Correlation Between Sox2 Gene and Glial Fibrillary Acidic

Protein (GFAP) in Type I Taste Cell of the Tongue

Julio Francisco1, Dave Harris2, and Eugene Delay3
1
Department of Biological Science, Florida Institute of Technology, 2 Neuroscience Graduate
Program, University of Vermont, 3 Department of Biology, University of Vermont
Abstract
The tongue can differentiate taste stimuli, which is advantageous since it can detect
hazardous substances and yet provides a positive eating experience. To differentiate these
stimuli, the tongue has taste buds, and each taste bud has taste sensory cells. There are three
types of taste cells: type I, II, and III. Type I taste cells are the main focus of this research.
We want to determine if a correlation exists between glial fibrillary acidic protein (GFAP)
and Sox2 gene in Type I taste cells. GFAP is an intermediate filament that provides structural
support. Sox2, on the other hand, regulates the development of taste buds and taste cells.
We attempted to visualize GFAP and Sox2 via immunohistochemistry but failed in
staining GFAP. Protocol changes must be made for future experiments, including different
protease or antigen-retrieval procedures. Overall, the experiment remains inconclusive.
Introduction
The tongue contains small, rounded protrusions called gustatory papillae. Circumvallate
papilla (CVP) is one of three types of gustatory papillae and is located at the rear of the tongue
(Purves et al., 2012). In 1989, Ahpin, Ellis, Arnott, and Kaufman highlighted the process of CVP
development in mouse. This process begins with the epithelial cells thickening, forming a
plate-like structure called placode on the surface of the tongue. Next, epithelial cells form a
downward growth in a process called invagination. This event occurs during the 13th day of
gestation, the period in which the embryo is carried inside the mothers womb. In Day 14, the
invaginated epithelial appears as an inverted cup with a dense nerve plexus within its core (pg.
41). The end-product is a fully developed CVP.
Furthermore, the development of CVP is mediated by many genes. SOX2, in particular,
encodes for B1-HMG-box transcription factor which binds DNA structure without specificity but
with high affinity (Thomas, 2001). By binding to DNA, Sox2 can control gene expression,
especially during embryonic development.This gene is highly expressed in both taste placode
and papillae (Doty, 2015). Also, since taste bud cells within each gustatory papillae have a life
cycle ranging from 2 days to over 3 weeks (Hamamichi, Asano-Miyoshi, & Emori, 2006), there is
a need for self-renewal of novel taste bud cells. Sox2 is important in regulating the
differentiation of progenitor cells on the tongue into taste bud sensory cells (Okubo, Pevny,
Hogan, 2006).
At the end of the initial development of CVP at about E18, CVP will contain
approximately 250 taste buds along its trench walls (Purves et al., 2012).There are three main
types of taste cells within each taste bud: Type I, II, and III. Type I cells are characterized for its
apical dark granules structure and possess tall microvilli (Finger, 2005). Also, Type I cells are
glial-like support cells which (Vandenbeuch, Clapp, & Kinnamon, 2008) express proteins, such
as glutamate-aspartate transporter (GLAST) and NTPDase2 (an enzyme that degrades ATP),
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responsible for clearing neurotransmitters (Dvoryanchikov, Sinclair, Perea-Martinez, Wang,
Chaudhari, 2009; Vandenbeuch et al., 2013).
Type II cells, on the other hand, possess large round nucleus and short microvilli (Finger,
2005). Type II cells express receptors for sweet, bitter, and umami. It also possess
voltage-gated Ca2+ channels and communicate with other cells via release of ATP.
Finally, Type III cells possess a narrow, spindle shaped and single, tall thick microvilli
(Finger, 2005). Type III cells form synapses with afferent nerves and release serotonin after
Type II stimulation (Vandenbeuch, Clapp, & Kinnamon, 2008).
In this experiment, we want to determine if there is a correlation between Sox2 and a
protein called GFAP. GFAP is an intermediate filament protein which provides structural support
mainly in astrocytes. When an injury occurs, astrocytes react by producing more GFAP (Eng,
Ghirnikar, & Lee, 2000). Still, little is known about the GFAP in Type I taste cells. We hope to
elucidate the correlation between Sox2 and GFAP in normal Type I taste cells through the use
of immunohistochemistry.
Experimental Procedures
Subjects
Adult mice, obtained from the Jackson Laboratory (Bar Harbor, Me, USA), were used.
The mice were maintained in a 12/12 hours of light and dark cycle with Purina Mouse Chow
available ad libitum. Protocols were approved by the Institutional Animal Care and Use
Committee (IACUC) at the University of Vermont, Burlington, VT.
Chemical Reagents
Rabbit Polyclonal Anti-Sox2 and Goat pAB GFAP (as known as Goat Anti-GFAP) were obtained
from Millipore (Temecal, CA) and Abcam, respectively. Alexa Fluor 546 Donkey Anti-goat IgG
and Alexa Fluor 546 goat Anti-rabbit were obtained from Invitrogen-Molecular Probe (Eugene,
OR, USA) and Life Technology (Eugene, OR, USA), respectively. Goat Serum was obtained
from MP Biomedicals, LLC (Solon, OH, USA).
Tissue Preparation
Adult mice were anesthetized with a lethal dose of sodium pentobarbital (Beuthanasia)
until pedal reflex activity was lost. The mouses abdominal and thoracic cavities were opened.
Once the heart was exposed, the right atrium was opened, and the left ventricle was perfused
with 0.1 M phosphate buffered saline (PBS) and 4% paraformaldehyde (Electron Microscopy
Sciences, PA, USA) in 0.1 M PBS, pH 7.4. The tongue was excised and cryoprotected with 20%
sucrose solution. After a day, the the tongue was cut into three pieces to separate circumvallate,
fungiform, and foliate papillae. Each block was placed in a mold, covered with OCT (Tissue Tek)
and frozen overnight at -80C. During the next day, the tissues were sectioned coronally at 6 m
thickness in a cryostat. The sections were collected and placed on slides.

Immunohistochemistry
The slides were washed with PBS (3x15 min). Next, the slides were immersed in the
sodium citrate and incubated in a water bath on a hot plate. Once the temperature water
reached 90, the hot plate was set to a low setting for 15 minutes. After 15 minutes, the coplin
jar was removed from the hot bath and allowed to cool for 40 minutes in the sodium citrate.
Once the slides were cooled, the slides were washed with PBS. After washing, the slides were

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placed in a moist chamber on micropipet tip racks. Then, 300 l of 5% goat serum-blocking
buffer solution was added to the slides and incubated for 1 hour. After an hour, the slides were
incubated overnight at 4 with a mixture of 1:200 dilution Rabbit Anti-Sox2 in 5% goat
serum-PBS solution.
The following day the slides were washed with PBST (PBS with 5% Triton) three times,
10-15 minutes per wash. Next, the slides were incubated in 300 l of Alexa Fluor 546 Goat
Anti-rabbit secondary antibodies at 1:1,000 dilution for an hour. The slides were then washed
three times with PBST, then incubated in 1:200 dilution of Rabbit Anti-Sox2 antibody in blocking
buffer solution for 1 hour. Following three PBS washes, the slides were immersed with nuclear
fluorescent dye Sytox Green for 3 minutes. Then, the slides were washed with PB for 15
minutes. Afterwards, coverslips were mounted with Fluoromount-G (Southern Biotech) and the
slides were stored in the refrigerator overnight at 4 prior to imaging.
Imaging
The stained sections were imaged using Nikon Eclipse E600 Scope (Spot RT KE Diagnostic
Instruments Inc.) and Spot software (Spot Advanced, Version 4.6). Using GIMP 2.8.14 (GNU
Image Manipulation Program) and ImageJ (NIH, Bethesda, MD, USA), individual nuclei and
cells positive for antibody were counted.
Results:

Figure 1. Section of circumvallate papillae labeled with Sox2 and GFAP antibodies. (A) The image on the
left shows the labeling of Sox2. The green stains are the cells nuclei, while the red marks are Sox2. (B)
The right image is labeling for GFAP. The green staining is the nuclear staining, while the weakly blue
signal could be GFAP.

Sox2 were labeled well (Figure 1A). However, GFAP labeling was unsuccessful (Figure
1B). The GFAP signal was not detectably above background noise detected, which made it
difficult to distinguish cellular components from GFAP. This suggests the need for enhancing
the GFAP signal, possibly through antigen retrieval.
There were other attempts to stain for GFAP, including different concentrations of Goat
Anti-GFAP antibody and the use of trypsin protease. Trypsin protease is a protein that hydrolyze
peptide bonds within a protein. With trypsin protease, we were hoping GFAP would be more
exposed in order to produce a strong signal. Unfortunately, the use of trypsin did not aided in

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making the GFAP signal visible neither did the concentrations of Goat Anti-GFAP antibody used
here.
Discussion:
Type I taste cells are glial-like, supporting cells which help maintain homeostasis of the
taste bud by clearing neurotransmitters using proteins such glutamate-aspartate transporter
(GLAST) (Finger, 2005). GLAST removes glutamate from the extracellular space (Lawton,
Furness, Lindemann, & Hackney, 2000). Since glial cells, such as astrocytes (Catalani et. al.,
2002) and ependyma (Roessmann, Velasco, Sindely, & Gambetti, 1980), express GFAP, we
hypothesized that Type I taste cell contain GFAP due to its glial-like nature; however, it
remained uncertain if Type I cells expressed GFAP. The main focus of this experiment was not
to determine if Type I taste cells express GFAP. The aim of this research was to determine if
there was a correlation between GFAP and Sox2. Since this experiment is a new venue of
scientific work, troubleshooting was the main issue of the research. Sox2 was labeled correctly
but GFAP failed to label. This is due to high background noise that made it difficult to
distinguish GFAP from other cellular components. As a result, the correlation between GFAP
and Sox2 was difficult to determine due to the lack of data. Therefore, other approaches must
be employ. Several ways to make GFAP signal more stronger may be to use a different
protease or incorporate other techniques such as biotinylation and different antigen-retrieval
process.
In addition, it should be taken into consideration that GFAP might not be expressed in
the tongue. In fact, there are different intermediate filaments found in the tongue from different
animals. For instance, Witt, Reutter, Ganchrow, and Ganchrow (2000) found two class of
intermediate filaments, cytokeratins and vimentin, in the taste buds of human, chick, and
Xenopus laevis. Therefore, it is possible that there are intermediate filaments other than GFAP
that might presence to provide structural support to type I taste cells. To determine if GFAP
does exist, a western blotting approach should be taken.
Overall, we conducted immunohistochemistry to stain for Sox2 and GFAP in type I cells
of CVP. The staining for Sox2 was performed well; however, GFAP staining was undetectable.
We must attempt other approaches to stain for GFAP. Also, one must also consider that GFAP
is not localized in Type I taste cells. If we do successfully label GFAP in the CVP, then we will
attempt to label for GFAP in fungiform papillae in future experiment to truly understand the
correlation between GFAP and Sox2.

Acknowledgement
I will like to thank Dr. Eugene Delay, Diane Morgan, and Dave Harris for helping me with the
research project.
Funding Statement
This project described was supported by a grant from NSF (1262786; REU Site:
Summer Neuroscience Undergraduate Research Fellowship Program at UVM), the College of
Medicine at UVM, and NIH-NINDS (1R01DC012829).

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