Genetic visualization of the secondary olfactory pathway in Tbx21 transgenic mice
© Mitsui et al; licensee BioMed Central Ltd. 2011
Received: 27 July 2010
Accepted: 6 December 2010
Published: 1 February 2011
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 and genetic 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 the brain, Tbx21 is specifically expressed in mitral and tufted cells of the olfactory bulb.
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 with various 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 is both 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 of neurons predominantly located along the olfactory pathway. Furthermore, we generated transgenic mice expressing the genetically-encoded fluorescent exocytosis indicator, synaptopHluorin, in mitral and tufted cells for visualization of presynaptic neural activities in the piriform cortex.
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 the bulb to the cortex.
Odour molecules emitted from objects enter into the nostrils, reach the olfactory epithelium, and bind odourant receptors (ORs) expressed on the cilia of olfactory sensory neurons (OSNs). The odour information is then converted into electrical signals, transmitted to glomeruli in the olfactory bulb (OB) via precisely wired neural circuitry and represented as topographic 'odour maps' on the glomerular array of the OB . After the OR multigene family was discovered , 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 molecular markers and transcriptional enhancers has facilitated the 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 to the olfactory cortex in mammals. For example, it is not known how odour information is transmitted from the OB to various regions of the olfactory cortex, including the piriform cortex, the cortical amygdaloid nuclei and the 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 from glomeruli to various regions of the olfactory cortex. Despite their functional significance, only a few 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 . We analysed transgenic mice that harboured transgenes with various lengths of Tbx21 5'-upstream regions fused to a fluorescent reporter. As a result, we identified a cis-regulatory enhancer element that 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.
Mitral and tufted cell-specific expression of Tbx21 protein in the mouse brain
Faithful expression of transgene in mitral and tufted cells in Tbx5.0gV transgenic mice
Variable patterns of transgene expression in Tbx2.6gV transgenic mice
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 detectable in 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
Transgenic mouse expressing spH in mitral and tufted cells
The piriform cortex was exposed in anaesthetized transgenic mice and the spH fluorescence was monitored with a CCD camera on a conventional fluorescence microscope connected to an image analysis system. In order to validate that the change in spH fluorescence reflects pre-synaptic activity of mitral and tufted cell axon 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 the pulse number (Figure 7 C1, C2 and C3) and amplitude (Figure 7 7D1 and D2) of electrical stimulation to the OB, indicating the positive correlation between the extent of activated 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 is represented in the piriform cortex, we first identified a glomerulus responsive to specific odourants by using the optical imaging method of intrinsic signals, then applied electrical stimuli onto the glomerulus and compared the pre-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 in the 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 the increased 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 fluorescence in the mitral and tufted cell axon terminals upon the odourant stimulation. Applications of TMT and HA to mouse 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 electrical stimulation of the respective glomeruli responsive to the two odourants. Further experiments of in vivo spH imaging in the olfactory cortex will enhance our understanding of neuroanatomical and functional architecture of the secondary olfactory system.
Members of the T-box family transcription factors play important roles in development and functions of various tissues including the brain . In the OB, Tbr1, Tbr2 and Tbx21 are expressed in the excitatory projection neurons, mitral and tufted cells [18, 24, 25]. In particular, the expression of Tbx21 is highly specific to the mitral and tufted cells, whereas the Tbr1 and Tbr2 are also present in various cell types in other brain regions. The strictly localized expression of Tbx21 gene prompted us to analyse its transcriptional enhancer activity using transgenic 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 a highly 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 and tufted 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 and tufted cells such as Tbr1, Tbr2 or Tbx21 itself. This notion is corroborated by the fact Tbx21 expression is completely absent in the OB of Tbr1-deficient mice . In addition, the MCE contains putative binding sites for Brn-3, Gli3, Klf15 and Six4. These transcription factors and related molecules belonging to the same families seem to play important roles in various aspects of the olfactory system development [27–31]. Further studies are required in order to identify the trans-binding factors crucial for the mitral and tufted cell-specific transcriptional regulation by MCE.
Previous studies described transgene expression in the mitral and tufted cells by using transcriptional enhancers of Pcdh21 , neurotensin  and AP-2ε . 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 and tufted cells do not persist into adulthood but begin to diminish soon after birth [17, 32]. Thus, the Tbx21 gene enhancer/promoter will become a more useful and convenient tool for genetic manipulations of the mitral and tufted cells because its highly specific and efficient enhancer activity with the shorter length of necessary sequence (~5 kb) guarantees the mitral and tufted cell-directed 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 was used as the enhancer/promoter. In many lines of Tbx2.6gV transgenic mice, gapVenus expression was detected frequently in various types of neurons along the olfactory 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 . Interestingly, the fluorescent OSNs projected their axons predominantly into a glomerular cluster onto which the fluorescent mitral cells send their dendrites, suggesting that the transgene was coincidently expressed in 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 coordinated expression of homophilic adhesion molecules in both pre- and post-synaptic neurons for the formation of synaptic 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 of regulatory 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 the axon 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 olfactory cortex. In addition to spH, any genetically-encoded molecular tools of interest will be expressed in the mitral and tufted cells by the use of Tbx21 enhancer.
We have identified the mitral and tufted cell-specific transcriptional enhancer upstream of the mouse Tbx21 gene. A short sequence of ~300 bp was necessary and sufficient for the transgene expression in these projection neurons of the olfactory bulb. spH expression in the mitral and tufted cells enabled us to monitor the presynaptic neural activities in the piriform cortex. Thus, this study paves a new avenue of the olfactory research into higher brain centres.
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 β-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 plasmid by 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 the transcription 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  was used instead of gapVenus.
The generation of transgenic mice was performed as previously described . Briefly, gel-purified transgenes were microinjected into the pronucleus of fertilized eggs that were obtained from crossing (C56BL/6J × DBA/2J) F1 mice. The manipulated eggs were cultured to the two-cell stage 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  were purchased from The Jackson Laboratory (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 of brain sections was carried out as previously described . The following primary antibodies were used: guinea pig anti-Tbx21 (1:10000) ; rat anti-GFP (1:1000, Nacalai Tesque, Kyoto, Japan); rabbit anti-Arx (1:1000 provided by Dr K Kitamura) ; rabbit anti-Tbr1 (1:1000, Abcam, Cambridge, UK), rat phycoerythrin-conjugated anti-Tbr2 (1:200, eBioscience, CA, USA), rabbit anti-Pcdh21 (1:1000) ; mouse anti-PGP9.5 (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). Fluorescent images were obtained with a fluorescent microscope (Axioplan, Carl Zeiss, Oberkochen, Germany) equipped with a CCD camera and an image analysis system (DP-70, Olympus, Tokyo, Japan) or a confocal laser scanning microscope (Fluoview FV1000, Olympus).
In vivospH 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 was maintained 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 exposed by removing a 5 × 4 mm area of skull as previously described . Agarose gel was mounted on the brain and covered with a glass coverslip to form a temporary chamber for the optical imaging. The piriform cortex was imaged using an Olympus BX51 microscope and epifluorescence 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. Optical signals 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 post-triggered by an initiation of respiration using a custom-built circuit so that the acquisition of a first post-stimulus frame was always time-locked with respiration initiation.
Electrical microstimulation (100 μA, 60 Hz, 5 pulses of 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 (in mineral oil) of TMT (Contech Inc, Delta, Canada) and HA (Tokyo Kasei, Tokyo, Japan) were used. Odourants were delivered using a computer-controlled custom-built olfactometer. Initiations of microstimulation and odour stimulation were time-locked with the respiration initiation that also triggered the acquisition of the first post-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 . 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 fluctuation and high-frequency shot noise of the differential image (cutoff frequencies, σ = 50.0/mm for the high cut-off and σ = 0.1/mm for the low cut-off). In order to achieve a better signal-to-noise ratio, these filtered images were averaged 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 the image 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 the piriform cortex with 0.5 s time bins. spH responses in different parts of the piriform cortex upon glomerular microstimulation were quantified from three mice and subjected to a cluster analysis.
In siliconucleotide 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) . 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. Calculation window: 100 base pairs; minimal conserved width: 100 base pairs; and conservation identity: 80%.
Potential transcription factor-binding sites within mouse MCE were predicted with the MatInspector analysis program free trial version (Genomatix, Germany: http://www.genomatix.de/en/index.html) . Selected groups for solution parameters of MatInspector used were as follows: general core promoter elements: 0.75/optimized; vertebrates: 0.75/optimized).
green fluorescent protein
mitral and tufted cell-specific enhancer
olfactory sensory neuron
polymerase chain reaction
Simian virus 40
We are grateful to A Miyawaki (RIKEN BSI) for the Venus cDNA; G Miesenböck (Memorial Sloan-Kettering Cancer Center) for the synaptopHluorin 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 and Technology of Japan.
- Mori K, Nagao H, Yoshihara Y: The olfactory bulb: coding and processing of odor molecule information. Science. 1999, 286: 711-715. 10.1126/science.286.5440.711.View ArticlePubMedGoogle Scholar
- Buck L, Axel R: A novel multigene familymay encode odorant receptors: a molecular basis for odor recognition. Cell. 1991, 65: 175-187. 10.1016/0092-8674(91)90418-X.View ArticlePubMedGoogle Scholar
- Chess A, Simon I, Cedar H, Axel R: Allelic inactivation regulates olfactory receptor gene expression. Cell. 1994, 78: 823-834. 10.1016/S0092-8674(94)90562-2.View ArticlePubMedGoogle Scholar
- Ressler KJ, Sullivan SL, Buck LB: Information coding in the olfactory system: evidence for a stereotyped and highly organized epitope ma in the olfactory bulb. Cell. 1994, 79: 1245-1255. 10.1016/0092-8674(94)90015-9.View ArticlePubMedGoogle Scholar
- Vassar R, Chao SK, Sitcheran R, Nunez JM, Vosshall LB, Axel R: Topographic organization of sensory projections to the olfactory bulb. Cell. 1994, 79: 981-991. 10.1016/0092-8674(94)90029-9.View ArticlePubMedGoogle Scholar
- Mombaerts P, Wang F, Dulac C, Chao SK, Nemes A, Mendelsohn M, Edmondson J, Axel R: Visualizing an olfactory sensory map. Cell. 1996, 87: 675-686. 10.1016/S0092-8674(00)81387-2.View ArticlePubMedGoogle Scholar
- Mori K, Mataga N, Imamura K: Differential specificities of single mitral cells in rabbit olfactory bulb for a homologous series of fatty acid odor molecules. J Neurophysiol. 1992, 67: 786-778.PubMedGoogle Scholar
- Rubin BD, Katz LC: Optical imaging of odorant representations in the mammalian olfactory bulb. Neuron. 1999, 23: 499-511. 10.1016/S0896-6273(00)80803-X.View ArticlePubMedGoogle Scholar
- Uchida N, Takahashi TK, Tanifuji M, Mori K: Odor maps in the mammalian olfactory bulb: domain organization and odorant structural features. Nat Neurosci. 2000, 3: 1035-1043. 10.1038/79857.View ArticlePubMedGoogle Scholar
- Yoshihara Y, Kawasaki M, Tamada A, Fujita H, Hayashi H, Kagamiyama H, Mori K: OCAM: a new member of the neural cell adhesion molecule family related to zone-to-zone projection of olfactory and vomeronasal axons. J Neurosci. 1997, 17: 5830-5842.PubMedGoogle Scholar
- Serizawa S, Miyamichi K, Nakatani H, Suzuki M, Saito M, Yoshihara Y, Sakano H: Negative feedback regulation ensures the one receptor-one olfactory neuron rule in mouse. Science. 2003, 302: 2088-2094. 10.1126/science.1089122.View ArticlePubMedGoogle Scholar
- Serizawa S, Miyamichi K, Takeuchi H, Yamagishi Y, Suzuki M, Sakano H: A neuronal identity code for the odorant receptor-specific and activity-dependent axon sorting. Cell. 2006, 127: 1057-1069. 10.1016/j.cell.2006.10.031.View ArticlePubMedGoogle Scholar
- Kobayakawa K, Kobayakawa R, Matsumoto H, Oka Y, Imai T, Ikawa M, Okabe M, Ikeda T, Itohara S, Kikusui T, et al: Innate versus learned odour processing in the mouse olfactory bulb. Nature. 2007, 450: 503-508. 10.1038/nature06281.View ArticlePubMedGoogle Scholar
- Kaneko-Goto T, Yoshihara S, Miyazaki H, Yoshihara Y: BIG-2 mediates olfactory axon convergence to target glomeruli. Neuron. 2008, 57: 834-846. 10.1016/j.neuron.2008.01.023.View ArticlePubMedGoogle Scholar
- Nagai Y, Sano H, Yokoi M: Transgenic expression of Cre recombinase in mitral/tufted cells of the olfactory bulb. Genesis. 2005, 43: 12-16. 10.1002/gene.20146.View ArticlePubMedGoogle Scholar
- Walz A, Omura M, Mombaerts P: Development and topography of the lateral olfactory tract in the mouse: imaging by genetically encoded and injected fluorescent markers. J Neurobiol. 2006, 66: 835-846. 10.1002/neu.20266.View ArticlePubMedGoogle Scholar
- Feng W, Simoes-de-Souza F, Finger TE, Restrepo D, Williams T: Disorganized olfactory bulb lamination in mice deficient for transcription factor AP-2ε. Mol Cell Neurosci. 2009, 42: 161-171. 10.1016/j.mcn.2009.06.010.PubMed CentralView ArticlePubMedGoogle Scholar
- Faedo A, Ficara F, Ghiani M, Aiuti A, Rubenstein JLR, Bulfone A: Developmental expression of the T-box transcription factor T-bet/Tbx21 during mouse embryogenesis. Mech Dev. 2002, 116: 157-160. 10.1016/S0925-4773(02)00114-4.View ArticlePubMedGoogle Scholar
- Yoshihara S, Omichi K, Yanazawa M, Kitamura K, Yoshihara Y: Arx homeobox gene is essential for development of mouse olfactory system. Development. 2005, 132: 751-762. 10.1242/dev.01619.View ArticlePubMedGoogle Scholar
- Miesenböck G, De Angelis DA, Rothman JE: Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins. Nature. 1998, 394: 192-195.View ArticlePubMedGoogle Scholar
- Bozza T, McGann JP, Mombaerts P, Wachowiak M: In vivo imaging of neuronal activity by targeted expression of a genetically encoded probe in the mouse. Neuron. 2004, 42: 9-21. 10.1016/S0896-6273(04)00144-8.View ArticlePubMedGoogle Scholar
- Li Z, Burrone J, Tyler WJ, Hartman KN, Albeanu DF, Murthy VN: Synaptic vesicle recycling studied in transgenic mice expressing synaptopHluorin. Proc Natl Acad Sci USA. 2005, 102: 6131-6136. 10.1073/pnas.0501145102.PubMed CentralView ArticlePubMedGoogle Scholar
- Naiche LA, Harrelson Z, Kelly RG, Papaioannou VE: T-box genes in vertebrate development. Annu Rev Genet. 2005, 39: 219-239. 10.1146/annurev.genet.39.073003.105925.View ArticlePubMedGoogle Scholar
- Bulfone A, Wang F, Hevner R, Anderson S, Cutforth T, Chen S, Meneses J, Pedersen R, Axel R, Rubenstein JLR: An olfactory sensory map develops in the absence of normal projection neurons or GABAergic interneurons. Neuron. 1998, 21: 1273-1282. 10.1016/S0896-6273(00)80647-9.View ArticlePubMedGoogle Scholar
- Kimura N, Nakashima K, Ueno M, Kiyama H, Taga T: A novel mammalian T-box-containing gene, Tbr2, expressed in mouse developing brain. Dev Brain Res. 1999, 115: 183-193. 10.1016/S0165-3806(99)00064-4.View ArticleGoogle Scholar
- Tada M, Smith JC: T-targets: clues to understanding the functions of T-box proteins. Dev Growth Differ. 2001, 43: 1-11. 10.1046/j.1440-169x.2001.00556.x.View ArticlePubMedGoogle Scholar
- Naruse I, Keino H: Apoptosis in the developing CNS. Prog Neurobiol. 1995, 47: 135-155. 10.1016/0301-0082(95)00024-P.View ArticlePubMedGoogle Scholar
- Hagino-Yamagishi K, Minamikawa-Tachino R, Ichikawa M, Yazaki K: Expression of Brain-2 in the developing olfactory bulb. Dev Brain Res. 1999, 113: 133-137. 10.1016/S0165-3806(98)00192-8.View ArticleGoogle Scholar
- Balmer CW, LaMantia AS: Loss of Gli3 and Shh function disrupts olfactory axon trajectories. J Comp Neurol. 2004, 472: 292-307. 10.1002/cne.20053.View ArticlePubMedGoogle Scholar
- Laub F, Dragomir C, Ramirez F: Mice without transcription factor KLF7 provide new insight into olfactory bulb development. Brain Res. 2006, 1103: 108-113. 10.1016/j.brainres.2006.05.065.View ArticlePubMedGoogle Scholar
- Chen B, Kim EH, Xu PX: Initiation of olfactory placode development and neurogenesis is blocked in mice lacking both Six1 and Six4. Dev Biol. 2009, 326: 75-85. 10.1016/j.ydbio.2008.10.039.View ArticlePubMedGoogle Scholar
- Feng W, Williams T: Cloning and characterization of the mouse AP-2ε gene: a novel family member expressed in the developing olfactory bulb. Mol Cell Neurosci. 2003, 24: 460-475. 10.1016/S1044-7431(03)00209-4.View ArticlePubMedGoogle Scholar
- Miyasaka N, Morimoto K, Tsubokawa T, Higashijima S, Okamoto H, Yoshihara H: From the olfactory bulb to higher brain centers: genetic visualization of secondary olfactory pathways in zebrafish. J Neurosci. 2009, 29: 4756-4767. 10.1523/JNEUROSCI.0118-09.2009.View ArticlePubMedGoogle Scholar
- Nagai T, Ibata K, Park ES, Kubota M, Mikoshiba K, Miyawaki A: A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications. Nat Biotechnol. 2002, 20: 87-90. 10.1038/nbt0102-87.View ArticlePubMedGoogle Scholar
- Matsuno H, Okabe S, Mishina M, Yanagida T, Mori K, Yoshihara Y: Telencephalin slows spine maturation. J Neurosci. 2006, 26: 1776-1786. 10.1523/JNEUROSCI.2651-05.2006.View ArticlePubMedGoogle Scholar
- Kobayashi K, Morita S, Mizuguchi T, Sawada H, Yamada K, Nagatsu I, Fujita K, Nagatu T: Functional and high level expression of human dopamine β-hydroxylase in transgenic mice. J Biol Chem. 1994, 269: 29725-29731.PubMedGoogle Scholar
- Szabo SJ, Sullivan BM, Stemmann C, Satoskar AR, Sleckman BP, Glimcher LH: Distinct effects of T-bet in TH1 lineage commitment and IFN-γ production in CD4 and CD8 T cells. Science. 2002, 295: 338-342. 10.1126/science.1065543.View ArticlePubMedGoogle Scholar
- Mitsui S, Saito M, Hayashi K, Mori K, Yoshihara Y: A novel phenylalanine-based targeting signal directs telencephalin to neuronal dendrite. J Neurosci. 2005, 25: 1122-1131. 10.1523/JNEUROSCI.3853-04.2005.View ArticlePubMedGoogle Scholar
- Kitamura K, Yanazawa M, Sugiyama N, Miura H, Iizuka-Kogo A, Kusaka M, Omichi K, Suzuki R, Kato-Fukai Y, Kamiirisa K, et al: Mutation of ARX causes abnormal development of forebrain and testes in mice and X-linked lissencephaly with abnormal genitalia in humans. Nat Genet. 2002, 32: 359-369. 10.1038/ng1009.View ArticlePubMedGoogle Scholar
- Yoshida I, Mori K: Odorant category profile selectivity of olfactory cortex neurons. J Neurosci. 2007, 27: 9105-9144. 10.1523/JNEUROSCI.2720-07.2007.View ArticlePubMedGoogle Scholar
- VISTA. [http://genome.lbl.gov/vista/index.shtml]
- Cartharius K, Frech K, Grote K, Klocke B, Haltmeier M, Klingenhoff A, Frisch M, Bayerlein M, Werner T: MatInspector and beyond: promoter analysis based on transcription factor binding sites. Bioinformatics. 2005, 21: 2933-2942. 10.1093/bioinformatics/bti473.View ArticlePubMedGoogle Scholar
- Neville KR, Haberly LB: Beta and gamma oscillations in the olfactory system of the urethane-anesthetized rat. J Neurophysiol. 2003, 90: 3921-3930. 10.1152/jn.00475.2003.View ArticlePubMedGoogle Scholar
This article is published under license to 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 in any medium, provided the original work is properly cited.