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First published online 4 August 2004
doi: 10.1242/dev.01281

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The Fgf8 signal causes cerebellar differentiation by activating the Ras-ERK signaling pathway

Tatsuya Sato^1,2,* and Harukazu Nakamura^1,2,

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

View larger version (69K): [in a new window]   Fig. 7. Distinct disruption of Fgf8a and Fgf8b by siRNA. (A) Alignment of partial sequence of Fgf8a and Fgf8b mRNA. The target sequence of Fgf8a- and Fgf8b-siRNA is underlined with red and green, respectively. The number indicates the number from the start codon. (B-E) Effects of Fgf8b-siRNA on Fgf8 expression. We could detect disruption of Fgf8 mRNA to some extent. Since the probe used for in-situ hybridization hybridized to both Fgf8a and Fgf8b, disruption of Fgf8b may be more than we can detect. (F-I) Effects of Fgf8a-siRNA on Fgf8 expression. We could not detect the effect of siRNA. This may be due to the fact that Fgf8b is predominantly expressed in the isthmus. (J-M) Application of both Fgf8a- and Fgf8bsiRNA. Electroporation of both Fgf8a- and Fgf8b-siRNA resulted in distinct reduction of Fgf8 mRNA in the isthmus. (N-P) Effects of siRNA on the activation of ERK. Fgf8b-siRNA decreased the activation level of ERK (N), although Fgf8a-siRNA hardly affected ERK activity (O). (Q-S) Effects of Fgf8b-siRNA on Otx2 expression. The arrows represent the caudal border of the Otx2 expression domain. Application of Fgf8b-siRNA by electroporation resulted in a caudal shift of the Otx2 expression domain; in other words, the mesencephalon extended caudally. (T-V) Effects of Fgf8a-siRNA on Otx2 expression. Fgf8a-siRNA did not affect Otx2 expression. Dorsal view (B,F,J,N-Q,T), Lateral view (C,D,G,H,K,L,R,U), GFP fluorescence to indicate the site of siRNA introduction (E,I,M,S,V). mes, mesencephalon; met, metencephalon; cont, control side; exp, experimental side. Scale bars: 200 µm.

View larger version (100K): [in a new window]   Fig. 1. ERK activation in the neural tube. (A) Whole-mount in-situ hybridization for Fgf8 at the 8-somite stage (HH9+). (B-E) Distribution of activated ERK revealed immunohistochemically with anti-dpERK antibody in normal embryos. (B) Eight-somite stage (HH9+). (C) Ten-somite stage (HH10). (D) Twelve-somite stage (HH11-). (E) Fourteen-somite stage (HH11+). ERK is activated around the isthmus (arrow on B) and the anterior tip of the neural tube at the 8-somite stage. By the 14-somite stage, ERK has been inactivated in the metencephalon.

View larger version (72K): [in a new window]   Fig. 2. ERK activation by Fgf8 signal. (A) Immunohistochemistry with anti-dpERK 1 hour after insertion of an SU5402-soaked bead (asterisk). ERK activation is repressed by SU5402, an inhibitor of the Fgf receptor. (B) Dorsal and (C) lateral view of an E14.5 brain after misexpression of Fgf8b. Instead of the tectum, cerebellum has differentiated in the mesencephalic region (arrow). (D) Dorsal view of an E6.5 brain after misexpression of Fgf8a. The tectum enlarged because the fate of the diencephalic alar plate was changed to tectum. (E) Misexpression of GFP at 3 hours after electroporation. The GFP misexpression site corresponds to that of Fgf8 shown in (F-H). (F-H) Immunohistochemistry with anti-dpERK antibody after misexpression of Fgf8a 1 µg/µl (F), Fgf8b 1 µg/µl (G), Fgf8b 0.01 µg/µl (H). ERK was activated only in the diencephalon by Fgf8a (F). ERK was activated at the site where Fgf8b was misexpressed through the diencephalon, mesencephalon and metencephalon (G). Misexpression of Fgf8b at a concentration of 0.01 µg/µl caused ERK activation only in the diencephalon (H), as was the case of Fgf8a misexpression. di, diencephalon; mes, mesencephalon; met, metencephalon; tel, telencephalon; tect, tectum; cer, cerebellum; cer-ect, ectopic cerebellum. Scale bars: 200 µm (A,F-H), 4 mm (B-D).

View larger version (90K): [in a new window]   Fig. 3. Fate change of the metencephalon to the mesencephalon by RasS17N misexpression. (A) Repression of ERK activity by misexpression of RasS17N. (B-D) Views from the dorsal (B), left (C) and right (D) of the E10.5 chick brain after misexpression of RasS17N. The tectum differentiated ectopically in place of the cerebellum (arrow on B,D). Posterior part differentiated into the cerebellum according to the original program (arrowheads on D). (E) A transverse section of the brain at the level of (E) indicated on (A). (F,G) Higher magnification of the cerebellum and ectopic tectum indicated as (F,G, respectively), on (D). The external granular layer is differentiating on the control side (arrowhead on F). (H) Higher magnification of the proper tectum. The ectopic tectum has nine layers (G), while the proper tectum has ten layers (H). (I,J,L,M) Whole-mount immunohistochemistry with anti-neurofilament antibody on E4 embryos. (K,N) Bright field of (J,M, respectively). An arrow and arrowhead on (K) indicate a large ectopic swelling and a small one in the metencephalon, respectively. In normal embryos (I), oculomotor (III) and trochlear (IV) nerves are discernible. In RasS17N-transfected embryos (J), trochlear nerve fibers arise from the posterior part of the large swelling and run dorsally (arrowheads). Additional nerve fibers arise from the posterior part of the small swelling (arrow). These fibers merge while running dorsally. In some cases, a nerve trunk originates from the ventral metencephalon and run a similar course to the oculomotor nerve (arrowheads on L). In another embryo that is transfected with RasS17N, several additional nerve fibers could be discerned posterior to the proper trochlear nerve. In some embryos, the nerve trunk that resembles oculomotor nerve arose from the metencephalic region (M, arrowheads). tel, telencephalon; di, diencephalon; tect, tectum; cer, cerebellum; cont, control side; exp, experimental side; egl, external granular layer; ne, neuroepithelium; III, oculomotor nerve; IV, trochlear nerve. Scale bars: 4 mm (B,C,D); 2 mm (E); 200 µm (A,F,G,H); 400 µm (I-N).

View larger version (91K): [in a new window]   Fig. 4. Effects of RasS17N misexpression on Otx2, Gbx2 and Fgf8. Effects on Otx2 (A-C,J,K), Gbx2 (D-F,L-N) and Fgf8 (G-I,OQ). Misexpression site of RasS17N (brown in C,F,I) is assessed by immunohistochemistry against HA-tag. Otx2, Gbx2 and Fgf8 expression is represented as blue by in-situ hybridization. Twenty-four and 42 hours after electroporation (A-I and J-Q, respectively). Left (control) side of the brain vesicles (A,D,G,L) and right (transfected) side (B,C,E,F,H,I,M,P). Dorsal view (J,O). Higher magnification (B',C',E',F',H',I',K,N,Q). The rectangular area on (E) corresponds to (E'). By 24 hours after electroporation, Otx2 was induced in the metencephalon by RasS17N misexpression (B,C,B',C'). Gbx2 was repressed in the metencephalon by RasS17N misexpression (E,F,E',F'). Endogenous Fgf8 expression was also repressed, but was induced in the periphery of RasS17N expression in the metencephalon (H,I,H',I'). By 42 hours after electroporation, the boundary of patchy expression of Otx2 in the metencephalic region became blurred (J,K), and Otx2 expression area expanded (arrowheads in K). Repression of Gbx2 and Fgf8 just posterior to the proper mesencephalon became wider, leaving a Gbx2- and Fgf8-free region (L-Q). di, diencephalon; mes, mesencephalon; met, metencephalon; cont, control side; exp, experimental side. Scale bars: 200 µm (AC,D-F,G-I,J,L,M,O,P), 100 µm (B',C',E',F',H',I',K,N,Q).

View larger version (74K): [in a new window]   Fig. 5. Effects of RasS17N misexpression on Wnt1, Pax2, Pax5, En1, En2 and Fgf8. Effects on Wnt1 (A,B), Pax2 (C-G), Pax5 (H-L), En1 (M-Q), En2 (R-V). Brown, immunohistochemical staining against HA-tag. Blue, signal for in-situ hybridization. Dorsal view (A,B), Left (control) side of the brain vesicles (C,H,M,R), right (transfected) side (D,E,I,J,N,O,S,T). Higher magnification (F,G,K,L,P,Q,U,V). The areas enclosed by the dashed line on (E,J,O,T) corresponds to (G,L,Q,V), respectively. Wnt1 was induced in the dorsal metencephalon (arrowheads on A). Expression of Pax2/5 and En1/2 were repressed by RasS17N misexpression. Scale bars: 200 µm.

View larger version (105K): [in a new window]   Fig. 6. Co-transfection of Fgf8b/a with RasS17N. (A-F) Dorsal view (A) and lateral view (B), parasagittal section at the line indicated on (A) as (C) and (E) (C,E) of E10.5 brain co-electroporated with Fgf8b and RasS17N at the 10-somite stage. (D,F) higher magnification of (C,E, respectively). The swelling in the mesencephalic region and anterior metencephalic region of the experimental side have sulci on the surface, and look like cerebellum (A,B). Histologically, however, the swelling in the mesencephalon and the anterior metencephalon of the experimental side do not have an external granular layer, which is seen in the cerebellum of the control side (C,D), and has tectal architecture (E,F). The posterior part of the metencephalic region consists of cerebellar structure (arrowheads on E). Antero-posterior direction is indicated by the double-headed arrow on (B,E). Dorsal view (G,I) and horizontal section (H) of the brain co-electroporated with Fgf8a and RasS17N. The tectum has extended to the diencephalic territory (G,H). Tectum differentiated ectopically in the metencephalon (I, arrow). tel, telencephalon; di, diencephalon; tect, tectum; cer, cerebellum; egl, external granular layer; tect, tectum; ne, neuroepithelial layer; roman numbers on (F) indicate the tectal layers. Scale bars: 4 mm (A,B,C,E,G,H,I); 200 µm (D,F).

View larger version (30K): [in a new window]   Fig. 8. Schematic drawing to show the organizing activity of Fgf8 and its signal transduction. Fgf8 is induced at the interface of Otx2 and Gbx2 expression, overlapping with Gbx2 expression. The site where Fgf8 mRNA is localized may receive a strong Fgf8 signal and cause the Ras-ERK pathway to be activated. Thus, this region may acquire the characteristics of rhombomere1 (r1), where cerebellum differentiates. By contrast, in the mesencephalon, the Fgf8-Ras-ERK pathway may be activated only weakly, which may play a role in rostrocaudal polarity formation of the tectum.