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First published online August 4, 2003
doi: 10.1242/10.1242/dev.00660

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Unique and combinatorial functions of Fgf3 and Fgf8 during zebrafish forebrain development

MRC Centre for Developmental Neurobiology, New Hunt's House, King's College London, Guy's Campus, London SE1 1UL, UK

View larger version (99K): [in a new window]   Fig. 1. Expression of fgf8 (B-F) and fgf3 (G-L) during development of the zebrafish embryo forebrain. Lateral (B-D,F,G,J-L) or dorsal (E,H,I) views with anterior to the left. The eye was removed in F and L to facilitate observation of expression in the brain. (A) Diagram depicting brain subdivsions and structures relevant to this study in a 24 hpf embryo. (B-F) fgf8 expression at tailbud stage (B), during early somitogenesis (C), late somitogenesis (D,E; E, dorsal view focussed on the hypothalamic region) and 30 hpf (F). (G-L) fgf3 expression. (G) onset at 80% epiboly (arrow). (H,I) Dorsal views at 90% epiboly (H) and 2s (I), showing fgf3 transcripts in the forebrain and in presumptive rhombomere 4. Early (J) and late (K) somitogenesis, and 30 hpf (L). ac, anterior commissure; d, diencephalon; dd, dorsal diencephalon; dt, dorsal thalamus; ep, epiphysis; fb, forebrain; h, hypothalamus; hb, hindbrain; hpf, hours postfertilisation; i, isthmus; os, optic stalk; poc, post-optic commisure; pt, pretectum; r, retina; s, somites; t, posterior tuberculum; tc, tectum; tel, telencephalon; tg, tegmentum; vt, ventral thalamus. Asterisk indicates the zona limitans intrathalamica.

View larger version (107K): [in a new window]   Fig. 2. Cell death and division in the forebrain are unaffected in embryos injected with Fgf morpholinos. (A-D) Dorsal views of the anterior neural plate at tailbud stage. Numbers of dividing cells within presumptive forebrain region (indicated by the rectangles) were counted and subjected to Student's t-test analyses. (E-H) Lateral views of whole-mounted 10s embryos, with anterior to the left, showing dividing cells. (I-L) Occasional apoptotic cells (arrowheads) detected dorsally in 10s embryos injected with control morpholino (I), Fgf8mo (J), Fgf3mo (K), or Fgf8 and Fgf3mo together (L). (M-P) Little cell death is detected in anterior neural tissue in 24 hpf embryos injected with control mo (M), Fgf8mo (N), Fgf3mo (O), or Fgf8 and Fgf3mo together (P).

View larger version (111K): [in a new window]   Fig. 3. Fgf3 and Fgf8 regulate telencephalic gene expression. Dorsal (A-D,M-P) or lateral (E-L,Q-T) views with anterior to the left. (A-D) emx1 expression at the anterior margin of the forebrain primordium at tailbud stage. (A) emx1 transcripts are detected in the presumptive anterior forebrain in control embryos but are reduced in embryos injected with Fgf8mo (B), and are undetectable in embryos injected with either Fgf3mo (C), or Fgf8mo and Fgf3mo (D). (E-H) emx1 expression at 24 hpf. (E) emxl expression is limited to the pallial telencephalon in control embryos. Arrow indicates expression boundary. emx1 transcripts are detected in both pallial and subpallial (arrow in F) telencephalon in embryos injected with Fgf8mo (F), Fgf3mo (G), or Fgf8mo and Fgf3mo (H). (I-L) tbr1 (red) and dlx2 (blue) expression at 24 hpf. (I) A control embryo: dlx2 transcripts are found in the ventral thalamus and subpallial telencephalon, and extend into the posterior region of the pallial telencephalon. tbr1 transcripts occupy the pallial telencephalon. (J) tbr1 expression extends into the anterior subpallial telencephalon, partially overlapping dlx2 expression, in embryos injected with Fgf8mo (compare arrows in I and J). tbr1 and dlx2 expression is reduced following injection with either Fgf3mo (K), or Fgf8mo and Fgf3mo (L). (M-P) Telencephalic eom expression at 24 hpf. (M) eom transcripts are detected in the telencephalon in control embryos. eom transcripts in the telencephalon are reduced or undetectable in embryos injected with Fgf8mo (N), Fgf3mo (O), or Fgf8mo and Fgf3mo (P). (Q-T) 28 hpf embryos, bisected along the AP axis, and mounted to show the internal brain surface following detection of fgf3 (blue) and dlx2 (red) transcripts. (Q) Control embryos express dlx2 in the telencephalon and ventral thalamus, and fgf3 in hypothalamus (and isthmus). (R) fgf3 transcripts (arrow) are upregulated in the telencephalon in the absence of Fgf8. Loss of Fgf3 (S), or both Fgf3 and Fgf8 (T), results in the absence of dlx2 expression. d, diencephalon; tel, telencephalon.

View larger version (124K): [in a new window]   Fig. 4. Diencephalic gene expression is altered in the absence of Fgf8 and Fgf3. Lateral views of embryos bisected along the AP axis and with eyes removed (A-D,E-P), frontal (A'-D') or dorsal (Q-T) views. (A-D) shh expression at 30 hpf. Embryos mounted to show internal surface of the brain. shh is expressed in the ventral midline of the brain, anterior hypothalamus and ZLI in control embryos (A). shh expression is unaffected in embryos injected with Fgf8mo (B), is slightly reduced after injection of Fgf3mo (C), but is reduced throughout the brain, especially in the hypothalamus (arrow) and ZLI, when both Fgf3mo and Fgf8mo are injected (D). (A'-D') As A-D, but frontal views with the ZLI in focus. Arrows indicated the dorsal extent of shh expression. shh expression in the ZLI extends into the dorsal diencephalon in control (A') and Fgf8mo-injected (B') embryos. Dorsal shh expression in the ZLI is severely reduced in embryos injected with either Fgf3mo (C'), or both Fgf8mo and Fgf3mo (D'). (E-H) nk2.1b expression at 30 hpf. nk2.1b is expressed in the subpallial telencephalon, anterior ventral thalamus and hypothalamus in control embryos (E). Expression is unaffected in embryos injected with Fgf8mo (F). Ventral thalamic expression is reduced in embryos injected with Fgf3mo (arrowhead; G), and absent (arrowhead) in embryos injected with both Fgf8mo and Fgf3mo (H). (IL) twhh expression at 30 hpf. twhh is expressed in the anterior ventral thalamus (arrowhead) in control embryos and those injected with either Fgf8mo (J) or Fgf3mo (K), but not in those co-injected with Fgf8mo and Fgf3mo (arrowhead; L). (M-P) pax6.1 expression at 28 hpf. In control embryos pax6.1 is expressed in the telencephalon, posterior ventral thalamus and dorsal thalamus (M). Expression is unaltered following injection with Fgf8mo (N). pax6.1 expression domains are smaller and merged following injection with either Fgf3mo (O), or both Fgf8mo and Fgf3mo (P). (Q-T) pax2.1 expression in developing optic stalks (and isthmic region) at 18 hpf. Two well-separated optic stalks are present in control embryos (Q). A slight reduction in pax2.1 expression is detected in embryos injected with Fgf8mo (R), whereas expression is relatively normal in embryos injected with Fgf3mo (S). The pax2.1 expression domains are fused (arrowhead) in embryos injected with both Fgf8mo and Fgf3mo (T). dt, dorsal thalamus; h, hypothalamus; os, optic stalk; vt, ventral thalamus; zli, zona limitans intrathalamica.

View larger version (88K): [in a new window]   Fig. 5. Lack of Fgf8 or Fgf3 results in aberrant axon trajectory and failure of commissure formation in the forebrain. (A-C) Diagrams depicting neuronal clusters (light brown) and axon tracts (dark brown) during zebrafish forebrain development [from information and diagrams in Wilson et al. and Ross et al. (Wilson et al., 1990; Ross et al., 1992)]. Lateral (D,G,J,M) or frontal (E,F,H,I,K,L,N,O) views focussed on the anterior and postoptic commissures following immunocytochemistry at 34 hpf with acetylated ß-tubulin antibodies. (D-F) Control embryos showing anterior and postoptic commissures. (G-I) Anterior commissure formation is defective in embryos injected with Fgf8mo. In some cases there is a complete failure of anterior commissure formation (H), and in other cases axons with abnormal trajectories (arrowhead) extend towards the midline (I). (J-L) Formation of both commissures is defective in embryos injected with Fgf3mo. In some cases, axons extend across the midline (arrowhead), partially forming commissures that are positioned abnormally close together (L). (M-O) There is a pronounced failure of commissure formation in the absence of both Fgf8 and Fgf3 (N). In severe cases, no axons enter the midline (O). ac, anterior commissure; drc, dorsorostral cluster; ep, epiphysis; npc, nuclei of the posterior commissure; poc, post-optic commissure; vcc, ventrocaudal cluster; vrc, ventrorostral cluster.

View larger version (92K): [in a new window]   Fig. 6. Expression of neuronal markers is altered in embryos lacking Fgf8 and Fgf3. Lateral views of whole-mounted embryos with anterior to the left. (A-D) zash1a expression at 18 hpf. In control embryos, zash1a expression is detected in the dorsorostral cluster in the telencephalon and in the ventrorostral cluster in the diencephalon, as well as in the presumptive epiphysis (A). Injection of Fgf8mo results in a slight reduction of zash1a expression in neurons of the ventral diencephalon. By contrast, expression within the presumptive epiphysis is expanded (B). zash1a expression is lost from dorsorostral and ventrorostral clusters, but is expanded in the presumptive epiphysis in embryos injected with either Fgf3mo alone (C), or with Fgf8mo and Fgf3mo (D). (E-H) isl1 expression at 30 hpf. In control embryos, isl1 is expressed in a subset of neurons within dorsorostral and ventrorostral clusters (E). There is very little effect on isl1 expression following fgf8mo injection (F). isl1 expression is reduced in the dorsorostral and ventrorostral clusters in embryos injected with Fgf3mo (G), and reduced in the dorsorostral cluster and virtually absent in the ventrorostral cluster in embryos injected with both Fgf8mo and Fgf3mo (H). (I-L) lim1 expression at 30 hpf. lim1 is expressed in a subset of neurons within the dorsorostral and ventrorostral clusters (I), and is unaffected following Fgf8mo injection (J). Injection of Fgf3mo results in reduced lim1 expression in many neuronal populations of the brain, including dorsorostral and ventrorostral clusters (K), whereas lim1 expression is lacking in most neuronal populations, including the dorsorostral and ventrorostral clusters, following injection with both Fgf8mo and Fgf3mo (L). d, diencephalon; drc, dorsorostral cluster; ep, presumptive epiphysis; tel, telencephalon; vrc, ventrorostral cluster.

View larger version (109K): [in a new window]   Fig. 7. FGF signalling is required for forebrain patterning during gastrulation and somitogenesis stages. Dorsal (A,D,E,H) or lateral (B,C,F,G,IT) views of whole-mounted control embryos (A-D,I,M,Q), or embryos treated with SU5402 (E-H,J-L,N-P,R-T) during the developmental periods indicated. (A-H) Sister embryos taken from each batch immediately following treatment with either DMSO (A-D) or SU5402 (E-H), and analysed for expression of erm, an FGF-responsive gene, to test the effectiveness of inhibition. Forebrain erm expression is efficiently blocked by SU5402 following treatment from 80% to tailbud stage (A,E), tailbud to 8s (B,F) and 13s to 18s (C,D,G,H). After treatment, remaining embryos were allowed to develop to 24 hpf (I-L) or 30 hpf (M-T). Control embryos express eom in the pallial telencephalon (I). SU5402 treatment from 50% epiboly to tailbud stage eliminates eom expression (J), whereas treatment from 80% epiboly to tailbud results in expansion of eom expression into the subpallial telencephalon (K; compare arrowheads in I and K). SU5402 treatment during somite stages does not alter the eom expression boundary (arrowhead; L). nk2.1b expression in the telencephalon and diencephalon at 30 hpf in a control emrbyo (M). SU5402 treatment from 80% epiboly to tailbud stage (N), or from tailbud to 8s (O), completely blocks expression of nk2.1b in the telencephalon and reduces expression in the ventral thalamus, whereas hypothalamic expression is less affected. Treatment from 13s to 18s results in reduced nk2.1b expression in the telencephalon and ventral thalamus but does not affect hypothalamic expression (P). isl1 expression in the forebrain at 30 hpf in a control embryo (Q). Although isl1 expression in the dorsal diencephalon is expanded, all other sites of expression are reduced or eliminated following SU5402 treatment from 80% epiboly to tailbud (R), or tailbud to 8s (S). There is little effect on isl1 expression following treatment from 13s to 18s (T). fb, forebrain; hb, hindbrain.

View larger version (34K): [in a new window]   Fig. 8. Diagram depicting sites of fgf3 and fgf8 expression relative to their proposed roles in forebrain patterning in the zebrafish embryo. (A) Inhibition of Fgf signalling by SU5402 treatment prior to the onset of fgf8 expression in the presumptive forebrain at tailbud stage results in forebrain patterning defects that correlate with those observed following injection of Fgf3mo. Based on these and other results it is proposed that fgf3 expression during gastrulation is required for subsequent correct regional specification of the pallial telencephalon (tbr1), the subpallial telencephalon (dlx2), the ventral thalamus (dlx2) and the ZLI (shh), and for gene expression in precursor and differentiated neurons throughout the forebrain (zash1a, lim1 and isl1). (B) fgf3 and fgf8 expression overlaps in the dorsal telencephalon during early somitogenesis, and these Fgfs are proposed to have combined roles in patterning the subpallial telencephalon (restriction of emx1 expression) and the anterior ventral thalamus (positive regulation of twhh and negative regulation of pax2.1 expression). In addition, Fgf8 is proposed to function independently of Fgf3 in some aspects of telencephalic patterning (e.g. repression of tbr1 and fgf3 expression). h, hypothalamus; tel, telencephalon; vt, ventral thalamus. Asterisks indicate the ZLI. In the diagrams, the early domain of Fgf3 expression is indicated in turquoise in A; the domain of Fgf3 and Fgf8 co-expression is indicated in dark blue in B. In both figures, forebrain regions with defects are indicated in pink and yolk is depicted in yellow.