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First published online 8 December 2004
doi: 10.1242/dev.01581

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Regulation of isthmic Fgf8 signal by sprouty2

Asuka Suzuki-Hirano¹, Tatsuya Sato^1,2,* and Harukazu Nakamura^1,2,

¹ Department of Molecular Neurobiology, Institute of Development, Aging and Cancer, Tohoku University, Seiryo-machi 4-1, Aobaku, Sendai 980-8575, Japan
² Graduate School of Life Sciences, Tohoku University, Seiryo-machi 4-1, Aoba-ku, Sendai 980-8575, Japan

View larger version (102K): [in a new window]   Fig. 1. sprouty2 and Fgf8 expression in normal embryos. (A,C,F) In situ hybridization for Fgf8; (B,D,G) in situ hybridization for sprouty2; (E) in situ hybridization for both Fgf8 and sprouty2, at HH 12 (A,B), HH 21 (C-E), HH 25 (F,G). In all the embryos examined, Sprouty2 was expressed overlapping Fgf8, the anterior neural ridge and the isthmus. The ages of the embryos are indicated. tel, telencephalon; di, diencephalon; mes, metencephalon; is, isthmus; met, metencephalon, HH represents the stage of Hamburger and Hamilton (Hamburger and Hamilton, 1951), scale bar: 200 µm.

View larger version (68K): [in a new window]   Fig. 2. Induction of Sprout2 by Fgf8. sprouty2 induction at 3 hours after Fgf8a misexpression (A-D, the same embryo), and 24 hours after electroporation (E-H). E and F are form the same embryo, and G and H show the same section at the mesencephalon. (I-L,M-R) sprouty2 induction at 3 hours after Fgf8b misexpression (I-L, the same embryo), and 24 hours after electroporation (M-R, M,N and Q,R are from the same embryo). O and P are higher magnifications of the areas indicated as O and P on M and N, respectively. (S) Electroporation of mouse Fgf8b and hybridization with cFgf8 and mFgf8 probes that do not cross hybridize each other. In situ hybridization for Fgf8 (A,C,E,G,I,K,M,O,Q), for sprouty2 (B,D,F,H,J,L,N,P,R). At 3 hours after electroporation with Fgf8a vector (HH 9), misexpression of Fgf8 is easily discernible (A,C), and sprouty2 is induced overlapping Fgf8 expression (B,D). At 24 hours after electroporation, Fgf8 misexpression can be discerned in a wide area (E). sprouty2 is expressed overlapping Fgf8 (E-H). At 3 hours after electroporation with Fgf8b expression vector, overlapping expression of sprouty2 and Fgf8 is easily discernible (I-L). At 24 hours after electroporation of chick Fgf8 vector (1 µg/µl, M,N) (HH 18), Fgf8 and sprouty2 expression is widely discernible from the metencephalon to the diencephalon at the lateral side of the mesencephalon (M,N). Strong line-like expression of Fgf8 and sprouty2 along the roof plate of the mesencephalon was also seen (arrows on M and N). Misexpression of mouse Fgf8 (purple, S) and hybridization with chick Fgf8 (red, S) show that the expression along the roof plate is of the transcripts from the embryonic gene. Higher magnification figures show that sprouty2 expression is induced slightly more widely than the Fgf8 expression area (O,P). The arrowhead on O and P indicate the same point. At 24 hours after electroporation of chick Fgf8 vector at a concentration of 0.1 µg/µl, V-shaped expression of Fgf8 and sprouty2 is seen (Q,R). The right-hand-side on the dorsal view represents the experimental side. tel, telencephalon; di, diencephalon; mes, mesencephalon; is, isthmus; met, metencephalon; cont, control side; exp, experimental side. Scale bar: 200 µm.

View larger version (83K): [in a new window]   Fig. 3. Reconstruction of the isthmus after mouse Fgf8b misexpression. (A) In situ hybridization for Otx2 (purple), in situ hybridization for mouse Fgf8 (red): A, part e and inlet of A, parts b and c. Arrowhead indicates the posterior limit of the Otx2 expression domain. Photos of A, parts c-f are from the same embryo. (B) in situ hybridization for Gbx2 (purple), in situ hybridizaition for mouse Fgf8 (red): B, part e and inlet of B, parts b and c. Arrowhead indicates anterior limit of Gbx2 expression domain. Photos of B, parts c-f are from the same embryo. (C) Double in situ hybridization with chick Fgf8 probe (purple), and with mouse Fgf8 probe (red). Arrowheads on C, part f indicate chick Fgf8 (endogenous) expression line. (D) in situ hybridization for Lmxb1 (purple), in situ hybridization for mouse Fgf8 (red): D, part d and inlet of D, parts b and c. Arrowheads on D, part g indicate Limx1b expression line. Otx2 expression in the mesencephalon retreats (A), and complementarily, Gbx2 expression extends rostrally (B). Fgf8 expression in the isthmus, that is expression from the embryonic gene (purple on C), disappears, and a new expression line appears by 36 hours after electroporation. Interaction of among Fgf8, Lmx1b and Wnt1 may be involved in reconstruction of the isthmus as indicated by Matsunaga et al. (Matsunaga et al., 2003). mes, mesencephalon; is, isthmus; met, metencephalon; exp, experimental side; cont, control side. Scale bar: 200 µm.

View larger version (99K): [in a new window]   Fig. 4. Function of Sprouty2 as a repressor of ERK activity. (A-D) Repression of ERK activity by sprouty2 misexpression. (E) Upregulation of ERK activity by morpholino antisense oligonucleotide against sprouty2, and (F-H) by misexpression of the dominant negative form of sprouty2. (I) Co-transfection of sprouty2 and sprouty2-DN. The effects of Sprouty2 were canceled by Sprouty2-DN. Immunohistochemical staining with anti-dpERK (brown). D', H' and E' are fluorescences of GFP and FITC, respectively, to indicate the misexpression site. (A,E,I) 3 hours after electroporation, (B,F) 6 hours after electroporation, (C,D,G,H) 9 hours after electroporation. G and H are higher magnifications of the isthmus region. Arrowhead indicates ERK activation zone. Scale bar: 200 µm.

View larger version (89K): [in a new window]   Fig. 5. Fate change of the presumptive cerebellum to the tectum. Dorsal view (A), control side (B), and experimental side (C) of the brain of E12.5 (HH 38) after electroporation with Sprouty2 expression vector around HH 9. (D) Horizontal section at the line indicated on A. Higher magnification of the area indicated on D (E,F,G), and the proper tectum (H). (I,J) Whole mount immunohistochemistry with anti-neurofilament antibody on HH 21 embryo, 3A10, to show trochlear nerve trajectory. After Sprouty2 misexpression, the swelling in the metencephalic region looks smooth (A,C). Histologically, the cerebellum has an external granular layer (E). The swelling on the experimental side does not have external granular layer, but has laminar structure characteristic of the tectum (compare F, G and H). A nerve bundle that resembles the trochlear nerve is added caudal to the ectopic swelling (arrows, J). cer; cerebrum; di, diencephalon; te, tectum; is, isthmus; cel, cerebellum; te-tect, tectum ectopically differentiated in the metencephalon; egl, external granular layer; fp, floor plate; exp, experimental side; cont, control side. Scale bar: 4 mm in A, 200 µm in D-J.

View larger version (81K): [in a new window]   Fig. 6. Effects of Sprouty2 misexpression on Otx2, Gbx2 and Fgf8 expression. In situ hybridization for Otx2 (A-D, purple), Gbx2 (E-H, purple) and Fgf8 (I-K, purple). Misexpression of Sprouty2 is assessed by immunohistochemistry with the antibody against HA-tag (A,E,I, brown); 24 hours after electroporation (HH 18; A-C,E-G,I-K); 48 hours after electroporation (HH 22; D,H). Sprouty2 induced Otx2 expression, and repressed Gbx2 expression in the metencephalic region. (D,H) By 48 hours after electroporation, regulation of Otx2 and Gbx2 expression may have occurred and the expression domain of these genes reduced. (J) The Fgf8 expression ring shifted caudally. mes, mesencephalon; is, isthmus; cont, control side; exp, experimental side. Scale bar: 200 µm.

View larger version (100K): [in a new window]   Fig. 7. Sprouty2-DN causes rostral shift of the caudal border of the tectum. (A,C,D) Dorsal view (A: HH 30; C: HH24; D: HH23). (B) Horizontal section of the level indicated by white line on A.(D) Whole mount in situ hybridization for Wnt1. Arrows indicate the caudal border of the tectum. The caudal border of the tectum at the experimental side locates rostrally to that at the control side. mes: mesencephalon, is: isthmus, met: metencephalon, fp, floor plate; cont, control side; exp, experimental side. Scale bar: 200 µm.

View larger version (82K): [in a new window]   Fig. 8. Effects of Sprouty2-DN misexpression on Otx2, Gbx2 and Fgf8 expression. In situ hybridization for Otx2 (A-C), Gbx2 (D-F) and Fgf8 (G,H). Misexpression of sprouty2-DN is assessed by immunohistochemistry with the antibody against HA-tag (A,D,G), 24 hours after electroporation (HH 18; A,B,D,E,G,H), 48 hours after electroporation (HH 22; C,F). Sprouty2-DN repressed Otx2 expression, and induced Gbx2 expression in the mesencephalic region. By 48 hours after electroporation, regulation of Otx2 and Gbx2 expression may have occurred, and the expression domain of these genes abuts (C,F). The Fgf8 expression ring is extended rostrally (arrows on G,H). mes, mesencephalon; is, isthmus; met, metencephalon; cont, control side; exp, experimental side. Scale bar: 200 µm.