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Expression of the Optx2 homeobox gene during mouse development
Mechanisms of Development 83 (1999) 183±186
Gene expression pattern
Expression of the Optx2 homeobox gene during mouse development Jeffrey Toy a, Olof H. Sundin a, b,* a
Department of Ophthalmology, The Johns Hopkins University School of Medicine, Baltimore, MD, USA Department of Molecular Biology and Genetics, The Johns Hopkins University School of Medicine, Baltimore, MD, USA
b
Received 4 January 1999; received in revised form 17 February 1999; accepted 22 February 1999
Abstract Optx2, a member of the sine oculis-Six family of homeobox genes, is ®rst expressed in the anterior neural plate of the mouse embryo, and subsequently in the optic vesicle and ventral forebrain. During later development, expression is further restricted to precursors of the neural retina, optic chiasm, adenohypophysis and neurohypophysis. In the adult mouse retina, Optx2 mRNA is found in cells within the ganglion cell layer and inner nuclear layer. q 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Optx2; Homeobox; Eye development; Retina; Forebrain; Sine oculis; Pituitary; Optic vesicle; Optic chiasm; Six3; Rathke's pouch
1. Results Optx2 is a mouse homeobox gene (Toy et al., 1998) belonging to the sine oculis-Six gene family (Cheyette et al., 1994;Serikaku and O'Tousa, 1994; Oliver et al., 1995). It is most closely related to mouse Six3 (Oliver et al., 1995), and its chicken counterpart, cSix3 (Bovolenta et al., 1998). Optx2 represents a distinct gene with its own human and chicken orthologs. The insect ortholog is optix, a novel gene that maps near sine oculis in the Drosophila genome. Expression of the chicken gene, cOptx2, begins in the anterior gastrula and neural plate, and is later restricted to the lens placode, optic cup, optic chiasm, and pituitary. cOptx2 is expressed throughout the adult retina and in the pituitary. Although the function of Optx2 remains unknown, ectopic expression of mouse Optx2 in pigment epithelial cells can induce these cells to express markers characteristic of retinal neuroblasts and neurons (Toy et al., 1998). The earliest expression of mouse Optx2 is detected at E8.25, with signal Localized to the most rostral portion of the neural plate (Fig.1A), in both neural and surface ectoderm (Fig. 1B). At E8.5, expression is Localized to the ventral forebrain and optic vesicles (Fig. 1C,D). At E9.5, Optx2 is expressed in the optic vesicle, optic chiasm and optic nerve (Fig. 1E,F). At E10.5, Optx2 expression * Corresponding author. Wilmer Eye Institute, The Johns Hopkins University School of Medicine, 600 North Wolfe Street, Baltimore, MD 21287±9289, USA. Tel: 1 1-410-614-1546; fax: 1 1-410-502-5382. E-mail address: [email protected] (O.H. Sundin)
continues in the anterior rim of the nasal placode (Fig. 1G,H) and neural retina, where it is most intense in the center (Figs. 1I and 2A). Unlike chick cOptx2 (Toy et al., 1998), mouse Optx2 is not expressed in the lens placode, although it is expressed in lens precursors at the neural plate stage. Along the ventral midline, caudal to the optic chiasm, Optx2 mRNA is detected in the ventral forebrain, underlying buccal ectoderm, and Rathke's pouch (Fig. 2B,D). The pars intermedia portion of the adenohypophysis arises from Rathke's pouch, which develops from ventral midline ectoderm, while the adjacent ventral forebrain generates the neurohypophysis of the pituitary (Kaufman, 1992). Expression is observed in the ventro-caudal portion of the early pallium, a region of the dorsal forebrain that gives rise to the hippocampus and dentate gyrus (solid arrow, Fig. 2C) (Yoshida et al., 1997). In the E13.5 eye, Optx2 mRNA is concentrated in cells of the neural retina near the pigment epithelium (Fig. 2E). No signal is observed in the retina of newborns (data not shown). In the adult mouse retina, Optx2 expression is restricted to cells in the vitreal face of the inner nuclear layer, and in the ganglion cell layer (Fig. 2F). Although chicken cOptx2 is expressed in mature photoreceptors, we did not observe Optx2 expression in this layer of the mouse retina.
2. Methods Wholemount in situ hybridization with digoxigeninlabeled riboprobes and sectioning of staged C3H mouse
0925-4773/99/$ - see front matterq 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0925-477 3(99)00049-0
184
J. Toy, O.H. Sundin / Mechanisms of Development 83 (1999) 183±186
Fig. 1. Optx2 expression during mouse development (10 mm sections). Blue/purple signal, Optx2 mRNA. (A) E8.25 neural fold embryo (lateral view, anterior right) showing signal in the anterior neural plate and head ectoderm. (B) E8.25, transverse section through forebrain of embryo in A. (C) E8.5 embryo, frontal view. Signal in ventral forebrain and optic vesicles (arrow). (D) E8.5, lateral view. (E) E9.5 embryo, ventro-lateral view, anterior right. Signal is restricted to the optic vesicle (®lled arrow), buccal roof/optic chiasm (open arrow), and optic nerve (signal beneath surface). (F) E10.5 embryo, transverse section showing signal in the ventral forebrain at the level of the optic chiasm. (G) E10.5 embryo, lateral view, anterior left. Optx2 signal in the eye and nasal placodes. Signal in telencephalon and otic vesicle are artifacts. (H) Ventral view of embryo in G. Signal in nasal placodes (arrows) buccal roof/optic chiasm, and entrance to Rathke's pouch. (I) E10.5 eye (faces left), transverse section of the head. Signal in the neural retina, none detected in pigment epithelium (®lled arrow) or lens placode (open arrow). Optic stalk, OS. Scale bar: A, C, D, E, H: 200 mm; B, F, I: 100 mm; G: 400 mm.
J. Toy, O.H. Sundin / Mechanisms of Development 83 (1999) 183±186
185
Fig. 2. Optx2 expression in the developing and adult mouse Sections: A±D: 10mM; E, F: 15mM. Blue/purple signal: Optx2 mRNA. (A) E10.5 embryo, horizontal section of the head, anterior left. Signal in the central neural retina. Filled arrow: pigment epithelium, Open arrow: lens placode. (B) E10.5 embryo, transverse section through the ventral forebrain (VF) at the level of the optic cup. Signal in buccal ectoderm anterior to Rathke's pouch indicated by arrowhead. Neural retina, NR. Arrow: pigment epithelium. (C) E10.5 embryo, sagittal section through forebrain. Signal in the ventral forebrain of the optic chiasm/ pituitary region (open arrow). Signal in dorsal telencephalic-diencephalic boundary (®lled arrow). (D) E10.5 embryo, transverse section. Rathke's pouch (arrow). (E) E13.5 mouse retina with retinal pigment epithelium (RPE). Signal in the neural retina (NR). (F) Adult mouse retina (C57BL6). Outer segments (OS) visible on upper edge of specimen. Signal in the ganglion cell (GC) and inner nuclear layer (INL). Outer nuclear layer, ONL. Scale bar: C: 200 mm; A, B, D: 100 mm; E, F: 50 mm.
embryos and C57BL6 adult retina were performed as described (Li et al., 1994). The Optx2 cDNA (GenBank accession number AF120273) was used to derive the riboprobes P1 (nts 723±1347), P2 (nts 723±1220), and P3 (nts 23± 480).
Acknowledgements This work was supported by N.I.H. grant EY10813 to O.H.S. J.T. received support from the Knights Templar Eye Foundation.
186
J. Toy, O.H. Sundin / Mechanisms of Development 83 (1999) 183±186
References Bovolenta, P., Mallamaci, A., Puelles, L., Boncinelli, E., 1998. Expression pattern of cSix3, a member of the Six/sine oculis family of transcription factors. Mech. Dev. 70, 201±203. Cheyette, B.N.R., Green, P.J., Martin, K., Garren, H., Hartenstein, V., Zipursky, S.L., 1994. The Drosophila sine oculis locus encodes a homeodomain-containing protein required for the development of the entire visual system. Neuron 12, 977±996. Kaufman, M.H., 1992. The Atlas of Mouse Development. Academic Press, London, UK. Li, H.S., Yang, J.M., Jacobson, R.D., Pasko, D., Sundin, O., 1994. Pax-6 is ®rst expressed in a region of ectoderm anterior to the early neural plate, implications for stepwise determination of the lens. Dev. Biol. 162, 181±194.
Oliver, G., Mailhos, A., Wehr, R., Copeland, N.G., Jenkins, N.A., Gruss, P., 1995. Six3, a murine homologue of the sine oculis gene, demarcates the most anterior border of the developing neural plate and is expressed during eye development. Development 121, 4045±4055. Serikaku, M.A., O'Tousa, J.E., 1994. Sine oculis is a homeobox gene required for Drosophila visual system development. Genetics 138, 1137±1150. Toy, J., Yang, J.M., Leppert, G.S., Sundin, O.H., 1998. The Optx2 homeobox gene is expressed in early precursors of the eye and activates retinaspeci®c genes. Proc. Natl. Acad. Sci. USA 95, 10643±10648. Yoshida, M., Suda, Y., Matsuo, I., Miyamoto, N., Takeda, N., Kuratani, S., Aizawa, S., 1997. Emx1 and Emx2 functions in development of dorsal telencephalon. Development 124, 101±111.
Gene expression pattern
Expression of the Optx2 homeobox gene during mouse development Jeffrey Toy a, Olof H. Sundin a, b,* a
Department of Ophthalmology, The Johns Hopkins University School of Medicine, Baltimore, MD, USA Department of Molecular Biology and Genetics, The Johns Hopkins University School of Medicine, Baltimore, MD, USA
b
Received 4 January 1999; received in revised form 17 February 1999; accepted 22 February 1999
Abstract Optx2, a member of the sine oculis-Six family of homeobox genes, is ®rst expressed in the anterior neural plate of the mouse embryo, and subsequently in the optic vesicle and ventral forebrain. During later development, expression is further restricted to precursors of the neural retina, optic chiasm, adenohypophysis and neurohypophysis. In the adult mouse retina, Optx2 mRNA is found in cells within the ganglion cell layer and inner nuclear layer. q 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Optx2; Homeobox; Eye development; Retina; Forebrain; Sine oculis; Pituitary; Optic vesicle; Optic chiasm; Six3; Rathke's pouch
1. Results Optx2 is a mouse homeobox gene (Toy et al., 1998) belonging to the sine oculis-Six gene family (Cheyette et al., 1994;Serikaku and O'Tousa, 1994; Oliver et al., 1995). It is most closely related to mouse Six3 (Oliver et al., 1995), and its chicken counterpart, cSix3 (Bovolenta et al., 1998). Optx2 represents a distinct gene with its own human and chicken orthologs. The insect ortholog is optix, a novel gene that maps near sine oculis in the Drosophila genome. Expression of the chicken gene, cOptx2, begins in the anterior gastrula and neural plate, and is later restricted to the lens placode, optic cup, optic chiasm, and pituitary. cOptx2 is expressed throughout the adult retina and in the pituitary. Although the function of Optx2 remains unknown, ectopic expression of mouse Optx2 in pigment epithelial cells can induce these cells to express markers characteristic of retinal neuroblasts and neurons (Toy et al., 1998). The earliest expression of mouse Optx2 is detected at E8.25, with signal Localized to the most rostral portion of the neural plate (Fig.1A), in both neural and surface ectoderm (Fig. 1B). At E8.5, expression is Localized to the ventral forebrain and optic vesicles (Fig. 1C,D). At E9.5, Optx2 is expressed in the optic vesicle, optic chiasm and optic nerve (Fig. 1E,F). At E10.5, Optx2 expression * Corresponding author. Wilmer Eye Institute, The Johns Hopkins University School of Medicine, 600 North Wolfe Street, Baltimore, MD 21287±9289, USA. Tel: 1 1-410-614-1546; fax: 1 1-410-502-5382. E-mail address: [email protected] (O.H. Sundin)
continues in the anterior rim of the nasal placode (Fig. 1G,H) and neural retina, where it is most intense in the center (Figs. 1I and 2A). Unlike chick cOptx2 (Toy et al., 1998), mouse Optx2 is not expressed in the lens placode, although it is expressed in lens precursors at the neural plate stage. Along the ventral midline, caudal to the optic chiasm, Optx2 mRNA is detected in the ventral forebrain, underlying buccal ectoderm, and Rathke's pouch (Fig. 2B,D). The pars intermedia portion of the adenohypophysis arises from Rathke's pouch, which develops from ventral midline ectoderm, while the adjacent ventral forebrain generates the neurohypophysis of the pituitary (Kaufman, 1992). Expression is observed in the ventro-caudal portion of the early pallium, a region of the dorsal forebrain that gives rise to the hippocampus and dentate gyrus (solid arrow, Fig. 2C) (Yoshida et al., 1997). In the E13.5 eye, Optx2 mRNA is concentrated in cells of the neural retina near the pigment epithelium (Fig. 2E). No signal is observed in the retina of newborns (data not shown). In the adult mouse retina, Optx2 expression is restricted to cells in the vitreal face of the inner nuclear layer, and in the ganglion cell layer (Fig. 2F). Although chicken cOptx2 is expressed in mature photoreceptors, we did not observe Optx2 expression in this layer of the mouse retina.
2. Methods Wholemount in situ hybridization with digoxigeninlabeled riboprobes and sectioning of staged C3H mouse
0925-4773/99/$ - see front matterq 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0925-477 3(99)00049-0
184
J. Toy, O.H. Sundin / Mechanisms of Development 83 (1999) 183±186
Fig. 1. Optx2 expression during mouse development (10 mm sections). Blue/purple signal, Optx2 mRNA. (A) E8.25 neural fold embryo (lateral view, anterior right) showing signal in the anterior neural plate and head ectoderm. (B) E8.25, transverse section through forebrain of embryo in A. (C) E8.5 embryo, frontal view. Signal in ventral forebrain and optic vesicles (arrow). (D) E8.5, lateral view. (E) E9.5 embryo, ventro-lateral view, anterior right. Signal is restricted to the optic vesicle (®lled arrow), buccal roof/optic chiasm (open arrow), and optic nerve (signal beneath surface). (F) E10.5 embryo, transverse section showing signal in the ventral forebrain at the level of the optic chiasm. (G) E10.5 embryo, lateral view, anterior left. Optx2 signal in the eye and nasal placodes. Signal in telencephalon and otic vesicle are artifacts. (H) Ventral view of embryo in G. Signal in nasal placodes (arrows) buccal roof/optic chiasm, and entrance to Rathke's pouch. (I) E10.5 eye (faces left), transverse section of the head. Signal in the neural retina, none detected in pigment epithelium (®lled arrow) or lens placode (open arrow). Optic stalk, OS. Scale bar: A, C, D, E, H: 200 mm; B, F, I: 100 mm; G: 400 mm.
J. Toy, O.H. Sundin / Mechanisms of Development 83 (1999) 183±186
185
Fig. 2. Optx2 expression in the developing and adult mouse Sections: A±D: 10mM; E, F: 15mM. Blue/purple signal: Optx2 mRNA. (A) E10.5 embryo, horizontal section of the head, anterior left. Signal in the central neural retina. Filled arrow: pigment epithelium, Open arrow: lens placode. (B) E10.5 embryo, transverse section through the ventral forebrain (VF) at the level of the optic cup. Signal in buccal ectoderm anterior to Rathke's pouch indicated by arrowhead. Neural retina, NR. Arrow: pigment epithelium. (C) E10.5 embryo, sagittal section through forebrain. Signal in the ventral forebrain of the optic chiasm/ pituitary region (open arrow). Signal in dorsal telencephalic-diencephalic boundary (®lled arrow). (D) E10.5 embryo, transverse section. Rathke's pouch (arrow). (E) E13.5 mouse retina with retinal pigment epithelium (RPE). Signal in the neural retina (NR). (F) Adult mouse retina (C57BL6). Outer segments (OS) visible on upper edge of specimen. Signal in the ganglion cell (GC) and inner nuclear layer (INL). Outer nuclear layer, ONL. Scale bar: C: 200 mm; A, B, D: 100 mm; E, F: 50 mm.
embryos and C57BL6 adult retina were performed as described (Li et al., 1994). The Optx2 cDNA (GenBank accession number AF120273) was used to derive the riboprobes P1 (nts 723±1347), P2 (nts 723±1220), and P3 (nts 23± 480).
Acknowledgements This work was supported by N.I.H. grant EY10813 to O.H.S. J.T. received support from the Knights Templar Eye Foundation.
186
J. Toy, O.H. Sundin / Mechanisms of Development 83 (1999) 183±186
References Bovolenta, P., Mallamaci, A., Puelles, L., Boncinelli, E., 1998. Expression pattern of cSix3, a member of the Six/sine oculis family of transcription factors. Mech. Dev. 70, 201±203. Cheyette, B.N.R., Green, P.J., Martin, K., Garren, H., Hartenstein, V., Zipursky, S.L., 1994. The Drosophila sine oculis locus encodes a homeodomain-containing protein required for the development of the entire visual system. Neuron 12, 977±996. Kaufman, M.H., 1992. The Atlas of Mouse Development. Academic Press, London, UK. Li, H.S., Yang, J.M., Jacobson, R.D., Pasko, D., Sundin, O., 1994. Pax-6 is ®rst expressed in a region of ectoderm anterior to the early neural plate, implications for stepwise determination of the lens. Dev. Biol. 162, 181±194.
Oliver, G., Mailhos, A., Wehr, R., Copeland, N.G., Jenkins, N.A., Gruss, P., 1995. Six3, a murine homologue of the sine oculis gene, demarcates the most anterior border of the developing neural plate and is expressed during eye development. Development 121, 4045±4055. Serikaku, M.A., O'Tousa, J.E., 1994. Sine oculis is a homeobox gene required for Drosophila visual system development. Genetics 138, 1137±1150. Toy, J., Yang, J.M., Leppert, G.S., Sundin, O.H., 1998. The Optx2 homeobox gene is expressed in early precursors of the eye and activates retinaspeci®c genes. Proc. Natl. Acad. Sci. USA 95, 10643±10648. Yoshida, M., Suda, Y., Matsuo, I., Miyamoto, N., Takeda, N., Kuratani, S., Aizawa, S., 1997. Emx1 and Emx2 functions in development of dorsal telencephalon. Development 124, 101±111.