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Ectopic Wnt signal determines the eyeless phenotype of zebrafish masterblind mutant

Sandra van de Water1, Marc van de Wetering2, Jos Joore1,{ddagger}, John Esseling1,§, Robert Bink, Hans Clevers2 and Danica Zivkovic1,*

1 Hubrecht Laboratory, Netherlands Institute for Developmental Biology, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands
2 Department of Immunology, University Hospital, PO Box 85500, 3508 GA Utrecht, The Netherlands
{ddagger} Present address: Westburg b.v., PO Box 214, 3830 AE Leusden, The Netherlands
§ Present address: Laboratory of Plant Cell Biology, Arboretumlaan 4, 6703 BD Wageningen, The Netherlands



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  		Fig. 1. Marker gene characterisation of "late" lithium phenotypes by in situ hybridisation of (A-C) tailbud stage and (D-H) 24 hpf (wild type, left) and lithium- treated (Li+, right) embryos (A,C,D,F,G,H, lateral view; B,E dorsal view; anterior to the left). (A,B) Anteriorly shifted wnt1 covers the anterior neural plate (Li+). (C) Shh is diminished in the anterior midline (Li+). (D,F) The mild phenotype is characterised by the loss of brain anterior to D2/3 diencephalic boundary as revealed by (D) pax6 and (F) otx2. (E,G) The severe phenotype is characterised by the loss of brain anterior to the MHB as revealed by (E) pax6 and (G) wnt1. MHB was absent in the most severe cases (*). (H) The hindbrain was unaffected by the treatment as shown by krox20 expression.

 


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  		Fig. 2. Overexpression of (A) dominant negative dn Xgsk3ß in wild-type embryos induced small eyes or loss of eyes as evaluated at 2 dpf. This phenotype was not induced either by overexpression of (B) full-length fl Xgsk3ß or by (C) frame shift fs Xgsk3ß. Numbers of embryos per treatment are indicated (top of graph) pooled from (A) 4, (B) 7 and (C) 4 independent experiments. (D-F) Phenotypes at 3 dpf or (G) at 24 hpf (lateral view, rostral to the left, dorsal up) of (D) wildtype, and upon overexpression of (E) dn Xgsk3ß that induced small eyes (left) or loss of eyes and forebrain (right), or (F,G) full-length fl Xgsk3ß that induced deletion of the ventral forebrain and partial eye fusion. (H-J) emx1 expression, at approx. 30 hpf, in (H) wild type is restricted to the telencephalon (arrow), but overexpression of dn Xgsk3ß (I,J) resulted in (I) eye reduction accompanied by an expanded emx1 domain in a few cases, and (J) in loss of eyes accompanied by normal emx1 expression in the majority of cases. (K,L) fgf8 expression in a squash preparation (dorsal view) at approx. 30 hpf in (K) wild type, and (L) an embryo overexpressing dn Xgsk3ß, showing small eyes accompanied by loss of optic stalk labelling (right panel). 1, MHB; 2, optic stalks; 4, dorsal diencephalon; 5, retina. mRNAs (320 pg/1 nl) were injected into one-cell stage embryos.

 


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  		Fig. 3. Sensitivity to lithium of mbl heterozygotes as compared to wildtypes. Eye phenotype frequencies in 2 dpf zebrafish embryos treated at 4 hpf with lithium (0.3 M for 5-10 minutes). (A) Heterozygous mbl+/- x HLwt outcrosses. A 6-minute treatment resulted in 50% eyeless and 50% wild-type embryos. (B) Wild-type crosses where a 6-minute treatment resulted in a less than 2% phenotype. Numbers of embryos pooled from 2 independent experiments are indicated at the top of the graph. Wt, wild type; se, small eyes; ne, no eyes; de, dead.

 


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  		Fig. 4. (A,B) Frequencies of phenotypes at 2 dpf of embryos from in-crosses of heterozygous mbl fish upon overexpression of (A) dominant negative dn Xgsk3ß that induced loss of eyes in approx. 80% of embryos, and (B) full-length fl Xgsk3ß, which rescued eyes of mbl-/- embryos. Numbers of embryos pooled from (A) 4 and (B) 5 independent experiments are indicated at the top of the graph; n.i., non-injected. (C-E) Eye phenotypes (lateral view, rostral to the left, dorsal up) at 3 dpf of (C) wild-type embryo from an eyeless clutch, (D) mbl embryo from an eyeless clutch, (E) overexpression of full-length fl Xgsk3ß rescued eyes in mbl. (F-H) flh expression at the tailbud stage. (G) in mbl, anterolateral borders of the neural plate (anterodorsal view) ectopically express flh compared to (F) wild type (arrowheads). (H) Overexpression of full-length fl Xgsk3ß reduced ectopic expression of flh (arrowhead, ectopic flh in mbl). mRNA (320 pg/1nl) was injected into one-cell stage embryos.

 


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  		Fig. 5. emx1 expression at approx. 30 hpf is restricted to the telencephalon in wild-type embryos (A). In mbl embryos with small eyes (B) and (C) eyeless, this expression is anteroventrally expanded. (D) Overexpression of full-length fl Xgsk3ß in mbl embryos rescued eyes but did not normalize the mutant emx1 domain. (E-H) fgf8 expression at approx. 30 hpf in (E) wild-type embryos and (F) eyeless mbl embryos that lost the expression in retinae and optic stalks and still showed weak expression in facial ectoderm (FEC) and dorsal diencephalon and a normal expression at the MHB. (G,H) Overexpression of full-length fl Xgsk3ß in mbl embryos rescued expression in one optic stalk and one eye (G) and (H) both eyes and fully rescued wild-type fgf8 expression. (A-H, lateral view, rostral to the left; G,H lower panels in a frontal view.) 1, MHB; 2, optic stalks; 3, FEC; 4, dorsal diencephalon; 5, retina. mRNAs (320 pg/1nl) were injected in 1-cell embryos.

 


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  		Fig. 6. (A) Position of axin1/mbl is at 62.8 cM on zebrafish LG 3 (59.7-83.2 cM) with respect to the position of marker z24851 that was used for genotyping. The LG 3 map was integrated from information available at http:/wwwmap.tuebingen.mpg.de and http:/zebrafish.mgh.harvard.edu/cgi_bin/ssr_map/view_lg.cgi. Left: scale in cM; right: scale in cR. (B) Point mutation A to T transversion in axin1 of mbl. (C) Wild-type uninjected embryo at ca 30 hpf and (D) embryo injected with 150 pg of mutant L399Q axin1 mRNA, which induced loss of telencephalon and laterally positioned eyes in 25% of 30 hpf embryos. (E,F) Whole-mount in situ hybridisation with axin1 riboprobe in 24 hpf embryos showing strong expression in the midbrain, ventral midhindbrain and ventral hindbrain, and a low signal in telencephalon, dorsal midbrain and midhindbrain boundary. The abnormal brain of mbl embryos (F) still shows expression of axin1 mRNA (arrows) comparable to that of the wild-type embryo (E).

 




© The Company of Biologists Ltd 2001