Auswahl der wissenschaftlichen Literatur zum Thema „Proneural gene dynamics“

Glucose transporter 2 (GLUT2; gene name SLC2A2) has a key role in the regulation of glucose dynamics in organs central to metabolism. Although GLUT2 has been studied in the context of its participation in peripheral and central glucose sensing, its role in the brain is not well understood. To decipher the role of GLUT2 in brain development, we knocked down slc2a2 ( glut2), the functional ortholog of human GLUT2, in zebrafish. Abrogation of glut2 led to defective brain organogenesis, reduced glucose uptake and increased programmed cell death in the brain. Coinciding with the observed localization of glut2 expression in the zebrafish hindbrain, glut2 deficiency affected the development of neural progenitor cells expressing the proneural genes atoh1b and ptf1a but not those expressing neurod. Specificity of the morphant phenotype was demonstrated by the restoration of brain organogenesis, whole-embryo glucose uptake, brain apoptosis, and expression of proneural markers in rescue experiments. These results indicate that glut2 has an essential role during brain development by facilitating the uptake and availability of glucose and support the involvement of glut2 in brain glucose sensing.

Identification of the mechanisms underlying the genetic control of spatial structure formation is among the relevant tasks of developmental biology. Both experimental and theoretical approaches and methods are used for this purpose, including gene network methodology, as well as mathematical and computer modeling. Reconstruction and analysis of the gene networks that provide the formation of traits allow us to integrate the existing experimental data and to identify the key links and intra-network connections that ensure the function of networks. Mathematical and computer modeling is used to obtain the dynamic characteristics of the studied systems and to predict their state and behavior. An example of the spatial morphological structure is the Drosophila bristle pattern with a strictly defined arrangement of its components – mechanoreceptors (external sensory organs) – on the head and body. The mechanoreceptor develops from a single sensory organ parental cell (SOPC), which is isolated from the ectoderm cells of the imaginal disk. It is distinguished from its surroundings by the highest content of proneural proteins (ASC), the products of the achaete-scute proneural gene complex (AS-C). The SOPC status is determined by the gene network we previously reconstructed and the AS-C is the key component of this network. AS-C activity is controlled by its subnetwork – the central regulatory circuit (CRC) comprising seven genes: AS-C, hairy, senseless (sens), charlatan (chn), scratch (scrt), phyllopod (phyl), and extramacrochaete (emc), as well as their respective proteins. In addition, the CRC includes the accessory proteins Daughterless (DA), Groucho (GRO), Ubiquitin (UB), and Seven-in-absentia (SINA). The paper describes the results of computer modeling of different CRC operation modes. As is shown, a cell is determined as an SOPC when the ASC content increases approximately 2.5-fold relative to the level in the surrounding cells. The hierarchy of the effects of mutations in the CRC genes on the dynamics of ASC protein accumulation is clarified. AS-C as the main CRC component is the most significant. The mutations that decrease the ASC content by more than 40 % lead to the prohibition of SOPC segregation.

Abstract Glioblastoma (GBM) is the most common primary malignant neoplasm with poor survival despite treatment. Developing effective therapies remains challenging due to intratumoral heterogeneity, which drives therapeutic resistance and recurrence. To understand how genetic events alter the epigenome to enhance clonal fitness, we developed an isogenic human neural progenitor cell (NPC)-based model of the proneural (PRO) GBM subtype. We introduced TERT promoter (TERTp) C228T and gain-of-function TP53 R248Q mutations in H1 human embryonic stem cells. Wildtype (WT) cells, single TERTp, and double TERTp/TP53 mutants underwent differentiation to NPCs. Lentiviral transduction of double mutants with PDGFRA D842V resulted in triple mutant PRO NPCs. Bulk and single cell transcriptomics of our model system revealed hundreds of gene expression changes with increased mesoderm and human GBM mesenchymal (MES) subtype signatures in single TERTp and double TERTp/TP53 mutants, versus WT cells. TERTp mutation increased telomerase expression and activity, conferring proliferative advantage and immortalization in NPCs and astrocytes. Additionally, TERT expression was further increased in TERTp/TP53 mutants. Surprisingly, triple mutant PRO NPCs, versus WT, displayed < 100 differentially expressed genes, associated with neurodevelopmental and dynamic cytoskeletal processes. Evolution analyses using gene counts signature and splicing dynamics revealed a developmental trajectory model from WT to additive mutants to PRO NPCs. Only triple mutant PRO NPCs formed tumors after intracranial injection in athymic nude mice, with mean survival of 100 days. Tumors presented histopathological features of GBM, and single cell transcriptomic analyses revealed evolution from immune-interacting to both neural progenitor- and neuronal-like subpopulations, with similar cell cycling signatures. Transcription factor genes related to WNT signaling and lineage commitment as well as glial and neuronal cytoskeletal genes exhibited epigenetic selection in vivo, signatures also observed in PRO NPCs in vitro. Our model thus provides opportunity for dissection of epigenetic and functional mechanisms underlying serial mutations during PRO tumor evolution.

Neural stem/precursor cells (NPCs) generate the large variety of neuronal phenotypes comprising the adult brain. The high diversity and complexity of this organ have its origin in embryonic life, during which NPCs undergo symmetric and asymmetric divisions and then exit the cell cycle and differentiate to acquire neuronal identities. During these processes, coordinated regulation of cell cycle progression/exit and differentiation is essential for generation of the appropriate number of neurons and formation of the correct structural and functional neuronal circuits in the adult brain. Cend1 is a neuronal lineage-specific modulator involved in synchronization of cell cycle exit and differentiation of neuronal precursors. It is expressed all along the neuronal lineage, from neural stem/progenitor cells to mature neurons, and is associated with the dynamics of neuron-generating divisions. Functional studies showed that Cend1 has a critical role during neurogenesis in promoting cell cycle exit and neuronal differentiation. Mechanistically, Cend1 acts via the p53-dependent/Cyclin D1/pRb signaling pathway as well as via a p53-independent route involving a tripartite interaction with RanBPM and Dyrk1B. Upon Cend1 function, Notch1 signaling is suppressed and proneural genes such as Mash1 and Neurogenins 1/2 are induced. Due to its neurogenic activity, Cend1 is a promising candidate therapeutic gene for brain repair, while theCend1minimal promoter is a valuable tool for neuron-specific gene delivery in the CNS. Mice withCend1genetic ablation display increased NPC proliferation, decreased migration, and higher levels of apoptosis during development. As a result, they show in the adult brain deficits in a range of motor and nonmotor behaviors arising from irregularities in cerebellar cortex lamination and impaired Purkinje cell differentiation as well as a paucity in GABAergic interneurons of the cerebral cortex, hippocampus, and amygdala. Taken together, these studies highlight the necessity for Cend1 expression in the formation of a structurally and functionally normal brain.

Neural cell fate specification is a multistep process in which stem cells undergo sequential changes in states, giving rise to particular lineages such as neurons and astrocytes. This process is accompanied by dynamic changes of chromatin and in transcription, thereby orchestrating lineage-specific gene expression programs. A pressing question is how these events are interconnected to sculpt cell fate. We show that altered chromatin due to loss of the chromatin remodeler Chd5 causes neural stem cell activation to occur ahead of time. This premature activation is accompanied by transcriptional derepression of ribosomal subunits, enhanced ribosome biogenesis, and increased translation. These untimely events deregulate cell fate decisions, culminating in the generation of excessive numbers of astrocytes at the expense of neurons. By monitoring the proneural factor Mash1, we further show that translational control is crucial for appropriate execution of cell fate specification, thereby providing new insight into the interplay between transcription and translation at the initial stages of neurogenesis.

Abstract Glioblastoma represents an aggressive, primary brain tumor with limited treatment options. Despite advances in molecularly characterizing glioblastoma, metabolic alterations driving its aggressive phenotype are only beginning to be recognized. Integrative cross-platform analyses coupling global metabolomic and gene expression profiling identified alterations in fatty acid β-oxidation (FAO) as a key metabolic node differentiating glioblastoma from low-grade astrocytoma, which was defined by an accumulation of acylcarnitines. Metabolic heterogeneity was observed within glioblastoma that could further define tumors as FAO ‘high’ and ‘low’, which were enriched with mesenchymal and proneural subtypes of glioblastoma, respectively. These findings were metabolomically and functionally recapitulated in molecular subtype-specific preclinical models, with the majority of baseline mitochondrial oxygen consumption being a result of enhanced FAO in these cells. The biologic consequence of enhanced FAO in glioblastoma is directly dependent upon tumor microenvironment. FAO serves as a metabolic cue to drive proliferation in a β-HB/GPR109A/cAMP-dependent autocrine manner in nutrient favorable conditions while providing an efficient, alternate source of ATP only in nutrient unfavorable conditions. Accordingly, inhibiting FAO alone in glioblastoma cells with etomoxir only led to modest anti-proliferative activity and minimal cytotoxicity. However, rational combinatorial strategies designed to target the dynamic roles FAO plays in gliomagenesis resulted in metabolic synthetic lethality in glioblastoma. Specifically, dual targeting of FAO (etomoxir) and glycolysis (2DG) resulted in robust energetic stress, necroptosis mediated cell death, and a significant improvement in survival in an orthotopic glioblastoma mouse model. In summary, we identified FAO as a dominant metabolic node in glioblastoma that provides metabolic plasticity, allowing these cells to adapt to their dynamic microenvironment. Combinatorial strategies designed to target these diverse roles FAO plays in gliomagenesis offers therapeutic potential in glioblastoma.

The spatial organization of Drosophila melanogaster epidermal structures in embryos and adults constitutes a classic model system for understanding how the two dimensional arrangement of particular cell types is generated. For example, the legs of the Drosophila melanogaster adult are covered with bristles, which in most segments are arranged in longitudinal rows. Here we elucidate the key roles of two regulatory genes, hairy and achaete, in setting up this periodic bristle pattern. We show that achaete is expressed during pupal leg development in a dynamic pattern which changes, by approximately 6 hours after puparium formation, into narrow longitudinal stripes of 3–4 cells in width, each of which represents a field of cells (proneural field) from which bristle precursor cells are selected. This pattern of gene expression foreshadows the adult bristle pattern and is established in part through the function of the hairy gene, which also functions in patterning other adult sense organs. In pupal legs, hairy is expressed in four longitudinal stripes, located between every other pair of achaete stripes. We show that in the absence of hairy function achaete expression expands into the interstripe regions that normally express hairy, fusing the two achaete stripes and resulting in extra-wide stripes of achaete expression. This misexpression of achaete, in turn, alters the fields of potential bristle precursor cells which leads to the misalignment of bristle rows in the adult. This function of hairy in patterning achaete expression is distinct from that in the wing in which hairy suppresses late expression of achaete but has no effect on the initial patterning of achaete expression. Thus, the leg bristle pattern is apparently regulated at two levels: a global regulation of the hairy and achaete expression patterns which partitions the leg epidermis into striped zones (this study) and a local regulation (inferred from other studies on the selection of neural precursor cells) that involves refinement steps which may control the alignment and spacing of bristle cells within these zones.

Abstract INTRODUCTION Primary cilium is a highly conserved, dynamic cellular organelle which plays several roles in embryonic development, intracellular signaling, and cell cycle. Structural alterations of primary cilium have been described in human gliomas including glioblastoma (GBM), however, its actual role in pathogenesis and treatment resistance of these tumors is largely unknown. METHODS We investigated cilium morphology and expression of cilium-related genes in human glioma of various WHO grade and in couples of patient-derived glioma stem-like cells (GSCs) that were established from the very same GBM at first diagnosis and at recurrence. Immunohistochemistry with anti-Arl13b antibody was used to assess cilium morphology. The expression levels of genes involved in ciliary disassembly complex (CDC) were analyzed by quantitative real-time PCR, using neural progenitor cells (NPCs) as control. Lastly, we assessed 3 GSC cultures that were treated with a drug inhibiting cilia disassembly (CCB-Cil). RESULTS Anaplastic oligodendroglioma and proneural GBM showed the highest percentage of ciliated cells. In GBM, we found the highest percentage of fragmented cilia. GSCs derived from newly diagnosed GBMs displayed lower percentages of ciliated cells than those derived from recurrent GBMs (20% vs 70%). Morphological analysis indicated that GSCs from recurrent GBM show cilia with extremely various morphology compared with GSCs from newly diagnosed GBM and NPCs. Gene analysis showed reduced expression of CDC-related genes in GSCs from newly diagnosed GBM with respect to those from recurrent GBMs. CCB-Cil treatment determined a global reduction of CDC-related genes, increased expression of differentiation markers (GFAP), and reduction of stemness markers (SOX2). CONCLUSIONS The increased percentage of ciliated cells in GSCs from recurrent GBM may be related to a compensatory response of CDC and to an accelerated ciliary turnover. Blocking cilia disassembly reduces stemness features and induces differentiation in GSCs, suggesting that this approach could represent a promising strategy for targeting GBM.

The timely transition from neural progenitor to post-mitotic neuron requires down-regulation and loss of the neuronal transcriptional repressor, REST. Here, we have used mice containing a gene trap in the Rest gene, eliminating transcription from all coding exons, to remove REST prematurely from neural progenitors. We find that catastrophic DNA damage occurs during S-phase of the cell cycle, with long-term consequences including abnormal chromosome separation, apoptosis, and smaller brains. Persistent effects are evident by latent appearance of proneural glioblastoma in adult mice deleted additionally for the tumor suppressor p53 protein (p53). A previous line of mice deleted for REST in progenitors by conventional gene targeting does not exhibit these phenotypes, likely due to a remaining C-terminal peptide that still binds chromatin and recruits co-repressors. Our results suggest that REST-mediated chromatin remodeling is required in neural progenitors for proper S-phase dynamics, as part of its well-established role in repressing neuronal genes until terminal differentiation.

The inner ear is the sensory organ for hearing and balance. Its functional unit is the sensory patch that comprises: i) hair cells, which are the mechano- transducers sensing the stimuli and are embedded in the supporting cell layer, and ii) sensory neurons, which conduct these stimuli to the hindbrain. The generation of hair cells and neurons occurs in the otic placode early during embryonic development. Cell fate specification relies on expression of proneural genes and is concomitant with organ growth and morphogenesis. We used zebrafish embryos and combined live imaging and genetic tools to investigate: i) the location of the different progenitor pools, ii) the potentialities they exhibit, and iii) the dynamic behavior of these cells in generating the different fates. We have generated progenitor maps for the different cell fates by lineage tracing and focused our analysis on the behavioral changes of progenitors upon depletion of a proneural gene and the spatial and temporal aspects of cell fate specification.
L'oïda interna és l'òrgan sensorial responsable de l'audició i l'equilibri. La seva unitat funcional és el parxe sensorial que contèn: i) les cèl.lules ciliades, que són els mecano-transductors que detecten, i ii) les neurones sensorials, que envien aquests estímuls al cervell posterior. La generació de cèl.lules ciliades i de neurones te lloc a la placoda òtica molt aviat durant el desenvolupament embrionari . L'especificació del destí cel.lular es basa en l'expressió dels gens proneurals i és concomitant amb el creixement de l’òrgan i la seva morfogènesi. Hem utilitzat embrions de peix zebra i combinat imatges en viu amb eines genètiques per investigar: i) la ubicació dels diferents grups de progenitors, ii) les potencialitats que presenten, i iii) el comportament dinàmic d'aquestes cèl.lules en la generació dels diferents destins. Hem generat mapes progenitors pels diferents destins cel.lulars a partir d’experiments de llinatge i hem centrat la nostra anàlisi en els canvis de comportament dels progenitors després de la inactivació d'un gen proneural i els aspectes espacials i temporals de l'especificació de destí cel.lular.

L’inhibition latérale par Notch est un mécanisme bien conservé au sein des espèces qui permet la formation de pattern de destins cellulaires1. Dans de nombreux tissus, la signalisation intercellulaire entre Delta et Notch coordonne dans le temps et l’espace des décisions de destin cellulaire binaires dont l’origine est proposée stochastique. Dans le contexte du développement des organes sensoriels chez la Drosophile, il a été proposé que la rupture de symétrie entre cellules équipotentes dépendait de fluctuations aléatoires dans le niveau d’expression de Delta/Notch2 (ou d’un de ses régulateurs en amont, par exemple YAP1 dans l’intestin de la souris3), avec des petites différences qui sont amplifiées et stabilisées pour générer des destins distincts. La décision cellulaire stochastique médiée par Notch peut aussi être biaisée par des facteurs intrinsèques (par exemple, l’histoire de la cellule4) ou des facteurs extrinsèques. Bien que l’inhibition latérale ait été largement étudiée dans de nombreux contextes développementaux, il manque toujours une analyse détaillée in vivo de la dynamique de l’acquisition du destin cellulaire et des signaux régulant cette décision. Ici, nous avons utilisé une approche quantitative d’imagerie en temps réel pour étudier la dynamique de spécification des organes sensoriels dans l’abdomen de la drosophile. Pour suivre la compétence des cellules à s’engager dans le destin neural et devenir une cellule précurseur des organes sensoriels (SOP), nous avons utilisé l’accumulation du facteur de transcription Scute, un régulateur majeur de la formation des organes sensoriels dans l’abdomen. Pour visionner Scute directement dans les pupes en développement, nous avons utilisé des pupes exprimant la protéine Scute taguée par une GFP. Nous avons généré des films haute résolution dans le temps et l’espace puis nous avons segmenté et traqué tous les noyaux grâce un pipeline personnalisé. Nous avons ainsi pu étudier quantitativement la dynamique de l’expression de Scute dans toutes les cellules. Après avoir défini un index de différence de destin cellulaire (FDI), nous avons trouvé que la rupture de symétrie était détectée tôt, quand les cellules exprimaient encore un niveau faible et hétérogène de Scute. Quelques rares cas de résolution tardive ont été observés c’est-à-dire quand deux cellules voisines accumulent toutes les deux un fort niveau de Scute avant d’être séparées. Il est aussi intéressant de noter que le niveau de Scute n’a pas rapidement diminué dans les cellules non sélectionnées, immédiatement après la rupture de symétrie. D’autre part, nous avons trouvé une corrélation positive entre la pente du FDI après la rupture de symétrie et l’hétérogénéité intercellulaire mesurée dans le niveau de Scute mais il reste à démontrer si l’augmentation de l’hétérogénéité est causalement liée à la rupture de symétrie. Nous avons ensuite voulu savoir si cette décision cellulaire stochastique était biaisée par l’ordre de naissance (comme proposé dans le contexte de décision AC/VU chez le C. elegans4) ou par la taille et la géométrie des contacts cellulaires (comme suggéré par une modélisation5). Nous avons trouvé qu’aucun des deux biais ne semblait influencer la décision cellulaire binaire médiée par Notch dans l’abdomen de la Drosophile. En conclusion, nos données d’imagerie fournissent une analyse quantitative détaillée de la dynamique des proneuraux pendant l’inhibition latérale chez la Drosophile
Lateral inhibition by Notch is a conserved mechanism that regulates the formation of regular patterns of cell fates1. In many tissues, intercellular Delta-Notch signaling coordinates in time and space binary fate decisions thought to be stochastic. In the context of sensory organ development in Drosophila, it has been proposed that fate symmetry breaking between equipotent cells relies on random fluctuations in the level of Delta/Notch2 (or one of their upstream regulators, e.g. YAP1 in the mouse gut3), with small differences being amplified and stabilized to generate distinct fates. Notch-mediated stochastic fate choices may also be biased by intrinsic, i.e. cell history4, or extrinsic factors. Although lateral inhibition has been extensively studied in many developmental contexts, a detailed in vivo analysis of fate and signaling dynamics is still lacking. Here, we used a quantitative live imaging approach to study the dynamics of sensory organ fate specification in the Drosophila abdomen. The accumulation of the transcription factor Scute (Sc), a key regulator of sensory organ formation in the abdomen, was used as a proxy to monitor proneural competence and SOP fate acquisition in developing pupae expressing GFP-tagged Sc. We generated high spatial and temporal resolution movies and segmented/tracked all nuclei using a custom-made pipeline. This allowed us to quantitatively study Sc dynamics in all cells. Having defined a fate difference index (FDI), we found that symmetry breaking can be detected early, when cells expressed very low and heterogeneous levels of Sc. We also observed rare cases of late fate resolution, e.g. when two cells close to each other accumulate high levels of GFP-Scute before being pulled away from each other. Interestingly, we did not observe a rapid decrease in GFP-Sc levels in non-selected cells right after symmetry breaking. Also, the rate of change of FDI values after symmetry breaking appeared to positively correlate with cell-to-cell heterogeneity in Sc levels. Whether increased heterogeneity is causally linked to symmetry breaking remains to be tested. We next addressed if this stochastic fate decision is biased by birth order (as proposed in the context of the AC/VU decision in worms4) or by the size and geometry of cell-cell contacts (as modeling suggested5). We found that neither appeared to significantly influence Notch-mediated binary fate decisions in the Drosophila abdomen. In conclusion, our live imaging data provide a detailed analysis of proneural dynamics during lateral inhibition in Drosophila

The Lower Rhombic Lip (LRL) is a transient neuroepithelial structure of dorsal hindbrain that gives rise to deep brainstem nuclei like the vestibular, auditory and precerebellar nuclei. In this work, we have followed the LRL-progenitor cell population through early steps of neurogenesis and hindbrain morphogenesis to understand proneural function and progenitor dynamics during neuronal specification. We provide information about the atoh1 gene regulatory network operating in the specification of LRL cells, and the kinetics of cell proliferation and behaviour of atoh1a-derivatives by using functional and in vivo imaging strategies in the zebrafish embryo. We propose that atoh1a and atoh1b have subfunctionalized: atoh1a acts as the fate selector gene in LRL-progenitors, whereas atoh1b acts as the downstream neuronal differentiation gene carrying out the neurogenic program. Moreover, our in vivo cell lineage approaches revealed a regionalization of modes of division within the LRL, orchestrating the balance between neuronal differentiation and progenitor cell self-renewal.
El Llavi Ròmbic Inferior (LRI) és una estructura neuroepitelial transient del romboencèfal dorsal que genera nuclis profunds del tronc de l’encèfal, com ara els nuclis vestibulars, auditius i precerebel·lars. En aquest treball hem seguit la població progenitora del LRI durant els primers estadis neurogènics i de morfogènesi per entendre la funció proneural i la dinàmica dels progenitors durant l’especificació neuronal. Informem sobre la xarxa genètica reguladora depenent d’atoh1 que opera al LRI, així com del comportament proliferatiu i migratori de les cèl·lules derivades del LRI servint-nos d’experiments funcionals i d’imatge in vivo d’embrions de peix zebra. Proposem que atoh1a i atoh1b estan subfuncionalitzats: atoh1a actua com a gen selector dels progenitors LRI, mentre que atoh1b funciona sota atoh1a mantenint el seu programa neurogènic. A més, els estudis de llinatge cel·lular in vivo mostren la regionalització dels diferents modes de divisió, orquestrant així l’equilibri entre la diferenciació neuronal i l’auto-renovació progenitora.