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First published online 15 March 2006
doi: 10.1242/dev.02329

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Zinc-finger gene Fez in the olfactory sensory neurons regulates development of the olfactory bulb non-cell-autonomously

Tsutomu Hirata^1,*, Masato Nakazawa¹, Sei-ichi Yoshihara², Hitoshi Miyachi³, Kunio Kitamura⁴, Yoshihiro Yoshihara² and Masahiko Hibi^1,

¹ Laboratory for Vertebrate Axis Formation, Center for Developmental Biology, RIKEN, Kobe 650-0047, Japan.
² Laboratory for Neurobiology of Synapse, Brain Science Institute, RIKEN, Wako-shi, Saitama 351-0198, Japan.
³ Laboratory for Animal Resources and Genetic Engineering, Center for Developmental Biology, RIKEN, Kobe 650-0047, Japan.
⁴ Department of Mental Retardation and Birth Defect Research, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Kodaira, Tokyo 187-8502, Japan.

View larger version (88K): [in a new window]   Fig. 1. Expression of Fez during mouse development. (A-K) Expression of Fez at E8.0 (A), E8.5 (B), E9.5 (C), E10.5 (D), E12.5 (E-I) and E15.5 (J,K). (A-D) Whole-mount in situ hybridization, lateral views with anterior to the left. (E-K) In situ hybridization of coronal sections from the anterior (E) to the posterior (H), and coronal sections of the olfactory epithelium (I-K) and the olfactory bulb (K). Fez transcripts were detected in the olfactory epithelium (black arrowheads; E,I,J,K), septum (black arrow, E), roof of the telencephalon (F), hypothalamus (asterisks; G,H), prethalamus (black arrowhead, G) and amygdala (black arrows; G,H), but not in the olfactory bulb (black arrows in K). (I) High-magnification view of the olfactory epithelium. Fez expression was detected in the olfactory epithelium, but was scarce in the vomeronasal organs (VNO, white arrowheads in E,I).

View larger version (10K): [in a new window]   Fig. 2. Generation of Fez-deficient mice. (A) Schematic diagram of the Fez gene locus (wild-type allele), targeting vector and targeted allele. Exons containing the zinc-finger domains are indicated by gray boxes. The targeting vector contains a phosphoglycerate kinase I promoter-linked neomycin-resistance gene (PGK-NEO) in place of part of the coding region (amino acids 1-350), and a thymidine kinase gene (TK) at the end of the 3' arm. E, EcoRI; H, HindIII; K, KpnI; X, XbaI; B, BamHI. (B) Southern blot analysis of the targeted ES clones. EcoRI- and BamHI-digested DNA from wild-type ES clones (+/+) and targeted clones (+/–) was hybridized with the probe shown in A (HindIII-KpnI fragment). The wild-type and targeted alleles showed 5.5- and 3.5-kb bands, respectively.

View larger version (64K): [in a new window]   Fig. 3. Abnormal morphology of the olfactory bulb in Fez-deficient mice. (A) The olfactory bulbs were smaller and farther apart in Fez-deficient embryos (right) than in wild-type embryos (WT, left), at P0. (B-E) Nissl staining of sagittal sections of the head region of E14.5 (B,C) or E18.5 (D,E) wild-type (B,D) and Fez-deficient (C,E) embryos; anterior to the right. Scale bars: 0.5 mm. (F-I) Nissl staining of coronal sections of olfactory bulbs from E14.5 (F,G) or E18.5 (H,I) wild-type (F,H) and Fez-deficient (G,I) mice; medial to the right. The olfactory bulb was smaller in Fez-deficient embryos than in wild-type embryos at E18.5 and P0. An irregular formation of the mitral cell layer (arrows, D,H,I) was detected in Fez-deficient embryos at E18.5.

View larger version (75K): [in a new window]   Fig. 4. Defects in the axonal tracts of olfactory sensory neurons in Fez-deficient mice. (A-F) Coronal sections of the frontal region, including the nasal epithelium and anterior telencephalon, of E12.5 wild-type (WT; A,C,E) and Fez-deficient (Fez–/–; B,D,F) embryos were stained with Hematoxylin and Eosin (HE; A,B), and analyzed by immunohistochemistry with anti-NCAM (C,D) or anti-GAP43 (E,F) antibodies. Immune complexes were visualized by FAST 3,3'-diaminobenzidine (DAB); medial to the right. The axons of OSNs, which express NCAM and GAP43, reached the olfactory bulb in wild-type embryos, but did not extend and generated a fibrocellular mass in Fez-deficient embryos.

View larger version (121K): [in a new window]   Fig. 5. Abnormal layer formation of the olfactory bulb in Fez-deficient mice. (A-T) Coronal sections of the olfactory bulb (A-D,I-T) or olfactory epithelium (E-H) from E18.5 wild-type (WT) and Fez-deficient (Fez–/–) embryos were analyzed by immunohistochemistry with anti-GAP43, anti-OMP, anti-NCAM, or a combination of anti-reelin and anti-TBX21 antibodies, or by in situ hybridization with reelin, Dlx1, Gad67 and tyrosine hydroxylase (Th) probes. Medial is to the right. Immune complexes were visualized with DAB (A-H), or Alexa488 (anti-reelin) and Alexa568 (anti-TBX21, M,N). In situ signals were stained with BM purple substrate. GAP43 and OMP were expressed in OSNs and in the olfactory nerve layer (ONL) in the wild-type bulb. (A-F) GAP43- and OMP-positive OSNs did not extend after the lamina cribrosa and formed the FCM in Fez-deficient embryos. The expression of GAP43 and OMP in the nasal epithelium was not affected in Fez-deficient embryos. Fez-deficient embryos showed an aberrantly wide mitral cell layer, which expresses reelin and TBX21 (K-N), and displayed a reduced number local circuit neurons, which express Gad67 and Th (Q-T), with aberrant positioning. Periglomerular cells and granule cells are marked by arrowheads and arrows, respectively (Q,S). (O,P) Progenitors for local circuit neurons were located in the ventricular/subventricular zone (VZ/SVZ) and expressed Dlx1; the number of Dlx1-expressing cells was reduced in the olfactory bulb of Fez-deficient embryos.

View larger version (189K): [in a new window]   Fig. 6. Reduction of interneurons in the olfactory bulb of Fez-deficient mice. (A-D) Neurons born at E14.5 (A,B) or E16.5 (C,D) in wild-type (WT, A,C) and Fez-deficient (Fez–/–, B,D) embryos were labeled with bromodeoxyuridine (BrdU) for two hours, and the proliferating neuronal cells analyzed the same day. Coronal sections of the olfactory bulb were stained with anti-BrdU antibody and DAB, and counter-stained with Hematoxylin; medial is to the right. The BrdU-positive local-circuit progenitors in the VZ/SVZ of the olfactory bulb appeared normal in Fez-deficient embryos. The number of neurons born at E16.5 was reduced in Fez-deficient embryos compared with wild type (55.5±7.6%). BrdU-positive cells from five sections were counted (n=3).

View larger version (94K): [in a new window]   Fig. 7. Defects in the rostral migratory stream (RMS) in Fez-deficient mice. (A,B) Neurons born at E14.5 in control (WT) and Fez-deficient (Fez–/–) embryos were labeled with BrdU and their positions were determined at E18.5; coronal sections, medial to the right. The total number of BrdU-positive neurons in Fez-deficient embryos was slightly reduced, and they were not localized to the glomerular layer but were located mainly on the medial side of the granule cell layer. (C-F) Expression of Dlx1 at E18.5 (C,D) and E15.5 (E,F); sagittal sections, with anterior to the right. Dlx1 is expressed in interneuron progenitors of the OB and marks the rostral migratory stream (RMS, arrows). The RMS from the ganglionic eminence to the olfactory bulb was shorter in Fez-deficient embryos than in wild type at E15.5 and E18.5. The distribution of Dlx1-expressing cells was affected in the Fez–/– olfactory bulb (arrowheads, C,D). (G,H) The expression of Slit1 in the septum and ganglionic eminence was not significantly affected in Fez-deficient embryos (H), compared with wild type (G).

View larger version (56K): [in a new window]   Fig. 8. Expression of Fez and Arx is mutually independent. (A-D) Expression of ARX in Fez+/– (A,C) and Fez–/– embryos (B,D) at E12.5 (A,B) and E14.5 (C,D) was detected by immunofluorescence staining. ARX proteins are detected in the ganglionic eminence and the RMS to the olfactory bulb (OB). (E-H) Expression of Arx in Fez+/– (E,G) and Fez–/– embryos (F,H) at E16.5 (E,F) and P0 (G,H). Expression of ARX and Arx was not affected at the developmental periods examined. (I-L) Expression of Fez in wild-type control (WT, I,K), and Arx-deficient (Arx KO, J,L) embryos at E12.5 (I,J) and E14.5 (K,L). Expression of Fez in the olfactory epithelium (OE) is not affected in Arx-deficient embryos.

View larger version (109K): [in a new window]   Fig. 9. Fez is required in olfactory sensory neurons for axon targeting and OB layer formation. (A-I) The OMP protein (A-C), and the reelin (D-F) and Gad67 (G-I) transcripts in control (WT) and Fez-deficient (Fez–/–) mice, and in Fez-deficient mice in which Fez expression was rescued under the control of the olfactory sensory neuron-specific #123 promoter (Fez–/–, Tg+), at postnatal day 0 (P0) were detected by immunohistochemistry or in situ hybridization. Fez+/– mice containing a transgene composed of #123 promoter-linked Fez-IRES GapVenus expression units [Fez+/–,Tg(#123p-Fez)] were crossed with Fez+/– mice to generate control mice (WT), Fez-deficient mice (Fez–/–) and rescued Fez-deficient mice (Fez–/–, Tg+). In some of the rescued Fez-deficient embryos, formation of the ONL (OMP-positive, C), the mitral/tufted cell layer (MCL, reelin-positive, F), and the layer of local circuit neurons (glomerular layer, GL; granule cell layer, GCL) was rescued in one or both bulbs. Three different transgenic alleles were used. The numbers of rescued and non-rescued embryos are shown in Table 1. (J) Transgene-mediated Fez expression in the olfactory epithelium (OE), but not the olfactory bulbs (OB), of Fez–/– mice partially rescued axon projection (arrowhead) of OSNs at E14.5. The OSNs expressing the transgene were monitored by immunohistochemistry with an anti-GFP (anti-Venus) antibody.