Expression of Genes Encoding Multi-Transmembrane Proteins in Specific Primate Taste Cell Populations

BACKGROUND: Using fungiform (FG) and circumvallate (CV) taste buds isolated by laser capture microdissection and analyzed using gene arrays, we previously constructed a comprehensive database of gene expression in primates, which revealed over 2,300 taste bud-associated genes. Bioinformatics analyses identified hundreds of genes predicted to encode multi-transmembrane domain proteins with no previous association with taste function. A first step in elucidating the roles these gene products play in gustation is to identify the specific taste cell types in which they are expressed. METHODOLOGY/PRINCIPAL FINDINGS: Using double label in situ hybridization analyses, we identified seven new genes expressed in specific taste cell types, including sweet, bitter, and umami cells (TRPM5-positive), sour cells (PKD2L1-positive), as well as other taste cell populations. Transmembrane protein 44 (TMEM44), a protein with seven predicted transmembrane domains with no homology to GPCRs, is expressed in a TRPM5-negative and PKD2L1-negative population that is enriched in the bottom portion of taste buds and may represent developmentally immature taste cells. Calcium homeostasis modulator 1 (CALHM1), a component of a novel calcium channel, along with family members CALHM2 and CALHM3; multiple C2 domains; transmembrane 1 (MCTP1), a calcium-binding transmembrane protein; and anoctamin 7 (ANO7), a member of the recently identified calcium-gated chloride channel family, are all expressed in TRPM5 cells. These proteins may modulate and effect calcium signalling stemming from sweet, bitter, and umami receptor activation. Synaptic vesicle glycoprotein 2B (SV2B), a regulator of synaptic vesicle exocytosis, is expressed in PKD2L1 cells, suggesting that this taste cell population transmits tastant information to gustatory afferent nerve fibers via exocytic neurotransmitter release. CONCLUSIONS/SIGNIFICANCE: Identification of genes encoding multi-transmembrane domain proteins expressed in primate taste buds provides new insights into the processes of taste cell development, signal transduction, and information coding. Discrete taste cell populations exhibit highly specific gene expression patterns, supporting a model whereby each mature taste receptor cell is responsible for sensing, transmitting, and coding a specific taste quality

Genome-Wide Analysis of Gene Expression in Primate Taste Buds Reveals Links to Diverse Processes

Efforts to unravel the mechanisms underlying taste sensation (gustation) have largely focused on rodents. Here we present the first comprehensive characterization of gene expression in primate taste buds. Our findings reveal unique new insights into the biology of taste buds. We generated a taste bud gene expression database using laser capture microdissection (LCM) procured fungiform (FG) and circumvallate (CV) taste buds from primates. We also used LCM to collect the top and bottom portions of CV taste buds. Affymetrix genome wide arrays were used to analyze gene expression in all samples. Known taste receptors are preferentially expressed in the top portion of taste buds. Genes associated with the cell cycle and stem cells are preferentially expressed in the bottom portion of taste buds, suggesting that precursor cells are located there. Several chemokines including CXCL14 and CXCL8 are among the highest expressed genes in taste buds, indicating that immune system related processes are active in taste buds. Several genes expressed specifically in endocrine glands including growth hormone releasing hormone and its receptor are also strongly expressed in taste buds, suggesting a link between metabolism and taste. Cell type-specific expression of transcription factors and signaling molecules involved in cell fate, including KIT, reveals the taste bud as an active site of cell regeneration, differentiation, and development. IKBKAP, a gene mutated in familial dysautonomia, a disease that results in loss of taste buds, is expressed in taste cells that communicate with afferent nerve fibers via synaptic transmission. This database highlights the power of LCM coupled with transcriptional profiling to dissect the molecular composition of normal tissues, represents the most comprehensive molecular analysis of primate taste buds to date, and provides a foundation for further studies in diverse aspects of taste biology

Three-dimensional aligned fibrillar scaffolds -- fabrication and characterization

Aligned fibrillar scaffolds (AFSs) have been widely studied for their application in regenerative medicine, providing possible transplantable tissue replacements for nerve, spinal cord, tendon, ligament, muscle, etc. However, researches in AFSs are technically challenging mainly due to the complex fabrication and characterization processes, especially when the AFSs are made to be fully three-dimensional (3D). As the structure is linked to the quality and function of the engineered tissue product, there is an urgent need for novel techniques to characterize AFSs non-invasively and non-destructively and to link their characteristics to their functions and outcome. In this thesis AFS fabrication and characterization were explored. By combining second harmonic generation (SHG) imaging, multiphoton microscopy (MPM), and various image processing tools, the whole process of 3D tissue characterization could be achieved in a non-invasive, precise, and quantitative way. A proof-of-concept AFS with blended fibers made of polycaprolactone and porcine gelatin was used to demonstrate the feasibility of implementing such a strategy. The data indicated that, in terms of scaffold characterization, the proposed MPM method was capable of measuring the porosity of homogenous scaffolds precisely from deconvolved 3D images. Furthermore, the method could also be used to illustrate the orientation of the aligned nanofibers. Next, when SH-SY5Y neurons were cultured on the AFS, the MPM imaging was capable of evaluating the cell viability ratio, cell-localization in AFS, and neurite outgrowth. This provided guidance for selecting the alignment method for AFS functional recovery. Lastly, when employing this non-invasive imaging-based characterization method, it was possible to illustrate the relationship between the alignment of collagen arrays in decellularized corneal stroma and the transparency. In summary, the proposed strategy can provide some essential scaffold/tissue properties (such as alignment of fiber, porosity of scaffold, and cell viability ratio) quantitatively and non-invasively, which will help both scaffold processing design and characterization

Three-dimensional aligned fibrillar scaffolds -- fabrication and characterization

Aligned fibrillar scaffolds (AFSs) have been widely studied for their application in regenerative medicine, providing possible transplantable tissue replacements for nerve, spinal cord, tendon, ligament, muscle, etc. However, researches in AFSs are technically challenging mainly due to the complex fabrication and characterization processes, especially when the AFSs are made to be fully three-dimensional (3D). As the structure is linked to the quality and function of the engineered tissue product, there is an urgent need for novel techniques to characterize AFSs non-invasively and non-destructively and to link their characteristics to their functions and outcome. In this thesis AFS fabrication and characterization were explored. By combining second harmonic generation (SHG) imaging, multiphoton microscopy (MPM), and various image processing tools, the whole process of 3D tissue characterization could be achieved in a non-invasive, precise, and quantitative way. A proof-of-concept AFS with blended fibers made of polycaprolactone and porcine gelatin was used to demonstrate the feasibility of implementing such a strategy. The data indicated that, in terms of scaffold characterization, the proposed MPM method was capable of measuring the porosity of homogenous scaffolds precisely from deconvolved 3D images. Furthermore, the method could also be used to illustrate the orientation of the aligned nanofibers. Next, when SH-SY5Y neurons were cultured on the AFS, the MPM imaging was capable of evaluating the cell viability ratio, cell-localization in AFS, and neurite outgrowth. This provided guidance for selecting the alignment method for AFS functional recovery. Lastly, when employing this non-invasive imaging-based characterization method, it was possible to illustrate the relationship between the alignment of collagen arrays in decellularized corneal stroma and the transparency. In summary, the proposed strategy can provide some essential scaffold/tissue properties (such as alignment of fiber, porosity of scaffold, and cell viability ratio) quantitatively and non-invasively, which will help both scaffold processing design and characterization

Expression of genes encoding multi-transmembrane proteins in specific primate taste cell populations.

BackgroundUsing fungiform (FG) and circumvallate (CV) taste buds isolated by laser capture microdissection and analyzed using gene arrays, we previously constructed a comprehensive database of gene expression in primates, which revealed over 2,300 taste bud-associated genes. Bioinformatics analyses identified hundreds of genes predicted to encode multi-transmembrane domain proteins with no previous association with taste function. A first step in elucidating the roles these gene products play in gustation is to identify the specific taste cell types in which they are expressed.Methodology/principal findingsUsing double label in situ hybridization analyses, we identified seven new genes expressed in specific taste cell types, including sweet, bitter, and umami cells (TRPM5-positive), sour cells (PKD2L1-positive), as well as other taste cell populations. Transmembrane protein 44 (TMEM44), a protein with seven predicted transmembrane domains with no homology to GPCRs, is expressed in a TRPM5-negative and PKD2L1-negative population that is enriched in the bottom portion of taste buds and may represent developmentally immature taste cells. Calcium homeostasis modulator 1 (CALHM1), a component of a novel calcium channel, along with family members CALHM2 and CALHM3; multiple C2 domains; transmembrane 1 (MCTP1), a calcium-binding transmembrane protein; and anoctamin 7 (ANO7), a member of the recently identified calcium-gated chloride channel family, are all expressed in TRPM5 cells. These proteins may modulate and effect calcium signalling stemming from sweet, bitter, and umami receptor activation. Synaptic vesicle glycoprotein 2B (SV2B), a regulator of synaptic vesicle exocytosis, is expressed in PKD2L1 cells, suggesting that this taste cell population transmits tastant information to gustatory afferent nerve fibers via exocytic neurotransmitter release.Conclusions/significanceIdentification of genes encoding multi-transmembrane domain proteins expressed in primate taste buds provides new insights into the processes of taste cell development, signal transduction, and information coding. Discrete taste cell populations exhibit highly specific gene expression patterns, supporting a model whereby each mature taste receptor cell is responsible for sensing, transmitting, and coding a specific taste quality

MCTP1 is expressed in TRPM5 cells.

<p>Expression of MCTP1 in LE, FG TB, and CV TB (A) as well as top and bottom portions of CV TB (B) by microarray analyses. * p<0.005 compared to LE (A) or CV TB bottom (B). Expression units are GC-RMA normalized average intensities of microarray signals. Double label <i>in situ</i> hybridization (ISH) for MCTP1 and TRPM5 (C–H). MCTP1 (C, F) and TRPM5 (D, G) are expressed in similar cells in the merged images (E, H). Double label ISH for MCTP1 and PKD1L3 (I–N). MCTP1 (I, L) and PKD1L3 (J, M) are expressed in different cells in the merged images (K, N). Single taste buds are illustrated in F–H and L–N. Scale bar is 30µm in E and represents scale for C–E and I–K. Scale bar is 25µm in H and represents scale for F–H and L–N. Images are from primate CV papilla. O, Pie chart illustrating fraction of cells expressing MCTP1, TRPM5, or both MCTP1 and TRPM5. Cells with only TRPM5 signals may contain MCTP1 transcripts below the detection limit of ISH. P, Pie chart illustrating fraction of cells expressing MCTP1, PKD1L3, or both MCTP1 and PKD1L3.</p

Genes encoding transmembrane proteins are expressed in human CV taste buds.

<p>Section of human CV papilla before (A) and after (B) laser capture microdissection of taste buds. Collected taste bud regions (C), were isolated from CV papilla and used for molecular analysis of gene expression. A laser beam was used to cut the perimeter of taste buds and physically separate them from surrounding lingual epithelium. Taste buds were next lifted away from the tissue section with an adhesive cap. Panel C is an image of six isolated taste bud regions, devoid of surrounding lingual epithelium and connective tissue, on the adhesive cap. Scale bar is 40µm. Semi-quantitative PCR (D) for known taste genes (TRPM5 and PKD2L1), genes predicted or known to encode transmembrane proteins, and the housekeeping gene GAPDH in isolated CV taste buds (black bars) or non-gustatory lingual epithelium (white bars) collected by laser capture microdissection. Relative expression is shown on a logarithmic scale.</p

ANO7 is expressed in TRPM5 cells in mouse.

<p>Double label <i>in situ</i> hybridization (ISH) for ANO7 and TRPM5 (A–F). ANO7 (A, D) and TRPM5 (B, E) are expressed in similar cells in the merged images (C, F). Double label ISH for ANO7 and PKD2L1 (G–L). ANO7 (G, J) and PKD2L1 (H, K) are expressed in different cells in the merged images (I, L). Images in D–F and J–L depict single taste buds at higher magnification. Scale bar is 40µm in C and represents scale for A–C and G–I. Scale bar is 10µm in F and represents scale for D–F and J–L. Images are from mouse CV papilla. M, Pie chart illustrating fraction of cells expressing ANO7, TRPM5, or both ANO7 and TRPM5. N, Pie chart illustrating fraction of cells expressing ANO7, PKD2L1, or both ANO7 and PKD2L1.</p