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Specification of an anterior neuroectoderm patterning by Frizzled8a-mediated Wnt8b signalling during late gastrulation in zebrafish

¹ Department of Genetic Engineering, Kyungpook National University, Daegu 702-701, Korea
² TG Biotech, KNU Technopark, Kyungpook National University, Daegu 702-701, Korea
³ Department of Biology, Chungnam National University, Daejeon 305-764, Korea
⁴ Laboratory of Molecular Genetics, NICHD, NIH, Bethesda, MD 20892, USA

View larger version (117K): [in a new window]   Fig. 1. The expressions of fz8a and wnt8b are overlapped during late gastrulation of zebrafish embryos. (A-D) Dorsolateral views of embryos that were double stained for fz8a (red) and wnt8b (blue) mRNA. (A) At 90% epiboly stage, wnt8b started its expression in the posterior boundary of fz8a expression domain. (B) At 100% epiboly stage, the posterior boundary of fz8a expression domain, where wnt8b expression was overlapped, was gradually decreased; thus, the two adjacent expression domains for fz8a and wnt8b were separated. (C,D) Magnified images of insets from A and B, respectively. The broken red and blue lines indicate the posterior boundary of fz8a expression and anterior boundary of wnt8b expression, respectively. Arrows indicate fz8a-expressing cells remained in the wnt8b expression domain. Scale bars: in A, 100 µm for A,B; in D, 100 µm for C,D.

View larger version (82K): [in a new window]   Fig. 2. LiCl treatment at gastrula mimics wnt8b overexpression. (A-D) Wild-type (control) embryos at 100% epiboly are stained with anterior neuroectoderm markers, opl, anf, six3 and fz8a. (E) Control embryo at 25 hpf. (F-I) One-cell embryos were injected with low dose of wnt8b mRNA (2 pg/embryo), further cultured to 100% epiboly and then their expression patterns of the anterior neuroectoderm markers analysed. (J) 25 hpf embryo injected with wnt8b. (K-N) Embryos were exposed to LiCl at the shield stage, allowed to grow until 100% epiboly and then their expression patterns of the anterior neuroectoderm markers were analysed. (O) 25 hpf embryo treated with LiCl at shield stage. Scale bars: in N, 250 µm for A-D,F-I,K-N; in O, 250 µm for E,J,O.

View larger version (98K): [in a new window]   Fig. 3. Gain of Wnt signalling is stage specific. Various gastrula stage embryos were exposed to LiCl and the gain of Wnt activity was analysed at the nine-somite stage embryos (A-L). Untreated embryos (control) (A,G), embryos treated with LiCl at shield (B,H), 70% epiboly (C,I), 80% epiboly (D,J), 90% epiboly (E,K) and 100% epiboly stage (F,L). The expansion of midbrain and loss of eye primordia phenotypes are verified with eng2 and rx1 expressions in embryos exposed to LiCl during gastrulation (H-L) and in untreated control (G). Embryos exposed to LiCl before the mid-gastrula stage exhibit a complete loss of eye primordia (B,C,H,I), while embryos exposed to LiCl at late gastrula onwards show partial loss of eye primordia (D,E,J,K). By contrast, embryos exposed to LiCl at the end of gastrulation show almost normal anterior brain structures including eye primordia (F,L). Arrowheads indicate caudal defect in eye primordia. Scale bar: 250 µm. Anterior brain patterning is regulated by posteriorizing activity of Wnt signalling at late gastrula (M-X'). Changes of pax6 expression (blue) in the diencephalon and pax2 expression (red) in the MHB were analysed in the one-somite (M-R) and seven-somite stage embryos (S-X'), respectively. In diencephalon, pax6 expression is most abundant in the posterior diencephalon domain (arrow) and gradually decreases anteriorly. Untreated embryos (control) (M,S,S'), embryos exposed to LiCl at 60% (N,T,T'), 70% (O,U,U'), 80% (P,V,V'), 90% (Q,W,W') and 100% epiboly (R,X,X'). In embryos treated with LiCl at early gastrula, the forebrain (telencephalon, ventral diencephalon and posterior diencephalon) and eye primordia fates are lost or severely reduced, but MHB domain is expanded anteriorly (N,O,T,U,T',U'). By contrast, embryos exposed to LiCl at 80% (P,V,V') and 90% epiboly (Q,W,W'), the posterior diencephalon fate is expanded without increasing MHB domain. Embryos exposed to LiCl at the 100% epiboly show almost normal anterior brain patterning at the seven-somite stage (X,X'). Scale bar: 125 µm.

View larger version (95K): [in a new window]   Fig. 4. Ectopic expression of fz8a reduces eye size, while Fz8a-CRDTM induces large eyes. Wild-type embryos (A,E) and embryos injected with fz8a (300 pg/embryo; B,F), fz8a-CRDTM (250 pg/embryo; C,G) and dNTCF3 (25 pg/embryo; D,H) were allowed to grow until the nine-somite stage. Changes of the eye size are shown in lateral view (upper panel) and anterior view (lower panel). Arrowhead indicates caudally reduced eye primordia in fz8a-injected embryos (F), and arrows denote the enlarged eye primordia in embryos injected with fz8a-CRDTM (G) or dNTCF3 (H). Scale bar: 250 µm. (I) Structure of cDNA constructs encoding dominant negative forms of mutant protein used for injection experiments. CRD, cysteine rich domain; TM, transmembrane domain; ß-BD, ß-catenin binding domain; NLS, nuclear localisation signal.

View larger version (111K): [in a new window]   Fig. 5. Fz8a synergies with Wnt8b. The co-injection of fz8a (5 pg/embryo) with wnt8b (0.5 pg/embryo) causes radialised phenotype (A); however, co-injection of higher doses of fz8a (50 pg/embryo) with wnt1 (5 pg/embryo) does not induce radialised phenotype (B). Dominant-negative form of Fz8a (fz8a-CRDTM) rescues loss of eye phenotype driven by wnt8b overexpression (D-F). 25 hpf embryos from uninjected control (C), wnt8b injection (D) and wnt8b/fz8a-CRDTM co-injection (E,F). Embryos were injected with fz8a-CRDTM, followed by either treatment with LiCl (or not) at the shield stage, and then further cultured to 27 hpf (J). The anterior brain defects caused by wnt8b overexpression are rescued by fz8a-CRDTM (G-I,K-R). The expression of anf (telencephalon and diencephalon), rx3 (eye primordia) and six3 (eye primordia), which disappeared at the putative anterior brain (H,L,P) of the 100% epiboly stage embryos, is restored by the co-injection of fz8a-CRDTM (I, M,N,Q,R). Scale bar: 100 µm in F; 250 µm in R.

View larger version (88K): [in a new window]   Fig. 6. Knockdown of wnt8b and fz8a alters the anterior brain patterning. The effects of morpholino oligonucleotides for fz8a (fz8a-MO) and wnt8b (wnt8b-MO), and LiCl treatment at late gastrula stage embryos on the expressions of anterior brain markers were analysed at the 100% epiboly stage (A-T). The putative telencephalon domain marked by opl is not affected by morpholinos (B, fz8a-MO; C, wnt8b-MO) or LiCl treatment (D). Note that caudal region of six3 expression domain (eye primordia), which is not affected by fz8a-MO (F), is expanded posteriorly by wnt8b-MO (G). By contrast, LiCl treatment at late gastrula reduces six3 expression (H). The posterior diencephalon marked by caudal domain of fkd5 (arrow) is remarkably reduced by fz8a-MO (J) and wnt8b-MO (K), but LiCl treatment expands fkd5 expression domain (L). The expression of eng2 in midbrain almost disappears after treatment with fz8a-MO (N) and wnt8b-MO (O). However, LiCl treatment does not alter eng2 expression in the putative midbrain domain (P). The putative MHB domain marked by pax2 expression is reduced by fz8a-MO (R), although it is not affected by wnt8b-MO (S) or LiCl treatment (T). Scale bar: 250 µm. The effects of fz8a-MO, wnt8b-MO and hsp-fz8a-CRD-EGFP on the anterior brain patterning were analysed at the seven-somite stage embryos (U-Z'). The expression domains for pax6 and pax2 are remarkably reduced by fz8a-MO (V). However, wnt8b-MO injection reduces pax6 expression domain without any changes of pax2 expression domain (W). In embryos expressing a secreted dominant-negative form of Fz8a DNA under the heat-inducible promoter (hsp-fz8a-CRD-EFGP), pax6 expression domain is reduced without any changes of pax2 expression domain (X), similar to the embryos injected with wnt8b-MO (W). The expression domain for eng2 is remarkably reduced by fz8a-MO (Z) or wnt8b-MO (Z'). Arrowheads and asterisks indicate pax6 expression in the putative posterior diencephalon and pax2 expression in MHB, respectively. Scale bars: in X, 125 µm for U-X; in Z', 125 µm for Y-Z'; 250 µm for A-T.

View larger version (68K): [in a new window]   Fig. 7. Specificity of fz8a-MO and wnt8b-MO. The ability of fz8a-MO and wnt8b-MO, which can block dorsalisation induced by a co-injection of fz8a and wnt8b, were analysed at the bud stage embryos (A-D,G-J). Uninjected control embryos (A) and embryos dorsalised by a co-injection of fz8a (10 pg/embryo) and wnt8b (1 pg/embryo) mRNAs (B). Injection of fz8a-MO (14 ng/embryo) rescues the dorsalised phenotype induced by a co-injection of fz8a and wnt8b mRNAs (C). Injection of wnt8b-MO (13 ng/embryo) also rescues the dorsalised phenotype induced by a mixture of fz8a and wnt8b mRNAs (D). The expression of pax2 and no tail (ntl) in uninjected control embryos (G). The radialised pax2 expression and laterally expanded ntl expression caused by a co-injection of fz8a and wnt8b mRNAs (H) are rescued by fz8a-MO (I) and wnt8b-MO (J). The anterior view of the radialised expression of pax2 is shown in an inset (H). In control morpholino experiment, neither 4misfz8a-MO (E,F) nor 4miswnt8b-MO (K,L) could rescue dorsalised phenotype induced by co-injection of fz8a and wnt8b. Scale bar: in L, 250 µm for G-L.

View larger version (54K): [in a new window]   Fig. 8. The expression of fz8a is negatively regulated by Wnt8b. (A) Schematic representation of the transplantation experiment. A small group of 5-10 cells that overexpresses wnt8b was drawn up from a labelled midblastula donor embryo and transplanted into the blastoderm of the midblastula host embryos. (B) The control donor cells cannot affect fz8a expression in the anterior neuroectoderm of the recipient embryo (left), while transplanted cells that overexpress wnt8b strongly suppress fz8a expression in the anterior neuroectoderm of the 100% epiboly stage of host embryos (right). The transplanted embryonic cells are visualised in brown. The expression of fz8a in wild type (WT) (left) and headless (hdl) mutant (right) at the 30% epiboly stage (C) and 100% epiboly stage (D). In hdl mutant embryos, fz8a expression in the neuroectoderm is remarkably decreased.

View larger version (19K): [in a new window]   Fig. 9. A proposed mechanism can generate a differential gradient of Fz8a-mediated Wnt8b signalling required for the anterior brain patterning. The spatial expression pattern of fz8a, wnt8b, pax6 and eng2 in the presumptive forebrain and midbrain domains of the 90% and 100% epiboly stage embryos is schematically illustrated. At the 90% epiboly stage, the posterior expression domain of fz8a overlaps that of wnt8b expression. At the 100% epiboly stage, fz8a expression within this overlapping domain gradually lost its expression by the repression Wnt8b that is secreted from presumptive midbrain. This repression might decrease Fz8a/Wnt8b signal activity in the presumptive posterior diencephalon, thus generating a sharp gradient of Fz8a/Wnt8b signal activity at 100% epiboly stage. As results, eng2 (light yellow) expression was probably induced by higher Fz8a/Wnt8b signal activity in the presumptive midbrain during late gastrulation. However, anteriorly transferred Wnt8b can establish lower Fz8a/Wnt8b signalling activity, thus defines territory of pax6 expression in the presumptive diencephalon (grey). Broken and unbroken brown lines indicate hypothetical gradient of Fz8a/Wnt8b signal activity at the 90% and 100% epiboly stage, respectively. T, telencephalon; E, eye primordium; vd, ventral diencephalon; M, midbrain.