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PathogenicACTBandACTG1gene variants, encoding the actin isoformsβCYA andγCYA, respectively, are frequently associated with theBaraitser-Winter-CerebroFrontoFacial syndrome (BWCFF-S) that includes malformations of cortical development. Here we explore whether cerebral organoids grown from BWCFF-S patient-derived induced pluripotent stem cells can provide insight into the pathogenesis underlying the cortical malformations of these patients. Cerebral organoids expressing either anACTBor anACTG1gene variant, each with a point mutation resulting in a single amino acid substitution, are reduced in size, showing smaller ventricle-like structures with a thinner ventricular zone (VZ). This decrease in VZ- progenitors is associated with a striking change in the orientation of their cleavage plane from predominantly vertical (control) to predominantly horizontal (BWCFF-S), which is incompatible with increasing VZ-progenitor abundance. The underlying cause appears to be an altered subcellular tubulin localization due to the actin mutations that affects mitotic spindle positioning of VZ-progenitors in BWCFF-S.

Since the ancestors of modern humans separated from those of Neandertals, around one hundred 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 the 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 the mouse, three substitutions in two of these proteins, KIF18a and KNL1, cause a prolongation of metaphase and a reduction in chromosome segregation errors in apical progenitors of the developing neocortex. Conversely, the ancestral substitutions cause a reduction in metaphase length and an increase in chromosome segregation errors in human brain organoids. Our data also show that, in these aspects, Neandertals were more similar to chimpanzees than to modern humans. Thus, the fidelity of chromosome segregation during neocortex development improved in modern humans after their divergence from Neandertals.

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.

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. DOI: http://dx.doi.org/10.7554/eLife.18683.001, eLife digest The human brain is about three times as big as the brain of our closest living relative, the chimpanzee. Moreover, a part of the brain called the cerebral cortex – which plays a key role in memory, attention, awareness and thought – contains twice as many cells in humans as the same region in chimpanzees. Networks of brain cells in the cerebral cortex also behave differently in the two species. How these species differences arise is not clear, but it likely occurs in the earliest phases of development when brain stem and progenitor cells divide and give rise to cerebral cortex cells in the growing brain. To study the earliest stages of brain development, researchers often use human brain cells grown in the laboratory. Under the right conditions, cells collected from adult humans and other animals can be reprogrammed to behave like brain stem cells. Recently, researchers have been able to use these reprogrammed cells to make tissue that resembles the brain in petri dishes, known as brain organoids. Mora-Bermúdez, Badsha, Kanton, Camp et al. have now analysed brain organoids grown from reprogrammed human, chimpanzee and orangutan cells. The experiments showed that the human and chimpanzee brain organoids were remarkably similar in many ways including in the mix of cell types and in how these cells were arranged. Mora-Bermúdez et al. then used live microscopy to show that progenitor cells that form the human cerebral cortex spend around 50% more time in a stage of the cell division process called metaphase compared to the same cells from chimpanzees or orangutans. Metaphase is the part of the division process when the cell makes sure that structures called chromosomes, which carry the cell’s DNA, can be separated and distributed equally between the two daughter cells. Mora-Bermúdez et al. also found that progenitor cells more likely to become neurons sooner had a shorter metaphase than progenitor cells more likely to remain proliferating as stem cells for longer. This suggests that a longer metaphase may be a feature of brain stem cells. Further studies are now needed to find out how the length of time these progenitor cells spend in metaphase affects how chimpanzee and human brains develop; and whether this can help explain why the human brain is so much larger. DOI: http://dx.doi.org/10.7554/eLife.18683.002

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.

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.

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.

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.

Quantitative characterization of protein interactions under physiological conditions is vital for systems biology. Fluorescence photobleaching/activation experiments of GFP-tagged proteins are frequently used for this purpose, but robust analysis methods to extract physicochemical parameters from such data are lacking. Here, we implemented a reaction-diffusion model to determine the contributions of protein interaction and diffusion on fluorescence redistribution. The model was validated and applied to five chromatin-interacting proteins probed by photoactivation in living cells. We found that very transient interactions are common for chromatin proteins. Their observed mobility was limited by the amount of free protein available for diffusion but not by the short residence time of the bound proteins. Individual proteins thus locally scan chromatin for binding sites, rather than diffusing globally before rebinding at random nuclear positions. By taking the real cellular geometry and the inhomogeneous distribution of binding sites into account, our model provides a general framework to analyze the mobility of fluorescently tagged factors. Furthermore, it defines the experimental limitations of fluorescence perturbation experiments and highlights the need for complementary methods to measure transient biochemical interactions in living cells.

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. DOI: http://dx.doi.org/10.7554/eLife.02875.001, eLife digest A stem cell can divide in two ways. Either it can split symmetrically into two identical daughter stem cells, or it can split asymmetrically into a stem cell and a specialist cell. The structure that forms inside the dividing cell to separate pairs of chromosomes—called the mitotic spindle—also partitions the molecules that determine what kind of cell each daughter cell will become. The mitotic spindle is made up of protein microtubules. Astral microtubules connect the spindle to a structure found at the inner face of the cell membrane called the cell cortex. This helps the spindle to orient itself correctly and control the plane of cell division. This is particularly important in cells that are different at their top and bottom, like polarized neural stem cells. To divide symmetrically, these cells need to split vertically from top to bottom. Then, to divide asymmetrically they tilt the cell division plane off-vertical. Classical studies on neuroblasts from the fruit fly Drosophila have shown that a big, 90° reorientation, from vertical to horizontal underlies this change. However, in the primary stem cells of the mammalian brain, subtle off-vertical tilting suffices for asymmetric divisions to occur. This tilting must be finely regulated: if not, neurodevelopmental disorders, such as microcephaly and lissencephaly, may arise. Mora-Bermúdez et al. investigated how mammalian cortical stem cells control such subtle spindle orientation changes by taking images of developing brain tissue from genetically modified mice. These show that not all astral microtubules affect whether the spindle reorients, as was previously thought. Instead, only those connecting the spindle to the cell cortex at the top and bottom of the cell—the apical/basal astrals—are involved. A decrease in the number of apical/basal astrals enables the spindle to undergo small reorientations. Mora-Bermúdez et al. therefore propose a model in which the spindle becomes less strongly anchored when the number of apical/basal astrals is reduced. This makes the spindle easier to tilt, allowing neural stem cells to undergo asymmetric divisions to produce neurons. The decrease in the number of apical/basal astrals appears to be caused by a reduction in the amount of a molecule that is known to help link the microtubules to the cell cortex. This reduction occurs only in the cortex at the top of the cell. Mora-Bermúdez et al. were also able to manipulate this process by adding very low doses of a microtubule inhibitor called nocodazole, which reduced the number of only the apical/basal astrals, increasing the ability of the spindle to reorient. DOI: http://dx.doi.org/10.7554/eLife.02875.002

The orientation of the mitotic spindle determines whether divisions of the polarized neural progenitors in the ventricular zone cause their expansion or lead to neurogenesis. A guanine nucleotide exchange factor for the small GTPase RhoA is now shown to tip this balance in favor of neurogenesis.

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 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.

The evaluation of fluorescence microscopy images acquired in high-throughput cell phenotype screens constitutes a substantial bottleneck and motivates the development of automated image analysis methods. Here we introduce a computational scheme to process 3D multi-cell time-lapse images as they are produced in large-scale RNAi experiments. We describe an approach to automatically segment, track, and classify cell nuclei into different mitotic phases. This enables automated analysis of the duration of single phases of the cell life cycle and thus the identification of cell cultures that show an abnormal mitotic behavior. Our scheme proves a high accuracy, suggesting a promising future for automating the evaluation of high-throughput experiments.

A bottleneck for high-throughput screening of live cells is the automated analysis of the generated image data. An important application in this context is the evaluation of the duration of cell cycle phases from confocal time-lapse microscopy image sequences, which typically involves a tracking step. The tracking step is an important part since it relates segmented cells from one time frame to the next. However, a main problem is that often the movement of single cells is superimposed with a global movement. The reason for the global movement lies in the high-throughput acquisition of the images and the repositioning of the microscope. If a tracking algorithm is applied to these images then only a superposition of the microscope movement and the cell movement is determined but not the real movement of the cells. In addition, since the displacements are generally larger, it is more difficult to determine the correspondences between cells. We have developed a phase-correlation based approach to compensate for the global movement of the microscope by registering each image of a sequence to a reference coordinate system. Our approach uses a windowing function in the spatial domain of the cross-power spectrum. This allows to determine the global movement by direct evaluation of the phase gradient, avoiding phase unwrapping. We present experimental results of applying our approach to synthetic and real image sequences. It turns out that the global movement can well be compensated and thus successfully decouples the global movement from the individual movement of the cells.

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.

In high-throughput cell phenotype screens large amounts of image data are acquired. The evaluation of these microscopy images constitutes a bottleneck and motivates the development of automated image analysis methods. Here we introduce a computational scheme to process 3D multi-cell image sequences as they are produced in large-scale RNAi experiments. We describe an approach to automatically segment, track, and classify cell nuclei into seven different mitotic phases. In particular, we present an algorithm based on a finite state machine to check the consistency of the resulting sequence of mitotic phases and to correct classification errors. Our approach enables automated determination of the duration of the single phases and thus the identification of cell cultures with delayed mitotic progression.

The evaluation of fluorescence microscopy images acquired in high-throughput cell phenotype screens constitutes a substantial bottleneck and motivates the development of automated image analysis methods. Here we introduce a computational scheme to process 3D multi-cell time-lapse images as they are produced in large-scale RNAi experiments. We describe an approach to automatically segment, track, and classify cell nuclei into different mitotic phases. This enables automated analysis of the duration of single phases of the cell life cycle and thus the identification of cell cultures that show an abnormal mitotic behavior. Our scheme proves a high accuracy, suggesting a promising future for automating the evaluation of high-throughput experiments.