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First published online 26 January 2006
doi: 10.1242/dev.02257

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Netrin 1 regulates ventral tangential migration of guidepost neurons in the lateral olfactory tract

¹ Division of Brain Function, National Institute of Genetics, Graduate University for Advanced Studies (SOKENDAI), Yata 1111, Mishima 411-8540, Japan.
² PREST, Japan Science and Technology Agency, 4-1-8 Honcho Kawaguchi, Saitama, Japan.

View larger version (103K): [in a new window]   Fig. 1. Ventral tangential cell migration in organotypic culture. (A) Schematics of organotypic culture. A strip was dissected from the E10.5 mouse telencephalon along the dorsoventral axis to include the presumptive LOT area (green area) around the middle. A small area in the dorsal neocortex of the strip was labeled with fluorescent dye rhodamine (asterisk). (B) The strip 3 hours after preparation. Rhodamine-labeled cells (magenta) do not migrate yet from the labeled position (asterisk). Boundary between the neocortex and the GE is visualized by immunostaining against reticulon 1 (green). (C) The strip after 36 hours in culture. Merged image of rhodamine (magenta) and immunostaining with mAb lot1 (green). One of the rhodamine-labeled cells migrating ventrally reacts with mAb lot1 (arrowhead). (D,E) The strip after 2 days in culture. Many rhodamine-labeled cells (magenta in D, white in E) migrate in the ventral direction from the dye injection point (asterisk) and stop before entering the GE. Reticulon 1 is immunostained in green in D. (F,G) High-magnification views of the upper (F) and lower (G) boxes in E. The processes of many rhodamine-labeled cells are directed in the ventral direction in F, but reoriented in the rostrocaudal direction at the presumptive LOT area in G. Left, rostral aspect; top, dorsal aspect; dcx, dorsal neocortex; vcx, ventral neocortex; GE, ganglionic eminence. Scale bars: 500 µm in B,D; 50 µm in C,F,G.

View larger version (113K): [in a new window]   Fig. 2. Tangential cell migration in organotypic co-culture. (A) Combination of the dorsal neocortex from a green mouse embryo with the ventral neocortex in a wild-type strip in the dorsoventrally ordered manner. Many GFP-expressing cells migrate through the ventral neocortex down to the presumptive LOT area. (B) Combination of the wild-type dorsal neocortex with the ventral neocortex in a green mouse strip in the dorsoventrally ordered manner. GFP-expressing cells do not migrate into the dorsal neocortex. (C) The isolated dorsal neocortex labeled with rhodamine (asterisk) and cultured alone for 2 days. Rhodamine-labeled cells migrate ventrally and accumulate in the ventral edge of the explant. (D) Co-culture of two dorsal neocortices ventrally facing each other, one of which was injected with rhodamine (asterisk). Rhodamine-labeled cells migrate and stop close to the combination boundary with the other explant. (E) Co-culture in which the dorsal neocortex of a green mouse embryo was combined with the lateral side of a wild-type strip. GFP-expressing cells preferentially penetrate in the LOT area and the neocortex but not the GE. (F,G) The dorsal neocortex prepared from a green mouse embryo (F) or labeled with rhodamine (G) was combined with the ventral side of the GE in a wild-type strip in the dorsoventrally reversed orientation. In neither of the co-cultures did cells of the combined dorsal neocortex penetrate into the GE. (H) High-magnification view around the combination boundary in G. Rhodamine-labeled cells perpendicularly change orientation and align along the boundary. Broken white lines outline the explants and broken green lines indicate the combination boundaries in A-H. The ventral direction of each explant is indicated by an arrow. (I) A possible scheme of regulation of the ventral tangential migration. The neocortex contains ventrally directing signals for migrating cells. The whole area of the GE has mechanisms to exclude lot cells. Scale bars: 500 µm in A-G; 100 µm in H.

View larger version (86K): [in a new window]   Fig. 3. Effect of XtJ mutation on tangential cell migration. (A,B) The E10.5 XtJ/XtJ telencephalon strip labeled with rhodamine (asterisk) and cultured for 2 days. The boundary between the neocortex and the GE is visualized with anti-reticulon 1 antibody (green in A). Rhodamine-labeled cells create an ectopic cluster (arrowhead) in the neocortex instead of migrating towards the presumptive LOT area. (C) Co-culture in which the dorsal neocortex from a green mouse is combined with the ventral neocortex in the XtJ/XtJ strip. GFP-expressing cells migrate into the ventral neocortex of the XtJ/XtJ mouse. (D) Co-culture in which the dorsal cortex from an XtJ/XtJ green mouse is combined with the ventral neocortex in the wild-type strip. XtJ/XtJ cells barely migrate into the wild-type ventral neocortex. Arrows indicate the ventral direction of the explants. The white broken lines outline the explants, and green broken lines show the borderlines between explants. Scale bars: 500 µm.

View larger version (83K): [in a new window]   Fig. 4. Attraction of tangentially migrating cells to netrin 1. (A-D) Co-cultures of rhodamine-labeled strips with aggregates (blue outline) of HEK cells expressing netrin 1 (A,B) and the mock control cells (C,D). The boundary between the neocortex and the GE is stained with anti-reticulon 1 antibody (green in A,C). Many rhodamine-labeled cells migrate toward the netrin 1-secreting cells, but not the control cells. (E) The Dcc mutant strip labeled with rhodamine and cultured for 2 days. The cells migrate normally to the LOT area. (F) Co-culture of the Dcc mutant strip with a cell aggregate expressing netrin 1 (blue outline). The cells are not attracted to the aggregate. (G) The Dcc mutant strip cultured with 10 µM cyclopamine. The migration of rhodamine-labeled cells is not affected by the treatment. (H) The percentage of dye-labeled cells that reside in the halves of strips proximal to cell aggregates. The average values and the standard errors (thin horizontal bars) are calculated from the wild-type strips cultured with mock-transfected cells (upper) and netrin 1-expressing cells (middle) and Dcc homozygous mutant strips culture with netrin 1-expressing cells (bottom). The numbers of strips used for quantification are indicated by n in the parentheses. Scale bars: 500 µm.

View larger version (152K): [in a new window]   Fig. 5. Expression of netrin 1 and Dcc in the developing telencephalon. (A-H) Expression of netrin 1 (A-D) and Dcc (E-H) mRNAs in coronal sections of E10.5 (A,E), E11.5 (B,F) and E12.5 (C,G) wild-type and E12.5 XtJ/XtJ telencephalons (D,H). Asterisks indicate the presumptive LOT area. In wild-type embryos, netrin 1 is strongly expressed in the neuroepithelium of the GE during all these embryonic stages and weakly on the surface of the olfactory tubercle at E12.5 (arrowhead in C). Dcc is expressed in the surface layer of the neocortex. The expression patterns are essentially similar in the XtJ/XtJ telencephalon. (I-M) Double-immunostaining of a coronal section of E12.5 telencephalon (I-L) and cultured cortical neurons (M) with mAb lot1 (white in K; magenta in I,J,M) and anti-Dcc antibody (white in L; green in I,J,M). (J-L) are enlargements of the boxed area in I. The cortical neurons in M were dissociated from the E11.0 neocortex and cultured for 36 hours before the immunostaining as described previously (Tozaki et al., 2002). All lot1-positive neurons express Dcc. Scale bars: 500 µm in A-I; 20 µm in J,M.

View larger version (123K): [in a new window]   Fig. 6. Distribution of lot cells in the telencephalon of netrin 1 mutant embryos. Telencephalons of heterozygous (A,C,D,F,G,I,K) and homozygous (B,E,H,J,L) netrin 1 knock-in mutant embryos stained with mAb lot1 (A,B,D,E,G-L), anti-neuropilin 1 antibody (K,L) and X-gal solution (C,F,G-J). (A,B) At E12.5, distributions of lot cells in heterozygous and homozygous mutants are indistinguishable. (D,E) At E13.0, lot cells spread ventrally in heterozygous, but not in homozygous mutant embryos. (C,F) Expression of lacZ reporter gene that presumably reflects endogenous netrin 1 expression. lacZ is only expressed in the neuroepithelium of the GE at E12.5 (C), but the expression expands onto the surface of the rostral olfactory tubercle at E13.5 (F). (G,H) E14.5 telencephalons double-stained with mAb lot1 and X-gal. (I,J) High-magnification views of boxed areas in G and H. (K,L) Coronal sections of E14.5 telencephalon including the LOT stained with mAb lot1 (magenta) and anti-neuropilin 1 antibody (green). The distribution of lot cells expands ventrally towards the lacZ-expressing region in the olfactory tubercle in the heterozygous telencephalons (arrow in I) but not in the homozygous telencephalons, where lot cells seem to be stacked in the ventral edge of the cellular array (arrowheads in J,L). All of 30 homozygous mutants showed the same disruptive phenotype. Scale bars: 500 µm.

View larger version (75K): [in a new window]   Fig. 7. Pathfinding of olfactory bulb axons in the telencephalon of Dcc mutant embryo. (A-D) Projection of olfactory bulb axons in E14.5 heterozygous (A,C) and homozygous (B,D) Dcc mutant embryos visualized with anti-neuropilin 1 antibody. The number of axons elongating from the ventral side of the olfactory bulb in the homozygous mutant (arrow in D) is much lower than that in the heterozygous mutant. The phenotype was apparent in all of the 20 homozygous mutant telencephalons. Left, rostral aspect; top, dorsal aspect. (E-H) Fluorescent dye labeling of medial olfactory bulb axons in E16.5 heterozygous (E,G) and homozygous (F,H) Dcc mutant embryos. (E,F) Lateral views of left hemispheres. Left, rostral aspect; top, dorsal aspect. (G,H) Ventral views of left hemispheres showing the dye-labeled ventral pathways; left, rostral aspect; top, lateral aspect. Dye-labeled axons split into the dorsal (white arrowheads in E,F) and ventral (arrows in E-H) pathways to enter into the LOT. In the homozygous telencephalon, axons projecting in the ventral pathway are tangled and misdirected back to the olfactory bulb. This aberrant projection of olfactory bulb axons was detected in all of 15 homozygous Dcc mutants and three out of five homozygous netrin 1 mutants. Scale bars: 1 mm in A,B; 500 µm in C-H.

View larger version (41K): [in a new window]   Fig. 8. Distribution of lot cells and projection of olfactory bulb axons in wild-type and netrin 1 or Dcc mutant embryos. Lateral views of hemispheres in wild-type (A-C) and netrin 1 or Dcc mutant embryos (D-F). (A,D) The initial phase of ventral tangential migration of lot cells (magenta) to the dorsal part of the LOT area by E12.5 is indistinguishable among all of the genotypes. (B) At E13.0, netrin 1 expression begins on the rostral surface of the olfactory tubercle in the wild-type telencephalon (blue shaded area), which presumably attracts lot cells further ventrally. (E) In the mutant telencephalon, without netrin 1-Dcc signal, lot cells remain absent from the ventral part of the LOT area. (C,F) From E13.5 onwards, olfactory bulb axons (green) project from the olfactory bulb, following the array of lot cells. In the mutant telencephalon, the ventral pathway of olfactory bulb axons, which is destined to project through the lot cell-vacant area, shows obvious projection errors.