The ciliary gene INPP5E confers dorsal telencephalic identity to human cortical organoids by negatively regulating Sonic hedgehog signaling.

Defects in primary cilia, cellular antennas that control multiple intracellular signaling pathways, underlie several neurodevelopmental disorders, but it remains unknown how cilia control essential steps in human brain formation. Here, we show that cilia are present on the apical surface of radial glial cells in human fetal forebrain. Interfering with cilia signaling in human organoids by mutating the INPP5E gene leads to the formation of ventral telencephalic cell types instead of cortical progenitors and neurons. INPP5E mutant organoids also show increased Sonic hedgehog (SHH) signaling, and cyclopamine treatment partially rescues this ventralization. In addition, ciliary expression of SMO, GLI2, GPR161, and several intraflagellar transport (IFT) proteins is increased. Overall, these findings establish the importance of primary cilia for dorsal and ventral patterning in human corticogenesis, indicate a tissue-specific role of INPP5E as a negative regulator of SHH signaling, and have implications for the emerging roles of cilia in the pathogenesis of neurodevelopmental disorders.

The Multifaceted Roles of Primary Cilia in the Development of the Cerebral Cortex.

The primary cilium, a microtubule based organelle protruding from the cell surface and acting as an antenna in multiple signaling pathways, takes center stage in the formation of the cerebral cortex, the part of the brain that performs highly complex neural tasks and confers humans with their unique cognitive capabilities. These activities require dozens of different types of neurons that are interconnected in complex ways. Due to this complexity, corticogenesis has been regarded as one of the most complex developmental processes and cortical malformations underlie a number of neurodevelopmental disorders such as intellectual disability, autism spectrum disorders, and epilepsy. Cortical development involves several steps controlled by cell-cell signaling. In fact, recent findings have implicated cilia in diverse processes such as neurogenesis, neuronal migration, axon pathfinding, and circuit formation in the developing cortex. Here, we will review recent advances on the multiple roles of cilia during cortex formation and will discuss the implications for a better understanding of the disease mechanisms underlying neurodevelopmental disorders.

A transient role of the ciliary gene Inpp5e in controlling direct versus indirect neurogenesis in cortical development.

During the development of the cerebral cortex, neurons are generated directly from radial glial cells or indirectly via basal progenitors. The balance between these division modes determines the number and types of neurons formed in the cortex thereby affecting cortical functioning. Here, we investigate the role of primary cilia in controlling the decision between forming neurons directly or indirectly. We show that a mutation in the ciliary gene Inpp5e leads to a transient increase in direct neurogenesis and subsequently to an overproduction of layer V neurons in newborn mice. Loss of Inpp5e also affects ciliary structure coinciding with reduced Gli3 repressor levels. Genetically restoring Gli3 repressor rescues the decreased indirect neurogenesis in Inpp5e mutants. Overall, our analyses reveal how primary cilia determine neuronal subtype composition of the cortex by controlling direct versus indirect neurogenesis. These findings have implications for understanding cortical malformations in ciliopathies with INPP5E mutations.

The mitochondrial calcium uniporter is crucial for the generation of fast cortical network rhythms.

The role of the mitochondrial calcium uniporter (MCU) gene (Mcu) in cellular energy homeostasis and generation of electrical brain rhythms is widely unknown. We investigated this issue in mice and rats using Mcu-knockout and -knockdown strategies in vivo and in situ and determined the effects of these genetic manipulations on hippocampal gamma oscillations (30-70 Hz) and sharp wave-ripples. These physiological network states require precise neurotransmission between pyramidal cells and inhibitory interneurons, support spike-timing and synaptic plasticity and are associated with perception, attention and memory. Absence of the MCU resulted in (i) gamma oscillations with decreased power (by >40%) and lower synchrony, including less precise neural action potential generation ('spiking'), (ii) sharp waves with decreased incidence (by about 22%) and decreased fast ripple frequency (by about 3%) and (iii) lack of activity-dependent pyruvate dehydrogenase dephosphorylation. However, compensatory adaptation in gene expression related to mitochondrial function and glucose metabolism was not detected. These data suggest that the neuronal MCU is crucial for the generation of network rhythms, most likely by influences on oxidative phosphorylation and perhaps by controlling cytoplasmic Ca2+ homeostasis. This work contributes to an increased understanding of mitochondrial Ca2+ uptake in cortical information processing underlying cognition and behaviour.

Gli3 controls the onset of cortical neurogenesis by regulating the radial glial cell cycle through Cdk6 expression.

The cerebral cortex contains an enormous number of neurons, allowing it to perform highly complex neural tasks. Understanding how these neurons develop at the correct time and place and in accurate numbers constitutes a major challenge. Here, we demonstrate a novel role for Gli3, a key regulator of cortical development, in cortical neurogenesis. We show that the onset of neuron formation is delayed in Gli3 conditional mouse mutants. Gene expression profiling and cell cycle measurements indicate that shortening of the G1 and S phases in radial glial cells precedes this delay. Reduced G1 length correlates with an upregulation of the cyclin-dependent kinase gene Cdk6, which is directly regulated by Gli3. Moreover, pharmacological interference with Cdk6 function rescues the delayed neurogenesis in Gli3 mutant embryos. Overall, our data indicate that Gli3 controls the onset of cortical neurogenesis by determining the levels of Cdk6 expression, thereby regulating neuronal output and cortical size.

Direct Interactions Between Gli3, Wnt8b, and Fgfs Underlie Patterning of the Dorsal Telencephalon.

A key step in the development of the cerebral cortex is a patterning process, which subdivides the telencephalon into several molecularly distinct domains and is critical for cortical arealization. This process is dependent on a complex network of interactions between signaling molecules of the Fgf and Wnt gene families and the Gli3 transcription factor gene, but a better knowledge of the molecular basis of the interplay between these factors is required to gain a deeper understanding of the genetic circuitry underlying telencephalic patterning. Using DNA-binding and reporter gene assays, we here investigate the possibility that Gli3 and these signaling molecules interact by directly regulating each other's expression. We show that Fgf signaling is required for Wnt8b enhancer activity in the cortical hem, whereas Wnt/ß-catenin signaling represses Fgf17 forebrain enhancer activity. In contrast, Fgf and Wnt/ß-catenin signaling cooperate to regulate Gli3 expression. Taken together, these findings indicate that mutual interactions between Gli3, Wnt8b, and Fgf17 are crucial elements of the balance between these factors thereby conferring robustness to the patterning process. Hence, our study provides a framework for understanding the genetic circuitry underlying telencephalic patterning and how defects in this process can affect the formation of cortical areas.

The ciliogenic transcription factor Rfx3 is required for the formation of the thalamocortical tract by regulating the patterning of prethalamus and ventral telencephalon.

Primary cilia are complex subcellular structures that play key roles during embryogenesis by controlling the cellular response to several signaling pathways. Defects in the function and/or structure of primary cilia underlie a large number of human syndromes collectively referred to as ciliopathies. Often, ciliopathies are associated with mental retardation (MR) and malformation of the corpus callosum. However, the possibility of defects in other forebrain axon tracts, which could contribute to the cognitive disorders of these patients, has not been explored. Here, we investigate the formation of the corticothalamic/thalamocortical tracts in mice mutant for Rfx3, which regulates the expression of many genes involved in ciliogenesis and cilia function. Using DiI axon tracing and immunohistochemistry experiments, we show that some Rfx3(-/-) corticothalamic axons abnormally migrate toward the pial surface of the ventral telencephalon (VT). Some thalamocortical axons (TCAs) also fail to leave the diencephalon or abnormally project toward the amygdala. Moreover, the Rfx3(-/-) VT displays heterotopias containing attractive guidance cues and expressing the guidance molecules Slit1 and Netrin1. Finally, the abnormal projection of TCAs toward the amygdala is also present in mice carrying a mutation in the Inpp5e gene, which is mutated in Joubert Syndrome and which controls cilia signaling and stability. The presence of identical thalamocortical malformations in two independent ciliary mutants indicates a novel role for primary cilia in the formation of the corticothalamic/thalamocortical tracts by establishing the correct cellular environment necessary for its development.

Gli3 controls corpus callosum formation by positioning midline guideposts during telencephalic patterning.

The corpus callosum (CC) represents the major forebrain commissure connecting the 2 cerebral hemispheres. Midline crossing of callosal axons is controlled by several glial and neuronal guideposts specifically located along the callosal path, but it remains unknown how these cells acquire their position. Here, we show that the Gli3 hypomorphic mouse mutant Polydactyly Nagoya (Pdn) displays agenesis of the CC and mislocation of the glial and neuronal guidepost cells. Using transplantation experiments, we demonstrate that agenesis of the CC is primarily caused by midline defects. These defects originate during telencephalic patterning and involve an up-regulation of Slit2 expression and altered Fgf and Wnt/ß-catenin signaling. Mutations in sprouty1/2 which mimic the changes in these signaling pathways cause a disorganization of midline guideposts and CC agenesis. Moreover, a partial recovery of midline abnormalities in Pdn/Pdn;Slit2(-/-) embryos mutants confirms the functional importance of correct Slit2 expression levels for callosal development. Hence, Gli3 controlled restriction of Fgf and Wnt/ß-catenin signaling and of Slit2 expression is crucial for positioning midline guideposts and callosal development.

Pax6 exerts regional control of cortical progenitor proliferation via direct repression of Cdk6 and hypophosphorylation of pRb.

The mechanisms by which early spatiotemporal expression patterns of transcription factors such as Pax6 regulate cortical progenitors in a region-specific manner are poorly understood. Pax6 is expressed in a gradient across the developing cortex and is essential for normal corticogenesis. We found that constitutive or conditional loss of Pax6 increases cortical progenitor proliferation by amounts that vary regionally with normal Pax6 levels. We compared the gene expression profiles of equivalent Pax6-expressing progenitors isolated from Pax6⁺/⁺ and Pax6⁻/⁻ cortices and identified many negatively regulated cell-cycle genes, including Cyclins and Cdks. Biochemical assays indicated that Pax6 directly represses Cdk6 expression. Cyclin/Cdk repression inhibits retinoblastoma protein (pRb) phosphorylation, thereby limiting the transcription of genes that directly promote the mechanics of the cell cycle, and we found that Pax6 inhibits pRb phosphorylation and represses genes involved in DNA replication. Our results indicate that Pax6's modulation of cortical progenitor cell cycles is regional and direct.

Gli3 is required in Emx1+ progenitors for the development of the corpus callosum.

The corpus callosum (CC) is the largest commissure in the forebrain and mediates the transfer of sensory, motor and cognitive information between the cerebral hemispheres. During CC development, a number of strategically located glial and neuronal guidepost structures serve to guide callosal axons across the midline at the corticoseptal boundary (CSB). Correct positioning of these guideposts requires the Gli3 gene, mutations of which result in callosal defects in humans and mice. However, as Gli3 is widely expressed during critical stages of forebrain development, the precise temporal and spatial requirements for Gli3 function in callosal development remain unclear. Here, we used a conditional mouse mutant approach to inactivate Gli3 in specific regions of the developing telencephalon in order to delineate the domain(s) in which Gli3 is required for normal development of the corpus callosum. Inactivation of Gli3 in the septum or in the medial ganglionic eminence had no effect on CC formation, however Gli3 inactivation in the developing cerebral cortex led to the formation of a severely hypoplastic CC at E18.5 due to a severe disorganization of midline guideposts. Glial wedge cells translocate prematurely and Slit1/2 are ectopically expressed in the septum. These changes coincide with altered Fgf and Wnt/ß-catenin signalling during CSB formation. Collectively, these data demonstrate a crucial role for Gli3 in cortical progenitors to control CC formation and indicate how defects in CSB formation affect the positioning of callosal guidepost cells.

Gli3 controls subplate formation and growth of cortical axons.

The formation of a functional cortical circuitry requires the coordinated growth of cortical axons to their target areas. While the mechanisms guiding cortical axons to their targets have extensively been studied, very little is known about the processes which promote their growth in vivo. Gli3 encodes a zinc finger transcription factor which is expressed in cortical progenitor cells and has crucial roles in cortical development. Here, we characterize the Gli3 compound mutant Gli3(Xt/Pdn), which largely lacks Neurofilament(+) fibers in the rostral and intermediate neocortex. DiI labeling and Golli-τGFP immunofluorescence indicate that Gli3(Xt/Pdn) cortical neurons form short and stunted axons. Using transplantation experiments we demonstrate that this axon growth defect is primarily caused by a nonpermissive cortical environment. Furthermore, in Emx1Cre;Gli3(Pdn/fl) conditional mutants, which mimic the reduction of Gli3 expression in the dorsal telencephalon of Gli3(Xt/Pdn) embryos, the growth of cortical axons is not impaired, suggesting that Gli3 controls this process early in telencephalic development. In contrast to cortical plate neurons, Gli3(Xt/Pdn) embryos largely lack subplate (SP) neurons which normally pioneer cortical projections. Collectively, these findings show that Gli3 specifies a cortical environment permissive to the growth of cortical axons at the progenitor level by controlling the formation of SP neurons.

Transcriptional analysis of Gli3 mutants identifies Wnt target genes in the developing hippocampus.

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/ß-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/ß-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.

The Gli3 hypomorphic mutation Pdn causes selective impairment in the growth, patterning, and axon guidance capability of the lateral ganglionic eminence.

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.

The alpha-chemokine CXCL14 is up-regulated in the sciatic nerve of a mouse model of Charcot-Marie-Tooth disease type 1A and alters myelin gene expression in cultured Schwann cells.

At present the pathogenesis of CMT1A neuropathy, caused by the overexpression of PMP22, has not yet been entirely understood. The PMP22-overexpressing C61 mutant mouse is a suitable animal model, which mimics the human CMT1A disorder. We observed that myelin gene expression in the sciatic nerve of the C61 mouse was up-regulated at postnatal day 4 to 7 (P4-P7). When investigating the morphology of peripheral nerves in C61 and wildtype mice at early stages of postnatal development, hypermyelination could be detected in the femoral quadriceps and sciatic nerve of transgenic animals at postnatal day 7 (P7). In order to identify genes, other than Pmp22, that are modulated in sciatic nerve of P7 transgenic mice, we applied microarray technology. Amongst the regulated genes, the gene encoding the alpha-chemokine CXCL14 was most prominently up-regulated. We report that Cxcl14 was expressed exclusively by Schwann cells of the sciatic nerve, as well as by cultured Schwann cells triggered to differentiate. Furthermore, in cultured Schwann cells CXCL14 modulated the expression of myelin genes and altered cell proliferation. Our findings demonstrate that early overexpression of PMP22, in a mouse model of CMT1A, results in a strong up-regulation of CXCL14, which seems to play a novel regulatory role in Schwann cell differentiation.

A crucial role for primary cilia in cortical morphogenesis.

Primary cilia are important sites of signal transduction involved in a wide range of developmental and postnatal functions. Proteolytic processing of the transcription factor Gli3, for example, occurs in primary cilia, and defects in intraflagellar transport (IFT), which is crucial for the maintenance of primary cilia, can lead to severe developmental defects and diseases. Here we report an essential role of primary cilia in forebrain development. Uncovered by N-ethyl-N-nitrosourea-mutagenesis, cobblestone is a hypomorphic allele of the IFT gene Ift88, in which Ift88 mRNA and protein levels are reduced by 70-80%. cobblestone mutants are distinguished by subpial heterotopias in the forebrain. Mutants show both severe defects in the formation of dorsomedial telencephalic structures, such as the choroid plexus, cortical hem and hippocampus, and also a relaxation of both dorsal-ventral and rostral-caudal compartmental boundaries. These defects phenocopy many of the abnormalities seen in the Gli3 mutant forebrain, and we show that Gli3 proteolytic processing is reduced, leading to an accumulation of the full-length activator isoform. In addition, we observe an upregulation of canonical Wnt signaling in the neocortex and in the caudal forebrain. Interestingly, the ultrastructure and morphology of ventricular cilia in the cobblestone mutants remains intact. Together, these results indicate a critical role for ciliary function in the developing forebrain.

Gene expression profiling reveals that peripheral nerve regeneration is a consequence of both novel injury-dependent and reactivated developmental processes.

One of the most striking features of the injured mature peripheral nervous system is the ability to regenerate. The lesioned peripheral nervous system displays stereotypic histopathological reactions indicating the activation of a co-ordinated lesion-induced gene expression programme. Previous research has already identified molecular components of this axonal switch from a mature transmitting to a regenerative growth mode. The observed alterations in gene expression within the lesioned distal nerve stump were largely attributed to recapitulated developmental processes. However, to our knowledge, this hypothesis has not been proven systematically. Most of the stereotypic molecular and cellular reactions during nerve development and repair can be assigned to specific time windows. Consequently, we have compared gene expression profiles of both paradigms at six different time-points each by means of cDNA array hybridization. Our data identified injury-specific molecular reactions and revealed to what extent developmental mechanisms are reactivated in response to nerve lesion. Ninety-one genes (47% of the regeneration-associated genes) were found to be significantly regulated in both paradigms, suggesting that regeneration only partially recapitulates development and that approximately half of the regulated genes are part of a regeneration-dependent programme. Interestingly, mainly genes encoding signal transducers or factors involved in processes such as cell death, immune response, transport and transcriptional regulation showed injury-specific gene expression.

Evidence for macrophage-mediated myelin disruption in an animal model for Charcot-Marie-Tooth neuropathy type 1A.

Charcot-Marie-Tooth neuropathy type 1A (CMT 1 A) is the most common inherited neuropathy in humans and is mostly caused by a 1.5-Mb tandem duplication of chromosome 17 comprising the gene for the peripheral myelin protein 22-kDa (PMP 22). Although there are numerous studies on the functional role of PMP 22, the mechanisms of myelin degeneration under PMP 22-overexpression conditions have not yet been fully understood. We have shown previously that in mouse mutants hetero- or homozygously deficient for two other myelin components, P0 and C x 32, respectively, immune cells contribute to the demyelinating neuropathy. To test this possibility for PMP 22 overexpression, we investigated a putative mouse model for CMT 1 A, i.e., the mouse strain C 6 1 mildly overexpressing human PMP 22 in peripheral nerves. Electron microscopic and electrophysiologic investigations revealed that this mouse strain develops pathologic features similar to those found in CMT 1 A patients. A novel finding, however, was the upregulation of CD8- and F4/80-positive lymphocytes and macrophages, respectively, in peripheral nerves. The observation that macrophages enter endoneurial tubes of the mutants and obviously phagocytose morphologically normal myelin strongly suggests that the myelin degeneration is mediated at least partially by these phagocytic cells. By gene array technology and quantitative RT-PCR of peripheral nerve homogenates from PMP 22 mutants, monocyte chemoattractant protein-1 (MCP-1; cc l2) could be identified as a putative factor to attract or activate macrophages that attack myelin sheaths in this model of CMT 1 A.