Francois Guillemot: Projects

Regulation of brain stem cells by extracellular signals

In the embryonic brain, progenitor cells adopt specific fates in response to signals from their environment, including morphogens such as sonic hedgehog or Wnts that act at long distances, and membrane-bound signals acting on neighbouring cells such as Notch ligands. In neurogenic regions in the adult brain (hippocampus and subventricular zone), in addition to morphogens and Notch signals, neurotransmitters released by local neurons or axons projecting from distant neurons provide feedback signals that adjust stem cell activity to the requirements of the surrounding neuronal circuits.

We wish to understand how neural stem cells interpret these various extracellular signals to select an appropriate cellular response. We are also asking whether stem cell populations in the embryonic and adult brain are heterogeneous in their response to signals and in their behaviour. For example, are there distinct pools of stem cells in the adult hippocampus or subventricular zone that receive different signals and have different probabilities of self-renewing or differentiating into neurons or glial cells?

To examine the interactions between neural stem cells and their surrounding niche and investigate the heterogeneity of the signaling state and cellular behaviour of stem cells, we use in vivo imaging and single cell genomic approaches in the embryonic and adult brain. We use primary cultures of neural stem cells to examine the response of stem cells to extracellular signals or combinations of signals with biochemical and genomic approaches.

To analyse the signaling activity of neural stem cells in vivo, we use reporter mice (transgenic mice in which fluorescent reporter proteins are induced by a specific pathway such as Shh, Wnt, Notch or BMP) and single cell transcript profiling. We are establishing electrophysiological recording techniques to analyse the response of stem cells of the adult hippocampus to neurotransmitters. We also examine the behavioural response of stem cells to extracellular signals by fate mapping stem cell subsets and by examining the impact of mutations in signaling pathway components on stem cell behaviour by in vivo clonal analysis.

To determine how extracellular signals regulate stem cell fates and whether different signals synergise or antagonize each other, we identify the signaling pathway mobilised by extracellular signals as well as the gene expression programmes they induce, and the resulting changes in cell proliferation and differentiation, by performing proteomics, transcriptomics and imaging of stem cell cultures. We are also developing imaging approaches in organotypic slices and in vivo to analyse stem cell behaviour in situ and in real time.

In the publications listed below, we have shown that the transcription factor Ascl1 is essential for the exit from quiescence and proliferation of stem cells of the adult hippocampus and subventricular zone. We also showed that Ascl1 expression is induced by kainic acid, a signal that mimics neuronal activity, and that it is repressed by Notch signaling, a pathway that maintains stem cell quiescence. We concluded that Ascl1 is an essential component of the machinery that integrates stimulatory and inhibitory signals from the environment and selects the resulting stem cell fate (Andersen et al., 2014).

We have shown that the stability of Ascl1 protein is regulated by the E3-ubiquitin ligase Huwe1 and that destabilisation of Ascl1 by Huwe1 is required for proliferating stem cells of the adult hippocampus to return to quiescence. When Huwe1 is mutated and Ascl accumulates, stem cells fail to return to quiescence and the proliferative stem cell pool becomes depleted in older mice. We deduced from the result the existence of two fundamentally different pools of quiescent stem cells in the adult mouse hippocampus: a pool of resting cells that have previously proliferated, have returned transiently to quiescence, and are preferentially re-activated by niche signals, and a pool of dormant stem cells that have never proliferated and are activated only infrequently and relatively independently from niche signals (Urban et al., 2016)

We have also shown that exposing cultures of mouse neural stem cells to different extracellular signals is sufficient to drive them to either an active or a quiescent state. While the growth factors EGF and FGF are known to promote neural stem cell proliferation, we showed that substituting EGF by BMP4 is sufficient to induce a state of quiescence (reversible cell cycle arrest), which is very similar, both physiologically and in gene expression, to the quiescent state of neural stem cells and other tissue stem cells in vivo (Martynoga et al., 2013).

Figure 1

Figure 1: model of the signaling interactions between the neural stem cells and the cellular components of the niche in the adult hippocampus. Mitogenic signals are symbolised with green arrows and quiescence signals with red bars.

Figure 2

Figure 2: Neural stem cell culture induced into quiescence by BMP4 and stained for the stem cell markers GFAP (green) and nestin (red) and the nuclear marker Dapi (blue).

Selected publications

Urban, N., van den Berg, D.L.C., Forget, A., Andersen, J., Demmers, J.A., Hunt, C., Ayrault, O., Guillemot, F. (2016). Return to quiescence of mouse neural stem cells by degradation of a pro-activation protein. Science 353, 292-295.

Andersen, J., Urbán, N., Achimastou, A., Ito, A., Simic, M., Ullom, K., Martynoga, B., Lebel, M., Göritz, C., Frisén, J., Nakafuku, M., Guillemot F. (2014) A transcriptional mechanism integrating inputs from extracellular signals to activate hippocampal stem cellsNeuron, 83, 1085-1097.

Martynoga, B., Mateo, J.L., Zhou, B., Andersen, J., Achimastou, A., Urbán, N., van den Berg, D., Georgopoulou, D. , Hadjur, S., Wittbrodt, J., Ettwiller,L., Piper, L., Gronostajski, R.M., Guillemot, F. (2013). Epigenomic enhancer annotation reveals a key role for NFIX in neural stem cell quiescenceGenes Dev. 27, 1769-1786.

Specification of cellular identities in the brain

We study the transcription factors that specify the neuronal or glial fates of multipotent stem cells in the embryonic and adult brain and characterise the mechanisms that underlie their activity, including the downstream transcriptional networks that induce and stabilise cellular identities, and the epigenetic mechanisms that determine the temporal competence of progenitors to adopt particular fates.

In the developing cerebral cortex, progenitors generate sequentially different subtypes of neurons that occupy progressively more superficial layers of the cortex. The competence of progenitors to produce neurons of a particular laminar identity becomes progressively restricted during embryonic development.

In the unpublished study listed below, we have identified a novel function of the proneural transcription factor Neurog2 in specification of a particular subtype of neurons in the layer 6 of the mouse cortex, cortico-thalamic neurons. Overexpression and silencing experiments by in utero electroporation in the embryonic cortex have shown that Neurog2 is required to specify cortico-thalamic neurons (projecting to the thalamus) and to suppress the alternative layer 6 cortico-cortical identity (neurons projecting to the contralateral cortical hemisphere).

We have identified several transcription factors that are directly regulated by Neurog2 and form a regulatory network that establishes the cortico-thalamic identity.

Layer 6 neurons are generated during a brief time window (at embryonic day 12.5 in the mouse). While forced expression of Neurog2 by in utero electroporation of cortical progenitors at embryonic day 12.5 generates extra cortico-thalamic neurons, the same experiment performed at embryonic day 13.5 has no such effect, demonstrating that progenitors have then lost the competence to respond to Neurog2 and differentiate into layer 6 neurons.

Chromatin immunoprecipitation in day 13.5 electroporated progenitors has shown that Neurog2 retains the capacity to bind to the regulatory elements of its target genes but that an histone modification that marks compact chromatin has accumulated at Neurog2 binding sites.

Inactivation of components of the Polycomb Repressive Complex, which is involved in chromatin compaction and transcriptional silencing, in embryonic day 13.5 progenitors restores the capacity of ectopic Neurog2 to induce the formation of cortico-thalamic neurons, demonstrating that the Polycomb Repressive Complex is in part responsible for the loss of competence of day 13.5 progenitors to generate layer 6 neurons.

Interestingly, if the same experiment is performed at embryonic day 14.5, no cortico-thalamic neuron is generated demonstrating that an additional mechanism, which is independent of the Polycomb Repressive Complex and remains unidentified, restricts progenitor competence at this stage.

Figure 1

Figure 1: scheme showing that progenitors at different stages of cortical development generate neurons destined to different layers of the cortex. This is thought to be due to sequential changes in their competence to generate neurons of different identities.

Figure 2

Figure 2: Experiment of in utero electroporation of Neurog2, a determinant of layer 6 cortico-thalamic neurons, demonstrating that progenitors electroporated at embryonic day 13.5 (marked by GFP) have lost the competence to respond to Neurog2 and produce cortico-thalamic neurons (marked by Zfpm2). However, silencing of Eed, a component of Polycomb Repressor Complex 2, restores the competence of Neurog2-expressing embryonic day 13.5 progenitors to differentiate into Zfpm2+ layer 6 cortico-thalamic neurons.

Selected publication

Oishi, K., van den Berg, D., Guillemot, F. (2015).Redundant mechanisms provide robust temporal control to the specification of cortico-thalamic neuron identity by proneural Neurogenin factors. In preparation.

Transcriptional programmes of neurogenesis

We study the gene expression programmes that underlie the acquisition of different fates by neural stem cells. Which genes are induced or repressed when stem cells remain dormant, or self-renew, or differentiate? How are these genes regulated in a coordinated manner? How is the timing of gene expression controlled during the progression of cellular differentiation?

We use large-scale genomic approaches (RNA-Seq, ChIP-Seq, proteomics) to characterise gene expression programmes in mouse and human neural stem cells and their differentiated progenies, obtained either by FACS-purification from the embryonic or postnatal brain or from stem cell culture. We examine the contribution of different families of transcription factors in the regulation of these gene expression programmes, asking for example if master transcription factors implement stem cell fates by mobilising networks of more specialised transcription regulators or by directly regulating effector molecules that are involved in the acquisition of cellular phenotypes.

We also investigate the molecular mechanisms that determine the range of target genes that are regulated by a master transcription factor in a particular context, including the mechanisms that control the switch of activity of the same factor in mitotic progenitors versus postmitotic neurons, and the mechanisms that control the regulation of different targets at different times during progression of a differentiation programme.

We focus in particular on epigenetic mechanisms, such as posttranslational modifications of histones influencing chromatin compaction at target loci, and on the role of master transcription factors and other components of neurogenic programmes in recruiting chromatin modifying enzymes. We also examine the role of post-translational modifications such as phosphorylations by signaling pathways, in the regulation of the binding of transcription factors to the DNA and their interactions with transcriptional co-regulators.

In the publications listed below, we have systematically characterised the regulatory elements (enhancers and promoters) active in self-renewing mouse neural stem cells, by genome-wide mapping of accessible DNA and of several histone modifications. We have predicted with motif search algorithms and machine learning, the transcription factor network recruited at these active elements, and we have validated these predictions by demonstrating a role of the transcription factor Olig2 in activation of proliferation genes and suppression of differentiation and quiescence genes (Mateo et al. 2015).

We have also compared the enhancer elements that are active in proliferating versus quiescent neural stem cells, by genome-wide mapping of a histone modification and a co-activator in mouse neural stem cells cultivated in the presence of different signaling molecules (EGF and BMP4, respectively). We then bioinformatically predicted that different transcription factor families are recruited at proliferation-specific and quiescence-specific enhancers, respectively, and identified NFIX as an essential transcription factor for regulation of quiescence-specific genes in culture and for maintenance of quiescence in stem cells of the adult mouse hippocampus in vivo (Martynoga et al., 2014).

We have identified on a genome scale the genes regulated by the proneural transcription factor Ascl1/Mash1 in the mouse embryonic brain and in neural stem cell cultures. Analysis of the regulated genes unexpectedly identified core cell cycle regulators (e.g. cyclins, cdks, E2f genes), which led to the demonstration of a novel function of this important regulator of neurogenesis in promoting the proliferation of progenitor cells in the embryonic brain (Castro et al., 2011).

Figure 1

Figure 1: clustering of regulatory regions in the genome of neural stem cells based on the recruitment of different transcription factors and the presence of different histone modifications.

Figure 2

Figure 2: example of an enhancer element in the Ccnd2 gene identified by the recruitment of the co-activator p300 and the histone modification H3K27ac that is occupied by the proneural factor Ascl1.

Selected publications

Mateo, J.L., van den Berg, D.L.C., Haeussler, M., Drechsel, D., Gaber, Z.B., Castro, D.S., Robson, P., Crawford, G.E., Flicek, P., Ettwiller, L., Wittbrodt, J., Guillemot, F., Martynoga, B. (2015). Characterisation of the neural stem cell gene regulatory network identifies Olig2 as a multi-functional regulator of self-renewalGenome Res. 25, 41-56.

Martynoga, B., Mateo, J.L., Zhou, B., Andersen, J., Achimastou, A., Urbán, N., van den Berg, D., Georgopoulou, D. , Hadjur, S., Wittbrodt, J., Ettwiller,L., Piper, L., Gronostajski, R.M., Guillemot, F. (2013). Epigenomic enhancer annotation reveals a key role for NFIX in neural stem cell quiescenceGenes Dev. 27, 1769-1786.

Castro, D.S., Martynoga, B., Parras, C., Ramesh, V., Pacary, E., Johnston, C., Drechsel, D., Lebel-Potter, M., Galinanes-Garcia, L., Hunt, C., Dolle, D., Bithell, A., Ettwiller, L., Buckley, N., Guillemot, F. (2011). A novel function of the proneural factor Ascl1 in progenitor proliferation identified by genome-wide characterization of its targetsGenes Dev. 25, 930-945.

Regulation of neuronal migration

We study neuronal migration as a powerful model to investigate how a cellular behaviour is controlled at the genomic level. The process of neuronal migration is complex but can be readily visualised and quantified, and many of the genes involved have already been identified in fibroblasts and other simple cell culture models. Our aim is to identify the transcriptional module that is activated in a newborn neuron to promote its migration, and to determine how regulatory modules for migration differ in neurons that use different modes of migration (e.g. radial migration of cortical projection neurons, tangential migration of cortical interneurons, chain migration of adult-born olfactory interneurons).

We use in utero electroporation and time-lapse video-microscopy of organotypic cortical slices to image neuronal migration and to study the regulation and function of candidate genes and identify the downstream pathways regulating this process.

In the publications listed below, we have identified the microcephaly gene Cenpj/Cpap as a transcriptional target of the proneural factor Ascl1 in progenitors and newborn neurons of the embryonic cerebral cortex. We have shown that the Cenpj protein, which is associated with the centrosome, regulates the orientation of the mitotic spindle during progenitor division and is required to destabilise the microtubule cytoskeleton and to couple the nucleus with the centrosome during radial migration. Cenpj is therefore an essential component of the programme neurogenesis activated by Ascl1, which regulates both progenitor divisions and neuronal migration (Garcez et al., 2015).

We have shown that Ascl1 also promotes the migration of cortical neurons by inducing the expression of the small GTP-binding protein Rnd3, and that an interaction of Rnd3 with the semaphoring receptor Plexin B2 fine-tunes the level of RhoA signaling in migrating neurons. Thus the convergence of a cell extrinsic Plexin pathway and a cell intrinsic proneural factor-small GTP-binding protein pathway determines the level of RhoA activity appropriate for neuronal migration (Azzarelli et al., 2014).

We have also found that the two proneural transcription factors co-expressed in cortical progenitors, Neurog2 and Ascl1, promote the migration of newborn neurons, each through induction of the expression of a different small GTP-binding protein, Rnd2 and Rnd3, respectively. Neurog2 and Ascl1 have distinct roles in migration because they induce the expression of Rnd2 and Rnd3 at different times in the migration process and because Rnd2 and Rnd3 are located and regulate RhoA activity in different compartments of the migrating neuron. Therefore proneural factors regulate neuronal migration transcriptionally by directly controlling the spatio-temporal activity of the cytoskeleton regulator RhoA (Pacary et al., 2011).

Figure 1

Figure 1: migrating neurons in the cerebral cortex of a mouse embryo, labelled by in utero electroporation of Green Fluorescent Protein.

Figure 2

Figure 2: analysis by Fluorescence Resonance Energy Transfer (FRET) of RhoA signaling in cortical neurons following silencing of proneural factor targets Rnd2 and Rnd3.

Selected publications

Garcez, P.P., Diaz-Alonso, J., Crespo-Enriquez, I., Castro, D., Bell, D., Guillemot, F. (2015). Cenpj/CPAP regulates progenitor divisions and neuronal migration in the cerebral cortex downstream of Ascl1Nat. Commun. In press. 6, 6474.

Azzarelli, R., Pacary, E., Garg, R., Garcez, P., van den Berg, D., Riou, P., Ridley, A.J., Friedel, R.H., Parsons, M., Guillemot, F. (2014). An antagonistic interaction between PlexinB2 and Rnd3 controls RhoA activity and cortical neuron migrationNat. Commun. 5, 3405.

Pacary, E., Heng, J., Azzarelli, R., Riou, P., Castro, D., Lebel-Potter, M., Parras, C., Bell, D.M., Ridley, A.J., Parsons, M., Guillemot. F. (2011). Proneural transcription factors regulate different steps of cortical neuron migration through Rnd-mediated inhibition of RhoA signallingNeuron 69, 1069-84.

Transcriptional and epigenetic regulators of human brain development associated with neurodevelopmental disorders

Neuronal activity in the cerebral cortex underlies cognition and other fundamental functions of the human brain. Mutations in genes with essential roles in development of the cerebral cortex result in neurodevelopmental disorders such as intellectual disability, autism and epilepsy. We study the role of several neurodevelopmental disease-causing genes that code for transcription factors and chromatin remodelling factors, to gain insights into both the normal development of the human brain and the mechanisms that underlie prevalent disorders of human cognition.

Large scale sequencing of individuals with intellectual disability has revealed that heterozygous mutations in genes encoding subunits of the BAF SWI/SNF complex, including ARID1B and BCL11A, are among the most common causes of this pathology. The BAF SWI/SNF complex is a major regulator of chromatin remodelling and transcriptional regulation.

We havebegun to elucidate the mechanisms by which loss of one copy of BCL11A or ARID1B affects brain development, using mouse models (Dias et al., 2016 and unpublished data). These mice recapitulate some of the anatomical and behavioural features of the disease in humans. Comparison of gene expression profiles in the brains of mutant and control mice has provided evidence for the involvement of these genes in transcriptional regulation in the developing and adult brain.

We are now extending these findings by studying the function of various disease-associated chromatin remodellers and transcription factors in human cerebral cortex development. We use human foetal brain explants and iPSC-derived cortical cell culture to identify the direct transcriptional targets of these factors and the cellular processes that they regulate during cortical development. We also characterise the mechanisms by which mutation of these factors results in dysregulation of target genes and in pathological development of the human cortex. 

Project 5 Figure 1

Figure 1: Illustration of our strategy to identify genes regulating human cerebral cortex development and to characterise the pathological mechanisms associated with mutations in these genes. We focus on disease-causing genes that encode transcription factors and chromatin remodellers. To study their normal developmental functions, we identify their transcriptional targets by ChIP-Seq and RNA-Seq and we characterise the cellular processes they regulate by inactivating them in iPSC-derived neuronal cultures. To identify the pathological mechanisms underlying the neurodevelopmental disorders associated with these genes, we introduce the disease-causing mutations in iPSC-derived cultures by CRISPR/Cas9-mediated mutagenesis and we characterise the resulting defects in gene expression and cellular behavior.

Selected publications

Dias, C., Estruch, S.B., Graham, S.A., McRae, J., Sawiak, S.J., Hurst, J.A., Joss, S.K., Holder, S.E., Morton, J.E., Turner, C., Thevenon, J., Mellul, K., Sánchez-Andrade, G., Ibarra-Soria, X., Deriziotis, P., Santos, R.F., Lee, S.C., Faivre, L., Kleefstra, T., Liu, P., Hurles, M.E.; DDD Study, Fisher, S.E., Logan, D.W. (2016) BCL11A Haploinsufficiency Causes an Intellectual Disability Syndrome and Dysregulates Transcription.  Am. J. Hum. Genet. 99, 253-274.

Francois Guillemot

francois.guillemot@crick.ac.uk
+44 (0)20 379 61458

  • Qualifications and history
  • 1989 PhD Institut d'Embryologie du CNRS, Paris, France
  • 1989-1991 Postdoctoral fellow Harvard Medical School, Boston, USA
  • 1991-1994 Postdoctoral fellow Mount Sinai Hospital, Toronto, Canada
  • 1994 Group Leader, IGBMC, Strasbourg, France
  • 2002 Group Leader, Medical Research Council National Institute for Medical Research, London, UK
  • 2015 Group Leader, the Francis Crick Institute, London, UK