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Inductive signal and tissue responsiveness defining the tectum and the cerebellum

Tatsuya Sato, Isato Araki and Harukazu Nakamura^*

Department of Molecular Neurobiology, Institute of Development, Aging and Cancer, Seiryo-machi 4-1, Aoba-ku, Sendai 980-8575, Japan
Present address: Department of Neurobiology, University of Heidelberg, Im Neuenheimer Feld 364, D-69120 Heidelberg, Germany

View larger version (48K): [in a new window]   Fig. 1. Analysis of Fgf8a and Fgf8b in the isthmus by RT-PCR. (A) Part of the Fgf8b cDNA and amino acid sequence showing the difference between Fgf8a and Fgf8b. Fgf8a lacks the boxed region (Crossley and Martin, 1995; MacArthur et al., 1995a). The primer pair for RT-PCR are underlined, expected length of Fgf8a and Fgf8b being 202 and 235, respectively. (B) Quantitative RT-PCR. The number above each lane indicates the number of the cycles of PCR. Lane M is the DNA size marker of a x174/HincII digest. Analysis by NIH Image based on this figure indicated that amplification was logarithmic until cycle 40. RT-PCR analysis shows that Fgf8a and Fgf8b are localized in the isthmic region, Fgf8b being predominant. Other Fgf8 isoforms were not detected.

View larger version (80K): [in a new window]   Fig. 2. Effects of Fgf8a misexpression. (A) Dorsal view of the E7.5 chick brain. Misexpression of Fgf8a enlarged the tectum (compare control side and the experimental side). In A-C the anterior limit of the tectum is indicated by white arrowheads and a black arrow (in B) on the control side, and by black arrowheads on the experimental side. (B) Horizontal section showing that the tectum has enlarged into the diencephalic territory. (C) Tectal swelling extended towards anterior at E3.5. (D,E) Whole-mount immunohistochemistry with anti-neurofilament antibody of an E3.5 embryo. On the control side, oculomotor (III) and trigeminal (V) nerves are present. On the experimental side (E), there are 3 nerve trunks that run similarly to the oculomotor nerve (arrowheads). The arrow indicates the oculomotor nerve on the control side. (F) Immunohistochemistry with anti-Islet1 antibody of an E3.5 embryo to show the motor nuclei. On the control side the oculomotor nucleus (III) at the mesencephalon and the trochlear nucleus (IV) at the isthmus are clearly visible. On the experimental side the oculomotor nucleus extends anteriorly into the diencephalic territory (arrowheads). cont: control side; exp: experimental side, tel: telencephalon, di: diencephalon, tect: tectum, mes: mesencephalon. Scale bars, 4 mm (A, B), 800 µm (C), 400 µm (D, E) and 200 µm (F).

View larger version (82K): [in a new window]   Fig. 3. Effects of Fgf8b misexpression. (A,B) Dorsal and lateral view of an E15.5 brain after misexpression of Fgf8b. The tectum has disappeared, and instead cerebellum has differentiated ectopically (arrow in A,B). (C) Misexpression on both sides has completely replaced the tectum with the cerebellum (cer). (D) Parasagittal section of the brain in B to show the cerebellum proper (cer) and the part that differentiated from the mesencephalic alar plate (arrow). (E,F) Higher magnification of the cerebellum proper (cer) and the cerebellum-like structure (indicated as E and F in D). The cerebellar structure in the mesencephalic region has an external granular layer (compare the cells indicated by an arrow in E and F), and the layer that is identical to the Purkinje cell layer (compare arrowheads in E and F). (G,H) Immunocytochemistry with anti-calbindin antibody. Parasagittal cryosection of the brain shown in C was stained with anti-calbindin antibody. H is a higher magnification micrograph of the area indicated in G. Anti-calbindin antibody specifically stains Purkinje cells in the cerebellum, and G and H show that Purukinje cells are differentiated in this structure. (I-L) E3.5 embryos after Fgf8b misexpression. (I) Lateral view; there is no tectal swelling (arrowhead). (J,K) Whole-mount immunohistochemistry with anti-neurofilament antibody. (L) Flat-mount specimen after immunohistochemistry with anti-islet-1 antibody. The oculomotor nerve trunk (arrowhead inK) and the nucleus (L) disappeared on the experimental side. cer: cerebellum, cont: control side, di: diencephalon, exp: experimental side, mes: mesencephalon, tect: tectum, tel: telencephalon, III: oculomotor nerve trunk or oculomotor nucleus, IV: trochlear nucleus, V: trigeminal nerve. The anteroposterior direction is indicated by the arrow on B, D and G. Scale bars, 4 mm (A-D,G), 800 µm (I), 400 µm (J,K), 200 µm (L) and 100 µm (E,F,H).

View larger version (90K): [in a new window]   Fig. 4. Effects of Fgf8a on down stream gene expression. Fgf8a did not affect Otx2 (A, B), Gbx2 (C,D), c-Irx2 (E,F), Pax2 (G,H), or Pax5 (I,J) expression. En 2 expression was upregulated by Fgf8a in the diencephalic region (K,L). En1 expression was also upregulated in the diencephalic region (M). Pax6 expression was repressed at the posterior diencephalon (N,O). Whole-mount in situ hybridization for Fgf8a (A,C,E,G,I, red), and for Otx2 (B), Gbx2 (D), c-Irx2 (F), Pax2 (H), Pax5 (J), En1 (M), and Pax6 (N,O), all in purple. (K,L) In situ hybridization for Fgf8a (purple) and immunohistochemistry showing En2 expression (brown). An arrow indicates the rostral limit of En2 (K), En1 (M), and Pax6 (N,O) expression, and the caudal limit of En2 (L) expression on the experimental side. An arrowhead indicates the limit of corresponding gene expression on the control side (K-O). Scale bars, 600 µm.

View larger version (70K): [in a new window]   Fig. 5. Effects of Fgf8b on down stream gene expression. Fgf8b repressed Otx2 (A-C), induced Gbx2 (D-F), Irx2 (G- I) and Pax2 (J-L) expression. Fgf8b upregulated En2 expression in the diencephalic region (M,N). Whole-mount in situ hybridization for Fgf8b (A,D,G,J, red), and for Otx2 (B,C), Gbx2 (E,F), Irx2 (H,I), Pax2 (K,L) in purple. (M,N) In situ hybridization for Fgf8b (purple) and immunohistochemistry showing En2 expression (brown), C,F,I and L show the control side. An arrow indicates the rostral (M) and caudal (N) limit of En2 expression on the experimental side, and an arrowhead indicates the rostral (M) and caudal (N) limit of En2 expression on the control side. Scale bars, 600 µm.

View larger version (58K): [in a new window]   Fig. 6. Semi-quantitative analysis of Fgf8b misexpression. Electroporation of pMiwFgf8b at a concentration of 1.0 µg/µl (A,B) and 0.1 µg/µl (C,D) resulted in fate change of the mesencephalic alar plate to the cerebellum. However, electroporation of pMiwFgf8b at a concentration of 0.01 µg/µl resulted in enlargement of the tectum (E,F), as seen with Fgf8a. Electroporation at a concentration of 0.001 µg/µl had no effect (G,H). Embryos were all fixed at E7.5. Scale bars are 4 mm.

View larger version (67K): [in a new window]   Fig. 7. Co-transfection of Fgf8b with Otx2. When Otx2 was co-electroporated, the effect of Fgf8b was converted to an Fgf8a-type effect at a concentration of 0.1 µg/µl, 10 times higher concentration than single Fgf8b electroporation. Arrowhead indicates ectopic tectal structure in the metencephalon (D,F). Embryos were fixed at E7.5. Scale bars, 4 mm.

View larger version (24K): [in a new window]   Fig. 8. Schematic drawing to show the inductive activity of Fgf8. In normal development, Fgf8 expression is induced at the interface of Otx2 and Gbx2 expression domains, overlapping with Gbx2. En1 and Pax2 are expressed both in the mesencephalon and the metencephalon, and they may be involved in the initiation of Fgf8 expression. In the metencephalic region, Fgf8 signal (red) is strong enough to support c-Irx2 and Gbx2 expression and to repress Otx2 expression, which may induce this region to develop as R1. Consequently, the alar plate of R1 differentiates as cerebellum. In contrast, in the mesencephalic region, Fgf8 signal may be too weak to repress Otx2 expression. The alar plate differentiates as tectum.