Review
Instructing neuronal identity during CNS development and
astroglial-lineage reprogramming: Roles of NEUROG2 and ASCL1
Malek Chouchane
a,b, Marcos R. Costa
a,⇑aBrain Institute, Federal University of Rio Grande do Norte, Natal 59072-970, Brazil b
Neurological Surgery Department, University of California, San Francisco 94158, USA
a r t i c l e i n f o
Article history:Received 20 November 2017
Received in revised form 16 February 2018 Accepted 27 February 2018
Available online xxxx Keywords:
Neural progenitors
Neuronal identity specification Astroglia lineage-reprogramming Neurog2
Ascl1
a b s t r a c t
The adult mammalian brain contains an enormous variety of neuronal types, which are generally catego-rized in large groups, based on their neurochemical identity, hodological properties and molecular mark-ers. This broad classification has allowed the correlation between individual neural progenitor populations and their neuronal progeny, thus contributing to probe the cellular and molecular mecha-nisms involved in neuronal identity determination during central nervous system (CNS) development. In this review, we discuss the contribution of the proneural genes Neurogenin2 (Neurog2) and Achaete-scute homolog 1 (Ascl1) for the specification of neuronal phenotypes in the developing neocor-tex, cerebellum and retina. Then, we revise recent data on astroglia cell lineage reprogramming into induced neurons using the same proneural proteins to compare the neuronal phenotypes obtained from astroglial cells originated in those CNS regions. We conclude that Ascl1 and Neurog2 have different con-tributions to determine neuronal fates, depending on the neural progenitor or astroglial population expressing those proneural factors. Finally, we discuss some possible explanations for these seemingly conflicting effects of Ascl1 and Neurog2 and propose future approaches to further dissect the molecular mechanisms of neuronal identity specification.
Ó 2018 Published by Elsevier B.V.
Contents
1. Introduction . . . 00
2. Influences of Ascl1 and Neurog2 expression for the acquisition of neuronal identities by neocortex neural progenitors and lineage-reprogrammed astrocytes . . . 00
2.1. Neural progenitors and neuronal types of the neocortex . . . 00
2.2. Neurog2 and neuronal specification in the developing neocortex . . . 00
2.3. Ascl1 and neuronal specification in the developing neocortex . . . 00
2.4. Neurog2 and Ascl1 instruct different neuronal phenotypes in lineage-reprogrammed neocortical astrocytes. . . 00
3. Influences of Ascl1 and Neurog2 expression for the acquisition of neuronal identities by cerebellum neural progenitors and lineage-reprogrammed astrocytes . . . 00
3.1. Neural progenitors and neuronal types of the cerebellum . . . 00
3.2. Neuronal identity determination by Neurog2 and Ascl1 in the developing cerebellum. . . 00
3.3. Neurog2 and Ascl1 instruct different neuronal phenotypes in lineage-reprogrammed cerebellar astrocytes. . . 00
4. Influences of Ascl1 and Neurog2 expression for the acquisition of neuronal identities by retinal neural progenitors and lineage-reprogrammed Müller Glia . . . 00
4.1. Neural progenitors and neuronal types of the retina . . . 00
4.2. Neuronal identity determination by Neurog2 and Ascl1 in the developing retina . . . 00
4.3. Ascl1 instruct multiple neuronal phenotypes in lineage-reprogrammed Müller Glia. . . 00
5. Concluding remarks . . . 00
https://doi.org/10.1016/j.brainres.2018.02.045 0006-8993/Ó 2018 Published by Elsevier B.V.
⇑ Corresponding author.
E-mail address:[email protected](M.R. Costa).
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j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / b r e s
reprogram-6. Perspectives . . . 00 References . . . 00
1. Introduction
The transcription factors (TFs) Neurogenin2 (Neurog2) and Achaete-scute homolog 1 (Ascl1) have been widely described in the developing central nervous system (CNS) as master regulators of neural fate and specification of neuronal identities (Bertrand et al., 2002). Numerous loss and gain of function studies have pro-vided evidence of their necessity and sufficiency to specify neu-ronal identities in the developing neocortex (Britz et al., 2006; Casarosa et al., 1999; Fode et al., 2000; Horton et al., 1999; Kovach et al., 2013; Mattar et al., 2004; Parras et al., 2002; Poitras et al., 2007; Schuurmans et al., 2004), cerebellum (Florio et al., 2012; Grimaldi et al., 2009) and retina (Akagi et al., 2004; Brzezinski et al., 2011). Within the telencephalon, Neurog2 is expressed in progenitor cells committed to the generation of gluta-matergic neurons, whereas Ascl1 is expressed in progenitors con-tributing to the generation of GABAergic interneurons. On the other hand, in the developing cerebellum, Neurog2 and Ascl1 are expressed by overlapping populations of progenitor cells that give rise to GABAergic neurons, such as cerebellar nuclei neurons and Purkinje cells. Finally, in the retina, progenitors expressing Neu-rog2 generate all neuronal types, whereas Ascl1 + progenitors gen-erate all neuronal types but retinal ganglion cells – RGCs (Brzezinski et al., 2011; Ma and Wang, 2006), suggesting that those proneural genes have redundant roles in neuronal specification in the developing retina (Akagi et al., 2004), but Neurog2 would have one particular role in RGC specification. Different contributions of Neurog2 and Ascl1 for the generation of specific cell types can also be found in the spinal cord (Helms et al., 2005; Mizuguchi et al., 2001; Scardigli et al., 2001) and other CNS structures (Andersson et al., 2006; Kele et al., 2006; Pattyn et al., 2004). In this work, we will review the multiple contributions of Neurog2 and Ascl1 to the specification of neuronal identity in the developing neocor-tex, cerebellum and retina. Next, we will discuss recent data on cell-lineage reprogramming of glial cells into neurons using Neu-rog2 and Ascl1 to address the question as to whether the origin (neocortex, cerebellum or retina) of the reprogrammed glial cell interferes with the probability to generate induced neurons with a specific phenotype.
2. Influences of Ascl1 and Neurog2 expression for the acquisition of neuronal identities by neocortex neural progenitors and lineage-reprogrammed astrocytes 2.1. Neural progenitors and neuronal types of the neocortex
The mammalian neocortex is a complex structure, containing hundreds of different types of neurons. In a broader manner, these neurons are classified into interneurons, which extend short axons and make synapses onto local cells, and projection neurons, which extend long axons and make synapses onto cells located at distant targets (Markram et al., 2004a,b). Projection neurons can be further classified according to their targets into commissural/callosal, cortico-pontine, cortico-tectal, cortico-spinal and cortico-thalamic neurons, which connect to other neurons in the contralateral neo-cortex, pons, tectum, spinal cord and thalamus, respectively (Molyneaux et al., 2007). Despite these different hodological prop-erties, all projection neurons of the neocortex share a glutamater-gic identity. Neocortical interneurons, however, can be either glutamatergic or GABAergic. For instance, layer IV spiny stellate
cells found in primary sensory areas of the neocortex are gluta-matergic, but connect only to neighboring cells, being therefore classified as interneurons (Markram et al., 2004a,b). The largest proportion of neocortical interneurons, however, is composed of aspiny GABAergic neurons, which can be subdivided into several subtypes according to their morphologies, physiology and molecu-lar signatures (Markram et al., 2004a,b). Therefore, most gluta-matergic neurons in the neocortex are excitatory projection neurons (with the exception of spiny stellate cells, which are exci-tatory interneurons) and most GABAergic neurons are inhibitory interneurons.
Despite their morphological differences, all neocortical gluta-matergic neurons are exclusively generated by progenitor cells located in the dorsal telencephalon, whereas neocortical GABAer-gic interneurons are solely generated in the ventral telencephalon (Molyneaux et al., 2007; Wonders and Anderson, 2006). Thus, two separate progenitor domains in the developing telencephalon con-tribute to the generation of neurons with distinct neurotransmitter identities in the neocortex. This patterning is also reflected in the expression of the proneural genes Neurog2 and Ascl1. While the former is expressed by progenitors in the dorsal telencephalon that generate glutamatergic neurons, the latter is mostly expressed by progenitors in the ventral telencephalon and regulates the acquisi-tion of a GABAergic identity (Schuurmans and Guillemot, 2002). These observations suggest that Neurog2 and Ascl1 could be instructive to the acquisition of glutamatergic and GABAergic fates, respectively. Below, we will discuss some of the work addressing this possibility.
2.2. Neurog2 and neuronal specification in the developing neocortex Neurog2 is a basic helix-loop-helix (bHLH) TF that regulates the expression of key genes encoding for proteins involved in several mechanisms during CNS development, such as progenitor cell pro-liferation and patterning, as well as neuronal differentiation, sub-type specification and migration (Florio et al., 2012; Hand et al., 2005; Heng et al., 2008; Imayoshi and Kageyama, 2014; Schuurmans et al., 2004). Pioneer studies have shown that the expression of several TFs involved in the specification of neocorti-cal neurons is severely altered in Neurog2 mutant mice (Schuurmans et al., 2004). In these animals, the expression of Neu-ronal Differentiation 1 and 2 (NeuroD1 and NeuroD2), T-box, brain 1 (Tbr1) and T-box, brain 2 (Tbr2) is decreased, whereas the expression of distal-less genes 1, 2 (Dlx1, Dlx2) is upregulated in the dorsal telencephalon. As a consequence, neocortical neurons exhibit a downregulation in the expression of vesicular glutamate transporters (Vglut1 and Vglut2) and an upregulation of biosyn-thetic enzymes for GABA such as glutamic acid decarboxylase 1 and 2 (GAD1, GAD2) and GABA transporter 1 (GABA T-1), sug-gesting a switch of neurotransmitter identities.
These observations suggest that Neurog2 is not only important for the initiation of a glutamatergic phenotype in neocortical neu-rons, but also to repress an alternative subcortical GABAergic dif-ferentiation program. According to this interpretation, chromatin immunoprecipitation studies have shown that Neurog2 binds directly to the E-box of Tbr2 promoter inducing its activation (Ochiai et al., 2009). In turn, Tbr2 acts as a transcriptional repressor of the ventral gene Early B-Cell Factor 1 (Ebf1), which positively regulates the expression of the ventral gene Ascl1 (Kovach et al.,
2013). As a consequence, Neurog2 indirectly represses the
reprogram-expression of Ascl1. Indeed, sustained overreprogram-expression of Neurog2 in the ventral telencephalon, achieved through electroporation, is sufficient to induce the expression of TFs involved in the specifica-tion of glutamatergic neurons, such as Nescient Helix-Loop-Helix 1 - Nscl1/2, Class B Basic Helix-Loop-Helix Protein 5 - bHLHb5, Neu-roD1/2/4/6 and Tbr1 (Kovach et al., 2013; Mattar et al., 2008). Interestingly, the kinetics of gene expression induced by Neurog2 overexpression in the dorsal and ventral telencephalon is distinct, with a significant temporal delay in the ventral region (Mattar et al., 2008). This delay can be partly explained by the presence of context-specific cofactors, such as Neuronal Differentiation 4 (NeuroD4 or Math3). Accordingly, co-expressed Neurog2 and Math3 synergistically bind to NeuroD1 and 2 genes promoters accelerating the kinetics of gene expression in the ventral telen-cephalon (Mattar et al., 2008). These regional differences in the transcriptional cascades elicited by Neurog2 may suggest that the acquisition of a glutamatergic identity in the dorsal telen-cephalon requires additional factors. In other words, Neurog2 would not suffice to specify a glutamatergic identity, but would rather act as permissive factor.
According to this view, analysis of transgenic mice in which the coding sequences of Ascl1 and Neurog2 are exchanged indicate that while Ascl11 has the capacity to re-specify the phenotype of neuronal populations normally derived from Neurog2-expressing progenitors in the dorsal telencephalon, mis-expression of Neu-rog2 in Ascl1-expressing progenitors does not result in any observ-able change in neuronal phenotype (Parras et al., 2002). Notably, ectopic expression of Neurog2 in the ventral telencephalon can rescue the neurogenesis defects of Ascl1 null mutants, indicating that the pro-neuronal activity of Neurog2 is indeed independent of its contribution to neuronal phenotype specification (Parras et al., 2002). Corroborating this interpretation, analysis of gene expression in the brain of Neurog2, Neurog1, Pax6 and Tlx1 knock-out (KO) mice suggests that Neurog2 and Neurog1 are required to specify the glutamatergic identity of early-born neocortical neu-rons, whereas later-born neocortical neurons are specified in a neurogenin-independent manner, requiring instead the synergistic activities of Pax6 and Tlx (Schuurmans et al., 2004).
It is important to keep in mind that electroporation of plasmids (Mattar et al., 2008) and knock-in strategies (Parras et al., 2002) likely leads to different expression levels of NEUROG2 and, there-fore, may not fully recapitulate its physiological roles in the speci-fication of neuronal fates in the dorsal telencephalon. Moreover, the endogenous expression of Neurog2 in dorsal progenitor cells shows and oscillatory pattern complimentary to the expression of the canonical Notch target Hes Family BHLH Transcription Factor 1 – Hes1 (Shimojo et al., 2008). This oscillation, and the consequent variations in the expression levels of Neurog2, is likely to induce different transcriptional network according to the affinities of reg-ulatory elements in target genes to the binding of Neurog2. Simi-larly, phosphorylation of NEUROG2 at multiple serine residues regulates its binding to and activation of target genes (Ali et al., 2011; Hindley et al., 2012). For instance, the cyclin-dependent p27 promotes neurogenesis by stabilizing NEUROG2 in cortical pro-genitors (Nguyen et al., 2006) and the glycogen synthase kinase 3 (GSK3) inhibits neurogenesis by phosphorylating NEUROG2 and decreasing its ability to bind to DNA E-box response elements (Li et al., 2012). Future experiments should clarify whether these post-transcriptional modifications and the different levels of Neu-rog2 expression may elicit the activation of separate gene networks modulating the acquisition of particular neuronal phenotypes.
Collectively, the expression pattern of Neurog2 and loss/gain of function studies strongly suggest that this proneural protein is functionally involved in the specification of glutamatergic neurons in the neocortex. However, lineage-tracing studies of Neurog2-expressing progenitors unambiguously showing their contribution
to generate all classes of glutamatergic neocortical neurons still lack. It would be interesting to evaluate the phenotype of GFP+ neurons in the neocortex of Neurog2-GFP mice (Winpenny et al., 2011) or use Neurog2-CreERT mice lines (Brzezinski et al., 2011; Florio et al., 2012) to map the fate of neurons generated from Neu-rog2+ progenitors at different developmental stages. Considering the similarities between the expression patterns of Neurog2 and Empty spiracle homeobox 1 (Emx1) in the dorsal telencephalon (Chan et al., 2001), it is tempting to speculate that Neurog2-lineage will comprise solely glutamatergic neurons in the neocor-tex, as it happens for the Emx1-lineage (Gorski et al., 2002). In con-trast, however, pallial astrocytes and oligodendrocytes, which also derive from Emx1-progenitors (Gorski et al., 2002), would not be expected in the Neuro2-lineage.
2.3. Ascl1 and neuronal specification in the developing neocortex Similar to Neurog2, the contribution of Ascl1 in the specification of neuronal identity has been firstly grasped through gain of func-tion studies. Using transgenic mouse embryos in which the Neurog2 sequence is replaced by Ascl1, it has been shown that cells in the dorsal telencephalon begin to express the GABAergic genes Dlx1 and the glutamate decarboxylase 67 (GAD67) at times when no expression of these markers can be observed in wild type animals (Fode et al., 2000). Conversely, production of GABAergic neurons is greatly decreased in Ascl1 knockout mice (Casarosa et al., 1999; Horton et al., 1999) and transient accumulation of Ascl1 in neural progenitor cells induces the expression of gene networks involved in GABAergic neuron differentiation, such as Dlx1/2/5/6 and LIM homeobox 6 – Lhx6 (Petryniak et al., 2007; Yun et al., 2002). Decreased differentiation of GABAergic interneurons in Ascl1 knockout mice is more pronounced in the medial ganglionic emi-nence than in the striatum, suggesting a regional impact of Ascl1 on neurogenesis (Wang et al., 2009). Intriguingly, the differentiation of neocortical GABAergic neurons can be partly rescued in Ascl1 null mice through ectopic expression of Neurog2 in the ventral telen-cephalon (Parras et al., 2002). Together with other lines of evidence discussed above, these data may indicate that neither Ascl1 is entirely required to specify a GABaergic fate, nor Neurog2 is suffi-cient to specify a glutamatergic fate in differentiating neurons.
Despite the well-defined contribution of Ascl1-expressing pro-genitors to generate GABAergic interneurons in the neocortex, this proneural factor cannot be simply interpreted as a GABAergic deter-minant. In fact, fate-mapping experiments have shown that Ascl1 progenitors give rise to diverse neuronal subtypes throughout the central nervous system including glutamatergic neurons in the retina (see below), noradrenergic neurons in the locus coeruleus and cholinergic neurons in the basal ganglia (Kim et al., 2008). Post-transcriptional modifications of Ascl1 affect its ability to acti-vate the expression of target genes and may help to explain how Ascl1 regulates the acquisition of alternative neuronal phenotypes (Ali et al., 2014; Castro et al., 2011; Li et al., 2014; Parras et al., 2002). Different concentrations of this proneural protein could also lead to the activation of distinct gene networks. Accordingly, the expression levels of Ascl1 oscillate in progenitor cells of the ventral telencephalon and maintenance of this oscillatory pattern is impor-tant for the activation of gene networks involved in progenitor cell proliferation and multipotency, whereas sustained expression of Ascl1 activate genes driving cell cycle exit and neuronal differenti-ation (Imayoshi and Kageyama, 2014; Jacob et al., 2013).
2.4. Neurog2 and Ascl1 instruct different neuronal phenotypes in lineage-reprogrammed neocortical astrocytes
Besides the pivotal roles of Ascl1 and Neurog2 for neocortical neurogenesis during development, both TFs have also been proved
reprogram-extremely powerful to convert postnatal neocortical astrocytes into induced neurons (Berninger et al., 2007; Blum et al., 2011; Chouchane et al., 2017; Heinrich et al., 2010). Pioneer in vitro stud-ies have suggested that, according to their developmental roles in the developing neocortex, Neurog2 and Ascl1 would convert post-natal cortical astrocytes into glutamatergic and GABAergic induced neurons (iNs), respectively (Berninger et al., 2007; Blum et al., 2011; Heinrich et al., 2010). Recent work in our laboratory corrob-orate these previous observations, but also show that the potential of Neurog2 and Ascl1 to specify glutamatergic and GABAergic fates in induced neurons is not absolute (Chouchane et al., 2017). In fact, we observed that about 60% of neocortical astrocytes lineage reprogrammed with Ascl1 adopted a GABAergic neuronal pheno-type, with the remaining 40% adopting glutamatergic fate. Simi-larly, Neurog2 converted neocortical astrocytes mostly into glutamatergic iNs, but a significant fraction (20%) of iNs showed a GABAeric identity (Chouchane et al., 2017). These results indicate that Neurog2 or Ascl1 drives the acquisition of a pan-neuronal
phe-notype and neuronal subtype specification by
lineage-reprogrammed astrocytes in an independent fashion. Interestingly, the combination of NeuroD4 and lnsm1, whose expression is upregulated following expression of ASCL1 or NEUROG2, is able to reprogram astrocytes into glutamatergic neurons (Masserdotti et al., 2015), further indicating that both proneural factors can eli-cit the expression of genes involved in the acquisition of the same neurotransmitter identity. In the future, it will be interesting to compare the transcriptional cascades activated by ASCL1 and NEU-ROG2 in lineage-reprogrammed astrocytes and neural progenitor cells isolated from the dorsal or ventral telencephalon. This could shed a new light on the mechanisms controlling the acquisition of different neuronal identities during neocortex development and lineage reprogramming.
Transplantation experiments of lineage-converted neocortical astrocytes brought an additional layer of difficulties in interpreting the roles of Ascl1 and Neurog2 for the specification of iNs pheno-types. They show that neocortical astrocytes converted into iNs adopt distinct neuronal phenotypes depending on the grafting region (Chouchane et al., 2017). For instance, iNs derived from NEUROG2 lineage-reprogrammed astrocytes and transplanted in the postnatal cerebral cortex mostly adopted a phenotype of pyra-midal spiny neurons, which are glutamatergic in this region. In contrast, when these cells were grafted in the postnatal subventric-ular zone (SVZ), iNs were found in the olfactory bulb (OB) and dis-played clear hallmarks of GABAergic interneurons within the granular cell layer (GCL) and glomerular layer (GL). Transplants of ASCL1 lineage-reprogrammed neocortical astrocytes in the post-natal cerebral cortex yield an extremely reduced number of iNs and these few cells morphologically resemble GABAergic interneu-rons. However, after transplantation in the SVZ, a large fraction of neocortical astrocytes nucleofected with Ascl1 differentiates into OB interneurons. These experiments indicate that neither NEU-ROG2 nor ASCL1 are sufficient to establish a definite neuronal
phe-notype in vivo. Rather, this phenomenon depends on
environmental signals present in the postnatal cerebral cortex or SVZ. According to this interpretation, retrovirally-mediated expression of Neurog2 and coadjutant administration of growth factors can convert glial cells of the adult mouse striatum and cere-bral cortex into GABAergic and glutamatergic neurons, respectively (Grande et al., 2013). Ascl1 expression alone is not sufficient to promote neurogenesis in those brain regions, but the combination of Ascl1 with other TFs can elicit neuronal reprogramming in vivo (Torper et al., 2013, 2015; Heinrich et al., 2010). Finally, expression of NeuroD1 (a Neurog2 target gene) is capable of inducing the con-version of astrocytes and NG2 cells into glutamatergic neurons, with a small fraction of NG2 cells (10%) adopting a GABAergic phe-notype, in the injured adult cerebral cortex (Guo et al., 2014).
3. Influences of Ascl1 and Neurog2 expression for the acquisition of neuronal identities by cerebellum neural progenitors and lineage-reprogrammed astrocytes 3.1. Neural progenitors and neuronal types of the cerebellum
The cerebellum is comprised by roughly eight types of neurons (Palay and Chan-Palay, 2012) that can be generally grouped into GABAergic and glutamatergic neurons. The first comprises Purk-inje, Golgi/Lugaro, basket and stellate cells, as well inhibitory cere-bellar nuclei neurons. Granule cells, unipolar brush cells and excitatory cerebellar nuclei neurons, in turn, are glutamatergic (Butts et al., 2014). During cerebellum development, the Rhombic lip, a germinative niche established in the interface between neural tissue and non-neural roof plate tissue, generates glutamatergic cerebellar nuclei neurons, unipolar brush cells and external gran-ule cell (EGC) progenitors, which are subsequently responsible for the generation of granule cells (Englund et al., 2006; Kita et al., 2013; Machold and Fishell, 2005). On the other hand, the ventricular zone (VZ) of the cerebellum anlage harbors progenitors that generate all GABAergic neuronal types of the cerebellum (Hoshino, 2006; Sudarov et al., 2011). Therefore, there is a clear segregation between progenitors committed to the generation of glutamatergic and GABAergic neurons of the cerebellum, just alike in the developing neocortex. In sharp contrast with this latter structure, however, Neurog2 and Ascl1 are expressed in VZ progen-itors of the cerebellum, which gives rise to GABAergic neurons, while their expression is not detected in the Rhombic lip or EGL, origins of all glutamatergic cerebellar neurons (Zordan et al., 2008). EGC progenitors, in turn, express the proneural TF Atonal Homolog 1 (Atoh1), which plays an important role in the differen-tiation of cerebellum granule cells (Gazit et al., 2004).
3.2. Neuronal identity determination by Neurog2 and Ascl1 in the developing cerebellum
Interestingly, domains of Neurog2 and Ascl1 expression within the VZ only partially overlap, suggesting that these two proneural factors may be expressed by separate progenitors and, therefore, contribute to the generation of distinct populations GABAergic neurons in the cerebellum (Zordan et al., 2008). Alternatively, Ascl1 and Neurog2 could be expressed at different stages of cell lineage progression and contribute to the generation of all cerebel-lar GABAergic neurons. According to this last possibility, Ascl1 expression is observed in early VZ progenitors that generate deep
cerebellar GABAergic neurons and Purkinje cells (Kim et al.,
2008), whereas Neurog2 expression can be distinguished only in progenitors undertaking the last round of cell division and postmi-totic neurons (Florio et al., 2012). In Purkinje cells, late-onset expression of Neurog2 plays a key role in dendrite morphogenesis (Florio et al., 2012), suggesting that sequential expression of Ascl1 and Neurog2 is necessary to Purkinje cell specification.
In contrast, other lines of evidence indicate some redundancy in the roles of Ascl1 and Neurog2 for neuronal specification in the cerebellum. For example, in Neurog2 null mice, the expression of Ascl1 is upregulated in VZ progenitors and the specification of GABAergic interneurons and Purkinje cells occur normally, despite the morphological alterations in these cells (Florio et al., 2012). Additionally, Ascl1 knock out mice show a reduction in the number of GABAergic interneurons, but Purkinje cells are unaffected (Grimaldi et al., 2009), suggesting that other proneural factor com-pensate the absence of Ascl1 at least for the specification of Purk-inje cells. It is unclear whether the expression of Ascl1 and Neurog2 oscillate in cerebellum progenitors hampering the detec-tion of a substantial overlap by convendetec-tional
reprogram-cal methods. Notwithstanding a redundant or sequential influence of Neurog2 and Ascl1 in cerebellum progenitors, the data available up to now allows inferring that both proneural factors contribute to specify GABAergic phenotypes.
3.3. Neurog2 and Ascl1 instruct different neuronal phenotypes in lineage-reprogrammed cerebellar astrocytes
In accordance with this developmental role, forced expression of either Neurog2 or Ascl1 in postnatal cerebellum astrocytes converts these cells into iNs adopting mostly (70%) a GABAergic phenotype (Chouchane et al., 2017). This ratio, especially for Neurog2, is very different from that observed with neocortical astrocytes, suggesting that this TF activates distinctive gene networks in astrocytes iso-lated from different CNS regions. Differences in the phenotype of iNs have also been observed after transplantation in the neocortex and the SVZ. Cerebellar astrocytes reprogrammed with NEUROG2 and transplanted in the postnatal neocortex, unlike their neocorti-cal counterparts, differentiate into a very small number of iNs with GABAergic interneuron phenotypes, suggesting that NEUROG2 expression alone is not sufficient to reprogram all astroglial popu-lations into pyramidal-like iNs in vivo. Following transplantation in the SVZ, cerebellum astroglia nucleofected with Ascl1 converted into OB interneurons. Interestingly, however, despite the instruc-tive role of the environment, ASCL1 lineage-reprogrammed cortical and cerebellar astroglia iNs generated GCL and PGL-like interneu-rons at different ratios, suggesting that the origin of the astroglial cell still play some role in fate determination.
4. Influences of Ascl1 and Neurog2 expression for the
acquisition of neuronal identities by retinal neural progenitors and lineage-reprogrammed Müller Glia
4.1. Neural progenitors and neuronal types of the retina
The vertebrate retina harbors seven principal cell types: rods and cones (primary sensory neurons); horizontal cells, bipolar cells and amacrine cells (interneurons); retinal ganglion cells (output neurons); and Müller glial cells. Horizontal cells and a subpopula-tion of amacrine cells are GABAergic, whereas bipolar cells, pho-toreceptors and retina ganglion cells (RGCs) are glutamatergic (M.D, 2003). Several studies have shown that these different cell types arise in temporally segregate waves (Rapaport et al., 2004; la Vail et al., 1991; Wong and Rapaport, 2009; Young, 1985). Clonal analysis experiments in the developing rodent retina, using retroviral-mediated transfection of progenitor cells, indicate that multipotent retinal progenitor cells (RPCs) contribute to generate all those different cell types (Turner and Cepko, 1987; Turner et al., 1990). However, it remains largely disputed whether intrin-sic differences among RPCs or extrinintrin-sic and/or stochastic signals act on equivalent RPCs or their progeny to specify different neu-ronal phenotypes (Cayouette et al., 2006; Cepko, 2014). For conci-sion purposes, we will here focus on how Ascl1 and Neurog2 expression in RPCs could contribute to the generation of distinct neuronal types in the developing retina.
4.2. Neuronal identity determination by Neurog2 and Ascl1 in the developing retina
The expression of Ascl1 and Neurog2 in RPCs defines separate subsets of RPCs (Brzezinski et al., 2011; Hufnagel et al., 2010). These cells may express only Ascl1, only Neurog2 or both TFs at the same time (Brzezinski et al., 2011), resembling the expression pattern described in the cerebellum VZ. In agreement with this pattern, Ascl1- and Neurog2-inducible expression fate mapping using the CreERTM/LoxP system reveals that Ascl1 and
Neurog2-lineage comprise mostly overlapping, but still slightly different, populations of neurons. While Neurog2-lineage is heavily biased towards cell types that exit the cell cycle shortly after tamoxifen treatment and encompasses all cell types, including RGCs (Brzezinski et al., 2011; Ma and Wang, 2006), Ascl1+ progenitors give rise to all retinal cell types except for RGCs (Brzezinski et al., 2011). These data indicate that Ascl1+ progenitors in the develop-ing retina have the competence to generate all but one cell type. They also indicate that both Neurog2 and Ascl1 may be expressed in cell lineages encompassing glutamatergic and GABAergic neu-rons. However, it is not clear whether those proneural proteins could be expressed in an oscillatory manner in retinal progenitor. In this scenario, we could postulate a hypothetical scenario in which, despite their overlapped expression in RPCs, the sustained expression of either Ascl1 or Neurog2 during cell differentiation could induce different neuronal fates.
4.3. Ascl1 instruct multiple neuronal phenotypes in lineage-reprogrammed Müller Glia
According to the cell lineage of Ascl1+ progenitors in the devel-oping retina, the Reh’s lab has shown in a series of key publications that forced expression of Ascl1 in Müller glial cells, both in vitro (Pollak et al., 2013; Wohl and Reh, 2016) and in vivo (Jorstad et al., 2017; Ueki et al., 2015), is able to convert these glial cells into different types of neurons, including photoreceptors, amacrine, bipolar and horizontal cells. Notably, lineage reprogramming of Müller glial cells into interneurons (bipolar and amacrine cells) can be achieved in the adult mammal injured retina through Ascl1 expression combined with histone deacetylases I and II inhibition (Jorstad et al., 2017). Also agreeing with developmental data, Ascl1 expression in MGCs is not sufficient to lineage-reprogram these cells into RGCs (Pollak et al., 2013; Wohl and Reh, 2016; Jorstad et al., 2017; Ueki et al., 2015).
Considering the differences observed in the Ascl1- and Neurog2-lineages in the developing retina, it is tempting to specu-late that forced expression of Neurog2 in MGC could convert these cells into RGCs. In fact, recent work in our laboratory shows that forced expression of Neurog2 in MGC in vitro is sufficient to lin-eage convert these cells into neurons expressing POU4F1, also known as BRN3A, a transcription factor expressed in RGCs. Still, according to the developmental roles of Neurog2 in the retina, all other neuronal populations are induced after lineage reprogram-ming of MGCs with this proneural factor (Guimarães and Costa, unpublished data).
5. Concluding remarks
Proneural proteins ASCL1 and NEUROG2 were first described as necessary and sufficient to confer a neuronal fate to progenitor cells in the developing nervous system (Bertrand et al., 2002). Recent lines of evidence obtained from direct lineage reprogram-ming experiments indicate that they are also sufficient to induce a neuronal fate in astroglial cells (Tables 1 and 2). ASCL1 is also suf-ficient to convert non-neural cells into neurons (Chanda et al., 2014), likely due to its capacity to bind to regulatory elements of target genes even in nucleosome-bound regions of the genome (Soufi et al., 2015; Wapinski et al., 2013). On the other hand, NEU-ROG2 requires additional factors to convert non-neural cells into neurons (Blanchard et al., 2014; Liu et al., 2012). This may suggest that, in contrast to ASCL1, NEUROG2 can only bind to accessible regulatory elements of its target genes.
According to this view, cultured neocortical and cerebellar astroglia progressively become less amenable to reprogramming
into neurons by ASCL1 or NEUROG2 (Chouchane et al., 2017;
reprogram-Masserdotti et al., 2015). In neocortical astrocytes, this can be par-tially explained by the activity of the repressor complex REST pre-venting NEUROG2 from binding to the NeuroD4 promoter (Masserdotti et al., 2015). Expression of NEUROD4 can bypass this repression and reprograms refractory astroglia (Masserdotti et al., 2015). Interestingly, NEUROD1, another NEUROG2 target gene, is also sufficient to reprogram reactive glial cells in the neocortex
of stab-injured or Alzheimer’s disease (AD) model mice (Guo
et al., 2014). Expression of NEUROG2 alone has a limited potential to reprogram glial cells into neurons in the adult mice brain, but this potential can be augmented by coadjutant treatment with basic fibroblast growth factor (FGF2) and epidermal growth factor (EGF) and previous ischemic lesion (Grande et al., 2013). This may suggest that both the lesion environment and high concentrations of FGF2/EGF act to remodel the chromatin of glial cells, allowing the binding of NEUROG2 in regulatory elements of its target genes. Supporting this notion, it has been shown that FGF2 induces Lysine 4 methylation and suppresses Lysine 9 methylation of histone H3 at the signal transducer and activator of transcription (STAT) bind-ing site (Song and Ghosh, 2004). Similarly, EGFR signal affects chromatin architecture at the regulatory element of cyclin D1 through a process involving Cre-binding protein (CBP), Histone deacetylase 1 (HDAC1) and Suv39h1 histone/chromatin remodel-ing complex (Lee et al., 2010).
Despite the common pro-neuronal effect of NEUROG2 and ASCL1, these proteins do not seem to have a fixed role in neuronal type specification both in the developing nervous system and in lineage-reprogrammed cells (Fig. 1). Various models could help to explain the different contributions of NEUROG2 and ASCL1 for the specification of GABAergic or glutamatergic neuronal fates, such as: i) Co-expression of additional TFs; ii) Activation of sepa-rate gene networks due to post-transcriptional modifications in the ASCL1 or NEUROG2 proteins; iii) Induction of different gene networks associated with oscillatory levels of expression. Also, dif-ferences in the epigenetic state of the cells may affect the outcome of Ascl1 and Neurog2 expression. In fact, reprogrammed somatic cells retain residual DNA methylation signatures characteristic of their somatic tissue of origin. These are called ‘‘memory’’ of origin and favor cell differentiation toward lineages related to the donor cells (Hu et al., 2010; Polo et al., 2010; Tian et al., 2011). Astroglial cells from separate regions of the CNS may present different chro-matin modifications in genes targeted by neurogenic TFs. These modifications are likely to occur in early progenitor cells, under influence of distinct morphogenetic signals at different domains of the developing CNS (Kiecker and Lumsden, 2005; Lupo et al., 2006) before generation of neurons and glial cells. This could explain why astroglia isolated from cerebellum or neocortex, and lineage-converted by Neurog2 or Ascl1 expression, generate iNs
Table 2
Phenotype of iNs generated in vivo following Ascl1- or Neurog2-induced lineage-reprogramming of astroglial cells.
Type of glial cells Treatment iNs Phenotype Reference
Resident striatal GFAP + cells (adult rats) Ascl1, Brn2, Myt1l Not determined Torper et al. (2013) Neocortical astrocytes (adult mice) Neurog2 and growth factors Glutamatergic Grande et al.
(2013) Striatal astrocytes (adult mice) Neurog2 and growth factors GABAergic
Reactive glia of the neocortex (adult mice) Ascl1 and Sox2 Immature neurons Heinrich et al. (2010) Muller Glia of damaged retina (postnatal and
adult mice)
Ascl1 Amacrine cells (GABAergic), bipolar cell and photoreceptors (glutamatergic)
Ueki et al. (2015) Muller Glia of damaged retina (adult mice) Ascl1 Amacrine cells (GABAergic) and bipolar cell (glutamatergic) Jorstad et al.
(2017) Striatal astrocytes (adult mice) NeuroD1, Ascl1, Lmx1a and
miR218
TH+, SLC6A3 + iNs Cervo et al.
(2017) Reactive glia of the neocortex (adult mice) Neurog2 + Bcl-2 Ctip2+ (80%) and Foxp2+ (20%) (deep layer spiny neurons) Gascón (2016) Table 1
Phenotype of iNs generated in vitro following Ascl1- or Neurog2-induced lineage-reprogramming of astroglial cells.
Type of glial cells Treatment iNs Phenotype Reference
Neocortex astrocytes (postnatal mice)
Neurog2 85% VglutT1+, 20% Tbr2 and 48% Tbr1 iNs Heinrich et al. (2010) Neocortex astrocytes
(postnatal mice)
Ascl1 + Dlx2 93% BIII-Tub+, 40% iNs with GABAergic interneuron firing pattern (stuttering) Neocortex astrocytes
(postnatal rats)
Neurog2 vGLUT1 + iNs Blum et al. (2011)
Neocortex astrocytes (postnatal mice) Ascl1, Lmx1b, Nurr1 50% TH + iNs Addis et al. (2011) Neocortex astrocytes
(postnatal mice)
Neurog2 + Bcl-2 vGLUT1 + iNs Gascón et al. (2016)
Neocortex astrocytes (postnatal mice)
Neurog2 80% Glutamate+, 20% GABA + iNs Chouchane et al. (2017) Cerebellum astrocytes
(postnatal mice)
Neurog2 40% Glutamate+, 60% GABA + iNs Neocortex astrocytes
(postnatal mice)
Ascl1 40% Glutamate+, 60% GABA + iNs Cerebellum astrocytes
(postnatal mice)
Ascl1 30% Glutamate+, 70% GABA + iNs
Müller Glia (postnatal mice) Ascl1 30% Otx2+ (early photoreceptor, bipolar cells); 20% Calretinin+ (amacrine and bipolar cells)
Pollak et al. (2013) Müller Glia (postnatal mice) Ascl1 + miR-124-9-9 Not determined Wohl and Reh (2016)
Human astrocytes Ascl1 GAD67+, GABA + iNs Corti et al. (2012)
Human astrocytes Ascl1, Brn2, Myt1l TH + iNs Torper et al. (2013)
Human astrocytes NeuroD1, Ascl1, Lmx1a and miR218 TH+, DDC+, SLC6A3 + iNs Cervo et al. (2017)
reprogram-with some hallmarks of neurons of these regions. Alternatively, astroglial cells obtained from different regions could express dif-ferent sets of microRNAs or long non-coding RNAs involved in
the specification of neuronal fates (Flynn and Chang, 2014;
Jönsson et al., 2015). 6. Perspectives
Future experiments using single-cell RNA sequencing (Macosko et al., 2015) and pseudotime analysis (Coelho et al., 2017; Trapnell et al., 2014) may contribute to elucidate the precise molecular mechanisms involved in neuronal fate specification during lineage
reprogramming. Indeed, comparison of the transcriptional profile of fibroblasts lineage-reprogrammed into neurons and differentiat-ing dorsal telencephalon neural progenitors reveals that both induced neurons and primary neurons display similar gene expres-sion arrays (Coelho et al., 2017; Treutlein et al., 2016). We advocate that a systematic analysis of the transcriptional profile of astroglial cells isolated from different CNS regions and transfected with either Neurog2 or Ascl1 might be a powerful tool to uncover the molecular mechanisms of neuronal fate specification. The reasons supporting this view are many: i) both ASCL1 and NEUROG2 con-vert astroglial cells into neurons with high efficiency (Chouchane et al., 2017; Heinrich et al., 2010) ii) the same proneural gene,
Fig. 1. Correlation between the neurochemical phenotype of neurons generated from neural progenitors and lineage-reprogrammed astroglia. Superior part of the scheme summarizes the contribution of ASCL1+ (green), NEUROG2+ (red) and ASCL1+/NEUROG2+ (yellow) neural progenitor cells in different regions of the developing CNS (neocortex, cerebellum and retina) to the generation of glutamatergic and GABAergic neurons. Observe that neural progenitor cells expressing the same proneural gene contribute to generate distinct classes of neurons. Bottom part of the scheme shows the neurochemical phenotypes observed in induced neurons derived from astroglial isolates from the same CNS regions and lineage-reprogrammed with ASCL1 or NEUROG2 in vitro. Note that NEUROG2 induces predominantly a glutamatergic phenotype in neural progenitor cells and neocortex astroglia, but induces mainly a GABAergic fate in their cerebellum counterparts. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
reprogram-e.g. Neurog2, may elicit different neuronal phenotypes in astroglial cells obtained from neocortex (glutamatergic) or cerebellum (GABAergic) (Chouchane et al., 2017); iii) microarray bulk cell anal-ysis shows that ASCL1 and NEUROG2 induce different gene
net-works in neocortex astrocytes (Masserdotti et al., 2015).
Therefore, comparing the transcriptional profile of cerebellum astrocytes and neocortex astrocytes transfected with Neurog2 will likely contribute to identify overlapping patterns of gene expres-sion associated with the acquisition of pan-neuronal features, as well as non-overlapping transcriptional profiles associated with the acquisition of separate neurochemical fates. This strategy could also contribute to built an algorithm capable of predicting lineage reprogramming outcomes, as it has been previously done to fibrob-lasts (Ronquist et al., 2017).
A comprehensive understanding of the molecular mechanisms involved in the specification of particular neuronal identities is piv-otal for the development of future cell-based therapies aiming at restoring neural circuits in acute and chronic neurological diseases. For instance, the observation that neocortex but not cerebellum astroglia lineage-reprogrammed with NEUROG2 can differentiate into iNs resembling pyramidal neurons after transplantation in the neonatal mouse cerebral cortex may indicate that not all start-ing cells are amenable for in vivo reprogrammstart-ing (Chouchane et al., 2017). In the same direction, the observation that resident glial cells transfected with Neurog2 or NeuroD1 differentiate into iNs with some features of local neurons (Grande et al., 2013; Guo et al., 2014) may suggest that efficiently genetic manipulations capable of instructing a pan-neuronal phenotype in resident glial cells could be sufficient to achieve some degree of functional improvement. References
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