Cortical Neurogenesis Requires Bcl6-Mediated Transcriptional Repression of Multiple Self-Renewal-Promoting Extrinsic Pathways.

During neurogenesis, progenitors switch from self-renewal to differentiation through the interplay of intrinsic and extrinsic cues, but how these are integrated remains poorly understood. Here, we combine whole-genome transcriptional and epigenetic analyses with in vivo functional studies to demonstrate that Bcl6, a transcriptional repressor previously reported to promote cortical neurogenesis, acts as a driver of the neurogenic transition through direct silencing of a selective repertoire of genes belonging to multiple extrinsic pathways promoting self-renewal, most strikingly the Wnt pathway. At the molecular level, Bcl6 represses its targets through Sirt1 recruitment followed by histone deacetylation. Our data identify a molecular logic by which a single cell-intrinsic factor represses multiple extrinsic pathways that favor self-renewal, thereby ensuring robustness of neuronal fate transition

Characterization of the neural stem cell gene regulatory network identifies OLIG2 as a multifunctional regulator of self-renewal

The gene regulatory network (GRN) that supports neural stem cell (NS cell) self-renewal has so far been poorly characterized. Knowledge of the central transcription factors (TFs), the noncoding gene regulatory regions that they bind to, and the genes whose expression they modulate will be crucial in unlocking the full therapeutic potential of these cells. Here, we use DNase-seq in combination with analysis of histone modifications to identify multiple classes of epigenetically and functionally distinct cis-regulatory elements (CREs). Through motif analysis and ChIP-seq, we identify several of the crucial TF regulators of NS cells. At the core of the network are TFs of the basic helix-loop-helix (bHLH), nuclear factor I (NFI), SOX, and FOX families, with CREs often densely bound by several of these different TFs. We use machine learning to highlight several crucial regulatory features of the network that underpin NS cell self-renewal and multipotency. We validate our predictions by functional analysis of the bHLH TF OLIG2. This TF makes an important contribution to NS cell self-renewal by concurrently activating pro-proliferation genes and preventing the untimely activation of genes promoting neuronal differentiation and stem cell quiescence.Welcome Trust grants: (WT095908, WT098051), FEBS Long-Term Fellowship, Medical Research Council Grant-in-Aid (U117570528)

PLZF regulates fibroblast growth factor responsiveness and maintenance of neural progenitors.

Distinct classes of neurons and glial cells in the developing spinal cord arise at specific times and in specific quantities from spatially discrete neural progenitor domains. Thus, adjacent domains can exhibit marked differences in their proliferative potential and timing of differentiation. However, remarkably little is known about the mechanisms that account for this regional control. Here, we show that the transcription factor Promyelocytic Leukemia Zinc Finger (PLZF) plays a critical role shaping patterns of neuronal differentiation by gating the expression of Fibroblast Growth Factor (FGF) Receptor 3 and responsiveness of progenitors to FGFs. PLZF elevation increases FGFR3 expression and STAT3 pathway activity, suppresses neurogenesis, and biases progenitors towards glial cell production. In contrast, PLZF loss reduces FGFR3 levels, leading to premature neuronal differentiation. Together, these findings reveal a novel transcriptional strategy for spatially tuning the responsiveness of distinct neural progenitor groups to broadly distributed mitogenic signals in the embryonic environment

PLZF Regulates Fibroblast Growth Factor Responsiveness and Maintenance of Neural Progenitors

<div><p>Distinct classes of neurons and glial cells in the developing spinal cord arise at specific times and in specific quantities from spatially discrete neural progenitor domains. Thus, adjacent domains can exhibit marked differences in their proliferative potential and timing of differentiation. However, remarkably little is known about the mechanisms that account for this regional control. Here, we show that the transcription factor Promyelocytic Leukemia Zinc Finger (PLZF) plays a critical role shaping patterns of neuronal differentiation by gating the expression of Fibroblast Growth Factor (FGF) Receptor 3 and responsiveness of progenitors to FGFs. PLZF elevation increases FGFR3 expression and STAT3 pathway activity, suppresses neurogenesis, and biases progenitors towards glial cell production. In contrast, PLZF loss reduces FGFR3 levels, leading to premature neuronal differentiation. Together, these findings reveal a novel transcriptional strategy for spatially tuning the responsiveness of distinct neural progenitor groups to broadly distributed mitogenic signals in the embryonic environment.</p></div

PLZF is broadly expressed by early neural progenitors and then becomes restricted to a central domain committed to ventral interneuron and astrocyte production.

<p>(A–H, K) Antibody costaining analysis shows that PLZF is initially expressed by a subset of SOX2⁺ progenitors in the open neural plate at e2 (HH 10), and then becomes broadly expressed by most NPCs. From e4–e6 (HH 21–28), PLZF becomes confined to a central domain of NPCs in the intermediate spinal cord that persists throughout the course of neurogenesis and early stages of gliogenesis. PLZF is also expressed by many differentiated LHX1/5⁺ neurons in the dorsal spinal cord. pMN, motor neuron progenitor domain; pOL, oligodendrocyte progenitor domain. (I, J) Mapping of PLZF expression at e6 (HH 28) relative to the homeodomain proteins that pattern the spinal cord reveals that the progenitor expression of PLZF is associated with the pdI6, p0, p1, p2, and p3 domains known to give rise to interneurons (INs) early in development followed by astrocytes (ASTs). (L) During early gliogenesis at e9 (HH 35), PLZF is expressed by SOX9⁺ astroglial progenitors in the VZ, but absent from migratory SOX9⁺ OLIG2⁻ astrocyte progenitors and SOX9⁺ OLIG2⁺ oligodendrocyte progenitors (OLPs). All of the PLZF⁺ SOX9⁻ cells at these later stages correspond to subsets of differentiated interneurons (unpublished data). (M, N) Summaries of the domain-restricted expression of PLZF within the spinal cord, and the timing of its expression relative to early markers of progenitor maintenance and gliogenesis.</p

Reduced PLZF activity compromises progenitor maintenance and promotes neuronal differentiation.

<p>(A, G) Electroporation of chick embryos with shRNA vectors against <i>PLZF</i> (U6::<i>shPLZF</i>) at e2 (HH 10) dramatically reduces the expression of PLZF protein in the developing spinal cord upon collection at e4 (HH 21). Insets show the extent of electroporation marked by nEGFP fluorescence. (B, H, M) PLZF knockdown reduces the area of the VZ. Chart indicates the mean VZ area ± SEM for both control and shPLZF-electroporated embryos relative to the untransfected contralateral sides of the spinal cord. Blue dotted lines demarcate the border of the contralateral VZ in each image. (B–D, H–J) PLZF knockdown reduces the expression of multiple genes and proteins associated with progenitor maintenance including SOX2, <i>HES5-2</i>, and <i>ID2</i>. (E, K) PLZF loss coincides with an increase in the number and density of cells expressing the proneural transcription factor NEUROG2 within the VZ. (F, L, Q) PLZF knockdown also increases the frequency of apoptotic cell death. (N) Chart displays the mean pixel intensity of SOX2 staining ± SEM in shPLZF-transfected cells relative to empty vector controls. (O) Chart displays the level of <i>HES5-2</i> and <i>ID2</i> mRNA in control and shPLZF-electroporated spinal cords, relative to the contralateral control sides. (P–Q) Charts display the mean number of shPLZF-transfected cells ± SEM expressing the indicated markers relative to empty vector controls. Data are representative of at least 10 images taken from ≥8 embryos electroporated in the same experiment. In all panels, *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001, and ****<i>p</i><0.0001.</p

PLZF misexpression promotes progenitor maintenance and reduces neuronal differentiation.

<p>(A–B, F–G) NPCs were electroporated with control IRES-<i>nEGFP</i> or <i>PLZF</i>-IRES-<i>nEGFP</i> vectors at e3 (HH 17) and analyzed at e5 (HH 25). PLZF-transfected cells display an increased expression of the progenitor marker SOX2 and reduced expression of the neuronal marker NEUN. Yellow dashed line indicates the approximate boundary between the ventricular and mantle zones. (C–D, H–I) Obligate activator and repressor forms of PLZF were created by attaching to PLZF's DNA binding domain either the VP16 transactivator domain or the Engrailed repressor (EnR) domain. EnR-PLZF phenocopies wild-type PLZF in increasing the proportion of transfected cells expressing SOX2. Conversely, VP16-PLZF had a strong antimorphic effect, promoting extensive differentiation of the transfected cells. (E, J) Charts display the mean proportion of PLZF-, EnR-PLZF-, and VP16-PLZF-transfected cells expressing the indicated markers ± SEM relative to empty vector controls. Data are representative of multiple sections taken from >8 embryos for each condition. (K–S) PLZF and EnR-PLZF misexpression reduce the expression of <i>ASCL1</i> and <i>NEUROG1</i> mRNA as well as NEUROG2 protein. Particularly notable changes in expression are indicated by boxes. (T–U) Charts display the mean level of <i>ASCL1</i> and <i>NEUROG1</i> mRNA ± SEM in control, PLZF, and EnR-PLZF-electroporated spinal cords, relative to the contralateral control sides. (V) Quantification of the mean number of transfected NPCs expressing NEUROG2 protein ± SEM relative to empty vector controls. In all panels, **<i>p</i><0.01, ***<i>p</i><0.001, and ****<i>p</i><0.0001. (W) Summary illustrating the repressive actions of PLZF on neuronal differentiation and presumed indirect positive effects on progenitor maintenance.</p