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First published online January 27, 2005
doi: 10.1242/10.1242/dev.01619

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Arx homeobox gene is essential for development of mouse olfactory system

Sei-ichi Yoshihara¹, Kayo Omichi², Masako Yanazawa², Kunio Kitamura^2,*, and Yoshihiro Yoshihara^1,

¹ Laboratory for Neurobiology of Synapse, RIKEN Brain Science Institute, Wako-shi, Saitama 351-0198, Japan
² Mitsubishi Kagaku Institute of Life Sciences, Machida-shi, Tokyo 194-8511, Japan

View larger version (77K): [in a new window]   Fig. 1. Expression of Arx in developing mouse OB. Parasagittal sections of E11.5 (A), E12.5 (B), E14.5 (C), E16.5 (D), P0 (E), and P60 (F) wild-type mice were labeled with anti-Arx antibody (red) and counterstained with PI (blue: nucleus). Note that Arx is expressed in the olfactory bulb (OB) and rostral migratory stream (RMS), but not in olfactory epithelium (OE), at all the stages. Arrowheads in B: OB anlagen. Signals in the OE at P60 are non-specific autofluorescence (F). (G-V) Coronal sections of P0 wild-type mouse OB. (G-J) A triple-labeled section for Arx (red in G,J), Tbx21 (green in H,J: mitral cells), and DAPI (blue in I,J: nucleus). Arx is not expressed in Tbx21(+) mitral cells. (K-R) A triple-labeled section for Arx (red in K,N,O,R), GABA (green in L,N,P,R), and TH (blue in M,N,Q,R). (O-R) A magnified view of the glomerular layer (GL). Arrowheads in (K-R): Arx(+), GABA(+), and TH(+) periglomerular cells in the GL. Arrows in (K-N): Arx(+) and GABA(+) granule cells in the granule cell layer (GCL). (S-V) A magnified view of the GCL. A triple-labeled section for Arx (red in S,V), GABA (green in T,V: granule cells), and GLAST (blue in U,V: radial glia). Arrows in (S-V): Arx(+) and GABA(+) granule cells. Arrowheads in (S-V): Arx(+) and GLAST(+) radial glia. GE, ganglionic eminence; LGE, lateral ganglionic eminence; MCL, mitral cell layer; MGE, medial ganglionic eminence; ONL, olfactory nerve layer. Scale bars: in F, 200 µm for A-F; in J, 100 µm for G-J; in N, 40 µm for K-N; in V, 20 µm for O-V.

View larger version (66K): [in a new window]   Fig. 2. Failure of OB morphogenesis in Arx-deficient mice. Whole-mount dorsal views of the anterior telencephalon at E12.5 (A,B), E16.5 (C,D), and P0 (E,F). At E12.5, the anterior telencephalon was indistinguishable between Arx-deficient (B) and wild-type (A) mice. At E16.5 and P0, mutant OB (D,F) was smaller than wild-type OB (C,E). Note that there is a wide empty space between the left and right OB of mutant mouse (asterisks in D,F). Scale bars: in B, 500 µm for A,B; in F, 1 mm for C-F.

View larger version (42K): [in a new window]   Fig. 3. Proliferation defect of OB interneuron progenitors in Arx-deficient mice. (A-F) Coronal sections of E14.5 olfactory bulb (OB; A,B), rostral migratory stream (RMS; C,D) and ganglionic eminence (E,F) of wild-type (A,C,E) and Arx-deficient (B,D,F) mice that incorporated BrdU for 1 hour were labeled with anti-BrdU antibody. BrdU-labeled cells were observed in the subventricular zone (SVZ) of wild-type and mutant mice. (G) Quantification of BrdU-positive cells in wild-type and mutant OB. Values are mean±s.e.m. (n=3, t-test, P<0.01, marked with double asterisks). (H) Quantification of BrdU-positive cells in wild-type and mutant RMS and SVZ. Values are mean±s.e.m. (n=3, t-test, P<0.05, marked with asterisk). Scale bar: 200 µm.

View larger version (66K): [in a new window]   Fig. 4. Impaired entry of interneuron progenitors into the OB in Arx-deficient mice. Nissl-stained parasagittal sections of wild-type (A,C,E) and Arx-deficient (B,D,F) mice at E12.5 (A,B), E16.5 (C,D), and P0 (E,F). At E16.5 and P0, the mutant mouse had a smaller olfactory bulb (OB) and shortened rostral migratory stream (RMS) (D,F). (G-J) In situ hybridization analysis of Dlx2 (G,H) and Dlx5 (I,J) expression in wild-type (G,I) and mutant (H,J) mice at P0. (K,L) Parasagittal sections of wild-type (K) and mutant (L) mice at P0 were labeled with anti-Arx antibody (red) and counterstained with PI (blue). In the Arx-deficient mouse (L), the antibody can recognize a truncated non-functional Arx protein. (M,N) Immunohistochemical labeling of GABA in wild-type (M) and mutant (N) mice at P0. While GABA is expressed in periglomerular cells in glomerular layer (GL) and granule cells in the granule cell layer (GCL) in the wild-type mouse (M), GABA-expressing cells are observed at the rostral end (arrowheads) and ventrally apposing region (asterisk) of the RMS (N). (O) Parasagittal section of P0 Arx heterozygous female brain was labeled with anti-LacZ antibody (red) and counterstained with DAPI (blue). Because Arx is located on the X chromosome, the Arx heterozygous female mouse is mosaic for Arx deficiency. LacZ(+) Arx-deficient cells failed to enter the OB. LGE, lateral ganglionic eminence; LV, lateral ventricle; MGE, medial ganglionic eminence; OE, olfactory epithelium. Scale bars: in F, 400 µm for A-F; in O, 400 µm for G-O.

View larger version (72K): [in a new window]   Fig. 5. Loss of TH(+) interneurons in the OB of Arx-deficient mice. (A-J) Parasagittal sections of E16.5 (A-D) and P0 (E-J) brains of wild-type (A,C,E,G,I) and Arx-deficient (B,D,F,H,J) mice were labeled with anti-TH antibody (red), anti-GABA antibody (green) and counterstained with DAPI (blue). In wild-type mice, TH immunoreactivity was observed in periglomerular cells (PG) in the olfactory bulb (OB) (A,E,I). However, no TH(+) cells were detected in the OB or the rostral migratory stream (RMS) of mutant mice (B,F,J). In contrast, differentiation of TH(+) cells in the substantia nigra (SN) was not affected (D). TH signals in the caudate putamen (CPu) are derived from axon terminals of dopaminergic neurons in the SN (E,F,I,J). (K,L) In situ hybridization analysis of Nurr1 expression on OB coronal sections of P0 wild-type (K) and mutant (L) mice. In the wild-type mouse (K), Nurr1(+) cells were observed in the glomerular layer (GL) and granule cell layer (GCL), whereas no Nurr1-positive cells were observed in mutant OB (L). MCL, mitral cell layer; OE, olfactory epithelium; ONL, olfactory nerve layer. Scale bars: in B, 400 µm for A,B; in D, 200 µm for C,D; in J, 800 µm for E-J; in L, 100 µm for K,L.

View larger version (117K): [in a new window]   Fig. 6. Abnormal distribution of mitral cells and their dendrites in Arx-deficient mice. (A-F) Immunohistochemical analysis of Tbx21 expression in the olfactory bulb (OB) parasagittal sections (A-D) and coronal sections (E,F). In Arx-deficient mice at E16.5 (B) and P0 (D,F), mitral cells were found widely scattered in the OB, compared to wild-type (A,C,E). In a coronal section of the mutant OB (F), the mitral cell layer (MCL) showed an irregular contour: the MCL in the medial side of the OB (the right side in F) was thicker than in the lateral side (the left side in F). (G,H) Immunohistochemical analysis of L1 expression in the lateral olfactory tract (LOT) in coronal sections of P0 wild-type (G) and mutant (H) mice. LOT formation was normal in both mice. (I-P) Immunohistochemical analysis of Thy-1 (blue: whole dendrites of mitral cells) (I,J,O,P), reelin (red: proximal dendrites of mitral cells) (K,L,O,P), and Tbx21 (green: somata of mitral cells) (M,N,O,P) expression. In wild-type mice, mitral cell dendrites labeled with Thy-1 and reelin extended in a radial direction (I,K, arrowheads in O). In mutant mice, the orientation of proximal dendrites was variable (L, arrowheads in P), but the distal dendrites tend to reach the apical region of the OB (J,P). Thy-1 immunoreactivity in proximal dendrites in mutant mice disappeared (J,P). EPL, external plexiform layer; GL, glomerular layer. Scale bars: in F, 200 µm for A-F; in H, 100 µm for G,H; in P, 50 µm for I-P.

View larger version (104K): [in a new window]   Fig. 7. Abnormal projection of olfactory axons in Arx-deficient mice. (A,B) Immunofluorescence labeling of NCAM (red: olfactory axons) and PI staining (blue: nucleus) on parasagittal sections of E12.5 wild-type (A) and Arx-deficient (B) mice. In both mice, pioneer olfactory axons reached the anterior tip of the telencephalon (T). (C,D) Coronal sections of Nissl-stained olfactory epithelium (OE) of E16.5 wild-type (C) and mutant (D) mice. (E,F) Immunohistochemical analysis of olfactory marker protein (OMP) expression in mature olfactory sensory neurons in the OE of E16.5 wild-type (E) and mutant (F) mice. (G,H) Immunofluorescence labeling of OMP (red: olfactory axons) and PI staining (blue: nucleus) on parasagittal sections of the olfactory bulb (OB) from E16.5 wild-type (G) and mutant (H) mice. In mutant mice, a small subpopulation of olfactory nerve (ON) kept contact with the OB (arrowheads in H). However, most of the ON did not reach the OB and terminated in an unusual axon-tangled structure, the FCM, in front of the OB (asterisk in H). Scale bars: in B, 100 µm for A,B; in D, 400 µm for C,D; in F, 600 µm in E,F; in H, 200 µm for G,H.

View larger version (83K): [in a new window]   Fig. 8. Disorganization of OB layer structure in Arx-deficient mice. (A-H) Immunohistochemical analysis of NCAM (blue: olfactory axons) (A,B,G,H), Tbx21 (green: mitral cells) (C,D,G,H), and GABA (red: interneurons) (E,F,G,H) expression on horizontal sections of olfactory bulb (OB) from wild-type (A,C,E,G) and Arx-deicient (B,D,F,H) mice at P0. In the wild-type mouse, the olfactory nerve layer (ONL) surrounded the whole surface of the OB (A,G). In contrast, the ONL was present only in the rostromedial side (right side in B) of the OB and most of the olfactory axons terminated in the FCM (asterisks in B,H) in the mutant mouse. Besides the ONL, the formation of the mitral cell layer (MCL in C,G), glomerular layer (GL in E,G), and granule cell layer (GCL in E,G) was affected in mutant OB (D,F,H). (I,J) Magnified images of NCAM (red) and DAPI (blue) labeling on OB horizontal sections of P0 wild-type (I) and mutant (J) mice. Protoglomeruli structures (arrowheads in I) were only observed in wild-type OB. (K,L) Immunohistochemical analysis of NCAM (red: olfactory axons) and neuropeptide Y (NPY) (green: olfactory ensheathing glia in the inner ONL) expression on horizontal sections of P0 wild-type (K) and mutant (L) mice. NPY immunoreactivity was observed in the inner portion of the ONL in both wild-type and mutant OB. (M,N) Immunohistochemical analysis of NCAM (red: olfactory axons) and S100 (green: olfactory ensheathing glia in the outer ONL) expression in P0 wild-type (M) and mutant (N) mice. S100 immunoreactivity was observed in the outer portion of the ONL in both wild-type and mutant OB and in the FCM (asterisk in N) of mutant mouse. (O,P) Immunohistochemical analysis of NCAM (red: olfactory axons) and GLAST (green: radial glia) expression in P0 wild-type (O) and mutant (P) mice. GLAST(+) radial processes were observed throughout the OB of the wild-type mouse (arrowheads in O). In contrast, radial processes were only found in the rostromedial region of mutant OB (arrowheads in P) where the innervation of olfactory axons occurred. (Q-V) Immunohistochemical analysis of expression patterns of cell recognition molecules on horizontal sections of P0 wild-type (Q,S,U) and Arx-deficient (R,T,V) mice. (Q,R) NCAM (red) and OCAM (green). (S,T) NCAM (red) and neuropilin-1 (Npn-1) (green). (U,V) NCAM (red) and neuropilin-2 (Npn-2) (green). Arrowheads in (Q,S,U) indicate the OCAM(+)/Npn-1(–)/Npn-2(+) tongue-shaped area in the wild-type mouse (Nagao et al., 2000). Arrowheads in R,T,V indicate a subpopulation of olfactory axons reaching the OB in the mutant mouse. Asterisks in R,T,V: FCM. Scale bar: in V, 200 µm for A-H and K-V; 50 µm for I,J.

View larger version (29K): [in a new window]   Fig. 9. A schematic representation of OB structures in wild-type and Arx-deficient mice at P0. Diagrams show horizontal sections of the olfactory bulb (OB) in wild-type (A) and Arx-deficient mice (B). Anterior is to the top and lateral is to the left. Mutant mice showed the impaired entry of interneuron progenitors (orange) into the OB, the loss of TH(+) periglomerular cells (dark pink), and the disorganization of OB layer structure. The innervation of olfactory axons (purple) and the apical projection of radial glial fibers (blue) were observed only at the rostromedial side of OB in mutant mice. The olfactory axons that failed to reach mutant OB formed the fibrocellular mass (FCM) in front of the OB. RMS, rostral migratory stream.