Frederick J. Livesey

Vertebrate neural cell-fate determination: Lessons from the retina

Postmitotic neurons are produced from a pool of cycling progenitors in an orderly fashion during development. Studies of cell-fate determination in the vertebrate retina have uncovered several fundamental principles by which this is achieved. Most notably, a model for vertebrate cell-fate determination has been proposed that combines findings on the relative roles of extrinsic and intrinsic regulators in controlling cell-fate choices. At the heart of the model is the proposal that progenitors pass through intrinsically determined competence states, during which they are capable of giving rise to a limited subset of cell types under the influence of extrinsic signals.

Human cerebral cortex development from pluripotent stem cells to functional excitatory synapses

Efforts to study the development and function of the human cerebral cortex in health and disease have been limited by the availability of model systems. Extrapolating from our understanding of rodent cortical development, we have developed a robust, multistep process for human cortical development from pluripotent stem cells: directed differentiation of human embryonic stem (ES) and induced pluripotent stem (iPS) cells to cortical stem and progenitor cells, followed by an extended period of cortical neurogenesis, neuronal terminal differentiation to acquire mature electrophysiological properties, and functional excitatory synaptic network formation. We found that induction of cortical neuroepithelial stem cells from human ES cells and human iPS cells was dependent on retinoid signaling. Furthermore, human ES cell and iPS cell differentiation to cerebral cortex recapitulated in vivo development to generate all classes of cortical projection neurons in a fixed temporal order. This system enables functional studies of human cerebral cortex development and the generation of individual-specific cortical networks ex vivo for disease modeling and therapeutic purposes.

Prox1 function controls progenitor cell proliferation and horizontal cell genesis in the mammalian retina

Retinal progenitor cells regulate their proliferation during development so that the correct number of each cell type is made at the appropriate time. We found that the homeodomain protein Prox1 regulates the exit of progenitor cells from the cell cycle in the embryonic mouse retina. Cells lacking Prox1 are less likely to stop dividing, and ectopic expression of Prox1 forces progenitor cells to exit the cell cycle. During retinogenesis, Prox1 can be detected in differentiating horizontal, bipolar and AII amacrine cells. Horizontal cells are absent in retinae of Prox1−/− mice and misexpression of Prox1 in postnatal progenitor cells promotes horizontal-cell formation. Thus, Prox1 activity is both necessary and sufficient for progenitor-cell proliferation and cell-fate determination in the vertebrate retina.

Directed differentiation of human pluripotent stem cells to cerebral cortex neurons and neural networks

Efficient derivation of human cerebral neocortical neural stem cells (NSCs) and functional neurons from pluripotent stem cells (PSCs) facilitates functional studies of human cerebral cortex development, disease modeling and drug discovery. Here we provide a detailed protocol for directing the differentiation of human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) to all classes of cortical projection neurons. We demonstrate an 80-d, three-stage process that recapitulates cortical development, in which human PSCs (hPSCs) first differentiate to cortical stem and progenitor cells that then generate cortical projection neurons in a stereotypical temporal order before maturing to actively fire action potentials, undergo synaptogenesis and form neural circuits in vitro. Methods to characterize cortical neuron identity and synapse formation are described.

The Level of the Transcription Factor Pax6 Is Essential for Controlling the Balance between Neural Stem Cell Self-Renewal and Neurogenesis

Neural stem cell self-renewal, neurogenesis, and cell fate determination are processes that control the generation of specific classes of neurons at the correct place and time. The transcription factor Pax6 is essential for neural stem cell proliferation, multipotency, and neurogenesis in many regions of the central nervous system, including the cerebral cortex. We used Pax6 as an entry point to define the cellular networks controlling neural stem cell self-renewal and neurogenesis in stem cells of the developing mouse cerebral cortex. We identified the genomic binding locations of Pax6 in neocortical stem cells during normal development and ascertained the functional significance of genes that we found to be regulated by Pax6, finding that Pax6 positively and directly regulates cohorts of genes that promote neural stem cell self-renewal, basal progenitor cell genesis, and neurogenesis. Notably, we defined a core network regulating neocortical stem cell decision-making in which Pax6 interacts with three other regulators of neurogenesis, Neurog2, Ascl1, and Hes1. Analyses of the biological function of Pax6 in neural stem cells through phenotypic analyses of Pax6 gain- and loss-of-function mutant cortices demonstrated that the Pax6-regulated networks operating in neural stem cells are highly dosage sensitive. Increasing Pax6 levels drives the system towards neurogenesis and basal progenitor cell genesis by increasing expression of a cohort of basal progenitor cell determinants, including the key transcription factor Eomes/Tbr2, and thus towards neurogenesis at the expense of self-renewal. Removing Pax6 reduces cortical stem cell self-renewal by decreasing expression of key cell cycle regulators, resulting in excess early neurogenesis. We find that the relative levels of Pax6, Hes1, and Neurog2 are key determinants of a dynamic network that controls whether neural stem cells self-renew, generate cortical neurons, or generate basal progenitor cells, a mechanism that has marked parallels with the transcriptional control of embryonic stem cell self-renewal.

Ezh2, the histone methyltransferase of PRC2, regulates the balance between self-renewal and differentiation in the cerebral cortex

Multipotent progenitor cells of the cerebral cortex balance self-renewal and differentiation to produce complex neural lineages in a fixed temporal order in a cell-autonomous manner. We studied the role of the polycomb epigenetic system, a chromatin-based repressive mechanism, in controlling cortical progenitor cell self-renewal and differentiation. We found that the histone methyltransferase of polycomb repressive complex 2 (PCR2), enhancer of Zeste homolog 2 (Ezh2), is essential for controlling the rate at which development progresses within cortical progenitor cell lineages. Loss of function of Ezh2 removes the repressive mark of trimethylated histone H3 at lysine 27 (H3K27me3) in cortical progenitor cells and also prevents its establishment in postmitotic neurons. Removal of this repressive chromatin modification results in marked up-regulation in gene expression, the consequence of which is a shift in the balance between self-renewal and differentiation toward differentiation, both directly to neurons and indirectly via basal progenitor cell genesis. Although the temporal order of neurogenesis and gliogenesis are broadly conserved under these conditions, the timing of neurogenesis, the relative numbers of different cell types, and the switch to gliogenesis are all altered, narrowing the neurogenic period for progenitor cells and reducing their neuronal output. As a consequence, the timing of cortical development is altered significantly after loss of PRC2 function.

A Human Stem Cell Model of Early Alzheimer’s Disease Pathology in Down Syndrome

Cultured cerebral cortex neurons generated from human Down syndrome induced pluripotent stem cells rapidly develop Alzheimer’s disease pathologies. A Window into the Alzheimer’s Disease Brain Alzheimer’s disease (AD) is a major global health problem for which there are no disease-modifying treatments. A human cellular model of AD would enable detailed functional studies of AD pathogenesis and would provide a simple way to screen for new drugs. An effective cellular model would use the appropriate cell type (in this case, glutamatergic projection neurons of the human cerebral cortex), would develop accurate pathology, and would do so in a reproducible manner over a time scale short enough for practical use. A pressing general question, however, is whether neurological diseases that take decades to become manifest in humans could be successfully modeled over a reasonable time scale in cultured cells. Shi et al. tackle this challenge using cerebral cortex neurons generated from stem cells derived from people with Down syndrome, who have a genetic predisposition to developing AD at a young age. They demonstrate that the cultured cortical neurons develop the two characteristic pathological hallmarks of AD in a few months, suggesting that complex neurodegenerative diseases that take decades to manifest in human patients can be modeled reliably in cultured neurons over a period of months. Individuals with Down syndrome (caused by trisomy of chromosome 21) have a very high risk of developing AD because they carry an extra copy of a major AD-associated gene, which encodes amyloid precursor protein (APP). First, Shi et al. made cortical neurons from induced pluripotent stem cells from Down syndrome patients. They then showed that these neurons in just a few months develop the two characteristic pathological hallmarks of AD: aggregates of amyloid peptides generated by misprocessing of APP and neurofibrillary tangles of hyperphosphorylated tau protein. These pathologies were developed by cortical neurons derived from not only Down syndrome induced pluripotent stem cells but also Down syndrome embryonic stem cells. This finding demonstrated that these pathologies were reproducible in cortical neurons derived from different sources and that their formation was not influenced by the cellular reprogramming strategy used to derive induced pluripotent stem cells from adult fibroblasts. The buildup of pathological amyloid aggregates in the cultured human cortical neurons could be blocked by a drug called γ-secretase, which is being tested in clinical trials for treating AD. The new work of Shi et al. suggests that human cortical neurons derived from stem cells from Down syndrome patients will be useful for screening new candidate drugs and for developing new disease intervention strategies for treating AD. Human cellular models of Alzheimer’s disease (AD) pathogenesis would enable the investigation of candidate pathogenic mechanisms in AD and the testing and developing of new therapeutic strategies. We report the development of AD pathologies in cortical neurons generated from human induced pluripotent stem (iPS) cells derived from patients with Down syndrome. Adults with Down syndrome (caused by trisomy of chromosome 21) develop early-onset AD, probably due to increased expression of a gene on chromosome 21 that encodes the amyloid precursor protein (APP). We found that cortical neurons generated from iPS cells and embryonic stem cells from Down syndrome patients developed AD pathologies over months in culture, rather than years in vivo. These cortical neurons processed the transmembrane APP protein, resulting in secretion of the pathogenic peptide fragment amyloid-β42 (Aβ42), which formed insoluble intracellular and extracellular amyloid aggregates. Production of Aβ peptides was blocked by a γ-secretase inhibitor. Finally, hyperphosphorylated tau protein, a pathological hallmark of AD, was found to be localized to cell bodies and dendrites in iPS cell–derived cortical neurons from Down syndrome patients, recapitulating later stages of the AD pathogenic process.

A Schwann cell mitogen accompanying regeneration of motor neurons

Motor neurons are the only adult mammalian neurons of the central nervous system to regenerate following injury. This ability is dependent on the environment of the peripheral nerve and an intrinsic capacity of motor neurons for regrowth. We report here the identification, using a technique known as messenger RNA differential display, of an extracellular signalling molecule, previously described as the pancreatic secreted protein Reg-2 (ref. 4), that is expressed solely in regenerating and developing rat motor and sensory neurons. Axon-stimulated Schwann cell proliferation is necessary for successful regeneration,, and we show that Reg-2 is a potent Schwann cell mitogen in vitro. In vivo, Reg-2 protein is transported along regrowing axons and inhibition of Reg-2 signalling significantly retards the regeneration of Reg-2-containing axons. During development, Reg-2 production by motor and sensory neurons is regulated by contact with peripheral targets. Strong candidates for peripheral factors regulating Reg-2 production are cytokines of the LIF/CNTF family, because Reg-2 is not expressed in developing motor or sensory neurons of mice carrying a targeted disruption of the LIF receptor gene, a common component of the receptor complexes for all of the LIF/CNTF family.

G&T-seq: parallel sequencing of single-cell genomes and transcriptomes

The simultaneous sequencing of a single cells genome and transcriptome offers a powerful means to dissect genetic variation and its effect on gene expression. Here we describe G&T-seq, a method for separating and sequencing genomic DNA and full-length mRNA from single cells. By applying G&T-seq to over 220 single cells from mice and humans, we discovered cellular properties that could not be inferred from DNA or RNA sequencing alone.

Netrin and netrin receptor expression in the embryonic mammalian nervous system suggests roles in retinal, striatal, nigral, and cerebellar development.

The netrins are laminin-like axon guidance molecules that are conserved among Caenorhabditis elegans, Drosophila, and vertebrates and that have chemoattractive and chemorepellant properties. To study the possible actions of this gene family in the developing and adult mammalian nervous systems, we have cloned a partial cDNA which corresponds to a region conserved among chick netrin-1, netrin-2, and unc-6 and studied its expression and that of a netrin receptor, dcc, the deleted in colorectal cancer gene, in the developing and adult rat CNS. The localization of cells expressing netrin or dcc suggests that these genes, in addition to their actions in defining the ventral midline, may act in controlling retinal ganglion cell axon guidance in the optic nerve, cell migration in the developing cerebellum and olfactory epithelium, and development and maintenance of connections to the substantia nigra and corpus striatum.