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First published online 1 March 2006
doi: 10.1242/dev.02295

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Essential and opposing roles of zebrafish ß-catenins in the formation of dorsal axial structures and neurectoderm

Gianfranco Bellipanni^1,*, Máté Varga^1,*, Shingo Maegawa^1,*, Yoshiyuki Imai², Christina Kelly¹, Andrea Pomrehn Myers², Felicia Chu², William S. Talbot² and Eric S. Weinberg^1,

¹ Department of Biology, University of Pennsylvania, Philadelphia, PA 19104, USA.
² Department of Developmental Biology, Stanford University School of Medicine, Stanford, CA 94305, USA.

View larger version (36K): [in a new window]   Fig. 1. Comparison of sequence of zebrafish ß-catenin-2 with other ß-catenins. (A) ClustalW-generated sequence alignment of the least conserved regions of Xenopus ß-catenin (Accession Number AAA49670), goldfish ß-catenin (AAP94282.1), zebrafish ß-catenin-1 (AAC59732.1), zebrafish ß-catenin-2 (AAM53438.1), and computationally predicted ß-catenin-1 and ß-catenin-2 proteins from Fugu and Tetraodon (using Ensembl genomic data sets). Of the 55 amino acid differences between the two zebrafish ß-catenins, five changes are at positions 105-109 and 28 are in the N-terminal end of the protein (the same region that shows high levels of divergence in the predicted pufferfish ß-catenin-2 proteins). Based on sequences in these two regions, zebrafish ß-catenin-1 clearly forms a closely related group with the Xenopus and goldfish ß-catenins, and with pufferfish ß-catenin-1, whereas zebrafish and pufferfish ß-catenin-2 are more distantly related. (B) Maximum parsimony analysis of vertebrate and Ciona ß-catenin cDNA nucleotide coding sequences. Numbers indicate the hits supporting the branching pattern from 1000 bootstraps.

View larger version (16K): [in a new window]   Fig. 2. ichabod maps near the ß-catenin-2 locus in the telomeric region of LG19. (A) Linkage map of the distal region of LG19 obtained from a map cross of an ichabod female and brass male (see text for description). Map positions (in cM) are obtained from the latest update of the integrated map of the zebrafish genome (see Day et al. at http://zfin.org). Two markers were tested for map position 91.5 cM (z68894/z24777) and for map position 91.6 (z1799/CA91.6-1) and single markers were tested at other map positions. Recombination frequencies (recombinants/meiosis tested) between ichabod and each of the markers are shown below the map. In addition to testing SSLP markers from the MGH microsatellite map (`Z' markers), we tested two additional markers: (1) CA85.6-3, a polymorphic CA-repeat marker located 1.5 kb from the non-polymorphic marker, Z26695, previously mapped to position 85.6 cM on LG19; and (2) CA(91.6)-1, a polymorphic CA-repeat marker found within the 3'UTR of ß-catenin-2 cDNA [an EST for ß-catenin-2, fc16f05/mgc:65770 had previously been mapped to 91.6 on the heat shock meiotic panel by Ian Woods and William Talbot (unpublished)]. The closest marker showing recombination with ichabod was CA(85.6)-3, located 1.1 cM away. No recombinants were obtained between ichabod and z68894, z24777, CA(91.6)-1, z1799, z25291 and z61401, even though these markers were spread over a region extending almost 10 cM from CA(85.6)-3. (B) Linkage map of SSLP markers determined from a completely independent map cross (generated from a WIK x OregonAB/TübingenWT cross). To test if the map positions in the non-recombinant region of the map cross shown in A were correct, we determined recombination frequencies using markers that spanned this region. As is indicated, in contrast to the ichabod map cross, we did observe recombination in this region of LG19 (except between markers z14236 and z25291).

View larger version (65K): [in a new window]   Fig. 3. Maternal ß-catenin-2 transcript is reduced in ichabod embryos. (A) RT-PCR assay of RNA expression levels in wild-type and ichabod embryos. For each stage shown, oligo dT-primed cDNA was used as a template for three separate PCR amplifications using primers for ß-catenin-1, ß-catenin-2 and ef1. Maternal ß-catenin-2 transcript was reduced in ichabod embryos, but reduction of maternal ß-catenin-1 transcript or zygotic transcripts of either gene was not observed. (B-M) Whole-mount in situ hybridization showing ubiquitous expression of both ß-catenin genes and low expression of maternal ß-catenin-2. One-cell stage (top row: B,E,H,K), sphere stage (middle row: C,F,I,L) and 90% epiboly (bottom row: D,G,J,M) wild-type (B-D, H-J) and ichabod (E-G, K-M) embryos were tested for expression of ß-catenin-1 (B-G) and ß-catenin-2 (H-M). Embryos in D,J are shown in lateral view with dorsal towards the right.

View larger version (55K): [in a new window]   Fig. 4. Injection of morpholino antisense oligonucleotides (MOs) directed against the translational initiation site of ß-catenin-2 can phenocopy the ichabod mutation, whereas MOs against ß-catenin-1 have no ventralizing effect. (A-C) Effect of injection of MO1 into wild-type embryos. Injection of 1 mM MO1 often results in a slight necrosis in the head (B) and 3 mM MO1 causes more severe necrosis and bent shortened tails (C), but in neither case were the embryos ventralized. A wild-type embryo at the same stage is shown for comparison (A). (D-G) Effect of injection of MO2 into wild-type embryos. Examples of Class 2 (E) and Class 1 (F) embryos obtained by injecting wild-type embryos with 3 mM MO2 are compared with a wild-type embryo (D) and an embryo injected with 3 mM MO2mis (G). (H) The effects of injection of 3 mM MO2 can be rescued by co-injection of ß-catenin-2* RNA (ß-catenin-2 RNA with an altered ribosome binding region that will not bind to MO2). Injection of 3 mM MO2 alone yielded a distribution of ventralized phenotypes (red bars). Injection of ß-catenin-2* RNA alone had no ventralizing effect (compare green and yellow bars). Co-injection of MO2 and the RNA yielded mostly wild-type-appearing embryos, with only a few embryos exhibiting weak ventralization (blue bars). In this experiment, we classified non-ventralized embryos into two classes, wild-type and C5, with C5 embryos exhibiting kinky notochords. (I) Injection of MO2 into ichabod embryos shifted the phenotypic distribution to more-severe ventralized classes.

View larger version (67K): [in a new window]   Fig. 5. ß-catenin-2, but not ß-catenin-1, is required for normal expression of the early dorsal markers bozozok and squint. Wild-type embryos were injected with 3 mM MO1 (B,E) or 3 mM MO2 (C,F) and compared with uninjected embryos (A,D) for bozozok (A-C) and squint (D-F) expression at 30% epiboly stage. bozozok expression is inhibited by MO2 and squint expression is partially inhibited by MO2; MO1 injection has no effect on expression of these two markers.

View larger version (100K): [in a new window]   Fig. 6. A new phenotype (`ciuffo') results from loss of function of both ß-catenin genes. (A-D) The `ciuffo' phenotype is apparent in ichabod embryos injected with 3 mM MO1 (B) and wild-type embryos injected with 3 mM MO1 and 3 mM MO2 (D). Uninjected ichabod (A) and wild-type (C) embryos are shown for comparison. (E-H) Co-injection of ß-catenin-1* RNA (ß-catenin-1 RNA with an altered ribosome binding region that will not bind to MO1) into ichabod embryos can suppress the `ciuffo' phenotype produced by MO1 injection. In comparison with uninjected ichabod embryos, which develop to C1 phenotypes (E), injection of 1 mM MO1 transforms approximately two-thirds of the embryos into `ciuffo' phenotypes (F), injection of the RNA alone causes rescue to wild-type and less ventralized C2-C4 phenotypes (G), but co-injection of both MO1 and RNA results in embryos with the original severe C1 phenotype and with C1a phenotype (H). Each of these panels shows a group of representative embryos of one of four repeats of this experiment, which all gave consistent results. (I-Z) Hindbrain and anterior neural markers and neuronal markers are not expressed in ichabod embryos (J,M,S) but are expressed in 3 mM MO1-injected ichabod (`ciuffo') embryos (K,N,T). The posterior neural marker hox6b6 is expressed in some ichabod embryos (P) and in `ciuffo' embryos (Q). Wild-type embryos are shown as positive controls (I,L,O,R). The following probes were used: krox20 (I-K), emx1 (L-N), hoxb6b (O-Q) and islet1 (R-T). All three types of embryos express myoD (U-Z), although the expression is radialized in both ichabod (V) and `ciuffo' (Z) embryos compared with wild type (U). Embryos in A-H and L-N are at 24 hpf and those in I-K,O-V are at 22 hpf. For I-Z, probes are indicated in the lower left corner and type of embryo in the lower right corner of each panel.

View larger version (59K): [in a new window]   Fig. 7. ß-Catenin-1 and ß-catenin-2 function redundantly to repress expression of chordin and goosecoid, but not bozozok or squint. Wild-type (A,D,G,J,M,P), ichabod (B,E,H,K,N,Q) or MO1-injected ichabod (C,F,I,L,O,R) embryos were assayed for expression of boz (A-C), sqt (D-F), gsc (G-L) or chd (M-R) at 30% epiboly (A-I,M-O) or 50% epiboly (J-L,P-R). MO1-injected and non-injected ichabod embryos of the same embryo clutch were compared for each marker. All embryos are shown in animal pole views.