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Unidirectional translocation of microbeads from the apical surface toward the basal region of the VZ. (A) Schematic of the method used to incorporate fluorescent microbeads (diameters are ∼2 μm) into embryonic mouse brain tissue using magnetic activity, followed by tracking of their movement in slice cultures using time-lapse microscopy. (B) Images of fluorescent beads in slice cultures (a, b; red) incorporated into E13.5 mouse brain tissue. The white dashed line indicates the apical surface. Beads were aligned at the apical surface at the starting time point (a). Several beads had detached from their original position after 24 h of incubation (b). Bar=50 μm. (C) Tracking of microbeads in brain slice cultures. Positions of fluorescent beads relative to the apical surface (y-coordinate) were measured at each time point (x-coordinate) from the time-lapse images. Movements of nine microbeads acquired from three independent brain slices are displayed (slice numbers (1–3) and beads (A–D) are indicated in the graph). (D) Immunostaining of mouse brain sections from cultured tissue slices after microbead incorporation (a, b; red). Antibody for proliferating cell nuclear antigen (PCNA) stains proliferative cells (a′; green), whereas Tuj1 stains post-mitotic neurons (a, a′; magenta). Note that fluorescent microbeads were observed only in the proliferative zone, the VZ (white arrowheads). Yellow dashed lines indicate the apical surface. Bar=10 μm. (E) Comparison of nuclear and microbead tracks in brain tissue slices. Plane positions of nuclei marked with NLS-GFP (a) or incorporated microbeads (b) were measured along the apical surface (x-coordinate) and apical–basal axis (y-coordinate). Slice numbers (1–3), nuclei and beads (A–D) are indicated in each graph.
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A hallmark of neurogenesis in the vertebrate brain is the apical-basal nuclear oscillation in polarized neural progenitor cells. Known as interkinetic nuclear migration (INM), these movements are synchronized with the cell cycle such that nuclei move basally during G1-phase and apically during G2-phase. However, it is unknown how the direction of m...
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... confirmed that TPX2-RNAi did not result in an increase of apoptotic cells with the 18-h time course of the experiment (Supplementary Figure S4C), and that the perturbed basal-to-apical nuclear migration by TPX2-RNAi was recovered by introduction of the human TPX2 gene (Supplementary Figure S4D). Similar perturbed nuclear movement was also observed using RNAi with a different target sequence (Supplementary Figure S5A), indicating that the perturbation in nuclear migration by TPX2 knockdown was not due to an off-target effect of RNAi. More directly, we analysed the dynamics of basal-to- apical nuclear migration in brain slices treated with TPX2 RNAi and observed (1) a decrease in velocity of migration and (2) a non-linear, stepwise movement during migration ( Figure 3G; Supplementary Movies S4 and S5). ...
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... TPX2 is knocked down, the average velocity of nuclear movement from basal-to-apical region (50-15 mm from the apical surface, respectively) showed a statistically significant de- crease ( Figure 3G, from 1.0 ± 0.11 (control) to 0.5 ± 0.07 (TPX2 RNAi) mm/min, P-value o0.001, t-test). In addition, the number of subapical mitotic cells was increased in the TPX2 RNAi-treated brain, suggesting that the nuclei had entered M-phase before reaching the apical surface (Supplementary Figure S5B). This can be attributed to slower nuclear movement in the apical direction. ...
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... next sought to elucidate how Tpx2 affects G2-phase nuclear migration. TPX2 RNAi might affect epithelial polarity or centrosomal activity; however, we did not find any aber- rant pattern using markers of epithelial characteristics such as ZO-1, Par-3 and b-catenin or using a marker for TACC3, a centrosomal protein involved in INM ( Xie et al, 2007), in the TPX2 knocked-down tissue (Supplementary Figure S5C-E). We then tested the possibility that microtubule organization in the apical processes of neural progenitor cells is regulated by Tpx2 in a cell cycle-dependent manner. ...
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... test whether the translocation system in the VZ is cell autono- mous or dependent on the activity of surrounding cells, we observed the behaviour of fluorescent microbeads in cultures of brain slices. Magnetic fluorescent beads were incorporated into the VZ from its apical surface by applying a magnet on the pial side, followed by brain slice culture and time-lapse imaging ( Figure 5A). Incorporated fluorescent beads were initially aligned on the apical surface. ...
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... fluorescent beads were initially aligned on the apical surface. Some beads were translocated to the basal region over the course of brain slice culture ( Figure 5B). HVEM imaging confirmed that this treatment did not impair normal cell-cell contact or disrupt normal tissue architecture (Supplementary Figure S6). ...
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... imaging confirmed that this treatment did not impair normal cell-cell contact or disrupt normal tissue architecture (Supplementary Figure S6). Once introduced, the beads tended to stay at the apical surface for a short time before beginning basally directed translocation ( Figure 5C, see Supplementary Movie S7). During the translocation, beads showed 'ratcheting,' back- and-forth movement as observed in the migration of G1-phase nuclei ( Figure 1B). ...
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... the translocation, beads showed 'ratcheting,' back- and-forth movement as observed in the migration of G1-phase nuclei ( Figure 1B). Upon reaching the basal region of the VZ, the fluorescent beads terminated their transloca- tion and never entered the neuronal layer ( Figure 5D). In addition and importantly, once the beads reached the basal end of the VZ, they remained stationary and did not show the return, basal-to-apical movement ( Figure 5C). ...
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... reaching the basal region of the VZ, the fluorescent beads terminated their transloca- tion and never entered the neuronal layer ( Figure 5D). In addition and importantly, once the beads reached the basal end of the VZ, they remained stationary and did not show the return, basal-to-apical movement ( Figure 5C). We then examined whether the basal translocation of beads was due to their association with nuclei migrating in the apical-to- basal direction. ...
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... then examined whether the basal translocation of beads was due to their association with nuclei migrating in the apical-to- basal direction. A comparison of the trajectories of NLS-GFP- labelled nuclei and fluorescent beads in the same brain slices (Supplementary Movie S8) clearly indicates that nuclei underwent radial migration within the elongated cell shape, whereas beads took more variable orientations ( Figure 5E). This result implies that beads move not by adhesion to migrating nuclei. ...
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... acute delay in apical-to-basal migration is most likely due to the lack of a decrease in basal nuclear density, and of an increase in apical nuclear density, both of which are simultaneously caused by individual nuclei migrating api- cally in the normal situation. To confirm that the perturbation of basally directed nuclear movement is not due to unex- pected effects of HU on G1-phase cells, but instead due to the physical displacement effect, we tested whether microbead translocation ( Figure 5) is also perturbed by the same drug treatment. Indeed, most fluorescent beads incorporated from the apical surface translocated shorter distances after treat- ment with 1 mM HU than in the control ( Figure 6D). ...
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... Each nucleus searches the area of the SR and then goes to the centre of gravity of an area within the SR that is unoccupied by nuclei. (3) The cell- cycle phase of each nucleus has a randomly provided initial value (colour codes are indicated in Figure 7Ab); the lengths of the cell-cycle phases were taken from values that have been experimentally measured in the developing mouse brain (Takahashi et al, 1995). If no commands are provided to After in utero electroporation of plasmids of p18 Ink4c and NLS-GFP into the E13.5 mouse brain, two thymidine analogues (CldU and IdU) were introduced into pregnant mice at different times (5 and 2 h, respectively) before fixing the embryos. ...
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... further evaluate the validity of the computational model, we tested this prediction in brain tissue. The cell cycle of neural progenitors, particularly the duration of the G1-phase, becomes progres- sively longer as development progresses (Takahashi et al, 1995). Thus, the effect of cell-cycle length can be tested in vivo by comparing two different developmental stages. ...
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... this cascade, the absence of Tpx2 might result in the aberrant localization of TACC ( Barr and Gergely, 2007). Although centrosomal proteins are known to be involved in INM ( Xie et al, 2007), we observed that the loss of Tpx2 function did not affect the localization of TACC3 protein at the centrosome during mitosis (Supplementary Figure S5E), suggesting that Tpx2 can act independently of centrosomal proteins. We instead found that microtubule distribution in the apical process becomes narrower in width in the G2-phase compared with the G1-phase and that this change depends on Tpx2 function. ...
Citations
... The issue that has generated the most controversy when establishing a common model that would explain the functioning of the INM in the different epithelia has undoubtedly been the origin of the forces that move the nuclei in each part of the INM (Bertipaglia et al., 2018;Strzyz et al., 2016). Although not all published work may entirely agree, it is widely accepted that the main force driving INM in shorter epithelia (zebrafish retina and hindbrain) is generated by actomyosin contraction acting during BA INM (Leung et al., 2011;Norden et al., 2009;Yanakieva et al., 2019), with the AB INM a passive stochastic movement resulting from the pressure generated by the cells doing the BA INM (Kosodo et al., 2011;Leung et al., 2011;Norden et al., 2009). In contrast, in longer epithelia like the embryonary rodent cortex, INM seems to depend on tubulin-bound dynein also acting during BA INM (Hu et al., 2013;Schenk et al., 2009;Tsai et al., 2010). ...
... Therefore, each phase takes place at specific apicobasal positions of the nucleus. It is now clear that INM requires cell cycle progression (Kosodo et al., 2011;Leung et al., 2011;Strzyz et al., 2015;Ueno et al., 2006); however, controversy remains on whether INM controls the cell cycle. So, we wondered whether cell cycle progression was affected or not by vinculin depletion. ...
... During INM, nuclei of proliferating NSCs occupy different positions along the apicobasal axis throughout different cell cycle phases. In G1/S phases, nuclei migrate from apical to basal in a passive and stochastic way (Kosodo et al., 2011;Leung et al., 2011;Norden et al., 2009). In contrast, during G2/M phases, nuclei migrate from basal to apical by a directed active process; however, the entity of the cytoskeleton supporting this apically directed movement prior to mitosis remains controversial. ...
Vinculin is an actin-binding protein (ABP) that strengthens the connection between the actin cytoskeleton and adhesion complexes. It binds to β-catenin/N-cadherin complexes in apical adherens junctions (AJs), which maintain cell-to-cell adhesions, and to talin/integrins in the focal adhesions (FAs) that attach cells to the basal membrane. Here, we demonstrate that β-catenin targets vinculin to the apical AJs and the centrosome in the embryonic neural tube (NT). Suppression of vinculin slows down the basal-to-apical part of interkinetic nuclear migration (BAINM), arrests neural stem cells (NSCs) in the G2 phase of the cell cycle, and ultimately dismantles the apical actin cytoskeleton. In the NSCs, mitosis initiates when an internalized centrosome gathers with the nucleus during BAINM. Notably, our results show that the first centrosome to be internalized is the daughter centrosome, where β-catenin and vinculin accumulate, and that vinculin suppression prevents centrosome internalization. Thus, we propose that vinculin links AJs, the centrosome, and the actin cytoskeleton where actomyosin contraction forces are required.
... During proliferative stages, neuroepithelial progenitor cells are known to actively make the space needed at the apical mitotic zone by displacing other non-mitotic progenitor nuclei 43,44 . However, we did not see many cells displacing the arrested emerging PRs or squeezing through them; this is probably because the columnar morphology of emerging PRs means that they form a less penetrable barrier as compared with progenitor cells. ...
The concomitant occurrence of tissue growth and organization is a hallmark of organismal development1–3. This often means that proliferating and differentiating cells are found at the same time in a continuously changing tissue environment. How cells adapt to architectural changes to prevent spatial interference remains unclear. Here, to understand how cell movements that are key for growth and organization are orchestrated, we study the emergence of photoreceptor neurons that occur during the peak of retinal growth, using zebrafish, human tissue and human organoids. Quantitative imaging reveals that successful retinal morphogenesis depends on the active bidirectional translocation of photoreceptors, leading to a transient transfer of the entire cell population away from the apical proliferative zone. This pattern of migration is driven by cytoskeletal machineries that differ depending on the direction: microtubules are exclusively required for basal translocation, whereas actomyosin is involved in apical movement. Blocking the basal translocation of photoreceptors induces apical congestion, which hampers the apical divisions of progenitor cells and leads to secondary defects in lamination. Thus, photoreceptor migration is crucial to prevent competition for space, and to allow concurrent tissue growth and lamination. This shows that neuronal migration, in addition to its canonical role in cell positioning⁴, can be involved in coordinating morphogenesis.
... Rosettes begin as tubular epithelial structures with a single layer of NPCs. NPCs in rosettes display apicobasal polarity, with the apical tight junction marker zonula occludens-1 (ZO1) expressed at the apical layer (33)(34)(35). Rosettes have a lumen that is demarcated by the interlinked NPCs in the apical layer (Fig. 1). ...
... Given the increased area of the lumen we observed, we wondered whether the integrity of the apical membrane was somehow compromised within the HCMV-infected rosettes. We stained for ZO1, a tight junction marker that has been used to demarcate the apical membrane of both mammalian neural tubes and rosettes (33)(34)(35). Earlier reports had observed loss of integrity and continuity of ZO1 staining after HCMV infection in two types of polarized cells, retinal pigment and intestinal epithelial cells, indicating impairment of the tight junctions of the apical membrane (40,41). ...
... In vitro, NID1 has been shown to act as a linker protein, connecting laminin to collagen IV (42)(43)(44)(45). Laminin is a major extracellular matrix protein found in the basement membranes in most vertebrate species, particularly in the pial membrane of the CNS (33)(34)(35). After we observed the downregulation of NID1 in HCMV-infected rosettes, we investigated the expression of laminin by IF analysis of the B1 subunit in these structures as well (as described in Materials and Methods). ...
Human cytomegalovirus (HCMV) is a leading cause of birth defects in humans. These birth defects include microcephaly, sensorineural hearing loss, vision loss, and cognitive impairment. The process by which the developing fetus incurs these neurological defects is poorly understood. To elucidate some of these mechanisms, we have utilized HCMV-infected induced pluripotent stem cells (iPSCs) to generate in vitro brain organoids, modeling the first trimester of fetal brain development. Early during culturing, brain organoids generate neural rosettes. These structures are believed to model neural tube formation. Rosette formation was analyzed in HCMV-infected and mock-infected brain organoids at 17, 24, and 31 days postinfection. Histological analysis revealed fewer neural rosettes in HCMV-infected compared to mock-infected organoids. HCMV-infected organoid rosettes incurred multiple structural deficits, including increased lumen area, decreased ventricular zone depth, and decreased cell count. Immunofluorescent (IF) analysis found that nidogen-1 (NID1) protein expression in the basement membrane surrounding neural rosettes was greatly reduced by virus infection. IF analysis also identified a similar downregulation of laminin in basement membranes of HCMV-infected organoid rosettes. Knockdown of NID1 alone in brain organoids impaired their development, leading to the production of rosettes with increased lumen area, decreased structural integrity, and reduced laminin localization in the basement membrane, paralleling observations in HCMV-infected organoids. Our data strongly suggest that HCMV-induced downregulation of NID1 impairs neural rosette formation and integrity, likely contributing to many of HCMV's most severe birth defects. IMPORTANCE HCMV infection in pregnant women continues to be the leading cause of virus-induced neurologic birth defects. The mechanism through which congenital HCMV (cCMV) infection induces pathological changes to the developing fetal central nervous system (CNS) remains unclear. Our lab previously reproduced identified clinical defects in HCMV-infected infants using a three dimensional (3D) brain organoid model. In this new study, we have striven to discover very early HCMV-induced changes in developing brain organoids. We investigated the development of neural tube-like structures, neural rosettes. HCMV-infected rosettes displayed multiple structural abnormalities and cell loss. HCMV-infected rosettes displayed reduced expression of the key basement membrane protein, NID1. We previously found NID1 to be specifically targeted in HCMV-infected fibroblasts and endothelial cells. Brain organoids generated from NID1 knockdown iPSCs recapitulated the structural defects observed in HCMV-infected rosettes. Findings in this study revealed HCMV infection induced early and dramatic structural changes in 3D brain organoids. We believe our results suggest a major role for infection-induced NID1 downregulation in HCMV-induced CNS birth defects.
... As the complex, dynamic 3D organisation of cells in growing epithelia is governed by simple physical concepts, computer simulations present powerful tools to understand the emergent properties of epithelia [66], including IKNM and its effects [67][68][69][70][71]. Cellular Potts models, which represent a generalisation of the Ising model to cells, have long been used to simulate complex 3D cell shapes [72][73][74]. ...
Pseudostratified epithelia have smooth apical and basal surfaces, yet along the apical-basal axis, cells assume highly irregular shapes, which we introduce as punakoids. They interact dynamically with many more cells than visible at the surface. Here, we review a recently developed new perspective on epithelial cell organisation. Seemingly random at first sight, the cell packing configurations along the entire apical-basal axis follow fundamental geometrical relationships, which minimise the lateral cell-cell contact energy for a given cross-sectional cell area variability. The complex 3D cell neighbour relationships in pseudostratified epithelia thus emerge from a simple physical principle. This paves the way for the development of data-driven 3D simulation frameworks that will be invaluable in the simulation of epithelial dynamics in development and disease.
... While kinesins 5 and 12 have not been explored in INM, a common regulator of them called TPX2 has been shown to be important for maintaining the polarity of neural progenitors and promoting apical nuclear migration during INM, possibly by regulating microtubule sliding (Kosodo et al., 2011). We previously showed that, when microtubule sliding is pharmacologically inhibited throughout the migratory neuron, the leading process becomes shorter, microtubules buckle, and the neuron deviates from its normal path (Rao et al., 2016). ...
During neuronal migration, forces generated by cytoplasmic dynein yank on microtubules extending from the centrosome into the leading process and move the nucleus along microtubules that extend behind the centrosome. Scaffolds, such as radial glia, guide neuronal migration outward from the ventricles, but little is known about the internal machinery that ensures that the soma migrates along its proper path rather than moving backward or off the path. Here we report that depletion of KIFC1, a minus-end-directed kinesin called HSET in humans, causes neurons to migrate off their appropriate path, suggesting that this molecular motor is what ensures fidelity of the trajectory of migration. For these studies, we used rat migratory neurons in vitro and developing mouse brain in vivo , together with RNA interference and ectopic expression of mutant forms of KIFC1. We found that crosslinking of microtubules into a nonsliding mode by KIFC1 is necessary for dynein-driven forces to achieve sufficient traction to thrust the soma forward. Asymmetric bouts of microtubule sliding driven by KIFC1 thereby enable the soma to tilt in one direction or another, thus providing midcourse corrections that keep the neuron on its appropriate trajectory. KIFC1-driven sliding of microtubules further assists neurons in remaining on their appropriate path by allowing the nucleus to rotate directionally as it moves, which is consistent with how we found that KIFC1 contributes to interkinetic nuclear migration at an earlier stage of neuronal development.
SIGNIFICANCE STATEMENT Resolving the mechanisms of neuronal migration is medically important because many developmental disorders of the brain involve flaws in neuronal migration and because deployment of newly born neurons may be important in the adult for cognition and memory. Drugs that inhibit KIFC1 are candidates for chemotherapy and therefore should be used with caution if they are allowed to enter the brain.
... RGCs expand their number and exhibit a much higher number of asymmetrical cell divisions as compared with NE cells (Subramanian et al., 2019). During cell expansion, RGC nuclei show a characteristic interkinetic nuclear migration (INM) synchronized with the cell cycle phases during proliferation (Kosodo et al., 2011). The RGC nuclei migrate toward the basal side of the developing cortex during G1 phase and remain there during S phase before they migrate apically during G2 phase and proceed with M-phase once they reach the ventricular surface (Kosodo et al., 2011;Miyata et al., 2014). ...
... During cell expansion, RGC nuclei show a characteristic interkinetic nuclear migration (INM) synchronized with the cell cycle phases during proliferation (Kosodo et al., 2011). The RGC nuclei migrate toward the basal side of the developing cortex during G1 phase and remain there during S phase before they migrate apically during G2 phase and proceed with M-phase once they reach the ventricular surface (Kosodo et al., 2011;Miyata et al., 2014). This pattern of migration during early neurogenesis requires functional microtubules and actin filaments (Götz and Huttner, 2005). ...
Microcephaly or reduced head circumference results from a multitude of abnormal developmental processes affecting brain growth and/or leading to brain atrophy. Autosomal recessive primary microcephaly (MCPH) is the prototype of isolated primary (congenital) microcephaly, affecting predominantly the cerebral cortex. For MCPH, an accelerating number of mutated genes emerge annually, and they are involved in crucial steps of neurogenesis. In this review article, we provide a deeper look into the microcephalic MCPH brain. We explore cytoarchitecture focusing on the cerebral cortex and discuss diverse processes occurring at the level of neural progenitors, early generated and mature neurons, and glial cells. We aim to thereby give an overview of current knowledge in MCPH phenotype and normal brain growth.
... Taken together, the data suggest that the orientation of cell division contributes to the apexbase localized expansion of the luminal side of the MEL. Luminal-basal nuclear movement in the flat region exhibits interkinetic nuclear migration (IKNM) behaviours, which have been observed in various pseudostratified epithelial tissues [33][34][35][36][37][38]. During this process, nuclei at the basal side of the cell move closer to the luminal surface prior to mitosis, and the daughter nuclei move back to the basal side, which results in even growth within the epithelial layer (figure 3hi). ...
... IKNM in pseudostratified tissues is dictated by the cytoskeletal machineries, including actomyosin and microtubules, during cell-cycle progression [35,36,38]. To examine the role of the cell cycle in IKNM, we perturbed the cell cycle using DNA synthesis inhibitor mitomycin C (MMC) and examined the morphological response of the MEL. ...
... Previous evaluations have shown that TAG-1 (transient axonal glycoprotein-1) knockdown impedes basalward nuclear movement during IKNM in the ventricular zone of murine embryonic brains, leading to overcrowding of the neural progenitor cells on the luminal side of the structures and severe cortical dysplasia [42]. It has also been shown that cytoskeletal machinery, including actomyosin and microtubules, regulates basalward nuclear movement [35,36,38]. These observations provide the basis for the identification of the molecules responsible for nuclear behaviour in the MEL, which need to be clarified in future studies. ...
The bending of epithelial tubes is a fundamental process in organ morphogenesis, driven by various multicellular behaviours. The cochlea in the mammalian inner ear is a representative example of spiral tissue architecture where the continuous bending of the duct is a fundamental component of its morphogenetic process. Although the cochlear duct morphogenesis has been studied by genetic approaches extensively, it is still unclear how the cochlear duct morphology is physically formed. Here, we report that nuclear behaviour changes are associated with the curvature of the pseudostratified epithelium during murine cochlear development. Two-photon live-cell imaging reveals that the nuclei shuttle between the luminal and basal edges of the cell is in phase with cell-cycle progression, known as interkinetic nuclear migration, in the flat region of the pseudostratified epithelium. However, the nuclei become stationary on the luminal side following mitosis in the curved region. Mathematical modelling together with perturbation experiments shows that this nuclear stalling facilitates luminal-basal differential growth within the epithelium, suggesting that the nuclear stalling would contribute to the bending of the pseudostratified epithelium during the cochlear duct development. The findings suggest a possible scenario of differential growth which sculpts the tissue shape, driven by collective nuclear dynamics.
... Cortical development is regulated by numerous mechanisms that discretely ensure that a series of temporally ordered events unfold in the correct order, generate the correct celltypes, and ultimately generate morphologically patterned tissue. This requires intact programming that emerges from a wide berth of biological processes, including the expression of specific genes [1] and molecules [2], patterns of sustained metabolic activity [3], the prevention of DNA damage [4], ongoing cell-cycle dynamics [5], regulation of cell survival mechanisms [6,7], as well as orchestrated cell fate decision making [8]. Should any of these processes become altered during in utero cortical development, neocortical neurogenesis may become attenuated and this may yield developmental disorders, disruptions and/or delays. ...
It is widely accepted that narcotic use during pregnancy and specific environmental factors (e.g., maternal immune activation and chronic stress) may increase risk of neuropsychiatric illness in offspring. However, little progress has been made in defining human-specific in utero neurodevelopmental pathology due to ethical and technical challenges associated with accessing human prenatal brain tissue. Here we utilized human induced pluripotent stem cells (hiPSCs) to generate reproducible organoids that recapitulate dorsal forebrain development including early corticogenesis. We systemically exposed organoid samples to chemically defined “enviromimetic” compounds to examine the developmental effects of various narcotic and neuropsychiatric-related risk factors within tissue of human origin. In tandem experiments conducted in parallel, we modeled exposure to opiates (μ-opioid agonist endomorphin), cannabinoids (WIN 55,212-2), alcohol (ethanol), smoking (nicotine), chronic stress (human cortisol), and maternal immune activation (human Interleukin-17a; IL17a). Human-derived dorsal forebrain organoids were consequently analyzed via an array of unbiased and high-throughput analytical approaches, including state-of-the-art TMT-16plex liquid chromatography/mass-spectrometry (LC/MS) proteomics, hybrid MS metabolomics, and flow cytometry panels to determine cell-cycle dynamics and rates of cell death. This pipeline subsequently revealed both common and unique proteome, reactome, and metabolome alterations as a consequence of enviromimetic modeling of narcotic use and neuropsychiatric-related risk factors in tissue of human origin. However, of our 6 treatment groups, human-derived organoids treated with the cannabinoid agonist WIN 55,212-2 exhibited the least convergence of all groups. Single-cell analysis revealed that WIN 55,212-2 increased DNA fragmentation, an indicator of apoptosis, in human-derived dorsal forebrain organoids. We subsequently confirmed induction of DNA damage and apoptosis by WIN 55,212-2 within 3D human-derived dorsal forebrain organoids. Lastly, in a BrdU pulse-chase neocortical neurogenesis paradigm, we identified that WIN 55,212-2 was the only enviromimetic treatment to disrupt newborn neuron numbers within human-derived dorsal forebrain organoids. Cumulatively this study serves as both a resource and foundation from which human 3D biologics can be used to resolve the non-genomic effects of neuropsychiatric risk factors under controlled laboratory conditions. While synthetic cannabinoids can differ from naturally occurring compounds in their effects, our data nonetheless suggests that exposure to WIN 55,212-2 elicits neurotoxicity within human-derived developing forebrain tissue. These human-derived data therefore support the long-standing belief that maternal use of cannabinoids may require caution so to avoid any potential neurodevelopmental effects upon developing offspring in utero.
... In the mouse embryo, expression of CyclinD1 and D2 begins in the epiblast and nascent primitive streak, respectively, just a few hours before gastrulation, possibly explaining the contraction of the cell cycle at the streak, where cells have a short G1 [77]. Interestingly, IKNM and the cell cycle can be uncoupled [78]. An in silico 2D pseudostratified epithelium model based on data collected from chick neuroepithelium revealed that slowing IKNM was sufficient to promote non-apical mitosis [79]. ...
Mitosis is a key process in development and remains critical to ensure homeostasis in adult tissues. Besides its primary role in generating two new cells, cell division involves deep structural and molecular changes that might have additional effects on cell and tissue fate and shape. Specific quantitative and qualitative regulation of mitosis has been observed in multiple morphogenetic events in different embryo models. For instance, during mouse embryo gastrulation, the portion of epithelium that undergoes epithelial to mesenchymal transition, where a static epithelial cell become mesenchymal and motile, has a higher mitotic index and a distinct localization of mitotic rounding, compared to the rest of the tissue. Here we explore the potential mechanisms through which mitosis may favor tissue reorganization in various models. Notably, we discuss the mechanical impact of cell rounding on the cell and its environment, and the modification of tissue physical parameters through changes in cell-cell and cell-matrix adhesion.
... This raises the possibility that these proteins may play a role in the mechanism driving interkinetic nuclear migration (IKNM). IKNM is thought to be mediated by actin and myosin in short pseudostratified epithelia such as the otic epithelium, while it involves microtubuledependent processes in pseudostratified epithelia with longer cells (Norden et al., 2009;Tsai et al., 2010;Kosodo et al., 2011;Leung et al., 2011;Strzyz et al., 2015;Norden, 2017). Cell polarity proteins have so far not been implicated in this process, but our data suggest that they may play a role, for example in anchoring cytoskeletal proteins to both the nucleus and the cell membrane or in regulating their dynamics. ...
Using immunostaining and confocal microscopy, we here provide the first detailed description of otic neurogenesis in Xenopus laevis . We show that the otic vesicle comprises a pseudostratified epithelium with apicobasal polarity (apical enrichment of Par3, aPKC, phosphorylated Myosin light chain, N-cadherin) and interkinetic nuclear migration (apical localization of mitotic, pH3-positive cells). A Sox3-immunopositive neurosensory area in the ventromedial otic vesicle gives rise to neuroblasts, which delaminate through breaches in the basal lamina between stages 26/27 and 39. Delaminated cells congregate to form the vestibulocochlear ganglion, whose peripheral cells continue to proliferate (as judged by EdU incorporation), while central cells differentiate into Islet1/2-immunopositive neurons from stage 29 on and send out neurites at stage 31. The central part of the neurosensory area retains Sox3 but stops proliferating from stage 33, forming the first sensory areas (utricular/saccular maculae). The phosphatase and transcriptional coactivator Eya1 has previously been shown to play a central role for otic neurogenesis but the underlying mechanism is poorly understood. Using an antibody specifically raised against Xenopus Eya1, we characterize the subcellular localization of Eya1 proteins, their levels of expression as well as their distribution in relation to progenitor and neuronal differentiation markers during otic neurogenesis. We show that Eya1 protein localizes to both nuclei and cytoplasm in the otic epithelium, with levels of nuclear Eya1 declining in differentiating (Islet1/2+) vestibulocochlear ganglion neurons and in the developing sensory areas. Morpholino-based knockdown of Eya1 leads to reduction of proliferating, Sox3- and Islet1/2-immunopositive cells, redistribution of cell polarity proteins and loss of N-cadherin suggesting that Eya1 is required for maintenance of epithelial cells with apicobasal polarity, progenitor proliferation and neuronal differentiation during otic neurogenesis.
