Différenciation et plasticité des cellules souches neurales

The study of cellular plasticity is a powerful tool to understand the mechanisms directing cell fate choice during differentiation and transformation of a normal cell into a cancerous one. In Drosophila, the transcription factor Gcm control glial fate determination. In gcm mutants, cells that normally develop into glia enter the path of neuronal differentiation, whereas ectopic expression of gcm in neural progenitors induces glia. These properties make gcm an important tool for understanding the basics of cellular plasticity. My thesis project aims to understand the mechanisms controlling the plasticity of neural stem cells (NSCs). Based on this aim, we showed that the ability of NSCs to be transformed into glia, after forced expression of Gcm, declines with age and that upon entry into quiescence or apoptosis, they cannot be converted. We also found that the process of fate conversion does not manifest itself only through the expression of glial markers but also by specific changes in the level of chromatin. Remarkably, we also showed that the stability of the protein Gcm is controlled by two opposite and interconnected loops, where Repo and the histone acetyltransferase CBP play a major role.L’étude de la plasticité cellulaire est un puissant outil pour comprendre le choix du destin cellulaire pendant la différenciation et dans les processus cancéreux lors de la transformation d’une cellule normale en une cellule maligne. Chez la drosophile, le facteur de transcription Gcm contrôle la détermination du destin glial. Dans des mutants gcm, les cellules qui se développent normalement en glie entrent dans la voie de différenciation neuronale alors que l’expression ectopique de gcm dans des progéniteurs neuronaux induit de la glie. Ces données font de Gcm un outil important pour comprendre les bases de la plasticité cellulaire. Mon projet de thèse vise à comprendre les mécanismes contrôlant la plasticité des cellules souches neurales. Nous avons ainsi montré que la capacité des CSNs à se convertir en glie après expression forcée de Glide/Gcm décline avec l'âge et que lors de l'entrée en phase quiescente ou apoptotique, ils ne peuvent plus être convertis. Nous avons aussi découvert que le processus de conversion du destin ne se manifeste pas uniquement par l’expression de marqueurs gliaux mais aussi par des changements spécifiques au niveau de la chromatine. D’une manière intéressante, nous avons aussi montré que la stabilité de la protéine Glide/Gcm est contrôlée par deux voies opposées, où Repo et l’histone acetyltransférase CBP jouent un rôle majeur

Neural stem cells plasticity and differentiation

L’étude de la plasticité cellulaire est un puissant outil pour comprendre le choix du destin cellulaire pendant la différenciation et dans les processus cancéreux lors de la transformation d’une cellule normale en une cellule maligne. Chez la drosophile, le facteur de transcription Gcm contrôle la détermination du destin glial. Dans des mutants gcm, les cellules qui se développent normalement en glie entrent dans la voie de différenciation neuronale alors que l’expression ectopique de gcm dans des progéniteurs neuronaux induit de la glie. Ces données font de Gcm un outil important pour comprendre les bases de la plasticité cellulaire. Mon projet de thèse vise à comprendre les mécanismes contrôlant la plasticité des cellules souches neurales. Nous avons ainsi montré que la capacité des CSNs à se convertir en glie après expression forcée de Glide/Gcm décline avec l'âge et que lors de l'entrée en phase quiescente ou apoptotique, ils ne peuvent plus être convertis. Nous avons aussi découvert que le processus de conversion du destin ne se manifeste pas uniquement par l’expression de marqueurs gliaux mais aussi par des changements spécifiques au niveau de la chromatine. D’une manière intéressante, nous avons aussi montré que la stabilité de la protéine Glide/Gcm est contrôlée par deux voies opposées, où Repo et l’histone acetyltransférase CBP jouent un rôle majeur.The study of cellular plasticity is a powerful tool to understand the mechanisms directing cell fate choice during differentiation and transformation of a normal cell into a cancerous one. In Drosophila, the transcription factor Gcm control glial fate determination. In gcm mutants, cells that normally develop into glia enter the path of neuronal differentiation, whereas ectopic expression of gcm in neural progenitors induces glia. These properties make gcm an important tool for understanding the basics of cellular plasticity. My thesis project aims to understand the mechanisms controlling the plasticity of neural stem cells (NSCs). Based on this aim, we showed that the ability of NSCs to be transformed into glia, after forced expression of Gcm, declines with age and that upon entry into quiescence or apoptosis, they cannot be converted. We also found that the process of fate conversion does not manifest itself only through the expression of glial markers but also by specific changes in the level of chromatin. Remarkably, we also showed that the stability of the protein Gcm is controlled by two opposite and interconnected loops, where Repo and the histone acetyltransferase CBP play a major role

Neural stem cells plasticity and differentiation

L étude de la plasticité cellulaire est un puissant outil pour comprendre le choix du destin cellulaire pendant la différenciation et dans les processus cancéreux lors de la transformation d une cellule normale en une cellule maligne. Chez la drosophile, le facteur de transcription Gcm contrôle la détermination du destin glial. Dans des mutants gcm, les cellules qui se développent normalement en glie entrent dans la voie de différenciation neuronale alors que l expression ectopique de gcm dans des progéniteurs neuronaux induit de la glie. Ces données font de Gcm un outil important pour comprendre les bases de la plasticité cellulaire. Mon projet de thèse vise à comprendre les mécanismes contrôlant la plasticité des cellules souches neurales. Nous avons ainsi montré que la capacité des CSNs à se convertir en glie après expression forcée de Glide/Gcm décline avec l'âge et que lors de l'entrée en phase quiescente ou apoptotique, ils ne peuvent plus être convertis. Nous avons aussi découvert que le processus de conversion du destin ne se manifeste pas uniquement par l expression de marqueurs gliaux mais aussi par des changements spécifiques au niveau de la chromatine. D une manière intéressante, nous avons aussi montré que la stabilité de la protéine Glide/Gcm est contrôlée par deux voies opposées, où Repo et l histone acetyltransférase CBP jouent un rôle majeur.The study of cellular plasticity is a powerful tool to understand the mechanisms directing cell fate choice during differentiation and transformation of a normal cell into a cancerous one. In Drosophila, the transcription factor Gcm control glial fate determination. In gcm mutants, cells that normally develop into glia enter the path of neuronal differentiation, whereas ectopic expression of gcm in neural progenitors induces glia. These properties make gcm an important tool for understanding the basics of cellular plasticity. My thesis project aims to understand the mechanisms controlling the plasticity of neural stem cells (NSCs). Based on this aim, we showed that the ability of NSCs to be transformed into glia, after forced expression of Gcm, declines with age and that upon entry into quiescence or apoptosis, they cannot be converted. We also found that the process of fate conversion does not manifest itself only through the expression of glial markers but also by specific changes in the level of chromatin. Remarkably, we also showed that the stability of the protein Gcm is controlled by two opposite and interconnected loops, where Repo and the histone acetyltransferase CBP play a major role.STRASBOURG-Bib.electronique 063 (674829902) / SudocSudocFranceF

Inhibition of SoxB2 or HDACs suppresses Hydractinia head regeneration by affecting blastema formation

Regeneration has long been known to occur in the cnidarian Hydractinia. This process refers to its ability to regrow structures, i.e a head, lost by injury, a phenomenon that depends on the migration of proliferative cells to the site of injury, and the formation of a blastema, a mass of undifferentiated cells that will restore the missing head tissues. In our study, we showed that members of SoxB transcription factors and HDACs are involved in the regulation of Hydractinia neurogenesis in tissue homeostasis and regeneration. Particularly, we revealed that knockdown of SoxB2 or Hdac2 (a class I HDAC) knockdown, or inhibition of HDAC activity, suppress head regeneration. Here, we show that SoxB2 knockdown, or the inhibition of HDACs activity by TSA, a HDAC Class I and II inhibitor, interfere with head regeneration by affecting the migration of proliferative cells and the formation of a proliferative blastema

Interlocked loops trigger lineage specification and stable fates in the Drosophila nervous system

Multipotent precursors are plastic cells that generate different, stable fates at the correct number, place and time, to allow tissue and organ formation. While fate determinants are known to trigger specific transcriptional programs, the molecular pathway driving the progression from multipotent precursors towards stable and specific identities remains poorly understood. Here we demonstrate that, in Drosophila neural precursors, the glial determinant glial cell missing (Gcm) acts as a 'time bomb' and triggers its own degradation once the glial programme is stably activated. This requires a sequence of transcriptional and post-transcriptional loops, whereby a Gcm target first affects the expression and then acetylation of the fate determinant, thus controlling Gcm levels and stability over time. Defective homeostasis between the loops alters the neuron: glia ratio and freezes cells in an intermediate glial/neuronal phenotype. In sum, we identify an efficient strategy triggering cell identity, a process altered in pathological conditions such as cancer

Interlocked loops trigger lineage specification and stable fates in the Drosophila nervous system

Multipotent precursors are plastic cells that generate different, stable fates at the correct number, place and time, to allow tissue and organ formation. While fate determinants are known to trigger specific transcriptional programs, the molecular pathway driving the progression from multipotent precursors towards stable and specific identities remains poorly understood. Here we demonstrate that, in Drosophila neural precursors, the glial determinant glial cell missing (Gcm) acts as a 'time bomb' and triggers its own degradation once the glial programme is stably activated. This requires a sequence of transcriptional and posttranscriptional loops, whereby a Gcm target first affects the expression and then acetylation of the fate determinant, thus controlling Gcm levels and stability over time. Defective homeostasis between the loops alters the neuron:glia ratio and freezes cells in an intermediate glial/neuronal phenotype. In sum, we identify an efficient strategy triggering cell identity, a process altered in pathological conditions such as cancer

An evolutionarily conserved soxb-hdac2 crosstalk regulates neurogenesis in a cnidarian

SoxB transcription factors and histone deacetylases (HDACs) are each major players in the regulation of neurogenesis, but a functional link between them has not been previously demonstrated. Here, we show that SoxB2 and Hdac2 act together to regulate neurogenesis in the cnidarian Hydractinia echinata during tissue homeostasis and head regeneration. We find that misexpression of SoxB genes modifies the number of neural cells in all life stages and interferes with head regeneration. Hdac2 was coexpressed with SoxB2, and its downregulation phe-nocopied SoxB2 knockdown. We also show that SoxB2 and Hdac2 promote each other\u27s transcript levels, but Hdac2 counteracts this amplification cycle by deacetylating and destabilizing SoxB2 protein. Finally, we present evidence for conservation of these interactions in human neural progenitors. We hypothesize that crosstalk between SoxB transcription factors and Hdac2 is an ancient feature of metazoan neurogenesis and functions to stabilize the correct levels of these multifunctional proteins

CRISPR/Cas9-mediated gene knockin in the hydroid Hydractinia symbiolongicarpus

Abstract Background Hydractinia symbiolongicarpus, a colonial cnidarian, is a tractable model system for many cnidarian-specific and general biological questions. Until recently, tests of gene function in Hydractinia have relied on laborious forward genetic approaches, randomly integrated transgenes, or transient knockdown of mRNAs. Results Here, we report the use of CRISPR/Cas9 genome editing to generate targeted genomic insertions in H. symbiolonigcarpus. We used CRISPR/Cas9 to promote homologous recombination of two fluorescent reporters, eGFP and tdTomato, into the Eukaryotic elongation factor 1 alpha (Eef1a) locus. We demonstrate that the transgenes are expressed ubiquitously and are stable over two generations of breeding. We further demonstrate that CRISPR/Cas9 genome editing can be used to mark endogenous proteins with FLAG or StrepII-FLAG affinity tags to enable in vivo and ex vivo protein studies. Conclusions This is the first account of CRISPR/Cas9 mediated knockins in Hydractinia and the first example of the germline transmission of a CRISPR/Cas9 inserted transgene in a cnidarian. The ability to precisely insert exogenous DNA into the Hydractinia genome will enable sophisticated genetic studies and further development of functional genomics tools in this understudied cnidarian model

A cellular and molecular analysis of SoxB-driven neurogenesis in a cnidarian

Neurogenesis is the generation of neurons from stem cells, a process that is regulated by SoxB transcription factors (TFs) in many animals. Although the roles of these TFs are well understood in bilaterians, how their neural function evolved is unclear. Here, we use Hydractinia symbiolongicarpus, a member of the early-branching phylum Cnidaria, to provide insight into this question. Using a combination of mRNA in situ hybridization, transgenesis, gene knockdown, transcriptomics, and in vivo imaging, we provide a comprehensive molecular and cellular analysis of neurogenesis during embryogenesis, homeostasis, and regeneration in this animal. We show that SoxB genes act sequentially at least in some cases. Stem cells expressing Piwi1 and Soxb1, which have broad developmental potential, become neural progenitors that express Soxb2 before differentiating into mature neural cells. Knockdown of SoxB genes resulted in complex defects in embryonic neurogenesis. Hydractinia neural cells differentiate while migrating from the aboral to the oral end of the animal, but it is unclear whether migration per se or exposure to different microenvironments is the main driver of their fate determination. Our data constitute a rich resource for studies aiming at addressing this question, which is at the heart of understanding the origin and development of animal nervous systems