The methyl binding domain 3/nucleosome remodelling and deacetylase complex regulates neural cell fate determination and terminal differentiation in the cerebral cortex.

BACKGROUND: Chromatin-modifying complexes have key roles in regulating various aspects of neural stem cell biology, including self-renewal and neurogenesis. The methyl binding domain 3/nucleosome remodelling and deacetylation (MBD3/NuRD) co-repressor complex facilitates lineage commitment of pluripotent cells in early mouse embryos and is important for stem cell homeostasis in blood and skin, but its function in neurogenesis had not been described. Here, we show for the first time that MBD3/NuRD function is essential for normal neurogenesis in mice. RESULTS: Deletion of MBD3, a structural component of the NuRD complex, in the developing mouse central nervous system resulted in reduced cortical thickness, defects in the proper specification of cortical projection neuron subtypes and neonatal lethality. These phenotypes are due to alterations in PAX6+ apical progenitor cell outputs, as well as aberrant terminal neuronal differentiation programmes of cortical plate neurons. Normal numbers of PAX6+ apical neural progenitor cells were generated in the MBD3/NuRD-mutant cortex; however, the PAX6+ apical progenitor cells generate EOMES+ basal progenitor cells in reduced numbers. Cortical progenitor cells lacking MBD3/NuRD activity generate neurons that express both deep- and upper-layer markers. Using laser capture microdissection, gene expression profiling and chromatin immunoprecipitation, we provide evidence that MBD3/NuRD functions to control gene expression patterns during neural development. CONCLUSIONS: Our data suggest that although MBD3/NuRD is not required for neural stem cell lineage commitment, it is required to repress inappropriate transcription in both progenitor cells and neurons to facilitate appropriate cell lineage choice and differentiation programmes.We wish to thank Nicola Reynolds for the help with figures; Aoife O’Shaughnessy for the critical reading of the manuscript; Peter Humphreys, the SCI Biofacility staff and Margaret McLeish for technical assistance; Stephanie Hall and Gerard Evan for access to the Laser Capture Microscope and Nathalie Saurat and members of the BH lab for useful discussions. This work was supported by a Wellcome Trust Senior Fellowship in the Basic Biomedical Sciences awarded to BH and a bourse de formation from the Fonds de la Recherche en Santé Québec awarded to EK.This is the final published version of the article. It was originally published in Neural Development (Knock E, et al., Neural Development, 2015, 10:13, doi:10.1186/s13064-015-0040-z). The final version is available at http://dx.doi.org/10.1186/s13064-015-0040-

NuRD suppresses pluripotency gene expression to promote transcriptional heterogeneity and lineage commitment

Transcriptional heterogeneity within embryonic stem cell (ESC) populations has been suggested as a mechanism by which a seemingly homogeneous cell population can initiate differentiation into an array of different cell types. Chromatin remodeling proteins have been shown to control transcriptional variability in yeast and to be important for mammalian ESC lineage commitment. Here we show that the Nucleosome Remodeling and Deacetylation (NuRD) complex, which is required for ESC lineage commitment, modulates both transcriptional heterogeneity and the dynamic range of a set of pluripotency genes in ESCs. In self-renewing conditions, the influence of NuRD at these genes is balanced by the opposing action of self-renewal factors. Upon loss of self-renewal factors, the action of NuRD is sufficient to silence transcription of these pluripotency genes, allowing cells to exit self-renewal. We propose that modulation of transcription levels by NuRD is key to maintaining the differentiation responsiveness of pluripotent cells

The Methyl-CpG Binding Proteins Mecp2, Mbd2 and Kaiso Are Dispensable for Mouse Embryogenesis, but Play a Redundant Function in Neural Differentiation

The precise molecular changes that occur when a neural stem (NS) cell switches from a programme of self-renewal to commit towards a specific lineage are not currently well understood. However it is clear that control of gene expression plays an important role in this process. DNA methylation, a mark of transcriptionally silent chromatin, has similarly been shown to play important roles in neural cell fate commitment in vivo. While DNA methylation is known to play important roles in neural specification during embryonic development, no such role has been shown for any of the methyl-CpG binding proteins (Mecps) in mice.. No evidence for functional redundancy between these genes in embryonic development or in the derivation or maintenance of neural stem cells in culture was detectable. However evidence for a defect in neuronal commitment of triple knockout NS cells was found.Although DNA methylation is indispensable for mammalian embryonic development, we show that simultaneous deficiency of three methyl-CpG binding proteins genes is compatible with apparently normal mouse embryogenesis. Nevertheless, we provide genetic evidence for redundancy of function between methyl-CpG binding proteins in postnatal mice

The methyl binding domain 3/nucleosome remodelling and deacetylase complex regulates neural cell fate determination and terminal differentiation in the cerebral cortex

Abstract Background Chromatin-modifying complexes have key roles in regulating various aspects of neural stem cell biology, including self-renewal and neurogenesis. The methyl binding domain 3/nucleosome remodelling and deacetylation (MBD3/NuRD) co-repressor complex facilitates lineage commitment of pluripotent cells in early mouse embryos and is important for stem cell homeostasis in blood and skin, but its function in neurogenesis had not been described. Here, we show for the first time that MBD3/NuRD function is essential for normal neurogenesis in mice. Results Deletion of MBD3, a structural component of the NuRD complex, in the developing mouse central nervous system resulted in reduced cortical thickness, defects in the proper specification of cortical projection neuron subtypes and neonatal lethality. These phenotypes are due to alterations in PAX6+ apical progenitor cell outputs, as well as aberrant terminal neuronal differentiation programmes of cortical plate neurons. Normal numbers of PAX6+ apical neural progenitor cells were generated in the MBD3/NuRD-mutant cortex; however, the PAX6+ apical progenitor cells generate EOMES+ basal progenitor cells in reduced numbers. Cortical progenitor cells lacking MBD3/NuRD activity generate neurons that express both deep- and upper-layer markers. Using laser capture microdissection, gene expression profiling and chromatin immunoprecipitation, we provide evidence that MBD3/NuRD functions to control gene expression patterns during neural development. Conclusions Our data suggest that although MBD3/NuRD is not required for neural stem cell lineage commitment, it is required to repress inappropriate transcription in both progenitor cells and neurons to facilitate appropriate cell lineage choice and differentiation programmes

Neural differentiation of 3KO NS Cells.

<p>NS cells were cultured for 15 days in neuronal differentiation conditions and immunostained for ß-tubulin III (green) and Gfap (red) in panels 1 and 2, Map2 in panels 3 and 4, and for Gad67 (green), a marker of Gabaergic neurons, in panels 5 and 6. Cells were counterstained in Dapi (blue). Panels 1, 3, and 5: wild-type NS cells; panels 2, 4, and 6: 3KO NS cells. B. Quantitation of the number of ß-tubulinIII expressing (white bars) and Gfap-expressing (grey bars) cells in 14 day neural differentiation cultures from wild-type (left) and 3KO (right) NS cell cultures. Error bars represent SEM from three independent experiments. C. Quantitation of the number of ß-tubulinIII positive cells present in NS cell cultures after seven days' neuronal differentiation. Data is shown for three different wild-type and three different 3KO NS cell lines. Error bars represent SEM from at least three independent experiments. The difference seen between the wild-type and 3KO lines is highly statistically significant (p<0.0024, Bonferroni). D. Images of 7 day neural cultures from wild-type (left) or 3KO (right) NS cells, stained for ß-tubulin III (green) and Dapi (blue). E. Relative percentage of ß-tubulin III positive cells present in NS cell cultures of indicated genotype after eight days' neuronal differentiation. Error bars represent SEM from at least four independent fields of cells. All cell lines were derived from E14.5 cortex except those labeled “<i>Zbtb33</i> Flox” and “<i>Zbtb33</i> KO”, which are ES cell-derived NS lines, with the <i>Zbtb33</i> KO line having been derived from the <i>Zbtb33</i> Flox line by Cre-transfection and gene deletion. Hence data for the <i>Zbtb33</i> Flox line is included as an appropriate wild-type control for the <i>Zbtb33</i> -null NS line.</p

Genetic interaction between Mecps in postnatal animals.

<p>A. Cumulative plot of percentage of Mecp2-null mice (solid black line), Mecp2/Kaiso-double null mice (dotted black line), Mecp2/Mbd2-double null mice (dotted grey line) and Mecp2/Mbd2/Kaiso-triple null mice (solid grey line) surviving over time. All mice used in this analysis were produced from mouse lines that had undergone at least 6 generations of backcrossing to C57Bl/6 mice. B. Statistical analysis of the data pictured in A. N refers to the number of mice in the sample. A two-tailed Mann-Whitney test (<a href="http://faculty.vassar.edu/lowry/utest.html" target="_blank">http://faculty.vassar.edu/lowry/utest.html</a>) was used to calculate p-values. p_Mecp2 gives the p-value as compared to the Mecp2 single-null data, and p_3KO gives the p-value as compared to the triple null data.</p

3KO Neural stem cells.

<p>A. Proliferation rates of three 3KO NS cell lines (grey lines) and two wild-type NS cell lines (black lines) were measured by the MTT assay over three days. Error bars represent SEM. B. Immunocytochemistry of the indicated NS cell markers (red staining) in one wild-type NS cell line (top panels) and three independent 3KO NS cell lines. Cells were counterstained with Dapi (blue). C. Expression levels of the genes indicated across the X-axis were measured in wild type, single mutant, and triple mutant NS cell lines (as indicated in the legend). Expression was monitored in triplicate in two biological replicates by quantitative PCR, and results of a representative experiment is shown. Expression levels were normalized to Sox2 expression and are shown relative to the levels found in an E14.5 cortex-derived wildtype NS cell line. The <i>Zbtb33^−/y</i> NS line was made by Cre transfection of the <i>Zbtb33^Flox/y</i> NS line, as described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004315#pone.0004315-Prokhortchouk2" target="_blank">[24]</a>, while the remaining mutant NS lines were all derived from E14.5dpc embryos. D. Immunostaining of NS cell cultures after four days in the presence of serum and removal of growth factors to induce Gfap activation. Left panels: bright field, right panels: Gfap (green), Dapi (blue). Top panels: WT cells, middle and bottom panels: two independent 3KO cell lines.</p

RT-PCR Primers used in this study.

<p>RT-PCR Primers used in this study.</p