Building brains in a dish: Prospects for growing cerebral organoids from stem cells

AbstractThe recent development of organoid techniques, in which embryonic brain-like tissue can be grown from human or mouse stem cells in vitro offers the potential to transform the way in which brain development is studied. In this review, we summarize key aspects of the embryonic development of mammalian forebrains, focussing in particular on the cerebral cortex and highlight significant differences between mouse and primates, including human. We discuss recent work using cerebral organoids that has revealed key similarities and differences between their development and that of the brain in vivo. Finally, we outline the ways in which cerebral organoids can be used in combination with CRISPR/Cas9 genome editing to unravel genetic mechanisms that control embryonic development of the cerebral cortex, how this can help us understand the causes of neurodevelopmental disorders and some of the key challenges which will have to be resolved before organoids can become a mainstream tool to study brain development

Expression Profiles of Wnt Genes during Neural Differentiation of Mouse Embryonic Stem Cells

The Wnt family of secreted signaling proteins regulates many aspects of animal development and the behavior of several types of stem cells, including embryonic stem (ES) cells. Activation of canonical Wnt signaling has been shown to either inhibit or promote the differentiation of ES cells into neurons, depending on the stage of differentiation. Here, we describe the expression of all 19 mouse Wnt genes during this process. Using the well-established retinoic acid induction protocol we found that all Wnt genes except Wnt8b are expressed as ES cells differentiate into neurons, many of them in dynamic patterns. The expression pattern of 12 Wnt genes was analyzed quantitatively at 2-day intervals throughout neural differentiation, showing that multiple Wnt genes are expressed at each stage. A large proportion of these, including both canonical and noncanonical Wnts, are expressed at highest levels during later stages of differentiation. The complexity of the patterns observed indicates that disentangling specific roles for individual Wnt genes in the differentiation process will be a significant challenge

Activation of Wnt/β-Catenin Signalling Affects Differentiation of Cells Arising from the Cerebellar Ventricular Zone

Development of the cerebellum proceeds under the precise spatio-temporal control of several key developmental signalling pathways, including the Wnt/β-catenin pathway. We recently reported the activity of Wnt/β-catenin signalling in the perinatal cerebellar ventricular zone (VZ), a germinal centre in the developing cerebellum that gives rise to GABAergic and glial cells. In order to investigate the normal function of Wnt/β-catenin signalling in the VZ and the cell lineages it gives rise to, we used a combination of ex vivo cerebellar slice culture and in vivo genetic manipulation to dysregulate its activity during late embryonic development. Activation of the pathway at the cerebellar ventricular zone led to a reduction in the number of cells expressing the glial lineage markers Sox9 and GFAP and the interneuron marker Pax2, but had no consistent effect on either proliferation or apoptosis. Our findings suggest that activation of the Wnt/β-catenin pathway in the cerebellar ventricular zone causes a shift in the cell types produced, most likely due to disruption of normal differentiation. Thus, we propose that regulation of Wnt/β-catenin signalling levels are required for normal development of cells arising from the cerebellar ventricular zone during late embryogenesis

Positive autoregulation of the transcription factor Pax6 in response to increased levels of either of its major isoforms, Pax6 or Pax6(5a), in cultured cells

<p>Abstract</p> <p>Background</p> <p>Pax6 is a transcription factor essential for normal development of the eyes and nervous system. It has two major isoforms, Pax6 and Pax6(5a), and the ratios between their expression levels vary within narrow limits. We tested the effects of overexpressing either one or other isoform on endogenous Pax6 expression levels in Neuro2A and NIH3T3 cells.</p> <p>Results</p> <p>We found that both isoforms caused an up-regulation of endogenous Pax6 expression in cells with (Neuro2A) or without (NIH3T3) constitutive Pax6 expression. Western blots showed that cells stably transfected with constructs expressing either Pax6 or Pax6(5a) contained raised levels of both Pax6 and Pax6(5a). Quantitative RT-PCR confirmed an increase in levels of <it>Pax6(5a) </it>mRNA in cells containing Pax6-expressing constructs and an increase in levels of <it>Pax6 </it>mRNA in cells containing Pax6(5a)-expressing constructs. The fact that the introduction of constructs expressing only one isoform increased the cellular levels of not only that isoform but also the other indicates that activation of the endogenous <it>Pax6 </it>locus occurred. The ratio between the levels of the two isoforms was maintained close to physiological values. The overexpression of either isoform in neuroblastoma (Neuro2A) cell lines also promoted morphological change and an increase in β-III-tubulin expression, indicating an increase in neurogenesis.</p> <p>Conclusion</p> <p>Our results demonstrate that Pax6 can up-regulate production of Pax6 protein from an entire intact endogenous <it>Pax6 </it>locus in its genomic environment. This adds to previous studies showing that Pax6 can up-regulate reporter expression driven by isolated <it>Pax6 </it>regulatory elements. Furthermore, our results suggest that an important function of positive feedback might be to stabilise the relative levels of Pax6 and Pax6(5a).</p

Adenomatous polyposis coli is required for early events in the normal growth and differentiation of the developing cerebral cortex

<p>Abstract</p> <p>Background</p> <p>Adenomatous polyposis coli (Apc) is a large multifunctional protein known to be important for Wnt/β-catenin signalling, cytoskeletal dynamics, and cell polarity. In the developing cerebral cortex, <it>Apc </it>is expressed in proliferating cells and its expression increases as cells migrate to the cortical plate. We examined the consequences of loss of Apc function for the early development of the cerebral cortex.</p> <p>Results</p> <p>We used <it>Emx1</it>^{<it>Cre </it>}to inactivate <it>Apc </it>specifically in proliferating cerebral cortical cells and their descendents starting from embryonic day 9.5. We observed reduction in the size of the mutant cerebral cortex, disruption to its organisation, and changes in the molecular identity of its cells. Loss of Apc leads to a decrease in the size of the proliferative pool, disrupted interkinetic nuclear migration, and increased apoptosis. β-Catenin, pericentrin, and N-cadherin proteins no longer adopt their normal high concentration at the apical surface of the cerebral cortical ventricular zone, indicating that cell polarity is disrupted. Consistent with enhanced Wnt/β-catenin signalling resulting from loss of Apc we found increased levels of TCF/LEF-dependent transcription and expression of endogenous Wnt/β-catenin target genes (<it>Axin2 </it>(<it>conductin</it>), <it>Lef1</it>, and <it>c-myc</it>) in the mutant cerebral cortex. In the <it>Apc </it>mutant cerebral cortex the expression of transcription factors <it>Foxg1</it>, <it>Pax6</it>, <it>Tbr1</it>, and <it>Tbr2 </it>is drastically reduced compared to normal and many cells ectopically express <it>Pax3</it>, <it>Wnt1</it>, and <it>Wt1 </it>(but not <it>Wnt2b</it>, <it>Wnt8b</it>, <it>Ptc</it>, <it>Gli1</it>, <it>Mash1</it>, <it>Olig2</it>, or <it>Islet1</it>). This indicates that loss of Apc function causes cerebral cortical cells to lose their normal identity and redirect to fates normally found in more posterior-dorsal regions of the central nervous system.</p> <p>Conclusion</p> <p>Apc is required for multiple aspects of early cerebral cortical development, including the regulation of cell number, interkinetic nuclear migration, cell polarity, and cell type specification.</p

Normal ventral telencephalic expression of Pax6 is required for normal development of thalamocortical axons in embryonic mice

<p>Abstract</p> <p>Background</p> <p>In addition to its well-known expression in dorsal telencephalic progenitor cells, where it regulates cell proliferation and identity, the transcription factor Pax6 is expressed in some ventral telencephalic cells, including many postmitotic neurons. Its functions in these cells are unknown.</p> <p>Results</p> <p>We generated a new floxed allele of <it>Pax6 </it>and tested the consequences of a highly specific ventral telencephalic depletion of Pax6. We used the <it>Six3</it>^{<it>A1A2</it>}-<it>Cre </it>allele that drives production of Cre recombinase in a specific region of Pax6-expression close to the internal capsule, through which thalamic axons navigate to cerebral cortex. Depletion in this region caused many thalamic axons to take aberrant routes, either failing to turn normally into ventral telencephalon to form the internal capsule or exiting the developing internal capsule ventrally. We tested whether these defects might have resulted from abnormalities of two structural features proposed to guide thalamic axons into and through the developing internal capsule. First, we looked for the early pioneer axons that project from the region of the future internal capsule to the thalamus and are thought to guide thalamocortical axons to the internal capsule: we found that they are present in conditional mutants. Second, we examined the development of the corridor of Islet1-expressing cells that guides thalamic axons through ventral telencephalon and found that it was broader and less dense than normal in conditional mutants. We also examined corticofugal axons that are thought to interact with ascending thalamocortical axons, resulting in each set providing guidance to the other, and found that some are misrouted to lateral telencephalon.</p> <p>Conclusion</p> <p>These findings indicate that ventral telencephalic Pax6 is important for formation of the Islet1-expressing corridor and the thalamic and cortical axons that grow through it. We suggest that Pax6 might affect thalamic axonal growth indirectly via its effect on the corridor.</p