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Sequence relationships and expression patterns of zebrafish zic2 and zic5 genes
Gene Expression Patterns 4 (2004) 345–350 www.elsevier.com/locate/modgep
Sequence relationships and expression patterns of zebrafish zic2 and zic5 genes Reiko Toyama, Diego M. Gomez, Miyeko D. Mana, Igor B. Dawid* Laboratory of Molecular Genetics, National Institute of Child Health and Human Development, National Institutes of Health, Bldg. 6B, Room 413, 9000 Rockville Pike, Bethesda, MD 20892, USA Received 7 July 2003; received in revised form 30 September 2003; accepted 30 September 2003
Abstract The zinc finger motif forms a DNA binding domain that is found in a wide variety of proteins. Among them, the members of the zic gene family are highly conserved throughout metazoans. We report here the isolation of two new members of this gene family in zebrafish, zic2.2 and zic5, isolated during random screening for tissue-specific genes. Zic2.2 is closely related to the previously reported zic2 gene, which we propose to rename zic2.1; these two genes form a subfamily with other vertebrate zic2 genes. We compare here the expression patterns of zic2.1, zic2.2, and zic5. All three genes showed dynamic expression patterns starting after the initiation of zygotic transcription, predominantly in the developing neural tube. Compared to zic2.1, zic2.2 was expressed in a similar but distinct manner during early development, particularly in the retina and the forming somites. A zic2.2 ortholog has not been identified in other vertebrate species, suggesting that the zic2.1/zic2.2 pair resulted from a genome duplication event during the evolution of the zebrafish lineage. q 2003 Elsevier B.V. All rights reserved. Keywords: Zinc finger gene; zic2; zic5; Zebrafish; Central nervous system; Eye; Somite
1. Results and discussion The zic family genes encode zinc finger proteins that play important roles in vertebrate development. The members of this family, including the Drosophila opa gene, contain five copies of the C2H2 zinc finger domain. Five zic genes, zic1 –5, have been identified in vertebrates. In the mouse, zic1 –3 are expressed in the developing and mature central nervous system in overlapping but distinct patterns (Aruga et al., 1996; Nagai et al., 1997). Mutation analysis has revealed critical functions for zic genes during early vertebrate development. Mutations in the human zic2 gene cause holoprosencephaly (HPE) in a haploinsufficient manner (Brown et al., 1998). Concordantly, mutant mice with reduced expression of the zic2 gene show a delay in neurulation resulting in HPE and spina bifida (Nagai et al., 2000). Recently, it is suggested that zic2 contributes retinal ganglion cell subtype identity to pattern binocular vision (Herrera et al., 2003). In Xenopus, the zic1 and 2 genes act as neural inducers (Mizuseki et al., 1998; Nakata et al., 1998; * Corresponding author. Tel.: þ 1-301-496-4448; fax: þ1-301-496-0243. E-mail address: [email protected] (I.B. Dawid). 1567-133X/$ - see front matter q 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.modgep.2003.09.011
Kuo et al., 1998), but under certain conditions, zic2 may also act as a neural inhibitor but a neural crest inducer (Brewster et al., 1998). Zic5 is considered to mediate neural crest development in Xenopus (Nakata et al., 2000). In the zebrafish, zic1 (also known as opl) has been used as an early forebrain marker (Grinblat et al., 1998; Rohr et al., 1999; Gamse et al., 2001), but the function of this gene during development remains to be elucidated. Here we report the identification of two additional zebrafish zic genes, zic2.2 and zic5, and the analysis of their temporal and spatial expression patterns in comparison to known zebrafish zic genes. These genes were identified during a random in situ-based expression screening project (Kudoh et al., 2001). Sequence analysis revealed that the zebrafish contains two isoforms of zic2, a common occurrence in zebrafish due to a genome duplication during evolution (Wittbrodt et al., 1998). One of the newly discovered zic genes is highly similar to the previously reported zebrafish zic2 gene (Genbank accession number AF151535, by Postlthwaite); we propose to designate AF151535 as zebrafish zic2.1 and the newly isolated gene as zic2.2 (accession number AF207751). Grinblat and Sive (2001) reported a zebrafish zic2 gene which is identical to
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zic2.1. The proteins encoded by the zic2.1 and zic2.2 genes share a high degree of amino acid identity (entire protein, 82.5%; zinc finger domain, 94.4%). Among zic2 genes from different species, the zinc finger domain is most highly conserved with . 93% amino acid identity (Fig. 1A,C). The second gene isolated in our screen falls into the zic5 subfamily, the most diverged among the zic gene family (Fig. 1B,C). The zinc finger domain of the zic5 gene product shows . 91% amino acid identity among zebrafish, mouse and Xenopus, with the zebrafish and Xenopus zic5 gene products sharing high similarity throughout their sequence. However, human zic5 contains several unique glycine, alanine, or proline rich domains which increase the size of this protein by almost 140 amino acids. The zic5 genes do not have the characteristic C-terminal sequence found in other zic family members.
1.1. Zic2.1 expression Despite a high degree of sequence similarity, the zic2.1 and zic2.2 genes were expressed in a clearly distinguishable pattern. Expression of zic2.1 was first observed on the future dorsal side of the embryo at the oblong/sphere stage (determined by double staining with vega2, which is a ventral marker (Kawahara et al., 2000); data not shown), and expanded to the entire margin by 30% epiboly (data not shown). During gastrulation, dorsal expression continued but circumferential expression on the ventral side was downregulated. Expression consisted of two domains: an area closer to the epiboly margin where zic2.1 was expressed in the involuting hypoblast and mesendoderm (Fig. 2A – C, asterisk and arrow), and an area near the animal pole where zic2.1 was expressed in the ectoderm (Fig. 2A – C arrowhead). At bud stage, zic2.1 mRNA was detected in the anterior dorso-lateral ectoderm extending to the anterior neural edge (Fig. 2D,E), in a pattern reminiscent of that of zic1/opl (Grinblat et al., 1998). However, the zic2.1 expression domain is broader and not as sharply delineated as that of zic1/opl. At the 5 somite stage, zic2.1 expression was detected in the dorsal forebrain and in three discrete domains located posterior to it (Fig. 2F). The middle domain overlapped with the pax2.1 expression domain (data not shown), indicating that it is located at the midbrainhindbrain boundary (Fig. 2F, arrowhead). More posteriorly, zic2.1 was expressed only at the lateral edge of the posterior neural keel (Fig. 2G). At the 10 somite stage and later, zic2.1 mRNA was found in the entire dorsal neural tube (Fig. 2I), with high expression in the telencephalon, retina, part of the optic stalk, and tectum (Fig. 2H –J). At 48 hpf, high levels of expression were maintained in the brain while expression in the spinal cord was greatly reduced (data not shown). At this stage expression in the retina was limited to the inner-most layer adjacent to the lens, and an additional site of zic2.1 expression emerged at the distal edge of the pectoral fin buds (Fig. 2K, arrow). 1.2. Zic2.2 expression
Fig. 1. Structural comparison among vertebrate Zic proteins. (A and B) Structures of zic2 (A) and zic5 (B) gene products from different vertebrate species. The zinc finger domain is colored in red. Amino acid identities within the zinc finger domain are indicated. (C) Phylogenetic tree based on full length amino acid sequences of the zic family gene products. Amino acid sequences were obtained from protein databases. Zic1: D. rerio (AAC25102), X. laevis (AAB99946), H. sapiens (Q15915), M. musculus (P46684); Zic2: D. rerio zic2.1 (AAF73190), Zic2.2 (this paper, AAG35717), X. laevis (BAA33407), H. sapiens (AAG28409), M. musculus (BAA11115); Zic3: X. laevis (BAA23874), H. sapiens (O60481), M. musculus (Q62521); Zic4: M. musculus (Q61467); Zic5: D. rerio (nucleotide accession number AY326458), X. laevis (BAA95699), H. sapiens (AKK55418).
No maternal zic2.2 transcripts were found, and expression was first detected in the entire blastoderm at the sphere stage with a slight dorso-ventral gradient (determined by double staining with vega2; data not shown, Fig. 3A). This gradient was transient so that zic2.2 was expressed ubiquitously at 30% epiboly (Fig. 3B). During gastrulation, zic2.2 expression was downregulated in a ventral-animal domain (Fig. 3C,D). Zic2.2 RNA was not detected in the germ ring (Fig. 3C), like zic2.1 RNA, but was expressed in the ectoderm as seen in a section in Fig. 3E. At the bud stage, the zic2.2 gene was expressed in almost the entire neural ectoderm (Fig. 3F, section data not shown.), except for a circular region at the posterior neural edge (Fig. 3F,H, asterisk). Although zic2.2 RNA was present over a wide area, its levels varied so that distinct patterns
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Fig. 2. Zic2.1 gene expression. (A–C) 75% epiboly, dorsal view (A). The lines a and b in (A) correspond to the plain of section shown in (B) (dorsal midline) and (C) (tilted equatorial), respectively. Asterisks indicate involuting hypoblast expression, and arrowheads indicate ectodermal expression in (A), (B), and (C). Arrows show lateral mesendodermal expression in the area closer to the margin in (A) and (C). (D,E) Bud stage, lateral (D) and dorsal (E) view. (F) 5 somite stage, anterior-dorsal view. The arrowheads indicate the presumptive midbrain-hindbrain boundary in (F) and (H). (G) 7 somite stage, posterior dorsal view. (H) 10 somite stage, anterior dorsal view. The arrows mark trigeminal ganglia. (I) 20 somite stage, lateral view. (J) 24 hpf, anterior dorsal view. Arrow indicates optic stalk. (K) 48 hpf, anterior dorsal view. The arrow indicates the fin bud. In all lateral views anterior is to the left, in all dorsal views anterior is up.
emerged. In the anterior part, a T-shaped area of low zic2.2 expression was seen, and in the middle of the embryo, ridges of zic 2.2 expression parallel to the midline were observed (Fig. 3G, arrows). At the five somite (Fig. 3I – K) and 10 somite stage (not shown), zic2.2 mRNA was found in the neural ectoderm along the anterior-posterior axis, mostly at the outer edge of the converging neural keel. At this stage, much weaker but distinct expression was also observed in the posterior paraxial mesoderm (Fig. 3I,J, arrowhead). At the 20 somite stage and at 24 hpf, zic2.2 expression was primarily seen in the dorsal neural tube except in the telencephalon and posterior midbrain (Fig. 3L – N, O-2, 3, P– R). Double in situ hybridization with krox20 revealed that the zic2.2 gene was not expressed in rhombomere 4 (Fig. 3M,N insert). Additional expression was found in posterior somites (Fig. 3L, arrowhead) which appeared to represent a continuation of the paraxial mesoderm expression seen at earlier stages (see above). Zic 2.2 mRNA was first observed in the eye at the 10 somite stage (not shown) and reached high levels in the retina at 24 hpf (Fig. 3P,R). Interestingly, the area around the choroid fissure was zic2.2-negative (Fig. 3P). At 48 hpf, the retinal expression of zic2.2 persisted only in the innermost cell layer adjacent to the lens (Fig. 3S,T). In both mouse and Xenopus, only one zic2 gene has been reported so far. In Xenopus, zic2 mRNA is detected in the dorsal ectoderm during the early gastrula to neurula
stages, and later forms stripes that do not overlap with those formed by primary neurons. In addition, the anterior neural folds express zic2 at a high level, while low levels of expression were observed in the floor plate and somites. Moreover, Xenopus zic2 expression was found in the presumptive cranial neural crest and later in the migrating hindbrain crest, and the ciliary marginal zone of the neural retina (Brewster et al., 1998; Nakata et al., 1998). In the mouse, zic2 expression was observed both in embryonic mesoderm and ectoderm, but it was higher in the neuroectoderm and limited to the dorsal region from 8.0 dpc onward. Zic2 mRNA is also expressed in the somites, developing limbs, the optic stalk, and the neural retina (Nagai et al., 1997). In Xenopus and mouse, zic2 was expressed in the dorsal part of the neuroectoderm. Also, recent paper showed that chick zic2 was expressed in the dorsal neural tube (Warner et al., 2003). Of the two zebrafish zic2 genes, the zic2.1 expression pattern resembled that of Xenopus and mouse zic2 most closely. The marginal expression at 30% epiboly and mesendodermal expression during gastrulation may reflect mesodermal expression found in the mouse. Expression in the dorsal neural tube, retina, and optic stalk were observed in all three species. The pectoral fin bud expression of zic2.1 may correspond to the expression in the limbs of the mouse. In addition to the high degree of amino acid sequence homology, this temporally and spatially
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Fig. 3. Zic2.2 gene expression. (A,B) Blastula stage, lateral view. Sphere stage (A) and 30% epiboly (B). (C –E) 75% epiboly, lateral view (C) and animal view, dorsal to the right (D). A line in (D) indicates the plain of section shown in (E). In (E), dorsal to the right. (F–H) Bud stage, lateral view (F) and dorsal view (G, H). Note that zic2.2 is not expressed in a posterior ectodermal domain (asterisks in (F) and (H)). (I–K) 5 somite stage, lateral (I) and dorsal (J,K) views. The arrowhead indicates paraxial mesoderm. (L) 20 somite stage, lateral view. The arrowhead indicates posterior somites. (M–R) 24 hpf. Double staining is shown with pax2.1 (M,N in red) or krox20 (N insert, the arrows indicate rhombomeres 3 and 5). The position of sections (O, 1– 3) are indicated in (N). (S,T) 48 hpf. Head in lateral (S) and dorsal (T) view. In all lateral views anterior is to the left, in all dorsal views anterior is up.
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conserved expression pattern suggests that zebrafish zic2.1 is the ortholog of zic2 genes of other vertebrates. The zic2 expression in somites along the rostral-caudal axis found in Xenopus and mouse was not observed with either zebrafish zic2.1 or 2.2, but zebrafish zic2.2 was expressed in a very restricted area of the developing somites. Thus, some aspects of the zic2 expression pattern has been assumed by zic2.2, in agreement with the view that duplicated genes in zebrafish display partly overlapping patterns while dividing some of the expression domains of their vertebrate homologs among themselves.
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1.3. Zic5 expression Zic5 mRNA was first detected at the bud stage overlapping with the anterior neural edge (Fig. 4A) in an area that was very restricted as compared to the zic2.1 and zic2.2 expression patterns at this stage. By the 5 somite stage, zic5 transcript was found in the dorsal neural keel/ plate throughout the embryonic axis (Fig. 4B), resembling the pattern of zic2.1. A high level of expression was observed in the presumptive hindbrain area (Fig. 4C,D bracket), while at the posterior region, a bilateral expression
Fig. 4. Zic5 gene expression. (A) Bud stage, dorsal view. (B– F) 5 somite stage, lateral view (B), dorsal views moving from anterior to posterior (C –E). The arrowhead indicates the midbrain-hindbrain boundary in (C). The brackets in (C) and (D) indicate same expression domain in the hindbrain. (F) Section through the position marked by arrowheads in (E). (G) 10 somite stage, double stained with pax2.1 in red. (H) 15 somite stage. (I –O) 24 hpf. The arrowhead indicates the midbrain-hindbrain boundary, the open triangle and arrow show the dorsal forebrain and telencephalon, respectively. The asterisk indicates expression in the ventral diencephalon near the eye. (L) shows double staining with pax2.1 in red. The positions of sections shown in (O) (1–3) are indicated in (I). (P) 48 hpf. The arrowhead indicates the midbrain-hindbrain boundary. In all lateral views anterior is to the left, in all dorsal views anterior is up.
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domain was found at the edge of the neural ectoderm (Fig. 4E, arrowheads, F). At 10 somite and later, zic5 expression was observed in the dorsal part of the CNS along the anterior-posterior axis (Fig. 4G –I), similar to zic2.1. At 24 hpf, the zic5 gene was expressed in a very complex manner in the brain, including the telencephalon (Fig. 4J –M, arrow), ventral diencephalon rostral to the eyes overlapping with part of the optic stalk (Fig. 4J –N, asterisk), dorsal diencephalon and midbrain (Fig. 4J –L, open triangle), the midbrain-hindbrain boundary (Fig. 4J– K, arrowhead), and dorsal hindbrain. The zic5 gene was also expressed in the inner layer of the retina, but at a lower level than zic2.1 and 2.2 (Fig. 4M,O2). By 48 hpf, most of zic5 expression was downregulated except in the midbrain-hindbrain boundary and part of the diencephalon (Fig. 4P). In Xenopus, zic5 mRNA is detected in the neural folds with enhanced expression in the presumptive midbrain and hindbrain starting from the late gastrula stage. At the tailbud stage, zic5 is expressed in the eyes, dorsal brain, and the posterior part of the dorsal spinal cord (Nakata et al., 2000). By comparison, zebrafish zic5 was expressed in the eye and the dorsal part of the CNS, with enhanced expression in the anterior and posterior regions. Thus, the overall expression profile of zebrafish zic5 is quite similar to that of Xenopus zic5, except that zebrafish zic5 exhibited a more complex expression pattern in the forebrain at 24 hpf. In sum, the two copies of the zic2 gene and the zic5 gene in the zebrafish show complex expression patterns in the neuroectoderm and developing CNS, similar to the patterns exhibited by their homologs in other vertebrates.
2. Materials and methods Zebrafish zic2.2 and zic5 cDNA were isolated during a random expression screening project (Kudoh et al., 2001). A pair of zic2.1 specific primers was designed based on the sequence of AF151535. DNA from a zebrafish normalized cDNA library (Kudoh et al., 2001) was amplified with these primers and the resulting cDNA was cloned into the pCS2(þ ) vector. Whole mount in situ hybridization was performed as described previously (Toyama et al., 1995; Toyama and Dawid, 1997). For sectioning, embryos were dehydrated and embedded in JB-4 plastic resin (Polyscience Inc.) after in situ hybridization.
Acknowledgements We thank E. Laver for fish husbandry assistance.
References Aruga, J., Nagai, T., Tokuyama, T., Hayashizaki, Y., Okazaki, Y., Chapman, V.M., Mikoshiba, K., 1996. The mouse zic gene family. Homologues of the Drosophila pair-rule gene odd-paired. J. Biol. Chem 271, 1043–1047. Brewster, R., Lee, J., Ruiz i Altaba, A., 1998. Gli/Zic factors pattern the neural plate by defining domains of cell differentiation. Nature 393, 579 –583. Brown, S.A., Warburton, D., Brown, L.Y., Yu, C.Y., Roeder, E.R., StengelRutkowski, S., et al., 1998. Holoprosencephaly due to mutations in ZIC2, a homologue of Drosophila odd-paired. Nat. Genet. 20, 180 –183. Gamse, J.T., Sive, H., 2001. Early anteroposterior division of the presumptive neurectoderm in Xenopus. Mech. Dev. 104, 21 –36. Grinblat, Y., Gamse, J., Patel, M., Sive, H., 1998. Determination of the zebrafish forebrain: induction and patterning. Development 125, 4403–4416. Grinblat, Y., Sive, H., 2001. zic Gene expression marks anteroposterior pattern in the presumptive neurectoderm of the zebrafish gastrula. Dev. Dyn. 222, 688–693. Herrera, E., Brown, L., Aruga, J., Rachel, R.A., Dolen, G., Mikoshiba, K., Brown, S., Mason, C.A., 2003. Zic2 patterns binocular vision by specifying the uncrossed retinal projection. Cell 114, 545 –557. Kawahara, A., Wilm, T., Solnica-Krezel, L., Dawid, I.B., 2000. Functional interaction of vega2 and goosecoid homeobox genes in zebrafish. Genesis 28, 58–67. Kudoh, T., Tsang, M., Hukriede, N.A., Chen, X., Dedekian, M., Clarke, C.J., et al., 2001. A gene expression screen in zebrafish embryogenesis. Genome Res. 11, 1979–1987. Kuo, J.S., Patel, M., Gamse, J., Merzdorf, C., Liu, X., Apekin, V., Sive, H., 1998. Opl: a zinc finger protein that regulates neural determination and patterning in Xenopus. Development 125, 2867–2882. Mizuseki, K., Kishi, M., Matsui, M., Nakanishi, S., Sasai, Y., 1998. Xenopus Zic-related-1 and Sox-2, two factors induced by chordin, have distinct activities in the initiation of neural induction. Development 125, 579–587. Nagai, T., Aruga, J., Takada, S., Gunther, T., Sporle, R., Schughart, K., Mikoshiba, K., 1997. The expression of the mouse Zic1, Zic2, and Zic3 gene suggests an essential role for Zic genes in body pattern formation. Dev. Biol. 182, 299 –313. Nagai, T., Aruga, J., Minowa, O., Sugimoto, T., Ohno, Y., Noda, T., Mikoshiba, K., 2000. Zic2 regulates the kinetics of neurulation. Proc. Natl Acad. Sci. USA 97, 1618–1623. Nakata, K., Nagai, T., Aruga, J., Mikoshiba, K., 1998. Xenopus Zic family and its role in neural and neural crest development. Mech. Dev. 75, 43 –51. Nakata, K., Koyabu, Y., Aruga, J., Mikoshiba, K., 2000. A novel member of the Xenopus Zic family, Zic5, mediates neural crest development. Mech. Dev. 99, 83–91. Rohr, K.B., Schulte-Merker, S., Tautz, D., 1999. Zebrafish zic1 expression in brain and somites is affected by BMP and hedgehog signalling. Mech. Dev. 85, 147–159. Toyama, R., O’Connell, M.L., Wright, C.V., Kuehn, M.R., Dawid, I.B., 1995. Nodal induces ectopic goosecoid and lim1 expression and axis duplication in zebrafish. Development 121, 383 –391. Toyama, R., Dawid, I.B., 1997. lim6, a novel LIM homeobox gene in the zebrafish: comparison of its expression pattern with lim1. Dev. Dyn. 209, 406–417. Warner, S.J., Hutson, M.R., Oh, S.H., Gerlach-Bank, L.M., Lomax, M.I., Barald, K.F., 2003. Expression of ZIC genes in the development of the chick inner ear and nervous system. Dev. Dyn. 226, 702 –712. Wittbrodt, J., Meyer, A., Schartl, M., 1998. More genes in fish? BioEssays 20, 511–515.
Sequence relationships and expression patterns of zebrafish zic2 and zic5 genes Reiko Toyama, Diego M. Gomez, Miyeko D. Mana, Igor B. Dawid* Laboratory of Molecular Genetics, National Institute of Child Health and Human Development, National Institutes of Health, Bldg. 6B, Room 413, 9000 Rockville Pike, Bethesda, MD 20892, USA Received 7 July 2003; received in revised form 30 September 2003; accepted 30 September 2003
Abstract The zinc finger motif forms a DNA binding domain that is found in a wide variety of proteins. Among them, the members of the zic gene family are highly conserved throughout metazoans. We report here the isolation of two new members of this gene family in zebrafish, zic2.2 and zic5, isolated during random screening for tissue-specific genes. Zic2.2 is closely related to the previously reported zic2 gene, which we propose to rename zic2.1; these two genes form a subfamily with other vertebrate zic2 genes. We compare here the expression patterns of zic2.1, zic2.2, and zic5. All three genes showed dynamic expression patterns starting after the initiation of zygotic transcription, predominantly in the developing neural tube. Compared to zic2.1, zic2.2 was expressed in a similar but distinct manner during early development, particularly in the retina and the forming somites. A zic2.2 ortholog has not been identified in other vertebrate species, suggesting that the zic2.1/zic2.2 pair resulted from a genome duplication event during the evolution of the zebrafish lineage. q 2003 Elsevier B.V. All rights reserved. Keywords: Zinc finger gene; zic2; zic5; Zebrafish; Central nervous system; Eye; Somite
1. Results and discussion The zic family genes encode zinc finger proteins that play important roles in vertebrate development. The members of this family, including the Drosophila opa gene, contain five copies of the C2H2 zinc finger domain. Five zic genes, zic1 –5, have been identified in vertebrates. In the mouse, zic1 –3 are expressed in the developing and mature central nervous system in overlapping but distinct patterns (Aruga et al., 1996; Nagai et al., 1997). Mutation analysis has revealed critical functions for zic genes during early vertebrate development. Mutations in the human zic2 gene cause holoprosencephaly (HPE) in a haploinsufficient manner (Brown et al., 1998). Concordantly, mutant mice with reduced expression of the zic2 gene show a delay in neurulation resulting in HPE and spina bifida (Nagai et al., 2000). Recently, it is suggested that zic2 contributes retinal ganglion cell subtype identity to pattern binocular vision (Herrera et al., 2003). In Xenopus, the zic1 and 2 genes act as neural inducers (Mizuseki et al., 1998; Nakata et al., 1998; * Corresponding author. Tel.: þ 1-301-496-4448; fax: þ1-301-496-0243. E-mail address: [email protected] (I.B. Dawid). 1567-133X/$ - see front matter q 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.modgep.2003.09.011
Kuo et al., 1998), but under certain conditions, zic2 may also act as a neural inhibitor but a neural crest inducer (Brewster et al., 1998). Zic5 is considered to mediate neural crest development in Xenopus (Nakata et al., 2000). In the zebrafish, zic1 (also known as opl) has been used as an early forebrain marker (Grinblat et al., 1998; Rohr et al., 1999; Gamse et al., 2001), but the function of this gene during development remains to be elucidated. Here we report the identification of two additional zebrafish zic genes, zic2.2 and zic5, and the analysis of their temporal and spatial expression patterns in comparison to known zebrafish zic genes. These genes were identified during a random in situ-based expression screening project (Kudoh et al., 2001). Sequence analysis revealed that the zebrafish contains two isoforms of zic2, a common occurrence in zebrafish due to a genome duplication during evolution (Wittbrodt et al., 1998). One of the newly discovered zic genes is highly similar to the previously reported zebrafish zic2 gene (Genbank accession number AF151535, by Postlthwaite); we propose to designate AF151535 as zebrafish zic2.1 and the newly isolated gene as zic2.2 (accession number AF207751). Grinblat and Sive (2001) reported a zebrafish zic2 gene which is identical to
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zic2.1. The proteins encoded by the zic2.1 and zic2.2 genes share a high degree of amino acid identity (entire protein, 82.5%; zinc finger domain, 94.4%). Among zic2 genes from different species, the zinc finger domain is most highly conserved with . 93% amino acid identity (Fig. 1A,C). The second gene isolated in our screen falls into the zic5 subfamily, the most diverged among the zic gene family (Fig. 1B,C). The zinc finger domain of the zic5 gene product shows . 91% amino acid identity among zebrafish, mouse and Xenopus, with the zebrafish and Xenopus zic5 gene products sharing high similarity throughout their sequence. However, human zic5 contains several unique glycine, alanine, or proline rich domains which increase the size of this protein by almost 140 amino acids. The zic5 genes do not have the characteristic C-terminal sequence found in other zic family members.
1.1. Zic2.1 expression Despite a high degree of sequence similarity, the zic2.1 and zic2.2 genes were expressed in a clearly distinguishable pattern. Expression of zic2.1 was first observed on the future dorsal side of the embryo at the oblong/sphere stage (determined by double staining with vega2, which is a ventral marker (Kawahara et al., 2000); data not shown), and expanded to the entire margin by 30% epiboly (data not shown). During gastrulation, dorsal expression continued but circumferential expression on the ventral side was downregulated. Expression consisted of two domains: an area closer to the epiboly margin where zic2.1 was expressed in the involuting hypoblast and mesendoderm (Fig. 2A – C, asterisk and arrow), and an area near the animal pole where zic2.1 was expressed in the ectoderm (Fig. 2A – C arrowhead). At bud stage, zic2.1 mRNA was detected in the anterior dorso-lateral ectoderm extending to the anterior neural edge (Fig. 2D,E), in a pattern reminiscent of that of zic1/opl (Grinblat et al., 1998). However, the zic2.1 expression domain is broader and not as sharply delineated as that of zic1/opl. At the 5 somite stage, zic2.1 expression was detected in the dorsal forebrain and in three discrete domains located posterior to it (Fig. 2F). The middle domain overlapped with the pax2.1 expression domain (data not shown), indicating that it is located at the midbrainhindbrain boundary (Fig. 2F, arrowhead). More posteriorly, zic2.1 was expressed only at the lateral edge of the posterior neural keel (Fig. 2G). At the 10 somite stage and later, zic2.1 mRNA was found in the entire dorsal neural tube (Fig. 2I), with high expression in the telencephalon, retina, part of the optic stalk, and tectum (Fig. 2H –J). At 48 hpf, high levels of expression were maintained in the brain while expression in the spinal cord was greatly reduced (data not shown). At this stage expression in the retina was limited to the inner-most layer adjacent to the lens, and an additional site of zic2.1 expression emerged at the distal edge of the pectoral fin buds (Fig. 2K, arrow). 1.2. Zic2.2 expression
Fig. 1. Structural comparison among vertebrate Zic proteins. (A and B) Structures of zic2 (A) and zic5 (B) gene products from different vertebrate species. The zinc finger domain is colored in red. Amino acid identities within the zinc finger domain are indicated. (C) Phylogenetic tree based on full length amino acid sequences of the zic family gene products. Amino acid sequences were obtained from protein databases. Zic1: D. rerio (AAC25102), X. laevis (AAB99946), H. sapiens (Q15915), M. musculus (P46684); Zic2: D. rerio zic2.1 (AAF73190), Zic2.2 (this paper, AAG35717), X. laevis (BAA33407), H. sapiens (AAG28409), M. musculus (BAA11115); Zic3: X. laevis (BAA23874), H. sapiens (O60481), M. musculus (Q62521); Zic4: M. musculus (Q61467); Zic5: D. rerio (nucleotide accession number AY326458), X. laevis (BAA95699), H. sapiens (AKK55418).
No maternal zic2.2 transcripts were found, and expression was first detected in the entire blastoderm at the sphere stage with a slight dorso-ventral gradient (determined by double staining with vega2; data not shown, Fig. 3A). This gradient was transient so that zic2.2 was expressed ubiquitously at 30% epiboly (Fig. 3B). During gastrulation, zic2.2 expression was downregulated in a ventral-animal domain (Fig. 3C,D). Zic2.2 RNA was not detected in the germ ring (Fig. 3C), like zic2.1 RNA, but was expressed in the ectoderm as seen in a section in Fig. 3E. At the bud stage, the zic2.2 gene was expressed in almost the entire neural ectoderm (Fig. 3F, section data not shown.), except for a circular region at the posterior neural edge (Fig. 3F,H, asterisk). Although zic2.2 RNA was present over a wide area, its levels varied so that distinct patterns
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Fig. 2. Zic2.1 gene expression. (A–C) 75% epiboly, dorsal view (A). The lines a and b in (A) correspond to the plain of section shown in (B) (dorsal midline) and (C) (tilted equatorial), respectively. Asterisks indicate involuting hypoblast expression, and arrowheads indicate ectodermal expression in (A), (B), and (C). Arrows show lateral mesendodermal expression in the area closer to the margin in (A) and (C). (D,E) Bud stage, lateral (D) and dorsal (E) view. (F) 5 somite stage, anterior-dorsal view. The arrowheads indicate the presumptive midbrain-hindbrain boundary in (F) and (H). (G) 7 somite stage, posterior dorsal view. (H) 10 somite stage, anterior dorsal view. The arrows mark trigeminal ganglia. (I) 20 somite stage, lateral view. (J) 24 hpf, anterior dorsal view. Arrow indicates optic stalk. (K) 48 hpf, anterior dorsal view. The arrow indicates the fin bud. In all lateral views anterior is to the left, in all dorsal views anterior is up.
emerged. In the anterior part, a T-shaped area of low zic2.2 expression was seen, and in the middle of the embryo, ridges of zic 2.2 expression parallel to the midline were observed (Fig. 3G, arrows). At the five somite (Fig. 3I – K) and 10 somite stage (not shown), zic2.2 mRNA was found in the neural ectoderm along the anterior-posterior axis, mostly at the outer edge of the converging neural keel. At this stage, much weaker but distinct expression was also observed in the posterior paraxial mesoderm (Fig. 3I,J, arrowhead). At the 20 somite stage and at 24 hpf, zic2.2 expression was primarily seen in the dorsal neural tube except in the telencephalon and posterior midbrain (Fig. 3L – N, O-2, 3, P– R). Double in situ hybridization with krox20 revealed that the zic2.2 gene was not expressed in rhombomere 4 (Fig. 3M,N insert). Additional expression was found in posterior somites (Fig. 3L, arrowhead) which appeared to represent a continuation of the paraxial mesoderm expression seen at earlier stages (see above). Zic 2.2 mRNA was first observed in the eye at the 10 somite stage (not shown) and reached high levels in the retina at 24 hpf (Fig. 3P,R). Interestingly, the area around the choroid fissure was zic2.2-negative (Fig. 3P). At 48 hpf, the retinal expression of zic2.2 persisted only in the innermost cell layer adjacent to the lens (Fig. 3S,T). In both mouse and Xenopus, only one zic2 gene has been reported so far. In Xenopus, zic2 mRNA is detected in the dorsal ectoderm during the early gastrula to neurula
stages, and later forms stripes that do not overlap with those formed by primary neurons. In addition, the anterior neural folds express zic2 at a high level, while low levels of expression were observed in the floor plate and somites. Moreover, Xenopus zic2 expression was found in the presumptive cranial neural crest and later in the migrating hindbrain crest, and the ciliary marginal zone of the neural retina (Brewster et al., 1998; Nakata et al., 1998). In the mouse, zic2 expression was observed both in embryonic mesoderm and ectoderm, but it was higher in the neuroectoderm and limited to the dorsal region from 8.0 dpc onward. Zic2 mRNA is also expressed in the somites, developing limbs, the optic stalk, and the neural retina (Nagai et al., 1997). In Xenopus and mouse, zic2 was expressed in the dorsal part of the neuroectoderm. Also, recent paper showed that chick zic2 was expressed in the dorsal neural tube (Warner et al., 2003). Of the two zebrafish zic2 genes, the zic2.1 expression pattern resembled that of Xenopus and mouse zic2 most closely. The marginal expression at 30% epiboly and mesendodermal expression during gastrulation may reflect mesodermal expression found in the mouse. Expression in the dorsal neural tube, retina, and optic stalk were observed in all three species. The pectoral fin bud expression of zic2.1 may correspond to the expression in the limbs of the mouse. In addition to the high degree of amino acid sequence homology, this temporally and spatially
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Fig. 3. Zic2.2 gene expression. (A,B) Blastula stage, lateral view. Sphere stage (A) and 30% epiboly (B). (C –E) 75% epiboly, lateral view (C) and animal view, dorsal to the right (D). A line in (D) indicates the plain of section shown in (E). In (E), dorsal to the right. (F–H) Bud stage, lateral view (F) and dorsal view (G, H). Note that zic2.2 is not expressed in a posterior ectodermal domain (asterisks in (F) and (H)). (I–K) 5 somite stage, lateral (I) and dorsal (J,K) views. The arrowhead indicates paraxial mesoderm. (L) 20 somite stage, lateral view. The arrowhead indicates posterior somites. (M–R) 24 hpf. Double staining is shown with pax2.1 (M,N in red) or krox20 (N insert, the arrows indicate rhombomeres 3 and 5). The position of sections (O, 1– 3) are indicated in (N). (S,T) 48 hpf. Head in lateral (S) and dorsal (T) view. In all lateral views anterior is to the left, in all dorsal views anterior is up.
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conserved expression pattern suggests that zebrafish zic2.1 is the ortholog of zic2 genes of other vertebrates. The zic2 expression in somites along the rostral-caudal axis found in Xenopus and mouse was not observed with either zebrafish zic2.1 or 2.2, but zebrafish zic2.2 was expressed in a very restricted area of the developing somites. Thus, some aspects of the zic2 expression pattern has been assumed by zic2.2, in agreement with the view that duplicated genes in zebrafish display partly overlapping patterns while dividing some of the expression domains of their vertebrate homologs among themselves.
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1.3. Zic5 expression Zic5 mRNA was first detected at the bud stage overlapping with the anterior neural edge (Fig. 4A) in an area that was very restricted as compared to the zic2.1 and zic2.2 expression patterns at this stage. By the 5 somite stage, zic5 transcript was found in the dorsal neural keel/ plate throughout the embryonic axis (Fig. 4B), resembling the pattern of zic2.1. A high level of expression was observed in the presumptive hindbrain area (Fig. 4C,D bracket), while at the posterior region, a bilateral expression
Fig. 4. Zic5 gene expression. (A) Bud stage, dorsal view. (B– F) 5 somite stage, lateral view (B), dorsal views moving from anterior to posterior (C –E). The arrowhead indicates the midbrain-hindbrain boundary in (C). The brackets in (C) and (D) indicate same expression domain in the hindbrain. (F) Section through the position marked by arrowheads in (E). (G) 10 somite stage, double stained with pax2.1 in red. (H) 15 somite stage. (I –O) 24 hpf. The arrowhead indicates the midbrain-hindbrain boundary, the open triangle and arrow show the dorsal forebrain and telencephalon, respectively. The asterisk indicates expression in the ventral diencephalon near the eye. (L) shows double staining with pax2.1 in red. The positions of sections shown in (O) (1–3) are indicated in (I). (P) 48 hpf. The arrowhead indicates the midbrain-hindbrain boundary. In all lateral views anterior is to the left, in all dorsal views anterior is up.
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domain was found at the edge of the neural ectoderm (Fig. 4E, arrowheads, F). At 10 somite and later, zic5 expression was observed in the dorsal part of the CNS along the anterior-posterior axis (Fig. 4G –I), similar to zic2.1. At 24 hpf, the zic5 gene was expressed in a very complex manner in the brain, including the telencephalon (Fig. 4J –M, arrow), ventral diencephalon rostral to the eyes overlapping with part of the optic stalk (Fig. 4J –N, asterisk), dorsal diencephalon and midbrain (Fig. 4J –L, open triangle), the midbrain-hindbrain boundary (Fig. 4J– K, arrowhead), and dorsal hindbrain. The zic5 gene was also expressed in the inner layer of the retina, but at a lower level than zic2.1 and 2.2 (Fig. 4M,O2). By 48 hpf, most of zic5 expression was downregulated except in the midbrain-hindbrain boundary and part of the diencephalon (Fig. 4P). In Xenopus, zic5 mRNA is detected in the neural folds with enhanced expression in the presumptive midbrain and hindbrain starting from the late gastrula stage. At the tailbud stage, zic5 is expressed in the eyes, dorsal brain, and the posterior part of the dorsal spinal cord (Nakata et al., 2000). By comparison, zebrafish zic5 was expressed in the eye and the dorsal part of the CNS, with enhanced expression in the anterior and posterior regions. Thus, the overall expression profile of zebrafish zic5 is quite similar to that of Xenopus zic5, except that zebrafish zic5 exhibited a more complex expression pattern in the forebrain at 24 hpf. In sum, the two copies of the zic2 gene and the zic5 gene in the zebrafish show complex expression patterns in the neuroectoderm and developing CNS, similar to the patterns exhibited by their homologs in other vertebrates.
2. Materials and methods Zebrafish zic2.2 and zic5 cDNA were isolated during a random expression screening project (Kudoh et al., 2001). A pair of zic2.1 specific primers was designed based on the sequence of AF151535. DNA from a zebrafish normalized cDNA library (Kudoh et al., 2001) was amplified with these primers and the resulting cDNA was cloned into the pCS2(þ ) vector. Whole mount in situ hybridization was performed as described previously (Toyama et al., 1995; Toyama and Dawid, 1997). For sectioning, embryos were dehydrated and embedded in JB-4 plastic resin (Polyscience Inc.) after in situ hybridization.
Acknowledgements We thank E. Laver for fish husbandry assistance.
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