Mitsui et al. Neural Systems & Circuits 2011, 1:5
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RESEARCH

Open Access

Genetic visualization of the secondary olfactory
pathway in Tbx21 transgenic mice
Sachiko Mitsui1, Kei M Igarashi2,3, Kensaku Mori2, Yoshihiro Yoshihara1*

Abstract
Background: Mitral and tufted cells are the projection neurons in the olfactory bulb, conveying odour information
to various regions of the olfactory cortex. In spite of their functional importance, there are few molecular andgenetic tools that can be used for selective labelling or manipulation of mitral and tufted cells. Tbx21 was first
identified as a T-box family transcription factor regulating the differentiation and function of T lymphocytes. In thebrain, Tbx21 is specifically expressed in mitral and tufted cells of the olfactory bulb.
Results: In this study, we performed a promoter/enhancer analysis of mouse Tbx21 gene by comparing nucleotide
sequence similarity of Tbx21 genes among several mammalian species and generating transgenic mouse lines withvarious lengths of 5’ upstream region fused to a fluorescent reporter gapVenus. We identified the cis-regulatory
enhancer element (~300 nucleotides) at ~ 3.0 kb upstream of the transcription start site of Tbx21 gene, which isboth necessary and sufficient for transgene expression in mitral and tufted cells. In contrast, the 2.6-kb 5’-flanking
region of mouse Tbx21 gene induced transgene expression with variable patterns in restricted populations ofneurons predominantly located along the olfactory pathway. Furthermore, we generated transgenic miceexpressing the genetically-encoded fluorescent exocytosis indicator, synaptopHluorin, in mitral and tufted cells forvisualization of presynaptic neural activities in the piriform cortex.
Conclusions: The transcriptional enhancer of Tbx21 gene provides a powerful tool for genetic manipulations of
mitral and tufted cells in studying the development and function of the secondary olfactory pathways from thebulb to the cortex.

Background
Odour molecules emitted from objects enter into thenostrils, reach the olfactory epithelium, and bind odourant receptors (ORs) expressed on the cilia of olfactory
sensory neurons (OSNs). The odour information is thenconverted into electrical signals, transmitted to glomeruli in the olfactory bulb (OB) via precisely wired neuralcircuitry and represented as topographic ‘odour maps’
on the glomerular array of the OB [1]. After the OR
multigene family was discovered [2], the basic principles
of olfactory axon wiring to the glomeruli were elucidated using molecular biological, genetic engineering,
electrophysiological and neural activity imaging studies[3-9]. In particular, the availability of various molecularmarkers and transcriptional enhancers has facilitated the* Correspondence: yoshihara@brain.riken.jp
1
Laboratory for Neurobiology of Synapse, RIKEN Brain Science Institute, 2-1
Hirosawa, Wako-shi, Saitama 351-0198, Japan
Full list of author information is available at the end of the article

distinguishing, labelling and manipulating of distinct
types of OSNs. This has greatly promoted the recent
progress in the understanding of the primary olfactory
system [10-14].
In contrast, little is known of the functional architecture of axonal projections that extend from the OB tothe olfactory cortex in mammals. For example, it is notknown how odour information is transmitted from the
OB to various regions of the olfactory cortex, including
the piriform cortex, the cortical amygdaloid nuclei andthe lateral entorhinal cortex, or how the topographic
OR map on the OB is decoded in the olfactory cortex
for the translating of odour inputs into olfactory perception, emotion, memory and behavioural responses.
Mitral and tufted cells are the excitatory projection neurons in the OB that relay the odour information fromglomeruli to various regions of the olfactory cortex.
Despite their functional significance, only a few© 2011 Mitsui et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction inany medium, provided the original work is properly cited.

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molecular genetic tools are available for the selective
labelling or manipulation of mitral and tufted cells[15-17].
In this study, we focused on the mouse Tbx21 (T-bet)
gene, which is specifically expressed in mitral and tufted
cells in the brain [18]. We analysed transgenic mice thatharboured transgenes with various lengths of Tbx21 5’upstream regions fused to a fluorescent reporter. As aresult, we identified a cis-regulatory enhancer elementthat is necessary and sufficient for transgene expression
in mitral and tufted cells. Furthermore, the enhancer
was utilized to visualize presynaptic neural activities in
the olfactory cortex by generating transgenic mice that
expressed the genetically-encoded fluorescent exocytosis
indicator, synaptopHluorin (spH), in mitral and tufted
cells.

Results
Mitral and tufted cell-specific expression of Tbx21 proteinin the mouse brain

It was previously reported that Tbx21 messenger RNA(mRNA) is expressed specifically in the mitral and tufted
cells in the developing mouse brain [18]. We generated
polyclonal antibodies in a guinea pig against a carboxylterminal 20-amino acid sequence of mouse Tbx21 [19]and analysed the distribution of Tbx21 protein in thedeveloping and adult brains by immunohistochemistry.
Tbx21 protein was specifically localized to the mitraland tufted cells in the OB, while no expression wasobserved in any other cell types in the brain (Figure 1A
and 1B). The expression level of Tbx21 protein in the
mitral and tufted cells was higher in the accessory OB(AOB) than that in the main OB (MOB; Figure 1B). The
antibody gave no signal on the OB section preparedfrom Tbx21 knockout mouse (Figure 1C), confirming its
specific reactivity to Tbx21 protein. A double immunofluorescence labelling clearly showed the presence ofTbx21 protein in the mitral and tufted cells but not in
the Arx-positive GABAergic interneurons (granule andperiglomerular cells; Figure 1D-F). A triple immunofluorescence labelling revealed that Tbx21 was colocalized with Tbr1, Tbr2, PGP9.5 and protocadherin-21(Pcdh21), the four markers for mitral and tufted
cells (Figure 1G-N). Quantification of the numbers of
Tbx21-, Tbr1- and Pcdh21-positive cells indicated thatTbx21 is expressed in a vast majority, if not all, of themitral cells (94.6% ± 0.6% of the Tbr1-positive mitralcells) and a large population of tufted cells (72.4% ±
3.4% of the Pcdh21-positive tufted cells) in the adult OB(n = 5; Additional File 1). During development, theexpression of Tbx21 protein was first detectable atembryonic day 14.5, gradually increased to reach the
maximal level in neonates (Figure 1O-R), and slightly
decreased into adulthood (Figure 1A and 1B), consistent

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with the previous study showing ontogenic expressionof Tbx21 mRNA [18].
Faithful expression of transgene in mitral and tufted cells
in Tbx5.0gV transgenic mice

The Tbx21 gene locates on the long arm of mouse chromosome 11, a region syntenic to the human chromosome 17, and consists of six exons and five introns(Figure 2A). A homology plot revealed highly conserved
nucleotide sequences in the 5’-flanking region of mouse,
human, rhesus, dog and rat Tbx21 genes (Figure 2B),
implying the presence of potential cis-regulatory enhancer elements. In order to identify the mitral and tufted
cell-specific transcriptional enhancer, we first constructed three transgenes that carry different lengths of
the 5’-flanking sequence of mouse Tbx21 gene (5.0, 2.6
and 1.0 kb) fused to the membrane-tethered fluorescentreporter gapVenus complementary DNA (cDNA; Figure2C). Several founders and lines of transgenic mice weregenerated with each construct and analysed for gapVenus expression in the brain. The results are summarizedin Additional File 2.
With the longest construct harbouring 5.0-kb 5’-flanking
region of the Tbx21 gene (Tbx5.0gV), gapVenus was faithfully expressed in the mitral and tufted cells of transgenic
founders and lines at a highly efficient rate (15/17: 88%),
although there are some variations in the expression patterns and/or levels which are possibly due to the positional
effects of transgene integration sites at distinct chromosomal loci (Figure 3 and Additional File 1). Consistent withthe difference in endogenous Tbx21 expression levels
between the AOB and MOB, the transgene was expressedmore robustly and frequently in the mitral and tufted cellsof the AOB than those of the MOB in most transgeniclines. In ventrally viewed images of whole-mount brains,the embossed gapVenus fluorescence was observed along
the secondary accessory olfactory pathway reaching themedial and posteromedial cortical amygdaloid nuclei(Figure 3 A2, B, C and D1). In order to examine the gapVenus expression in more detail, the brain sections of
Tbx5.0gV mouse (line #4) were immunohistochemicallystained with anti-green fluorescent protein (GFP) antibody. The gapVenus expression clearly delineated the trajectory of mitral and tufted cell axons projecting from the
OB to various regions of the olfactory cortex, including
the anterior olfactory nucleus, the olfactory tubercle,
the piriform cortex, the medial and posteromedial
cortical amygdaloid nuclei and the lateral entorhinal cortex (Figure 3D2-D12). The ontogenic expression of gapVenus transgene in the mitral and tufted cells was paralleledwith that of the endogenous Tbx21, further confirming
the faithfulness of this enhancer/promoter activity (Additional File 3). These results indicate that the 5.0-kb
5’-flanking region of the Tbx21 gene can drive transgene

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Figure 1 Mitral and tufted cell-specific expression of Tbx21 protein. (A) A parasagittal section of adult mouse brain labelled with anti-Tbx21
antibody. Strong fluorescent signals are specifically observed in the olfactory bulb (OB). (B) A higher magnification of the OB. (C) No
immunoreactivity in the OB section from Tbx21-deficient mouse. (D-F) Double immunofluorescence labelling of OB section with anti-Tbx21 (D, F(magenta)) and anti-Arx (E and F (green)) antibodies. Tbx21 protein is expressed in the mitral and tufted cells but not in Arx-positive
interneurons. (G-J) Triple immunofluorescence labelling of OB section with anti-Tbx21 (G and J (red)), anti-Tbr1 (H and J (green)) and anti-Tbr2 (Iand J (blue)) antibodies. The three T-box family transcription factors are expressed in mostly overlapping, but some differential, subpopulationsof mitral and tufted cells. (K-N) Triple immunofluorescence labelling of the OB section with anti-Tbx21 (K and N (red)), anti-PGP9.5 (L and N(green)) and anti-protocadherin 21 (Pcdh21) (M and N (blue)) antibodies. All three molecules are expressed in the mitral and tufted cells but
localized to different subcellular compartments (Tbx21 in nuclei; PGP9.5 in cell bodies and proximal dendrites; Pcdh21 in cell bodies andglomeruli). (O-R) Developmental expression of Tbx21 protein in OB coronal sections from E14.5 (O), E16.5 (P), P0 (Q) and P7 (R) mice. EPL,
external plexiform layer; GCL, granule cell layer; GL, glomerular layer; IPL, internal plexiform layer; MCL, mitral cell layer.

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Figure 2 Organization of mouse Tbx21 gene and transgene constructs. (A) Genomic organization of Tbx21 gene on mouse chromosome
11. Exons are indicated by boxes with numbers: purple boxes indicate the open reading frame; and light blue boxes represent 5’- and 3’-nontranslated regions. (B) Nucleotide sequence identity plots of human, rhesus, dog and rat Tbx21 genes compared with mouse ortholog by using
the VISTA program [41]. Highly conserved regions (> 80% identity) in 5’-flanking sequences and introns are shown in pink. Mitral and tufted cellspecific enhancer (MCE; 307 bp) is present at ~3 kb upstream from the transcription start site of Tbx21 gene. (C) Transgene constructs. 5’flanking regions of Tbx21 gene with different lengths (5.0, 2.6 and 1.0 kb: yellow) were fused to a membrane-targeted Venus complementary
DNA (cDNA) (gV: green) to generate Tbx5.0gV, Tbx2.6gV and Tbx1.0gV transgenes. Highly conserved 307-bp sequence (MCE: red) and SV40
minimal promoter (blue) were fused with gapVenus cDNA to generate MCE-gV transgene. 5.0-kb 5’-flanking region of Tbx21 gene was fusedwith synaptopHluorin cDNA (spH: dark blue) in order to generate Tbx-spH transgene.

expression efficiently and reproducibly in the mitral andtufted cells.
Variable patterns of transgene expression in Tbx2.6gVtransgenic mice

Next, we examined Tbx2.6gV transgenic mice in which a
2.6-kb 5’-flanking region was used to direct gapVenus
expression. The analysis of Tbx2.6gV mice yielded twointeresting findings. First, the transgene expression was
hardly detectable in the mitral and tufted cells (2/43: 5%).
Among the four founders and 39 lines analysed, only twolines (#127: Figure 4K and #123: Figure 4L, Additional

File 2) showed a relatively broad pattern of gapVenus
expression in many types of neurons in the brain, including the mitral and tufted cells. Second, the transgene
expression was frequently observed in various types ofneurons along the olfactory neural pathways (27/43:63%), although the expression patterns and/or levels differed from line to line. For instance, gapVenus was
expressed in the OSNs in the olfactory epithelium in two
founders and 21 lines (Figure 4A, E1, F1, G-M, Additional
File 4), among which one founder and six lines also
showed the transgene expression in the vomeronasal sensory neurons in the vomeronasal organ. In most of theMitsui et al. Neural Systems & Circuits 2011, 1:5
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Figure 3 Mitral and tufted cell-specific transgene expression in Tbx5.0gV transgenic mice. (A1-A4) gapVenus fluorescence in whole-mountbrain of Tbx5.0gV transgenic mouse line #1 viewed from dorsal (A1), ventral (A2), lateral (A3) and medial (A4) sides. Strong gapVenus fluorescenceis observed in the mitral and tufted cell axons projecting from the olfactory bulb (OB) to various regions of the olfactory cortex. (B, C, D1) Ventral
views of whole-mount brains of Tbx5.0gV line #2 (B), #3 (C), and #4 (D1). In all the Tbx5.0gV lines, the accessory OB (AOB) mitral/tufted cells
display a higher level of gapVenus expression than the main OB (MOB) mitral and tufted cells, clearly delineating the secondary vomeronasal
pathway from the AOB to the amygdaloid nuclei. (D2-D12) Parasagittal (D2, D3) and coronal (D4-D12) brain sections of Tbx5.0gV mouse line #4
immunostained with anti-green fluorescent protein antibody. (D3) A higher magnification of OB. AON, anterior olfactory nucleus; LEnt, lateralentorhinal cortex; lot, lateral olfactory tract; Me, medial amygdaloid nucleus; OTu, olfactory tubercle; Pir, piriform cortex; PMCo, posteromedial
cortical amygdaloid nucleus.

lines with ectopic transgene expression in OSNs, gapVenus was observed in a restricted subset of OSNs in theventrolateral zone of the olfactory epithelium, which
project axons to the ventrolateral OB (Figure 4A, E1, F1,
G-M, Additional File 4). This pattern of gapVenus
expression in the primary olfactory pathway was complementary to that of NAD(P)H:quinone oxidoreductase(NQO1)-positive OSNs in the dorsomedial zone (Additional File 4). Other examples of gapVenus expression
along the olfactory neural pathways included the granulecells of OB (founder #1: Figure 4B), the neurons in theanterior olfactory nucleus (line #1: Figure 4C), theneurons in the nucleus of lateral olfactory tract and the
granule cells in AOB (line #4: Figure 4D), a subset ofneurons in the lateral entorhinal cortex (founder #3:
Figure 4E) and the neurons in the ventromedial hypothalamic nucleus (line #103: Figure 4F, line #129: Figure 4I).
In several lines which showed labelling of certain types ofneurons along the olfactory pathways, the transgene wasalso expressed in cells of non-olfactory-related areas(Additional File 2) such as the mammillary nucleus (line#105: Figure 4J), the pontine nucleus and the optic nerve(line #123: Figure 4L), the facial nucleus (line #114:
Figure 4M), the thalamus, the hippocampus, theMitsui et al. Neural Systems & Circuits 2011, 1:5
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Figure 4 Variable patterns of transgene expression in Tbx2.6gV transgenic mice. (A1, A2) Line #008: a zonal subset of olfactory sensory
neurons (OSNs) projecting axons to the ventrolateral region of the olfactory bulb (OB). (B1-B3) Founder #1: the granule cells (Gr) in the main OB.
(C1-C6) Line #001: the neurons in the anterior olfactory nucleus (AON) projecting axons to the contralateral AON and OB via the anterior
commissure (ac) (C2-C5) and the neurons in the central nucleus of amygdala (Ce) (C6). (D1-D4) Line #004: the granule cells in the accessory OB(AOB) (D2) and the neurons in the nucleus of lateral olfactory tract (NLOT) (D3, D4). (E1-E5) Founder #3: a small population of neurons in thelateral entorhinal cortex (LEnt) projecting axons to the dentate gyrus of the hippocampus (E2-E5) and a ventrolateral zonal subset of OSNs. PMCo,
posteromedial cortical amygdaloid nucleus. (F1-F4) Line #103: the neurons in the ventromedial hypothalamic nucleus (VMH) (F2-F4) and aventrolateral zonal subset of OSNs. (G-N) Other representative examples of Tbx2.6gV transgenic lines with various patterns of gapVenus
expression. MM, medial mammillary nucleus.

cerebellum, the area postrema and the spinal cord (datanot shown). In only two instances, gapVenus expression
was detected restrictedly in non-olfactory-related areas:
the cerebellum (founder #4: data not shown) and thebrainstem oligodendrocytes and the optic nerve (line#117: Figure 4N). These results suggest that the 2.6-kb 5’flanking region of Tbx21 gene does not contain the mitraland tufted cell enhancer but can drive the transgene
expression preferentially in some neurons along the olfactory neural pathways.
In contrast, the shortest fragment with 1.0-kb
upstream region of the Tbx21 gene showed little

enhancer/promoter activity. In all three lines of
Tbx1.0gV transgenic mice, gapVenus was not detectablein any type of cells along the olfactory neural pathways
including the mitral and tufted cells (Additional File 2).
MCE: a transcriptional enhancer for transgene expression
in mitral and tufted cells

The above results indicate that a crucial cis-regulatory
element for the transcription in mitral and tufted cells
resides between -5.0 and -2.6 kb from the transcription
start site of mouse Tbx21 gene. By a scrutinized homology search, we identified an extremely conserved 307-bp

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sequence (putative mitral and tufted cell-specific enhancer; MCE) at ~ 3.0 kb upstream of the transcription
start site of the mouse Tbx21 gene, which showed morethan 80% nucleotide identities with the human, monkey,
dog, and rat orthologs (Figure 2B and 5). In order toexamine whether this region can act as a transcriptional
enhancer to direct gene expression in the mitral andtufted cells, we constructed a transgene consisting ofMCE, SV40 minimal promoter, gapVenus and SV40
polyadenylation signal (Figure 2C: MCE-gV) and generated transgenic mice. Among the three founders andseven lines of MCE-gV transgenic mice examined, gapVenus expression was observed in the mitral and tufted
cells in the line #1 (Figure 6A), line #5 (Figure 6B),
founder #3 (Figure 6C) and line #8 (data not shown;
4/10: 40%, Additional File 2). Although the fluorescenceintensity of gapVenus in the mitral and tufted cells was
weak on whole-mount brain preparations (Figure 6 A1,
B1 and C 1) and ectopic transgene expression was frequently observed in other types of cells in these lines,
anti-GFP immunohistochemistry of the brain sections
clearly demonstrated the transgene expression in the
mitral and tufted cells (Figure 6A 2,3 , B 2, 3 and C 2-7 ).
Together with the results of Tbx5.0gV, Tbx2.6gV andTbx1.0gV transgenic mice, these findings indicate thatthe conserved sequence of 307 bp, designated as MCE,

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is required for and capable of inducing transgene
expression in the mitral and tufted cells.
Transgenic mouse expressing spH in mitral andtufted cells

Finally, we made use of the Tbx21 enhancer/promoter
for targeted expression of spH in the mitral and tufted
cells. spH is a genetically-encoded fluorescent protein
which can be used as an indicator of exocytosis, reflecting the presynaptic neural activities [20-22]. Five transgenic mouse lines (Tbx-spH) were generated thatharbour the 5.0-kb Tbx21 enhancer/promoter region
fused to spH (Figure 2C). Among them, one line (TbxspH #1) showed significant fluorescence of spH in
mitral and tufted cell axons and their terminal fields inthe olfactory cortex. Although a single Tbx-spH allele
on heterozygous background showed only weak fluorescence, the generation of homozygous transgenic mice
resulted in a marked increase of spH fluorescence thatwas easily observed on a whole-mount brain preparation
under a fluorescence stereomicroscope (Figure 7A and
7B). Therefore, the homozygous Tbx-spH #1 transgenic
mice were used in the subsequent in vivo experiments.
The piriform cortex was exposed in anaesthetizedtransgenic mice and the spH fluorescence was monitoredwith a CCD camera on a conventional fluorescence

Figure 5 Nucleotide sequence alignment of mitral and tufted cell-specific enhancer (MCE) from various mammalian species. Nucleotide
sequence alignment of MCE from mouse, rat, dog, cow, monkey and human Tbx21 gene upstream regions. Putative transcription factor-bindingsites are shown with upper bars. Two T-box sites (red) are present, which may act as targets of Tbr1, Tbr2 or Tbx21 expressed in the mitral andtufted cells.

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Figure 6 Mitral and tufted cell-specific enhancer (MCE) directs transgene expression in mitral and tufted cells. (A1-A3) MCE-gV transgenic
mouse line #1 (A1-A3), line #5 (B1-B3) and founder #3 (C1-C7). (A1, B1, C1) gapVenus fluorescence of whole-mount brains viewed from ventral side.
(A2, B2) Parasagittal brain sections of MCE-gV lines #1 (A2) and #5 (B2) labelled with anti-green fluorescent protein (GFP) antibody. (A3, B3)
Magnified views of the olfactory bulb. (C2-C7) Coronal sections of MCE-gV founder #3 labelled with anti-GFP antibody. In these three MCE-gV
transgenic lines, gapVenus expression is detected in the mitral and tufted cells.

microscope connected to an image analysis system. In
order to validate that the change in spH fluorescencereflects pre-synaptic activity of mitral and tufted cellaxon terminals, electrical stimulation was focally applied
to a small spot in the glomerular layer of the OB where
the dendrites of mitral and tufted cells receive odour
information from the OSNs. The intensity of spH fluorescence in the piriform cortex was dependent on thepulse number (Figure 7 C 1 , C 2 and C 3 ) and amplitude(Figure 7 7D1 and D2) of electrical stimulation to the OB,
indicating the positive correlation between the extent ofactivated mitral and tufted cells and the spH signal intensity in axon terminals of the mitral and tufted cells.
In order to examine how the glomerular map in OB isrepresented in the piriform cortex, we first identified aglomerulus responsive to specific odourants by using theoptical imaging method of intrinsic signals, then applied
electrical stimuli onto the glomerulus and compared thepre-synaptic neural activities of the mitral and tufted
cells in the piriform cortex. As odourant stimuli, we used
two compounds: 2,4,5-trimethylthiazoline (TMT: a component of fox odour) and heptanoic acid (HA: a component of apple odour). TMT activates a few glomeruli inthe posterior region (class II domain) of the dorsal zone
(Figure 7E1), while HA activates glomeruli in the anterior
region (class I domain; Figure 77F1). Electrical stimulation of these two distinct glomerular foci resulted in theincreased spH fluorescence in overlapping, but different,

regions of the piriform cortex: the ventroposterior region
by TMT (Figure 7E2 and E3) and the dorsal region by HA(Figure 7F2 and F3). A quantitative analysis of spH signals
revealed that identical stimulations evoked similar spatial
patterns of spH responses, whereas different stimulations
evoked distinct pattern of spH responses in the piriform
cortex (Figure 7G1 and G2).
Finally, we measured the change of spH fluorescencein the mitral and tufted cell axon terminals upon theodourant stimulation. Applications of TMT and HA tomouse nostril induced a significant increase in spH signals in the anterior piriform cortex with partly overlapping but mostly distinct patterns (Figure 7H1 and H2).
The regions activated by TMT and HA applications
roughly corresponded to those activated by the electricalstimulation of the respective glomeruli responsive to thetwo odourants. Further experiments of in vivo spH imaging in the olfactory cortex will enhance our understanding of neuroanatomical and functional architectureof the secondary olfactory system.

Discussion
Members of the T-box family transcription factors play
important roles in development and functions of various
tissues including the brain [23]. In the OB, Tbr1, Tbr2
and Tbx21 are expressed in the excitatory projection
neurons, mitral and tufted cells [18,24,25]. In particular,
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Figure 7 Transgenic mouse expressing synaptopHluorin (spH) in mitral and tufted cells. (A, B) Fluorescence of whole-mount brain of TbxspH transgenic mouse viewed from ventral (A) and lateral side (B). (C1-C3) spH signal increase in the piriform cortex is dependent on the pulse
numbers of electrical stimulation onto olfactory bulb (OB) glomeruli. ΔF/F fluorescence change images (C1), time course (C2) and pulse-response
relationship (C3). (D1, D2) spH signal increase is dependent on the amplitude of electrical stimulation onto OB glomeruli. Time course (D1) andamplitude-response relationship (D2). (E1-F3) spH signal increase upon the electrical stimulation of trimethylthiazoline (TMT; E1-E3) and heptanoic
acid (HA)-responsive (F1-F3) glomeruli. Odourant-responsive glomeruli were identified by the intrinsic signal imaging of the dorsal surface of OB
and stimulated with a microelectrode (E1, F1). The electrical stimulation of these two glomeruli leads to the spH signal increase in distinct
subregions of the piriform cortex (E2, E3, F2, F3). (G1) Similarity and dissimilarity between fluorescent responses evoked by the OB electricalmicrostimulation. The imaged region was divided into four subregions (APCd, APCv, PPCd, PPCv: dorsal and ventral parts of the anterior andposterior piriform cortex) [40,43]. Significantly responsive areas (P < 0.01, yellow-to-red areas in E2-3 and F2-3) evoked by electrical stimulation of
TMT- and HA-responsive glomeruli were plotted (n = 3). (G2) Cluster analysis of spH responses. The linkage distance was 4.95 ± 1.70 between
any two TMT stimulation images and 22.4 ± 4.53 between any two HA stimulation images. In contrast, the distance between any TMT
stimulation image and any HA stimulation image was significantly larger (58.0 ± 1.95; P< 0.00001). (H1, H2) spH signal increase in the piriform
cortex upon odourant stimulation. TMT (H1) or HA (H2) applied into mouse nostril leads to the spH signal increase in overlapping but distinct
regions of the piriform cortex.

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and tufted cells, whereas the Tbr1 and Tbr2 are alsopresent in various cell types in other brain regions. The
strictly localized expression of Tbx21 gene prompted us
to analyse its transcriptional enhancer activity usingtransgenic mouse approach. Transgene expression in the
mitral and tufted cells was efficiently induced with the
5’-flanking 5.0-kb region, but neither with the 2.6-kb
nor 1.0-kb regions, indicating that the crucial enhancer
activity resides within the 5.0 - 2.6 kb upstream
sequence (Figure 2 and 3). In silico homology search of
Tbx21 genes from various animal species identified ahighly conserved ~300-bp sequence located at 3.2-kb
upstream from the transcriptional start site of mouse
Tbx21 gene. When combined with the SV40 minimal
promoter, this ~300-bp sequence could direct the transgene expression in the mitral and tufted cells (Figure 6).
Hence, we designated this sequence as the mitral andtufted cell enhancer, MCE.
The nucleotide sequences of MCE in various mammalian species are extremely conserved with higher than
80% identities (Figure 5). The MCE contains several
characteristic motifs of transcription factor-binding sites.
Intriguingly, two putative T-box-binding sites are present in the mouse MCE, which are similar to the consensus T-box motif (TCACACCT) with only one
nucleotide substitution (Figure 5) [23,26], suggesting a
possibility that Tbx21 gene expression is regulated by
T-box transcription factors expressed in the mitral andtufted cells such as Tbr1, Tbr2 or Tbx21 itself. This
notion is corroborated by the fact Tbx21 expression iscompletely absent in the OB of Tbr1-deficient mice[18]. In addition, the MCE contains putative bindingsites for Brn-3, Gli3, Klf15 and Six4. These transcription
factors and related molecules belonging to the samefamilies seem to play important roles in various aspectsof the olfactory system development [27-31]. Further
studies are required in order to identify the transbinding factors crucial for the mitral and tufted cellspecific transcriptional regulation by MCE.
Previous studies described transgene expression in the
mitral and tufted cells by using transcriptional enhancers of Pcdh21 [15], neurotensin [16] and AP-2ε [17].
For neurotensin and AP-2ε, the gene targeting strategy
was utilized in order to make knock-in mice expressing
tauGFP and Cre recombinase, respectively. For Pcdh21,
a long upstream region encompassing ~10 kb was
required for the expression of Cre and tetracycline activator in transgenic mice. However, in the cases of
Pcdh21 and neurotensin, the transgene expression is not
specific to the mitral and tufted cells but is also
observed in other types of neurons in the brain [15,16].
For AP-2ε, the transgene expressions in the mitral andtufted cells do not persist into adulthood but begin todiminish soon after birth [17,32]. Thus, the Tbx21 gene

Page 10 of 14

enhancer/promoter will become a more useful and convenient tool for genetic manipulations of the mitral andtufted cells because its highly specific and efficientenhancer activity with the shorter length of necessary
sequence (~5 kb) guarantees the mitral and tufted celldirected transgene expression persistently into
adulthood.
Unexpectedly, we observed curious patterns of gapVenus expression in many transgenic founders and lines,
when the 2.6-kb upstream region of Tbx21 gene wasused as the enhancer/promoter. In many lines of
Tbx2.6gV transgenic mice, gapVenus expression was
detected frequently in various types of neurons along theolfactory neural pathways, such as the OSNs, vomeronasal sensory neurons, granule cells in the OB, neurons in
the olfactory cortex including the anterior olfactory
nucleus, piriform cortex, nucleus of the lateral olfactory
tract, lateral entorhinal cortex and the ventromedial
hypothalamic nucleus. We have encountered a similar
phenomenon with transgene zebrafish in which a 10-kb
upstream region of Lhx2a gene was used as an enhancer/
promoter. Although the endogenous Lhx2a gene is specifically expressed in a small subset of OSNs in zebrafish,
fluorescent reporters were detected faithfully not only in
the OSNs but also in a subset of mitral cells ectopically[33]. Interestingly, the fluorescent OSNs projected theiraxons predominantly into a glomerular cluster onto
which the fluorescent mitral cells send their dendrites,suggesting that the transgene was coincidently expressedin the pre-synaptic and post-synaptic neurons. Thus, we
speculate the existence of a putative transcriptional regulatory system that is active along selective neural circuits.
Such a mechanism would, for example, help coordinatedexpression of homophilic adhesion molecules in bothpre- and post-synaptic neurons for the formation ofsynaptic connections. Further studies are in progress to
identify the olfactory circuit-specific transcriptional
enhancer elements in both mice and zebrafish and to elucidate their evolutional and physiological significance ofregulatory gene expression along selective neural circuits.
We have applied the MCE-containing Tbx21 enhancer
for specific expression of spH in the mitral and tufted
cells and succeeded in in vivo imaging of presynaptic
activities in the piriform cortex of transgenic mice.
Upon either the electrical stimulation of OB glomeruli
or the odourant application into nostril, we observed
the significant increase in spH signals in the piriform
cortex, reflecting the active exocytosis events at theaxon termini of the mitral and tufted cells. Further comprehensive analysis with a systematic battery of various
odour molecules will help us to decipher how the odour
information represented on the glomerular array of OB
is transferred to the third-order neurons in the olfactorycortex. In addition to spH, any genetically-encodedMitsui et al. Neural Systems & Circuits 2011, 1:5
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molecular tools of interest will be expressed in the
mitral and tufted cells by the use of Tbx21 enhancer.

Conclusions
We have identified the mitral and tufted cell-specifictranscriptional enhancer upstream of the mouse Tbx21
gene. A short sequence of ~300 bp was necessary andsufficient for the transgene expression in these projection neurons of the olfactory bulb. spH expression in
the mitral and tufted cells enabled us to monitor thepresynaptic neural activities in the piriform cortex.
Thus, this study paves a new avenue of the olfactory
research into higher brain centres.
Methods
Plasmid construction

The membrane-targeted fluorescent reporter, gapVenus,
consists of a variant of yellow fluorescent protein with
fast and efficient maturation (Venus) and an N-terminal
palmitoylation signal of growth-associated protein-43(GAP-43) [34,35]. gapVenus cDNA was inserted into
EcoRI site of pBstN vector (containing human b-globin
gene intron and Simian virus 40 (SV40) polyadenylation
signal) to construct pBstN-gapVenus. The 5.0-kb 5’flanking region of mouse Tbx21 gene was amplified
from mouse tail DNA as a template with a pair of primers (F002-SacII: 5’-TTCCCCGCGGATAAGTGGCCTGACGTACAGGCGAG-3’; R002-XbaI: 5’-ATTCTCTAGACCGAGGCGGGCTGGGCGCCTTCCAG-3’) using
Expand High Fidelity polymerase chain reaction (PCR)
System (Roche Applied Science, IN, USA) and inserted
into the SacII/XbaI site of pBstN-gapVenus to generate
pTbx5.0gV. Tbx5.0gV, Tbx2.6gV and Tbx1.0gV transgenes with different lengths of the Tbx21 promoter/enhancer region were excised form pTbx5.0gV plasmidby digestion with SacII/KpnI, EcoRV/KpnI and AgeI/
KpnI, respectively. In order to construct pMCE-gV, a
307-bp fragment (-3462 ~ -3045) located upstream of thetranscription initiation site of mouse Tbx21 gene was
PCR-amplified and inserted into pBstN-gapVenus plasmid. In order to construct pTbx-spH, a similar procedure
was used as described above for pTbx5.0gV, except that
cDNA encoding spH [20] was used instead of gapVenus.
Animals

The generation of transgenic mice was performed as previously described [36]. Briefly, gel-purified transgenes weremicroinjected into the pronucleus of fertilized eggs thatwere obtained from crossing (C56BL/6J × DBA/2J) F 1
mice. The manipulated eggs were cultured to the two-cellstage and transferred into oviducts of pseudo-pregnant foster females (ICR strain). Integration of the transgenes was
screened by PCR of tail DNA. The Tbx21 (T-bet) mutant
mice [37] were purchased from The Jackson Laboratory

Page 11 of 14(Maine, USA). All animal experiments were approved by
the Animal Care and Use Committee of RIKEN and conformed to National Institutes of Health guidelines.
Immunohistochemistry

Immunohistochemistry of brain sections was carried out
as previously described [38]. The following primary antibodies were used: guinea pig anti-Tbx21 (1:10000) [19];rat anti-GFP (1:1000, Nacalai Tesque, Kyoto, Japan); rabbit anti-Arx (1:1000 provided by Dr K Kitamura) [39];rabbit anti-Tbr1 (1:1000, Abcam, Cambridge, UK), rat
phycoerythrin-conjugated anti-Tbr2 (1:200, eBioscience,
CA, USA), rabbit anti-Pcdh21 (1:1000) [14]; mouse antiPGP9.5 (1:100, Abcam); goat anti-NQO1 (1:100, Abcam).
Secondary antibodies labelled with Cy3, Cy5 or Alexa488
were purchased from Jackson ImmunoResearch (PA,USA) and Molecular Probes (OR, USA). Fluorescentimages were obtained with a fluorescent microscope(Axioplan, Carl Zeiss, Oberkochen, Germany) equippedwith a CCD camera and an image analysis system (DP70, Olympus, Tokyo, Japan) or a confocal laser scanningmicroscope (Fluoview FV1000, Olympus).
In vivo spH imaging

Imaging was performed in homozygous Tbx-spH mice
aged 6-7 weeks in accordance with the guidelines of the
Physiological Society of Japan and the animal experiment committee of the University of Tokyo. Animals
were anaesthetized by intraperitoneal injection of medetomidine (0.5 mg/kg) and urethane (0.6 g/kg). Additional doses of urethane were given if necessary.
Animals were placed in a custom-built stereotaxic apparatus (Narishige, Tokyo, Japan). Body temperature wasmaintained at 37.5°C using a homeothermic heatpad
system (MK-900, Muromachi, Tokyo, Japan). Respiratory rhythms were detected using a piezo transducer(MLT 1010, ADInstruments Japan Inc, Nagoya, Japan).
The anterior and posterior piriform cortex was exposedby removing a 5 × 4 mm area of skull as previously
described [40]. Agarose gel was mounted on the brain
and covered with a glass coverslip to form a temporarychamber for the optical imaging. The piriform cortex
was imaged using an Olympus BX51 microscope andepifluorescence condenser, with 4 × (0.28 NA) Olympus
objective. Illumination was provided by 75W Xenon arc
lamp (Olympus). A filter set of 490-500HP (exciter), 505(dichroic) and 515-560HQ (emitter) was used. Opticalsignals were recorded using a cooled CCD camera(ORCA-ER, Hamamatsu Photonics, Hamamatsu, Japan)
at 336 × 256 pixel resolution and a frame rate of 5 Hz.
Data acquisition was performed with a digital frame
grabber board (NI PCI-1424, National Instruments, TX,
USA) controlled by custom software written in LabVIEW (National Instruments). Image acquisition wasMitsui et al. Neural Systems & Circuits 2011, 1:5
http://www.neuralsystemsandcircuits.com/content/1/1/5

post-triggered by an initiation of respiration using a custom-built circuit so that the acquisition of a first poststimulus frame was always time-locked with respiration
initiation.
Electrical microstimulation (100 μA, 60 Hz, 5 pulsesof 0.2 ms, except for stimulus-response experiments)
was performed using an iridium electrode (MicroProbes,
MD, USA), a stimulator (Master-8, AMPI, Jerusalem,
Israel) and a stimulus isolator (SS501J, Nihon Kohden,
Tokyo, Japan). For odour stimulations, 10% dilutions (inmineral oil) of TMT (Contech Inc, Delta, Canada) andHA (Tokyo Kasei, Tokyo, Japan) were used. Odourants
were delivered using a computer-controlled custombuilt olfactometer. Initiations of microstimulation andodour stimulation were time-locked with the respiration
initiation that also triggered the acquisition of the firstpost-stimulus fame.
Images were analysed using IDL (Research Systems,
CO, USA) software. Raw traces were corrected for photobleaching by subtracting no-stimulus trials before further
analysis [21]. A differential image was obtained by dividing the temporal average of signals acquired during electrical or odour stimulations (from 0.5 to 1.5 s or from 0.5
to 2.0 s after stimulus onset, respectively) by 2-s temporal
average acquired before stimulation. A Gaussian spatial
filter was used to eliminate nonspecific global fluctuationand high-frequency shot noise of the differential image(cutoff frequencies, s = 50.0/mm for the high cut-off ands = 0.1/mm for the low cut-off). In order to achieve a
better signal-to-noise ratio, these filtered images wereaveraged for 10 trials. The pixel intensities of the images
with stimulus were compared by pixel-by-pixel t-test (P <0.01, two-tailed t-test, n = 10 trials) with those with blank(pure air) stimulus. Significant pixels were overlaid on theimage of the blood vessels. Final images were imported
into Adobe Photoshop 6.0 for cropping and display. In
stimulus-response experiments, the increase of fluorescence (ΔF/F) was calculated for all exposed regions in thepiriform cortex with 0.5 s time bins. spH responses indifferent parts of the piriform cortex upon glomerular
microstimulation were quantified from three mice andsubjected to a cluster analysis.
In silico nucleotide sequence analysis

The nucleotide sequence of mouse Tbx21 gene was compared with those of human, rhesus, dog and rat orthologs
by using the VISTA program (http://genome.lbl.gov/vista/
index.shtml, http://pipeline.lbl.gov/cgi-bin/gateway2) [41].
We compared the following sequences. Mouse chromosome 11: 96,956,048 - 96,983,806; human chromosome 17:
43,176,917 - 43,186,235; rhesus chromosome 16: 31,955,925- 31,961,651; dog chromosome 9: 27,412,274 - 27,418,981;
and rat chromosome 10: 85,814,742 - 85,819,229. We used
the following default settings of VISTA parameters.

Page 12 of 14

Calculation window: 100 base pairs; minimal conserved
width: 100 base pairs; and conservation identity: 80%.
Potential transcription factor-binding sites withinmouse MCE were predicted with the MatInspector analysis program free trial version (Genomatix, Germany:
http://www.genomatix.de/en/index.html) [42]. Selectedgroups for solution parameters of MatInspector usedwere as follows: general core promoter elements: 0.75/
optimized; vertebrates: 0.75/optimized).

Additional material
Additional file 1: Quantification of numbers of Tbx21-positive mitraland tufted cells in comparison with Tbr1 and protocadhein 21(Pcdh21). Five coronal sections at different levels along the anteroposterior
axis (600 μm interval) were prepared from five olfactory bulbs (OBs) anddouble-labelled with anti-Tbx21 and anti-Tbr1 antibodies or with anti-Tbx21
and anti-Pcdh21 antibodies. For mitral cells, the numbers of Tbx21- and/or
Tbr1-positive cells in the mitral cell layer were counted. For tufted cells, thenumbers of Tbx21- and/or Pcdh21-positive cells in the external plexiform
layer were counted. Tbx21 is expressed in a vast majority, if not all, of mitralcells (94.6 ± 0.6% of the Tbr1-positive mitral cells) and in a large populationof tufted cells (72.4 ± 3.4% of the Pcdh21-positive tufted cells).
Additional file 2: Summary of gapVenus expression in Tbx21
transgenic mice. Transgenic founders and lines with neuronal gapVenus
expression are summarized. Highly efficient rate of gapVenus expression in
the mitral and tufted cells is obvious in Tbx5.0gV founders and lines. On
the other hand, variable patterns of gapVenus expression are observed
among Tbx2.6gV founders and lines. Especially, most of the Tbx2.6gV
founders and lines show gapVenus expression in some neurons along theolfactory neural pathways. gapVenus expression in mitral and tufted cells is
detected in four of the five MCE-gV founder and lines. Red squares indicate
the presence of gapVenus-expressing neurons in olfactory-related regions(dark red) and other brain regions (light red); light blue squares indicate
gapVenus-expressing non-neuronal cells. M/T, miral and tufted cells; Gr,
granule cells; OC, olfactory cortex; MOE, main olfactory epithelium; VNO,
vomeronasal organ; MOB, main olfactory bulb; AOB, accessory olfactorybulb; AON, anterior olfactory nucleus; OTu, olfactory tubercle; Pir, piriform
cortex; NLOT, nucleus of lateral olfactory tract; Amyg, amygdala; LEnt, lateralentorhinal cortex.
Additional file 3: Ontogenic expression of endogenous Tbx21 andtransgenic gapVenus proteins in the olfactory bulb (OB) of Tbx5.0gV
mice [line #3(2)]. Double immunofluorescence labelling of OB coronal
sections from Tbx5.0gV transgenic mice [line #3(2)] at different
developmental stages (A, E, I) E14.5; (B, F, J) E16.5; (C, G, K) P0; (D, H, L) P7
with anti-Tbx21 (A-D (white), I-L (magenta)) and anti-green fluorescentprotein (E-H (white), I-L (green)) antibodies. Both the endogenous Tbx21 andtransgenic gapVenus proteins first appear in the mitral cells at E14.5 andgradually increase thereafter until P7. As the tufted cells develop much later
than the mitral cells, the presence of both proteins in the tufted cells
becomes obvious after birth. An apparent dissociation of two
immunoreactive signals in the merged images (J-L) results from the different
intracellular localization of the two proteins (Tbx21: cellular nuclei, gapVenus:
plasma membranes mainly of axons) in the mitral and tufted cells.
Additional file 4: Zonal expression of gapVenus in the primaryolfactory pathway of Tbx2.6gV transgenic mouse (line #120). Double
immunofluorescence labelling of coronal sections of the olfactory
epithelium (A-F) and olfactory bulb (G-L) from Tbx2.6gV transgenic mice(line #120) with anti-green fluorescent protein (A, D, G, J (white), C, F, I, L
(green)) and anti-NAD(P)H:quinone oxidoreductase (NQO1) (B, E, H, K
(white), C, F, I, L (magenta)) antibodies. The regions indicated by rectangles
in (A-C) and (G-I) are magnified in (D-F) and (J-L), respectively. The
expression of gapVenus is restricted to the ventral zone of the olfactory
epithelium, complementary to that of the dorsal zone-specific NQO1. A
similar pattern of gapVenus expression is observed in other Tbx2.6gVtransgenic lines (#006, 008, 103, 105, 124, 127 and 129; data not shown).

Mitsui et al. Neural Systems & Circuits 2011, 1:5
http://www.neuralsystemsandcircuits.com/content/1/1/5

Abbreviations
AOB: accessory OB; cDNA: complementary DNA; GAP-43: growth-associatedprotein-43; GFP: green fluorescent protein; HA: heptanoic acid; MCE: mitraland tufted cell-specific enhancer; MOB: main OB; mRNA: messenger RNA;
NQO1: NAD(P)H:quinone oxidoreductase; OB: olfactory bulb; OR: odourant
receptors; OSN: olfactory sensory neuron; Pcdh21: protocadherin 21; PCR:
polymerase chain reaction; spH: synaptopHluorin; SV40: Simian virus 40; TMT:
2,3,5-trimethylthiazoline.

Page 13 of 14

11.

12.

13.
Acknowledgements
We are grateful to A Miyawaki (RIKEN BSI) for the Venus cDNA; G
Miesenböck (Memorial Sloan-Kettering Cancer Center) for thesynaptopHluorin cDNA; K Kitamura (National Institute of Neuroscience) for
the anti-Arx antibody. We thank N Miyasaka for valuable discussions, S
Yoshihara and T Kaneko-Goto for their technical help and R Mizuguchi for
critical reading of the manuscript. We also thank the BSI Research Resource
Center for the generation and maintenance of transgenic mouse lines. This
study was supported in part by a Grant-in-Aid for Scientific Research (B), a
Grant-in-Aid for Scientific Research on Priority Area (Cellular Sensor) and a
Grant-in-Aid for Scientific Research on Innovative Area (Systems Molecular
Ethology) to YY from the Ministry of Education, Culture, Sports, Science andTechnology of Japan.

14.

15.
16.

17.

18.
Author details
1
Laboratory for Neurobiology of Synapse, RIKEN Brain Science Institute, 2-1
Hirosawa, Wako-shi, Saitama 351-0198, Japan. 2Department of Physiology,
Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyoku, Tokyo 113-0033, Japan. 3Kavli Institute for Systems Neuroscience andCentre for the Biology of Memory, Norwegian University of Science andTechnology, Trondheim, Norway.
Authors’ contributions
SM performed the molecular biological and immunohistochemical
experiments and drafted the manuscript. KMI and KM carried out thephysiological experiments and helped to draft the manuscript. YY conceived
the study, carried out molecular biological and immunohistochemical
experiments and drafted the manuscript. All authors read and approved thefinal manuscript.
Competing interests
The authors declare that they have no competing interests.
Received: 27 July 2010 Accepted: 6 December 2010
Published: 1 February 2011
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doi:10.1186/2042-1001-1-5
Cite this article as: Mitsui et al.: Genetic visualization of the secondary
olfactory pathway in Tbx21 transgenic mice. Neural Systems & Circuits
2011 1:5.

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