Guillemot lab Neural Stem Cell Biology Laboratory
: Regulation of brain stem cells by extracellular signals
Standard content
We wish to understand how neural stem cells interpret extracellular signals to select an appropriate cellular response.
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.
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.
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 signalling 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.
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)
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 signalling 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 signalling, 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 these results 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).
