Loss of Llgl1 in retinal neuroepithelia reveals links between apical domain size, Notch activity and neurogenesis.
ABSTRACT: To gain insights into the cellular mechanisms of neurogenesis, we analyzed retinal neuroepithelia deficient for Llgl1, a protein implicated in apicobasal cell polarity, asymmetric cell division, cell shape and cell cycle exit. We found that vertebrate retinal neuroepithelia deficient for Llgl1 retained overt apicobasal polarity, but had expanded apical domains. Llgl1 retinal progenitors also had increased Notch activity and reduced rates of neurogenesis. Blocking Notch function by depleting Rbpj restored normal neurogenesis. Experimental expansion of the apical domain, through inhibition of Shroom3, also increased Notch activity and reduced neurogenesis. Significantly, in wild-type retina, neurogenic retinal progenitors had smaller apical domains compared with proliferative neuroepithelia. As nuclear position during interkinetic nuclear migration (IKNM) has been previously linked with cell cycle exit, we analyzed this phenomenon in cells depleted of Llgl1. We found that although IKNM was normal, the relationship between nuclear position and neurogenesis was shifted away from the apical surface, consistent with increased pro-proliferative and/or anti-neurogenic signals associated with the apical domain. These data, in conjunction with other findings, suggest that, in retinal neuroepithelia, the size of the apical domain modulates the strength of polarized signals that influence neurogenesis.
Project description:Nuclei in the proliferative pseudostratified epithelia of vastly different organisms exhibit a characteristic dynamics - the so-called interkinetic nuclear migration (IKNM). Although these movements are thought to be intimately tied to the cell cycle, little is known about the relationship between IKNM and distinct phases of the cell cycle and the role that this association plays in ensuring balanced proliferation and subsequent differentiation. Here, we perform a quantitative analysis of modes of nuclear migration during the cell cycle using a marker that enables the first unequivocal differentiation of all four phases in proliferating neuroepithelial cells in vivo. In zebrafish neuroepithelia, nuclei spend the majority of the cell cycle in S phase, less time in G1, with G2 and M being noticeably shorter still in comparison. Correlating cell cycle phases with nuclear movements shows that IKNM comprises rapid apical nuclear migration during G2 phase and stochastic nuclear motion during G1 and S phases. The rapid apical migration coincides with the onset of G2, during which we find basal actomyosin accumulation. Inhibiting the transition from G2 to M phase induces a complete stalling of nuclei, indicating that IKNM and cell cycle continuation cannot be uncoupled and that progression from G2 to M is a prerequisite for rapid apical migration. Taken together, these results suggest that IKNM involves an actomyosin-driven contraction of cytoplasm basal to the nucleus during G2, and that the stochastic nuclear movements observed in other phases arise passively due to apical migration in neighboring cells.
Project description:The different cell types in the central nervous system develop from a common pool of progenitor cells. The nuclei of progenitors move between the apical and basal surfaces of the neuroepithelium in phase with their cell cycle, a process termed interkinetic nuclear migration (INM). In the retina of zebrafish mikre oko (mok) mutants, in which the motor protein Dynactin-1 is disrupted, interkinetic nuclei migrate more rapidly and deeply to the basal side and more slowly to the apical side. We found that Notch signaling is predominantly activated on the apical side in both mutants and wild-type. Mutant progenitors are, thus, less exposed to Notch and exit the cell cycle prematurely. This leads to an overproduction of early-born retinal ganglion cells (RGCs) at the expense of later-born interneurons and glia. Our data indicate that the function of INM is to balance the exposure of progenitor nuclei to neurogenic versus proliferative signals.
Project description:Progenitor cell nuclei in the rapidly expanding epithelium of the embryonic vertebrate central nervous system undergo a process called interkinetic nuclear migration (IKNM). Movements of IKNM are generally believed to involve smooth migration of nuclei from apical to basal and back during the G1 and G2 phases of the cell cycle, respectively. Yet, this has not been formally demonstrated, nor have the molecular mechanisms that drive IKNM been identified. Using time-lapse confocal microscopy to observe nuclear movements in zebrafish retinal neuroepithelial cells, we show that, except for brief apical nuclear translocations preceding mitosis, IKNM is stochastic rather than smooth and directed. We also show that IKNM is driven largely by actomyosin-dependent forces as it still occurs when the microtubule cytoskeleton is compromised but is blocked when MyosinII activity is inhibited.
Project description:Apicobasal polarity plays an important role in regulating asymmetric cell divisions by neural progenitor cells (NPCs) in invertebrates, but the role of polarity in mammalian NPCs is poorly understood. Here, we characterize the function of the PDZ domain protein MALS-3 in the developing cerebral cortex. We find that MALS-3 is localized to the apical domain of NPCs. Mice lacking all three MALS genes fail to localize the polarity proteins PATJ and PALS1 apically in NPCs, whereas the formation and maintenance of adherens junctions appears normal. In the absence of MALS proteins, early NPCs progressed more slowly through the cell cycle, and their daughter cells were more likely to exit the cell cycle and differentiate into neurons. Interestingly, these effects were transient; NPCs recovered normal cell cycle properties during late neurogenesis. Experiments in which MALS-3 was targeted to the entire membrane resulted in a breakdown of apicobasal polarity, loss of adherens junctions, and a slowing of the cell cycle. Our results suggest that MALS-3 plays a role in maintaining apicobasal polarity and is required for normal neurogenesis in the developing cortex.
Project description:A complete account of the whole developmental process of neurogenesis involves understanding a number of complex underlying molecular processes. Among them, those that govern the crucial transition from proliferative (self-replicating) to neurogenic neural progenitor (NP) cells remain largely unknown. Due to its sequential rostro-caudal gradients of proliferation and neurogenesis, the prospective spinal cord of the chick embryo is a good experimental system to study this issue. We report that the NOTCH ligand DELTA-1 is expressed in scattered cycling NP cells in the prospective chick spinal cord preceding the onset of neurogenesis. These Delta-1-expressing progenitors are placed in between the proliferating caudal neural plate (stem zone) and the rostral neurogenic zone (NZ) where neurons are born. Thus, these Delta-1-expressing progenitors define a proliferation to neurogenesis transition zone (PNTZ). Gain and loss of function experiments carried by electroporation demonstrate that the expression of Delta-1 in individual progenitors of the PNTZ is necessary and sufficient to induce neuronal generation. The activation of NOTCH signalling by DELTA-1 in the adjacent progenitors inhibits neurogenesis and is required to maintain proliferation. However, rather than inducing cell cycle exit and neuronal differentiation by a typical lateral inhibition mechanism as in the NZ, DELTA-1/NOTCH signalling functions in a distinct manner in the PNTZ. Thus, the inhibition of NOTCH signalling arrests proliferation but it is not sufficient to elicit neuronal differentiation. Moreover, after the expression of Delta-1 PNTZ NP continue cycling and induce the expression of Tis21, a gene that is upregulated in neurogenic progenitors, before generating neurons. Together, these experiments unravel a novel function of DELTA-NOTCH signalling that regulates the transition from proliferation to neurogenesis in NP cells. We hypothesize that this novel function is evolutionary conserved.
Project description:A vast number of apicobasal polarity proteins play essential roles in the polarization and morphogenesis of the neuroepithelia. Crumbs (Crb) type I transmembrane cell-cell adhesion proteins are among these proteins. Five crb genes have been identified in zebrafish. However, their expressional and functional differences during early neural development remain to be fully elucidated. Here, we study the spatial-temporal expression patterns and functions of Crb1, Crb2a, and Crb2b in the central nervous system (CNS) during the neurulation period. We show that: 1, the optic vesicle and undifferentiated retinal neuroepithelium only express Crb2a; 2, Crb1 and Crb2a expressions overlap extensively in the undifferentiated neural tube epithelium; 3, Crb2b expression is the weakest of the three and is restricted to the ventral-most regions of the anterior CNS; and 4, Nok and Crb proteins require each other for their apical localization in neuroepithelium. The commencements of Crb1, Crb2a, and Crb2b expressions follow a spatial-temporal spread from anterior to posterior and from ventral to dorsal and lag behind that of adherens junction components, such as ZO-1 and actin bundles. Genetic and morpholino suppression analyses suggest that in regions where these Crb expressions overlap, they are functionally redundant in maintaining apicobasal polarity of the undifferentiated neuroepithelium.
Project description:Development of nervous tissue is a coordinated process of neural progenitor cell (NPC) proliferation and neuronal differentiation. Intracellular signalling events that regulate the balance between NPC proliferation and neuronal differentiation, therefore, determine the size and composition of nervous tissues. Here, we demonstrate that negative regulation of phosphoinosite 3-kinase (PI3K)-Akt signalling by phosphatase tensin homologue (Pten) is essential for maintaining NPC population in mouse retina. We found that mouse retinal progenitor cells (RPCs) lacking the Pten gene complete neurogenesis earlier than their normal developmental schedule, resulting in their premature depletion in the mature retina. We further discover that Notch intracellular domain (NICD) fails to form transcription activator complex in Pten-deficient RPCs, and thereby unable to support RPC maintenance. Taken together, our results suggest that Pten plays a pivotal role in retinal neurogenesis by supporting Notch-driven RPC maintenance against neurogenic PI3K-Akt signalling.
Project description:We have previously demonstrated that Llgl1 loss results in a gain of mesenchymal phenotypes and a loss of apicobasal and planar polarity. We now demonstrate that these changes represent a fundamental shift in cellular phenotype. Llgl1 regulates the expression of multiple cell identity markers, including CD44, CD49f, and CD24, and the nuclear translocation of TAZ and Slug. Cells lacking Llgl1 form mammospheres, where survival and transplantability is dependent upon the Epidermal Growth Factor Receptor (EGFR). Additionally, Llgl1 loss allows cells to grow in soft-agar and maintain prolonged survival as orthotopic transplants in NOD-SCIDmice. Lineage tracing and wound healing experiments demonstrate that mammosphere survival is due to enhanced EGF-dependent migration. The loss of Llgl1 drives EGFR mislocalization and an EGFR mislocalization point mutation (P667A) drives these same phenotypes, including activation of AKT and TAZ nuclear translocation. Together, these data indicate that the loss of Llgl1 results in EGFR mislocalization, promoting pre-neoplastic changes.
Project description:Cell adhesion and apicobasal polarity together maintain epithelial tissue organization and homeostasis. Loss of adhesion has been described as a prerequisite for the epithelial to mesenchymal transition. However, what role misregulation of apicobasal polarity promotes tumor initiation and/or early progression remains unclear. We find that human low-grade endometrial cancers are associated with disrupted localization of the apical polarity protein Par3 and Ezrin while, the adhesion molecule E-cadherin remains unchanged, accompanied by decreased Notch signaling, and altered Notch receptor localization. Depletion of Par3 or Ezrin, in a cell-based model, results in loss of epithelial architecture, differentiation, increased proliferation, migration and decreased Notch signaling. Re-expression of Par3 in endometrial cancer cell lines with disrupted Par3 protein levels blocks proliferation and reduces migration in a Notch dependent manner. These data uncover a function for apicobasal polarity independent of cell adhesion in regulating Notch-mediated differentiation signals in endometrial epithelial cells.
Project description:The adult zebrash brain has a remarkable constitutive neurogenic capacity. The regulation and maintenance of its adult neurogenic niches are poorly understood. In mammals, Notch signaling is involved in stem cell maintenance both in embryonic and adult CNS. To better understand how Notch signaling is involved in stem cell maintenance during adult neurogenesis in zebrafish we analysed Notch receptor expression in five neurogenic zones of the adult zebrafish brain. Combining proliferation and glial markers we identified several subsets of Notch receptor expressing cells. We found that 90 [Formula: see text] of proliferating radial glia express notch1a, notch1b and notch3. In contrast, the proliferating non-glial populations of the dorsal telencephalon and hypothalamus rarely express notch3 and about half express notch1a/1b. In the non-proliferating radial glia notch3 is the predominant receptor throughout the brain. In the ventral telencephalon and in the mitotic area of the optic tectum, where cells have neuroepithelial properties, notch1a/1b/3 are expressed in most proliferating cells. However, in the cerebellar niche, although progenitors also have neuroepithelial properties, only notch1a/1b are expressed in a high number of PCNA [Formula: see text] cells. In this region notch3 expression is mostly in Bergmann glia and at low levels in few PCNA [Formula: see text] cells. Additionally, we found that in the proliferation zone of the ventral telencephalon, Notch receptors display an apical high to basal low gradient of expression. Notch receptors are also expressed in subpopulations of oligodendrocytes, neurons and endothelial cells. We suggest that the partial regional heterogeneity observed for Notch expression in progenitor cells might be related to the cellular diversity present in each of these neurogenic niches.