Academic literature on the topic 'Neurogenesis; ubiquitin; embryonic stem cell'

The procedure of neurogenesis has made numerous achievements in the past decades, during which various molecular biomarkers have been emerging and have been broadly utilized for the investigation of embryonic and adult neural stem cell (NSC). Nevertheless, there is not a consistent and systematic illustration to depict the functional characteristics of the specific markers expressed in distinct cell types during the different stages of neurogenesis. Here we gathered and generalized a series of NSC biomarkers emerging during the procedures of embryonic and adult neural stem cell, which may be used to identify the subpopulation cells with distinguishing characters in different timeframes of neurogenesis. The identifications of cell patterns will provide applications to the detailed investigations of diverse developmental cell stages and the extents of cell differentiation, which will facilitate the tracing of cell time-course and fate determination of specific cell types and promote the further and literal discoveries of embryonic and adult neurogenesis. Meanwhile, via the utilization of comprehensive applications under the aiding of the systematic knowledge framework, researchers may broaden their insights into the derivation and establishment of novel technologies to analyze the more detailed process of embryogenesis and adult neurogenesis.

ABSTRACT The JNKs are components of stress signaling pathways but also regulate morphogenesis and differentiation. Previously, we invoked a role for the JNKs in nerve growth factor (NGF)-stimulated PC12 cell neural differentiation (L. Marek et al., J. Cell. Physiol. 201:459-469, 2004; E. Zentrich et al., J. Biol. Chem. 277:4110-4118, 2002). Herein, the role for JNKs in neural differentiation and transcriptional regulation of the marker gene, NFLC, modeled in mouse embryonic stem (ES) cells was studied. NFLC-luciferase reporters revealed the requirement for NFLC promoter sequences encompassing base pairs −128 to −98 relative to the transcriptional start site as well as a proximal cyclic AMP response element-activating transcription factor binding site at −45 to −38 base pairs for transcriptional induction in NGF-treated PC12 cells and neurally differentiated ES cells. The findings reveal common promoter sequences that integrate conserved signal pathways in both PC12 cell and ES cell systems. To test the requirement for the JNK pathway in ES cell neurogenesis, ES cell lines bearing homozygous disruptions of the jnk1, jnk2, or jnk3 genes were derived and submitted to an embryoid body (EB) differentiation protocol. Neural differentiation was observed in wild-type, JNK2−/−, and JNK3−/− cultures but not in JNK1−/− EBs. Rather, an outgrowth of cells with epithelial morphology and enhanced E-cadherin expression but low NFLC mRNA and protein was observed in JNK1−/− cultures. The expression of wnt-4 and wnt-6, identified inhibitors of ES cell neurogenesis, was significantly elevated in JNK1−/− cultures relative to wild-type, JNK2−/−, and JNK3−/− cultures. Moreover, the Wnt antagonist, sFRP-2, partially rescued neural differentiation in JNK1−/− cultures. Thus, a genetic approach using JNK-deficient ES cells reveals a novel role for JNK1 involving repression of Wnt expression in neural differentiation modeled in murine ES cells.

Abstract Quiescent neural stem cells (NSCs) in the adult mouse brain are the source of neurogenesis that regulates innate and adaptive behaviors. Adult NSCs in the subventricular zone (SVZ) are derived from a subpopulation of embryonic neural stem-progenitor cells (NPCs) that is characterized by a slower cell cycle relative to the more abundant rapid cycling NPCs that build the brain. We have previously shown that slow cell cycle can cause the establishment of adult NSCs at the SVZ, although the underlying mechanism remains unknown. We found that Notch and an effector Hey1 form a module that is upregulated by cell cycle arrest in slowly dividing NPCs. In contrast to the oscillatory expression of the Notch effectors Hes1 and Hes5 in fast cycling progenitors, Hey1 displays a non-oscillatory stationary expression pattern and contributes to the long-term maintenance of NSCs. These findings reveal a novel division of labor in Notch effectors where cell cycle rate biases effector selection and cell fate. I will also discuss the heterogeneity of slowly dividing embryonic NPCs and the lineage relationship between adult NSCs and ependymal cells, which together form the niche for adult neurogenesis at the SVZ.

Ubiquitination regulates nearly every aspect of cellular events in eukaryotes. It modifies intracellular proteins with 76-amino acid polypeptide ubiquitin (Ub) and destines them for proteolysis or activity alteration. Ubiquitination is generally achieved by a tri-enzyme machinery involving ubiquitin activating enzymes (E1), ubiquitin conjugating enzymes (E2) and ubiquitin ligases (E3). E1 activates Ub and transfers it to the active cysteine site of E2 via a transesterification reaction. E3 coordinates with E2 to mediate isopeptide bond formation between Ub and substrate protein. The E1-E2-E3 cascade can create diverse types of Ub modifications, hence effecting distinct outcomes on the substrate proteins. Dysregulation of ubiquitination results in severe consequences and human diseases. There include cancers, developmental defects and immune disorders. In this review, we provide an overview of the ubiquitination machinery and discuss the recent progresses in the ubiquitination-mediated regulation of embryonic stem cell maintenance and cancer biology.

SMG6 is an endonuclease, which cleaves mRNAs during nonsense-mediated mRNA decay (NMD), thereby regulating gene expression and controling mRNA quality. SMG6 has been shown as a differentiation license factor of totipotent embryonic stem cells. To investigate whether it controls the differentiation of lineage-specific pluripotent progenitor cells, we inactivated Smg6 in murine embryonic neural stem cells. Nestin-Cre-mediated deletion of Smg6 in mouse neuroprogenitor cells (NPCs) caused perinatal lethality. Mutant mice brains showed normal structure at E14.5 but great reduction of the cortical NPCs and late-born cortical neurons during later stages of neurogenesis (i.e., E18.5). Smg6 inactivation led to dramatic cell death in ganglionic eminence (GE) and a reduction of interneurons at E14.5. Interestingly, neurosphere assays showed self-renewal defects specifically in interneuron progenitors but not in cortical NPCs. RT-qPCR analysis revealed that the interneuron differentiation regulators Dlx1 and Dlx2 were reduced after Smg6 deletion. Intriguingly, when Smg6 was deleted specifically in cortical and hippocampal progenitors, the mutant mice were viable and showed normal size and architecture of the cortex at E18.5. Thus, SMG6 regulates cell fate in a cell type-specific manner and is more important for neuroprogenitors originating from the GE than for progenitors from the cortex.

ABSTRACT Ubiquitination and the degradation of the large subunit of RNA polymerase II, Rpb1, is not only involved in DNA damage-induced arrest but also in other transcription-obstructing events. However, the ubiquitin ligases responsible for DNA damage-independent processes in mammalian cells remain to be identified. Here, we identified Wwp2, a mouse HECT domain ubiquitin E3 ligase, as a novel ubiquitin ligase of Rpb1. We found that Wwp2 specifically interacted with mouse Rpb1 and targeted it for ubiquitination both in vitro and in vivo. Interestingly, the interaction with and ubiquitination of Rpb1 was dependent neither on its phosphorylation state nor on DNA damage. However, the enzymatic activity of Wwp2 was absolutely required for its ubiquitin modification of Rpb1. Furthermore, our study indicates that the interaction between Wwp2 and Rpb1 was mediated through WW domain of Wwp2 and C-terminal domain of Rpb1, respectively. Strikingly, downregulation of Wwp2 expression compromised Rpb1 ubiquitination and elevated its intracellular steady-state protein level significantly. Importantly, we identified six lysine residues in the C-terminal domain of Rpb1 as ubiquitin acceptor sites mediated by Wwp2. These results indicate that Wwp2 plays an important role in regulating expression of Rpb1 in normal physiological conditions.

One of the biggest challenges currently facing ES cell biology is to understand the mechanisms involved in the differentiation of ES cells to specific lineages. Pure populations of a specific cell lineage cannot be achieved without selection, such as fluorescence activated cell sorting (FACS), immunopanning or growing cells in a selective environment following genetic manipulation. However, such techniques do not address why ES cells do and do not differentiate to particular lineages and cell types. To achieve greater understanding of the mechanism of neuronal differentiation from ES cells, an ES cell line was generated with eGFP driven by the neuronal specific gene tau. Tau is expressed exclusively in all neurons from the earliest stages of neuronal commitment, we find that a neural differentiation of this line results in eGFP expressing neurons. Using FACS, a pure population of neurons can be obtained from a heterogeneous population of differentiated cells. Neuronal differentiation can be quantified either by fluorescent microscopy or flow cytometry. These ES cells have been used to analyse the effect that density and the addition of exogenous factors have on neuronal differentiation, a transcriptome analysis experiment was performed by microarray analysis. Genes already known to be important during mammalian neural development were analysed for their involvement in ES cell neurogenesis. This comparison revealed a strong correlation between events of ES cells differentiation and normal embryonic development. The microarray analysis of ES cell neurogenesis also identified genes with an expression profile suggestive of a role in ES cell neurogenesis and development of the murine nervous system.

The Hedgehog (Hh) signalling pathway is one of the key signalling pathways orchestrating intricate organogenesis, including the development of neural tube, heart and skeletal muscle. Yet, insufficient mechanistic understanding of its diverse roles is available. Here, we show the molecular mechanisms regulating the neurogenic, cardiogenic and myogenic properties of Hh signalling, via effector protein Gli2, in embryonic and adult stem cells. In Chapter 2, we show that Gli2 induces neurogenesis, whereas a dominant-negative form of Gli2 delays neurogenesis in P19 embryonal carcinoma (EC) cells, a mouse embryonic stem (ES) cell model. Furthermore, we demonstrate that Gli2 associates with Ascl1/Mash1 gene elements in differentiating P19 cells and activates the Ascl1/Mash1 promoter in vitro. Thus, Gli2 mediates neurogenesis in P19 cells at least in part by directly regulating Ascl1/Mash1 expression. In Chapter 3, we demonstrate that Gli2 and MEF2C bind each other’s regulatory elements and regulate each other’s expression while enhancing cardiomyogenesis in P19 cells. Furthermore, dominant-negative Gli2 and MEF2C proteins downregulate each other’s expression while imparing cardiomyogenesis. Lastly, we show that Gli2 and MEF2C form a protein complex, which synergistically activates cardiac muscle related promoters. In Chapter 4, we illustrate that Gli2 associates with MyoD gene elements while enhancing skeletal myogenesis in P19 cells and activates the MyoD promoter in vitro. Furthermore, inhibition of Hh signalling in muscle satellite cells and in proliferating myoblasts leads to reduction in MyoD and MEF2C expression. Finally, we demonstrate that endogenous Hh signalling is important for MyoD transcriptional activity and that Gli2, MEF2C and MyoD form a protein complex capable of inducing skeletal muscle-specific gene expression. Thus, Gli2, MEF2C and MyoD participate in a regulatory loop and form a protein complex capable of inducing skeletal muscle-specific gene expression. Our results provide a link between the regulation of tissue-restricted factors like Mash1, MEF2C and MyoD, and a general signal-regulated Gli2 transcription factor. We therefore provide novel mechanistic insights into the neurogenic, cardiogenic and myogenic properties of Gli2 in vitro, and offer novel plausible explanations for its in vivo functions. These results may also be important for the development of stem cell therapy strategies.

The cerebral cortex is one of the most complex and divergent of all biological structures and is composed of hundreds of different types of highly interconnected neurons. This complexity underlies its ability to perform exceedingly complex neural processes. One of the most important questions in developmental neurobiology is how such a vast degree of diversity and specificity is achieved during embryogenesis. Furthermore, understanding the cellular and genetic basis of cortical development may yield insights into the mechanisms underlying human disorders such as mental retardation, autism, epilepsies and brain tumors.

During this Phd-project, we set out to identify novel transcription factors involved in cortical neurogenesis. Therefore, we initially took advantage of a model of in vitro embryonic stem cell (ESC)-derived corticogenesis that was previously established in the lab (Gaspard et al. 2008) and from several previously generated ESC lines that allow overexpression of specific transcription factors potentially involved in corticogenesis (van den Ameele et al. 2012).

Among the genes tested, Bcl6, a B-cell lymphoma oncogene known to be expressed during cortical development but without well-characterized function in this context, displayed a strong proneurogenic activity and thus became the main focus of this thesis.

During neurogenesis, neural stem/progenitor cells (NPCs) undergo an irreversible fate transition to become neurons. The Notch pathway is well known to be important for this process, and repression of Notch-dependent Hes genes is essential for triggering differentiation. However, Notch signalling often remains active throughout neuronal differentiation, implying a change in the transcriptional responsiveness to Notch during the neurogenic transition.

We showed that Bcl6 starts to be expressed specifically during the transition from progenitors to postmitotic neurons and is required for proper neurogenesis of the mouse cerebral cortex. Bcl6 promotes this neurogenic conversion by switching the composition of Notch-dependent transcriptional complexes at the Hes5 promoter. Bcl6 triggers exclusion of the co-activator Mastermind-like 1 and recruitment of the NAD+-dependent deacetylase Sirt1, which we showed to be required for Bcl6-dependent neurogenesis in vitro. The resulting epigenetic silencing of Hes5 leads to neuronal differentiation despite active Notch signalling. These findings thus suggest a role for Bcl6 as a novel proneurogenic factor and uncover Notch-Bcl6-Sirt1 interactions that may affect other aspects of physiology and disease (Tiberi et al. 2012a).

A subsequent yet unpublished part of this Phd-project focused on unraveling roles for Bcl6 in regionalization of the cerebral cortex. In all mammals, the three major areas of the neocortex are the motor, somatosensory and visual areas, each subdivided in secondary domains and complemented with species-specific additional areas. All these domains comprise of neurons with different functionality, molecular profiles, electrical activity and connectivity. Spatial patterning of the cortex is mainly under the control of diffusible molecules produced by organizing centers, but is also regulated by intrinsic, cell-autonomous programs (Tiberi et al. 2012b).

Since Bcl6 expression is confined to frontal and parietal regions of the developing cerebral cortex and remains high in postmitotic neurons, also after completion of neurogenesis, we hypothesized it would be involved in acquisition of motor and somatosensory identity. As expected from the neurogenesis defect in these regions, we observed a trend towards a reduced size of the frontal areas in the Bcl6 mutant cortex. Preliminary data from cDNA microarray profiling after gain- and loss-of-function of Bcl6 and from in situ hybridization on mouse cortex however do not show dramatic changes in molecular markers of different cortical areas. Similarly, the coarse-grained pattern of thalamocortical and efferent projections of motor and somatosensory neurons appears to be spared. These preliminary findings thus suggest that Bcl6 is not strictly required for proper acquisition of motor and somatosensory areal identity.
Doctorat en Sciences médicales
info:eu-repo/semantics/nonPublished

Neural Progenitor Cells (NPCs) are the primordial cells of central nervous system (CNS). Understanding how they are regulated benefits our knowledge of normal development, the pathology of neurological disorders, and of therapeutic designs. One gene identified as a putative regulator of stem cell (SC) populations, including NPCs, by virtue of displaying commonly elevated expression in representative SC populations is USP9X. During development, USP9X mRNA is found highly expressed in the ventricular zones of the developing murine CNS. On this basis the USP9X protein, a substrate specific deubiquitylating enzyme, was hypothesized to be highly expressed in NPCs, and to function in the control of NPC behavior. USP9X protein was found enriched within the apical end-feet structures of NPCs, where it partially co-localised with N-cadherin. This expression domain is common to highly conserved NPC polarity imparting complexes, and to fate determinant networks, which are functionally integrated together to control NPC fate. Thus USP9X protein expression was consistent with a putative regulatory role in NPC fate. To identify cellular processes regulated by USP9X in NPCs, the effect of USP9X over-expression was analysed in embryonic stem cell (ESC)-derived NPCs. ESC lines were generated housing transgenes encoding USP9X under the transcriptional control of the human Nestin 2nd intron, and differentiated into neurons via NPC intermediates. The nestin-USP9X transgene expression resulted in two phenomena. First, it produced a dramatically altered cellular architecture wherein the majority (over 80%) of NPCs were arranged into ‘rosette’ colonies. These NPCs expressed markers of Radial Glial cells, named radial progenitors (RPs) thereafter, and were highly polarised akin to their in-vivo counterparts. Second, USP9X over expression caused a five-fold percentage increase of RPs and neurons. BrdU labelling, as well as the examination of the RP:neuron ratio indicated that nestin-USP9X enhanced the self-renewal of RPs but did not block their subsequent differentiation to neurons and astrocytes. nestin-USP9X RPs reformed rosette colonies following passage as single cells whereas control cells did not, suggesting it aids the establishment of polarity. From these data it was proposed that USP9X-induced polarisation of NPCs, provides an environment conducive for self-renewal. The nestin-USP9X transgene was subsequently used to generate transgenic embryos. Initially, three founding embryos were analysed. In two of three nestin-USP9X embryos, thickening, convolution, and disorganised CNS tissues were observed. A further four nestin- USP9X transgenic embryos generated through the breeding of a transgenic line revealed relatively milder defects of thickened CNS tissues. These effects are speculated to result from expansion of the NPC population, but await further experimental investigation. Together this study identifies USP9X as a regulator of NPC function. In-vivo, USP9X was found highly enriched in the apical end-feet structures, common to molecular networks that regulate NPC fate, and that also have components known to be USP9X substrates. In ESC derived NPCs, USP9X over-expression promoted polarity and self-renewal, which was also speculated to occur in NPCs in-vivo upon limited analysis. This work affirms the intrinsic relationship between polarity and NPC fate decisions, which is here suggested to be coordinated by USP9X regulated pathways.
Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Sciences, 2010

Evidence reports the key roles of lncRNAs in several regulatory mechanisms of neurons and other brain cells. Neuronal lncRNAs are crucial for NSCs mediated-neuronal developmental stages like neurogenesis, neuronal differentiation, and synaptogenesis. Moreover, multilineage properties of NSCs and their association to specific cell types render them to identify the commonly accepted biomarkers for the brain. It is important to delineate the correlation between lncRNAs and NSCs fate decisions during neuronal development stages. In this review, we will summarize how NSCs fabricate embryonic tissue architecture of the central nervous system (CNS) and act as residuum in subventricular zone (SVZ) nearby the lateral wall of the lateral ventricles and the subgranular zone (SGZ) of hippocampus dentate gyrus (DG) of the adult brain. Additionally, describe the roles and molecular mechanisms of lncRNAs involved in NSCs self-renewal, neurogenesis, gliogenesis and synaptogenesis over the course of neural development. This will help us to better understand neuronal physiology.