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Prox1 is differentially localized during lens development
Mechanisms of Development 112 (2002) 195–198 www.elsevier.com/locate/modo
Gene expression pattern
Prox1 is differentially localized during lens development Melinda K. Duncan a,*, Wenwu Cui a, Dong-Jin Oh a, Stanislav I. Tomarev b a
b
Department of Biological Sciences, University of Delaware, Newark, DE 19716, USA Section on Molecular Genetics, Laboratory of Molecular and Developmental Biology, National Eye Institute, Bethesda, MD 20892, USA Received 17 September 2001; received in revised form 23 November 2001; accepted 29 November 2001
Abstract Prox1, the vertebrate cognate of Drosophila Prospero, is a homeodomain protein essential for the development of the lens, liver and lymphatic system. While it is well established that the subcellular distribution of Prospero changes during development, this had not been demonstrated for Prox1. Here, high-resolution confocal microscopy demonstrated that Prox1 protein is predominately cytoplasmic in the lens placode as well as the lens epithelium and germinative zone throughout development. However during fiber cell differentiation, Prox1 protein redistributes to cell nuclei. Finally, as lens fiber cells condense their chromatin in response to lens denucleation, Prox1 remains in the nucleus but does not appear to interact with DNA. Thus, it appears that the function of Prox1, like that of its Drosophila cognate Prospero, is at least partially controlled by changes in its subcellular distribution during development. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Lens development; Prox1; Confocal microscopy; Crystallin; Transcription; Promoter; Chromatin; Subcellular distribution
1. Results Prox1 is an atypical homeodomain protein related to Drosophila prospero. Homozygous Prox1 knockout mice have defects in the elongation of lens fiber cells (Wigle et al., 1999), the migration of hepatocytes during liver development (Sosa-Pineda et al., 2000) and the development of the lymphatic system (Wigle and Oliver, 1999). Prox1 mRNA is first detected in the lens placode and later in the lens of mouse, chicken and zebrafish embryos (Glasgow and Tomarev, 1998; Oliver et al., 1993; Tomarev et al., 1996). Later, the expression pattern of Prox1 becomes complex with expression in the neural retina, developing brain, liver and lymphatic system (Belecky-Adams et al., 1997; Sosa-Pineda et al., 2000; Torii et al., 1999; Wigle and Oliver, 1999). In mice, Prox1 protein is first detected at 9.5 dpc in the cytoplasm of the lens placode and adjacent head ectoderm (Fig. 1a, b). At 10.5 dpc, significant nuclear Prox1 is detected in the posterior portion of the lens pit (Fig. 1c), while Prox1 is found in both cytoplasm and nuclei in the walls of the lens pit which give rise to the lens epithelium (Fig. 1d). Note that the staining in the head ectoderm that will give rise to the corneal epithelium has decreased substantially. At 11.5 and 12.5 dpc, intense Prox1 staining is detected in the nuclei (excluding the nucleoli) in the posterior portion of the lens vesicle (Fig.
* Corresponding author. Tel.: 11-302-831-0533; fax: 11-302-831-2281. E-mail address: [email protected] (M.K. Duncan).
1e,h,i) while both cytoplasmic and nuclear Prox1 staining is still detectable in the presumptive lens epithelium (Fig. 1f,g). At 16.5 dpc (Fig. 1j,k), the pattern is similar to that at 12.5 dpc, however, Prox1 staining in lens fiber cell nuclei in deep layers is seen in discrete nuclear zones excluded from DNA (Fig. 1l). Further, this general pattern was observed in the mouse lens well into adulthood (data not shown). Thus, it appears that Prox1 protein is found in the cytoplasm (and to a much lesser extent the nucleus) of mouse lens epithelial cells, in the nucleus associated with DNA in the transition zone, and in nuclear regions excluded from DNA in mouse lens fiber cells which have initiated chromatin condensation. In order to determine whether the differential localization of Prox1 in the lens is observed in other vertebrates, Prox1 immunohistochemistry was performed on rat (Fig. 2a,d,g–i), human (Fig. 2b,e) and chicken lens (Fig. 2c,f). In all three cases, Prox1 protein was detected in both the cytoplasm and nucleus of lens epithelial cells (Fig. 2a–c) and appeared to both increase in levels and concentrate in the nuclei of newly differentiating lens fiber cells (Fig. 2d–f). Further, in all four species examined, Prox1 protein was excluded from chromatin in deep cortical fibers undergoing nuclear condensation (Fig. 2g–i; data not shown). In contrast, cMaf, another protein known to be essential for lens fiber cell differentiation (Kawauchi et al., 1999; Ring et al., 2000) was strictly associated with the nuclei of lens epithelial cells (Fig. 2j–l). Notably, Prospero, the Drosophila cognate of Prox1, is also often found in the cytoplasm of proliferating and undif-
0925-4773/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0925-477 3(01)00645-1
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Fig. 1. Changes in Prox1 protein localization during mouse lens development. (A) Presumptive eye of a 9.5 dpc embryo; note that Prox1 protein is primarily cytoplasmic in the head ectoderm. Bar ¼ 60 mm. (B) Higher magnification of the lens placode and optic vesicle shown in A. Bar ¼ 10 mm (C) Eye of a 10.5 dpc embryo; note that Prox1 protein is primarily nuclear in the posterior portion of the lens pit. Bar ¼ 60 mm. (D) Higher magnification of the tip of the closing lens pit boxed in panel B; note that Prox1 is detected in both the cytoplasm and nucleus of cells that will give rise to lens epithelium. Bar ¼ 6 mm. (E) Eye of a late 10.5 dpc embryo showing that Prox1 protein is predominately localized to the posterior portion of the lens vesicle. Bar ¼ 60 mm. (F) Higher magnification of the area boxed in white in panel E; note that significant amounts of cytoplasmic Prox1 are detected in both the presumptive lens epithelium and presumptive corneal epithelium. Bar ¼ 10 mm. (G) Same image shown in F with the DNA signal subtracted. Note that nuclear Prox1 levels vary between nuclei in the presumptive epithelium. Bar ¼ 10 mm. (H) Higher magnification of the area boxed in yellow in panel E, note that Prox1 is co-localized with the nuclear DNA excluding the nucleolus in presumptive lens fiber cells. Bar ¼ 6 mm. (I) Eye of a 12.5 dpc embryo, note the high levels of Prox1 protein detected in the nuclei of primary lens fibers. Bar ¼ 60 mm. (J) Eye of a 16.5 dpc mouse embryo; note the upregulation of nuclear Prox1 in the lens fibers at the transition zone. Bar ¼ 100 mm. (K) Same image shown in (J) with the DNA signal subtracted; note that some nuclear Prox1 is found in the lens epithelial cells at this stage. (I) Higher magnification of the area boxed in panel (J), note that significant nuclear Prox 1 in lens fiber cells beginning terminal differentiation is not co-localized with chromatin. Bar ¼ 6 mm. Green, DNA; Red, Prox1; Yellow, overlap. Abbreviations: ov, optic vesicle; pl, lens placode; pr, presumptive retina; lp, lens pit; pc, presumptive cornea; tp, tip of the lens pit; lv, lens vesicle; pe, presumptive lens epithelium; pf, primary lens fibers; e, lens epithelium; c, cornea; r, retina; f, lens fibers; t, transition zone. Arrowheads, regions of nuclear Prox1 excluded from DNA.
ferentiated precursors, relocalizing to the nucleus of postmitotic, differentiating cells (Li and Vaessin, 2000). In the larval central nervous system, Prospero is first seen in the cytoplasm of the neuroblast and relocalizes to the cortical crescent, which is asymmetrically distributed to the ganglion mother cell (Hirata et al., 1995; Knoblich et al., 1995; Spana and Doe, 1995). Since the amino acids responsible for the localization of Prospero to the cortical crescent are not conserved in Prox1 (Hirata et al., 1995; Tomarev et al., 1996), it is not surprising that Prox1 does not localize to a discrete region of cytoplasm. However, in Drosophila adult
sensory organ precursors, Prospero protein is evenly distributed in the cytoplasm of proliferating cells while nuclear Prospero was only found in postmitotic thecogen cells (Manning and Doe, 1999; Reddy and Rodrigues, 1999). Interestingly, the control of Prospero localization has been recently attributed to the presence of a nuclear export signal in the homeodomain whose function is controlled by the conformation of the prospero domain (Demidenko et al., 2001). Since the homeo- and Prospero domains are well conserved between Prox1 and Prospero (Tomarev et al., 1996), it seems likely that Prox1 uses a similar mechanism.
M.K. Duncan et al. / Mechanisms of Development 112 (2002) 195–198
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Fig. 2. Prox1 is found in both the cytoplasm and nucleus of lens epithelial cells in multiple species. (A,D,G–I) Lens from a 14-day-old rat stained for Prox1. (B,E) Lens from a 21-year-old human stained for Prox1. (C,F) Lens from an 11-day embryonic chicken stained for Prox1. Note that Prox1 is found in the cytoplasm and to a lesser extent the nucleus of lens epithelial cells (A–C; bar ¼ 20 mm) and becomes predominantly nuclear in lens fiber cells (D–F; bar ¼ 60 mm). (G–I) Nuclei from terminally differentiating 14 day rat lens fiber cells bar ¼ 10 mm. (G) Prox1 (H) DNA. (I) Merged. Note that nuclear Prox1 does not co-localize with DNA in lens fibers with condensing chromatin (arrow). (J–L) cMaf protein localization in the lens epithelium from a 14-dayold rat. Note that cMaf is predominantly a nuclear protein in the lens epithelium bar ¼ 20 mm. Green, DNA; Red, cMaf; Yellow, overlap. Abbreviations: e, lens epithelium; f, lens fiber cells; t, transition zone; ap, annular pad.
2. Materials and methods
Human lenses were isolated from donor cadaver eyes by the Oregon Lions Eye Bank (Portland, OR).
2.1. Tissue collection All mouse embryos were obtained from timed pregnant matings of FVB/N mice (Taketo et al., 1991) with noon of the date that the vaginal plug was observed defined to be 0.5 days post coitum (dpc). Rat eyes were isolated from 14-day post natal (dpn) Long–Evans rats. Chicken lenses were isolated from 11 day single comb white leghorn embryos.
2.2. Immunohistochemistry An affinity purified rabbit polyclonal antibody raised against the C-terminal homeo- and Prospero domains of human Prox1 (Belecky-Adams et al., 1997) was used at a 1:200 dilution to detect Prox1 protein and a polyclonal anticMaf antibody (Santa Cruz Biotechnology, Santa Cruz, CA)
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was used at a 1:50 dilution to detect cMaf. The primary antibodies were detected using goat anti-rabbit IgG conjugated to Alexafluor 568 (Molecular Probes, Eugene, OR) and nuclei were detected using the nucleic acid stain Syto13 (Molecular Probes). A detailed protocol may be found in Reed et al. (2001)). Acknowledgements We thank Dr Kirk Czymmek of the Core Microscopy Facilities of the University of Delaware for technical support. This work was supported by a NIH RO1 grant (EY12221) to MKD, a SigmaXI-National Academy of Science grant-in-aid award to W.C. and NEI intramural research funds to S.I.T. References Belecky-Adams, T., Tomarev, S., Li, H.S., Ploder, L., McInnes, R.R., Sundin, O., Adler, R., 1997. Pax-6, Prox 1, and Chx10 homeobox gene expression correlates with phenotypic fate of retinal precursor cells. Invest. Ophthalmol. Vis. Sci. 38, 1293–1303. Demidenko, Z., Badenhorst, P., Jones, T., Bi, X., Mortin, M.A., 2001. Regulated nuclear export of the homeodomain transcription factor. Prospero. Dev. 128, 1359–1367. Glasgow, E., Tomarev, S.I., 1998. Restricted expression of the homeobox gene prox1 in developing zebrafish. Mech. Dev. 76, 175–178. Hirata, J., Nakagoshi, H., Nabeshima, Y., Matsuzaki, F., 1995. Asymmetric segregation of the homeodomain protein Prospero during Drosophila development. Nature 377, 627–630. Kawauchi, S., Takahashi, S., Nakajima, O., Ogino, H., Morita, M., Nishizawa, M., Yasuda, K., Yamamoto, M., 1999. Regulation of lens fiber cell differentiation by transcription factor c-Maf. J. Biol. Chem. 274, 19254–19260. Knoblich, J.A., Jan, L.Y., Jan, Y.N., 1995. Asymmetric segregation of Numb and Prospero during cell division. Nature 377, 624–627.
Li, L., Vaessin, H., 2000. Pan-neural Prospero terminates cell proliferation during Drosophila neurogenesis. Genes Dev. 14, 147–151. Manning, L., Doe, C.Q., 1999. Prospero distinguishes sibling cell fate without asymmetric localization in the Drosophila adult external sense organ lineage. Development 126, 2063–2071. Oliver, G., Sosa-Pineda, B., Geisendorf, S., Spana, E.P., Doe, E.Q., Gruss, P., 1993. Prox1, a prospero-related homeobox gene expressed during mouse development. Mech. Dev. 44, 3–16. Reddy, G.V., Rodrigues, V., 1999. Sibling cell fate in the Drosophila adult external sense organ lineage is specified by prospero function, which is regulated by Numb and Notch. Development 126, 2083–2092. Reed, N.A., Oh, D., Czymmek, K.J., Duncan, M.K., 2001. An immunohistochemical method for the detection of proteins in the vertebrate lens. J. Immunol. Methods 253, 243–252. Ring, B.Z., Cordes, S.P., Overbeek, P.A., Barsh, G.S., 2000. Regulation of mouse lens fiber cell development and differentiation by the Maf gene. Development 127, 307–317. Sosa-Pineda, B., Wigle, J.T., Oliver, G., 2000. Hepatocyte migration during liver development requires Prox1. Nat. Genet. 25, 254–255. Spana, E.P., Doe, C.Q., 1995. The prospero transcription factor is asymmetrically localized to the cell cortex during neuroblast mitosis in Drosophila. Development 121, 3187–3195. Taketo, M., Schroeder, A.C., Mobraaten, L.E., Gunning, K.B., Hanten, G., Fox, R.R., Roderick, T.H., Stewart, C.L., Lilly, F., Hansen, C.T., et al., 1991. FVB/N: an inbred mouse strain preferable for transgenic analyses. Proc. Natl Acad. Sci. USA 88, 2065–2069. Tomarev, S.I., Sundin, O., Banerjee-Basu, S., Duncan, M.K., Yang, J.-M., Piatigorsky, J., 1996. Chicken homeobox gene Prox1 related to Drosophila prospero is expressed in the developing lens and retina. Dev. Dyn. 206, 354–367. Torii, M., Matsuzaki, F., Osumi, N., Kaibuchi, K., Nakamura, S., Casarosa, S., Guillemot, F., Nakafuku, M., 1999. Transcription factors Mash-1 and Prox-1 delineate early steps in differentiation of neural stem cells in the developing central nervous system. Development 126, 443– 456. Wigle, J.T., Chowdhury, K., Gruss, P., Oliver, G., 1999. Prox1 function is crucial for mouse lens-fibre elongation. Nat. Genet. 21, 318– 322. Wigle, J.T., Oliver, G., 1999. Prox1 function is required for the development of the murine lymphatic system. Cell 98, 769–778.
Gene expression pattern
Prox1 is differentially localized during lens development Melinda K. Duncan a,*, Wenwu Cui a, Dong-Jin Oh a, Stanislav I. Tomarev b a
b
Department of Biological Sciences, University of Delaware, Newark, DE 19716, USA Section on Molecular Genetics, Laboratory of Molecular and Developmental Biology, National Eye Institute, Bethesda, MD 20892, USA Received 17 September 2001; received in revised form 23 November 2001; accepted 29 November 2001
Abstract Prox1, the vertebrate cognate of Drosophila Prospero, is a homeodomain protein essential for the development of the lens, liver and lymphatic system. While it is well established that the subcellular distribution of Prospero changes during development, this had not been demonstrated for Prox1. Here, high-resolution confocal microscopy demonstrated that Prox1 protein is predominately cytoplasmic in the lens placode as well as the lens epithelium and germinative zone throughout development. However during fiber cell differentiation, Prox1 protein redistributes to cell nuclei. Finally, as lens fiber cells condense their chromatin in response to lens denucleation, Prox1 remains in the nucleus but does not appear to interact with DNA. Thus, it appears that the function of Prox1, like that of its Drosophila cognate Prospero, is at least partially controlled by changes in its subcellular distribution during development. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Lens development; Prox1; Confocal microscopy; Crystallin; Transcription; Promoter; Chromatin; Subcellular distribution
1. Results Prox1 is an atypical homeodomain protein related to Drosophila prospero. Homozygous Prox1 knockout mice have defects in the elongation of lens fiber cells (Wigle et al., 1999), the migration of hepatocytes during liver development (Sosa-Pineda et al., 2000) and the development of the lymphatic system (Wigle and Oliver, 1999). Prox1 mRNA is first detected in the lens placode and later in the lens of mouse, chicken and zebrafish embryos (Glasgow and Tomarev, 1998; Oliver et al., 1993; Tomarev et al., 1996). Later, the expression pattern of Prox1 becomes complex with expression in the neural retina, developing brain, liver and lymphatic system (Belecky-Adams et al., 1997; Sosa-Pineda et al., 2000; Torii et al., 1999; Wigle and Oliver, 1999). In mice, Prox1 protein is first detected at 9.5 dpc in the cytoplasm of the lens placode and adjacent head ectoderm (Fig. 1a, b). At 10.5 dpc, significant nuclear Prox1 is detected in the posterior portion of the lens pit (Fig. 1c), while Prox1 is found in both cytoplasm and nuclei in the walls of the lens pit which give rise to the lens epithelium (Fig. 1d). Note that the staining in the head ectoderm that will give rise to the corneal epithelium has decreased substantially. At 11.5 and 12.5 dpc, intense Prox1 staining is detected in the nuclei (excluding the nucleoli) in the posterior portion of the lens vesicle (Fig.
* Corresponding author. Tel.: 11-302-831-0533; fax: 11-302-831-2281. E-mail address: [email protected] (M.K. Duncan).
1e,h,i) while both cytoplasmic and nuclear Prox1 staining is still detectable in the presumptive lens epithelium (Fig. 1f,g). At 16.5 dpc (Fig. 1j,k), the pattern is similar to that at 12.5 dpc, however, Prox1 staining in lens fiber cell nuclei in deep layers is seen in discrete nuclear zones excluded from DNA (Fig. 1l). Further, this general pattern was observed in the mouse lens well into adulthood (data not shown). Thus, it appears that Prox1 protein is found in the cytoplasm (and to a much lesser extent the nucleus) of mouse lens epithelial cells, in the nucleus associated with DNA in the transition zone, and in nuclear regions excluded from DNA in mouse lens fiber cells which have initiated chromatin condensation. In order to determine whether the differential localization of Prox1 in the lens is observed in other vertebrates, Prox1 immunohistochemistry was performed on rat (Fig. 2a,d,g–i), human (Fig. 2b,e) and chicken lens (Fig. 2c,f). In all three cases, Prox1 protein was detected in both the cytoplasm and nucleus of lens epithelial cells (Fig. 2a–c) and appeared to both increase in levels and concentrate in the nuclei of newly differentiating lens fiber cells (Fig. 2d–f). Further, in all four species examined, Prox1 protein was excluded from chromatin in deep cortical fibers undergoing nuclear condensation (Fig. 2g–i; data not shown). In contrast, cMaf, another protein known to be essential for lens fiber cell differentiation (Kawauchi et al., 1999; Ring et al., 2000) was strictly associated with the nuclei of lens epithelial cells (Fig. 2j–l). Notably, Prospero, the Drosophila cognate of Prox1, is also often found in the cytoplasm of proliferating and undif-
0925-4773/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0925-477 3(01)00645-1
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Fig. 1. Changes in Prox1 protein localization during mouse lens development. (A) Presumptive eye of a 9.5 dpc embryo; note that Prox1 protein is primarily cytoplasmic in the head ectoderm. Bar ¼ 60 mm. (B) Higher magnification of the lens placode and optic vesicle shown in A. Bar ¼ 10 mm (C) Eye of a 10.5 dpc embryo; note that Prox1 protein is primarily nuclear in the posterior portion of the lens pit. Bar ¼ 60 mm. (D) Higher magnification of the tip of the closing lens pit boxed in panel B; note that Prox1 is detected in both the cytoplasm and nucleus of cells that will give rise to lens epithelium. Bar ¼ 6 mm. (E) Eye of a late 10.5 dpc embryo showing that Prox1 protein is predominately localized to the posterior portion of the lens vesicle. Bar ¼ 60 mm. (F) Higher magnification of the area boxed in white in panel E; note that significant amounts of cytoplasmic Prox1 are detected in both the presumptive lens epithelium and presumptive corneal epithelium. Bar ¼ 10 mm. (G) Same image shown in F with the DNA signal subtracted. Note that nuclear Prox1 levels vary between nuclei in the presumptive epithelium. Bar ¼ 10 mm. (H) Higher magnification of the area boxed in yellow in panel E, note that Prox1 is co-localized with the nuclear DNA excluding the nucleolus in presumptive lens fiber cells. Bar ¼ 6 mm. (I) Eye of a 12.5 dpc embryo, note the high levels of Prox1 protein detected in the nuclei of primary lens fibers. Bar ¼ 60 mm. (J) Eye of a 16.5 dpc mouse embryo; note the upregulation of nuclear Prox1 in the lens fibers at the transition zone. Bar ¼ 100 mm. (K) Same image shown in (J) with the DNA signal subtracted; note that some nuclear Prox1 is found in the lens epithelial cells at this stage. (I) Higher magnification of the area boxed in panel (J), note that significant nuclear Prox 1 in lens fiber cells beginning terminal differentiation is not co-localized with chromatin. Bar ¼ 6 mm. Green, DNA; Red, Prox1; Yellow, overlap. Abbreviations: ov, optic vesicle; pl, lens placode; pr, presumptive retina; lp, lens pit; pc, presumptive cornea; tp, tip of the lens pit; lv, lens vesicle; pe, presumptive lens epithelium; pf, primary lens fibers; e, lens epithelium; c, cornea; r, retina; f, lens fibers; t, transition zone. Arrowheads, regions of nuclear Prox1 excluded from DNA.
ferentiated precursors, relocalizing to the nucleus of postmitotic, differentiating cells (Li and Vaessin, 2000). In the larval central nervous system, Prospero is first seen in the cytoplasm of the neuroblast and relocalizes to the cortical crescent, which is asymmetrically distributed to the ganglion mother cell (Hirata et al., 1995; Knoblich et al., 1995; Spana and Doe, 1995). Since the amino acids responsible for the localization of Prospero to the cortical crescent are not conserved in Prox1 (Hirata et al., 1995; Tomarev et al., 1996), it is not surprising that Prox1 does not localize to a discrete region of cytoplasm. However, in Drosophila adult
sensory organ precursors, Prospero protein is evenly distributed in the cytoplasm of proliferating cells while nuclear Prospero was only found in postmitotic thecogen cells (Manning and Doe, 1999; Reddy and Rodrigues, 1999). Interestingly, the control of Prospero localization has been recently attributed to the presence of a nuclear export signal in the homeodomain whose function is controlled by the conformation of the prospero domain (Demidenko et al., 2001). Since the homeo- and Prospero domains are well conserved between Prox1 and Prospero (Tomarev et al., 1996), it seems likely that Prox1 uses a similar mechanism.
M.K. Duncan et al. / Mechanisms of Development 112 (2002) 195–198
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Fig. 2. Prox1 is found in both the cytoplasm and nucleus of lens epithelial cells in multiple species. (A,D,G–I) Lens from a 14-day-old rat stained for Prox1. (B,E) Lens from a 21-year-old human stained for Prox1. (C,F) Lens from an 11-day embryonic chicken stained for Prox1. Note that Prox1 is found in the cytoplasm and to a lesser extent the nucleus of lens epithelial cells (A–C; bar ¼ 20 mm) and becomes predominantly nuclear in lens fiber cells (D–F; bar ¼ 60 mm). (G–I) Nuclei from terminally differentiating 14 day rat lens fiber cells bar ¼ 10 mm. (G) Prox1 (H) DNA. (I) Merged. Note that nuclear Prox1 does not co-localize with DNA in lens fibers with condensing chromatin (arrow). (J–L) cMaf protein localization in the lens epithelium from a 14-dayold rat. Note that cMaf is predominantly a nuclear protein in the lens epithelium bar ¼ 20 mm. Green, DNA; Red, cMaf; Yellow, overlap. Abbreviations: e, lens epithelium; f, lens fiber cells; t, transition zone; ap, annular pad.
2. Materials and methods
Human lenses were isolated from donor cadaver eyes by the Oregon Lions Eye Bank (Portland, OR).
2.1. Tissue collection All mouse embryos were obtained from timed pregnant matings of FVB/N mice (Taketo et al., 1991) with noon of the date that the vaginal plug was observed defined to be 0.5 days post coitum (dpc). Rat eyes were isolated from 14-day post natal (dpn) Long–Evans rats. Chicken lenses were isolated from 11 day single comb white leghorn embryos.
2.2. Immunohistochemistry An affinity purified rabbit polyclonal antibody raised against the C-terminal homeo- and Prospero domains of human Prox1 (Belecky-Adams et al., 1997) was used at a 1:200 dilution to detect Prox1 protein and a polyclonal anticMaf antibody (Santa Cruz Biotechnology, Santa Cruz, CA)
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M.K. Duncan et al. / Mechanisms of Development 112 (2002) 195–198
was used at a 1:50 dilution to detect cMaf. The primary antibodies were detected using goat anti-rabbit IgG conjugated to Alexafluor 568 (Molecular Probes, Eugene, OR) and nuclei were detected using the nucleic acid stain Syto13 (Molecular Probes). A detailed protocol may be found in Reed et al. (2001)). Acknowledgements We thank Dr Kirk Czymmek of the Core Microscopy Facilities of the University of Delaware for technical support. This work was supported by a NIH RO1 grant (EY12221) to MKD, a SigmaXI-National Academy of Science grant-in-aid award to W.C. and NEI intramural research funds to S.I.T. References Belecky-Adams, T., Tomarev, S., Li, H.S., Ploder, L., McInnes, R.R., Sundin, O., Adler, R., 1997. Pax-6, Prox 1, and Chx10 homeobox gene expression correlates with phenotypic fate of retinal precursor cells. Invest. Ophthalmol. Vis. Sci. 38, 1293–1303. Demidenko, Z., Badenhorst, P., Jones, T., Bi, X., Mortin, M.A., 2001. Regulated nuclear export of the homeodomain transcription factor. Prospero. Dev. 128, 1359–1367. Glasgow, E., Tomarev, S.I., 1998. Restricted expression of the homeobox gene prox1 in developing zebrafish. Mech. Dev. 76, 175–178. Hirata, J., Nakagoshi, H., Nabeshima, Y., Matsuzaki, F., 1995. Asymmetric segregation of the homeodomain protein Prospero during Drosophila development. Nature 377, 627–630. Kawauchi, S., Takahashi, S., Nakajima, O., Ogino, H., Morita, M., Nishizawa, M., Yasuda, K., Yamamoto, M., 1999. Regulation of lens fiber cell differentiation by transcription factor c-Maf. J. Biol. Chem. 274, 19254–19260. Knoblich, J.A., Jan, L.Y., Jan, Y.N., 1995. Asymmetric segregation of Numb and Prospero during cell division. Nature 377, 624–627.
Li, L., Vaessin, H., 2000. Pan-neural Prospero terminates cell proliferation during Drosophila neurogenesis. Genes Dev. 14, 147–151. Manning, L., Doe, C.Q., 1999. Prospero distinguishes sibling cell fate without asymmetric localization in the Drosophila adult external sense organ lineage. Development 126, 2063–2071. Oliver, G., Sosa-Pineda, B., Geisendorf, S., Spana, E.P., Doe, E.Q., Gruss, P., 1993. Prox1, a prospero-related homeobox gene expressed during mouse development. Mech. Dev. 44, 3–16. Reddy, G.V., Rodrigues, V., 1999. Sibling cell fate in the Drosophila adult external sense organ lineage is specified by prospero function, which is regulated by Numb and Notch. Development 126, 2083–2092. Reed, N.A., Oh, D., Czymmek, K.J., Duncan, M.K., 2001. An immunohistochemical method for the detection of proteins in the vertebrate lens. J. Immunol. Methods 253, 243–252. Ring, B.Z., Cordes, S.P., Overbeek, P.A., Barsh, G.S., 2000. Regulation of mouse lens fiber cell development and differentiation by the Maf gene. Development 127, 307–317. Sosa-Pineda, B., Wigle, J.T., Oliver, G., 2000. Hepatocyte migration during liver development requires Prox1. Nat. Genet. 25, 254–255. Spana, E.P., Doe, C.Q., 1995. The prospero transcription factor is asymmetrically localized to the cell cortex during neuroblast mitosis in Drosophila. Development 121, 3187–3195. Taketo, M., Schroeder, A.C., Mobraaten, L.E., Gunning, K.B., Hanten, G., Fox, R.R., Roderick, T.H., Stewart, C.L., Lilly, F., Hansen, C.T., et al., 1991. FVB/N: an inbred mouse strain preferable for transgenic analyses. Proc. Natl Acad. Sci. USA 88, 2065–2069. Tomarev, S.I., Sundin, O., Banerjee-Basu, S., Duncan, M.K., Yang, J.-M., Piatigorsky, J., 1996. Chicken homeobox gene Prox1 related to Drosophila prospero is expressed in the developing lens and retina. Dev. Dyn. 206, 354–367. Torii, M., Matsuzaki, F., Osumi, N., Kaibuchi, K., Nakamura, S., Casarosa, S., Guillemot, F., Nakafuku, M., 1999. Transcription factors Mash-1 and Prox-1 delineate early steps in differentiation of neural stem cells in the developing central nervous system. Development 126, 443– 456. Wigle, J.T., Chowdhury, K., Gruss, P., Oliver, G., 1999. Prox1 function is crucial for mouse lens-fibre elongation. Nat. Genet. 21, 318– 322. Wigle, J.T., Oliver, G., 1999. Prox1 function is required for the development of the murine lymphatic system. Cell 98, 769–778.