Mutations in C9ORF72 are the most common cause of familial amyotrophic lateral sclerosis (ALS). Here, through a combination of RNA-Seq and electrophysiological studies on induced pluripotent stem cell (iPSC)-derived motor neurons (MNs), we show that increased expression of GluA1 AMPA receptor (AMPAR) subunit occurs in MNs with C9ORF72 mutations that leads to increased Ca2+-permeable AMPAR expression and results in enhanced selective MN vulnerability to excitotoxicity. These deficits are not found in iPSC-derived cortical neurons and are abolished by CRISPR/Cas9-mediated correction of the C9ORF72 repeat expansion in MNs. We also demonstrate that MN-specific dysregulation of AMPAR expression is also present in C9ORF72 patient post-mortem material. We therefore present multiple lines of evidence for the specific upregulation of GluA1 subunits in human mutant C9ORF72 MNs that could lead to a potential pathogenic excitotoxic mechanism in ALS.

Regional patterning of the cerebral cortex is initiated by morphogens secreted by patterning centers that establish graded expression of transcription factors within cortical progenitors. Here, we show that Dmrt5 is expressed in cortical progenitors in a high-caudomedial to low-rostrolateral gradient. In its absence, the cortex is strongly reduced and exhibits severe abnormalities, including agenesis of the hippocampus and choroid plexus and defects in commissural and thalamocortical tracts. Loss of Dmrt5 results in decreased Wnt and Bmp in one of the major telencephalic patterning centers, the dorsomedial telencephalon, and in a reduction of Cajal-Retzius cells. Expression of the dorsal midline signaling center-dependent transcription factors is downregulated, including Emx2, which promotes caudomedial fates, while the rostral determinant Pax6, which is inhibited by midline signals, is upregulated. Consistently, Dmrt5 −/− brains exhibit patterning defects with a dramatic reduction of the caudomedial cortex. Dmrt5 is increased upon the activation of Wnt signaling and downregulated in Gli3 xt/xt mutants. We conclude that Dmrt5 is a novel Wnt-dependent transcription factor required for early cortical development and that it may regulate initial cortical patterning by promoting dorsal midline signaling center formation and thereby helping to establish the graded expression of the other transcription regulators of cortical identity.

The corpus callosum (CC) is the major commissure that bridges the cerebral hemispheres. Agenesis of the CC is associated with human ciliopathies, but the origin of this default is unclear. Regulatory Factor X3 (RFX3) is a transcription factor involved in the control of ciliogenesis, and Rfx3–deficient mice show several hallmarks of ciliopathies including left–right asymmetry defects and hydrocephalus. Here we show that Rfx3–deficient mice suffer from CC agenesis associated with a marked disorganisation of guidepost neurons required for axon pathfinding across the midline. Using transplantation assays, we demonstrate that abnormalities of the mutant midline region are primarily responsible for the CC malformation. Conditional genetic inactivation shows that RFX3 is not required in guidepost cells for proper CC formation, but is required before E12.5 for proper patterning of the cortical septal boundary and hence accurate distribution of guidepost neurons at later stages. We observe focused but consistent ectopic expression of Fibroblast growth factor 8 (Fgf8) at the rostro commissural plate associated with a reduced ratio of GLIoma-associated oncogene family zinc finger 3 (GLI3) repressor to activator forms. We demonstrate on brain explant cultures that ectopic FGF8 reproduces the guidepost neuronal defects observed in Rfx3 mutants. This study unravels a crucial role of RFX3 during early brain development by indirectly regulating GLI3 activity, which leads to FGF8 upregulation and ultimately to disturbed distribution of guidepost neurons required for CC morphogenesis. Hence, the RFX3 mutant mouse model brings novel understandings of the mechanisms that underlie CC agenesis in ciliopathies.

Rodent‐based studies have shown that the membrane properties of oligodendrocytes play prominent roles in their physiology and shift markedly during their maturation from the oligodendrocyte precursor cell (OPC) stage. However, the conservation of these properties and maturation processes in human oligodendrocytes remains unknown, despite their dysfunction being implicated in human neurodegenerative diseases such as multiple sclerosis (MS) and amyotrophic lateral sclerosis (ALS). Here, we have defined the membrane properties of human oligodendrocytes derived from pluripotent stem cells as they mature from the OPC stage, and have identified strong conservation of maturation‐specific physiological characteristics reported in rodent systems. We find that as human oligodendrocytes develop and express maturation markers, they exhibit a progressive decrease in voltage‐gated sodium and potassium channels and a loss of tetrodotoxin‐sensitive spiking activity. Concomitant with this is an increase in inwardly rectifying potassium channel activity, as well as a characteristic switch in AMPA receptor composition. All these steps mirror the developmental trajectory observed in rodent systems. Oligodendrocytes derived from mutant C9ORF72‐carryng ALS patient induced pluripotent stem cells did not exhibit impairment to maturation and maintain viability with respect to control lines despite the presence of RNA foci, suggesting that maturation defects may not be a primary feature of this mutation. Thus, we have established that the development of human oligodendroglia membrane properties closely resemble those found in rodent cells and have generated a platform to enable the impact of human neurodegenerative disease‐causing mutations on oligodendrocyte maturation to be studied. Stem Cells 2016;34:1040–1053

Early development of the hippocampus, which is essential for spatial memory and learning, is controlled by secreted signaling molecules of the Wnt gene family and by Wnt/β-catenin signaling. Despite its importance, little is known, however, about Wnt-regulated genes during hippocampal development. Here, we used the Gli3 mutant mouse extra-toes (Xt(J)), in which Wnt gene expression in the forebrain is severely affected, as a tool in a microarray analyses to identify potential Wnt target genes. This approach revealed 53 candidate genes with restricted or graded expression patterns in the dorsomedial telencephalon. We identified conserved Tcf/Lef-binding sites in telencephalon-specific enhancers of several of these genes, including Dmrt3, Gli3, Nfia, and Wnt8b. Binding of Lef1 to these sites was confirmed using electrophoretic mobility shift assays. Mutations in these Tcf/Lef-binding sites disrupted or reduced enhancer activity in vivo. Moreover, ectopic activation of Wnt/β-catenin signaling in an ex vivo explant system led to increased telencephalic expression of these genes. Finally, conditional inactivation of Gli3 results in defective hippocampal growth. Collectively, these data strongly suggest that we have identified a set of direct Wnt target genes in the developing hippocampus and provide inside into the genetic hierarchy underlying Wnt-regulated hippocampal development.

Developing thalamocortical axons traverse the subpallium to reach the cortex located in the pallium. We tested the hypothesis that descending corticofugal axons are important for guiding thalamocortical axons across the pallial-subpallial boundary, using conditional mutagenesis to assess the effects of blocking corticofugal axonal development without disrupting thalamus, subpallium or the pallial-subpallial boundary. We found that thalamic axons still traversed the subpallium in topographic order but did not cross the pallial-subpallial boundary. Co-culture experiments indicated that the inability of thalamic axons to cross the boundary was not explained by mutant cortex developing a long-range chemorepulsive action on thalamic axons. On the contrary, cortex from conditional mutants retained its thalamic axonal growth-promoting activity and continued to express Nrg-1, which is responsible for this stimulatory effect. When mutant cortex was replaced with control cortex, corticofugal efferents were restored and thalamic axons from conditional mutants associated with them and crossed the pallial-subpallial boundary. Our study provides the most compelling evidence to date that cortical efferents are required to guide thalamocortical axons across the pallial-subpallial boundary, which is otherwise hostile to thalamic axons. These results support the hypothesis that thalamic axons grow from subpallium to cortex guided by cortical efferents, with stimulation from diffusible cortical growth-promoting factors.

Evolutionary differences in gene regulation between humans and lower mammalian experimental systems are incompletely understood, a potential translational obstacle that is challenging to surmount in neurons, where primary tissue availability is poor. Rodent-based studies show that activity-dependent transcriptional programs mediate myriad functions in neuronal development, but the extent of their conservation in human neurons is unknown. We compared activity-dependent transcriptional responses in developing human stem cell-derived cortical neurons with those induced in developing primary- or stem cell-derived mouse cortical neurons. While activity-dependent gene-responsiveness showed little dependence on developmental stage or origin (primary tissue vs. stem cell), notable species-dependent differences were observed. Moreover, differential species-specific gene ortholog regulation was recapitulated in aneuploid mouse neurons carrying human chromosome-21, implicating promoter/enhancer sequence divergence as a factor, including human-specific activity-responsive AP-1 sites. These findings support the use of human neuronal systems for probing transcriptional responses to physiological stimuli or indeed pharmaceutical agents.DOI: http://dx.doi.org/10.7554/eLife.20337.001

Previous studies have defined a requirement for Sonic hedgehog (Shh) signaling in patterning the ventral telencephalon, a major source of the neuronal diversity found in the mature telencephalon. The zinc finger transcription factor Gli3 is a critical component of the Shh signaling pathway and its loss causes major defects in telencephalic development. Gli3 is expressed in a graded manner along the dorsoventral axis of the telencephalon but it is unknown whether Gli3 expression levels are important for dorsoventral telencephalic patterning. To address this, we used the Gli3 hypomorphic mouse mutant Polydactyly Nagoya (Pdn). We show that in Pdn/Pdn embryos, the telencephalic expression of Gli3 remains graded, but Gli3 mRNA and protein levels are reduced, resulting in an upregulation of Shh expression and signaling. These changes mainly affect the development of the lateral ganglionic eminence (LGE), with some disorganization of the medial ganglionic eminence mantle zone. The pallial/subpallial boundary is shifted dorsally and the production of postmitotic neurons is reduced. Moreover, LGE pioneer neurons that guide corticofugal axons into the LGE do not form properly, delaying the entry of corticofugal axons into the ventral telencephalon. Pdn/Pdn mutants also show severe pathfinding defects of thalamocortical axons in the ventral telencephalon. Transplantation experiments demonstrate that the intrinsic ability of the Pdn ventral telencephalon to guide thalamocortical axons is compromised. We conclude that correct Gli3 levels are particularly important for the LGE's growth, patterning, and development of axon guidance capabilities.