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First published online August 18, 2003
doi: 10.1242/10.1242/dev.00671

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Zebrafish fgf24 functions with fgf8 to promote posterior mesodermal development

Bruce W. Draper1,*,{dagger}, David W. Stock2 and Charles B. Kimmel1

1 Institute of Neuroscience, University of Oregon, Eugene, OR 97403, USA
2 Department of Environmental, Population and Organismic Biology, University of Colorado, Boulder, CO 80309, USA



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  		Fig. 3. fgf24 splice-blocking morpholino oligonucleotides knock down functional fgf24 mRNA. (A) Genomic structure of the fgf24 gene. Translation initiation and termination codons are indicated. Exons are shown as boxes and intron sizes are not to scale. Splice donor site targeted by the fgf24-E3I3 morpholino oligo is shown. The colored line indicates the major splice variant observed following fgf24-E3I3 injection. Primers used for RT-PCR analysis in B are shown as arrows. (B) In addition to wild type (upper band in 5 ng lane), RT-PCR analysis detects two splice variants (arrows). (C) cDNA sequence comparison reveals that the major splice variant (bottom band in B) caused by fgf24-E3I3 results from aberrant splicing to an upstream cryptic slice donor site (underlined in wild-type sequence) that is present in exon 3. The sequences derived from exon 4 in the aberrantly spliced form are italicized. Note that use of the cryptic splice site results in a coding frame shift. (D) Position of the fgf24 antisense RNA probe (bold horizontal line) that was used for RNase protection assays in E is indicated relative to the wild-type fgf24 mRNA splice junctions (vertical lines). (E) The amount of wild-type fgf24 mRNA in MO injected and control embryos was determined by RNase protection, using odc levels as an internal control. The amount of MO injected per embryo is indicated above lane. (F) Relative levels of wild-type fgf24 mRNA in E was determined after amounts were normalized using the odc control.

 


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  		Fig. 1. fgf24 is a member of the fgf8/17/18 subfamily and is expressed during gastrulation. (A) Sequence comparison of the predicted amino acid sequences of zebrafish (Dr) Fgf24, Fgf18, Fgf8 and Fgf17, with human (Hs) FGF18, FGF8 and FGF17. Periods indicate identical residues; dashes indicate introduced gaps; arrows indicate exon boundaries. Only partial sequence for zebrafish Fgf18 is shown as the 5' end of the gene has not been identified. (B) Phylogenetic tree comparing the relatedness of zebrafish Fgf18 and Fgf24 with other members of the Fgf8 subfamily. Zebrafish Fgf10 is distantly related to the Fgf8 subfamily, and was included as an outgroup. Numbers indicate bootstrap support for the nodes. (C) fgf18 and fgf24 map to LG 14. Map position of fgf18 and fgf24, as determined by screening the T51 radiation hybrid panel is shown relative to representative zmarkers (left) and ESTs (right, listed by GenBank Accession Numbers). The entire linkage group is not shown. Zebrafish genes that are closely linked to zebrafish fgf18 have human homologs that are closely linked to human FGF18. (D) Temporal expression profiles for zebrafish fgf18 and fgf24 in comparison with fgf8 as determined by RT-PCR. The zebrafish ornithinedecarboxylase (odc) gene was used as an internal control (see Draper et al., 2001Go). cR, centiRay; MWM, molecular weight marker; hpf, hours post fertilization.

 


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  		Fig. 2. fgf24 is expressed in mesodermal precursors during gastrulation in a pattern that overlaps with the expression of fgf8, ntl and spt. Dorsal (A,C,E) and vegetal (B,D,F; dorsal is upwards) views of embryos showing expression of fgf24 at 4 hpf (A,B), 6 hpf (C,D) and 9 hpf (E,F). In addition to the germ ring, fgf24 has weak expression in the developing neural ectoderm (arrow in E), as determined by section analysis (not shown). (G) In situ hybridization and immunohistochemistry show the relationship between the expression of fgf24 (purple) and Ntl (brown) in one-somite-stage embryos. Asterisk indicates the tail bud. The expression pattern of fgf24 (H,L) is compared with that of fgf8 (I,M), spt (J,N) and ntl (K,O) in mid-gastrulation stage embryos (8.5 hpf) by whole-mount in situ hybridization (H-K; dorsal views) and in parasagittal sections (L-O). Arrowheads in H-K indicate approximate position of section though germ ring, and approximate division between the epiblast and hypoblast cell layers in L-O is indicated with a broken line. (L) fgf24 expression is higher in the hypoblast layer (arrow) relative to the epiblast, similar to the localization of spt (N). By contrast, fgf8 expression is highest in the epiblast (M) similar to the localization of ntl (O). In addition to the germ ring staining (arrowheads in H-O), fgf8 is also expressed in the developing hindbrain (arrow in I,M), spt in presomitic mesoderm (arrow in J,N) and ntl in the developing notochord (arrow in K). Scale bars: in A, 100 µm for A-K; in O, 50 µm for L-O.

 


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  		Fig. 4. Functional analysis reveals that fgf8 and fgf24 are together required for posterior mesodermal development. In situ hybridization and immunohistochemistry in 10-somite stage wild-type (A), fgf24MO embryos (B), fgf8- (C) and fgf8-;fgf24MO embryos (D). In A-D, pax2.1, krx20 and myod are stained purple, and Ntl protein is stained brown. At this stage in wild-type embryos, pax2.1 is expressed in the mid-hindbrain boundary (MHB), the otic placode and precursors of the pronephric ducts (black asterisks), krx20 in rhombomeres 3 and 5 (white asterisks), myod in adaxial cells (arrowhead) and a subset of cells in the forming somites (arrow), and Ntl protein in the developing notochord. At this stage, fgf24MO embryos (B) are indistinguishable from wild type, while fgf8 mutants (C) have reduced expression of pax2.1 in the MHB, and a reduced number of cells expressing myod in the forming somites. By contrast, fgf8-;fgf24MO embryos (D) have significantly reduced numbers of myod- (arrow), pax2.1- (asterisks) and Ntl-expressing cells relative to wild-type, fgf24MO and fgf8-;fgf24MO embryos. (E-H) Live wild-type and mutant embryos at 24 hpf. fgf24MO embryos (F) are morphologically indistinguishable from wild-type embryos (E), while fgf8- embryos (G) have a slightly shorter tail and a prominent MHB defect (arrowhead). fgf8-;fgf24MO embryos (H) have MHB defect (arrowhead), and produce significantly less posterior tissue than either fgf8 mutant or fgf24MO embryos. Scale bars: in A, 50 µm for A-D; in E, 100 µm for E-H.

 


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  		Fig. 5. Fgfs and T-box genes interact during posterior mesoderm development. Expression of ntl (purple) and Spt (brown) in 10-somite stage wild-type (A), fgf8 mutant (B) and fgf8-;fgf24MO embryos (C) reveals that fgf8-;fgf24MO embryos no longer have mesodermal precursors that in wild-type (A) and fgf8 mutants (B) are located in the tail bud (white asterisks) and presomitic mesoderm (arrows). In addition, analysis of these markers reveals that, at this stage, the tail buds of fgf8 mutant embryos (B) contain significantly less presomitic mesoderm precursors (Spt-expressing cells) in comparison with wild-type embryos (A; see also supplemental Fig. S1 at http://dev.biologists.org/supplemental/), and in the posterior notochord have a gap in the ntl expression domain (arrowhead). Dorsal (upper) and vegetal (lower) views showing expression of ntl (D,E), spt (F,G), fgf8 (H-K) and fgf24 (L-O) in mid-gastrula-stage (75-80% epiboly; 8.5 hpf) wild-type and mutant embryos (asterisks and arrows indicate dorsal and ventral tissues, respectively). fgf8-;fgf24MO embryos (E,G) have reduced expression of ntl and spt in mesodermal precursors relative to wild-type embryos (D,F). Expression of fgf8 in dorsal mesoderm is reduced in ntl (I), spt (J) and spt;ntl (K) mutant embryos relative to wild-type embryos (H). fgf24 expression is reduced dorsally in ntl embryos (M) ventrally in spt embryos (N) and dorsally and ventrally, but not laterally in spt;ntl embryos (O), relative to wild-type embryos (L). Scale bars: in A, 50 µm for AC; in D, 100 µm for D-O.

 


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  		Fig. 6. Double mutant analysis reveals that fgf8 genetically interacts with ntl and spt. Markers used for in situ hybridization and immunohistochemistry in A-E and K-N, as well as identifiers (e.g. arrows) are as described for Fig. 4A. Representative pictures of stained 12- to 13-somite-stage embryos (A-E,K-N) and live 24 hpf embryos (F-J,O-R) are shown. Relative to wild-type embryos (A), neither fgf8 (B) nor ntl (D) mutant embryos have severe deficiencies in the production of myod-expressing paraxial mesoderm (arrows), whereas fgf8;ntl double mutants (E) produce very little paraxial mesoderm and have significantly shorter tails at 24 hpf (J) in comparison with fgf8 (G) or ntl (I) single mutants. By contrast, fgf8;ntl double mutants appear to produce relatively normal amounts of pronephric tissue (E; asterisk). In addition to the single and double mutant phenotypes observed, fgf8-;ntl+/- embryos (C) produce less axial (Ntl expressing) and paraxial (myod expressing) mesoderm than do fgf8-;ntl+/+ embryos (B), and at 24 hpf (H) have tail lengths that are intermediate between embryos single mutant for either fgf8 (G) or ntl (I). Although neither fgf8 (L) nor spt (M) mutant embryos have severe deficiencies in the production of axial mesoderm, spt;fgf8 double mutant embryos produce a truncated notochord (N) and have shorter tails at 24 hpf (R) than either fgf8 (P) or spt (Q) single mutants. By contrast, spt;fgf8 double mutants appear to produce relatively normal amounts of pronephric tissue (N; asterisk).Wild-type sibling embryos (K,O) are shown for comparison. Scale bars: in A, 50 µm for A-E,K-N; in F, 100 µm for F-J,O-R.

 


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  		Fig. 7. fgf24 expression during later embryonic and larval development. In all panels, anterior is towards the left unless specified, and fgf24 expression is visualized in purple. (A-D) 12 hpf (six-somite stage), dorsal views. (A) fgf24 is expressed in the nasal placode (asterisk), otic placode (arrow), lateral mesoderm (arrowhead) and tail bud mesenchyme surrounding Kupffer's vesicle. Expression of fgf24 in the otic placode was confirmed by co-labeling embryos with either krx20 (red), which labels rhombomere 3 (r3) and r5 (B) or pax2.1 (red), which labels the otic placode and the mid-hindbrain boundary (C). (D) fgf24 expression in lateral mesoderm (arrowhead) is in cells that lie adjacent and medial to those expressing pax2.1 (red). (E,F) 18 hpf. (E) fgf24 is expressed in nasal ectoderm and in a discrete domain of the retina (arrow, dorsal view). (F) Lateral view of fgf24-expressing cells in the posterior gut (arrow), in tail bud mesenchyme and in the caudal fin primordium (arrowhead). (G) 20 hpf, dorsal view. fgf24expression in early pectoral fin bud mesenchyme (arrow). (H-J) 24 hpf. (H) fgf24 expression persists in the posterior gut (arrow) and caudal fin primordium (arrowhead), but is no longer detected in the tail bud (lateral view, yolk extension removed). (I) fgf24 is expressed in the pharyngeal endoderm (arrowheads) and in the pectoral fin bud mesenchyme (arrow), and in a posterior domain of the otic epithelium (not in focus). Inset in I shows sagittal section through the otic vesicle (outlined), showing more clearly the expression of fgf24 in the posterior otic epithelium (arrow) and pharyngeal endoderm (arrowheads). (J) Transverse section (dorsal upwards) showing fgf24 expression in fin bud mesenchyme (arrow) and gut (arrowhead). (K-O) 52 hpf. (K) At this stage, fgf24 is no longer expressed in pectoral fin mesenchyme, but instead is strongly expressed in the apical ectodermal ridge. (L) Lateral view of head showing fgf24 expression in the first and second pharyngeal pouches (pp1, ppl2), and the posterior ectodermal margin (pem, arrow) of the second pharyngeal arch. A ventral view (M) shows fgf24 expression in all pharyngeal pouches (pp1 and pp2-6, small arrows), and the olfactory bulb. Additionally, fgf24 is expressed in tooth germs, which develop on only the most posterior (seventh) pharyngeal arch. (N) A close-up ventral view shows fgf24 expression in bilateral domains (arrowheads) adjacent to the lateral edges of the mouth. (O) fgf24 is expressed in the olfactory organ and the olfactory bulb (dorsal view, anterior is upwards). dis, distal; e, eye; kv, Kupffer's vesicle; mhb, mid-hindbrain boundary; mo, mouth; nec, nasal ectoderm; nc, notochord; nt, neural tube; ob, olfactory bulb; olf, olfactory organ; op, otic placode; ov, otic vesicle; pem, posterior ectodermal margin; psm, presomitic mesoderm; pro, proximal; ret, retina; tb, tail bud; tg, tooth germ; ye, yolk extension. Scale bars: 100 µm in A,G; 50 µm in B-F,H-O.

 


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  		Fig. 8. fgf24 is required for pectoral fin development. (A,B) Dorsal views of shh expression in 24 hpf wild-type (A) and fgf24-E3I3 morpholino-injected (B) embryos. shh is detected in the pectoral fin buds (arrows) and floor plate (arrowhead) of wild-type embryos (A), but only in the floor plate of fgf24-E3I3 morpholino-injected embryos (B). Skeletal preparations of one-month-old wild-type (C,D) and fgf24-E3I3 morpholino-injected fish (E,F) shown in lateral (C,E) and ventral (D,F) views. Bone is stained red and cartilage blue. In wild-type fish, exoskeletal (cleithrum and postcleithrum) and endoskeletal (scapula, distal radials and lepidotrichs) components of the pectoral fin are visible (C). By contrast, only exoskeletal components can be identified in fgf24-E3I3 injected fish (E). cl, cleithrum; dr, distal radials; lep, lepidotrichs; pcl, postcleithrum; sc, scapula. Scale bar: in A, 50 µm for A,B.

 

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© The Company of Biologists Ltd 2003