Recipient Organization
UNIVERSITY OF GEORGIA
200 D.W. BROOKS DR
ATHENS,GA 30602-5016
Performing Department
Animal & Dairy Science
Non Technical Summary
In livestock animals, taste buds are the sensory organs for taste that are essential for life and quality of life, as they guide nutritive choices and motivate feed intake. Taste sensitivity and quality are determined by taste bud number and taste receptor gene expression in different taste cell types. Thus, studies on taste bud formation and cell differentiation will help us better understand animals' nutrient sensing and feeding behaviors. Our preliminary data suggested that the genetic regulation of taste organ formation is species-specific comparing chickens with mice. Therefore, in order to understand chickens' feeding behaviors, it is necessary to first characterize chicken taste bud formation, which is the focus of this proposal. We will define which genetic programs control and regulate: (1) taste bud number, and thus taste sensitivity (Aim 1), and (2) taste receptor gene expression in different taste cell types, and thus taste quality (Aim 2). Combining transcriptomic, morphological and physiological, as well as in vitro and in vivo approaches, will enable us to analyze mechanisms underlying chicken taste bud formation at molecular, cellular and ultracellular levels. Findings will provide beneficial information for increasing feed intake, improving health and performance, and creating new feedstuff for chickens. Moreover, the studies will set a realistic model for defining mechanisms of how taste buds are formed and regulated in other livestock animals.
Animal Health Component
30%
Research Effort Categories
Basic
50%
Applied
30%
Developmental
20%
Goals / Objectives
In the proposed studies we will address fundamental issues about the genetic and molecular basis of chicken taste buds that guide feed intake. The information from the studies will contribute to the understanding of which factors determine: (1) the taste bud number and thus taste sensitivity (Aim 1); and (2) the taste receptor gene expressions and thus taste modality (Aim 2). We will use modern techniques combining transcriptomic, morphological and physiological, as well as in vitro and in vivo approaches to analyze mechanisms underlying taste bud formation at molecular, cellular and ultracellular levels.
Project Methods
Phenotypic analyses and quantifications We will focus on the primary gustatory tissue, i.e., palate and the base of oral cavity. Western blotting and RT-PCR will be performed to screen gene expression patterns in different tissue compartments. In order to visualize GFP+ cells in all TBs of chimeric chickens, and to monitor distribution patterns and phenotypic changes of taste buds (TBs), we will use a highly efficient method recently developed in my lab for labeling TBs in oral epithelial sheets using two molecular markers: Vimentin and a-Gustducin [15, 25, 34]. Additionally, we will perform immunohistochemistry (IHC) and in situ hybridization on sections to analyze the distribution of labeled cells in detail. We have working protocols available for all antibodies to be used. Primers and probes will be carefully designed for different specific domains of selected genes.Quantification of numbers of TBs, TB clusters, and labeled cells will be made. In each condition, at least 3 samples will be used. TB number will be quantified from images of whole epithelial sheet immunoreacted with Vimentin and a-Gustducin. To quantify labeled TB cells, a set of serial sagittal sections from half of the palate or base of the oral cavity (lateral border to midline) will be immunoreacted with Vimentin and a-Gustducin. We will locate and quantify labeled cells in the epithelium, mesenchyme, and TBs. Laser-scanning confocal microscopy will be used to identify localization and/or co-localization of fluorescent immunosignals in a single cell. Appropriate analysis of variance or t-test will be performed. This general approach for phenotypic analyses and quantification will be adapted to specific experiments in all Aims.Identify genes important for taste bud formation and renewal using RNA-Seq analysisRNA-Seq is an invaluable tool for gene discovery and gene expression analysis as it can simultaneously provide transcriptome sequence information and digital gene expression data. We have demonstrated our capability to perform RNA-Seq studies. In the proposed studies, broiler-type, male and female chickens (P1, P3 and P5) will be used. The lower beak will be dissected. An mixture of Collagenase A (1 mg/ml) and Dispase II (2.5 mg/ml) will be injected into the sub-epithelial space of the base of oral cavity followed by incubation at 37°C for 2 hr. After enzymatic tissue digestion, the base of oral cavity will be dissected in sterile PBS. Under a stereomicroscope, the tissue will be trimmed to include epithelium and lamina propria. TB-containing and surrounding non-TB regions will be separated. The epithelial sheets will be peeled from the underlying CT. RNA will be extracted for RNA-Seq analysis. TopHat2 v2.0.12 will be used for reads mapping, followed by Cufflinks and DESeq2 analyses for identifying DEGs, and potentially, novel transcripts.Using Cuffdiff method, the DEG profile between the chicken GE and NGE will be examined. DEGs will be detected as significantly different by Cuffdiff (Fold_change>2, p< 0.05, FDR q< 0.05, GE vs NGE and GE vs GM. Special attention will be paid to DEGs regarding specific signaling pathways, e.g., Wnt and BMP pathway genes (e.g., Wnt3a, Wnt4, Wnt10a, SAMD8, BMP2, BMP7).Chicken TB numbers vary among gender and lines, e.g., our preliminary data showed that Broiler-type males have more TBs than females, and that the number continues to increase after hatching. Therefore, we will use tissue samples from both genders at key stages in order to generate more informative data.Validate expression of important genes in chicken taste buds, surrounding epithelium, and underlying connective tissueIn the proposed studies, we will analyze the expression of important genes extensively with qRT-PCR and in situ hybridization at the RNA level, and also Western blotting and IHC at the protein level.Broiler-type male and female chickens (P1, P3, and P5) will be used. For the qRT-PCR and Western blotting analyses, tissue dissections and processes for RNA and protein extraction from different tissue compartments will be similar to Aim 1. For in situ hybridization and IHC, tissue samples will be collected. At least two cross-exon primers and probes for each gene will be carefully designed for specific labeling.