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Role of Lmx1b and Wnt1 in mesencephalon and metencephalon development

Eiji Matsunaga^*, Tatsuya Katahira and Harukazu Nakamura

Department of Molecular Neurobiology, Institute of Development, Aging & Cancer, and Graduate School of Life Sciences, Tohoku University, Seiryo-machi 4-1, Aoba-ku, Sendai 980-8575, Japan
^* Present address: INSERM U106, Bâtiment de Pédiatrie, Hôpital de la Salpêtrière, 47 Boulevard de l'Hôpital, 75013 Paris, France

View larger version (81K): [in a new window]   Fig. 1. Normal expression patterns of Fgf8, Lmx1b and Wnt1. In situ hybridization of serial sections of the same embryos at stage 10 (A,C,E) and stage 12 (B,D,F), for Fgf8 (A,B), Lmx1b (C,D) and Wnt1 (E,F). At stage 10, Lmx1b and Wnt1 are expressed in the mesencephalon and metencephalon. Their expression overlaps with Fgf8 expression in the metencephalon. At stage 12, Lmx1b is expressed strongly in the mesencephalon, but in the metencephalon its expression is weak. Wnt1 expression in the metencephalon has almost disappeared. (G,H) Whole-mount in situ hybridization for Lmx1b (blue) and Fgf8 (red) (G), and for Wnt1 (blue) and Fgf8 (red) (H). Both Lmx1b and Wnt1 are expressed next to Fgf8 expression at the mes-metencephalic boundary. Scale bars: 250 µm. mes, mesencephalon; met, metencephalon.

View larger version (66K): [in a new window]   Fig. 2. Morphology after Lmx1b and Wnt1 misexpression. (A-C) Morphology of Lmx1b-misexpressed embryo at E7.5. Dorsal view (A); view from the caudal side (B); transverse section stained with Hematoxylin-Eosin (C). Expansion of the tectum, torus semicircularis and rhombic lip are seen on the experimental side. (D-H) Morphology after Wnt1 misexpression. E3.5 (48 hours after electroporation; D,E). E14.5 (13 days after electroporation; F-H). Dorsal view (D,F,G); view from the caudal side (E); horizontal section stained with Hematoxylin-Eosin (H). Extra folia (arrows on H) were formed in the cerebellum by Wnt1 misexpression. Scale bars: 2 mm (F), 1 mm (B,G), 500 µm (C,E,H). cer, cerebellum; cont., control side; exp., experimental side; met, metencephalon; rl, rhombic lip; tec, tectum; ts, torus semicircularis.

View larger version (78K): [in a new window]   Fig. 3. BrdU incorporation after Wnt1 misexpression. (A) GFP fluorescence micrograph to show misexpression site (marked by curved bar). Arrowheads indicate nonspecific fluorescence caused by blood cells. (B-D) Fluorescence micrographs for BrdU incorporation. Rectangles on low-power micrograph (B) indicate the area of C and D. The cryosections include surface ectoderm (arrows in C and D indicate the border between neuroepithelium and the surface ectoderm). V, ventricle; E, surface ectoderm; N, neuroepithelium; exp, experimental side; cont, control side.

View larger version (83K): [in a new window]   Fig. 6. Effects of Lmx1b misexpression on downstream gene expression. (A-F) Effects on Otx2 expression. In situ hybridization for Otx2 (blue) and Lmx1b (red) on the same embryo at 12 hours (A-C') and 24 hours (D-F) after electroporation. (B',C') High-power magnification of boxed areas in B,C. Otx2 is induced by Lmx1b misexpression, but only weakly induced in the isthmus at 24 hours after electroporation (E,F, arrows). (G-I) Effects of Otx2 misexpression on Fgf8 expression. In situ hybridization for Fgf8 (blue) and Otx2 (red) at 12 hours after Otx2 misexpression. Repression of Fgf8 is hardly observed at 12 hours after electroporation (H). (J-M) Effects of Lmx1b-EnR misexpression on Fgf8 expression. In situ hybridization for Fgf8 (J, control; K, experimental side). (L) Section of the specimen shown in J and K, in situ hybridization for Fgf8 (blue) and immunohistochemical staining for HA tag (tagged to Lmx1b-EnR). (M) High power magnification of area in L. As Lmx1b is revealed by immunohistochemical staining, it is localized in the nucleus. Fgf8 signal is localized in the cytoplasm. Fgf8 is induced in Lmx1b-EnR misexpressing cells (M) in the caudal mesencephalon (K,L). (N-P) Effects of co-electroporation of Lmx1b-EnR and Lmx1b on Fgf8 expression. In situ hybridization for Fgf8 at 24 hours after electroporation (blue, N,O). (P) In situ hybridization for Fgf8 (blue) and immunohistochemical staining for HA tag (tagged to Lmx1b-EnR). Ectopic expression of Fgf8 in the caudal mesencephalon is canceled by co-electroporation with wild-type Lmx1b. (Q-T) Effects of Lmx1b misexpression on Grg4 expression. In situ hybridization for Grg4 (blue) (Q,R), and immunostaining for HA (tagged to Lmx1b, brown) (S) at 6 hours after electroporation of pMiw-Lmx1b. Grg4 is induced in the metencephalon on the experimental side (R,S). (T) High-power magnification of the metencephalic region to show that Grg4 is induced in the Lmx1b-misexpressed cells. (A,D,G,J,N,Q) Views from the control side. (B,C,E,F,H,I,K-M,O,P,R-T) Views from the experimental side. Scale bars: 250 µm in F; 200 µm in C,I,K,L,P; 100 µm in S; 25 µm in C'; 10 µm in M,T.

View larger version (131K): [in a new window]   Fig. 4. Repression of Cash1 by Lmx1b and Wnt1 misexpression. (A) Lmx1b misexpression represses Cash1 expression at 48 hours after electroporation. The right-hand side is the experimental side. Rostral is towards the top. Black arrows indicate dorsal midline. By 48 hours after electroporation, some regulation may have occurred, and repression sites are patchy (red arrows). (B) Wnt1 misexpression represses Cash1 expression at 24 hours after electroporation. Both panels are dorsal views of the mesencephalic region of embryos. (A) Flat mount; (B) dorsal view. (C,D) Higher magnification of the areas indicated in B. At 24 hours after electroporation, Cash1 expression is repressed uniformly by Wnt1. Scale bars: 200 µm in A,B; 100 µm in C,D.

View larger version (69K): [in a new window]   Fig. 5. Regulation of Fgf8 by Lmx1b. (A-C) Cell-autonomous repression of Fgf8 by Lmx1b. Whole-mount in situ hybridization for Fgf8 (blue) at 12 hours after electroporation (A, control; B, experimental side). Section of the same embryo stained for Fgf8 (blue) and Lmx1b (red) (C). Note that Fgf8 expression was repressed in the Lmx1b-expressing cells (C). (D-F) Non cell-autonomous induction of Fgf8 by Lmx1b. Whole-mount in situ hybridization for Fgf8 (blue) at 24 hours after electroporation (D, control; E, experimental side). Section of the same specimen stained for Fgf8 (blue) and Lmx1b (red) (F). Fgf8 is still repressed in Lmx1b-misexpressing cells (asterisks indicated by red arrows, E), but around the Lmx1b-expressing cells (asterisks), Fgf8 is induced ectopically in the caudal metencephalon (black arrows in E). Scale bars: 100 µm. Views from the control side are printed in reverse for the comparison with the experimental side throughout the paper.

View larger version (84K): [in a new window]   Fig. 7. Effects of Lmx1b and Wnt1 misexpression on isthmus-related genes. (A-E) Wnt1 induction by Lmx1b misexpression. In situ hybridization for Wnt1 (blue; A,B,D) and Lmx1b (red; C,E) on the same embryo 24 hours after electroporation. (A) View from the control side; (B,C) view from the experimental side. (D,E) High-power magnifications of boxed areas in B,C, respectively. Wnt1 is expressed in the Lmx1b-expressing cells (D,E). (F-Q) Effects of Wnt1 misexpression on Fgf8, Otx2, Gbx2 and Lmx1b. In situ hybridization for Fgf8 (F,G), Lmx1b (I,J), Otx2 (L,M) and Gbx2 (O,P). (F,I,L,O) View from the control sides. (G,J,M,P) View from the experimental side. (H,K,N,Q) Fluorescence micrograph of GFP to show transfection efficiency. Fgf8 expression expanded caudally by Wnt1 misexpression (G). Lmx1b, Otx2 and Gbx2 expression is not affected (J,M,P). Scale bars: 500 µm in H,K,N,Q; 200 µm in C; 50 µm in E.

View larger version (102K): [in a new window]   Fig. 8. Effects of Otx2, Fgf8 and Gbx2 on Lmx1b expression. (A-C) In situ hybridization for Lmx1b (blue) and Otx2 (red) on the same embryo at 24 hours after electroporation of pMiw-Otx2. Lmx1b is induced by Otx2 misexpression in the metencephalon (B). (D-F) In situ hybridization for Lmx1b (blue) and Fgf8 (red) on the same embryo at 24 hours after electroporation of pMiw-Fgf8b. Lmx1b is induced by Fgf8b misexpression in the diencephalon and mesencephalon broadly (E). (G,H) In situ hybridization for Lmx1b (blue) on the embryo at 36 hours after electroporation of pMiw-Fgf8b. (H) Lmx1b expression appears as a half ring in the diencephalon. In addition, endogenous Lmx1b expression in the isthmus is also disappeared (H). (I-K) In situ hybridization for Lmx1b (blue) and Gbx2 (red) on the same embryo at 24 hours after electroporation of pMiw-Gbx2. (K) High-power magnification of J. Lmx1b was repressed by Gbx2 misexpression (indicated by arrows on K). (A,D,G,I) Views from the control side. (B,C,E,F,H,J,K) Views from the experimental sides. Scale bars: 500 µm in F,H; 250 µm in C,J; 100 µm in K.

View larger version (23K): [in a new window]   Fig. 9. Role of Lmx1b and Wnt1 in isthmus organizing activity. At 10-somite stage, Lmx1b and Wnt1 (blue) are expressed in the mesencephalon and metencephalon (A). Their expression overlaps with Fgf8 (red) expression around the isthmic region. Lmx1b represses Fgf8 cell-autonomously; however, it induced Fgf8 expression non cell-autonomously in adjacent cells. In cell-autonomous repression of Fgf8 by Lmx1b, Grg4 may intervene. In non cell-autonomous induction of Fgf8 by Lmx1b, Wnt1 may be involved. Fgf8 could induce Lmx1b. Gbx2 and Fgf8 induces each other's expression, and Gbx2 repressed Lmx1b expression. Otx2 and Gbx2 repress each other's expression. As a result of this complicated gene expression cascade, Fgf8 expression may be set and kept in the isthmic region just rostral to the Lmx1b and Wnt1 expression ring by E2.5 (B).