Robust derivation of transplantable dopamine neurons from human pluripotent stem cells by timed retinoic acid delivery.

Stem cell therapies for Parkinson's disease (PD) have entered first-in-human clinical trials using a set of technically related methods to produce mesencephalic dopamine (mDA) neurons from human pluripotent stem cells (hPSCs). Here, we outline an approach for high-yield derivation of mDA neurons that principally differs from alternative technologies by utilizing retinoic acid (RA) signaling, instead of WNT and FGF8 signaling, to specify mesencephalic fate. Unlike most morphogen signals, where precise concentration determines cell fate, it is the duration of RA exposure that is the key-parameter for mesencephalic specification. This concentration-insensitive patterning approach provides robustness and reduces the need for protocol-adjustments between hPSC-lines. RA-specified progenitors promptly differentiate into functional mDA neurons in vitro, and successfully engraft and relieve motor deficits after transplantation in a rat PD model. Our study provides a potential alternative route for cell therapy and disease modelling that due to its robustness could be particularly expedient when use of autologous- or immunologically matched cells is considered.

A Shh/Gli-driven three-node timer motif controls temporal identity and fate of neural stem cells.

How time is measured by neural stem cells during temporal neurogenesis has remained unresolved. By combining experiments and computational modeling, we define a Shh/Gli-driven three-node timer underlying the sequential generation of motor neurons (MNs) and serotonergic neurons in the brainstem. The timer is founded on temporal decline of Gli-activator and Gli-repressor activities established through down-regulation of Gli transcription. The circuitry conforms an incoherent feed-forward loop, whereby Gli proteins not only promote expression of Phox2b and thereby MN-fate but also account for a delayed activation of a self-promoting transforming growth factor-ß (Tgfß) node triggering a fate switch by repressing Phox2b. Hysteresis and spatial averaging by diffusion of Tgfß counteract noise and increase temporal accuracy at the population level, providing a functional rationale for the intrinsically programmed activation of extrinsic switch signals in temporal patterning. Our study defines how time is reliably encoded during the sequential specification of neurons.

Sequentially acting SOX proteins orchestrate astrocyte- and oligodendrocyte-specific gene expression.

SOX transcription factors have important roles during astrocyte and oligodendrocyte development, but how glial genes are specified and activated in a sub-lineage-specific fashion remains unknown. Here, we define glial-specific gene expression in the developing spinal cord using single-cell RNA-sequencing. Moreover, by ChIP-seq analyses we show that these glial gene sets are extensively preselected already in multipotent neural precursor cells through prebinding by SOX3. In the subsequent lineage-restricted glial precursor cells, astrocyte genes become additionally targeted by SOX9 at DNA regions strongly enriched for Nfi binding motifs. Oligodendrocyte genes instead are prebound by SOX9 only, at sites which during oligodendrocyte maturation are targeted by SOX10. Interestingly, reporter gene assays and functional studies in the spinal cord reveal that SOX3 binding represses the synergistic activation of astrocyte genes by SOX9 and NFIA, whereas oligodendrocyte genes are activated in a combinatorial manner by SOX9 and SOX10. These genome-wide studies demonstrate how sequentially expressed SOX proteins act on lineage-specific regulatory DNA elements to coordinate glial gene expression both in a temporal and in a sub-lineage-specific fashion.

EviNet: a web platform for network enrichment analysis with flexible definition of gene sets.

The new web resource EviNet provides an easily run interface to network enrichment analysis for exploration of novel, experimentally defined gene sets. The major advantages of this analysis are (i) applicability to any genes found in the global network rather than only to those with pathway/ontology term annotations, (ii) ability to connect genes via different molecular mechanisms rather than within one high-throughput platform, and (iii) statistical power sufficient to detect enrichment of very small sets, down to individual genes. The users' gene sets are either defined prior to upload or derived interactively from an uploaded file by differential expression criteria. The pathways and networks used in the analysis can be chosen from the collection menu. The calculation is typically done within seconds or minutes and the stable URL is provided immediately. The results are presented in both visual (network graphs) and tabular formats using jQuery libraries. Uploaded data and analysis results are kept in separated project directories not accessible by other users. EviNet is available at https://www.evinet.org/.

Differential requirement of SUFU in tissue development discovered in a hypomorphic mouse model.

Suppressor of Fused (SUFU) is an essential negative regulator of the Hedgehog (HH) pathway and involved in GLI transcription factor regulation. Due to early embryonic lethality of Sufu-/- mice, investigations of SUFU's role later in development are limited to conditional, tissue-specific knockout models. In this study we developed a mouse model (SufuEx456(fl)/Ex456(fl)) with hypomorphic features where embryos were viable up to E18.5, although with a spectrum of developmental defects of varying severity, including polydactyly, exencephaly and omphalocele. Development of certain tissues, like the skeleton, was more affected than that of others such as skin, which remained largely normal. Interestingly, no apparent changes in the dorso-ventral patterning of the neural tube at E9.0 could be seen. Thus, this model provides an opportunity to globally study SUFU's molecular function in organogenesis beyond E9.5. Molecularly, SufuEx456(fl)/Ex456(fl) embryos displayed aberrant mRNA splicing and drastically reduced levels of Sufu wild-type mRNA and SUFU protein in all tissues. As a consequence, at E9.5 the levels of all three different GLI proteins were reduced. Interestingly, despite the reduction of GLI3 protein levels, the critical ratio of the GLI3 full-length transcriptional activator versus GLI3 truncated repressor remained unchanged compared to wild-type embryos. This suggests that the limited amount of SUFU protein present is sufficient for GLI processing but not for stabilization. Our data demonstrate that tissue development is differentially affected in response to the reduced SUFU levels, providing novel insight regarding the requirements of different levels of SUFU for proper organogenesis.

Lmx1a and Lmx1b regulate mitochondrial functions and survival of adult midbrain dopaminergic neurons.

The LIM-homeodomain transcription factors Lmx1a and Lmx1b play critical roles during the development of midbrain dopaminergic progenitors, but their functions in the adult brain remain poorly understood. We show here that sustained expression of Lmx1a and Lmx1b is required for the survival of adult midbrain dopaminergic neurons. Strikingly, inactivation of Lmx1a and Lmx1b recreates cellular features observed in Parkinson's disease. We found that Lmx1a/b control the expression of key genes involved in mitochondrial functions, and their ablation results in impaired respiratory chain activity, increased oxidative stress, and mitochondrial DNA damage. Lmx1a/b deficiency caused axonal pathology characterized by α-synuclein(+) inclusions, followed by a progressive loss of dopaminergic neurons. These results reveal the key role of these transcription factors beyond the early developmental stages and provide mechanistic links between mitochondrial dysfunctions, α-synuclein aggregation, and the survival of dopaminergic neurons.

The novel enterochromaffin marker Lmx1a regulates serotonin biosynthesis in enteroendocrine cell lineages downstream of Nkx2.2.

Intestinal hormone-producing cells represent the largest endocrine system in the body, but remarkably little is known about enteroendocrine cell type specification in the embryo and adult. We analyzed stage- and cell type-specific deletions of Nkx2.2 and its functional domains in order to characterize its role in the development and maintenance of enteroendocrine cell lineages in the mouse duodenum and colon. Although Nkx2.2 regulates enteroendocrine cell specification in the duodenum at all stages examined, it controls the differentiation of progressively fewer enteroendocrine cell populations when deleted from Ngn3(+) progenitor cells or in the adult duodenum. During embryonic development Nkx2.2 regulates all enteroendocrine cell types, except gastrin and preproglucagon. In developing Ngn3(+) enteroendocrine progenitor cells, Nkx2.2 is not required for the specification of neuropeptide Y and vasoactive intestinal polypeptide, indicating that a subset of these cell populations derive from an Nkx2.2-independent lineage. In adult duodenum, Nkx2.2 becomes dispensable for cholecystokinin and secretin production. In all stages and Nkx2.2 mutant conditions, serotonin-producing enterochromaffin cells were the most severely reduced enteroendocrine lineage in the duodenum and colon. We determined that the transcription factor Lmx1a is expressed in enterochromaffin cells and functions downstream of Nkx2.2. Lmx1a-deficient mice have reduced expression of Tph1, the rate-limiting enzyme for serotonin biosynthesis. These data clarify the function of Nkx2.2 in the specification and homeostatic maintenance of enteroendocrine populations, and identify Lmx1a as a novel enterochromaffin cell marker that is also essential for the production of the serotonin biosynthetic enzyme Tph1.

Dopamine Receptor Antagonists Enhance Proliferation and Neurogenesis of Midbrain Lmx1a-expressing Progenitors.

Degeneration of dopamine neurons in the midbrain causes symptoms of the movement disorder, Parkinson disease. Dopamine neurons are generated from proliferating progenitor cells localized in the embryonic ventral midbrain. However, it remains unclear for how long cells with dopamine progenitor character are retained and if there is any potential for reactivation of such cells after cessation of normal dopamine neurogenesis. We show here that cells expressing Lmx1a and other progenitor markers remain in the midbrain aqueductal zone beyond the major dopamine neurogenic period. These cells express dopamine receptors, are located in regions heavily innervated by midbrain dopamine fibres and their proliferation can be stimulated by antagonizing dopamine receptors, ultimately leading to increased neurogenesis in vivo. Furthermore, treatment with dopamine receptor antagonists enhances neurogenesis in vitro, both from embryonic midbrain progenitors as well as from embryonic stem cells. Altogether our results indicate a potential for reactivation of resident midbrain cells with dopamine progenitor potential beyond the normal period of dopamine neurogenesis.

Dopaminergic control of autophagic-lysosomal function implicates Lmx1b in Parkinson's disease.

The role of developmental transcription factors in maintenance of neuronal properties and in disease remains poorly understood. Lmx1a and Lmx1b are key transcription factors required for the early specification of ventral midbrain dopamine (mDA) neurons. Here we show that conditional ablation of Lmx1a and Lmx1b after mDA neuron specification resulted in abnormalities that show striking resemblance to early cellular abnormalities seen in Parkinson's disease. We found that Lmx1b was required for the normal execution of the autophagic-lysosomal pathway and for the integrity of dopaminergic nerve terminals and long-term mDA neuronal survival. Notably, human LMX1B expression was decreased in mDA neurons in brain tissue affected by Parkinson's disease. Thus, these results reveal a sustained and essential requirement of Lmx1b for the function of midbrain mDA neurons and suggest that its dysfunction is associated with Parkinson's disease pathogenesis.

Nkx2.2 and Nkx2.9 are the key regulators to determine cell fate of branchial and visceral motor neurons in caudal hindbrain.

Cranial motor nerves in vertebrates are comprised of the three principal subtypes of branchial, visceral, and somatic motor neurons, which develop in typical patterns along the anteroposterior and dorsoventral axes of hindbrain. Here we demonstrate that the formation of branchial and visceral motor neurons critically depends on the transcription factors Nkx2.2 and Nkx2.9, which together determine the cell fate of neuronal progenitor cells. Disruption of both genes in mouse embryos results in complete loss of the vagal and spinal accessory motor nerves, and partial loss of the facial and glossopharyngeal motor nerves, while the purely somatic hypoglossal and abducens motor nerves are not diminished. Cell lineage analysis in a genetically marked mouse line reveals that alterations of cranial nerves in Nkx2.2; Nkx2.9 double-deficient mouse embryos result from changes of cell fate in neuronal progenitor cells. As a consequence progenitors of branchiovisceral motor neurons in the ventral p3 domain of hindbrain are transformed to somatic motor neurons, which use ventral exit points to send axon trajectories to their targets. Cell fate transformation is limited to the caudal hindbrain, as the trigeminal nerve is not affected in double-mutant embryos suggesting that Nkx2.2 and Nkx2.9 proteins play no role in the development of branchiovisceral motor neurons in hindbrain rostral to rhombomere 4.

A conserved regulatory logic controls temporal identity in mouse neural progenitors.

Neural progenitors alter their output over time to generate different types of neurons and glia in specific chronological sequences, but this process remains poorly understood in vertebrates. Here we show that Casz1, the vertebrate ortholog of the Drosophila temporal identity factor castor, controls the production of mid-/late-born neurons in the murine retina. Casz1 is expressed from mid/late stages in retinal progenitor cells (RPCs), and conditional deletion of Casz1 increases production of early-born retinal neurons at the expense of later-born fates, whereas precocious misexpression of Casz1 has the opposite effect. In both cases, cell proliferation is unaffected, indicating that Casz1 does not control the timing of cell birth but instead biases RPC output directly. Just as Drosophila castor lies downstream of the early temporal identity factor hunchback, we find that the hunchback ortholog Ikzf1 represses Casz1. These results uncover a conserved strategy regulating temporal identity transitions from flies to mammals.

Tgfß signaling regulates temporal neurogenesis and potency of neural stem cells in the CNS.

How the sequential specification of neurons and progressive loss of potency associated with aging neural progenitors are regulated in vertebrate brain development is poorly understood. By examining a temporal differentiation lineage in the hindbrain, we here identify Tgfß as a switch signal that executes the transition between early and late phases of neurogenesis and concurrently constrains progenitor potency. Young progenitors have inherent competence to produce late-born neurons, but implementation of late-differentiation programs requires suppression of early identity genes achieved through temporally programmed activation of Tgfß downstream of Shh signaling. Unexpectedly, we find that sequentially occurring fate-switch decisions are temporally coupled, and onset of Tgfß signaling appears thereby to impact on the overall lifespan of the temporal lineage. Our study establishes Tgfß as a regulator of temporal identity and potency of neural stem cells, and provides proof of concept that Tgfß can be applied to modulate temporal specification of neurons in stem cell engineering.

Sox6 and Otx2 control the specification of substantia nigra and ventral tegmental area dopamine neurons.

Distinct midbrain dopamine (mDA) neuron subtypes are found in the substantia nigra pars compacta (SNc) and the ventral tegmental area (VTA), but it is mainly SNc neurons that degenerate in Parkinson's disease. Interest in how mDA neurons develop has been stimulated by the potential use of stem cells in therapy or disease modeling. However, very little is known about how specific dopaminergic subtypes are generated. Here, we show that the expression profiles of the transcription factors Sox6, Otx2, and Nolz1 define subpopulations of mDA neurons already at the neural progenitor cell stage. After cell-cycle exit, Sox6 selectively localizes to SNc neurons, while Otx2 and Nolz1 are expressed in a subset of VTA neurons. Importantly, Sox6 ablation leads to decreased expression of SNc markers and a corresponding increase in VTA markers, while Otx2 ablation has the opposite effect. Moreover, deletion of Sox6 affects striatal innervation and dopamine levels. We also find reduced Sox6 levels in Parkinson's disease patients. These findings identify Sox6 as a determinant of SNc neuron development and should facilitate the engineering of relevant mDA neurons for cell therapy and disease modeling.

CtBPs sense microenvironmental oxygen levels to regulate neural stem cell state.

Bone morphogenetic proteins (BMPs) secreted by the dorsal neural tube and overlying ectoderm are key signals for the specification of the roof plate and dorsal interneuron populations. However, the signals that confer nonneurogenic character to the roof plate region are largely unknown. We report that the roof plate region shows elevated oxygen levels compared to neurogenic regions of the neural tube. These high oxygen levels are required for the expression of the antineuronal transcription factor Hes1 in the roof plate region. The transcriptional corepressor CtBP is a critical mediator of the oxygen-sensing response. High oxygen promotes a decrease in the CtBP occupancy of the promoter of Hes1. Furthermore, under conditions of high oxygen and BMP, CtBP associates with HES1 and represses neurogenesis. We propose that CtBP integrates signals originating from microenvironmental levels of oxygen and BMP to confer nonneurogenic character to the roof plate region.

Transcription factor-induced lineage programming of noradrenaline and motor neurons from embryonic stem cells.

An important goal in stem cell biology is to develop methods for efficient generation of clinically interesting cell types from relevant stem cell populations. This is particularly challenging for different types of neurons of the central nervous system where hundreds of distinct neuronal cell types are generated during embryonic development. We previously used a strategy based on forced transcription factor expression in embryonic stem cell-derived neural progenitors to generate specific types of neurons, including dopamine and serotonin neurons. Here, we extend these studies and show that noradrenergic neurons can also be generated from pluripotent embryonic stem cells by forced expression of the homeobox transcription factor Phox2b under the signaling influence of fibroblast growth factor 8 (FGF8) and bone morphogenetic proteins. In neural progenitors exposed to FGF8 and sonic hedgehog both Phox2b and the related Phox2a instead promoted the generation of neurons with the characteristics of mid- and hindbrain motor neurons. The efficient generation of these neuron types enabled a comprehensive genome-wide gene expression analysis that provided further validation of the identity of generated cells. Moreover, we also demonstrate that the generated cell types are amenable to drug testing in vitro and we show that variants of the differentiation protocols can be applied to cultures of human pluripotent stem cells for the generation of human noradrenergic and visceral motor neurons. Thus, these studies provide a basis for characterization of yet an additional highly clinically relevant neuronal cell type.

Detailed expression analysis of regulatory genes in the early developing human neural tube.

Studies in model organisms constitute the basis of our understanding of the principal molecular mechanisms of cell fate determination in the developing central nervous system. Considering the emergent applications in stem cell-based regenerative medicine, it is important to demonstrate conservation of subtype specific gene expression programs in human as compared to model vertebrates. We have examined the expression patterns of key regulatory genes in neural progenitor cells and their neuronal and glial descendants in the developing human spinal cord, hindbrain, and midbrain, and compared these with developing mouse and chicken embryos. As anticipated, gene expression patterns are highly conserved between these vertebrate species, but there are also features that appear unique to human development. In particular, we find that neither tyrosine hydroxylase nor Nurr1 are specific markers for mesencephalic dopamine neurons, as these genes also are expressed in other neuronal subtypes in the human ventral midbrain and in human embryonic stem cell cultures directed to differentiate towards a ventral mesencephalic identity. Moreover, somatic motor neurons in the ventral spinal cord appear to be produced by two molecularly distinct ventral progenitor populations in the human, raising the possibility that the acquisition of unique ventral progenitor identities may have contributed to the emergence of neural subtypes in higher vertebrates.

SoxB1-driven transcriptional network underlies neural-specific interpretation of morphogen signals.

The reiterative deployment of a small cadre of morphogen signals underlies patterning and growth of most tissues during embyogenesis, but how such inductive events result in tissue-specific responses remains poorly understood. By characterizing cis-regulatory modules (CRMs) associated with genes regulated by Sonic hedgehog (Shh), retinoids, or bone morphogenetic proteins in the CNS, we provide evidence that the neural-specific interpretation of morphogen signaling reflects a direct integration of these pathways with SoxB1 proteins at the CRM level. Moreover, expression of SoxB1 proteins in the limb bud confers on mesodermal cells the potential to activate neural-specific target genes upon Shh, retinoid, or bone morphogenetic protein signaling, and the collocation of binding sites for SoxB1 and morphogen-mediatory transcription factors in CRMs faithfully predicts neural-specific gene activity. Thus, an unexpectedly simple transcriptional paradigm appears to conceptually explain the neural-specific interpretation of pleiotropic signaling during vertebrate development. Importantly, genes induced in a SoxB1-dependent manner appear to constitute repressive gene regulatory networks that are directly interlinked at the CRM level to constrain the regional expression of patterning genes. Accordingly, not only does the topology of SoxB1-driven gene regulatory networks provide a tissue-specific mode of gene activation, but it also determines the spatial expression pattern of target genes within the developing neural tube.

Mechanistic differences in the transcriptional interpretation of local and long-range Shh morphogen signaling.

Morphogens orchestrate tissue patterning in a concentration-dependent fashion during vertebrate embryogenesis, yet little is known of how positional information provided by such signals is translated into discrete transcriptional outputs. Here we have identified and characterized cis-regulatory modules (CRMs) of genes operating downstream of graded Shh signaling and bifunctional Gli proteins in neural patterning. Unexpectedly, we find that Gli activators have a noninstructive role in long-range patterning and cooperate with SoxB1 proteins to facilitate a largely concentration-independent mode of gene activation. Instead, the opposing Gli-repressor gradient is interpreted at transcriptional levels, and, together with CRM-specific repressive input of homeodomain proteins, comprises a repressive network that translates graded Shh signaling into regional gene expression patterns. Moreover, local and long-range interpretation of Shh signaling differs with respect to CRM context sensitivity and Gli-activator dependence, and we propose that these differences provide insight into how morphogen function may have mechanistically evolved from an initially binary inductive event.

Genome-wide characterization of Foxa2 targets reveals upregulation of floor plate genes and repression of ventrolateral genes in midbrain dopaminergic progenitors.

The transcription factors Foxa1 and Foxa2 promote the specification of midbrain dopaminergic (mDA) neurons and the floor plate. Whether their role is direct has remained unclear as they also regulate the expression of Shh, which has similar roles. We characterized the Foxa2 cis-regulatory network by chromatin immunoprecipitation followed by high-throughput sequencing of mDA progenitors. This identified 9160 high-quality Foxa2 binding sites associated with 5409 genes, providing mechanistic insights into Foxa2-mediated positive and negative regulatory events. Foxa2 regulates directly and positively key determinants of mDA neurons, including Lmx1a, Lmx1b, Msx1 and Ferd3l, while negatively inhibiting transcription factors expressed in ventrolateral midbrain such as Helt, Tle4, Otx1, Sox1 and Tal2. Furthermore, Foxa2 negatively regulates extrinsic and intrinsic components of the Shh signaling pathway, possibly by binding to the same enhancer regions of co-regulated genes as Gli1. Foxa2 also regulates the expression of floor plate factors that control axon trajectories around the midline of the embryo, thereby contributing to the axon guidance function of the floor plate. Finally, this study identified multiple Foxa2-regulated enhancers that are active in the floor plate of the midbrain or along the length of the embryo in mouse and chick. This work represents the first comprehensive characterization of Foxa2 targets in mDA progenitors and provides a framework for elaborating gene regulatory networks in a functionally important progenitor population.