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Expression of three Rx homeobox genes in embryonic and adult zebrafish
Mechanisms of Development 84 (1999) 195±198
Gene expression pattern
Expression of three Rx homeobox genes in embryonic and adult zebra®sh Jui Chang Chuang a, Peter H. Mathers c, Pamela A. Raymond a, b,* a
Program in Cell, Developmental and Neural Biology, University of Michigan Medical School, Ann Arbor, MI 48109-0616, USA b Department of Anatomy and Cell Biology, University of Michigan Medical School, Ann Arbor, MI 48109-0616, USA c Departments of Otolaryngology, Biochemistry and Ophthalmology, West Virginia University School of Medicine, Morgantown, WV, 26506-9200, USA Received 5 February 1999; received in revised form 18 March 1999; accepted 23 March 1999
Abstract The paired-class homeobox gene, Rx, is important in eye development. In this study we analyze expression patterns of three zebra®sh Rx genes (Zrx1, 2, 3) in embryos and adults. All three genes show dynamic spatiotemporal patterns of expression. Zrx3 is expressed earliest, in the anteriormost region of the neural plate, in regions that give rise to ventral diencephalon and retinae. As development proceeds, Zrx3 expression is reduced in the lateral optic primordia, and is absent in the optic cup, but is retained at the ventral midline of the diencephalon, and is expressed in hypothalamus in the adult. As the neural retina begins to differentiate, Zrx3 is re-expressed in a subset of cells in the inner nuclear layer, presumably bipolar cells, and this expression is retained in the adult. In contrast, Zrx1/2 have a slightly later onset of expression, are initially coincident with Zrx3, but then become complementary, remaining on in the optic primordia but disappearing from the ventral midline of the diencephalon. Zrx1/2 are down-regulated as the retina differentiates, except in the outer nuclear layer where they continue to be expressed at high levels in cone, but not rod, photoreceptors. This is the ®rst transcription factor described that distinguishes between cone and rod photoreceptors. q 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Paired-class homeobox gene; Rx; Eye; Retina; Cone photoreceptors; Inner nuclear layer; Germinal zone; Hypothalamus; Development; Zebra®sh
1. Introduction and results Rx class homeobox genes have been identi®ed from Drosophila, zebra®sh, Xenopus, mouse, and humans (Casarosa et al., 1997; Furukawa et al., 1997a ; Mathers et al., 1997; Eggert et al., 1998). Three Rx genes have been described in zebra®sh (Zrx1, 2 and 3), and two (Xenopus) or one (Drosophila, mammals) in other species. Zrx1 and Zrx2 are more homologous to each other (82% identical amino acid sequence in the homeodomain and the more conserved C-terminal region) than to Zrx3 (58% identity in the same region). In this paper we examine in detail the expression patterns of the three Zrx genes in embryos and in adult ®sh. Expression of Zrx3 is ®rst detected by whole-mount in situ hybridization at 8±9 h post-fertilization (hpf) in the anteriormost neural plate, a region which gives rise to forebrain and retina (Fig. 1A). As neurulation proceeds, Zrx3 expression becomes restricted to the lateral optic primordia and ventral medial diencephalon (Fig. 1B,C). A few hours later, Zrx3 expression is gradually reduced in the lateral * Corresponding author. Tel.: 1 1-734-647-0811; fax: 1 1-734-7631166. E-mail address: [email protected] (P.A. Raymond)
optic primordia, but is maintained at the midline in the presumptive anterior hypothalamus (Fig. 1D,E). When the neural retina begins to differentiate during the second day of development, Zrx3 is again detected in a small patch of cells located in the ventronasal region of the presumptive inner nuclear layer (INL) of the retina (Schmitt and Dowling, 1999). Expression gradually spreads across the retina as differentiation progresses, but remains con®ned to a subset of cells in the INL (Fig. 1F,G). In retinal sections, Zrx3 expression is primarily localized to cells in the outer tier of the INL, which likely represent a sub-population of bipolar cells (Fig. 1H,I). The onset of expression (10 hpf) of Zrx2 is delayed by about 1±2 h compared with Zrx3, but the initial patterns of expression are identical. The onset of expression of Zrx1 (about 11 hpf) is slightly later than Zrx2, but their subsequent patterns of expression are identical. From 12 to 14 hpf, expression of both Zrx1/2 gradually becomes concentrated in the optic primordia, disappearing ®rst from the ventral diencephalon (presumptive anterior hypothalamus; Fig. 2A) and later from the medial region of the diencephalon between the optic primordia (Fig. 2B,C). As morphogenesis of the optic primordia continues, expression of Zrx1/ 2 becomes restricted to the presumptive neural retina (Fig. 2D) and disappears from the maturing retinal pigmented
0925-4773/99/$ - see front matterq 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0925-477 3(99)00077-5
196
J.C. Chuang et al. / Mechanisms of Development 84 (1999) 195±198
Fig. 1. In situ hybridization of Zrx3 in embryos and adults. (A±D) Anterior view of neurula stage embryos (9±18 hpf). op, optic primordia; oc, optic cup; h, presumptive hypothalamus. (E) Expression is restricted to the hypothalamus by 36 hpf. (F±H) Zrx3 reappears in the differentiating retina in the outer tier of the inner nuclear layer (INL) by 41 hpf. (I) Expression is maintained in the INL of adults (Ad). (E±G) Ventral views; (H) Horizontal section; (I) Radial section. l, lens; ONL, outer nuclear layer; GCL, ganglion cell layer.
epithelium (Fig. 2E). As the neural retina begins to differentiate during the second day, Zrx1/2 expression is gradually down-regulated, but it remains in proliferating cells in the circumferential germinal zone (Fig. 2F). However, when photoreceptors in the outer nuclear layer (ONL) of the retina begin to differentiate, Zrx1/2 expression increases in the ONL (Fig. 2G±I), following the spatiotemporal pattern of differentiation which originates in ventronasal retina (Raymond et al., 1995; Schmitt and Dowling, 1999). The level of expression of Zrx2 is greater than Zrx1 in immature photoreceptors at comparable stages (Fig. 2H,I), whereas
the expression of Zrx1 is enhanced during maturation of photoreceptors (Fig. 2J, black arrows). Expression of Zrx1/2 continues in adult retina, exclusively in cone, but not rod, photoreceptors (Fig. 2K,L). All four spectral classes of cones (red, green, blue and ultraviolet; Raymond et al., 1993) express Zrx1/2. The demonstration that Zrx genes are expressed in mature retinal cells is novel. In Xenopus and mouse, the Rx genes are down-regulated as the retina differentiates, and their expression has not been reported in the mature retina. A related member of the paired-class homeodomain family,
J.C. Chuang et al. / Mechanisms of Development 84 (1999) 195±198
197
Fig. 2. In situ hybridization of Zrx1 and Zrx2 in embryos and adults. (A±D) Anterior views of embryos (12±18 hpf).The arrow indicates the midline. (E) Horizontal section of embryo at 18 hpf. Zrx1 expression is restricted to neural retina (nr); optic stalk (os) and retinal pigmented epithelium (rpe) are negative. (F) Ventral view of embryo at 36 hpf. Zrx1 expression is in the circumferential germinal zone (gz) and around the choroid ®ssure (cf). (G) Lateral view of right eye at 46 hpf showing Zrx2 expression in the differentiating ventronasal retina and gz (white asterisks). V, ventral; D, dorsal. (H±J) Sections of zebra®sh eyes from 3 to 10 days (10 d). Note Zrx1/2 expression in the ONL and gz. (K±L) In adult retina, Zrx1/2 is expressed only in cone photoreceptors and the gz. These sections are cut slightly obliquely, so the cones appear in staggered tiers. OPL, outer plexiform layer; osm, outer segments of the rod and cone photoreceptors. For other abbreviations see Fig. 1.
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J.C. Chuang et al. / Mechanisms of Development 84 (1999) 195±198
Crx, is expressed in both rod and cone photoreceptors (Chen et al., 1997; Furukawa et al., 1997b), but Zrx1/2 is the ®rst transcription factor described whose expression is restricted to cone photoreceptor cells. 2. Materials and methods Zebra®sh embryos were maintained at 28.58C as described by Wester®eld (1995). Some embryos were raised in PTU (1-phenyl-2-thiourea, 0.003%) starting at 12 hpf to prevent pigment formation. Whole mount in situ hybridization was performed using digoxigenin-labeled RNA probes as described by Jowett and Lettice (1994), with the following modi®cations: the temperature of the hybridization was 568C, the hybridization buffer was 50% formamide, 0.1 M Tris±HCl (pH 7.5), 0.3 M NaCl, 1 mM EDTA, 10% dextran sulfate and 1% blocking solution (Boehringer Mannheim, Indianapolis, IN), and after hybridization, embryos were incubated in 50% formamide in 2 £ SSC at 608C for 30 min. In situ hybridization on cryosections (5-10 mm) was performed as described (Raymond et al., 1993). The RNA probes were generated from cDNA clones, approximately 2.5 kb in length, including 1.5 kb of 3 0 UTR (Zrx1 and Zrx2), or 1.3 kb in length, including 0.4 kb of 3 0 UTR (Zrx3). Probes used in Fig. 2A±G were generated from 550 bp PCR fragments containing homeodomain sequences. Probes were visualized with NBT/BCIP (Nitro-blue tetrazolium/5-Bromo-4chloro-3-indolyl phosphate), and the color reaction was carried to completion. No signals could be detected with sense RNA probes. Acknowledgements This work was supported by grants from NIH (EY04318) to P.A.R. and NIH (EY12152) to P.H.M.
References Casarosa, S., Andreazzoli, M., Simeone, A., Barsacchi, G., 1997. Xrx1, a novel Xenopus homeobox gene expressed during eye and pineal gland development. Mech. Dev. 61, 187±198. Chen, S., Wang, Q.L., Nie, Z., Sun, H., Lennon, G., Copeland, N.G., Gilbert, D.J., Jenkins, N.A., Zack, D.J., 1997. Crx, a novel Otx-like paired-homeodomain protein, binds to and transactivates photoreceptor cell-speci®c genes. Neuron 19, 1017±1030. Eggert, T., Hauck, B., Hildebrandt, N., Gehring, W.J., Walldorf, U., 1998. Isolation of a Drosophila homolog of the vertebrate homeobox gene Rx and its possible role in brain and eye development. Proc. Natl. Acad. Sci. USA 95, 2343±2348. Furukawa, T., Kozak, C., Cepko, C.L., 1997. Rax, a novel paired-like homeobox gene, shows expression in the anterior neural fold and developing retina. Proc. Natl. Acad. Sci. USA 94, 3088±3093. Furukawa, T., Morrow, E.M., Cepko, C.L., 1997. Crx, a novel otx-like homeobox gene shows photoreceptor-speci®c expression and regulates photoreceptor differentiation. Cell 91, 531±541. Jowett, T., Lettice, L., 1994. Whole-mount in situ hybridization on zebra®sh embryos using a mixture of digoxigenin-and ¯uorescein-labelled probes. Trends Genet. 10, 73±74. Mathers, P.H., Grinberg, A., Mahon, K.A., Jamrich, M., 1997. The Rx homeobox gene is essential for vertebrate eye development. Nature 387, 603±607. Raymond, P.A., Barthel, L.K., Rounsifer, M.E., Sullivan, S.A., Knight, J.K., 1993. Expression of rod and cone visual pigments in gold®sh and zebra®sh: a rhodopsin-like gene is expressed in cones. Neuron 10, 1161±1174. Raymond, P.A., Barthel, L.K., Curran, G.A., 1995. Developmental patterning of rod and cone photoreceptors in embryonic zebra®sh. J. Comp. Neurol. 359, 537±550. Schmitt, E.A., Dowling, J.E., 1999. Early retinal development in the zebra®sh. Danio rerio: light and electron microscopic analysis. J. Comp. Neurol. 404, 515±536. Wester®eld, M., 1995. The Zebra®sh Book. A Guide for the Laboratory Use of Zebra®sh (Brachydanio rerio). 3rd edn. University of Oregon Press, Eugene, OR.
Gene expression pattern
Expression of three Rx homeobox genes in embryonic and adult zebra®sh Jui Chang Chuang a, Peter H. Mathers c, Pamela A. Raymond a, b,* a
Program in Cell, Developmental and Neural Biology, University of Michigan Medical School, Ann Arbor, MI 48109-0616, USA b Department of Anatomy and Cell Biology, University of Michigan Medical School, Ann Arbor, MI 48109-0616, USA c Departments of Otolaryngology, Biochemistry and Ophthalmology, West Virginia University School of Medicine, Morgantown, WV, 26506-9200, USA Received 5 February 1999; received in revised form 18 March 1999; accepted 23 March 1999
Abstract The paired-class homeobox gene, Rx, is important in eye development. In this study we analyze expression patterns of three zebra®sh Rx genes (Zrx1, 2, 3) in embryos and adults. All three genes show dynamic spatiotemporal patterns of expression. Zrx3 is expressed earliest, in the anteriormost region of the neural plate, in regions that give rise to ventral diencephalon and retinae. As development proceeds, Zrx3 expression is reduced in the lateral optic primordia, and is absent in the optic cup, but is retained at the ventral midline of the diencephalon, and is expressed in hypothalamus in the adult. As the neural retina begins to differentiate, Zrx3 is re-expressed in a subset of cells in the inner nuclear layer, presumably bipolar cells, and this expression is retained in the adult. In contrast, Zrx1/2 have a slightly later onset of expression, are initially coincident with Zrx3, but then become complementary, remaining on in the optic primordia but disappearing from the ventral midline of the diencephalon. Zrx1/2 are down-regulated as the retina differentiates, except in the outer nuclear layer where they continue to be expressed at high levels in cone, but not rod, photoreceptors. This is the ®rst transcription factor described that distinguishes between cone and rod photoreceptors. q 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Paired-class homeobox gene; Rx; Eye; Retina; Cone photoreceptors; Inner nuclear layer; Germinal zone; Hypothalamus; Development; Zebra®sh
1. Introduction and results Rx class homeobox genes have been identi®ed from Drosophila, zebra®sh, Xenopus, mouse, and humans (Casarosa et al., 1997; Furukawa et al., 1997a ; Mathers et al., 1997; Eggert et al., 1998). Three Rx genes have been described in zebra®sh (Zrx1, 2 and 3), and two (Xenopus) or one (Drosophila, mammals) in other species. Zrx1 and Zrx2 are more homologous to each other (82% identical amino acid sequence in the homeodomain and the more conserved C-terminal region) than to Zrx3 (58% identity in the same region). In this paper we examine in detail the expression patterns of the three Zrx genes in embryos and in adult ®sh. Expression of Zrx3 is ®rst detected by whole-mount in situ hybridization at 8±9 h post-fertilization (hpf) in the anteriormost neural plate, a region which gives rise to forebrain and retina (Fig. 1A). As neurulation proceeds, Zrx3 expression becomes restricted to the lateral optic primordia and ventral medial diencephalon (Fig. 1B,C). A few hours later, Zrx3 expression is gradually reduced in the lateral * Corresponding author. Tel.: 1 1-734-647-0811; fax: 1 1-734-7631166. E-mail address: [email protected] (P.A. Raymond)
optic primordia, but is maintained at the midline in the presumptive anterior hypothalamus (Fig. 1D,E). When the neural retina begins to differentiate during the second day of development, Zrx3 is again detected in a small patch of cells located in the ventronasal region of the presumptive inner nuclear layer (INL) of the retina (Schmitt and Dowling, 1999). Expression gradually spreads across the retina as differentiation progresses, but remains con®ned to a subset of cells in the INL (Fig. 1F,G). In retinal sections, Zrx3 expression is primarily localized to cells in the outer tier of the INL, which likely represent a sub-population of bipolar cells (Fig. 1H,I). The onset of expression (10 hpf) of Zrx2 is delayed by about 1±2 h compared with Zrx3, but the initial patterns of expression are identical. The onset of expression of Zrx1 (about 11 hpf) is slightly later than Zrx2, but their subsequent patterns of expression are identical. From 12 to 14 hpf, expression of both Zrx1/2 gradually becomes concentrated in the optic primordia, disappearing ®rst from the ventral diencephalon (presumptive anterior hypothalamus; Fig. 2A) and later from the medial region of the diencephalon between the optic primordia (Fig. 2B,C). As morphogenesis of the optic primordia continues, expression of Zrx1/ 2 becomes restricted to the presumptive neural retina (Fig. 2D) and disappears from the maturing retinal pigmented
0925-4773/99/$ - see front matterq 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0925-477 3(99)00077-5
196
J.C. Chuang et al. / Mechanisms of Development 84 (1999) 195±198
Fig. 1. In situ hybridization of Zrx3 in embryos and adults. (A±D) Anterior view of neurula stage embryos (9±18 hpf). op, optic primordia; oc, optic cup; h, presumptive hypothalamus. (E) Expression is restricted to the hypothalamus by 36 hpf. (F±H) Zrx3 reappears in the differentiating retina in the outer tier of the inner nuclear layer (INL) by 41 hpf. (I) Expression is maintained in the INL of adults (Ad). (E±G) Ventral views; (H) Horizontal section; (I) Radial section. l, lens; ONL, outer nuclear layer; GCL, ganglion cell layer.
epithelium (Fig. 2E). As the neural retina begins to differentiate during the second day, Zrx1/2 expression is gradually down-regulated, but it remains in proliferating cells in the circumferential germinal zone (Fig. 2F). However, when photoreceptors in the outer nuclear layer (ONL) of the retina begin to differentiate, Zrx1/2 expression increases in the ONL (Fig. 2G±I), following the spatiotemporal pattern of differentiation which originates in ventronasal retina (Raymond et al., 1995; Schmitt and Dowling, 1999). The level of expression of Zrx2 is greater than Zrx1 in immature photoreceptors at comparable stages (Fig. 2H,I), whereas
the expression of Zrx1 is enhanced during maturation of photoreceptors (Fig. 2J, black arrows). Expression of Zrx1/2 continues in adult retina, exclusively in cone, but not rod, photoreceptors (Fig. 2K,L). All four spectral classes of cones (red, green, blue and ultraviolet; Raymond et al., 1993) express Zrx1/2. The demonstration that Zrx genes are expressed in mature retinal cells is novel. In Xenopus and mouse, the Rx genes are down-regulated as the retina differentiates, and their expression has not been reported in the mature retina. A related member of the paired-class homeodomain family,
J.C. Chuang et al. / Mechanisms of Development 84 (1999) 195±198
197
Fig. 2. In situ hybridization of Zrx1 and Zrx2 in embryos and adults. (A±D) Anterior views of embryos (12±18 hpf).The arrow indicates the midline. (E) Horizontal section of embryo at 18 hpf. Zrx1 expression is restricted to neural retina (nr); optic stalk (os) and retinal pigmented epithelium (rpe) are negative. (F) Ventral view of embryo at 36 hpf. Zrx1 expression is in the circumferential germinal zone (gz) and around the choroid ®ssure (cf). (G) Lateral view of right eye at 46 hpf showing Zrx2 expression in the differentiating ventronasal retina and gz (white asterisks). V, ventral; D, dorsal. (H±J) Sections of zebra®sh eyes from 3 to 10 days (10 d). Note Zrx1/2 expression in the ONL and gz. (K±L) In adult retina, Zrx1/2 is expressed only in cone photoreceptors and the gz. These sections are cut slightly obliquely, so the cones appear in staggered tiers. OPL, outer plexiform layer; osm, outer segments of the rod and cone photoreceptors. For other abbreviations see Fig. 1.
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J.C. Chuang et al. / Mechanisms of Development 84 (1999) 195±198
Crx, is expressed in both rod and cone photoreceptors (Chen et al., 1997; Furukawa et al., 1997b), but Zrx1/2 is the ®rst transcription factor described whose expression is restricted to cone photoreceptor cells. 2. Materials and methods Zebra®sh embryos were maintained at 28.58C as described by Wester®eld (1995). Some embryos were raised in PTU (1-phenyl-2-thiourea, 0.003%) starting at 12 hpf to prevent pigment formation. Whole mount in situ hybridization was performed using digoxigenin-labeled RNA probes as described by Jowett and Lettice (1994), with the following modi®cations: the temperature of the hybridization was 568C, the hybridization buffer was 50% formamide, 0.1 M Tris±HCl (pH 7.5), 0.3 M NaCl, 1 mM EDTA, 10% dextran sulfate and 1% blocking solution (Boehringer Mannheim, Indianapolis, IN), and after hybridization, embryos were incubated in 50% formamide in 2 £ SSC at 608C for 30 min. In situ hybridization on cryosections (5-10 mm) was performed as described (Raymond et al., 1993). The RNA probes were generated from cDNA clones, approximately 2.5 kb in length, including 1.5 kb of 3 0 UTR (Zrx1 and Zrx2), or 1.3 kb in length, including 0.4 kb of 3 0 UTR (Zrx3). Probes used in Fig. 2A±G were generated from 550 bp PCR fragments containing homeodomain sequences. Probes were visualized with NBT/BCIP (Nitro-blue tetrazolium/5-Bromo-4chloro-3-indolyl phosphate), and the color reaction was carried to completion. No signals could be detected with sense RNA probes. Acknowledgements This work was supported by grants from NIH (EY04318) to P.A.R. and NIH (EY12152) to P.H.M.
References Casarosa, S., Andreazzoli, M., Simeone, A., Barsacchi, G., 1997. Xrx1, a novel Xenopus homeobox gene expressed during eye and pineal gland development. Mech. Dev. 61, 187±198. Chen, S., Wang, Q.L., Nie, Z., Sun, H., Lennon, G., Copeland, N.G., Gilbert, D.J., Jenkins, N.A., Zack, D.J., 1997. Crx, a novel Otx-like paired-homeodomain protein, binds to and transactivates photoreceptor cell-speci®c genes. Neuron 19, 1017±1030. Eggert, T., Hauck, B., Hildebrandt, N., Gehring, W.J., Walldorf, U., 1998. Isolation of a Drosophila homolog of the vertebrate homeobox gene Rx and its possible role in brain and eye development. Proc. Natl. Acad. Sci. USA 95, 2343±2348. Furukawa, T., Kozak, C., Cepko, C.L., 1997. Rax, a novel paired-like homeobox gene, shows expression in the anterior neural fold and developing retina. Proc. Natl. Acad. Sci. USA 94, 3088±3093. Furukawa, T., Morrow, E.M., Cepko, C.L., 1997. Crx, a novel otx-like homeobox gene shows photoreceptor-speci®c expression and regulates photoreceptor differentiation. Cell 91, 531±541. Jowett, T., Lettice, L., 1994. Whole-mount in situ hybridization on zebra®sh embryos using a mixture of digoxigenin-and ¯uorescein-labelled probes. Trends Genet. 10, 73±74. Mathers, P.H., Grinberg, A., Mahon, K.A., Jamrich, M., 1997. The Rx homeobox gene is essential for vertebrate eye development. Nature 387, 603±607. Raymond, P.A., Barthel, L.K., Rounsifer, M.E., Sullivan, S.A., Knight, J.K., 1993. Expression of rod and cone visual pigments in gold®sh and zebra®sh: a rhodopsin-like gene is expressed in cones. Neuron 10, 1161±1174. Raymond, P.A., Barthel, L.K., Curran, G.A., 1995. Developmental patterning of rod and cone photoreceptors in embryonic zebra®sh. J. Comp. Neurol. 359, 537±550. Schmitt, E.A., Dowling, J.E., 1999. Early retinal development in the zebra®sh. Danio rerio: light and electron microscopic analysis. J. Comp. Neurol. 404, 515±536. Wester®eld, M., 1995. The Zebra®sh Book. A Guide for the Laboratory Use of Zebra®sh (Brachydanio rerio). 3rd edn. University of Oregon Press, Eugene, OR.