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Fgf signalling through MAPK cascade is required for development of the subpallial telencephalon in zebrafish embryos

Minori Shinya^1,2, Sumito Koshida³, Atsushi Sawada^1,2, Atsushi Kuroiwa¹ and Hiroyuki Takeda^2,*,

¹ Division of Biological Science, Graduate School of Science, Nagoya University, Chikusa-ku, Nagoya, 464-8602 Japan
² Early Embryogenesis, Department of Developmental Genetics, National Institute of Genetics, Yata 1111, Mishima, 411-8540 Japan
³ Kondoh Differentiation Signalling Project ERATO, JST, Kinkichihou Hatsumei Centre, 14 Kawaramachi, Yoshida, Sakyo-ku, Kyoto, 606-8305 Japan
^* Department of Biological Sciences, Graduate School of Science, University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-0033, Japan

View larger version (87K): [in a new window]   Fig. 1. ERK activation detected by anti-dpERK antibody in wild-type embryos. (A-C,E) Lateral view, dorsal towards the right. (F) Dorsal view of the head region. (A) 75% epiboly stage; ERK activation is seen in the marginal mesoderm. (B) Three-somite stage; ERK activation is seen in the MHB and tailbud, and weakly in the ANB. (C) Five-somite stage; ERK activation in the ANB becomes strong. (D) Sagittal section of the embryo shown in C. ERK is strongly activated in the ANB (C,D). (E,F) Eight-somite stage; staining in the ANB is faint. Asterisks indicate the staining in the ANB and arrowheads indicate the staining in the MHB. Scale bar: 100 µm in A-C,E,F; 30 µm in D.

View larger version (54K): [in a new window]   Fig. 2. ERK activation in embryos treated with inhibitors to Fgf signalling. All embryos were analysed at the five-somite stage. (A-C) Lateral view, dorsal towards the right; ERK activation observed in GFP-injected embryo (A) is suppressed by injection of RasN17 (B) and bFR4 (C) RNAs. (D) Schematic representation of the SU5402 injection; SU5402 (F) or DMSO as a control (E) was injected into the head region at the tailbud stage. (E,F) Dorsal view of the head region; ERK activation in the ANB is suppressed by the injection of SU5402 (F). (G,H) Lateral view of the head region; BSA- (G) or Fgf8b- (H) soaked beads were transplanted in the thick head region at tailbud stage. Ectopic ERK activation is found around the Fgf8b beads (H). (I) Western blot analysis of activated ERK after RNA injections. Homogenates prepared from injected embryos at 75% epiboly, tailbud (bud) and six-somite (6s) stages are loaded. Western blot detects one major band of about 50 kDa (arrow), which is sensitive to inhibitor injections. Relative intensity of the major band is shown at the bottom of each lane. (J) Western blot analysis of activated ERK after SU5402 treatment (soak method, see Fig. 8E,F). Homogenates prepared from control- and SU5402-treated embryos at the three-somite stage are loaded. Relative intensity of the major band is shown at the bottom of each lane. Relative intensities in (I,J) were normalised by a nonspecific minor band (asterisk) that is insensitive to any treatments. Asterisks in A-H indicate endogenous staining in the ANB and arrows indicate the transplanted beads. Scale bar: 100 µm in A-F; 50 µm in G,H.

View larger version (89K): [in a new window]   Fig. 3. Immunostaining and western blot for activated ERK in ace mutants. (A,B) Dorsal views of the head region at the five-somite embryos stained with anti-dpERK antibody. ERK activation in the ANB is detected in both the wild-type (A) and ace homozygous mutant (B). Asterisks indicate the anterior edge of the neural plate. (C) Western blot analysis of activated ERK; the equal amount of homogenates prepared from wild-type and ace mutant at the six- to seven-somite stage were loaded in each lane. Relative intensity of the major band is shown at the bottom of each lane. Relative intensity was normalised by a non-specific minor band (asterisk) that is insensitive to any Fgf inhibitors (see Fig. 2). Scale bar: 50 µm in A,B.

View larger version (50K): [in a new window]   Fig. 4. Expression pattern and the activity of zebrafish fgf3. (A-E) Expression pattern of fgf3. (A,C,E) Dorsal view of the head region. (A,B,E) Tailbud stage and (C,D) five-somite stage, wild-type embryos (A-D) and ace mutant embryo (E). ace homozygous mutants were recognised by fragmented myoD staining in adaxial cells. (B,D) Sagittal sections taken from the embryo shown in A,C, respectively. fgf3 expression in the ANB is maintained normally in ace mutant embryo. (F,G) Lateral view, dorsal towards the right; ERK is activated in the whole embryo when injected fgf3 (F); the effect is cancelled by co-injection of RasN17 RNAs (G). As shown in F, Fgf3 exhibits strong posteriorizing and dorsalizing effects when overexpressed at early stages (S. K. et al., unpublished). Asterisks indicate positive staining in the ANB and arrowheads indicate that in the MHB. Scale bar: 100 µm in A,C,E-G; 30 µm in B,D.

View larger version (115K): [in a new window]   Fig. 5. Patterning and neurogenesis in the telencephalon in RasN17-injected embryos. Injected RNA is shown in the upper right and the probe used is indicated at the bottom. (A,B) Lateral view of the head region; RasN17-injected embryo exhibits the turbid telencephalon, truncation of a part of the eye and no MHB (arrowheads in A,B) in the head region. A' and B' are high magnification views of the telencephalic region of A,B respectively. (C-F) Sagittal section of the embryos detected for DNA fragmentation at 33 hpf (C,D) and 26 hpf (E,F); apoptotic cells are detected in the telencephalon of the RasN17-injected embryo at 33 hpf (arrows in D) but not at 26 hpf (F). (G-X) Lateral views (G-R,U-X) and dorsal views (S,T) of the head region; whole-mount in situ hybridisation was performed at the 26 hpf (G-P,U,V), 14-somite (Q,R), 15-somite (S,T) and 12-somite (W,X) stages. Subpallial telencephalic expressions of dlx2 (G,H), nk2.1b (I,J,Q,R), islet-1 (U,V) and zash1a (W,X) are lost in the RasN17-injected embryo while the pallial telencephalic markers, emx1 (G,H,S,T), eom (M,N) and tbr1 (O,P) cover the entire telencephalon. (Y,Z) Sagittal sections of the telencephalon showing BrdU labelling at the seven- to eight-somite stage after injection of GFP (Y) and RasN17RNAs (Z). The labelled cells are indicated by arrows. The telencephalic region is marked by broken red lines. The ventral (V) and dorsal (D) regions we defined in the sections are separated by broken blue lines. The ‘ventral’ corresponds approximately to the region showing strong ERK staining. Arrows in A,B,G,I,Q,U,W indicate the subpallial telencephalon, arrowhead in K,M,O,S indicate the subpallial telencephalon which is negative for emx1, eom or tbr1. Dots indicate the boundary between the telencephalon and ventral diencephalon. di, diencephalon; tel, telencephalon. Scale bar: 50 µm in A,B,Q,R,W,X; 10 µm in C-F,Y,Z; 30 µm in G-P.

View larger version (70K): [in a new window]   Fig. 6. Telencephalic territory is normally specified in RasN17-injected embryo. Injected RNA is shown in the upper right and the probe used is indicated at the bottom. (A-D) Dorsal views of the head region at tailbud stage: emx1 expression in the prospective telencephalon is not altered by RasN17 injection (A,B); gsc expression in the prechordal plate is not altered by RasN17 injection (C,D). (E,F) Lateral views of the head region at the five-somite stage, anterior towards the left; bf-1 expression in the telencephalon is normally seen in RasN17-injected embryo. (G,H) Lateral views of the head region at 15-somite stage, anterior towards the left; otx2 expression in the anterior forebrain is normally downregulated in RasN17-injected embryos. Arrows indicate the mid-diencephalon. Scale bar: 75 µm in A-D; 50 µm in E-H.

View larger version (106K): [in a new window]   Fig. 7. Patterning and neurogenesis in the telencephalon in the bFR4-and XFD-injected embryos. Injected RNA is shown in the upper right and the probe used is indicated at the bottom. Lateral view (A-M) and dorsal view (N,O) of the head region are shown. Whole-mount in situ hybridisation was performed at the 26 hpf (A-K), 14-somite (L,M) and 15-somite (N,O) stages. Subpallial telencephalic expressions of dlx2 (A,B), nk2.1b (C,D,L,M) and islet-1 (E,F) are suppressed in the bFR4-injected embryo, while the pallial telencephalic markers, emx1 (G,H,N,O), and tbr1 (I,J) cover the entire telencephalon. Similarly, in the XFD-injected embryo, tbr1 expression is expanded (K). Arrows indicate the subpallial telencephalon, and arrowheads indicate the subpallial telencephalon, which is negative for emx1, eom or tbr1. Dots indicate the boundary between the telencephalon and ventral diencephalon. Scale bar: 30 µm in A-K,N; 50 µm in L,M.

View larger version (120K): [in a new window]   Fig. 8. Patterning and neurogenesis in the telencephalon after treatment of SU5402. The probe used is indicated at the bottom. All embryos are lateral views of the head region at 26 hpf. Embryos were injected with SU5402 (B,D) or DMSO (A,C) in the head region at tailbud stage (A-D). In E,F, the embryos were soaked in medium containing SU5402 at the five- and 10-somite stages, respectively. (A-D) dlx2 expression in the subpallial telencephalon is abolished by SU5402, while emx1 reaches the edge of the telencephalon after SU5402 treatment. Note the smaller telencephalon is frequently seen in treated embryos (B,D). (E,F) dlx2 expression is greatly reduced when the embryos are treated with SU5402 at the five-somite stage (E), while the expression is relatively normal when treated at the 10-somite stage (F). Arrows indicate the gene expression in the subpallial telencephalon. Dots indicate the boundary between the telencephalon and ventral diencephalon, while arrowheads indicate the ventral region that is negative for emx1. Scale bar: 50 µm.

View larger version (109K): [in a new window]   Fig. 9. Gene expression in the embryos injected with morpholino-modified (MO) antisense oligonucleotides. Injected MO is shown in the upper right and the probe used is indicated at the bottom. Lateral views of the head region at 26 hpf. Embryos were injected with 4mis-MO (A,F,I,K), fgf8-MO (B,G), fgf3-MO (C,H,J,L), fgf8-MO and fgf3-MO (D), and fgf3-MO and fgf3 mRNA (E). (A-E) Wild-type embryos were used. Injection of either fgf8-MO (B) or fgf3-MO (C) reduces nk2.1b expression. However, reduction of nk2.1b is further enhanced when both fgf8-MO and fgf3-MO was co-injected (D). (A'-C') en2 expression after 4mis-MO (A'), fgf8-MO (B') and fgf3-MO (C') injection. en2 expression is reduced in fgf8-MO-injected embryo but remains unchanged in fg3-MO-injected embryo. (E) nk2.1b expression is rescued by co-injection of fgf3-MO and fgf3 RNAs. (F-H) Embryos from ace heterozygous parents were used; fgf3-MO but not fgf8-MO injection enhances the reduction of nk2.1b in some embryos (17/58). (I-L) Injected embryos from ace heterozygous parents were double-stained with en2 and emx1 (I,J) or dlx2 (K,L), and homozygous mutants were identified by a lack of en2 expression in the MHB. An arrowhead in I indicates the subpallial telencephalon, which is negative for emx1. Dots indicate the boundary between the telencephalon and ventral diencephalon. Scale bar: 50 µm. (M) Western blot analyses of translation of injected fgf3-myc. RNA injections were carried out at the one-cell stage. The homogenates were prepared from injected blastula. Anti-Myc antibody detects a major band about 50 kDa which is not present in control injection (GFP RNAs). Relative intensity of the major band is shown at the bottom of each lane. Relative intensity was normalised by a nonspecific minor band that appears in all lanes and insensitive to morpholino injection. (N) Morpholino oligonucleotides used in the present study. Red letters in 4mis-MO indicate mismatch nucleotides to fgf3-MO.