zic1 and zic4 are expressed in both the neural tissue and somite. While the function of zic1/zic4 is well known in the neural tissues (Aruga et al. 1998, 2002; Grinberg et al. 2004), reports about their function in the somites are relatively few. Aruga et al. reported that the deletion of Zic1 caused abnormalities in dorsal structures of the vertebrae in mouse (Aruga et al. 1999). However, the function in other somite- derived tissues was largely unknown. In this section, we introduce the function of zic1/zic4 in the somite revealed by analyses of a medaka spontaneous mutant double anal fin (Da).
Da is an enhancer mutant of zic1/zic4 in which a large DNA fragment is inserted 8.6 kb downstream of zic4 (Moriyama et al. 2012) (Fig. 8.3a). This fragment was found to be a DNA-based transposable element, and its size (180 kb) is larger than any other transposon reported to date (Inoue et al. 2017). The transposon is named Teratorn after the name of one of the extinct gigantic birds of prey. In Da, the expression of zic1/zic4 in the dorsal somite is lost, while the expression in the neural tissues is less affected, suggesting that the somite enhancer of zic1/zic4 is specifi- cally disrupted (Ohtsuka et al. 2004) (Fig. 8.2b, d, and f). In Da, the dorsal morphol- ogy in the trunk region is ventralized, and the dorsal half of the trunk appears to be a mirror image of the ventral half across the lateral midline (Fig. 8.3b, c). Thus, zic1/
zic4 was revealed to play an important role in dorsoventral patterning of the verte- brate (at least teleost) trunk during late development, which was a long-standing mystery in developmental biology. Interestingly, the ventral duplication phenotypes are seen not only in the somite-derived tissues but also in the ectoderm- and the neural crest-derived tissues surrounding the somites. Transplantation of the wild- type somites to Da demonstrated that the loss of zic1/zic4 expression in the somites is responsible for the Da phenotypes (Kawanishi et al. 2013). The following detailed phenotypes of Da are mainly based on Ohtsuka et al. 2011.
8.3.1 Embryonic Phenotypes
The phenotypes of the Da embryo can be classified into five features: (1) localiza- tion of the pigment cells, (2) shape of the dorsal fin fold, (3) shape of the dorsal myotome, (4) caudal structures, and (5) positioning of the neuromast.
In the wild-type medaka embryo, the dorsal melanocytes of the trunk and tail region are arranged in a single line along the dorsal midline, whereas those on the ventral side are arranged in two lines along the ventral midline. In contrast, the dor- sal melanocytes in Da are arranged in two lines in the same way as the ventral melanocytes (Fig. 8.3d, e). In addition to the melanophores, the location of other chromatophores, namely, xanthophores and leucophores, is altered in Da. Numerous
8 Zic Genes in Teleosts: Their Roles in Dorsoventral Patterning in the Somite
b c
j
zic1 zic4
180 kb Teratorn
8.6 kb a
H
H H
H
E E
d e
h i
k
m
wt Da
f g
l
n o
* *
adult
hatching stage
hatching stage
hatching stage
adult
adult
adult Fig. 8.3 Phenotypes of the Da mutant. (a) A huge transposon, Teratorn, is inserted 8.6 kb down- stream of zic4 in the Da genome. Adult wild-type (b) and Da mutant (c) medaka. Da shows a ven- tralized pigmentation (white arrowhead) and dorsal fin (black arrowhead), as well as a rhombic
147
xanthophores and leucophores appear in a single row along the dorsal midline of the wild-type, while only a few of these chromatophores are seen in Da.
The median fins, including the dorsal, caudal, and anal fins, develop directly from the continuous epidermal fold surrounding the trunk and tail region. In the wild-type medaka embryo, the anterior end of the dorsal fin fold is situated seven somites posterior to that of the ventral fin fold (Fig. 8.3f). However, the anterior end of the dorsal fin fold in Da has shifted anteriorly toward the position of the ventral fin fold (Fig. 8.3g).
In the wild-type medaka embryo, the tops of the dorsal myotomes elongate and come in contact at the dorsal midline. Subsequently, the top of the dorsal myotome thickens, covering the neural tube (Fig. 8.3h). In contrast, in Da, the top of the dor- sal myotomes remain at both sides of the neural tube, and each myotome grows to protrude in the dorsal direction without covering the neural tube (Fig. 8.3i) (Tamiya et al. 1997).
Teleosts including medaka have a homocercal caudal fin. Although it is superfi- cially symmetric along DV axis, the caudal part of the vertebral column tilts strongly dorsally so that the fin expanse is purely a ventral structure. The notochord terminus of a wild-type embryo starts to bend dorsally at stage 32 (somite-completion stage), whereas it extends straight into the tail in Da (Moriyama et al. 2012). This leads to the rhombic caudal fin of adult Da (Fig. 8.3b, c).
Neuromasts, which make up a small cluster of sensory cells that are part of the lateral line system, allow the sensing of mechanical changes in water. These cells are normally localized in the lateral and ventral regions in the wild-type medaka.
However, they are mislocated at the dorsal region of Da.
8.3.2 Adult Phenotypes
The phenotypes of the adult Da mutant include (1) shapes of the anal fin and caudal fin, (2) mislocalization of iridophores, (3) an altered body shape, and (4) an irregular axial skeleton.
Fig. 8.3 (continued) caudal fin (red arrowhead) and teardrop body shape. (d, e) Distribution of melanocytes in the tail region of hatching stage embryo (dorsal view). In wild-type, melanocytes are arranged in a single line along the dorsal midline (arrow in d). In contrast, those in Da are arranged in two lines in the same manner as ventral melanocytes (arrows in e). (f, g) Position of the dorsal fin fold at hatching stage (lateral view). The anterior limit of the dorsal fin fold (arrowhead) is shifted anteriorly in Da. (h, i) Myotomal morphology at hatching stage. Dashed lines delineate the edge of the myotome. A double-headed arrow indicates the gap between the dorsal myotomes in Da. (j, k) Caudal skeleton morphology of adult wild-type and Da medaka. An arrow indicates enlarged epurals in Da. E epural, H hypural. (l–o) Phenotypes of the trunk axial skeleton. The neural spines of Da are also deformed and larger (arrowheads in m) than those of wild-type medaka (l). The epipleurals, which are normally attached to the proximal region of the ribs in the wild-type (arrow in n), are missing or truncated in Da (o). Scale bars: 1 cm for b, c, j, and k;
200 μm for d–i; 10 mm for l, m; 1 mm for n and o (b–i are modified from Kawanishi et al. 2013) 8 Zic Genes in Teleosts: Their Roles in Dorsoventral Patterning in the Somite
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).