Tuning of neocortical astrogenesis rates by Emx2 in meural stem cells

Generation of astrocytes within the murine developing cerebral cortex mainly takes place during the first postnatal week, after neuronogenesis and prior to the bulk of oligogenesis. This process involves a great variety of highly complex regulatory mechanisms. Astrocytic outputs depend on two primary factors: progressive commitment of multipotent precursors to astroglial fates and proper tuning of proliferation of astrocyte-committed progenitors. To date, several regulatory mechanisms have been identified for the former process, while very little is known about modulation of astroblast proliferation (reviewed in Mallamaci, 2013). Intriguingly, astrogenic rates remain very low during the whole neuronogenic phase, although the mouse cortex is already able to generate astrocytes at E14.5–E15.5, thanks to specific chromatin reconfiguration (Fan et al., 2005). Poor proliferation of astroblasts may contribute to this effect (Seuntjens et al., 2009). Among different factors modulating astrocyte-committed proliferation, the Egf-receptor (EgfR) and the secreted ligand Fgf9 both specifically promote it (Viti et al., 2003; Lum et al., 2009). Emx2, a pleiotropic hub (Gangemi et al., 2006) controlling a variety of neurodevelopmental processes, is highly expressed in the early neuronogenic pallium, while it fades out together with neuronogenesis ending. This temporal progression is possibly linked to the progressive decline of Wnt signals supporting Emx2 expression (Theil et al, 2002) and late arousal of Fgf8 (http://developingmouse.brain-map.org/) antagonizing it (Garel et al., 2003). In a previous in vitro study (Brancaccio et al., 2010), we reported that Emx2 overexpression in neural stem cells (NSCs) leads to a reduction of their astrocytic outputs, due to unknown mechanisms. In the paper highlighted here (Falcone et al., 2014), we showed that this phenomenon occurs also in vivo and dissected its cellular and molecular mechanisms. At the beginning of our study, we verified that the decrease of the ultimate glial output of NSCs induced by Emx2 overexpression takes place also in vivo and it is due to a shrinkage of the proliferating astrogenic pool. We injected a plasmid expressing Emx2 into the lateral ventricular cavity of P0 pups and electroporated it into the cortex. The analysis of P4 mice electroporated cortices revealed a decrease of S100β+ astrocytes and S100β+Ki67+ astroglial proliferating progenitors in Emx2-gain of function (GOF) samples, by about 30% and 50%, respectively (Figure 1A). [Frequencies of these cell types were conversely upregulated in the posterior cortex of E17.5 Emx2+/- embryos, suggesting that the inhibition of astrogenesis elicited by gain-of-function manipulations was not due to a dominant negative effect (Figure 1B)]. Then, these results were replicated in an in vitro model, set up to dissect molecular mechanisms involved in Emx2 antiastrogenic function. E12.5 cortico-cerebral precursors were engineered for conditional Emx2 overexpression, which was activated at the in vitro equivalent of P0. Following this manipulation, the final astroglial output was reduced approximately as much as in vivo. Moreover, it was associated to a prominent shrinkage of the astrogenic proliferating pool. Interestingly, Emx2 impact on astrogenesis depended mainly on cell-autonomous mechanisms. This was verified by mixing a small amount of lentivirus-engineered precursors with an excess of isochronic wild type precursors, conditioning the medium. Even in this situation, Emx2-engineered cells expressing S100β were significantly reduced upon transgene activation.Figure 1: Altered astrocytogenesis upon Emx2 manipulation in vivo.(A) Distribution of S100β+ astrocytes and S100β+Ki67+ astroglial proliferating progenitors in the posterior parietal cortex of P4 pups electroporated at P0 with a control (NC) and, alternatively, a constitutive Emx2 expressor plasmid (Emx2-GOF). (B) Distribution of S100β+ astrocytes and S100β+Ki67+ astroglial proliferating progenitors in the posterior parietal cortex of E17.5 embryos heterozygous for an Emx2-null allele and their littermate wild type controls.To cast light on molecular mechanisms mediating Emx2 anti-astrogenic effect, we looked at a few well-known genes promoting the expansion of the astrogenic proliferating pool, including EgfR and Fgf9. We found that Emx2 overexpression downregulates both EgfR and Fgf9. Consistently, the same genes were upregulated in Emx2+/- cultures. Functional relevance of EgfR and Fgf9 to Emx2 action was tested by delivering a lentivector driving EgfR expression and, alternatively, the Fgf9 ligand to Emx2-GOF cultures at in vitro equivalent of P0. Both EgfR and Fgf9 were able not only to rescue the normal astroglial output, but to restore wild type astrogenic proliferating rates too. The reconstruction of the pathways leading to EgfR and Fgf9 downregulation represented a step forward in the understanding of Emx2 anti-astrogenic activity. Both EgfR and Fgf9 levels showed scarce sensitivity to exogenous Fgf9 addition and EgfR overexpression, respectively, thus suggesting that Emx2 regulation of astrogenesis may occur along two separated pathways. Regarding EgfR regulation, we suspected that it could be mediated by Bmp signaling. Indeed, Emx2 promotes such signaling (Shimogori et al., 2004), which, in turn, inhibits EgfR expression (Lillien and Raphael, 2000). Interestingly, Emx2 was able to upregulate two established endogenous reporters of Bmp signaling, Id3 and Msx1. Moreover Bmp inhibition by LDN193189 rescued EgfR expression levels in Emx2-GOF samples, while not perturbing them in controls. As for Fgf9, we hypothesized that its regulation might depend on Sox2 repression. We found that Emx2 overexpression almost abolishes Sox2 expression in cortico-cerebral precursors at astrogenesis peak time. Moreover, Sox2 overexpression rescued Fgf9-mRNA levels in Emx2-GOF cultures. Interestingly, a sort of upstream crosslink among these two regulatory branches exists. In fact, on one hand Bmp inhibition restored also Fgf9 expression, on the other hand Sox2 overexpression rescued EgfR levels. Besides, Emx2, while downregulating Fgf9 in control conditions, increased Fgf9 upon Bmp signaling inhibition. This suggests that Bmp signaling could inhibit Fgf9 expression by counteracting an Emx2-dependent stimulatory pathway (Figure 2).Figure 2: Epistatic relationships among Emx2 and mediators of its antiastrogenic activity.The question mark highlights a hypothetical regulatory branch accounting for the divergent effects exerted by Emx2 overexpression on Fgf9-mRNA levels in control conditions and upon Bmp signalling inhibition.Finally, we evaluated the physiological relevance of Emx2 to the confinement of the bulk of astrogenesis to postnatal life. First, we rigorously documented that both Emx2 mRNA and protein levels progressively decrease in cortico-cerebral stem cells from embryonic towards perinatal stages. High Emx2 levels are associated to the neuronogenic phase, whereas Emx2 is barely detectable concomitantly with the arousal of astrogenesis. Then, we assessed consequences of short-term Emx2 overexpression in embryonic NSCs on the size of the astrogenic lineage, unveiled by a Lif-supplemented, pro-differentiative medium. As expected, Emx2 overexpression led to a reduction of the final output of both S100β+ and GFAP+ cells, a result mirrored by cultures loss-of-function for Emx2. In summary: (1) Emx2 overexpression in cortico-cerebral stem cells inhibits astrogenesis in vivo as well as in vitro, by shrinking the proliferating astrogenic pool and thus provoking a pronounced decrease of its ultimate astroglial output (Figure 1); (2) Emx2 exerts its anti-astrogenic function by downregulating EgfR and Fgf9, via promotion of Bmp signaling and Sox2 suppression (Figure 2). These phenomena may be instrumental to fine tuning of cortico-cerebral histogenesis, i.e., the in vivo temporal progression of Emx2 expression levels can help to hold back astrogliogenesis during the neuronogenic phase of development. On the other side, the sensitivity of astrogenic rates to Emx2 expression levels points to Emx2 as an appealing therapeutic tool, suitable for control of reactive gliosis as well as for selective channelling of neural precursors to neuronogenesis in processes of brain repair. The work highlighted in this manuscript was fully supported by SISSA in tramurary funding. The subject of this “Highlight” has been presented at the ISDN 2014 meeting (19-24 July, 2014, Montreal, Canada).

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