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

First published online 1 March 2006
doi: 10.1242/dev.02290

This Article Summary Full Text Full Text (PDF) Supplementary Material 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 Nomura, T. Articles by Osumi, N. Search for Related Content PubMed PubMed Citation Articles by Nomura, T. Articles by Osumi, N.

Pax6-dependent boundary defines alignment of migrating olfactory cortex neurons via the repulsive activity of ephrin A5

¹ Division of Developmental Neuroscience, Center for Translational and Advanced Animal Research (CTTAR), Tohoku University School of Medicine, 2-1, Seiryo-machi, Aoba-ku, 980-8575, Japan.
² Department of Cell and Molecular Biology, Karolinska Institute, SE-171 77 Stockholm, Sweden.
³ Core Research for Evolutional Science and Technology, Japan Science and Technology Agency, Kawaguchi, 332-0012, Japan.

View larger version (78K): [in a new window]   Fig. 1. Migration pattern and characteristics of olfactory cortex neurons. (A,B) The dorsal part of the telencephalon in E11.75 rat embryos is labeled by electroporation of GFP-expressing vector (arrow in B). After electroporation, the embryos were cultured for 48 hours in whole-embryo culture system. (C,C') During WEC, GFP-labeled cells migrate dorsoventrally in the telencephalon (arrows in C,C'), and stop at the pallium-subpallium boundary (PSB, open arrow in C'). Immunostaining with anti-Pax6 antibody indicates that Pax6 is not detected in the GFP-positive migrating cells (arrows in C,C'). Inset in C is at higher magnification in C'. (D-F'') Immunostaining with anti-type III ß-tubulin (Tuj1, D-D''), anti-calbindin (CB, E-E'') and anti-reelin (Rln, F-F'') antibodies of GFP-labeled telencephalon. GFP-positive migrating neurons (green cells in D-F) are positive for ß-tubulin (D-D''), CB (E-E'') and Rln (F-F''). Di, diencephalons; Tel, telencephalon. Scale bars: 500 µm in B; 100 µm in C'; 20 µm in F.

View larger version (88K): [in a new window]   Fig. 2. Abnormal migration of olfactory cortex neurons in Pax6 mutant telencephalon. Lateral views (A,B) and coronal sections (C-H) of the wild-type (A,C-E) and Pax6 mutant (B,F-H) telencephalon with GFP-labeled olfactory cortex neurons. Olfactory cortex neurons stop and align at the PSB in the wild type (arrows in A,C-E), whereas these neurons invaded the Dlx1-positive more ventral part of the Pax6 mutant telencephalon (arrowhead in B,F-H). r, rostral; c, caudal. Open arrows indicate the PSB. Scale bars: 500 µm.

View larger version (43K): [in a new window]   Fig. 3. Abnormal migration in Pax6 mutants is due to non-cell autonomous defects of migrating cells. (A) Experimental procedures for the cell transplantation between wild type and mutants using whole-embryo culture. (B,C) Lateral views of the wild-type (B) and Pax6 mutant telencephalon (C) after 48 hours in whole-embryo culture. The Pax6 mutant-derived cells stopped at the PSB (arrows in B,B'), whereas wild-type-derived cells invaded the ventral part of the mutant telencephalon (arrowhead in C,C'). (D) Comparison of migratory distance of the GFP-positive cells in the wild type and Pax6 mutant telencephalon. The number of GFP-positive cells was calculated at each distance and quantified as a percentage of total number of migrating cells. In the Pax6 mutant telencephalon, the number of GFP-positive cells derived from wild type was significantly increased in the area over 1200 µm distant from the injection point. Data are presented as percentage of the labeled cells in each area against the total number of labeled cells (mean±s.d. of three samples in each group). **P<0.01. Scale bars: 500 µm in B,C; 50 µm in B',C'.

View larger version (92K): [in a new window]   Fig. 4. Downregulation of ephrin A5 expression in the Pax6 mutant telencephalon. (A-H) Expression patterns of ephrin A5 (A,C,E,G) and Pax6 (B,F) in wild type (A-D) and Pax6 mutant (E-H) telencephalon. In situ hybridization was performed at E11.5 (A,B,E,F) and E12.5 (C,D,G,H) telencephalon. In wild type, ephrin A5 is expressed at the ventral part of the telencephalon (A,C). GFP-labeled cells stop at the PSB, corresponding to the expression border of ephrin A5 (white arrow in D). Open arrows in C,D indicate the PBS. In the Pax6 mutant, expression of ephrin A5 is reduced in the ventral part of the telencephalon (E,G), where the GFP-labeled cells invaded (arrowhead in H). (I-M) EphA receptor expression in the wild type E12.5 telencephalon. Expression of Eph receptors is detected by incubation with soluble-ephrin A5-Fc protein (I). No signal is detected with Fc control protein (J). (K-M) Immunohistochemistry with anti-EphA4 antibody in the embryo labeled by GFP plasmid electroporation. EphA4 is expressed in the dorsal and ventral parts of the telencephalon (K), including GFP-positive cells (L,M). LV, lateral ventricle. Scale bars: 500 µm in A,C,D,J; 200 µm in D; 20 µm in M.

View larger version (50K): [in a new window]   Fig. 5. Blockade of EphA/ephrin A interaction induced abnormal migration of olfactory cortex neurons. (A) Experimental procedures for EphA/ephrin A signaling blockade. (B,C) Migration patterns of GFP-positive cells in media containing Fc (B) or EphA3 (C). In Fc-containing media, the GFP-labeled neurons stopped at the PSB (arrows in B), whereas in the Eph-A3-Fc-containing media, the GFP-labeled neurons crossed the PSB and invaded the ventral part of the telencephalon (arrowheads in C). (D) Comparison of the number of GFP-positive cells in the ventral part of the telencephalon (the purple area) in Fc- and EphA3-treated telencephalon. Because in control (Fc treated) samples most of the GFP-positive cells aligned at 1.0-1.2 mm distant from the dorsal margin of the telencephalon, we marked this point as `prospective PSB' and compared with the number of GFP-positive cells ventral of this point. Data are presented as percentage of the labeled cells in the ventral telencephalon against the total number of labeled cells (mean±s.d. of four samples in each group). **P<0.01. r, rostral; c, caudal; d, dorsal; v, ventral. Scale bar: 500 µm.

View larger version (63K): [in a new window]   Fig. 6. Altered migration patterns of ventrally migrating neurons by overexpression of ephrin A5. (A) Experimental procedures for ephrin A5 misexpression in whole-embryo culture system. (B-D) Migration patterns of DiI-labeled neurons in the wild-type (B,C) and Pax6 mutant (D) embryos, in which ephrin A5 and/or GFP expression vector were electroporated. DiI-labeled neurons pass through the area in which GFP expression vector was electroporated (B,B'), whereas these neurons stop at the border area with misexpressed ephrin A5 expression vector and GFP vector (arrowheads, C-C'). (D,D') DiI-labeled neurons stop at the border area with misexpressed ephrin A5 in the Pax6 mutant embryo (arrowheads in D). Scale bars: 500 µm in D; 100 µm in D'.

View larger version (48K): [in a new window]   Fig. 7. Altered migration patterns of ventrally migrating neurons by loss of ephrin A5. (A,B) Altered migration pattern of olfactory cortex neurons in ephrin A5-deficient embryos. In the ephrin A5+/– mice, the dorsally derived GFP-labeled neurons stop at the PSB (arrows in A). However, in ephrin A5 homozygous mutant embryos, the GFP-labeled neurons invaded the ventral part of the telencephalon (arrowheads in B). (C) Comparison of migratory distance of GFP-positive cells in between and ephrin A5+/– and ephrin A5–/– mice. In ephrin A5–/– mice the number of GFP-positive cells was significantly increased in the area over 1200 µm distant from the injection point. Data are presented as percentage of the labeled cells in each area against the total number of labeled cells (mean±s.d. of three samples in each group). *P<0.05, **P<0.01. Scale bar: 500 µm.

View larger version (73K): [in a new window]   Fig. 8. Altered distributions of early-born olfactory cortex neurons in the Pax6 and ephrin A5 mutants. (A) Nissl staining of the E18.5 rat telencephalon. (B-E) Immunostaining with anti-BrdU antibody in E18.5 wild-type rat (B), Pax6 mutant rat (C), E18.5 ephrin A5+/– mouse (D) and ephrin A5–/– mouse (E) telencephalon. BrdU was injected at E11.75 (rat) or E9.75 (mice). (B-E) LOT area of the olfactory cortex. In the Pax6 mutant and ephrin A5–/–, fewer BrdU-labeled cells are present in the LOT area (arrowheads in C,E) compared with the wild type or ephrin A5+/– (arrows in B,D). (F) The number of BrdU-labeled cells in the LOT area of the wild-type, Pax6 mutant rats and ephrin A5-deficient mice. BrdU-labeled cells are decreased in Pax6 mutant rats and ephrin A5–/– mice, compared with wild-type rats and ephrin A5+/– mice. Data are presented as percentage of the BrdU-labeled cells in the Pax6 mutant rat or ephrin A5–/– mouse against the total number of labeled cells in the wild-type rat or ephrin A5+/– mouse (mean±s.d. of four animals in each group). **P<0.01. (G,H) Distribution of BrdU-labeled cells in E12.5 ephrin A5+/– and ephrin A5–/– mice, in which BrdU was injected at E9.75. In ephrin A5–/– mice, the number of BrdU-labeled cells was increased at the ventral part of the telencephalon (arrowheads in H). (I-K) Distribution of BrdU-labeled cells in the olfactory tubercle (inset in I) of wild-type rats and Pax6 mutant rat embryos at E18.5, in which BrdU was injected at E11.75. The number of BrdU-positive cells was increased in the Pax6 mutant olfactory tubercle (arrowheads in K). (L) Comparison of the number of BrdU-labeled cells in the olfactory tubercle of wild type, Pax6 mutant rats and ephrin A5-deficient mice. BrdU-labeled cells are increased in Pax6 mutant rats and ephrin A5–/– mice, compared with wild-type rats and ephrin A5+/– mice. Data are presented as percentage of the BrdU-labeled cells in the Pax6 mutant rat or ephrin A5–/– mouse against the total number of labeled cells in the wild-type or ephrin A5+/– mouse (mean±s.d. of three animals in each group). *P<0.05, **P<0.01. Scale bars: 500 µm in B,C,G; 100 µm in J. LOT, lateral olfactory tract; Ncx, neocortex; Str, striatum.

View larger version (26K): [in a new window]   Fig. 9. Schematic diagram of Pax6/ephrin A5-dependent mechanisms for alignment of olfactory cortex neurons at PSB. (A) Olfactory cortex neurons are generated in the dorsal part of the rat telencephalon at around E11.75 (corresponding to E9.75 in mouse embryos). These neurons migrate ventrally and align at the PSB through ephrin A5 repulsive activity. In the Pax6 mutant, reduced ephrin A5 expression allows these neurons to invade the ventral part of the telencephalon. (B) The ventrally migrating neurons include several distinct subtypes such as lot cells and olfactory cortex neurons. They migrate ventrally, probably owing to some attractant distributed in the dorsal part of the telencephalon and/or secreted from the ventral part of the telencephalon. Netrin 1 acts as an attractant for lot cells (Kawasaki et al., 2006). Ephrin A5 and other factors act as contact-dependent repellents for these neurons.