Neocortical neurogenesis in development and evolution-Human-specific features.

In this review, we focus on human-specific features of neocortical neurogenesis in development and evolution. Two distinct topics will be addressed. In the first section, we discuss the expansion of the neocortex during human evolution and concentrate on the human-specific gene ARHGAP11B. We review the ability of ARHGAP11B to amplify basal progenitors and to expand a primate neocortex. We discuss the contribution of ARHGAP11B to neocortex expansion during human evolution and its potential implications for neurodevelopmental disorders and brain tumors. We then review the action of ARHGAP11B in mitochondria as a regulator of basal progenitor metabolism, and how it promotes glutaminolysis and basal progenitor proliferation. Finally, we discuss the increase in cognitive performance due to the ARHGAP11B-induced neocortical expansion. In the second section, we focus on neocortical development in modern humans versus Neanderthals. Specifically, we discuss two recent findings pointing to differences in neocortical neurogenesis between these two hominins that are due to a small number of amino acid substitutions in certain key proteins. One set of such proteins are the kinetochore-associated proteins KIF18a and KNL1, where three modern human-specific amino acid substitutions underlie the prolongation of metaphase during apical progenitor mitosis. This prolongation in turn is associated with an increased fidelity of chromosome segregation to the apical progenitor progeny during modern human neocortical development, with implications for the proper formation of radial units. Another such key protein is transketolase-like 1 (TKTL1), where a single modern human-specific amino acid substitution endows TKTL1 with the ability to amplify basal radial glia, resulting in an increase in upper-layer neuron generation. TKTL1's ability is based on its action in the pentose phosphate pathway, resulting in increased fatty acid synthesis. The data imply greater neurogenesis during neocortical development in modern humans than Neanderthals due to TKTL1, in particular in the developing frontal lobe.

Longer metaphase and fewer chromosome segregation errors in modern human than Neanderthal brain development.

Since the ancestors of modern humans separated from those of Neanderthals, around 100 amino acid substitutions spread to essentially all modern humans. The biological significance of these changes is largely unknown. Here, we examine all six such amino acid substitutions in three proteins known to have key roles in kinetochore function and chromosome segregation and to be highly expressed in the stem cells of the developing neocortex. When we introduce these modern human-specific substitutions in mice, three substitutions in two of these proteins, KIF18a and KNL1, cause metaphase prolongation and fewer chromosome segregation errors in apical progenitors of the developing neocortex. Conversely, the ancestral substitutions cause shorter metaphase length and more chromosome segregation errors in human brain organoids, similar to what we find in chimpanzee organoids. These results imply that the fidelity of chromosome segregation during neocortex development improved in modern humans after their divergence from Neanderthals.

What Are the Human-Specific Aspects of Neocortex Development?

When considering what makes us human, the development of the neocortex, the seat of our higher cognitive abilities, is of central importance. Throughout this complex developmental process, neocortical stem and progenitor cells (NSPCs) exert a priming role in determining neocortical tissue fate, through a series of cellular and molecular events. In this Perspective article, we address five questions of relevance for potentially human-specific aspects of NSPCs, (i) Are there human-specific NSPC subtypes? (ii) What is the functional significance of the known temporal differences in NSPC dynamics between human and other great apes? (iii) Are there functional interactions between the human-specific genes preferentially expressed in NSPCs? (iv) Do humans amplify certain metabolic pathways for NSPC proliferation? and finally (v) Have differences evolved during human evolution, notably between modern humans and Neandertals, that affect the performance of key genes operating in NSPCs? We discuss potential implications inherent to these questions, and suggest experimental approaches on how to answer them, hoping to provide incentives to further understand key issues of human cortical development.

From stem and progenitor cells to neurons in the developing neocortex: key differences among hominids.

Comparing the biology of humans to that of other primates, and notably other hominids, is a useful path to learn more about what makes us human. Some of the most interesting differences among hominids are closely related to brain development and function, for example behaviour and cognition. This makes it particularly interesting to compare the hominid neural cells of the neocortex, a part of the brain that plays central roles in those processes. However, well-preserved tissue from great apes is usually extremely difficult to obtain. A variety of new alternative tools, for example brain organoids, are now beginning to make it possible to search for such differences and analyse their potential biological and biomedical meaning. Here, we present an overview of recent findings from comparisons of the neural stem and progenitor cells (NSPCs) and neurons of hominids. In addition to differences in proliferation and differentiation of NSPCs, and maturation of neurons, we highlight that the regulation of the timing of these processes is emerging as a general foundational difference in the development of the neocortex of hominids.

Brain organoids as models to study human neocortex development and evolution.

Since their recent development, organoids that emulate human brain tissue have allowed in vitro neural development studies to go beyond the limits of monolayer culture systems, such as neural rosettes. We present here a review of organoid studies that focuses on cortical wall development, starting with a technical comparison between pre-patterning and self-patterning brain organoid protocols. We then follow neocortex development in space and time and list those aspects where organoids have succeeded in emulating in vivo development, as well as those aspects that continue to be pending tasks. Finally, we present a summary of medical and evolutionary insight made possible by organoid technology.

Differences and similarities between human and chimpanzee neural progenitors during cerebral cortex development.

Human neocortex expansion likely contributed to the remarkable cognitive abilities of humans. This expansion is thought to primarily reflect differences in proliferation versus differentiation of neural progenitors during cortical development. Here, we have searched for such differences by analysing cerebral organoids from human and chimpanzees using immunohistofluorescence, live imaging, and single-cell transcriptomics. We find that the cytoarchitecture, cell type composition, and neurogenic gene expression programs of humans and chimpanzees are remarkably similar. Notably, however, live imaging of apical progenitor mitosis uncovered a lengthening of prometaphase-metaphase in humans compared to chimpanzees that is specific to proliferating progenitors and not observed in non-neural cells. Consistent with this, the small set of genes more highly expressed in human apical progenitors points to increased proliferative capacity, and the proportion of neurogenic basal progenitors is lower in humans. These subtle differences in cortical progenitors between humans and chimpanzees may have consequences for human neocortex evolution.

Non-canonical features of the Golgi apparatus in bipolar epithelial neural stem cells.

Apical radial glia (aRG), the stem cells in developing neocortex, are unique bipolar epithelial cells, extending an apical process to the ventricle and a basal process to the basal lamina. Here, we report novel features of the Golgi apparatus, a central organelle for cell polarity, in mouse aRGs. The Golgi was confined to the apical process but not associated with apical centrosome(s). In contrast, in aRG-derived, delaminating basal progenitors that lose apical polarity, the Golgi became pericentrosomal. The aRG Golgi underwent evolutionarily conserved, accordion-like compression and extension concomitant with cell cycle-dependent nuclear migration. Importantly, in line with endoplasmic reticulum but not Golgi being present in the aRG basal process, its plasma membrane contained glycans lacking Golgi processing, consistent with direct ER-to-cell surface membrane traffic. Our study reveals hitherto unknown complexity of neural stem cell polarity, differential Golgi contribution to their specific architecture, and fundamental Golgi re-organization upon cell fate change.

Novel insights into mammalian embryonic neural stem cell division: focus on microtubules.

During stem cell divisions, mitotic microtubules do more than just segregate the chromosomes. They also determine whether a cell divides virtually symmetrically or asymmetrically by establishing spindle orientation and the plane of cell division. This can be decisive for the fate of the stem cell progeny. Spindle defects have been linked to neurodevelopmental disorders, yet the role of spindle orientation for mammalian neurogenesis has remained controversial. Here we explore recent advances in understanding how the microtubule cytoskeleton influences mammalian neural stem cell division. Our focus is primarily on the role of spindle microtubules in the development of the cerebral cortex. We also highlight unique characteristics in the architecture and dynamics of cortical stem cells that are tightly linked to their mode of division. These features contribute to setting these cells apart as mitotic "rule breakers," control how asymmetric a division is, and, we argue, are sufficient to determine the fate of the neural stem cell progeny in mammals.

Sustained Pax6 Expression Generates Primate-like Basal Radial Glia in Developing Mouse Neocortex.

The evolutionary expansion of the neocortex in mammals has been linked to enlargement of the subventricular zone (SVZ) and increased proliferative capacity of basal progenitors (BPs), notably basal radial glia (bRG). The transcription factor Pax6 is known to be highly expressed in primate, but not mouse, BPs. Here, we demonstrate that sustaining Pax6 expression selectively in BP-genic apical radial glia (aRG) and their BP progeny of embryonic mouse neocortex suffices to induce primate-like progenitor behaviour. Specifically, we conditionally expressed Pax6 by in utero electroporation using a novel, Tis21-CreERT2 mouse line. This expression altered aRG cleavage plane orientation to promote bRG generation, increased cell-cycle re-entry of BPs, and ultimately increased upper-layer neuron production. Upper-layer neuron production was also increased in double-transgenic mouse embryos with sustained Pax6 expression in the neurogenic lineage. Strikingly, increased BPs existed not only in the SVZ but also in the intermediate zone of the neocortex of these double-transgenic mouse embryos. In mutant mouse embryos lacking functional Pax6, the proportion of bRG among BPs was reduced. Our data identify specific Pax6 effects in BPs and imply that sustaining this Pax6 function in BPs could be a key aspect of SVZ enlargement and, consequently, the evolutionary expansion of the neocortex.

Specific polar subpopulations of astral microtubules control spindle orientation and symmetric neural stem cell division.

Mitotic spindle orientation is crucial for symmetric vs asymmetric cell division and depends on astral microtubules. Here, we show that distinct subpopulations of astral microtubules exist, which have differential functions in regulating spindle orientation and division symmetry. Specifically, in polarized stem cells of developing mouse neocortex, astral microtubules reaching the apical and basal cell cortex, but not those reaching the central cell cortex, are more abundant in symmetrically than asymmetrically dividing cells and reduce spindle orientation variability. This promotes symmetric divisions by maintaining an apico-basal cleavage plane. The greater abundance of apical/basal astrals depends on a higher concentration, at the basal cell cortex, of LGN, a known spindle-cell cortex linker. Furthermore, newly developed specific microtubule perturbations that selectively decrease apical/basal astrals recapitulate the symmetric-to-asymmetric division switch and suffice to increase neurogenesis in vivo. Thus, our study identifies a novel link between cell polarity, astral microtubules, and spindle orientation in morphogenesis.

A genomic toolkit to investigate kinesin and myosin motor function in cells.

Coordination of multiple kinesin and myosin motors is required for intracellular transport, cell motility and mitosis. However, comprehensive resources that allow systems analysis of the localization and interplay between motors in living cells do not exist. Here, we generated a library of 243 amino- and carboxy-terminally tagged mouse and human bacterial artificial chromosome transgenes to establish 227 stably transfected HeLa cell lines, 15 mouse embryonic stem cell lines and 1 transgenic mouse line. The cells were characterized by expression and localization analyses and further investigated by affinity-purification mass spectrometry, identifying 191 candidate protein-protein interactions. We illustrate the power of this resource in two ways. First, by characterizing a network of interactions that targets CEP170 to centrosomes, and second, by showing that kinesin light-chain heterodimers bind conventional kinesin in cells. Our work provides a set of validated resources and candidate molecular pathways to investigate motor protein function across cell lineages.

The protein phosphatase 1 regulator PNUTS is a new component of the DNA damage response.

The function of protein phosphatase 1 nuclear-targeting subunit (PNUTS)--one of the most abundant nuclear-targeting subunits of protein phosphatase 1 (PP1c)--remains largely uncharacterized. We show that PNUTS depletion by small interfering RNA activates a G2 checkpoint in unperturbed cells and prolongs G2 checkpoint and Chk1 activation after ionizing-radiation-induced DNA damage. Overexpression of PNUTS-enhanced green fluorescent protein (EGFP)--which is rapidly and transiently recruited at DNA damage sites--inhibits G2 arrest. Finally, γH2AX, p53-binding protein 1, replication protein A and Rad51 foci are present for a prolonged period and clonogenic survival is decreased in PNUTS-depleted cells after ionizing radiation treatment. We identify the PP1c regulatory subunit PNUTS as a new and integral component of the DNA damage response involved in DNA repair.

SETDB1 is involved in postembryonic DNA methylation and gene silencing in Drosophila.

DNA methylation is fundamental for the stability and activity of genomes. Drosophila melanogaster and vertebrates establish a global DNA methylation pattern of their genome during early embryogenesis. Large-scale analyses of DNA methylation patterns have uncovered revealed that DNA methylation patterns are dynamic rather than static and change in a gene-specific fashion during development and in diseased cells. However, the factors and mechanisms involved in dynamic, postembryonic DNA methylation remain unclear. Methylation of lysine 9 in histone H3 (H3-K9) by members of the Su(var)3-9 family of histone methyltransferases (HMTs) triggers embryonic DNA methylation in Arthropods and Chordates. Here, we demonstrate that Drosophila SETDB1 (dSETDB1) can mediate DNA methylation and silencing of genes and retrotransposons. We found that dSETDB1 tri-methylates H3-K9 and binds methylated CpA motifs. Tri-methylation of H3-K9 by dSETDB1 mediates recruitment of DNA methyltransferase 2 (Dnmt2) and Su(var)205, the Drosophila ortholog of mammalian "Heterochromatin Protein 1", to target genes for dSETDB1. By enlisting Dnmt2 and Su(var)205, dSETDB1 triggers DNA methylation and silencing of genes and retrotransposons in Drosophila cells. DSETDB1 is involved in postembryonic DNA methylation and silencing of Rt1b{} retrotransposons and the tumor suppressor gene retinoblastoma family protein 1 (Rb) in imaginal discs. Collectively, our findings implicate dSETDB1 in postembryonic DNA methylation, provide a model for silencing of the tumor suppressor Rb, and uncover a role for cell type-specific DNA methylation in Drosophila development.

Automatic analysis of dividing cells in live cell movies to detect mitotic delays and correlate phenotypes in time.

Live-cell imaging allows detailed dynamic cellular phenotyping for cell biology and, in combination with small molecule or drug libraries, for high-content screening. Fully automated analysis of live cell movies has been hampered by the lack of computational approaches that allow tracking and recognition of individual cell fates over time in a precise manner. Here, we present a fully automated approach to analyze time-lapse movies of dividing cells. Our method dynamically categorizes cells into seven phases of the cell cycle and five aberrant morphological phenotypes over time. It reliably tracks cells and their progeny and can thus measure the length of mitotic phases and detect cause and effect if mitosis goes awry. We applied our computational scheme to annotate mitotic phenotypes induced by RNAi gene knockdown of CKAP5 (also known as ch-TOG) or by treatment with the drug nocodazole. Our approach can be readily applied to comparable assays aiming at uncovering the dynamic cause of cell division phenotypes.

Cytokinesis of neuroepithelial cells can divide their basal process before anaphase.

Neuroepithelial (NE) cells, the primary stem and progenitor cells of the vertebrate central nervous system, are highly polarized and elongated. They retain a basal process extending to the basal lamina, while undergoing mitosis at the apical side of the ventricular zone. By studying NE cells in the embryonic mouse, chick and zebrafish central nervous system using confocal microscopy, electron microscopy and time-lapse imaging, we show here that the basal process of these cells can split during M phase. Splitting occurred in the basal-to-apical direction and was followed by inheritance of the processes by either one or both daughter cells. A cluster of anillin, an essential component of the cytokinesis machinery, appeared at the distal end of the basal process in prophase and was found to colocalize with F-actin at bifurcation sites, in both proliferative and neurogenic NE cells. GFP-anillin in the basal process moved apically to the cell body prior to anaphase onset, followed by basal-to-apical ingression of the cleavage furrow in telophase. The splitting of the basal process of M-phase NE cells has implications for cleavage plane orientation and the relationship between mitosis and cytokinesis.

Maximal chromosome compaction occurs by axial shortening in anaphase and depends on Aurora kinase.

Eukaryotic cells must first compact their chromosomes before faithfully segregating them during cell division. Failure to do so can lead to segregation defects with pathological consequences, such as aneuploidy and cancer. Duplicated interphase chromosomes are, therefore, reorganized into tight rods before being separated and directed to the newly forming daughter cells. This vital reorganization of chromatin remains poorly understood. To address the dynamics of mitotic condensation of single chromosomes in intact cells, we developed quantitative assays based on confocal time-lapse microscopy of live mammalian cells stably expressing fluorescently tagged core histones. Surprisingly, maximal compaction was not reached in metaphase, but in late anaphase, after sister chromatid segregation. We show that anaphase compaction proceeds by a mechanism of axial shortening of the chromatid arms from telomere to centromere. Chromatid axial shortening was not affected in condensin-depleted cells, but depended instead on dynamic microtubules and Aurora kinase. Acute perturbation of this compaction resulted in failure to rescue segregation defects and in multilobed daughter nuclei, suggesting functions in chromosome segregation and nuclear architecture.

Measuring structural dynamics of chromosomes in living cells by fluorescence microscopy.

Mitotic and meiotic chromosomes are the compact packages that faithfully transport the genetic and epigenetic information to the following cell generations. How chromatin dynamically cycles between the decompacted interphase state that supports transcription and replication and the compacted state required for chromosome segregation is not understood. To address this long-standing problem, the structure of chromatin should ideally be studied in the physiological context of intact cells and organisms. We discuss here, the contributions that live-cell imaging can and has made to the study of mitotic chromosome compaction and highlight the power and limitations of this approach. We review methodologies used and suggest that combinatorial approaches and developing new imaging technologies will be key to shedding light on this long-standing question in cell biology.