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The homeobox genes vox and vent are redundant repressors of dorsal fates in zebrafish

Yoshiyuki Imai1, Michael A. Gates1,*, Anna E. Melby2, David Kimelman2, Alexander F. Schier3 and William S. Talbot1,{ddagger}

1 Department of Developmental Biology, Stanford University School of Medicine, Stanford, CA 94305, USA
2 Department of Biochemistry, University of Washington, Seattle, WA 98195, USA
3 Developmental Genetics Program, Skirball Institute of Biomolecular Medicine, Department of Cell Biology, New York University School of Medicine, New York, NY 10016, USA
* Present address: Molecular and Cellular Biology Program, University of Washington, Seattle, WA 98195, USA



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  		Fig. 1. Morphological analysis of dorsalized mutants. Pictures of live embryos at approximately 28-32 hours. (A) Wild type. (B) C4 dorsalized Dfst7 mutant. (C) Extensive degeneration in Dfst7 mutant in TL/WIK hybrid background. (D) C3 dorsalized embryo from cross of wild-type male and Dfst7/+ female. (E) C4 dorsalized Dfst8 mutant. (F) Most homozygous voxst9 embryos have a wild-type appearance. (G-I) Dfst7/voxst9 mutants have variable phenotype, ranging from wild-type to strongly dorsalized. C4 (G), C3 (H) and C1 (I) phenotypes are shown. Arrowhead in I indicates reduced ventral tail fin.

 


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  		Fig. 2. Mapping and molecular analysis of Dfst7, Dfst8 and voxst9. (A) Genomic DNA from pools of wild type, Dfst7, Dfst8 and Dfst4 embryos were tested with LG13 SSLP markers and primers for vox and vent. These mutations remove distinct but overlapping sets of markers on LG13. All markers were also tested in individual embryos to confirm the presence or absence of the PCR products (data not shown). (B) Linkage map of LG13 showing the positions of vox and vent and the regions deleted in Dfst7, Dfst8 and Dfst4 (vertical lines indicate deleted regions, broken vertical lines denote uncertainty in the locations of the breakpoints). Marker distances are based on Shimoda et al. (Shimoda et al., 1999). We localized vox and vent by scoring polymorphisms in both genes in the HS meiotic mapping panel (Kelly et al., 2000). (C) The voxst9 mutation is tightly linked to vox. All dorsalized embryos (n=291) had only the mutant vox allele, indicating that their genotype was Dfst7/voxst9. Three different genotypes were observed in embryos with a wild-type phenotype: only the wild-type allele (+/+ or Dfst7/+), both wild-type and mutant alleles (voxst9/+; arrowhead), and only the mutant allele (Dfst7/voxst9; asterisk). The Dfst7/voxst9 genotype is only partially penetrant, because some of these embryos have a wild-type phenotype. Analysis of SSLP markers confirmed that the voxst9 mutation resides in the distal region of LG13 (data not shown). (D) Schematic representation of Vox protein and the lesion in the voxst9 mutation. The voxst9 mutation changes the initiation codon (ATG) of the vox open reading frame to GTG. The second in-frame ATG occurs at codon 170, near the end of the homeodomain.

 


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  		Fig. 3. Overexpression of vox or vent but not voxst9 can counteract the expansion of dorsal mesodermal fates in Dfst7 mutants. Expression of gsc in wild-type (+) and Dfst7 embryos injected with synthetic mRNA for lacZ, vox, voxst9 or vent. (A,E) 50 pg lacZ, (B,F) 50 pg vox, (C,G) 50 pg voxst9 or (D,H) 30 pg vent mRNA was injected into 1-4-cell stage embryos from Dfst7/+ intercrosses. Embryos were fixed at 40% epiboly (5 hours) and expression of gsc was examined by in situ hybridization. In clutches injected with lacZ (n=36) or voxst9 (n=41), gsc expression was normal in about three-quarters of the embryos and strongly expanded in about a quarter of the embryos. In contrast, no embryos had strongly expanded gsc expression in clutches injected with vox (n=38) or vent (n=54); gsc expression was weakly or partially expanded in about 13% (vent) or 16% (vox) of these embryos, while expression was normal or reduced in the other embryos of these clutches. In B,D,F,H, genotypes (+ or Dfst7) were determined by PCR assay after the photographs were taken. Animal pole views, dorsal towards the right. Similar results were obtained using chd expression to monitor the expansion of dorsal fates (data not shown).

 


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  		Fig. 4. Dorsalized phenotype of embryos lacking vox and vent function, owing to morpholino oligonucleotide injection. (A) Uninjected wild-type control embryo. Wild-type (B-D) or voxst9 (E,F) embryos were injected at the 1-4-cell stage with 840 pg vox antisense MO (B,F), 840 pg vent MO (C,E), or co-injected with 420 pg vox and 420 pg vent MO (D). Photographs of living embryos were taken at approximately 30 hours. Lateral views, anterior towards the left.

 


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  		Fig. 5. Expression of goosecoid and chordin is ventrally expanded in embryos lacking vox and vent function. Expression of gsc (A-H) and chd (I-P) at 40% epiboly (5 hours) was examined by whole-mount in situ hybridization in wild type (A,I), voxst9 mutants (B,J), Dfst7/voxst9 mutants (C,K), Dfst7 mutants (D,L), control MO-injected embryos (E,M), vox MO-injected embryos (F,N), vent MO-injected embryos (G,O), and in embryos co-injected with vox and vent MO (H,P). In B,C,J,K, genotypes were determined by PCR assay after the photographs were taken. Animal pole views, dorsal towards the right.

 


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  		Fig. 6. Asymmetry of dorsal and ventral gene expression is established but not maintained in Dfst7 mutants. Expression of gsc (A-D), chd (E-H), boz (I-L) and bmp2b (M-P) was examined by in situ hybridization at the indicated stages in wild-type and Dfst7 mutants (genotypes shown at the bottom of each panel). In A,B,E,F,I,J,M,N, genotypes were determined by PCR assay after the photographs were taken. (A-L) Animal pole views, dorsal towards the right. (M-P) Lateral views, dorsal towards the right. We obtained similar results to those shown in N and P when we examined bmp2b expression in wild-type embryos co-injected with vox and vent MO.

 


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  		Fig. 7. Loss of ventrolateral mesoderm and expansion of neural territories in embryos that lack vox and vent function. Expression of pax2.1 (A-D), gata1 (E-H), nkx2.5 (I-M) and krox20 (N-Q) was examined by in situ hybridization at the (A-D,N-Q) three-somite stage (11 hours) or (E-M) 9-10 somite stage (~14 hours). (A,E,I,N) Wild type. (B,F,J,O) Dfst7 mutants. (C,G,K,P) Wild-type embryos injected with vox MO. (D,H,L,M,Q) Wild-type embryos co-injected with vox and vent MO. (A-D) pax2.1 is expressed in the pronephros and pronephric duct (arrowheads in wild-type embryos) and at the mid-hindbrain junction. (K-M) Arrowheads mark expression of nkx2.5. (A-J) Dorsal views, anterior towards the left. (K,L,N-Q) Lateral views, anterior towards the left. (M) Posterior view, dorsal towards the top.

 


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  		Fig. 8 Distinct neural patterning phenotypes in bmp2b/swr mutants and embryos lacking vox and vent function. Expression of dkk1 (A-D), otx2 (E-H), gata2 (I-L), emx1 (M-P) and six3.2 (Q-T) was examined by in situ hybridization in wild-type (A,E,I,M,Q), swr mutant (B,F,J,N,R), Dfst7 mutant (C,G,K,O,S) embryos and MO vox + vent embryos (D,H,L,P,T). Arrowheads in E-H mark the vegetal limit of otx2 expression. (A-D) Shield stage (6 hours), animal pole views, dorsal towards the right. (E-L) 75% epiboly (8 hours), lateral views, dorsal towards the right. (M-T) three-somite stage (11 hours), lateral views, anterior towards the left. In A,B, genotypes were determined by PCR assay after the photographs were taken.

 


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  		Fig. 9. Interactions among vox, vent, chordin and bozozok. The genotype of the embryos is shown at the top of the panel, and the use of MO, when injected, is shown at the bottom of the panel. The gene analyzed by in situ hybridization is shown on the left side of each row. Embryos were analyzed at 75% epiboly (A-H), 40% epiboly (I-L), or 50% epiboly (M-P). In boz mutant embryos co-injected with vox and vent MO, gsc expression resembled that shown in P (data not shown). In Dfst7;chd double mutants, domains of gata2 (D) and gsc (L) expression are both expanded when compared with wild type (A,I). In each of several cases examined, gata2 expression was slightly less expanded in Dfst7; chd mutants (D) than in chd single mutants (C); a similar result was observed in comparing chd mutants injected with vox and vent MO (H) and chd mutants injected with vox MO (G). The reduced expansion of the ventral marker gata2 (D,H) correlates with the increase in gsc expression in chd mutants lacking vox and vent function (L). (A-D,I-P) Animal pole views, dorsal towards the right. (E-H) Lateral views, dorsal towards the right. In A-D,I-P, genotypes were determined by PCR assay after the photographs were taken.

 


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  		Fig. 10. Model of interactions between vox, vent and other genes that pattern the dorsoventral axis. (A) In the late blastula boz represses vox, vent and bmp2b in parallel. vox and vent act redundantly to repress chd, gsc and other genes expressed in dorsal mesendoderm. (B) During gastrulation, bmp2b activates expression of ventral genes, such as gata2, bmp4 and bmp2b itself, and represses expression of neural genes in ventral ectoderm. By mid-gastrulation, vox/vent and bmp2b establish a positive feedback loop in which bmp2b activates expression of vox and vent, and vox and vent activate expression of bmp2b indirectly, via repression of chd.

 

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© The Company of Biologists Ltd 2001