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First published online 28 April 2004
doi: 10.1242/dev.01059

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The zebrafish iguana locus encodes Dzip1, a novel zinc-finger protein required for proper regulation of Hedgehog signaling

¹ Department of Biological Science, Graduate School of Science, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
² RIKEN Center for Developmental Biology, Kobe, 650-0047, Japan
³ Graduate School of Frontier Biosciences, Osaka University, Osaka 565-0871, Japan
⁴ Biology Department, University of Massachusetts, Amherst, MA 01003-9297, USA

View larger version (37K): [in a new window]   Fig. 6. The zinc-finger gene dzip1 is disrupted in igu mutants. (A) Genetic map of igu mutation relative to known genes in the region. P1 artificial chromosome (PAC) clones (below) and predicted ORFs (arrows in the middle) are shown. (B) Sequence ferograms showing nonsense mutations in iguts294e and igutm79a alleles. (C) Schematic view of the structure of predicted Igu/Dzip1 protein. A conserved domain (yellow box) has 51% identity with the human DZIP1. Putative nuclear localization signal (NLS), zinc finger (red boxes) and PEST domains (blue boxes) are shown. (D) Amino acid sequence of zebrafish Igu/Dzip1 protein. The NLS (gray), zinc finger (yellow) and positions of nonsense mutations (green) are highlighted. The N-terminal conserved domain is boxed. PEST sequences are underlined.

View larger version (92K): [in a new window]   Fig. 1. Aberrant expression of Hh target genes in igu mutants and comparison with four other Hh signaling mutants. (A1-A7) Wild-type, (B1-B7) igu, (C1-C7) syu/shh, (D1-7) dtr/gli1, (E1-E7) yot/gli2 and (F1-F7) con embryos at 30 hpf. (A1-F1,A2-F2) Expression of nk2.2 in the ventral neural tube is reduced in all mutants. Only igu embryos retain a punctate nk2.2 expression (arrowheads in B1, inset is a higher magnification of boxed area). Lateral views are shown in A1-F1 and the respective cross-sections at the middle of the yolk extension are shown in A2-F2. (A3-F3) Expression of pax6 is ventrally expanded in yot/gli2 (E3) and con (F3), but not in other mutants. (A4-F4,A5-F5) Expression of en1 in somites is shown in lateral views and in cross-sections. en1 expression in MPs and MFFs is completely lost in con (F4, F5). A low level of expression persists in syu/shh (C4) and yot/gli2 (E4), and no defect of en1 expression is seen in dtr/gli1 mutants (D4,D5). In contrast to the other four mutants, en1 expression in somites is upregulated in igu embryos (B4,B5). (A6-F7,A7-F7) Expression of ptc1 is shown in whole mount and in cross-sections. Expression of ptc1 is reduced in syu/shh, dtr/gli1, yot/gli2 and con mutants (C7-F7). Expression is most severely reduced in con mutants, and is mildly reduced in the neural tube of syu/shh, dtr/gli and yot/gli2 mutants. Note that ptc1 expression is not maintained in somites of dtr/gli1 embryos, despite the presence of en1 expression, and that the ptc1 expression persists in the sclerotome regions of yot/gli2 (E7). In igu embryos, ptc1 expression is upregulated in the entire somites (B6, B7), whereas the expression in neural tube is unaffected or slightly reduced in the trunk, and slightly more reduced in the brain.

View larger version (84K): [in a new window]   Fig. 2. Hh-independent target gene expression in igu embryos. (A-D) In control wild-type embryos treated with 5% DMSO, expression patterns of ptc1 at 14 hpf (A), myod at 14 hpf (B), en1 at 30hpf (C), ptc1 at 30 hpf (D) and nk2.2 at 30 hpf (E) are all normal. (F-J) Wild-type embryos treated with cyclopamine. Expression of ptc1 at 14 hpf (F), adaxial myod at 14 hpf (G), en1 at 30hpf (H), ptc1 at 30 hpf (I) and nk2.2 (J) at 30 hpf, is efficiently suppressed. Note that a low level of ptc1 expression in the neural tube in I appears to be independent of Smo-mediated Hh signaling, since similar ptc1 activation is seen in smu/smo mutants at 30 hpf (data not shown). (K-O) igu embryos treated with cyclopamine. A broad activation of ptc1 expression in igu mutants at 14 hpf (K) is not affected by cyclopamine. Arrows indicate the adaxial cells. Expression of myod in adaxial cells is indistinguishable from normal wild-type embryos, and is not affected by cyclopamine (L). Likewise, en1 (M) and ptc1 (N) expression in 30 hpf embryos is not affected by cyclopamine. Despite the defect of nk2.2 expression in igu mutants, the residual expression of nk2.2 in the neural tube is not eliminated by cyclopamine (O). The genotypes of these embryos were confirmed using tightly linked PCR-based genetic markers after in situ staining.

View larger version (118K): [in a new window]   Fig. 3. Decreased Gli activator function and/or increased Gli repressor function represses ectopic Hh target gene expression in igu embryos. (A-I) Introduction of dtr/gli1 mutant alleles into the igu genetic background suppressed ectopic Hh target gene expressions in somites. In dtr/gli1–/– embryos (A-C), the expression of nk2.2 and ptc1 is reduced, but en1 expression is normal. In dtr/gli1–/+;igu–/– embryos (D-F), nk2.2 expression is similar to dtr/gli1–/– embryos (D), with no nk2.2 expression in the posterior neural tube. By contrast, the expression of en1 (E) and ptc1 (F) are similar to that seen in igu–/– mutants; however, en1 expression is reduced in anterior somites. In dtr/gli1–/–;igu–/– embryos (G-I), the decrease of en1 and ptc1 is more evident (H,I), and the overall phenotype including nk2.2 (G) is stronger than in dtr/gli1–/– embryos. Insets show higher magnification views of ptc1 expression in the trunk region. (J-O) A drastic reduction of Hh target gene expression is caused by the introduction of a yot/gli2 mutant allele into the igu genetic background. One copy of yot/gli2 completely suppressed ectopic target gene expression (M-O), and the resulting phenotype is similar to or even stronger than that seen in yot/gli2–/– embryos (J-L). An arrowhead marks a spot of nk2.2 expression, which is consistently seen only in yot/gli2–/– embryos (J) but not in yot/gli2–/+;igu–/– embryos (M). The genotypes of these embryos were confirmed by PCR after in situ staining.

View larger version (89K): [in a new window]   Fig. 4. Inhibition of PKA does not activate Hh target genes. (A-C) Injection of 250 pg of dnPKA mRNA into wild-type embryos induced the ectopic expression of myod at 12 hpf (A), ptc1 at 30 hpf (B) and nk2.2at 30 hpf (C). (D-F) In igu embryos, overexpression of dnPKA did not induce the expression of myod (D), nk2.2 (E) and ptc1 (F), and Hh target gene expression is similar to that in igu mutants. Genotypes were confirmed by PCR after in situ analysis.

View larger version (85K): [in a new window]   Fig. 5. Activation of PKA does not efficiently suppress Hh target gene expression. (A-D) In wild-type embryos, the expression of myod at 14 hpf (A), nk2.2 at 30 hpf (B), en1 at 30 hpf (C) and ptc1 at 30 hpf (D) is repressed by 75 µM forskolin treatment. (E-H) In igu embryos, treatment with 75 µM forskolin did not block Hh target gene expression, with expression patterns similar to those seen in untreated igu mutants. Genotypes were confirmed by PCR after in situ analysis.

View larger version (109K): [in a new window]   Fig. 7. Subcellular localization of Igu/Dzip1 proteins in cultured cells. (A-C) Transfection of a construct encoding the His-tagged Igu/Dzip1 protein into NIH3T3 cells. Wild-type Igu/Dzip1 proteins (A) are located in the cytoplasm and enriched in punctate vesicles. These vesicles are also enriched with the lysosomal protein Lamp1 (B, C). (D-F) Transfection of a construct encoding the His-tagged Igu/Dzip1(ts294e) mutant protein. The truncated mutant Igu/Dzip1 proteins (D) are distributed in the cytoplasm and enriched in nuclei. (C,F) Merged images, in which nuclei are stained with 4',6-diamidino-2-phenylindole (DAPI).

View larger version (70K): [in a new window]   Fig. 8. Rescue of igu mutant phenotype by igu/dzip1 mRNA and permissive nature of Igu/Dzip1 proteins. (A-J) Injection of wild-type igu/dzip1 mRNA. Nearly all igu embryos injected with wild-type igu/dzip1 mRNA completely recovered the normal expression of Hh target genes (A-E), including: ptc1 expression at 14 hpf (A), nk2.2 expression at 30 hpf (B), ptc1 expression at 30 hpf (C, D) and en1 at 30 hpf (E) (compare with Fig. 1). Wild-type embryos injected with wild type igu/dzip1 mRNA did not show any defects in Hh target gene expression (F-J). (K-T) Injection of mutant mRNA that encodes truncated igu/dzip1 proteins. Expression of mutant Igu/Dzip1 does not affect Hh target gene expression in igu mutants (K-O) or wild-type embryos (P-T). (A,D,F,I,K,N,P,S) Dorsal views and other panels are side views. Expression patterns in A-J are identical to those seen in wild-type embryos, while those in K-O are identical to igu mutant embryos (see Fig. 1 for comparison).

View larger version (34K): [in a new window]   Fig. 9. Model of possible Igu function in Hh signaling. (A) In wild-type embryos, Igu function is required for the positive and negative regulation of Gli protein function in response to Hh signals. Components in green have activating functions, and those in red have repressor functions. (B) Without the normal Igu/Dzip1 protein function (light blue), Hh-dependent regulation is lost and Gli proteins cannot fully activate the expression of Hh target genes. In addition, the negative regulation of Hh signaling is also reduced. igu mutations could act directly on PKA-mediated negative regulation of Hh signals (broken arrows and lines in B) and/or could act indirectly, possibly by causing the constitutive nuclear import of Gli proteins (thin arrows). The resulting constitutive, but weak, activating function of Gli proteins is sufficient to induce en1 and ptc1 in broad regions in somites. This same low level of activator function is not sufficient to activate genes such as nk2.2 in the ventral neural tube the transcription of which requires a higher level of Hh signals.

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