The dorsal fin of adult Da is enlarged and similar in shape to the anal fin as if this mutant has two anal fins (Fig. 8.3b, c). The Da (double anal fin) nomenclature refers to this severe phenotype. In the case of the caudal fin, the shape becomes rhombic instead of triangular. The triangular caudal fin of wild-type medaka, called the homocercal caudal fin, exhibits dorsoventrally asymmetric morphology; the uro- style bends dorsally, and the hypurals are formed on the ventral side to support the caudal fin rays. However, in Da, the urostyle does not bend, and both the hypurals and epurals become equally larger in size. The fin rays articulate with both hypurals and epurals, which results in the dorsoventrally symmetric morphology of the cau- dal fin of Da (Moriyama et al. 2012) (Fig. 8.3j, k).
The iridophores, which are normally found in the abdominal region of wild-type medaka, are abundant at the dorsal side as well as the ventral side of the Da trunk region (Fig. 8.3b, c). Furthermore, Da exhibits a teardrop body shape, instead of a dorsally flattened one. The body shape can be mostly attributed to the shape of the myotome. The dorsal myotome of Da grows abnormally without filling the gap over the neural tube.
The axial skeleton is also morphologically altered in Da (Fig. 8.3l–o). The trunk neural arches of Da are irregularly shaped, and, in some cases, the midline fusion of several neural arches is disturbed, resulting in spina bifida occulta. The neural spines of Da are deformed and larger than those of wild-type medaka (Fig. 8.3l, m). The epipleurals, which are normally attached to the proximal region of the ribs in the wild-type, are missing or truncated in Da (Fig. 8.3n, o). However, the centrums, ribs, and hemal spines appear normal in this mutant. These axial skeleton pheno- types are quite similar to those reported in the Zic1−/− mouse (Aruga et al. 1999).
8.4 Transcriptional Regulation of zic1/zic4
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distantly related vertebrates including human (Fig. 8.4a). Transgenic medaka, which only contains these four CNEs, shows a reporter signal in the neural tube and the somite, indicating that the somite and the neural enhancers reside in the region of the CNEs.
Furthermore, because the state of epigenetic modifications, namely, histone modifications and DNA methylation, around zic1/zic4 locus is not altered in Da (Nakamura et al. 2014), it is suggested that physical interactions between the pro- moter and somite enhancer of zic1/zic4 may be disrupted in Da. Another Da mutant, which exhibits almost identical phenotypes to those of Da, was also isolated and is named Da-2 (Moriyama et al. 2012). Da-2 was genetically mapped to the region (74 kb) containing zic1/zic4. Teratorn is also associated with this allele, but the insertion point is within the intergenic region between zic1 and zic4 in this mutant.
Despite the different insertion point of Teratorn, the expressions of both zic1 and zic4 in somite are significantly reduced like original Da. These results suggest that Teratorn may affect the transcriptional regulation of zic1/zic4 not simply by extend- ing the distance between the regulatory elements and the promoter but by affecting the higher-order structure of chromatin and disrupting the regulatory landscape
Zebrafish Xenopus Mouse Human
zic1 zic4 plscr1
BAC clone Ola1-162B06: 142 kb Medaka vs
CNEs
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DNA me H3K27me3 CpG 1
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Dorsal Ventral Blastulamyotomemyotome 1
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Fig. 8.4 Transcriptional regulation of zic1 and zic4. (a) Homology comparison of the zic1/zic4 region. The location of the Teratorn transposon insertion in the Da mutant genome is indicated by a red arrow. (b) Active-gene-expression-state-dependent K27HMD shortening at the zic1/zic4 locus in the adult myotome. Patterns of DNA methylation and H3K27me3 are shown for the blas- tula embryo, and for the adult dorsal and ventral myotome. The red box indicates a large K27HMD identified in the blastula embryo, and the blue box indicates a shortened HMD in the adult dorsal myotome (b is modified from Nakamura et al. 2014)
8 Zic Genes in Teleosts: Their Roles in Dorsoventral Patterning in the Somite
around the zic1/zic4 locus. Indeed, the distance between the promoter region of zic1/zic4 and the most distant CNE is about 50 kb in medaka and about 400 kb in human, suggesting the existence of some mechanisms that mediate such long-range interaction. This needs to be addressed in the future.
8.4.2 Two Distinct Modes of Regulation of zic1/zic4 Transcription
The dorsal-specific expression of zic1/zic4 in somites is established in a cell- nonautonomous manner (Kawanishi et al. 2013). However, as embryos grow rap- idly, the signaling relationship between the somite and surrounding tissues could be changed, and so could be the case for gene regulation. Notably, expression of zic1/
zic4 is maintained even in somite-derived tissues of adult, suggesting that zic1/zic4 expression becomes less dependent on external signals as development proceeds (Fig. 8.2g). This idea is supported by in vitro culture experiments of somite-derived tissues (Kawanishi et al. 2013). While the cells derived from the somite at the seg- mentation stage lose zic1 expression within 1 day, those derived from later embry- onic stages and adult tissues maintained the expression at least for 1 week. This indicates that the regulation of the zic1 expression is changed from a signal- dependent induction by surrounding tissues to cell-autonomous maintenance.
Epigenetic modification is thought to play a role in the maintenance of zic1/zic4 expression. DNA methylation is one of the essential epigenetic modifications in vertebrates, and a small fraction of the genome is hypomethylated. Importantly, key developmental genes including zic1/zic4 are marked by especially large hypometh- ylated domains harboring the repressive H3K27me3 histone modification (large K27HMD), before activation (Nakamura et al. 2014) (Fig. 8.4b). The large K27HMD, including zic1/zic4, is shortened in the adult dorsal myotome, but not in ventral, suggesting that the large K27HMD strictly represses the activation of zic1/
zic4 in a pluripotent state, whereas its shortening consolidates long-term gene expression in adult differentiated cells (Fig. 8.4b). This is also the case with other key developmental genes (Nakamura et al. 2014).
Taken together, zic1/zic4 expression in dorsal somite is initially established by signals derived from surrounding tissues; however, it is later maintained in a cell- autonomous manner. Changes in epigenetic modifications may be involved in the long-term maintenance of gene expression.
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