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First published online 15 April 2009
doi: 10.1242/dev.031161

Development 136, 1675-1685 (2009)
Published by The Company of Biologists 2009
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Short- and long-range functions of Goosecoid in zebrafish axis formation are independent of Chordin, Noggin 1 and Follistatin-like 1b

Monica Dixon Fox and Ashley E. E. Bruce*

Department of Cell and Systems Biology, University of Toronto, 25 Harbord Street, Toronto, ON, M5S 3G5, Canada.


Figure 1
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  		Fig. 1. Ventral gsc overexpression induces complete secondary axes. (A-I) Live zebrafish embryo injected with gfp (A-C) or gsc (D-I) RNA. (A-F,H,I) DIC and fluorescence merge; (G) DIC. (A,D) Shield (arrowhead), animal view. (B,F) Bud; arrowheads indicate notochord, asterisk indicates prechordal plate. (C) 1 dpf, side view. (E) Bud; side view, secondary prechordal plate (asterisk). (G-K) 1 dpf, dorsal view. (J,K) dlx2a and shha (red). (J) gfp RNA injected. (K) gsc RNA injected showing duplicated dlx2a expression (brackets): staining differs from the control because the embryo had a ventral third eye. (L) Schematic of injection procedure, clones in green; one clone was generated per embryo. dien, diencephalon; e, eye; fp, floor plate; hg, hatching gland; myo, myotomes; noto, notochord; tele, telencephalon.

 

Figure 2
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  		Fig. 2. Dorsal gsc overexpression induces excess dorsal tissue. (A-H) Merged images of live zebrafish embryo injected with gfp (A-D) or gsc (E-H) RNA. (A,E) Shield (arrowhead). (B,F) Bud, arrowheads indicate notochord. (C,D,G,H) 1 dpf. (I) Notochord cell counts with standard deviations, five embryos per treatment. (J) Bud, anterior to the top. Anti-Ntl staining is brown, injected RNA is listed in lower right. hypo, hypochord; peri, periderm.

 

Figure 3
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  		Fig. 3. gsc recruits unlabeled cells to secondary axes. (A-K) Zebrafish embryos injected with gsc RNA. (A-C) Lateral views, (A) bud; (B,C) 1 dpf. (D,E) Animal views; (F-K) dorsal views, 1 dpf. (A,B,F,I) DIC; (G,J) fluorescence; (C,H,K) merge. (A) Secondary notochord with somites (arrowheads) at bud. (B,C) Myotomes (arrowheads) with GFP-labeled notochord (dashed lines). (D,E) Shield stage gsc RNA-injected embryos stained for membrane-GFP (brown) and chd (purple), with ectopic chd (arrowheads). (F-H) Secondary neural tube with GFP-labeled (arrow) and unlabeled cells; arrowhead marks anterior GFP limit. (I-K) Low gsc RNA dose ventral clone produces partial secondary axis. (K) Arrows indicate unlabeled cells in neural tissue (right inset) and in myotomes (left inset). (L-O) Shield (arrowheads). (L,N) gfp RNA injected; (M,N) gsc RNA injected, with wnt8 (L,M) and chd (N,O) in situ probe. nt, neural tissue.

 

Figure 4
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  		Fig. 4. gsc induces secondary axes in the absence of Chd. (A-G) Live chdtt250 embryos injected ventrally with 24 pg (A-D) or 48 pg (E-G) gsc RNA. (A,C,E) Shield, dorsal to the right. (B,D) 1 dpf, anterior to the top. (F,G) 1 dpf, anterior to the left. (B) Secondary axis with head (2/7, 29%). (D) Secondary axis without notochord (5/7, 71%). (F,G) Two notochords (arrows, 9/12, 75%). nt, neural tissue.

 

Figure 5
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  		Fig. 5. chd induces partial secondary axes. (A-H) Live zebrafish embryo injected with chd RNA. Shield (A), 1 dpf (B-H). (B) Partial secondary axis (arrow, 13/21, 62%). GFP-labeled neural tissue (C,D) (13/13, 100%), myotomes (E,F) (9/13, 69%) and ectopic otic vesicle (G,H) (6/13, 46%). Some injected embryos had beating cardiac tissue (4/13, 31%). (I-L) Live chdtt250 embryos injected ventrally with 48 pg gsc and nog1- and fstl1b-MOs. (I,J) Partial GFP-labeled secondary axis with neural (arrow) and somitic (arrowheads) tissue. (K,L) Secondary axis containing GFP-labeled notochord and neural tissue (3/5, 60%). For an example of an uninjected chd mutant embryo see Fig. 7D. (M,N) Wild-type embryo injected ventrally with chd, fstl1b and nog1 mRNAs. Arrowhead marks neural tissue. ov, otic vesicle.

 

Figure 6
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  		Fig. 6. gsc elicits distinct short- and long-range signaling activities. Injected construct is listed in lower left. (A-D,H,I) Live embryos at 1 dpf. (A,B) Embryo with two notochords (arrows). (C,D) Embryo with partial secondary axis, with neural (arrow) and somitic (arrowheads) tissue. (E-G) Shield stage embryos stained for chd. (E) Control; (F,G) gsc-VP2-injected embryos. Arrowheads indicate ectopic chd. (H,I) Dorsalized embryo with partial secondary axis.

 

Figure 7
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  		Fig. 7. gsc and chd morpholinos affect DV patterning. Injected construct or genotype is listed in lower right. (A-H) Live embryos, 1 dpf. (I-P) Tails, 1 dpf; stained with anti-Ntl antibody, anterior to the left. Arrowheads indicate thin notochords, arrows indicate truncated notochords. (Q-S) 60% epiboly embryos stained for wnt8; arrowhead marks dorsal. (Q) Control. (R) gsc-MO-injected embryo with ectopic wnt8 expression dorsally (11/81, 14%). (S) Ectopic staining in embryo injected with gsc-MO and a low concentration of chd-MO (46/106, 43%).

 

Figure 8
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  		Fig. 8. Model of Gsc function. Gsc directly or indirectly inhibits wnt8 and bmp transcription at high doses, whereas low Gsc doses predominantly inhibit bmp transcription. Gsc indirectly activates expression of chd, the product of which acts together with Nog1 and Fstl1b to inhibit the function of BMP proteins. See text for details.

 

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© The Company of Biologists Ltd 2009