Project description:Dopaminergic (DA) neurons are the predominant cell type in the midbrain that synthesize dopamine, a neurotransmitter implicated in various behavioural processes, including motor function, the reward pathway, and satiety. In diseases affecting these neurons, such as in Parkinson’s disease (PD), there is growing evidence that the gut-brain axis and selective vulnerability of DA neurons plays a crucial role in disease. Most investigations relating to DA neurons in the gut rely on immunoreactivity to tyrosine hydroxylase (TH) - a rate-limiting enzyme in the production of dopamine. However, the reliability of TH staining as a marker of DA neurons has been questioned in recent years. Our aim is to perform a comprehensive characterization of DA neurons in the gut using a well-accepted reporter mouse line, expressing a fluorescent protein under the dopamine transporter promoter (DAT). Our findings confirm a unique localization of DA neurons in the gut, and unveil that there are discrete subtypes of DA neurons in the gut, which we characterized using both immunofluorescence and single-cell transcriptomics. We observed distinct subtypes of DAT neurons expressing co-transmitters and modulators across both plexuses; some of them likely co-releasing acetylcholine, and a smaller population likely releasing nitric oxide; while others were positive for a slew of canonical DA markers (Vmat2, Girk2, Foxa2). Given the clear heterogeneity of DA gut neurons, further investigation is warranted to define their functional signatures and discover their inherent biological differences that predispose these cells to neurodegeneration.
Project description:Comparative transcriptomics between prf3, Prf-SBP-FLAG complemented lines as controls and tft3 2-2 line (CRISPR/Cas9 tomato mutant line in Rio Grande-prf3 Prf-SBP-FLAG complemented background), treated with either buffer (as control) or P. syringae DC3000 6 hours post infiltration.
Project description:On going efforts are directed at understanding the mutualism between the gut microbiota and the host in breast-fed versus formula-fed infants. Due to the lack of tissue biopsies, no investigators have performed a global transcriptional (gene expression) analysis of the developing human intestine in healthy infants. As a result, the crosstalk between the microbiome and the host transcriptome in the developing mucosal-commensal environment has not been determined. In this study, we examined the host intestinal mRNA gene expression and microbial DNA profiles in full term 3 month-old infants exclusively formula fed (FF) (n=6) or breast fed (BF) (n=6) from birth to 3 months. Host mRNA microarray measurements were performed using isolated intact sloughed epithelial cells in stool samples collected at 3 months. Microbial composition from the same stool samples was assessed by metagenomic pyrosequencing. Both the host mRNA expression and bacterial microbiome phylogenetic profiles provided strong feature sets that clearly classified the two groups of babies (FF and BF). To determine the relationship between host epithelial cell gene expression and the bacterial colony profiles, the host transcriptome and functionally profiled microbiome data were analyzed in a multivariate manner. From a functional perspective, analysis of the gut microbiota's metagenome revealed that characteristics associated with virulence differed between the FF and BF babies. Using canonical correlation analysis, evidence of multivariate structure relating eleven host immunity / mucosal defense-related genes and microbiome virulence characteristics was observed. These results, for the first time, provide insight into the integrated responses of the host and microbiome to dietary substrates in the early neonatal period. Our data suggest that systems biology and computational modeling approaches that integrate “-omic” information from the host and the microbiome can identify important mechanistic pathways of intestinal development affecting the gut microbiome in the first few months of life. KEYWORDS: infant, breast-feeding, infant formula, exfoliated cells, transcriptome, metagenome, multivariate analysis, canonical correlation analysis 12 samples, 2 groups