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doi: 10.1242/10.1242/dev.00156

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HrzicN, a new Zic family gene of ascidians, plays essential roles in the neural tube and notochord development

Shuichi Wada* and Hidetoshi Saiga{dagger}

Department of Biological Sciences, Graduate School of Science, Tokyo Metropolitan University, 1-1 Minamiohsawa, Hachiohji, Tokyo 192-0397, Japan
* Present address: Department of Zoology, Graduate School of Science, Kyoto University, Kitashirakawa-oiwakecho, Sakyo-ku, Kyoto, 606-8502 Japan



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  		Fig. 1. Structure and phylogenetic analysis of HrzicN. (A) The putative amino acid sequence of HrzicN. Zinc finger motifs are italicized. (B) Molecular phylogenetic tree of Zic genes constructed using the amino acid sequences of the zinc finger motifs by the Neighbor-Joining method and the computer program, ClustalW. Numbers at the nodes represent bootstrap values. macho-1 is another Halocynthia Zic gene isolated as a muscle determinant. Cszicr1a, Cszicr1b and Cszicr2 are Zic genes of Ciona savignyi, another ascidian species. opa (odd-paired) is a Zic gene of Drosophila. Amphizic is an amphioxus Zic gene registered on the database. xGli1, hGli2, xGli3, hGli3 and xGli4 are members of vertebrate Gli family of zinc finger protein. Other genes given are vertebrate Zic family genes. DDBJ/GenBank accession numbers: AB092643(HrZicN), BAB19958(macho-1), AB057747(Cszicrla), AB057748(Cszicr1b), AB057740(Cszicr2), NP524228(opa), AJ252245(Amphizic), Q91690(xGli1), BAA25668(hGli2), Q91660(xGli3), CAB59315(hGli3), Q91661(xGli4), NM_009573(mZic1), NM_003412(hZic1), AB009564(xZic1), NM_009574(mZic2), NM_007129(hZic2), AB009565(xZic2), NM_131558(zZic2), NM_009575(mZic3), NM_003413(hZic3), AB005292(xZic3), NM_009576(mZic4), AK006747(mZic5), AF378304(hZic5), AB034983(xZic5).

 


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  		Fig. 2. Expression of HrzicN in development. (A-E,K-O) The expression pattern of HrzicN and (F-J,PQ) its schematic representation at, the early 32-cell stage (A,F), 44-cell stage (B,G), 64-cell stage (C,H), 76-cell stage (D,I), 110-cell stage (E,J), early gastrula stage (K,P), middle gastrula stage (L,Q), neural plate stage (M), early neurula stage (N) and early tailbud stage (O). (R) Summary of HrzicN expression in the various cell lineages up to the 110-cell stage. Lineages positive for HrzicN expression are indicated by bold lines.

 


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  		Fig. 3. Phenotypes of HrzicN knockdown embryos. (A,B) An embryo injected with HrzicNMO to give a final concentration of 10 µM. Inset in (B) shows an embryo injected with HrzicNMO2 to give a final concentration of 10 µM. (C,D) Embryos injected with the standard control oligo to give a final concentration of 10 µM. (A,C) Tailbud equivalent stage embryos. (B,D) Swimming larvae equivalent stage embryos. Arrowheads indicate protrusions of the adhesive organ. (A) Dorsal view. (B-D) Lateral view.

 


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  		Fig. 6. Expression of mesodermal markers in HrzicN knockdown embryos. (A,C,E) Embryos injected with HrzicNMO. (B) An embryo co-injected with lacZ/HrzicN mRNA and HrzicNMO. Arrowheads indicate rescued expression of HrBra. (F-H,J) Control uninjected embryos. Expression of (A,B,F,G) HrBra, (C,H) Hrsna and (E,J) the actin gene, at the 110-cell stage. D and I are schematic representation of C and H, respectively; dots indicate blastomeres that exhibited Hrsna expression. All specimens, vegetal view.

 


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  		Fig. 4. Expression of epidermal and neuroectodermal markers in HrzicN knockdown embryos at the early tailbud equivalent stage. (A-C,G-I) Embryos injected with HrzicNMO. (D-F,J-L) Control uninjected embryos. Expression of HrETR-1 (A,D), HrTBB2 (B,E), HrTRP (C,F), Hroth (G,J), HrEpiD (H,K) and HrzicN (I,L) was shown. Arrowheads in D-F,J indicate gene expression in the neural tube, which is lost or severely reduced in HrzicN knockdown embryos. Arrowhead in H indicates putative neural tube precursor cells without HrEpiD expression. Arrows in A,B,D,E indicate gene expression in the adhesive organ, which is unaffected in HrzicN knockdown embryos. (A-E,G-J) A dorsal view. (F,K,L) A lateral view.

 


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  		Fig. 5. Expression of epidermal and neuroectodermal markers in HrzicN knockdown embryos examined before the tailbud stage. (A-C,E,K-O) Embryos injected with HrzicNMO. (F-H,J,P-T) Control uninjected embryos. (A,B,F,G,L,Q) Expression of HrETR-1, (C,E,H,J,M,R) Hroth, (K,P) HrEpiG, (N,S) HrTRP and (O,T) HrzicN. D and I are schematic representations of C and H, respectively; dots indicate blastomeres that exhibited Hroth expression. (A,C,F,H) Vegetal view of 110-cell stage embryos. (B,E,G,J) Animal view of 110-cell stage embryos. (K-T) Vegetal views. (K,P) Early gastrula equivalent stage embryos. (L-N,Q-S) Neural plate equivalent stage embryos. (O,T) Middle gastrula equivalent stage embryos. Arrowheads in B,E,G,J indicate gene expression in the sensory vesicle precursors. Arrows in C indicate ectopic expression of Hroth in the notochord precursors. Arrows in F show HrETR-1 expression in the A-line neural tube precursors, which is absent in HrzicN knockdown embryos (A). Brackets and braces in K and P indicate the region of the neural tube precursor cells of a-line and b-line, respectively, from which HrEpiG expression is excluded. Braces in L and O indicate A-line neural tube precursors without HrETR-1 expression and with HrzicN expression, respectively.

 


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  		Fig. 7. Expression of markers in embryos overexpressing HrzicN. (A-C) Embryos injected with HrzicN mRNA. (D,E) Vegetal view of control uninjected embryos. Expression of HrETR-1 (A,B,D) and HrBra (C,E) at the middle gastrula equivalent stage. A and B show opposite sides of the same specimen.

 


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  		Fig. 8. Effect of a MEK inhibitor, U0126, on the expression of HrzicN and HrBra. (A-D) Embryos cultured in Millipore-filtered seawater containing 2 µM U0126. (E-H) Control embryos cultured in Millipore-filtered seawater containing 0.02% DMSO. Expression of HrzicN (A,B,E,F) and HrBra (C,D,G,H). (A,C,E,G) 76-cell stage embryos. (B,D,F,H) Middle gastrula equivalent stage embryos. All specimens, vegetal view.

 


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  		Fig. 9. Summary of HrzicN function in three cell lineages. (A) The a-line neural tube precursors, (B) A6.2 and A6.4 lineages and (C) the B6.2 lineage. See Discussion for details.

 




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