Search for Meis1 target genes in embryonic sympathetic neurons
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE54144 | GEO | 2015/09/30
SECONDARY ACCESSION(S): PRJNA235300
REPOSITORIES: GEO
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Project description:Understanding factors that drive development of the sympathetic neuron is crucial to development of potential therapies for neuroblastoma. Here, we identify a key cell autonomous role for the LIM homeodomain transcription factor ISL1 for survival, proliferation and differentiation of sympathetic neurons throughout development. Chromatin immunoprecipitation assays performed utilizing antibody to ISL1 in chromatin extracts from sympathetic neurons demonstrated that ISL1 directly binds genomic regions within several genes critical for sympathetic neuron development and function, including subunits of the Insm1, Lmo1,Tlx3 and Prox1. Our studies represent in vivo ChIP-seq studies for sympathetic neurons which provide a basis for further exploration of factors critical to sympathetic neurons development and function .
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Project description:Gut-brain connections monitor the intestinal tissue and its microbial and dietary content1, regulating both intestinal physiological functions such as nutrient absorption and motility2,3, and brain–wired feeding behaviour2. It is therefore plausible that circuits exist to detect gut microbes and relay this information to central nervous system (CNS) areas that, in turn, regulate gut physiology4. We characterized the influence of the microbiota on enteric–associated neurons (EAN) by combining gnotobiotic mouse models with transcriptomics, circuit–tracing methods, and functional manipulation. We found that the gut microbiome modulates gut–extrinsic sympathetic neurons; while microbiota depletion led to increased cFos expression, colonization of germ-free mice with short-chain fatty acid–producing bacteria suppressed cFos expression in the gut sympathetic ganglia. Chemogenetic manipulations, translational profiling, and anterograde tracing identified a subset of distal intestine-projecting vagal neurons positioned to play an afferent role in microbiota–mediated modulation of gut sympathetic neurons. Retrograde polysynaptic neuronal tracing from the intestinal wall identified brainstem sensory nuclei activated during microbial depletion, as well as efferent sympathetic premotor glutamatergic neurons that regulate gastrointestinal transit. These results reveal microbiota–dependent control of gut extrinsic sympathetic activation through a gut-brain circuit.
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Project description:The objective of this study was to identify genes that are differentially regulated in cultured sympathetic neurons during BMP-induced dendritic growth and to determine the temporal expression patterns of these genes. We determined that inhibiting transcription within the first 24 hours after adding exogenous BMP-7 blocks BMP-induced dendritic growth in cultured sympathetic neurons dissociated from the superior cervical ganglia of embryonic rat pups. Thus, we determined the transcriptional profiles of cultured sympathetic neurons at 6 and 24 hours after addition of BMP-7 to identify early and later transcriptional responses that may drive dendrite formation. We isolated total RNA from sympathetic neurons exposed to three different experimental conditions: (1) no BMP-7 treatment; (2) treatment with BMP-7 for 6 hours; and (3) treatment with BMP-7 for 24 hours. Total RNA was labeled and hybridized to Affymetrix Rat Genome U34A oligonucleotide microarrays. This experiment was independently repeated three times using cultures derived from three independent dissections, such that a total of nine arrays were used. The nine samples were processed over two batches and the batching was balanced across the treatment conditions. Image data was processed using Affymetrix GCOS software to generate numeric, probe level data (CEL) and CEL data was then pre-processed using the RMA algorithm implemented by Partek Genomics Suite (St. Louis, MO). Following batch correction of the probe set level, normalized data using Partek, differential gene expression was determined by two-way ANOVA and linear contrasts between all treatment conditions were applied to generate fold-change values along with the statistical p-value. Lists of significantly changed transcripts were uploaded to MetaCore (GeneGo, ) for mining biological annotation and network analysis.
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Project description:Neurons derived from human pluripotent stem cells (hPSCs) are a remarkable tool for modeling human neural development and diseases. However, it remains largely unknown whether the hPSC-derived neurons can be functionally coupled with their target tissues in vitro, which is essential for understanding inter-cellular physiology and further translational studies. Here, we demonstrate that hPSC-derived sympathetic neurons can be obtained from hPSCs and that the resulting neurons form physical and functional connections with cardiac muscle cells. By use of multiple hPSC reporter lines, we recapitulated human autonomic neuron development in vitro, and successfully isolated PHOX2B::eGFP+ neurons exhibiting sympathetic marker expression, electrophysiological properties, and norepinephrine secretion. With pharmacological and optogenetic manipulations, the PHOX2B::eGFP+ neurons controlled the beating rates of cardiomyocytes, and their physical interaction led to neuronal maturation. Our study lays a foundation for the specification of human sympathetic neurons and for the hPSC-based neuronal control of end organs in a dish. Using the four genetic reporter systems (OCT4::eGFP, SOX10::eGFP, ASCL1::eGFP, and PHOX2B::eGFP reporter hESC lines), we were able to purify discrete cell populations at four differentiation stages, recapitulating the sympathoadrenal differentiation process in vitro with purified and defined populations in four specific differentiation stages. We performed transcriptome analysis of OCT4::eGFP+ cells (3 biological replicates, representing undifferentiated hESCs), SOX10::eGFP+ cells (3 biological replicates, multi-potent neural crest), ASCL1::eGFP+ cells (3 biological replicates, putative sympathoadrenal progenitors), and PHOX2B::eGFP+ cells (2 biological replicates, putative sympathetic neuronal precursors).
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Project description:The cardiac autonomic nervous system is crucial in controlling cardiac function, such as heart rate and cardiac contractility, and is divided into sympathetic and parasympathetic branches. Normally, there is a balance between these two branches to maintain homeostasis. However, cardiac disease states such as myocardial infarction, heart failure, and hypertension can induce the remodeling of cells involved in cardiac innervation, which is associated with an adverse clinical outcome. Although there are vast amounts of data for the histological structure and function of the cardiac autonomic nervous system, its molecular biological architecture in health and disease is still enigmatic in many aspects. Novel technologies such as single-cell RNA sequencing (scRNA-seq) hold promise for the genetic characterization of tissues at single-cell resolution. However, the relatively large size of neurons may impede the standardized use of these techniques. Here, this protocol exploits droplet-based single-nucleus RNA sequencing (snRNA-seq), a method to characterize the biological architecture of cardiac sympathetic neurons in health and disease. A stepwise approach is demonstrated to perform snRNA-seq of the bilateral superior cervical (SCG) and stellate ganglia (StG) dissected from adult mice. This method enables long-term sample preservation, maintaining an adequate RNA quality when samples from multiple individuals/experiments cannot be collected all at once within a short period of time. Barcoding the nuclei with hashtag oligos (HTOs) enables demultiplexing and the trace-back of distinct ganglionic samples post sequencing. Subsequent analyses revealed successful nuclei capture of neuronal, satellite glial, and endothelial cells of the sympathetic ganglia, as validated by snRNA-seq. In summary, this protocol provides a stepwise approach for snRNA-seq of sympathetic extrinsic cardiac ganglia, a method that has the potential for broader application in studies of the innervation of other organs and tissues.
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