QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 16 February 2005
doi: 10.1242/dev.01698

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Miyasaka, N. Articles by Yoshihara, Y. Search for Related Content PubMed PubMed Citation Articles by Miyasaka, N. Articles by Yoshihara, Y.

Robo2 is required for establishment of a precise glomerular map in the zebrafish olfactory system

Nobuhiko Miyasaka¹, Yuki Sato¹, Sang-Yeob Yeo^2,*, Lara D. Hutson^3,, Chi-Bin Chien³, Hitoshi Okamoto^2,4 and Yoshihiro Yoshihara^1,

¹ Laboratory for Neurobiology of Synapse, RIKEN Brain Science Institute, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan
² Laboratory for Developmental Gene Regulation, RIKEN Brain Science Institute, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan
³ Department of Neurobiology and Anatomy, University of Utah Medical Center, Salt Lake City, UT 84132, USA
⁴ Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Corporation (JST), 3-4-5 Nihonbashi, Chuo-ku, Tokyo 103-0027, Japan

View larger version (120K): [in a new window]   Fig. 1. Transient expression of robo2 mRNA in the developing olfactory placode, shown by whole-mount in situ hybridization. (A-E) Dorsal views with anterior to the top; (F) ventral view with anterior to the top. As the olfactory placode moves from a dorsal to a more ventral location during development, it is more easily viewed from the ventral side at 48 hpf. Arrowheads (both open and closed) denote the position of the olfactory placode. robo2 mRNA was detected in the olfactory placode in embryos at stages between 20-36 hpf (closed arrowheads in B-E). Scale bar: 50 µm.

View larger version (54K): [in a new window]   Fig. 2. ast embryos show OSN axon pathfinding defects. OSN axons in wild-type and homozygous ast embryos were labeled by external application of DiI at 3.5 days postfertilization (dpf) and viewed by confocal microscopy. (A) Diagrams representing frontal and dorsal views of the 3.5-dpf zebrafish head. ob, olfactory bulb; oe, olfactory epithelium; tel, telencephalon; di, diencephalon; a, anterior; p, posterior; d, dorsal; v, ventral. Broken rectangles indicate the region observed in B-G. (B-G) One wild-type (B,C) and two ast (D,E; F,G) examples are shown in frontal (B,D,F) and dorsal (C,E,G) views, as composite images generated from the series of optical sections. (D-G) In ast mutants, many axons reach the OB, but some fibers misroute posteriorly and penetrate into the diencephalon without reaching the OB (arrowheads and arrows). The posteriorly projecting fibers occasionally cross the midline (arrows in F,G). Scale bar: 100 µm.

View larger version (42K): [in a new window]   Fig. 3. Time-lapse observation of YFP-labeled olfactory axons in living embryos. Dynamic axon behaviors in representative ast/+;omp:yfp/+ (A-E) and ast/ast;omp:yfp/+ (F-J) embryos are shown. YFP expression under the control of the OMP promoter is observed in unipolar neurons (closed arrowheads in A,F), as well as in OSNs (open arrowheads in A,F). In the ast homozygote, several axons misroute ventromedially soon after exiting the olfactory placode at 1.5 dpf (short arrows in G). These misrouted axons form two fascicles at 2 dpf (short arrows in H), and further extend posteriorly to reach the diencephalon at 3 dpf (short arrows in I,J). A single aberrant fascicle that directly misprojects to the diencephalon is marked by the long arrows in G-J. Note that the YFP-labeled lateral glomeruli observed in the ast heterozygote (thick arrows in D) are not present in the homozygote (I). A-D,F-I, frontal views; E,J, dorsal views. Scale bar: 100 µm.

View larger version (100K): [in a new window]   Fig. 4. The unipolar neurons express robo2 and make pathfinding errors in ast embryos. (A,B) Frontal views of the olfactory placode of 30-hpf omp:yfp/+ embryos. (A) Whole-mount in situ hybridization. Blue dots are signals for robo2 mRNA on the focal plane. Broad blue staining in the olfactory placode is derived from signals on cells that are out of the focal plane. (B) Double stainning for robo2 mRNA and YFP antigen. Early developing OSNs (arrows) and unipolar neurons (arrowheads) are stained in brown with anti-GFP antibody. Hybridization signals for robo2 mRNA (blue dots) are seen on the somata of both cell types. White dotted lines indicate the boundary between the olfactory placode and telencephalon. (C,D) Frontal views of the head of wild-type (C) and ast homozygous (D) embryos stained with zns-2 antibody at 36 hpf. The zns-2-positive axons misroute medially or ventromedially immediately after exiting the olfactory placode in ast embryos (arrows in D). Asterisks mark the position of the olfactory pit. d, dorsal; m, medial. Scale bar: 50 µm.

View larger version (64K): [in a new window]   Fig. 5. Spatial expression patterns of four Slit mRNAs are consistent with a function as repulsive cues for the olfactory axons. Heads of 30-hpf whole-mounted embryos hybridized with slit1a (A-C), slit1b (D-F), slit2 (G-I) and slit3 (J-L) probes. The olfactory axon trajectory of a Tg(OMP2k:gap-YFP)rw032a transgenic embryo stained with anti-GFP antibody is shown in M-O. slit1a, slit1b and slit2 are expressed in bilateral clusters of cells located near the boundary between the telencephalon and diencephalon (arrowheads in C,F,I). slit1a and slit1b are also expressed bilaterally in the telencephalon (arrows in B,C,E,F). slit2 and slit3 are expressed along the midline in the ventral forebrain (thick arrows in H,K). The correlation between the regions of Slit expression and the olfactory axon trajectory (green) is schematized in P. A,D,G,J,M, lateral views with anterior to the left; B,E,H,K,N, frontal views with dorsal to the top; C,F,I,L,O, dorsal views with anterior to the top. The focal planes of frontal (B,E,H,K,N) and dorsal (C,F,I,L,O) views are indicated in the corresponding leftmost panels (A,D,G,J,M). Asterisks mark the position of the olfactory pit. Scale bar: 100 µm.

View larger version (66K): [in a new window]   Fig. 6. Ubiquitous misexpression of Slit2 causes posterior pathfinding errors of OSN axons. (A) Schematic of the experimental procedure. (B) A heterozygous hsp:slit2-gfp transgenic embryo overexpressing a Slit2-GFP fusion protein (green) and wild-type siblings at 24 hpf, which had received heat-shock treatment at 19 hpf. (C-H) One wild-type and two hsp:slit2-gfp/+ examples are shown. In Slit2-overexpressing embryos, some axonal fibers are deviated from the normal pathway and extend posteriorly (arrowheads and arrows in E-H), occasionally crossing the midline (arrows in E,F). C,E,G, frontal views with dorsal to the top; D,F,H, dorsal views with anterior to the left. Scale bar: 100 µm.

View larger version (139K): [in a new window]   Fig. 7. ast embryos show defasciculation of the olfactory nerve and impaired proto-glomerular organization. Frontal views of the head of wild-type (A-C,G-I) and ast homozygous (D-F,J-L) 72-hpf embryos stained with antibodies against PCAM (A,D,G,J; green in C,F,I,L), calretinin (B,E; magenta in C,F) and SV2 (H,K; magenta in I,L). (A-F) In wild type, olfactory axons maintain a tightly fasciculated state until they enter the OB (brackets in A,C). By contrast, olfactory axons in ast are defasciculated before reaching the OB (brackets in D,F) and some fibers enter the OB from improper entry sites (arrows in D,F). Calretinin-positive axons mainly project laterally to form two discrete proto-glomeruli in wild type (thick arrows in B,C), whereas in ast, only one irregularly shaped proto-glomerulus is seen at the lateralmost position in the OB (thick arrows in E,F). (G-L) Proto-glomeruli stained with antibodies against PCAM and SV2 in ast embryos (J-L) are less clearly defined than those in wild type (G-I). d, dorsal; v, ventral; m, medial. Scale bar: 50 µm.

View larger version (55K): [in a new window]   Fig. 8. ast adults exhibit a disorganized glomerular map of the OB. The projection pattern of a subpopulation of OSNs was visualized by the OMP promoter-directed YFP expression in omp:yfp/+ and ast/ast;omp:yfp/+ adults. (A,C) Whole-mount lateral views of OBs under a fluorescence stereomicroscope. Note that the posterolaterally located glomeruli observed in wild type (arrowheads in A) are not present in ast mutants (C), whereas ectopic glomerulus-like structures are seen in the posteroventral portion of the OB in ast mutants (arrows in C). (B,D) Double immunostaining with antibodies against YFP (green) and SV2 (magenta) of sagittal sections through the entrance point of the olfactory nerve at the anterior tip of OB. YFP-labeled axons ectopically innervate anteroventral and posteroventral glomeruli in ast mutants (arrows in D). In addition, aberrant axonal fascicles penetrate through the cellular layer of the OB (arrowheads in D). (E) Whole-mount lateral view of the left telencephalic hemisphere in an ast adult. A single fluorescent fascicle passes through the posteroventral surface of the OB and penetrates into the ventral telencephalic area (arrowheads). (F) Higher magnification view of the misprojecting fascicle shown in E. ob, olfactory bulb; R, right; L, left; D-tel, dorsal telencephalon; V-tel, ventral telencephalon; a, anterior; d, dorsal. Scale bar: in C, 250 µm for A,C; in D, 100 µm for B,D; in E, 250 µm; in F, 100 µm.