Single-cell RNA Sequencing Reveals Midbrain Dopamine Neuron Diversity Emerging During Mouse Brain Development
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE116138 | GEO | 2019/02/08
REPOSITORIES: GEO
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Project description:Pitx3 is a transcription factor that is expressed in all midbrain dopaminergic (mDA) neurons during early development, but later becomes restricted in dopaminergic subsets of substantia nigra compacta (SNc) and of the ventral tegmental are (VTA) that are vulnerable to neurodegenerative stress (MPTP, 6-OHDA, rotenone, Parkinson's disease). Overall, in mice, Pitx3 is required for developmental survival of ventral SNc neurons and for postnatal survival of VTA neurons (after postnatal day 40). With the aim of determining the gene networks that distinguish Pitx3-vulnerable (Pitx3-positive) from Pitx3-resistant (Pitx3-negative) subsets of SNc and VTA, we performed a comparison at the transcriptome level between FAC-sorted mDA neurons of SNc and VTA that were obtained from wild-type and Pitx3-/- newborn mice. The latter mice have already lost the majority of their TH+Calb1- mDA neurons of ventral SNc (Pitx3-dependent), but their TH+Calb1+ neurons of dorsal SNc (Pitx3-independent), including all of VTA neurons (50% are Pitx3-dependent and 50% Pitx3-independent), are unaffected by Pitx3 deletion. At postnatal day 40, Pitx3-/- mice display a marked loss of dopaminergic subsets of VTA that normally co-express Pitx3 and Calb1 (Pitx3-dependent neurons of VTA).
2016-01-31 | GSE75777 | GEO
Project description:Midbrain dopamine (mDA) neurons comprise a diverse group of cells with unique innervation targets and functions. This is illustrated by the selective sensitivity of mDA neurons of the substantia nigra compacta (SNc) in patients with Parkinson’s disease, while those in the ventral tegmental area (VTA) are relatively spared. Here we used single nuclei RNA sequencing (snRNA-seq) of approximately 70,000 mouse midbrain cells to build a high-resolution atlas of mouse mDA neuron diversity at the molecular level. The results showed that differences between mDA neuron groups could best be understood as a continuum without sharp differences between subtypes. Thus, we assigned mDA neurons to several “territories” and “neighborhoods” within a shifting gene expression landscape where boundaries are gradual rather than discrete. Based on the enriched gene expression patterns of these territories and neighborhoods, we were able to localize them in the adult mouse midbrain. Moreover, because the underlying mechanisms for the variable sensitivities of diverse mDA neurons to pathological insults are not well understood, we analyzed surviving neurons after partial 6-hydroxydopamine (6-OHDA) lesions to unravel gene expression patterns that correlate with mDA neuron vulnerability and resilience. Together, this atlas provides a basis for further studies on the neurophysiological role of mDA neurons in health and disease.
2023-10-26 | GSE233866 | GEO
Project description:Induced pluripotent stem cells (iPSCs) hold great promise for in vitro disease modeling and cell replacement therapy for Parkinson’s disease (PD). Both applications crucially require an in-depth profiling of the disease-relevant, iPSC-derived cell type. Midbrain dopaminergic (mDA) neurons derived from pluripotent stem cells are of substantial interest because of their instrumental value for PD therapy. IPSC-derived mDA neuron-like cells have been generated, however, detailed genetic and epigenetic characterization of strictly purified in vitro generated DA neurons has so far lagged behind. We generated mouse Pitx3gfp/+ iPSC-derived DA neurons that, after fluorescent activated cell sorting (FACS) allowed comprehensive comparison to mesodiencephalic dopaminergic (mdDA) neurons from Fac-sorted Pitx3gfp/+ ventral midbrains. We performed detailed analysis of global gene expression and genome-scale DNA methylation of CpG islands (CGIs) by reduced representation bisulfite sequencing. The reprogramming pathway from fibroblasts to iPSCs left parental cell footprints for both gene expression and DNA methylation. However, most gene expression patterns of iPSC-derived DA neurons closely resembled that of primary mdDA neurons with the strongest correlations for mdDA specific genes. Also, for DNA methylation patterns, high similarities were found for the vast majority of CGIs when comparing primary mdDA neurons with iPSC-derived DA neurons. Additionally, we found de novo DNA methylation during in vitro differentiation for hundreds of genes specifically in lineage-committed neural precursors that persisted in iPSC-derived DA neurons. Our study provides novel detailed characteristics of iPSC-derived DA neurons in comparison to the primary cell type. These findings add important information to our knowledge about these biomedically highly valuable, in vitro generated neurons. Microarray expression study comparing 3 samples of facs-sorted, Pitx3-gfp positive cells from each experimental group to a common reference consisting of adult mouse midbrain RNA. Each sample was analysed in normal and opposite dye orientation.
2014-05-13 | E-GEOD-47178 | biostudies-arrayexpress
Project description:Development of meso-diencephalic dopamine (mdDA) neurons requires the combined actions of the orphan nuclear receptor Nurr1 and the paired-like homeobox transcription factor Pitx3. Whereas all mdDA neurons require Nurr1 for expression of Th and survival, dependence on Pitx3 is only displayed by the mdDA subpopulation that will form the substantia nigra (SNc). Previously, we demonstrated that Pitx3-/- embryos lack the expression of the retinoic acid (RA)-generating enzyme Ahd2, which is normally selectively expressed in the Pitx3-dependent DA neurons of the SNc. Restoring RA-signaling in Pitx3-/- embryos revealed a selective dependence of SNc neurons on the presence of RA for differentiation into Th-positive neurons and maintenance throughout embryonic development. Whereas these data are suggestive of an important developmental role for RA in neurons of the SNc, it remained unclear whether other Nurr1 and Pitx3 target genes depend on RA signaling in a manner similar to Th. In search for genes that were affected in Pitx3-deficient mdDA neurons and restored upon embryonic RA treatment, we provide evidence that Delta-like 1, D2R (Drd2) and TH are regulated by Pitx3 and RA signaling, influencing the mdDA terminal differentiated phenotype. Furthermore, we show that regulation of Ahd2-mediated RA-signaling represents only one aspect of the Pitx3 downstream cascade, since Vmat2, Dat, Ahd2 (Aldh1a1), En1, En2 and Cck were unaffected by RA treatment and are (subset) specifically modulated by Pitx3. In conclusion, our data reveal several RA-dependent and -independent aspects of the Pitx3-regulated gene cascade suggesting that Pitx3 acts on multiple levels in the molecular subset-specification of mdDA neurons. RNA was isolated from dissected ventral midbrains of E14.5 Pitx3-/- and Pitx3+/+ mouse embryos. 3 Experimental samples each consisting of 3 Pitx3-/- ventral midbrains were hybridized to reference RNA derived from 10 Pitx3+/+ ventral midbrains
2011-11-22 | E-GEOD-32940 | biostudies-arrayexpress
Project description:Development of meso-diencephalic dopamine (mdDA) neurons requires the combined actions of the orphan nuclear receptor Nurr1 and the paired-like homeobox transcription factor Pitx3. Whereas all mdDA neurons require Nurr1 for expression of Th and survival, dependence on Pitx3 is only displayed by the mdDA subpopulation that will form the substantia nigra (SNc). Previously, we demonstrated that Pitx3-/- embryos lack the expression of the retinoic acid (RA)-generating enzyme Ahd2, which is normally selectively expressed in the Pitx3-dependent DA neurons of the SNc. Restoring RA-signaling in Pitx3-/- embryos revealed a selective dependence of SNc neurons on the presence of RA for differentiation into Th-positive neurons and maintenance throughout embryonic development. Whereas these data are suggestive of an important developmental role for RA in neurons of the SNc, it remained unclear whether other Nurr1 and Pitx3 target genes depend on RA signaling in a manner similar to Th. In search for genes that were affected in Pitx3-deficient mdDA neurons and restored upon embryonic RA treatment, we provide evidence that Delta-like 1, D2R (Drd2) and TH are regulated by Pitx3 and RA signaling, influencing the mdDA terminal differentiated phenotype. Furthermore, we show that regulation of Ahd2-mediated RA-signaling represents only one aspect of the Pitx3 downstream cascade, since Vmat2, Dat, Ahd2 (Aldh1a1), En1, En2 and Cck were unaffected by RA treatment and are (subset) specifically modulated by Pitx3. In conclusion, our data reveal several RA-dependent and -independent aspects of the Pitx3-regulated gene cascade suggesting that Pitx3 acts on multiple levels in the molecular subset-specification of mdDA neurons.
2011-11-22 | GSE32940 | GEO
Project description:Human pluripotent stem cells are a promising source of diverse cells for developmental studies, cell transplantation, disease modeling, and drug testing. However, their widespread use even for intensely studied cell types like spinal motor neurons, is hindered by the long duration and low yields of existing protocols for in vitro differentiation and by the molecular heterogeneity of the populations generated. We report a combination of small molecules that induce up to 50% motor neurons within 3 weeks from human pluripotent stem cells with defined subtype identities that are relevant to neurodegenerative diseases. Despite their accelerated differentiation, motor neurons expressed combinations of HB9, ISL1 and column-specific markers that mirror those observed in vivo in human fetal spinal cord. They also exhibited spontaneous and induced activity, and projected axons towards muscles when grafted into developing chick spinal cord. Strikingly, this novel protocol preferentially generates motor neurons expressing markers of limb-innervating lateral motor column motor neurons (FOXP1+/LHX3-). Access to high-yield cultures of human limb-innervating motor neuron subtypes will facilitate in-depth study of motor neuron subtype-specific properties, disease modeling, and development of large-scale cell-based screening assays. We analyze 3 samples including 2 positive samples and 1 negative sample. Descriptions are as follow: a) Positive Sample 1: SHH-derived, day 21 GFP-high FACS purified motor neurons.b) Positive Sample 2: S+P-derived, day 21 GFP-high FACS purified motor neurons. c) Negative: S+P condition, day 21 no GFP FACS purified motor neurons
2013-02-06 | E-GEOD-41795 | biostudies-arrayexpress
Project description:Meso-diencephalic dopaminergic (mdDA) neurons are critical for motor control and cognitive functioning and their loss or dysfunction is associated with disorders such as Parkinson’s disease (PD), schizophrenia and addiction. However, relatively little is known about the molecular mechanisms underlying mdDA neuron development and maintenance. Here, we determined the spatiotemporal map of genes involved in the development of mdDA neurons to gain further insights into their molecular programming. Genome-wide gene expression profiles of the developing ventral mesencephalon (VM) were compared at different developmental stages leading to the identification of novel regulatory roles of neuronal signaling through nicotinic acthylcholine receptors (Chrna6 and Chrnb3 subunits) and the identification of novel transcription factors (OC1 and 2)) involved in the generation of mdDA neuronal field. We show here that Pitx3 in cooperation with Nurr1 are the critical components in the activation of the Chrna6 and b3 subunits in mdDA neurons. Furthermore, we provide evidence of two divergent regulatory pathways resulting in the expression of Chrna6 and Chrnb3 respectively.
2012-07-09 | GSE35326 | GEO
Project description:Meso-diencephalic dopaminergic (mdDA) neurons are critical for motor control and cognitive functioning and their loss or dysfunction is associated with disorders such as Parkinson’s disease (PD), schizophrenia and addiction. However, relatively little is known about the molecular mechanisms underlying mdDA neuron development and maintenance. Here, we determined the spatiotemporal map of genes involved in the development of mdDA neurons to gain further insights into their molecular programming. Genome-wide gene expression profiles of the developing ventral mesencephalon (VM) were compared at different developmental stages leading to the identification of novel regulatory roles of neuronal signaling through nicotinic acthylcholine receptors (Chrna6 and Chrnb3 subunits) and the identification of novel transcription factors (OC1 and 2)) involved in the generation of mdDA neuronal field. We show here that Pitx3 in cooperation with Nurr1 are the critical components in the activation of the Chrna6 and b3 subunits in mdDA neurons. Furthermore, we provide evidence of two divergent regulatory pathways resulting in the expression of Chrna6 and Chrnb3 respectively. Embryonic ventral midbrain (VM) tissue was used to generate RNA samples for microarray analysis. Each sample consists of pooled RNA from three embryonic VMs, and is hybridized twice (in both dye orientations) against a common reference RNA (KC001-ref) consisting of pooled RNA from adult VMs. Each embryonic stages E10.5, E11.5, E12.5 and E13.5 is analysed from three independant samples.
2012-07-08 | E-GEOD-35326 | biostudies-arrayexpress
Project description:Human pluripotent stem cell (hPSC)-based replacement therapy holds great promise in treating Parkinson’s disease (PD). However, the heterogeneity of hPSC-derived donor cells and the low yield of midbrain dopaminergic (mDA) neurons after transplantation hinder its broad clinical application. Here, we depicted the single-cell molecular landscape during mDA neuron differentiation. We found that this process recapitulated the development of multiple but adjacent fetal brain regions including ventral midbrain, isthmus, and ventral hindbrain, resulting in heterogenous donor cell population. We reconstructed the differentiation trajectory of mDA lineage and identified CLSTN2 and PTPRO as specific surface markers of mDA progenitors, which were predictive of mDA neuron differentiation and could facilitate highly enriched mDA neurons (up to 80%) following progenitor sorting and transplantation. Marker sorted progenitors exhibited higher therapeutic potency in correcting motor deficits of PD mice. Different marker sorted grafts had a strikingly consistent cellular composition, in which mDA neurons were enriched, while off-target neuron types were mostly depleted, suggesting stable graft outcomes. Our study provides a better understanding of cellular heterogeneity during mDA neuron differentiation, and establishes a strategy to generate highly purified donor cells to achieve stable and predictable therapeutic outcomes, raising the prospect of hPSC-based PD cell replacement therapies.
2022-06-14 | GSE204796 | GEO
Project description:Human pluripotent stem cell (hPSC)-based replacement therapy holds great promise in treating Parkinson’s disease (PD). However, the heterogeneity of hPSC-derived donor cells and the low yield of midbrain dopaminergic (mDA) neurons after transplantation hinder its broad clinical application. Here, we depicted the single-cell molecular landscape during mDA neuron differentiation. We found that this process recapitulated the development of multiple but adjacent fetal brain regions including ventral midbrain, isthmus, and ventral hindbrain, resulting in heterogenous donor cell population. We reconstructed the differentiation trajectory of mDA lineage and identified CLSTN2 and PTPRO as specific surface markers of mDA progenitors, which were predictive of mDA neuron differentiation and could facilitate highly enriched mDA neurons (up to 80%) following progenitor sorting and transplantation. Marker sorted progenitors exhibited higher therapeutic potency in correcting motor deficits of PD mice. Different marker sorted grafts had a strikingly consistent cellular composition, in which mDA neurons were enriched, while off-target neuron types were mostly depleted, suggesting stable graft outcomes. Our study provides a better understanding of cellular heterogeneity during mDA neuron differentiation, and establishes a strategy to generate highly purified donor cells to achieve stable and predictable therapeutic outcomes, raising the prospect of hPSC-based PD cell replacement therapies.
2022-06-14 | GSE204795 | GEO