortex upon Emx1-Cre mediated dicer deletion [68], suggesting that radial migration of cortical glutamatergic neurons was impaired upon miRNA depletion. Consistent with this finding, misplacement of neurons in dicer-deleted embryonic or postnatal neocortex was reported in a number of studies [36, 69, 71, 75, 76]. Several mechanisms might explain the various defects in neuronal migration that have been reported in dicer-deleted cortex. For example, loss of radial RG cells, which has been observed upon conditional dicer deletion, might affect the integrity of the scaffold used during somatic translocation of radially migrating cortical projection neurons (Fig. 1a), thus causing their retention in the deep cortex [36, 68]. Another example is the loss of Cajal-Retzius cells, which have been shown to impair radial migration of cortical neurons [85]. Indeed, lost or misplaced Cajal-Retzius cells in the developing cortex have been observed upon conditional deletion of dicer [71, 75]. Finally, defective coordination of leading process extension and branching in neurons has also been observed upon dicer deletion [78, 86]. Given the intricacy of the signaling and pathways involved in control of the radial migration of neurons in developing cortex, it is very difficult to gain insight into the role of miRNAs in the regulation of radial migration of cortical projection neurons by using conditional deletion of dicer. This aspect therefore still remains poorly understood. Conditional deletion of Dgcr8 in embryonic neocortex In addition to dicer deletion, depletion of miRNAs can also be obtained by conditional deletion of the Dgcr8 gene. A recent study compared the phenotypes in the postnatal brain of mice in which the conditional deletion of dicer and Dgcr8 in post-mitotic neurons of the embryonic and postnatal cortex was mediated by Cre-recombinase driven by Camk2 promoter [72]. The loss of Dgcr8 resulted in a later lethality, milder structural abnormalities, and less apoptosis relative to that from dicer loss. Deep sequencing of small RNAs from postnatal hippocampus and cortex isolated from the conditional Dgcr8 deleted mice identified multiple non-canonical miRNAs, including miRtrons that were differentially expressed relative to dicer-deleted animals, suggesting a diverse population of highly expressed noncanonical miRNAs that together are likely to play important functional roles in post-mitotic neurons. It would be interesting to see whether the conditional deletion of Dgcr8 in NSCs/NPCs during corticogenesis will also result in a milder phenotype compared to conditional deletion of dicer. Recently, Dgcr8 gene has also gained momentum in the physiopathology of neural defects observed in the DiGeorge syndrome, a genetic disorder that in humans originates from the micro deletion of the region q11.2 of chromosome 22 encoding for about 30 genes, including the DGCR8 gene [87–89]. These studies further highlight the importance of miRNAs and miRNA biogenesis pathway genes in complex neural diseases. Limitations of the global miRNA-depletion approach Taken together, the in vivo studies of miRNA function, by conditional deletion of essential genes for miRNA biogenesis, revealed that miRNAs are crucial regulators of cortical development, and also underscored that miRNAs are part of a potent (and still underestimated) mechanism that can greatly impact on behavior and may predispose to brain disorders. On the other hand, this approach has some inconveniences, such as the subtle differences in the phenotypes observed in embryonic cortices of dicer-deleted mice that might be due to the promoter used to induce Creexpression (see for example [68, 69]), or by the fact that dicer ablation might be incomplete/delayed in certain cell types, or developmental times. Moreover, complete depletion of some miRNAs might require a long time upon dicer deletion [79]. Furthermore, one should also consider that additional functions of the microprocessor might contribute to the observed phenotypes. In agreement with the latter possibility, a recent study reported that knockdown of genes encoding for components of the microprocessor complex in NPCs of the developing mouse neocortex resulted in a loss of stem cell character and in their precocious differentiation, whereas knockdown of dicer did not, suggesting that the microprocessor regulates neurogenesis in a miRNA-independent way [90]. Finally, given the overwhelming amount of apoptosis observed upon dicer ablation in some studies, and that clearance of the apoptotic cells in the developing tissues is typically very rapid, it is sometimes very difficult to draw conclusions. Despite these limitations, which might complicate the analysis and interpretation of results, the approach of conditional deletion of essential moieties for miRNA biogenesis still remains widely used to investigate the functional role of miRNAs in mouse CNS, and indeed, several studies have identified specific miRNAs and targets responsible for some of the dicer phenotypes [66, 70, 73, 74, 91–93]. Convergent miRNA actions on targets coordinate gene networks in the developing cortex Proper cortical development requires the generation of the appropriate numbers and types of cells from common progenitor pools, and the right positioning of neurons in the cortical plate. This is achieved by integrating multiple intrinsic and extrinsic signals governing the relationships of 2983 Convergent microRNA actions coordinate neocortical development 1 3 self-renewal, commitment, survival, differentiation, delamination/migration, attractions/repulsions, etc. Accumulating evidence shows that a single miRNA can act in parallel on several targets either in one cell, in different cells, or at different developmental time points, thus coordinating the intrinsic and extrinsic signals that control cortical development. On the other hand, by using a proteomic approach, it was demonstrated that the extent of repression mediated by a single miRNA is surprisingly mild [94, 95]. with such a mild regulation, it is therefore unclear how a miRNA can provoke a meaningful functional change in a biological process. An attractive possibility to solve this apparent paradox is that cooperation between co-expressed miRNAs might compensate the fine-tuned mRNA regulation mediated by a single miRNA, thus exerting a broader impact on gene expression compared to a single miRNA (Fig. 2). This scenario is supported by experimental evidence indicating that different miRNA binding sites in the same 3′UTR can potentiate the degree of translational repression of a single miRNA [96–100]. In a recent review from Schouten and colleagues [18], the positive interaction of two or more individual miRNAs, or one individual miRNA, acting on multiple seed regions on the same 3′UTR was defined as “miRNA cooperativity,” a concept that highly resembles the “cooperative binding” of ligands to a single receptor protein, or of transcription factors to a single promoter in DNA. Although we agree with the strict definition of miRNA cooperativity [18], it does not account for the abundant evidence of individual miRNAs co-regulating different target genes in parallel (e.g., see Lim et al. [101] and Conaco et al. [102]), and that some of the co-regulated genes might exert redundant functions (see below). we therefore propose the wider concept of “convergent miRNA action,” which we define as “the synergic action of one or more individual miRNAs that by acting on different seed regions in one or more target genes results in a regulatory effect”. In our definition, the synergic action of miRNAs might affect a single gene, molecular pathway, and in turn different biological functions. examples of convergent miRNA actions include, but are not limited to, the simultaneous co-regulation of a single target mRNA by two or more miRNAs (Fig. 2a, previously defined by Schouten and colleagues as “miRNA cooperativity” [18] and here referred to as convergence on target, for internal consistency); coordinated regulation of different targets encoding for proteins that are acting on the same pathway (Fig. 2b, referred to as convergence on pathway); or coordinated regulation of targets encoding for proteins that are exerting similar functions (Fig. 2c, referred to as convergence on function). In order to be effective, convergent miRNA actions must occur not only between miRNAs with shared seed sequences (e.g., members of the same family of miRNAs, Fig. 2 examples of convergent miRNA actions on targets. miRNAs might act through convergent actions to orchestrate intrinsic and extrinsic signals during cortical development. examples of convergent miRNA actions include, but are not limited to, the simultaneous co-regulation of a single target by one or more co-expressed miRNAs [convergence on target, previously defined by Schouten and colleagues as miRNA cooperativity (a)]; coordinated regulation of different target encoding for proteins that are acting in the same pathway [convergence on pathway, (b)]; or coordinated regulation of target encoding for proteins that are exerting redundant functions [convergence on function, (c)]. 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