Alessandro Sessa

Tbr2 Directs Conversion of Radial Glia into Basal Precursors and Guides Neuronal Amplification by Indirect Neurogenesis in the Developing Neocortex

T-brain gene-2 (Tbr2) is specifically expressed in the intermediate (basal) progenitor cells (IPCs) of the developing cerebral cortex; however, its function in this biological context has so far been overlooked due to the early lethality of Tbr2 mutant embryos. Conditional ablation of Tbr2 in the developing forebrain resulted in the loss of IPCs and their differentiated progeny in mutant cortex. Intriguingly, early loss of IPCs led to a decrease in cortical surface expansion and thickness with a neuronal reduction observed in all cortical layers. These findings suggest that IPC progeny contribute to the correct morphogenesis of each cortical layer. Our observations were confirmed by tracing Tbr2+ IPC cell fate using Tbr2::GFP transgenic mice. Finally, we demonstrated that misexpression of Tbr2 is sufficient to induce IPC identity in ventricular radial glial cells (RGCs). Together, these findings identify Tbr2 as a critical factor for the specification of IPCs during corticogenesis.

CDKL5 ensures excitatory synapse stability by reinforcing NGL-1–PSD95 interaction in the postsynaptic compartment and is impaired in patient iPSC-derived neurons

Mutations of the cyclin-dependent kinase-like 5 (CDKL5) and netrin-G1 (NTNG1) genes cause a severe neurodevelopmental disorder with clinical features that are closely related to Rett syndrome, including intellectual disability, early-onset intractable epilepsy and autism. We report here that CDKL5 is localized at excitatory synapses and contributes to correct dendritic spine structure and synapse activity. To exert this role, CDKL5 binds and phosphorylates the cell adhesion molecule NGL-1. This phosphorylation event ensures a stable association between NGL-1 and PSD95. Accordingly, phospho-mutant NGL-1 is unable to induce synaptic contacts whereas its phospho-mimetic form binds PSD95 more efficiently and partially rescues the CDKL5-specific spine defects. Interestingly, similarly to rodent neurons, iPSC-derived neurons from patients with CDKL5 mutations exhibit aberrant dendritic spines, thus suggesting a common function of CDKL5 in mice and humans.

Direct Conversion of Fibroblasts into Functional Astrocytes by Defined Transcription Factors

Summary Direct cell reprogramming enables direct conversion of fibroblasts into functional neurons and oligodendrocytes using a minimal set of cell-lineage-specific transcription factors. This approach is rapid and simple, generating the cell types of interest in one step. However, it remains unknown whether this technology can be applied to convert fibroblasts into astrocytes, the third neural lineage. Astrocytes play crucial roles in neuronal homeostasis, and their dysfunctions contribute to the origin and progression of multiple human diseases. Herein, we carried out a screening using several transcription factors involved in defining the astroglial cell fate and identified NFIA, NFIB, and SOX9 to be sufficient to convert with high efficiency embryonic and postnatal mouse fibroblasts into astrocytes (iAstrocytes). We proved both by gene-expression profiling and functional tests that iAstrocytes are comparable to native brain astrocytes. This protocol can be then employed to generate functional iAstrocytes for a wide range of experimental applications.

Tbr2-positive intermediate (basal) neuronal progenitors safeguard cerebral cortex expansion by controlling amplification of pallial glutamatergic neurons and attraction of subpallial GABAergic interneurons

Little is known about how, during its formidable expansion in development and evolution, the cerebral cortex is able to maintain the correct balance between excitatory and inhibitory neurons. In fact, while the former are born within the cortical primordium, the latter originate outward in the ventral pallium. Therefore, it remains to be addressed how these two neuronal populations might coordinate their relative amounts in order to build a functional cortical network. Here, we show that Tbr2-positive cortical intermediate (basal) neuronal progenitors (INPs) dictate the migratory route and control the amount of subpallial GABAergic interneurons in the subventricular zone (SVZ) through a non-cell-autonomous mechanism. In fact, Tbr2 interneuron attractive activity is moderated by Cxcl12 chemokine signaling, whose forced expression in the Tbr2 mutants can rescue, to some extent, SVZ cell migration. We thus propose that INPs are able to control simultaneously the increase of glutamatergic and GABAergic neuronal pools, thereby creating a simple way to intrinsically balance their relative accumulation.

Rapid Conversion of Fibroblasts into Functional Forebrain GABAergic Interneurons by Direct Genetic Reprogramming

Transplantation of GABAergic interneurons (INs) can provide long-term functional benefits in animal models of epilepsy and other neurological disorders. Whereas GABAergic INs can be differentiated from embryonic stem cells, alternative sources of GABAergic INs may be more tractable for disease modeling and transplantation. We identified five factors (Foxg1, Sox2, Ascl1, Dlx5, and Lhx6) that convert mouse fibroblasts into induced GABAergic INs (iGABA-INs) possessing molecular signatures of telencephalic INs. Factor overexpression activates transcriptional networks required for GABAergic fate specification. iGABA-INs display progressively maturing firing patterns comparable to cortical INs, form functional synapses, and release GABA. Importantly, iGABA-INs survive and mature upon being grafted into mouse hippocampus. Optogenetic stimulation demonstrated functional integration of grafted iGABA-INs into host circuitry, triggering inhibition of host granule neuron activity. These five factors also converted human cells into functional GABAergic INs. These properties suggest that iGABA-INs have potential for disease modeling and cell-based therapeutic approaches to neurological disorders.

The Apical Complex Couples Cell Fate and Cell Survival to Cerebral Cortical Development

Cortical development depends upon tightly controlled cell fate and cell survival decisions that generate a functional neuronal population, but the coordination of these two processes is poorly understood. Here we show that conditional removal of a key apical complex protein, Pals1, causes premature withdrawal from the cell cycle, inducing excessive generation of early-born postmitotic neurons followed by surprisingly massive and rapid cell death, leading to the abrogation of virtually the entire cortical structure. Pals1 loss shows exquisite dosage sensitivity, so that heterozygote mutants show an intermediate phenotype on cell fate and cell death. Loss of Pals1 blocks essential cell survival signals, including the mammalian target of rapamycin (mTOR) pathway, while mTORC1 activation partially rescues Pals1 deficiency. These data highlight unexpected roles of the apical complex protein Pals1 in cell survival through interactions with mTOR signaling.

Arx acts as a regional key selector gene in the ventral telencephalon mainly through its transcriptional repression activity

The homeobox-containing gene Arx is expressed during ventral telencephalon development and required for correct GABAergic interneuron tangential migration from the ganglionic eminences to the olfactory bulbs, cerebral cortex and striatum. Its human ortholog is associated with a variety of neurological clinical manifestations whose symptoms are compatible with the loss of cortical interneurons and altered basal ganglia-related activities. Herein, we report the identification of a number of genes whose expression is consistently altered in Arx mutant ganglionic eminences. Our analyses revealed a striking ectopic expression in the ganglionic eminences of several of these genes normally at most marginally expressed in the ventral telencephalon. Among them, Ebf3 was functionally analyzed. Thus, its ectopic expression in ventral telencephalon was found to prevent neuronal tangential migration. Further, we showed that Arx is sufficient to repress Ebf3 endogenous expression and that its silencing in Arx mutant tissues partially rescues tangential cell movement. Together, these data provide new insights into the molecular pathways regulated by Arx during telencephalon development.

Wnt Signaling Has Opposing Roles in the Developing and the Adult Brain That Are Modulated by Hipk1

The canonical Wnt/Wingless pathway is implicated in regulating cell proliferation and cell differentiation of neural stem/progenitor cells. Depending on the context, β-Catenin, a key mediator of the Wnt signaling pathway, may regulate either cell proliferation or differentiation. Here, we show that β-Catenin signaling regulates the differentiation of neural stem/progenitor cells in the presence of the β-Catenin interactor Homeodomain interacting protein kinase-1 gene (Hipk1). On one hand, Hipk1 is expressed at low levels during the entire embryonic forebrain development, allowing β-Catenin to foster proliferation and to inhibit differentiation of neural stem/progenitor cells. On the other hand, Hipk1 expression dramatically increases in neural stem/progenitor cells, residing within the subventricular zone (SVZ), at the time when the canonical Wnt signaling induces cell differentiation. Analysis of mouse brains electroporated with Hipk1, and the active form of β-Catenin reveals that coexpression of both genes induces proliferating neural stem/progenitor cells to escape the cell cycle. Moreover, in SVZ derive neurospheres cultures, the overexpression of both genes increases the expression of the cell-cycle inhibitor P16Ink4. Therefore, our data confirm that the β-Catenin signaling plays a dual role in controlling cell proliferation/differentiation in the brain and indicate that Hipk1 is the crucial interactor able to revert the outcome of β-Catenin signaling in neural stem/progenitor cells of adult germinal niches.

ARX Regulates Cortical Intermediate Progenitor Cell Expansion and Upper Layer Neuron Formation Through Repression of Cdkn1c

Mutations in the Aristaless-related homeobox (ARX) gene are found in a spectrum of epilepsy and X-linked intellectual disability disorders. During development Arx is expressed in pallial ventricular zone (VZ) progenitor cells where the excitatory projection neurons of the cortex are born. Arx(-/Y) mice were shown to have decreased proliferation in the cortical VZ resulting in smaller brains; however, the basis for this reduced proliferation was not established. To determine the role of ARX on cell cycle dynamics in cortical progenitor cells, we generated cerebral cortex-specific Arx mouse mutants (cKO). The loss of pallial Arx resulted in the reduction of cortical progenitor cells, particularly the proliferation of intermediate progenitor cells (IPCs) was affected. Later in development and postnatally cKO brains showed a reduction of upper layer but not deeper layer neurons consistent with the IPC defect. Transcriptional profile analysis of E14.5 Arx-ablated cortices compared with control revealed that CDKN1C, an inhibitor of cell cycle progression, is overexpressed in the cortical VZ and SVZ of Arx KOs throughout corticogenesis. We also identified ARX as a direct regulator of Cdkn1c transcription. Together these data support a model where ARX regulates the expansion of cortical progenitor cells through repression of Cdkn1c.

MyT1 Counteracts the Neural Progenitor Program to Promote Vertebrate Neurogenesis

Summary The generation of neurons from neural stem cells requires large-scale changes in gene expression that are controlled to a large extent by proneural transcription factors, such as Ascl1. While recent studies have characterized the differentiation genes activated by proneural factors, less is known on the mechanisms that suppress progenitor cell identity. Here, we show that Ascl1 induces the transcription factor MyT1 while promoting neuronal differentiation. We combined functional studies of MyT1 during neurogenesis with the characterization of its transcriptional program. MyT1 binding is associated with repression of gene transcription in neural progenitor cells. It promotes neuronal differentiation by counteracting the inhibitory activity of Notch signaling at multiple levels, targeting the Notch1 receptor and many of its downstream targets. These include regulators of the neural progenitor program, such as Hes1, Sox2, Id3, and Olig1. Thus, Ascl1 suppresses Notch signaling cell-autonomously via MyT1, coupling neuronal differentiation with repression of the progenitor fate.