Capicua regulates neural stem cell proliferation and lineage specification through control of Ets factors

Capicua (Cic) is a transcriptional repressor mutated in the brain cancer oligodendroglioma. Despite its cancer link, little is known of Cic’s function in the brain. We show that nuclear Cic expression is strongest in astrocytes and neurons but weaker in stem cells and oligodendroglial lineage cells. Using a new conditional Cic knockout mouse, we demonstrate that forebrain-specific Cic deletion increases proliferation and self-renewal of neural stem cells. Furthermore, Cic loss biases neural stem cells toward glial lineage selection, expanding the pool of oligodendrocyte precursor cells (OPCs). These proliferation and lineage effects are dependent on de-repression of Ets transcription factors. In patient-derived oligodendroglioma cells, CIC re-expression or ETV5 blockade decreases lineage bias, proliferation, self-renewal, and tumorigenicity. Our results identify Cic as an important regulator of cell fate in neurodevelopment and oligodendroglioma, and suggest that its loss contributes to oligodendroglioma by promoting proliferation and an OPC-like identity via Ets overactivity

TRANSCRIPTIONAL REGULATION OF NEURONAL MIGRATION AND CELL FATE SPECIFICATION IN THE NEOCORTEX

The events that lead to the generation of a functional neocortex are tightly regulated and require the coordination of multiple events, including progenitor proliferation and maturation, neuronal differentiation and neuronal migration. I began my PhD studies by investigating the role of the zinc finger gene Zac1 in neocortical development. Zac1 was initially identified in the Schuurmans’ lab in a subtractive screen designed to isolate downstream effectors of the proneural gene Neurog2, which is a critical regulator of neocortical development. Previous studies had identified a role for Zac1 in the developing retina and cerebellum. I hypothesized that Zac1 would have essential roles in regulating the key events during the development of a functional neocortex. We tested the sufficiency of Zac1 in progenitor cell proliferation, progenitor cell maturation, neuronal differentiation and neuronal differentiation. We have demonstrated that Zac1 functions in regulating the radial glial cell to intermediate neuronal precurssor transition, neuronal differentiation and neuronal migration in the embryonic neocortex, acting in part through the regulation of Pac1 (Chapter 2). In the next stage of my PhD project, I tested the requirement and sufficiency of of two other members of the Plag family of transcription factors, Plag1 and Plagl2 in the developing neocortex (Chapter 3). Previously, it was established that Plag1 and Plagl2 (as well as Zac1) are associated with intrauterine growth restriction (IUGR) and intellectual disabilities. I found that Plag1 and Plagl2 have complementary roles in maintaining the ventral and dorsal boundaries of gene expression in the developing telencephalon, respectively, and that Plag1 is required while Plagl2 is sufficient to induce neocortical progenitor proliferation. Finally, in Chapter 4, I switched gears and focused my study on a disease of the neocortex, which is oligodendroglioma (ODG). My goal was to elucidate how ODG cells influence the behavior of mouse neural stem cells (mNSCs), and I hypothesized that the communication between ODG cells and mNSCs was , in part, EV dependent. I was able to show that ODG cells secrete factors that have dosage-specific effects on the growth of normal neural cells. In particular I demonstrated that ODG cells communicate with mNSCs by secreting EVs that carry several signaling molecules as cargo, including EGF, which acts in a dose-dependent manner to influence mNSC proliferation. I also demonstrated that inhibition of EV secretion by blocking the nSMase pathway increases ODG ‘tumoursphere’ number and size, while ectopic expression of this pathway lead to decrease in the generation of ‘tumour sphere’. Taken together, through my PhD work new molecular insights into how neocortical development is regulated have been gained, and an onset to understanding one of the means of a disease to influence the behavior of mNSCs in a non-cell autonomous manner have been studied. These studies bring us closer to understanding the molecular mechanisms that underlie normal neocortical development, and possibly open up new avenues for testing various biomarkers and therapies for ODG

Extracellular Vesicles as Conduits of Non-Coding RNA Emission and Intercellular Transfer in Brain Tumors

Non-coding RNA (ncRNA) species have emerged in as molecular fingerprints and regulators of brain tumor pathogenesis and progression. While changes in ncRNA levels have been traditionally regarded as cell intrinsic there is mounting evidence for their extracellular and paracrine function. One of the key mechanisms that enables ncRNA to exit from cells is their selective packaging into extracellular vesicles (EVs), and trafficking in the extracellular space and biofluids. Vesicular export processes reduce intracellular levels of specific ncRNA in EV donor cells while creating a pool of EV-associated ncRNA in the extracellular space and biofluids that enables their uptake by other recipient cells; both aspects have functional consequences. Cancer cells produce several EV subtypes (exosomes, ectosomes), which differ in their ncRNA composition, properties and function. Several RNA biotypes have been identified in the cargo of brain tumor EVs, of which microRNAs are the most studied, but other species (snRNA, YRNA, tRNA, and lncRNA) are often more abundant. Of particular interest is the link between transforming oncogenes and the biogenesis, cargo, uptake and function of tumor-derived EV, including EV content of oncogenic RNA. The ncRNA repertoire of EVs isolated from cerebrospinal fluid and serum is being developed as a liquid biopsy platform in brain tumors

Non-isotopic RNA in situ hybridization on embryonic sections

This unit describes methods for non-isotopic RNA in situ hybridization on embryonic mouse sections. These methods can be used to follow the spatiotemporal dynamics of gene expression in an embryonic tissue of interest. They involve the use of labeled (e.g., digoxygenin, FITC) antisense riboprobes that hybridize to a specific mRNA in the target tissue. The probes are detected using an alkaline phosphatase-conjugated antibody recognizing the label and a chromogenic substrate. This method can be used to: (1) assess the expression of a single gene within a tissue, (2) compare the expression profiles of two genes within a tissue, or (3) compare the distribution of a transcript and protein within a tissue. While this approach is not quantitative, it provides a qualitative assessment of the precise cell types where a gene is expressed, which is not easily achievable with other more quantitative methods such as quantitative PCR

Plag1 and Plagl2 have overlapping and distinct functions in telencephalic development

The Plag gene family has three members; Plagl1/Zac1, which is a tumor suppressor gene, and Plag1 and Plagl2, which are proto-oncogenes. All three genes are known to be expressed in embryonic neural progenitors, and Zac1 regulates proliferation, neuronal differentiation and migration in the developing neocortex. Here we examined the functions of Plag1 and Plagl2 in neocortical development. We first attempted, and were unable to generate, E12.5 Plag1;Plagl2 double mutants, indicating that at least one Plag1 or Plagl2 gene copy is required for embryonic survival. We therefore focused on single mutants, revealing a telencephalic patterning defect in E12.5 Plagl2 mutants and a proliferation/differentiation defect in Plag1 mutant neocortices. Specifically, the ventral pallium, a dorsal telencephalic territory, expands into the ventral telencephalon in Plagl2 mutants. In contrast, Plag1 mutants develop normal regional territories, but neocortical progenitors proliferate less and instead produce more neurons. Finally, in gain-of-function studies, both Plag1 and Plagl2 reduce neurogenesis and increase BrdU-uptake, indicative of enhanced proliferation, but while Plagl2 effects on proliferation are more immediate, Plag1 effects are delayed. Taken together, we found that the Plag proto-oncogenes genes are essential regulators of neocortical development and although Plag1 and Plagl2 functions are similar, they do not entirely overlap. This article has an associated First Person interview with the first author of the paper

Hamartoma-like lesions in the mouse retina: an animal model of Pten hamartoma tumour syndrome

PTEN hamartoma tumour syndrome (PHTS) is a heterogeneous group of rare, autosomal dominant disorders associated with PTEN germline mutations. PHTS patients routinely develop hamartomas, which are benign tissue overgrowths comprised of disorganized ‘normal’ cells. Efforts to generate PHTS animal models have been largely unsuccessful due to the early lethality of homozygous germline mutations in Pten, together with the lack of hamartoma formation in most conditional mutants generated to date. We report herein a novel PHTS mouse model that reproducibly forms hamartoma-like lesions in the central retina by postnatal day 21. Specifically, we generated a Pten conditional knockout (cKO) using a retinal-specific Pax6::Cre driver that leads to a nearly complete deletion of Pten in the peripheral retina but produces a mosaic of ‘wild-type’ and Pten cKO cells centrally. Structural defects were only observed in the mosaic central retina, including in Müller glia and in the outer and inner limiting membranes, suggesting that defective mechanical integrity partly underlies the hamartoma-like pathology. Finally, we used this newly developed model to test whether rapamycin, an mTOR inhibitor that is currently the only PHTS therapy, can block hamartoma growth. When administered in the early postnatal period, prior to hamartoma formation, rapamycin reduces hamartoma size, but also induces new morphological abnormalities in the Pten cKO retinal periphery. In contrast, administration of rapamycin after hamartoma initiation fails to reduce lesion size. We have thus generated and used an animal model of retinal PHTS to show that, although current therapies can reduce hamartoma formation, they might also induce new retinal dysmorphologies. This article has an associated First Person interview with the first author of the paper

Capicua regulates neural stem cell proliferation and lineage specification through control of Ets factors.

Capicua (Cic) is a transcriptional repressor mutated in the brain cancer oligodendroglioma. Despite its cancer link, little is known of Cic's function in the brain. We show that nuclear Cic expression is strongest in astrocytes and neurons but weaker in stem cells and oligodendroglial lineage cells. Using a new conditional Cic knockout mouse, we demonstrate that forebrain-specific Cic deletion increases proliferation and self-renewal of neural stem cells. Furthermore, Cic loss biases neural stem cells toward glial lineage selection, expanding the pool of oligodendrocyte precursor cells (OPCs). These proliferation and lineage effects are dependent on de-repression of Ets transcription factors. In patient-derived oligodendroglioma cells, CIC re-expression or ETV5 blockade decreases lineage bias, proliferation, self-renewal, and tumorigenicity. Our results identify Cic as an important regulator of cell fate in neurodevelopment and oligodendroglioma, and suggest that its loss contributes to oligodendroglioma by promoting proliferation and an OPC-like identity via Ets overactivity

Proneural genes define ground-state rules to regulate neurogenic patterning and cortical folding

International audienceAsymmetric neuronal expansion is thought to drive evolutionary transitions between lissencephalic and gyrencephalic cerebral cortices. We report that Neurog2 and Ascl1 proneural genes together sustain neurogenic continuity and lissencephaly in rodent cortices. Using transgenic reporter mice and human cerebral organoids, we found that Neurog2 and Ascl1 expression defines a continuum of four lineage-biased neural progenitor cell (NPC) pools. Double+ NPCs, at the hierarchical apex, are least lineage restricted due to Neurog2-Ascl1 cross-repression and display unique features of multipotency (more open chromatin, complex gene regulatory network, G2 pausing). Strikingly, selectively eliminating double+ NPCs by crossing Neurog2-Ascl1 split-Cre mice with diphtheria toxin-dependent "deleter" strains locally disrupts Notch signaling, perturbs neurogenic symmetry, and triggers cortical folding. In support of our discovery that double+ NPCs are Notch-ligand-expressing "niche" cells that control neurogenic periodicity and cortical folding, NEUROG2, ASCL1, and HES1 transcript distribution is modular (adjacent high/low zones) in gyrencephalic macaque cortices, prefiguring future folds