Co-culture system of human skin equivalents with mouse neural spheroids.

This study describes a co-culture system of human skin equivalents (HSEs) and dorsal root ganglion (DRG) neurons. We prepared spheroids of mouse DRG neurons with or without Schwann cells (SCs). Spheroids comprising DRG neurons and SCs showed longer neurite extensions than those comprising DRG neurons alone. Neurite extension of more than 1 mm was observed from spheroids cultured inside HSEs, whereas neurite extension was primarily observed on the surface of HSEs from spheroids cultured on HSEs. We propose that our model may be a useful tool for studying neurite extension in the human skin.

Subcellular mRNA localization and local translation of Arhgap11a in radial glial progenitors regulates cortical development.

mRNA localization and local translation enable exquisite spatial and temporal control of gene expression, particularly in polarized, elongated cells. These features are especially prominent in radial glial cells (RGCs), which are neural and glial precursors of the developing cerebral cortex and scaffolds for migrating neurons. Yet the mechanisms by which subcellular RGC compartments accomplish their diverse functions are poorly understood. Here, we demonstrate that mRNA localization and local translation of the RhoGAP ARHGAP11A in the basal endfeet of RGCs control their morphology and mediate neuronal positioning. Arhgap11a transcript and protein exhibit conserved localization to RGC basal structures in mice and humans, conferred by the 5' UTR. Proper RGC morphology relies upon active Arhgap11a mRNA transport and localization to the basal endfeet, where ARHGAP11A is locally synthesized. This translation is essential for positioning interneurons at the basement membrane. Thus, local translation spatially and acutely activates Rho signaling in RGCs to compartmentalize neural progenitor functions.

CROCCP2 acts as a human-specific modifier of cilia dynamics and mTOR signaling to promote expansion of cortical progenitors.

The primary cilium is a central signaling component during embryonic development. Here we focus on CROCCP2, a hominid-specific gene duplicate from ciliary rootlet coiled coil (CROCC), also known as rootletin, that encodes the major component of the ciliary rootlet. We find that CROCCP2 is highly expressed in the human fetal brain and not in other primate species. CROCCP2 gain of function in the mouse embryonic cortex and human cortical cells and organoids results in decreased ciliogenesis and increased cortical progenitor amplification, particularly basal progenitors. CROCCP2 decreases ciliary dynamics by inhibition of the IFT20 ciliary trafficking protein, which then impacts neurogenesis through increased mTOR signaling. Loss of function of CROCCP2 in human cortical cells and organoids leads to increased ciliogenesis, decreased mTOR signaling, and impaired basal progenitor amplification. These data identify CROCCP2 as a human-specific modifier of cortical neurogenesis that acts through modulation of ciliary dynamics and mTOR signaling.

The tyrosine capsid mutations on retrograde adeno-associated virus accelerates gene transduction efficiency.

Adeno-associated virus (AAV) vector is a critical tool for gene delivery through its durable transgene expression and safety profile. Among many serotypes, AAV2-retro is typically utilized for dissecting neural circuits with its retrograde functionality. However, this vector requires a relatively long-term incubation period (over 2 weeks) to obtain enough gene expression levels presumably due to low efficiency in gene transduction. Here, we aimed to enhance transgene expression efficiency of AAV2-retro vectors by substituting multiple tyrosine residues with phenylalanines (YF mutations) in the virus capsid, which is previously reported to improve the transduction efficiency of AAV2-infected cells by evading host cell responses. We found that AAV2-retro with YF mutations (AAV2-retroYF)-mediated transgene expression was significantly enhanced in the primary culture of murine cortical neurons at 1 week after application, comparable to that of the conventional AAV2-retro at 2 week after application. Moreover, transgene expressions in the retrogradely labeled neurons mediated by AAV2-retroYF were significantly increased both in the cortico-cortical circuits and in the subcortical circuits in vivo, while the retrograde functionality of AAV2-retroYF was equally effective as that of AAV2-retro. Our data indicate that YF mutations boost AAV2-retro-mediated retrograde gene transduction in vivo and suggest that the AAV2-retroYF should be useful for efficient targeting of the projection-defined neurons, which is suited to applications for dissecting neural circuits during development as well as future clinical applications.

Evolutionary innovations of human cerebral cortex viewed through the lens of high-throughput sequencing.

Humans had acquired a tremendously enlarged cerebral cortex containing a huge quantity and variety of cells during evolution. Such evolutionary uniqueness offers a neural basis of our cognitive innovation and human-specific features of neurodevelopmental and psychiatric disorders. Since human brain is hardly examined in vivo with experimental approaches commonly applied on animal models, the recent advancement of sequencing technologies offers an indispensable viewpoint of human brain anatomy and development. This review introduces the recent findings on the unique features in the adult and the characteristic developmental processes of the human cerebral cortex, based on high-throughput DNA sequencing technologies.

Spatial and temporal diversity of DCLK1 isoforms in developing mouse brain.

Doublecortin-like kinase 1 (DCLK1) is a Doublecortin family kinase involved in a range of brain development processes including cell migration, axon/dendrite growth, and synapse development. The Dclk1 gene potentially generates multiple splicing isoforms, but the detailed expression patterns in the brain as well as in vivo functions of each isoform are still incompletely understood. Here we assessed expression patterns of DCLK1 isoforms using multiple platforms including in silico, in situ, and in vitro datasets in the developing mouse brain, and show quantitative evidence that among the four DCLK1 isoforms, DCLK1-L and DCL are mainly expressed in the embryonic cortex whereas DCLK1-L and CPG16 become dominant compared to DCL and CARP in the postnatal cortex. We also provide compelling evidence that DCLK1 isoforms are distributed in the partially distinct brain regions in the embryonic and the postnatal stages. We further show that overexpression of DCLK1-L, but not the other isoforms, in neural progenitors causes severe migration defects in the cortex, and that the migration defects are dependent on the kinase activity of DCLK1-L. Our data thus uncover partially segregated localization of DCLK1 isoforms in the developing mouse brain and suggest different roles for distinct DCLK1 isoforms in the brain development and function.

Molecular drivers of human cerebral cortical evolution.

One of the most important questions in human evolutionary biology is how our ancestor has acquired an expanded volume of the cerebral cortex, which may have significantly impacted on improving our cognitive abilities. Recent comparative approaches have identified developmental features unique to the human or hominid cerebral cortex, not shared with other animals including conventional experimental models. In addition, genomic, transcriptomic, and epigenomic signatures associated with human- or hominid-specific processes of the cortical development are becoming identified by virtue of technical progress in the deep nucleotide sequencing. This review discusses ontogenic and phylogenetic processes of the human cerebral cortex, followed by the introduction of recent comprehensive approaches identifying molecular mechanisms potentially driving the evolutionary changes in the cortical development.

Long-read sequencing identifies GGC repeat expansions in NOTCH2NLC associated with neuronal intranuclear inclusion disease.

Neuronal intranuclear inclusion disease (NIID) is a progressive neurodegenerative disease that is characterized by eosinophilic hyaline intranuclear inclusions in neuronal and somatic cells. The wide range of clinical manifestations in NIID makes ante-mortem diagnosis difficult1-8, but skin biopsy enables its ante-mortem diagnosis9-12. The average onset age is 59.7 years among approximately 140 NIID cases consisting of mostly sporadic and several familial cases. By linkage mapping of a large NIID family with several affected members (Family 1), we identified a 58.1 Mb linked region at 1p22.1-q21.3 with a maximum logarithm of the odds score of 4.21. By long-read sequencing, we identified a GGC repeat expansion in the 5' region of NOTCH2NLC (Notch 2 N-terminal like C) in all affected family members. Furthermore, we found similar expansions in 8 unrelated families with NIID and 40 sporadic NIID cases. We observed abnormal anti-sense transcripts in fibroblasts specifically from patients but not unaffected individuals. This work shows that repeat expansion in human-specific NOTCH2NLC, a gene that evolved by segmental duplication, causes a human disease.

Human-Specific NOTCH2NL Genes Expand Cortical Neurogenesis through Delta/Notch Regulation.

The cerebral cortex underwent rapid expansion and increased complexity during recent hominid evolution. Gene duplications constitute a major evolutionary force, but their impact on human brain development remains unclear. Using tailored RNA sequencing (RNA-seq), we profiled the spatial and temporal expression of hominid-specific duplicated (HS) genes in the human fetal cortex and identified a repertoire of 35 HS genes displaying robust and dynamic patterns during cortical neurogenesis. Among them NOTCH2NL, human-specific paralogs of the NOTCH2 receptor, stood out for their ability to promote cortical progenitor maintenance. NOTCH2NL promote the clonal expansion of human cortical progenitors, ultimately leading to higher neuronal output. At the molecular level, NOTCH2NL function by activating the Notch pathway through inhibition of cis Delta/Notch interactions. Our study uncovers a large repertoire of recently evolved genes active during human corticogenesis and reveals how human-specific NOTCH paralogs may have contributed to the expansion of the human cortex.

Stress-induced unfolded protein response contributes to Zika virus-associated microcephaly.

Accumulating evidence support a causal link between Zika virus (ZIKV) infection during gestation and congenital microcephaly. However, the mechanism of ZIKV-associated microcephaly remains unclear. We combined analyses of ZIKV-infected human fetuses, cultured human neural stem cells and mouse embryos to understand how ZIKV induces microcephaly. We show that ZIKV triggers endoplasmic reticulum stress and unfolded protein response in the cerebral cortex of infected postmortem human fetuses as well as in cultured human neural stem cells. After intracerebral and intraplacental inoculation of ZIKV in mouse embryos, we show that it triggers endoplasmic reticulum stress in embryonic brains in vivo. This perturbs a physiological unfolded protein response within cortical progenitors that controls neurogenesis. Thus, ZIKV-infected progenitors generate fewer projection neurons that eventually settle in the cerebral cortex, whereupon sustained endoplasmic reticulum stress leads to apoptosis. Furthermore, we demonstrate that administration of pharmacological inhibitors of unfolded protein response counteracts these pathophysiological mechanisms and prevents microcephaly in ZIKV-infected mouse embryos. Such defects are specific to ZIKV, as they are not observed upon intraplacental injection of other related flaviviruses in mice.

Is this a brain which I see before me? Modeling human neural development with pluripotent stem cells.

The human brain is arguably the most complex structure among living organisms. However, the specific mechanisms leading to this complexity remain incompletely understood, primarily because of the poor experimental accessibility of the human embryonic brain. Over recent years, technologies based on pluripotent stem cells (PSCs) have been developed to generate neural cells of various types. While the translational potential of PSC technologies for disease modeling and/or cell replacement therapies is usually put forward as a rationale for their utility, they are also opening novel windows for direct observation and experimentation of the basic mechanisms of human brain development. PSC-based studies have revealed that a number of cardinal features of neural ontogenesis are remarkably conserved in human models, which can be studied in a reductionist fashion. They have also revealed species-specific features, which constitute attractive lines of investigation to elucidate the mechanisms underlying the development of the human brain, and its link with evolution.

A common developmental plan for neocortical gene-expressing neurons in the pallium of the domestic chicken Gallus gallus domesticus and the Chinese softshell turtle Pelodiscus sinensis.

The six-layered neocortex is a unique characteristic of mammals and likely provides the neural basis of their sophisticated cognitive abilities. Although all mammalian species share the layered structure of the neocortex, the sauropsids exhibit an entirely different cytoarchitecture of the corresponding pallial region. Our previous gene expression study revealed that the chicken pallium possesses neural subtypes that express orthologs of layer-specific genes of the mammalian neocortex. To understand the evolutionary steps leading toward animal group-specific neuronal arrangements in the pallium in the course of amniote diversification, we examined expression patterns of the same orthologs and a few additional genes in the pallial development of the Chinese softshell turtle Pelodiscus sinensis, and compared these patterns to those of the chicken. Our analyses highlighted similarities in neuronal arrangements between the two species; the mammalian layer 5 marker orthologs are expressed in the medial domain and the layer 2/3 marker orthologs are expressed in the lateral domain in the pallia of both species. We hypothesize that the mediolateral arrangement of the neocortical layer-specific gene-expressing neurons originated in their common ancestor and is conserved among all sauropsid groups, whereas the neuronal arrangement within the pallium could have highly diversified independently in the mammalian lineage.

Novel and robust transplantation reveals the acquisition of polarized processes by cortical cells derived from mouse and human pluripotent stem cells.

Current stem cell technologies have enabled the induction of cortical progenitors and neurons from embryonic stem cells (ESCs) and induced pluripotent stem cells in vitro. To understand the mechanisms underlying the acquisition of apico-basal polarity and the formation of processes associated with the stemness of cortical cells generated in monolayer culture, here, we developed a novel in utero transplantation system based on the moderate dissociation of adherens junctions in neuroepithelial tissue. This method enables (1) the incorporation of remarkably higher numbers of grafted cells and (2) quantitative morphological analyses at single-cell resolution, including time-lapse recording analyses. We then grafted cortical progenitors induced from mouse ESCs into the developing brain. Importantly, we revealed that the mode of process extension depends on the extrinsic apico-basal polarity of the host epithelial tissue, as well as on the intrinsic differentiation state of the grafted cells. Further, we successfully transplanted cortical progenitors induced from human ESCs, showing that our strategy enables investigation of the neurogenesis of human neural progenitors within the developing mouse cortex. Specifically, human cortical cells exhibit multiple features of radial migration. The robust transplantation method established here could be utilized both to uncover the missing gap between neurogenesis from ESCs and the tissue environment and as an in vivo model of normal and pathological human corticogenesis.

Neocortical neurogenesis is not really "neo": a new evolutionary model derived from a comparative study of chick pallial development.

The neocortex facilitates mammalian adaptive radiation by conferring highly sophisticated cognitive and motor abilities. A unique feature of the mammalian neocortex is its laminar structure in which similar neuronal subtypes are arranged in tangential layers and construct columnar circuits via interlaminar connections. The neocortical layer structure is completely conserved among all mammalian species, including monotremes and marsupials. However, this structure is missing in non-mammalian sister groups, such as birds and reptiles. The evolutionary origins of neocortical layers and cytoarchitectural borders have been the subject of debate over the past century. Using the chicken embryos as a model of evolutionary developmental biology (evo-devo model), we recently provided evidence suggesting that the evolutionary origin of layer-specific neuron subtypes predates the emergence of laminar structures. Based on this finding, we review the evolutionary conservation and divergence of neocortical development between mammals and non-mammals and discuss how the layered cytoarchitecture of the mammalian neocortex originated during evolution.

Evolutionary conservation of neocortical neurogenetic program in the mammals and birds.

The unique innovation of the layered neocortex in mammalian evolution is believed to facilitate adaptive radiation of mammalian species to various ecological environments by furnishing high information processing ability. There are no transitional states from the non-mammalian simple brain to the mammalian multilayered neocortex, and thus it is totally a mystery so far how this brain structure has been acquired during evolution. In our recent study, we found the evidence showing that the evolutionary origin of the neocortical neuron subtypes predates the actual emergence of layer structure. Our comparative developmental analysis of the chick pallium, homologous to the mammalian neocortex, revealed that mammals and avians fundamentally share the neocortical neuron subtypes and their production mechanisms, suggesting that their common ancestor already possessed a similar neuronal repertory. We further demonstrated that the neocortical layer-specific neuron subtypes are arranged as mediolaterally separated domains in the chick, but not as layers in the mammalian neocortex. These animal group-specific neuronal arrangements are accomplished by spatial modulation of the neurogenetic program, suggesting an evolutionary hypothesis that the regulatory changes in the neurogenetic program innovated the mammalian specific layered neocortex.

The temporal sequence of the mammalian neocortical neurogenetic program drives mediolateral pattern in the chick pallium.

The six-layered neocortex permits complex information processing in all mammalian species. Because its homologous region (the pallium) in nonmammalian amniotes has a different architecture, the ability of neocortical progenitors to generate an orderly sequence of distinct cell types was thought to have arisen in the mammalian lineage. This study, however, shows that layer-specific neuron subtypes do exist in the chick pallium. Deep- and upper-layer neurons are not layered but are segregated in distinct mediolateral domains in vivo. Surprisingly, cultured chick neural progenitors produce multiple layer-specific neuronal subtypes in the same chronological sequence as seen in mammals. These results suggest that the temporal sequence of the neocortical neurogenetic program was already inherent in the last common ancestor of mammals and birds and that mammals use this conserved program to generate a uniformly layered neocortex, whereas birds impose spatial constraints on the sequence to pattern the pallium.