Interkinetic Nuclear Migration Research Articles

Balancing competing effects of tissue growth and cytoskeletal regulation during Drosophila wing disc development

How a developing organ robustly coordinates the cellular mechanics and growth to reach a final size and shape remains poorly understood. Through iterations between experiments and model simulations that include a mechanistic description of interkinetic nuclear migration, we show that the local curvature, height, and nuclear positioning of cells in the Drosophila wing imaginal disc are defined by the concurrent patterning of actomyosin contractility, cell-ECM adhesion, ECM stiffness, and interfacial membrane tension. We show that increasing cell proliferation via different growth-promoting pathways results in two distinct phenotypes. Triggering proliferation through insulin signaling increases basal curvature, but an increase in growth through Dpp signaling and Myc causes tissue flattening. These distinct phenotypic outcomes arise from differences in how each growth pathway regulates the cellular cytoskeleton, including contractility and cell-ECM adhesion. The coupled regulation of proliferation and cytoskeletal regulators is a general strategy to meet the multiple context-dependent criteria defining tissue morphogenesis.

Vinculin is required for interkinetic nuclear migration (INM) and cell cycle progression.

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.

Role of Nesprin-2 and RanBP2 in BICD2-associated brain developmental disorders.

Bicaudal D2 (BICD2) is responsible for recruiting cytoplasmic dynein to diverse forms of subcellular cargo for their intracellular transport. Mutations in the human BICD2 gene have been found to cause an autosomal dominant form of spinal muscular atrophy (SMA-LED2), and brain developmental defects. Whether and how the latter mutations are related to roles we and others have identified for BICD2 in brain development remains little understood. BICD2 interacts with the nucleoporin RanBP2 to recruit dynein to the nuclear envelope (NE) of Radial Glial Progenitor cells (RGPs) to mediate their well-known but mysterious cell-cycle-regulated interkinetic nuclear migration (INM) behavior, and their subsequent differentiation to form cortical neurons. We more recently found that BICD2 also mediates NE dynein recruitment in migrating post-mitotic neurons, though via a different interactor, Nesprin-2. Here, we report that Nesprin-2 and RanBP2 compete for BICD2-binding in vitro. To test the physiological implications of this behavior, we examined the effects of known BICD2 mutations using in vitro biochemical and in vivo electroporation-mediated brain developmental assays. We find a clear relationship between the ability of BICD2 to bind RanBP2 vs. Nesprin-2 in controlling of nuclear migration and neuronal migration behavior. We propose that mutually exclusive RanBP2-BICD2 vs. Nesprin-2-BICD2 interactions at the NE play successive, critical roles in INM behavior in RGPs and in post-mitotic neuronal migration and errors in these processes contribute to specific human brain malformations.

A human dynein heavy chain mutation impacts cortical progenitor cells causing developmental defects, reduced brain size and altered brain architecture

Dynein heavy chain (DYNC1H1) mutations can either lead to severe cerebral cortical malformations, or alternatively may be associated with the development of spinal muscular atrophy with lower extremity predominance (SMA-LED). To assess the origin of such differences, we studied a new Dync1h1 knock-in mouse carrying the cortical malformation p.Lys3334Asn mutation. Comparing with an existing neurodegenerative Dync1h1 mutant (Legs at odd angles, Loa, p.Phe580Tyr/+), we assessed Dync1h1's roles in cortical progenitor and especially radial glia functions during embryogenesis, and assessed neuronal differentiation.p.Lys3334Asn /+ mice exhibit reduced brain and body size. Embryonic brains show increased and disorganized radial glia: interkinetic nuclear migration occurs in mutants, however there are increased basally positioned cells and abventricular mitoses. The ventricular boundary is disorganized potentially contributing to progenitor mislocalization and death. Morphologies of mitochondria and Golgi apparatus are perturbed in vitro, with different effects also in Loa mice. Perturbations of neuronal migration and layering are also observed in p.Lys3334Asn /+ mutants. Overall, we identify specific developmental effects due to a severe cortical malformation mutation in Dync1h1, highlighting the differences with a mutation known instead to primarily affect motor function.

Modeling study of the impact of actomyosin contractility on cell proliferation in the Drosophila wing imaginal disc.

A fundamental open question in developmental biology is deciphering how the regulation of cell proliferation and the actomyosin cytoskeleton coordinate proper formation and maintenance of tissue shape and size. To address this question, a close integration between experimental work and simulations from a novel multi-scale computational model are used. Specifically, a subcellular element (SCE) model of the Drosophila wing imaginal disc cross-section along the anterior-posterior axis is developed and calibrated using experimental data. This model accounts for actomyosin contractility in different cell regions and incorporates a detailed cell division sub model capable of capturing interkinetic nuclear migration, a fundamental process for proper cell division. Coupled model simulations and experiments reveal that anisotropic patterning of actomyosin contractility can regulate basal local curvature of the wing disc and positioning of nuclei. Moreover, it was found that cell proliferation also plays an important role in regulating basal curvature. However, this model has a simplified representation of actomyosin contractility which is not able to capture the micro level interactions between actin filaments and myosin motors that make up the actomyosin network and thus, the direction of the contractile forces cannot be accounted for explicitly. Therefore, in order to have a more biological representation of actomyosin, a coarse-grained stochastic model of subcellular level actomyosin network dynamics in two-dimensions is being developed. This model describes the actomyosin network via a large system of nodes with their dynamics represented by stochastic ordinary differential equations. The multi-scale SCE model, coupled with the stochastic model, will be used to gain insight into the detailed mechanism of how actomyosin contractility facilitates interkinetic nuclear migration, cell proliferation and formation of the wing shape.

Assessment of Dynein-Mediated Nuclear Migration in the Developing Cortex by Live-Tissue Microscopy.

During development of the cerebral cortex, neuroepithelial and radial glial cells undergo an oscillatory nuclear movement throughout their cell cycle, termed interkinetic nuclear migration. The nucleus of postmitotic neurons derived from these neural stem cells also translocates in a saltatory manner to enable neuronal migration toward the cortical plate. In these processes, various molecular motors, including cytoplasmic dynein, myosin II, and kinesins, are the driving force for nuclear migration at different stages. Despite efforts made to understand the mechanism regulating cortical development over decades, novel gene mutations discovered in neurodevelopmental disorders indicate that missing pieces still remain. Gene manipulation by in utero electroporation combined with live microscopy of neural stem cells in brain slices provides a powerful method to capture their detailed behaviors during proliferation and migration. The procedures described in this chapter enable the monitoring of cell cycle progression, mitosis, morphological changes, and migratory patterns in situ. This approach facilitates the elucidation of gene functions in cortical development and neurodevelopmental disorders.

In vitro characterization of the full-length human dynein-1 cargo adaptor BicD2

Cargo adaptors are crucial in coupling motor proteins with their respective cargos and regulatory proteins. BicD2 is a prominent example within the cargo adaptor family. BicD2 is able to recruit the microtubule motor dynein to RNA, viral particles, and nuclei. The BicD2-mediated interaction between the nucleus and dynein is implicated in mitosis, interkinetic nuclear migration (INM) in radial glial progenitor cells, and neuron precursor migration during embryonic neocortex development. Invitro studies involving full-length cargo adaptors are difficult to perform due to the hydrophobic character, low-expression levels, and intrinsic flexibility of cargo adaptors. Here, we report the recombinant production of full-length human BicD2 and confirm its biochemical activity by interaction studies with RanBP2. We also describe pH-dependent conformational changes of BicD2 using cryoelectron microscopy (cryo-EM), template-free structure predictions, and biophysical tools. Our results will help define the biochemical parameters for the invitro reconstitution of higher-order BicD2 protein complexes.

Functional Cooperation of α-Synuclein and Tau Is Essential for Proper Corticogenesis.

Alpha-synuclein (αSyn) and tau are abundant multifunctional neuronal proteins, and their intracellular deposits have been linked to many neurodegenerative diseases, including Alzheimer's disease and Parkinson's disease. Despite the disease relevance, their physiological roles remain elusive, as mice with knock-out of either of these genes do not exhibit overt phenotypes. To reveal functional cooperation, we generated αSyn-/-tau-/- double-knock-out mice and characterized the functional cross talk between these proteins during brain development. Intriguingly, deletion of αSyn and tau reduced Notch signaling and accelerated interkinetic nuclear migration of G2 phase at early embryonic stage. This significantly altered the balance between the proliferative and neurogenic divisions of progenitor cells, resulting in an overproduction of early born neurons and enhanced neurogenesis, by which the brain size was enlarged during the embryonic stage in both sexes. On the other hand, a reduction in the number of neural progenitor cells in the middle stage of corticogenesis diminished subsequent gliogenesis in the αSyn-/-tau-/- cortex. Additionally, the expansion and maturation of macroglial cells (astrocytes and oligodendrocytes) were suppressed in the αSyn-/-tau-/- postnatal brain, which in turn reduced the male αSyn-/-tau-/- brain size and cortical thickness to less than the control values. Our study identifies important functional cooperation of αSyn and tau during corticogenesis.SIGNIFICANCE STATEMENT Correct understanding of the physiological functions of αSyn and tau in CNS is critical to elucidate pathogenesis involved in the etiology of neurodegenerative diseases including Alzheimer's disease and Parkinson's disease. We show here that αSyn and tau are cooperatively involved in brain development via maintenance of progenitor cells. αSyn and tau double-knock-out mice exhibited an overproduction of early born neurons and accelerated neurogenesis at early corticogenesis. Furthermore, loss of αSyn and tau also perturbed gliogenesis at later embryonic stage, as well as the subsequent glial expansion and maturation at postnatal brain. Our findings provide new mechanistic insights and extend therapeutic opportunities for neurodegenerative diseases caused by aberrant αSyn and tau.

Impaired Cell Cycle Progression and Self-Renewal of Fetal Neural Stem and Progenitor Cells in a Murine Model of Intrauterine Growth Restriction

Individuals with intrauterine growth restriction (IUGR) are at an increased risk for neurodevelopmental impairment. Fetal cortical neurogenesis is a time-sensitive process in which fetal neural stem cells (NSCs) follow a distinct pattern of layer-specific neuron generation to populate the cerebral cortex. Here, we used a murine maternal hypoxia-induced IUGR model to study the impact of IUGR on fetal NSC development. In this model, timed-pregnant mice were exposed to hypoxia during the active stage of neurogenesis, followed by fetal brain collection and analysis. In the IUGR fetal brains, we found a significant reduction in cerebral cortical thickness accompanied by decreases in layer-specific neurons. Using EdU labeling, we demonstrated that cell cycle progression of fetal NSCs was delayed, primarily observed in the G2/M phase during inward interkinetic nuclear migration. Following relief from maternal hypoxia exposure, the remaining fetal NSCs re-established their neurogenic ability and resumed production of layer-specific neurons. Surprisingly, the newly generated neurons matched their control counterparts in layer-specific marker expression, suggesting preservation of the fetal NSC temporal identity despite IUGR effects. As expected, the absolute number of neurons generated in the IUGR group remained lower compared to that in the control group due to a reduced fetal NSC pool size as a result of cell cycle defect. Transcriptome analysis identified genes related to energy expenditure and G2/M cell cycle progression being affected by maternal hypoxia-induced IUGR. Taken together, maternal hypoxia-induced IUGR is associated with a defect in cell cycle progression of fetal NSCs, and has a long-term impact on offspring cognitive development.

Phosphatidylinositol transfer protein/planar cell polarity axis regulates neocortical morphogenesis by supporting interkinetic nuclear migration.

SUMMARYThe neocortex expands explosively during embryonic development. The earliest populations of neural stem cells (NSCs) form a thin pseudostratified epithelium whose contour determines that of the adult neocortex. Neocortical complexity is accompanied by disproportional expansion of the NSC layer in its tangential dimension to increase tissue surface area. How such disproportional expansion is controlled remains unknown. We demonstrate that a phosphatidylinositol transfer protein (PITP)/non-canonical Wnt planar cell polarity (ncPCP) signaling axis promotes tangential expansion of developing neocortex. PITP signaling supports trafficking of specific ncPCP receptors from the NSC Golgi system to potentiate actomyosin activity important for cell-cycle-dependent interkinetic nuclear migration (IKNM). In turn, IKNM promotes lateral dispersion of newborn NSCs and tangential growth of the cerebral wall. These findings clarify functional roles for IKNM in NSC biology and identify tissue dysmorphogenesis resulting from impaired IKNM as a factor in autism risk, developmental brain disabilities, and neural tube birth defects.

Micronutrient Balance Related to Neural Tube Defects and Prevention

Neural tube defects (NTDs) are a common congenital disorder resulting from failed neural tube formation, the precursor of the brain and spinal cord. The complex etiology of NTDs involve both genetic and environmental factors, thus investigating gene‐environment interactions is critical to understanding how NTDs occur or how NTDs may be prevented. For example, iron deficiency is among the most prevalent micronutrient deficiencies in pregnancy and there is evidence that iron deficiency can increase the risk of NTDs. Folic acid (FA) fortification in grain was implemented in the US in 1998 and now in many countries for the purpose of prevention of neural tube defects (NTDs). In 2017, the US Preventive Services Task Force further recommended the consumption of FA‐containing multivitamin/minerals (MVM) for women of childbearing age. Despite the great benefit of FA on NTD prevention, NTDs remain a serious risk.Our studies focus on mouse models of NTDs and I will discuss two projects. First, the benefits of MVM supplementation are emphasized, but a comparison between MVM supplementation and FA alone on neural tube closure and organ development has not been evaluated, particularly in the context of genetic mutations that lead to NTDs. Using a set of NTD models we have compared MMV to FA supplementation. MVM supplementation shows better improvement than FA alone in the penetrance of spinal and cranial NTDs. We are also using human iPSCs to generate organoids that assume a shape similar to the human spinal neural tube, and display neural tube characteristics, for example apical constriction and interkinetic nuclear migration. Subjecting the organoids to various molecular perturbations, such as Rho‐kinase inhibitor‐induced defects in apical constriction, we have found that both MVM and FA largely prevents this molecular deficit. The goal is to use the mouse NTD model and organoid system to better understand how FA/MVM act to prevent NTDs.Second, we are striving to move beyond the relatively non‐specific recommendations of FA/MVM supplementation to more personalized therapies that take into account the molecular underpinning of the NTD. For example, Iron homeostasis is not only controlled by the concentration of iron in diet, but by proteins responsible for its cellular uptake and metabolism. Sorting nexin 3 (Snx3) is known to regulate recycling of the transferrin receptor (TFRC), a mechanism necessary for cellular uptake of iron, and when Snx3 is knocked out in mice, embryos develop cranial NTDs. We hypothesize that in Snx3 mutants, mistrafficking of TFRC results in disrupted iron homeostasis which contributes to the failure of NT closure. Our current studies are evaluating whether intracellular iron levels are altered in Snx3 mutants and whether maternal diets supplemented with iron can prevent NTDs in the Snx3 background. The hope is that by taking into account the biological process that is disrupted, more tailored therapies can be envisioned to better prevent NTDs.

Mechanical constraints to cell-cycle progression in a pseudostratified epithelium

As organs and tissues approach their normal size during development or regeneration, growth slows down, and cell proliferation progressively comes to a halt. Among the various processes suggested to contribute to growth termination,1-10 mechanical feedback, perhaps via adherens junctions, has been suggested to play a role.11-14 However, since adherens junctions are only present in a narrow plane of the subapical region, other structures are likely needed to sense mechanical stresses along the apical-basal (A-B) axis, especially in a thick pseudostratified epithelium. This could be achieved by nuclei, which have been implicated in mechanotransduction in tissue culture.15 In addition, mechanical constraints imposed by nuclear crowding and spatial confinement could affect interkinetic nuclear migration (IKNM),16 which allows G2 nuclei to reach the apical surface, where they normally undergo mitosis.17-25 To explore how mechanical constraints affect IKNM, we devised an individual-based model that treats nuclei as deformable objects constrained by the cell cortex and the presence of other nuclei. The model predicts changes in the proportion of cell-cycle phases during growth, which we validate with the cell-cycle phase reporter FUCCI (Fluorescent Ubiquitination-based Cell Cycle Indicator).26 However, this model does not preclude indefinite growth, leading us to postulate that nuclei must migrate basally to access a putative basal signal required for S phase entry. With this refinement, our updated model accounts for the observed progressive slowing down of growth and explains how pseudostratified epithelia reach a stereotypical thickness upon completion of growth.

KIFC1 Regulates the Trajectory of Neuronal Migration

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.

PDK1 Regulates the Lengthening of G1 Phase to Balance RGC Proliferation and Differentiation during Cortical Neurogenesis.

During cortical development, the balance between progenitor self-renewal and neurogenesis is critical for determining the size/morphology of the cortex. A fundamental feature of the developing cortex is an increase in the length of G1 phase in RGCs over the course of neurogenesis, which is a key determinant of progenitor fate choice. How the G1 length is temporally regulated remains unclear. Here, Pdk1, a member of the AGC kinase family, was conditionally disrupted by crossing an Emx1-Cre mouse line with a Pdk1fl/fl line. The loss of Pdk1 led to a shorter cell cycle accompanied by increased RGC proliferation specifically at late rather than early/middle neurogenic stages, which was attributed to impaired lengthening of G1 phase. Coincidently, apical-to-basal interkinetic nuclear migration was accelerated in Pdk1 cKO cortices. Consequently, we detected an increased neuronal output at P0. We further showed the significant upregulation of the cell cycle regulator cyclin D1 and its activator Myc in the cKO cortices relative to those of control animals. Overall, we have identified a novel role for PDK1 in cortical neurogenesis. PDK1 functions as an upstream regulator of the Myc-cyclin D1 pathway to control the lengthening of G1 phase and the balance between RGC proliferation and differentiation.

Stalling interkinetic nuclear migration in curved pseudostratified epithelium of developing cochlea.

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.

Otic Neurogenesis in Xenopus laevis: Proliferation, Differentiation, and the Role of Eya1.

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.

Maternal sevoflurane exposure induces temporary defects in interkinetic nuclear migration of radial glial progenitors in the fetal cerebral cortex through the Notch signalling pathway.

ObjectivesThe effects of general anaesthetics on fetal brain development remain elusive. Radial glial progenitors (RGPs) generate the majority of neurons in developing brains. Here, we evaluated the acute alterations in RGPs after maternal sevoflurane exposure.MethodsPregnant mice were exposed to 2.5% sevoflurane for 6 hours on gestational day 14.5. Interkinetic nuclear migration (INM) of RGPs in the ventricular zone (VZ) of the fetal brain was evaluated by thymidine analogues labelling. Cell fate of RGP progeny was determined by immunostaining using various neural markers. The Morris water maze (MWM) was used to assess the neurocognitive behaviours of the offspring. RNA sequencing (RNA‐Seq) was performed for the potential mechanism, and the potential mechanism validated by quantitative real‐time PCR (qPCR), Western blot and rescue experiments. Furthermore, INM was examined in human embryonic stem cell (hESC)‐derived 3D cerebral organoids.ResultsMaternal sevoflurane exposure induced temporary abnormities in INM, and disturbed the cell cycle progression of RGPs in both rodents and cerebral organoids without cell fate alternation. RNA‐Seq analysis, qPCR and Western blot showed that the Notch signalling pathway was a potential downstream target. Reactivation of Notch by Jag1 and NICD overexpression rescued the defects in INM. Young adult offspring showed no obvious cognitive impairments in MWM.ConclusionsMaternal sevoflurane exposure during neurogenic period temporarily induced abnormal INM of RGPs by targeting the Notch signalling pathway without inducing long‐term effects on RGP progeny cell fate or offspring cognitive behaviours. More importantly, the defects of INM in hESC‐derived cerebral organoids provide a novel insight into the effects of general anaesthesia on human brain development.

Issue Information

Esophageal epithelium exhibits interkinetic nuclear migration. The EdU‐labeled nuclei were basal at 1 hour (top left), apical at 6 hours (top right), and basal side at 12 hours (bottom). Further details can be seen in the article by Dereje et al. in this issue. image

Colonic epithelial adaptation to EGFR-independent growth induces chromosomal instability and is accelerated by prior injury

Although much is known about the gene mutations required to drive colorectal cancer (CRC) initiation, the tissue-specific selective microenvironments in which neoplasia arises remains less characterized. Here, we determined whether modulation of intestinal stem cell niche morphogens alone can exert a neoplasia-relevant selective pressure on normal colonic epithelium. Using adult stem cell-derived murine colonic epithelial organoids (colonoids), we employed a strategy of sustained withdrawal of epidermal growth factor (EGF) and epidermal growth factor receptor (EGFR) inhibition to select for and expand survivors. EGFR-signaling-independent (iEGFR) colonoids emerged over rounds of selection and expansion. Colonoids derived from a mouse model of chronic mucosal injury showed an enhanced ability to adapt to EGFR inhibition. Whole-exome and transcriptomic analyses of iEGFR colonoids demonstrated acquisition of deleterious mutations and altered expression of genes implicated in EGF signaling, pyroptosis, and CRC. iEGFR colonoids acquired dysplasia-associated cytomorphologic changes, an increased proliferative rate, and the ability to survive independently of other required niche factors. These changes were accompanied by emergence of aneuploidy and chromosomal instability; further, the observed mitotic segregation errors were significantly associated with loss of interkinetic nuclear migration, a fundamental and dynamic process underlying intestinal epithelial homeostasis. This study provides key evidence that chromosomal instability and other phenotypes associated with neoplasia can be induced ex vivo via adaptation to EGF withdrawal in normal and stably euploid colonic epithelium, without introducing cancer-associated driver mutations. In addition, prior mucosal injury accelerates this evolutionary process.

An early cell shape transition drives evolutionary expansion of the human forebrain

SummaryThe human brain has undergone rapid expansion since humans diverged from other great apes, but the mechanism of this human-specific enlargement is still unknown. Here, we use cerebral organoids derived from human, gorilla, and chimpanzee cells to study developmental mechanisms driving evolutionary brain expansion. We find that neuroepithelial differentiation is a protracted process in apes, involving a previously unrecognized transition state characterized by a change in cell shape. Furthermore, we show that human organoids are larger due to a delay in this transition, associated with differences in interkinetic nuclear migration and cell cycle length. Comparative RNA sequencing (RNA-seq) reveals differences in expression dynamics of cell morphogenesis factors, including ZEB2, a known epithelial-mesenchymal transition regulator. We show that ZEB2 promotes neuroepithelial transition, and its manipulation and downstream signaling leads to acquisition of nonhuman ape architecture in the human context and vice versa, establishing an important role for neuroepithelial cell shape in human brain expansion.