Polymicrogyria is a relatively common but poorly understood defect of cortical development characterized by numerous small gyri and a thick disorganized cortical plate lacking normal lamination. Here we report de novo mutations in a beta-tubulin gene, TUBB2B, in four individuals and a 27-gestational-week fetus with bilateral asymmetrical polymicrogyria. Neuropathological examination of the fetus revealed an absence of cortical lamination associated with the presence of ectopic neuronal cells in the white matter and in the leptomeningeal spaces due to breaches in the pial basement membrane. In utero RNAi-based inactivation demonstrates that TUBB2B is required for neuronal migration. We also show that two disease-associated mutations lead to impaired formation of tubulin heterodimers. These observations, together with previous data, show that disruption of microtubule-based processes underlies a large spectrum of neuronal migration disorders that includes not only lissencephaly and pachygyria, but also polymicrogyria malformations.
Summary Diverse subsets of cortical interneurons play vital roles in higher-order brain functions. To investigate how this diversity is generated, we used single cell RNA-seq to profile the transcriptomes of murine cells collected along a developmental timecourse. Heterogeneity within mitotic progenitors in the ganglionic eminences is driven by a highly conserved maturation trajectory, alongside eminence-specific transcription factor expression that seeds the emergence of later diversity. Upon becoming postmitotic, progenitors diverge and differentiate into transcriptionally distinct states, including an interneuron precursor state. By integrating datasets across developmental timepoints, we identified shared sources of transcriptomic heterogeneity between adult interneurons and their precursors, revealing the embryonic emergence of interneuron cardinal subtypes. Our analysis revealed that the ASD-associated transcription factor Mef2c delineates early Pvalb-precursors, and is essential for their development. These findings shed new light on the molecular diversification of early inhibitory precursors, and identify gene modules that may influence the specification of human subtypes.
Mutations in the TUBB3 gene, encoding β-tubulin isotype III, were recently shown to be associated with various neurological syndromes which all have in common the ocular motility disorder, congenital fibrosis of the extraocular muscle type 3 (CFEOM3). Surprisingly and in contrast to previously described TUBA1A and TUBB2B phenotypes, no evidence of dysfunctional neuronal migration and cortical organization was reported. In our study, we report the discovery of six novel missense mutations in the TUBB3 gene, including one fetal case and one homozygous variation, in nine patients that all share cortical disorganization, axonal abnormalities associated with pontocerebellar hypoplasia, but with no ocular motility defects, CFEOM3. These new findings demonstrate that the spectrum of TUBB3-related phenotype is broader than previously described and includes malformations of cortical development (MCD) associated with neuronal migration and differentiation defects, axonal guidance and tract organization impairment. Complementary functional studies revealed that the mutated βIII-tubulin causing the MCD phenotype results in a reduction of heterodimer formation, yet produce correctly formed microtubules (MTs) in mammalian cells. Further to this, we investigated the properties of the MT network in patients' fibroblasts and revealed that MCD mutations can alter the resistance of MTs to depolymerization. Interestingly, this finding contrasts with the increased MT stability observed in the case of CFEOM3-related mutations. These results led us to hypothesize that either MT dynamics or their interactions with various MT-interacting proteins could be differently affected by TUBB3 variations, thus resulting in distinct alteration of downstream processes and therefore explaining the phenotypic diversity of the TUBB3-related spectrum.
Diverse subsets of cortical interneurons play a particularly important role in the stability of the neural circuits underlying cognitive and higher order brain functions, yet our understanding of how this diversity is generated is far from complete. We applied massively parallel single-cell RNA-seq to profile a developmental time course of interneuron development, measuring the transcriptomes of over 60,000 progenitors during their maturation in the ganglionic eminences and embryonic migration into the cortex. While diversity within mitotic progenitors is largely driven by cell cycle and differentiation state, we observed sparse eminence-specific transcription factor expression, which seeds the emergence of later cell diversity. Upon becoming postmitotic, cells from all eminences pass through one of three precursor states, one of which represents a cortical interneuron ground state. By integrating datasets across developmental timepoints, we identified transcriptomic heterogeneity in interneuron precursors representing the emergence of four cardinal classes (Pvalb, Sst, Id2 and Vip), which further separate into subtypes at different timepoints during development. Our analysis revealed that the ASD-associated transcription factor Mef2c discriminates early Pvalb-precursors in E13.5 cells, and removal of Mef2c confirms its essential role for Pvalb interneuron development. These findings shed new light on the molecular diversification of early inhibitory precursors, and suggest gene modules that may link developmental specification with the etiology of neuropsychiatric disorders.
SUMMARY The formation of the mammalian cortex requires the generation, migration, and differentiation of neurons. The vital role that the microtubule cytoskeleton plays in these cellular processes is reflected by the discovery that mutations in various tubulin isotypes cause different neurodevelopmental diseases, including lissencephaly (TUBA1A), polymicrogyria (TUBA1A, TUBB2B, TUBB3), and an ocular motility disorder (TUBB3). Here, we show that Tubb5 is expressed in neurogenic progenitors in the mouse and that its depletion in vivo perturbs the cell cycle of progenitors and alters the position of migrating neurons. We report the occurrence of three microcephalic patients with structural brain abnormalities harboring de novo mutations in TUBB5 (M299V, V353I, and E401K). These mutant proteins, which affect the chaperone-dependent assembly of tubulin heterodimers in different ways, disrupt neurogenic division and/or migration in vivo. Our results provide insight into the functional repertoire of the tubulin gene family, specifically implicating TUBB5 in embryonic neurogenesis and microcephaly.
The medial ganglionic eminence (MGE) gives rise to the majority of mouse forebrain interneurons. Here, we examine the lineage relationship among MGE-derived interneurons using a replication-defective retroviral library containing a highly diverse set of DNA barcodes. Recovering the barcodes from the mature progeny of infected progenitor cells enabled us to unambiguously determine their respective lineal relationship. We found that clonal dispersion occurs across large areas of the brain and is not restricted by anatomical divisions. As such, sibling interneurons can populate the cortex, hippocampus striatum and globus pallidus. The majority of interneurons appeared to be generated from asymmetric divisions of MGE progenitor cells, followed by symmetric divisions within the subventricular zone. Altogether, our findings uncover that lineage relationships do not appear to determine interneuron allocation to particular regions. As such, it is likely that clonally-related interneurons have considerable flexibility as to the particular forebrain circuits to which they can contribute.
Summary We demonstrate that cortical interneurons derived from ventral eminences, including the caudal ganglionic eminence, undergo programmed cell death. Moreover, with the exception of VIP interneurons, this occurs in a manner that is activity-dependent. In addition, we demonstrate that, within interneurons, Calcineurin, a calcium-dependent protein phosphatase, plays a critical role in sequentially linking activity to maturation (E15–P5) and survival (P5–P20). Specifically, embryonic inactivation of Calcineurin results in a failure of interneurons to morphologically mature and prevents them from undergoing apoptosis. By contrast, early postnatal inactivation of Calcineurin increases apoptosis. We conclude that Calcineurin serves a dual role of promoting first the differentiation of interneurons and, subsequently, their survival.
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