SUMMARY Autism spectrum disorder (ASD) is a complex developmental syndrome of unknown etiology. Recent studies employing exome- and genome-wide sequencing have identified nine high-confidence ASD (hcASD) genes. Working from the hypothesis that ASD-associated mutations in these biologically pleiotropic genes will disrupt intersecting developmental processes to contribute to a common phenotype, we have attempted to identify time periods, brain regions, and cell types in which these genes converge. We have constructed coexpression networks based on the hcASD “seed” genes, leveraging a rich expression data set encompassing multiple human brain regions across human development and into adulthood. By assessing enrichment of an independent set of probable ASD (pASD) genes, derived from the same sequencing studies, we demonstrate a key point of convergence in midfetal layer 5/6 cortical projection neurons. This approach informs when, where, and in what cell types mutations in these specific genes may be productively studied to clarify ASD pathophysiology.

Summary The mechanisms underlying Zika virus (ZIKV)-related microcephaly and other neurodevelopment defects remain poorly understood. Here, we describe the derivation and characterization, including single-cell RNA-seq, of neocortical and spinal cord neuroepithelial stem (NES) cells to model early human neurodevelopment and ZIKV-related neuropathogenesis. By analyzing human NES cells, organotypic fetal brain slices and a ZIKV-infected micrencephalic brain, we show that ZIKV infects both neocortical and spinal NES cells and their fetal homolog, radial glial cells (RGCs), causing disrupted mitoses, supernumerary centrosomes, structural disorganization and cell death. ZIKV infection of NES cells and RGCs causes centrosomal depletion and mitochondrial sequestration of phospho-TBK1 during mitosis. We also found that nucleoside analogs inhibit ZIKV replication in NES cells, protecting them from ZIKV-induced pTBK1 relocalization and cell death. We established a model system of human neural stem cells to reveal cellular and molecular mechanisms underlying neurodevelopmental defects associated with ZIKV infection and its potential treatment.

The corticospinal (CS) tract is involved in controlling discrete voluntary skilled movements in mammals. The CS tract arises exclusively from layer (L) 5 projection neurons of the cerebral cortex, and its formation requires L5 activity of Fezf2 (Fezl, Zfp312). How this L5-specific pattern of Fezf2 expression and CS axonal connectivity is established with such remarkable fidelity had remained elusive. Here we show that the transcription factor TBR1 directly binds the Fezf2 locus and represses its activity in L6 corticothalamic projection neurons to restrict the origin of the CS tract to L5. In Tbr1 null mutants, CS axons ectopically originate from L6 neurons in a Fezf2-dependent manner. Consistently, misexpression of Tbr1 in L5 CS neurons suppresses Fezf2 expression and effectively abolishes the CS tract. Taken together, our findings show that TBR1 is a direct transcriptional repressor of Fezf2 and a negative regulator of CS tract formation that restricts the laminar origin of CS axons specifically to L5.neocortex | pyramidal neuron | axon guidance | transcriptional repression S patial specificity of axonal connections is one of the most important prerequisites for normal development (1-3). In mammals, this is especially crucial for axons of the corticospinal (CS) system (4-7). Development of the CS tract is an intricate process that involves the molecular specification of CS neurons and axon pathfinding. All long-range subcortical axons projecting to the brainstem and spinal cord, including those that form the CS tract, originate exclusively from layer (L) 5 projection (pyramidal) neurons of the cerebral cortex (8-11). Projection neurons in other cortical layers give rise to axons that project within the cortex (L2-4) or to the thalamus (L6). How this highly conserved laminar pattern of projections is formed with such perfect accuracy remains elusive.Previous work revealed that the transcription factor FEZF2 (FEZL, ZFP312) is highly enriched in L5 CS neurons and is critical to the development of the CS tract (12-14). Inactivation of Fezf2 disrupts formation of the CS tract (12-14), whereas misexpression of Fezf2 in upper layer projection neurons induces ectopic subcortical projections (13). These findings indicate that Fezf2 transcription is tightly regulated during development, and that the integrity of normal Fezf2 expression is critical to proper development of the CS tract. Interestingly, Fezf2 is transiently expressed in L6 neurons during early embryonic development, where its transcription is directly repressed by SOX5, thereby establishing a high-in-L5, low-in-L6 postnatal pattern (15, 16). Paradoxically, in Sox5 null mutants, the number of axon projections reaching the brainstem and spinal cord is severely reduced despite increased cortical Fezf2 expression (15). This suggests that Sox5 is required for the formation of these connections independent of its regulation of Fezf2. Furthermore, SOX5 is normally expressed in L5 Fezf2-expressing CS neurons (15). Therefore, the down-regulation of Fezf2 in L6 neu...

Despite extensive studies on mammalian neurogenesis, its post-transcriptional regulation remains under-explored. Here we report that neural-specific inactivation of two murine post-transcriptional regulators, Pumilio 1 (Pum1) and Pum2, severely reduced the number of neural stem cells (NSCs) in the postnatal dentate gyrus (DG), drastically increased perinatal apoptosis, altered DG cell composition, and impaired learning and memory. Consistently, the mutant DG neurospheres generated fewer NSCs with defects in proliferation, survival, and differentiation, supporting a major role of Pum1 and Pum2 in hippocampal neurogenesis and function. Cross-linking immunoprecipitation revealed that Pum1 and Pum2 bind to thousands of mRNAs, with at least 694 common targets in multiple neurogenic pathways. Depleting Pum1 and/or Pum2 did not change the abundance of most target mRNAs but up-regulated their proteins, indicating that Pum1 and Pum2 regulate the translation of their target mRNAs. Moreover, Pum1 and Pum2 display RNA-dependent interaction with fragile X mental retardation protein (FMRP) and bind to one another's mRNA. This indicates that Pum proteins might form collaborative networks with FMRP and possibly other post-transcriptional regulators to regulate neurogenesis.

SUMMARY Exome sequencing analysis of over 2,000 children with complex malformations of cortical development identified 5 independent homozygous deleterious mutations in KATNB1, encoding the regulatory subunit of the microtubule severing enzyme katanin. Mitotic spindle formation is defective in patient-derived fibroblasts, a consequence of disrupted interactions of mutant KATNB1 with KATNA1, the catalytic subunit of katanin, and other microtubule associated proteins. Loss of KATNB1 orthologs in zebrafish (katnb1) and flies (kat80) results in microcephaly, recapitulating the human phenotype. In the developing Drosophila optic lobe, kat80 loss specifically affects the asymmetrically dividing neuroblasts, which display supernumerary centrosomes and spindle abnormalities during mitosis, leading to cell cycle progression delays and reduced cell numbers. Furthermore, kat80 depletion results in dendritic arborization defects in sensory and motor neurons, affecting neural architecture. Taken together, we provide insight into the mechanisms by which KATNB1 mutations cause human cerebral cortical malformations, demonstrating its fundamental role during brain development.