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| Status |
Public on May 28, 2021 |
| Title |
Single cell gene expression analysis of neurons in the right atrial ganglionic plexus in the pig |
| Organism |
Sus scrofa |
| Experiment type |
Expression profiling by RT-PCR
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| Summary |
As part of the NIH SPARC program efforts to study the intrinsic cardiac nervous system, we developed the first comprehensive molecular phenotyping data on the right atrial ganglionated plexus (RAGP) within the intrinsic cardiac nervous system of the pig. We collected hundreds of single neurons from the RAGP and assayed these for expression of a wide range of genes relevant to neuronal functions. The sinoatrial node (SAN) of 4 pigs were injected with FastBlue, a retrograde tracer, labeling neurons projecting to the SAN. The RAGP of those pigs were then collected and sectioned. Both SAN-projecting and non-SAN-projecting single neurons in RAGP were collected through laser capture microdissection (LCM), allowing for spatial mapping of all collected samples. Over 400 single neurons were collected (n=4 animals) and each neuron was assayed for over 300 genes using high throughput microfluidic qPCR using BioMark. Neuronal phenotypes were distinguished by multivariate analysis showing patterns of network activity between multiple genes. Interestingly, these phenotypes show no spatial preferences within the RAGP. Our results revealed extensive combinatorial expression of neurotransmitters across the RAGP neuronal phenotypes. Additionally, there was a large overlap in expression profiles of SAN-projecting and non-SAN-projecting neurons, without any single gene or module acting as a distinguishing marker between the connectionally labeled groups. Previous studies have focused on cholinergic and catecholaminergic processes, showing evidence for extensive protein expression of cholinergic markers in RAGP. Our findings are in stark contrast to these results and demonstrate high gene expression correlation between cholinergic and catecholaminergic markers such as tyrosine hydroxylase (Th) and choline acetyltransferase (ChAT) in single neurons. This finding suggests that cells are poised for a rapid shift in neurotransmitter signaling as well as the possibility for post transcriptional regulation of neuron phenotype plasticity. Our present findings significantly expand the list of neuromodulatory peptides and their receptors known to be expressed in RAGP. For example, neuropeptide Y and somatostatin showed distinct co-expression patterns with their respective receptors with significant non-overlapping expression of transmitters and cognate receptors across single neurons. Taken together, our results reveal a complex organization of neuronal networks within RAGP-SAN complex that is not entirely governed by connectivity or spatial location, with combinatorial patterns of local paracrine networks with potential influence on cardiac function.
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| Overall design |
Over 400 single neurons were collected (n=4 animals) and each neuron was assayed for over 200 genes using high throughput microfluidic qPCR using BioMark.
Twenty one 96 × 96 BioMark Arrays were used to measure gene expression across the (422 samples before QC) single-cell samples. The same serial dilution sample set was included in each chip to verify reproducibility and test for technical variability. Samples from each animal were run across three chips to obtain data on 283 genes per sample. Each set of chip runs for a given animal contained overlapping assays that served as technical replicates to evaluate chip-to-chip variability.
Data Normalization: Individual qRT-PCR results were examined to determine the quality of the qRT-PCR based on melt-curve analysis. Following this initial quality control, samples with >30% failed reactions and genes with >20% failed reactions were excluded from present analysis. A further 10 samples were determined to be outliers due overall gene expression distributions and were removed from the present analysis. Upon filtering based on these criteria, a total of 405 single-cell samples (152 non SAN-projecting neurons, 169 SAN-projecting neurons, and 84 neurons without reliable connectivity information) and 241 different gene assays were carried forward in the present analysis, with 211 genes showing >60% detectable expression across all RAGPs. Raw Ct values for individual samples were normalized against a median expression level of a subset of 140 robustly expressed genes (genes with greater than 60% working reactions) across all animals to obtain -DCt values.The vector of median sample expression value was chosen over potential reference genes based on comparison of stable expression across all samples against known housekeeping genes using the ‘selectHKs’ function in the NormqPCR package in R. The following equation was used to calculate -DCt values for each gene: -DCtgene = (median sample expression) - Ctgene.
The -DCt data were then rescaled using the median across all samples within a gene using the following equation:
-DDCtgene=-(DCtsample- DCtacross-sample-median).
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| Contributor(s) |
Moss A, Achanta S, Robbins S, Nieves S, Turick S, Hanna P, Ardell J, Shivkumar K, Schwaber J, Vadigepalli R |
| Citation(s) |
34337356 |
| NIH grant(s) |
| Grant ID |
Grant title |
Affiliation |
Name |
| OT2 OD023848 |
Comprehensive Structural and Functional Mapping of the Mammalian Cardiac Nervous System |
UNIVERSITY OF CALIFORNIA LOS ANGELES |
KALYANAM SHIVKUMAR |
| U01 HL133360 |
Multiscale Model of the Vagal Outflow to the Heart |
THOMAS JEFFERSON UNIVERSITY |
JAMES SCHWABER |
| U01 HL133360 |
Multiscale Model of the Vagal Outflow to the Heart |
THOMAS JEFFERSON UNIVERSITY |
Rajanikanth Vadigepalli |
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| Submission date |
Apr 23, 2020 |
| Last update date |
Aug 11, 2021 |
| Contact name |
Rajanikanth Vadigepalli |
| E-mail(s) |
Rajanikanth.Vadigepalli@jefferson.edu
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| Phone |
2159550576
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| Organization name |
Jefferson University
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| Department |
Pathology
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| Lab |
Daniel Baugh Institute
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| Street address |
1020 Locust St, 314C
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| City |
Philadelphia |
| State/province |
PA |
| ZIP/Postal code |
19107 |
| Country |
USA |
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| Platforms (1) |
| GPL28449 |
Fluidigm BioMark Sus Scrofa qPCR |
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| Samples (415)
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| This SubSeries is part of SuperSeries: |
| GSE154411 |
A spatially-tracked single cell transcriptomics map of neuronal networks in the intrinsic cardiac nervous system |
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| Relations |
| BioProject |
PRJNA627677 |
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