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Developmental expression of DCC in the rat retina
Developmental Brain Research 130 (2001) 133–138 www.elsevier.com / locate / bres
Short communication
Developmental expression of DCC in the rat retina ¨ ˚ Kjell Johansson*, Marie Torngren, Johan Wasselius, Lina Mansson, Berndt Ehinger Department of Ophthalmology, Wallenberg Retina Center, BMC, B13, SE-221 84 Lund, Sweden Accepted 26 June 2001
Abstract The protein product of the deleted in colorectal cancer (DCC) gene possesses netrin-binding activity and may be involved in axonal guidance during retinal development. The temporal and spatial expression of DCC was analyzed in developing rat retina by means of immunoblotting and immunohistochemistry as well as by reverse transcription-polymerase chain reaction. Transient DCC protein expression is evident on ganglion cell axons in embryonic and neonatal retina. Double labeling experiments demonstrate DCC immunolabeling on processes that stratify in the inner plexiform layer and are derived from cholinergic amacrine cells. This pattern is maintained during the early postnatal period. DCC immunolabeling in the inner plexiform layer declines with age and is not observed in adult retina. The down-regulation of the DCC protein is confirmed by Western blot analysis. mRNA for DCC is expressed in embryonic, postnatal and adult retina and shows no correlation with the protein down-regulation. We suggest that DCC expression may be correlated with the functional segregation of the inner plexiform layer. 2001 Elsevier Science B.V. All rights reserved. Theme: Development and regeneration Topic: Process outgrowth, growth cones, and sprouting Keywords: DCC; ChAT; Retina; Axon guidance; Development
The extracellular environment provides developing axons with diffusible factors that control the accurate guidance and the appropriate establishment of neuronal connections. These factors act as chemoattractive or chemorepulsive guidance cues, and a gradient of a cue may promote or inhibit outgrowing axons into target areas of the embryonic nervous system [19]. One set of diffusible guidance cues for developing neurons is a family of laminin-related molecules referred to as netrins. A transmembrane protein encoded by the gene ‘deleted in colorectal cancer’ (DCC) acts as guidance receptor for netrins [9,10]. Both netrin and DCC mRNA transcripts and the corresponding proteins are present in various regions of the embryonic brain at the time when axons are actively extending [3,4,7,10,13,17]. Low levels of DCC transcript have also been found by reverse transcription-polymerase chain reaction (RT-PCR) in adult mouse brain [15]. Retinal ganglion cells have an embryonic birthday [1,16], and their axons are guided centrally towards the *Corresponding author. Tel.: 146-46-222-0765; fax: 146-46-2220774. E-mail address: [email protected] (K. Johansson).
optic disc by different molecular cues [18]. The postnatal period of retinal development includes the growth of bipolar cell axons towards ganglion cell dendrites with which they establish connections. During this period, amacrine cells mature and their processes attain their adult connectivity pattern. There is a dynamic period of retinal development before eye opening, and it is not known if specific molecules govern axons within the plexiform layers. Our main objectives were to determine the temporal sequence of DCC mRNA and protein expression in the rat retina and correlate DCC expression with developmental events. Eyes from embryonic day 20 (E20) and postnatal days 0, 7, 14, 21 (P0, P7, P14, P21, respectively) and adult pigmented rats (B&K Universal, Sollentuna, Sweden) were fixed in 4% paraformaldehyde in 0.1 M phosphate buffer (PB) for 4 h at 48C. Cryo-sections were incubated with a monoclonal antibody raised against an epitope in the intracellular domain of the DCC receptor (Pharmingen, San Diego, CA, USA) and were used at 1:1000. The primary antibody was detected with a Texas Red-conjugated donkey anti-mouse IgG (Jackson, West Grove, PA, USA) diluted 1:200.
0165-3806 / 01 / $ – see front matter 2001 Elsevier Science B.V. All rights reserved. PII: S0165-3806( 01 )00221-8
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K. Johansson et al. / Developmental Brain Research 130 (2001) 133 – 138
Sections of P7, P14, P21 and adult retinas were doublelabeled for DCC and choline acetyltransferase (ChAT: ¨ dilution 1:2000) (Biometra, Gottingen, Germany). Sections of P10 retinas were double-labeled for DCC and the vesicular acetylcholine transporter protein (VAChT) (Chemicon, Temecula, CA, USA). Colocalization analysis of DCC and VAChT immunoreactivities was examined using a confocal laser-scanning microscope imaging system as described previously [21]. For immunoblotting, neural retina from three to four eyes in each developmental stage was pooled and an equal amount of protein (48.7 mg) from each stage was subjected to sodium dodecyl sulfatepolyacrylamide gel (7.5%). The immunoreactivity was visualized using the ECL detection method according to the manufacturer (Amersham, Buckinghamshire, UK). Total RNA was prepared using TRIzol solution (GibcoBRL, Paisley, UK). First strand cDNA was transcribed from 2 mg total RNA using oligo-dT and MuLV transcriptase (RNA PCR Core kit protocol; Perkin-Elmer, Foster City, CA, USA). cDNA was amplified (35 cycles) using an Eppendorf thermocycler in a reaction containing DCC-specific primers in the RNA PCR Core kit. Primers specific for the rat homologue of DCC were selected from Genebank sequences and synthesized (DNA Technology A / S, Aarhus, Denmark) as follows: forward primer, 59ACC CTT TGC TAC CTC CAC CT 39; and reverse primer, 59TGT TCG CTC AAG TCA TCC TG 39. A 380-bp glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA was amplified (35 cycles) from each developmental stage as an internal. The PCR products were subjected to electrophoresis on a 1.5% agarose gel and a SYBR Gold nucleic acid gel stain (Molecular Probes; Eugene, OR, USA) was used to visualize cDNAs. At E20 and P0, retinal ganglion cell axons in the nerve fiber layer (NFL) and optic nerve showed distinct DCC immunolabeling (Fig. 1A–D). At P0, DCC labeling was mainly observed in central parts of the retina (Fig. 1C and D). No or very weak DCC labeling was observed in the NFL or in the optic disc at P7, indicating a rapid downregulation of the protein on ganglion cell axons shortly after birth. DCC labeling was also observed within the developing inner plexiform layer (IPL) of both embryonic and postnatal retinas (Fig. 1B and D). At P0, the DCC labeling appeared diffuse and evenly distributed within this layer, but at P7 was localized to processes that ramified in two strata in the immature IPL (Fig. 2A). DCC labeling was observed in the IPL during postnatal development but declined from P10 and was down-regulated in the IPL of adult retinas (Fig. 2G). In retinal homogenate examined for DCC protein expression by immunoblotting, a major band was recognized at the predicted size of approximately 175–190 kDa [15] at E20, P0 and P7 (Fig. 1E). No DCC protein was detected in adult retina (Fig. 1E). Because the DCC antibody produced a labeling pattern similar to that displayed by cholinergic amacrine cells, a
double-labeling analysis was performed using an antibody directed against choline acetyltransferase (ChAT). At P7, very weak ChAT immunolabeling was evident in amacrine cell somata and terminals that ramified in two mirrorimaged strata in the IPL (Fig. 2B). In terms of temporal and spatial development, colocalization of DCC and ChAT immunoreactivities was evident during postnatal development to P10. The DCC expression declined with age (Fig. 2A, C, E and G), whereas the cholinergic strata continued to display strong ChAT immunolabeling intensities (Fig. 2D, F and H). Expression of DCC immunoreactivity on cholinergic terminals was further demonstrated by confocal microscopy of DCC and VAChT double-labeled P10 retinas. P10 retinas displayed optimal DCC staining and the cholinergic terminals were well developed (Fig. 3A and B). In the composite image of separate confocal images superimposed on each other, the immunolabeling of the terminals appeared to be co-localized (Fig. 3C). By using RT-PCR and primers specific for DCC, a 244-bp PCR product of the expected size for DCC was amplified from E20, P0, P7 and adult retina (Fig. 1F). RT-PCR analysis for GAPDH was performed on the same cDNAs and showed comparable efficiency between the samples (Fig. 1F). The data indicate that DCC protein can be down-regulated during development without necessarily decreasing the amounts of DCC mRNA. The emergence of DCC protein and mRNA expression was examined in the developing and adult rat retina. DCC protein expression predominated in two separate compartments during retinal development: ganglion cell axons and cholinergic amacrine cell processes. Immunoblotting demonstrated a band of the expected size for DCC [15] in embryonic and early postnatal tissues that was correlated with the immunohistochemical demonstration of DCC protein. The band was absent in simultaneously assayed extracts of adult rat retina, which do not show any DCC immunoreactivity. Small immunoreactive products were also detected in retina of E20 and P0, and may correspond to DCC degradation products as described previously [15]. mRNA for DCC was found in all investigated stages including adult retina. Whether or not this continued expression of DCC mRNA in adult retina relates to regulatory mechanisms at the post-transcriptional level is currently not known. However, low levels of mRNA for DCC are known to be present in some areas of the adult CNS [13], and are sometimes correlated with protein expression [15]. Our results suggest a regulated expression of DCC protein during two temporally distinct phases of rat retinal development. First, the distinct expression of DCC observed in retinal ganglion cell axons before and shortly after birth correlates with its presumed role as a guiding factor for ganglion cell axons [3,5,8]. DCC protein in axons was down-regulated before P7, which correlates well in time with completed ganglion cell axon guidance [2,14]. The decline of DCC expression in ganglion cell
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Fig. 1. DCC protein expression in embryonic day 20 (E20) and neonatal (P0) retinas. (A and B) By E20, DCC immunolabeling is present in the nerve fiber layer (NFL) and in the optic nerve (ON). (B) Higher magnification reveals a diffuse distribution of DCC immunolabeling in the embryonic inner plexiform layer (IPL). (C and D) Similar distribution pattern of DCC protein is observed in the nerve fiber layer (NFL), the optic nerve (ON) and the inner plexiform layer (IPL) by P0. (D) Micrographs at a higher magnification of the peripheral retina demonstrating the absence of DCC immunoreactivity in the nerve fiber layer (NFL). (E) Temporal profile of DCC protein expression in the retina by immunoblotting. Immunoreactivity for DCC is present in embryonic (E20) and postnatal (P0 and P7) retinas, but undetectable in adult (Ad) retinas. A size marker for 130 kDa is shown (arrow). Note also the presence of small immunoreactive products in E20 and P0 retinas. (F) Amplification of DCC cDNA from developing and mature rat retina. RNA was extracted from retina of the ages shown and amplified for 35 cycles. Size markers in base pairs are shown to the left. The arrow indicates the position of the 244-bp band. RT-PCR for GAPDH demonstrating that all RNA specimens were of comparable quality. Abbreviations: GCL, ganglion cell layer; IPL, inner plexiform layer; NBL, neuroblastic layer; NFL, nerve fiber layer; ON, optic nerve. Scale bars (A and C) 30 mm, (B and D) 10 mm.
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Fig. 2. Photomicrographs of P7 (A and B), P14 (C and D), P21 (E and F) and adult (G and H) retinas. The development of DCC (left column) and ChAT (right column) immunoreactivities is illustrated, as detected by double-labeled sections. (A–F) DCC and ChAT immunoreactivities are localized to the cholinergic strata in the IPL during postnatal stages (P7–P21). (G) The cholinergic strata of adult rats (Ad) are devoid of DCC immunoreactivity. Scale bar 15 mm.
axons begins at their distal parts after finishing their targeting phase and it continues proximally to their axons within the retina [7]. This may explain our observation of continued immunohistochemical expression of DCC in the optic nerve and nerve fiber layer after birth. Second, DCC protein is present in the IPL before birth, but persists in diminishing amounts from the end of the second postnatal week. In the mature rat retina, no DCC immunoreactivity was observed in this layer. The expression of DCC immunolabeling in the IPL coincides with the period in postnatal development when the amacrine cell populations
migrate and extend neurites into the IPL [11,14]. This feature is supported by previous observations of distinct DCC protein expression in differentiating amacrine cells [8,20]. Since DCC expression correlates with cell migration and the active guidance of neuronal processes [10], it may also govern the migration of neuroblasts and the navigation of neurites within the inner plexiform layer of the postnatal retina. Our data revealed a specific and transient expression of DCC on cholinergic amacrine cell processes during postnatal development. This DCC expression is an early
K. Johansson et al. / Developmental Brain Research 130 (2001) 133 – 138
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Committee for the Blind, as well as the Knut and Alice Wallenberg, the Crafoord, and the Clas Groschinsky Foundations.
References
Fig. 3. Confocal images of double-labeled P10 retina. (A and B) Localization of DCC (red) and VAChT (green) immunoreactivities to the cholinergic strata. (C) Micrographs of the DCC and VAChT positive strata shown merged, visualizing the coexpression of these markers. Scale bar 10 mm.
phenomenon that also temporally coincides when the cholinergic system emerges and establishes its connectivity [11,12]. In addition, cholinergic amacrine cells display spontaneous activity during early postnatal development and have been suggested to be crucial for the formation of appropriate connections in the immature IPL [6]. Whether or not DCC is involved in the formation of the cholinergic plexus in the developing rat retina remains to be established.
Acknowledgements This study was supported by the Swedish Medical Research Council (13012-01A and 14X-2321), the Foundation Fighting Blindness, Crown Princess Margrets
[1] C.L. Cepko, C.P. Austin, X. Yang, M. Alexiades, D. Ezzeddine, Cell fate determination in the vertebrate retina, Proc. Natl. Acad. Sci. 93 (1996) 589–595. [2] D. Crespo, D.D.M. O’Leary, W.M. Cowan, Changes in the number of optic nerve fibers during late prenatal and late postnatal development in the albino rat, Dev. Brain Res. 19 (1985) 129–134. [3] M.S. Deiner, T.E. Kennedy, A. Fazeli, T. Serafini, M. TessierLavigne, D.W. Sretavan, Netrin-1 and DCC mediate axon guidance locally at the optic disc: loss of function leads to optic nerve hypoplasia, Neuron 19 (1997) 575–589. ¨ [4] J.R. de la Torre, V.H. Hopker, G.-L. Ming, M.-M. Poo, M. TessierLavigne, A. Hemmati-Brivanlou, C.E. Holt, Turning of retinal growth cones in a netrin-1 gradient mediated by the netrin receptor DCC, Neuron 19 (1997) 1211–1224. [5] A. Fazeli, S.L. Dickinson, M.L. Hermiston, R.V. Tighe, R.G. Steen, C.G. Small, E.T. Stoeckli, K. Keino-Masu, M. Masu, H. Rayburn, J. Simons, R.T. Bronson, J.I. Gordon, M. Tessier-Lavigne, R.A. Weinberg, Phenotype of mice lacking functional Deleted in colorectal cancer (DCC) gene, Nature 386 (1997) 796–804. [6] M.B. Feller, D.P. Wellis, D. Stellwagen, F.S. Werblin, C.J. Shatz, Requirement for cholinergic synaptic transmission in the propagation of spontaneous retina waves, Science 272 (1996) 1182–1187. [7] J.M. Gad, S.L. Keeling, A.F. Wilks, T. Seong-Seng, H.M. Cooper, The patterns of guidance receptors, DCC and neogenin, are spatially and temporally distinct throughout mouse embryogenesis, Dev. Biol. 192 (1997) 258–273. [8] J.M. Gad, S.L. Keeling, T. Shu, L.J. Richards, H.M. Cooper, The spatial and temporal expression of netrin receptors, DCC and neogenin, in the developing mouse retina, Exp. Eye Res. 70 (2000) 711–722. [9] K. Keino-Masu, M. Masu, L. Hinck, E.D. Leonardo, S.S.-Y. Chan, J.G. Culotti, M. Tessier-Lavigne, Deleted in colorectal cancer (DCC) encodes a netrin receptor, Cell 87 (1996) 175–185. [10] T.E. Kennedy, T. Serafini, J.R. de la Torre, M. Tessier-Lavigne, Netrins are diffusible chemotropic factors for commissural axons in the embryonic spinal cord, Cell 78 (1994) 425–435. [11] I.-B. Kim, E.-J. Lee, M.-K. Kim, D.-K. Park, M.-H. Chun, Choline acetyltransferase-immunoreactive neurons in the developing rat retina, J. Comp. Neurol. 27 (2000) 604–616. [12] P. Koulen, Vesicular acetylcholine transporter (VAChT) a cellular marker in rat retinal development, NeuroReport 8 (1997) 2845– 2848. [13] F.J. Livesey, S.P. Hunt, Netrin and netrin receptor expression in the embryonic mammalian nervous system suggests roles in retinal, striatal, nigral, and cerebellar development, Mol. Cell. Neurosci. 8 (1997) 417–429. [14] V.H. Perry, Z. Henderson, R. Linden, Postnatal changes in retinal ganglion cell and optic axon populations in the pigmented rat, J. Comp. Neurol. 219 (1983) 356–368. [15] M.R. Reale, G. Hu, A.I. Zafar, R.H. Getzenberg, S.M. Levine, E.R. Fearon, Expression and alternative splicing of the deleted in colorectal cancer (DCC) gene in normal and malignant tissues, Cancer Res. 54 (1994) 4493–4501. [16] B.E. Reese, R.J. Colello, Neurogenesis in the retinal ganglion cell layer of the rat, Neuroscience 46 (1992) 419–429. [17] T. Shu, K.M. Valentino, C. Seaman, H.M. Cooper, L.J. Richards, Expression of the netrin-1 receptor, deleted in colorectal cancer
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(DCC), is largely confined to projecting neurons in the developing forebrain, J. Comp. Neurol. 416 (2000) 201–212. [18] C.A.O. Stuermer, M. Bastmeyer, The retinal axon’s pathfinding to the optic disc, Prog. Neurobiol. 62 (2000) 197–214. [19] M. Tessier-Lavigne, C. Goodman, The molecular biology of axon guidance, Science 274 (1996) 1123–1133.
[20] L.-C. Wang, R.A. Rachel, R.A. Marcus, C.A. Mason, Chemosuppression of retinal axon growth by mouse optic chiasm, Neuron 17 (1996) 979–990. ´ [21] J. Wasselius, K. Johansson, A. Bruun, C. Zucker, B. Ehinger, Correlations between cholinergic neurons and m2 receptors in the rat retina, NeuroReport 9 (1998) 1799–1802.
Short communication
Developmental expression of DCC in the rat retina ¨ ˚ Kjell Johansson*, Marie Torngren, Johan Wasselius, Lina Mansson, Berndt Ehinger Department of Ophthalmology, Wallenberg Retina Center, BMC, B13, SE-221 84 Lund, Sweden Accepted 26 June 2001
Abstract The protein product of the deleted in colorectal cancer (DCC) gene possesses netrin-binding activity and may be involved in axonal guidance during retinal development. The temporal and spatial expression of DCC was analyzed in developing rat retina by means of immunoblotting and immunohistochemistry as well as by reverse transcription-polymerase chain reaction. Transient DCC protein expression is evident on ganglion cell axons in embryonic and neonatal retina. Double labeling experiments demonstrate DCC immunolabeling on processes that stratify in the inner plexiform layer and are derived from cholinergic amacrine cells. This pattern is maintained during the early postnatal period. DCC immunolabeling in the inner plexiform layer declines with age and is not observed in adult retina. The down-regulation of the DCC protein is confirmed by Western blot analysis. mRNA for DCC is expressed in embryonic, postnatal and adult retina and shows no correlation with the protein down-regulation. We suggest that DCC expression may be correlated with the functional segregation of the inner plexiform layer. 2001 Elsevier Science B.V. All rights reserved. Theme: Development and regeneration Topic: Process outgrowth, growth cones, and sprouting Keywords: DCC; ChAT; Retina; Axon guidance; Development
The extracellular environment provides developing axons with diffusible factors that control the accurate guidance and the appropriate establishment of neuronal connections. These factors act as chemoattractive or chemorepulsive guidance cues, and a gradient of a cue may promote or inhibit outgrowing axons into target areas of the embryonic nervous system [19]. One set of diffusible guidance cues for developing neurons is a family of laminin-related molecules referred to as netrins. A transmembrane protein encoded by the gene ‘deleted in colorectal cancer’ (DCC) acts as guidance receptor for netrins [9,10]. Both netrin and DCC mRNA transcripts and the corresponding proteins are present in various regions of the embryonic brain at the time when axons are actively extending [3,4,7,10,13,17]. Low levels of DCC transcript have also been found by reverse transcription-polymerase chain reaction (RT-PCR) in adult mouse brain [15]. Retinal ganglion cells have an embryonic birthday [1,16], and their axons are guided centrally towards the *Corresponding author. Tel.: 146-46-222-0765; fax: 146-46-2220774. E-mail address: [email protected] (K. Johansson).
optic disc by different molecular cues [18]. The postnatal period of retinal development includes the growth of bipolar cell axons towards ganglion cell dendrites with which they establish connections. During this period, amacrine cells mature and their processes attain their adult connectivity pattern. There is a dynamic period of retinal development before eye opening, and it is not known if specific molecules govern axons within the plexiform layers. Our main objectives were to determine the temporal sequence of DCC mRNA and protein expression in the rat retina and correlate DCC expression with developmental events. Eyes from embryonic day 20 (E20) and postnatal days 0, 7, 14, 21 (P0, P7, P14, P21, respectively) and adult pigmented rats (B&K Universal, Sollentuna, Sweden) were fixed in 4% paraformaldehyde in 0.1 M phosphate buffer (PB) for 4 h at 48C. Cryo-sections were incubated with a monoclonal antibody raised against an epitope in the intracellular domain of the DCC receptor (Pharmingen, San Diego, CA, USA) and were used at 1:1000. The primary antibody was detected with a Texas Red-conjugated donkey anti-mouse IgG (Jackson, West Grove, PA, USA) diluted 1:200.
0165-3806 / 01 / $ – see front matter 2001 Elsevier Science B.V. All rights reserved. PII: S0165-3806( 01 )00221-8
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Sections of P7, P14, P21 and adult retinas were doublelabeled for DCC and choline acetyltransferase (ChAT: ¨ dilution 1:2000) (Biometra, Gottingen, Germany). Sections of P10 retinas were double-labeled for DCC and the vesicular acetylcholine transporter protein (VAChT) (Chemicon, Temecula, CA, USA). Colocalization analysis of DCC and VAChT immunoreactivities was examined using a confocal laser-scanning microscope imaging system as described previously [21]. For immunoblotting, neural retina from three to four eyes in each developmental stage was pooled and an equal amount of protein (48.7 mg) from each stage was subjected to sodium dodecyl sulfatepolyacrylamide gel (7.5%). The immunoreactivity was visualized using the ECL detection method according to the manufacturer (Amersham, Buckinghamshire, UK). Total RNA was prepared using TRIzol solution (GibcoBRL, Paisley, UK). First strand cDNA was transcribed from 2 mg total RNA using oligo-dT and MuLV transcriptase (RNA PCR Core kit protocol; Perkin-Elmer, Foster City, CA, USA). cDNA was amplified (35 cycles) using an Eppendorf thermocycler in a reaction containing DCC-specific primers in the RNA PCR Core kit. Primers specific for the rat homologue of DCC were selected from Genebank sequences and synthesized (DNA Technology A / S, Aarhus, Denmark) as follows: forward primer, 59ACC CTT TGC TAC CTC CAC CT 39; and reverse primer, 59TGT TCG CTC AAG TCA TCC TG 39. A 380-bp glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA was amplified (35 cycles) from each developmental stage as an internal. The PCR products were subjected to electrophoresis on a 1.5% agarose gel and a SYBR Gold nucleic acid gel stain (Molecular Probes; Eugene, OR, USA) was used to visualize cDNAs. At E20 and P0, retinal ganglion cell axons in the nerve fiber layer (NFL) and optic nerve showed distinct DCC immunolabeling (Fig. 1A–D). At P0, DCC labeling was mainly observed in central parts of the retina (Fig. 1C and D). No or very weak DCC labeling was observed in the NFL or in the optic disc at P7, indicating a rapid downregulation of the protein on ganglion cell axons shortly after birth. DCC labeling was also observed within the developing inner plexiform layer (IPL) of both embryonic and postnatal retinas (Fig. 1B and D). At P0, the DCC labeling appeared diffuse and evenly distributed within this layer, but at P7 was localized to processes that ramified in two strata in the immature IPL (Fig. 2A). DCC labeling was observed in the IPL during postnatal development but declined from P10 and was down-regulated in the IPL of adult retinas (Fig. 2G). In retinal homogenate examined for DCC protein expression by immunoblotting, a major band was recognized at the predicted size of approximately 175–190 kDa [15] at E20, P0 and P7 (Fig. 1E). No DCC protein was detected in adult retina (Fig. 1E). Because the DCC antibody produced a labeling pattern similar to that displayed by cholinergic amacrine cells, a
double-labeling analysis was performed using an antibody directed against choline acetyltransferase (ChAT). At P7, very weak ChAT immunolabeling was evident in amacrine cell somata and terminals that ramified in two mirrorimaged strata in the IPL (Fig. 2B). In terms of temporal and spatial development, colocalization of DCC and ChAT immunoreactivities was evident during postnatal development to P10. The DCC expression declined with age (Fig. 2A, C, E and G), whereas the cholinergic strata continued to display strong ChAT immunolabeling intensities (Fig. 2D, F and H). Expression of DCC immunoreactivity on cholinergic terminals was further demonstrated by confocal microscopy of DCC and VAChT double-labeled P10 retinas. P10 retinas displayed optimal DCC staining and the cholinergic terminals were well developed (Fig. 3A and B). In the composite image of separate confocal images superimposed on each other, the immunolabeling of the terminals appeared to be co-localized (Fig. 3C). By using RT-PCR and primers specific for DCC, a 244-bp PCR product of the expected size for DCC was amplified from E20, P0, P7 and adult retina (Fig. 1F). RT-PCR analysis for GAPDH was performed on the same cDNAs and showed comparable efficiency between the samples (Fig. 1F). The data indicate that DCC protein can be down-regulated during development without necessarily decreasing the amounts of DCC mRNA. The emergence of DCC protein and mRNA expression was examined in the developing and adult rat retina. DCC protein expression predominated in two separate compartments during retinal development: ganglion cell axons and cholinergic amacrine cell processes. Immunoblotting demonstrated a band of the expected size for DCC [15] in embryonic and early postnatal tissues that was correlated with the immunohistochemical demonstration of DCC protein. The band was absent in simultaneously assayed extracts of adult rat retina, which do not show any DCC immunoreactivity. Small immunoreactive products were also detected in retina of E20 and P0, and may correspond to DCC degradation products as described previously [15]. mRNA for DCC was found in all investigated stages including adult retina. Whether or not this continued expression of DCC mRNA in adult retina relates to regulatory mechanisms at the post-transcriptional level is currently not known. However, low levels of mRNA for DCC are known to be present in some areas of the adult CNS [13], and are sometimes correlated with protein expression [15]. Our results suggest a regulated expression of DCC protein during two temporally distinct phases of rat retinal development. First, the distinct expression of DCC observed in retinal ganglion cell axons before and shortly after birth correlates with its presumed role as a guiding factor for ganglion cell axons [3,5,8]. DCC protein in axons was down-regulated before P7, which correlates well in time with completed ganglion cell axon guidance [2,14]. The decline of DCC expression in ganglion cell
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Fig. 1. DCC protein expression in embryonic day 20 (E20) and neonatal (P0) retinas. (A and B) By E20, DCC immunolabeling is present in the nerve fiber layer (NFL) and in the optic nerve (ON). (B) Higher magnification reveals a diffuse distribution of DCC immunolabeling in the embryonic inner plexiform layer (IPL). (C and D) Similar distribution pattern of DCC protein is observed in the nerve fiber layer (NFL), the optic nerve (ON) and the inner plexiform layer (IPL) by P0. (D) Micrographs at a higher magnification of the peripheral retina demonstrating the absence of DCC immunoreactivity in the nerve fiber layer (NFL). (E) Temporal profile of DCC protein expression in the retina by immunoblotting. Immunoreactivity for DCC is present in embryonic (E20) and postnatal (P0 and P7) retinas, but undetectable in adult (Ad) retinas. A size marker for 130 kDa is shown (arrow). Note also the presence of small immunoreactive products in E20 and P0 retinas. (F) Amplification of DCC cDNA from developing and mature rat retina. RNA was extracted from retina of the ages shown and amplified for 35 cycles. Size markers in base pairs are shown to the left. The arrow indicates the position of the 244-bp band. RT-PCR for GAPDH demonstrating that all RNA specimens were of comparable quality. Abbreviations: GCL, ganglion cell layer; IPL, inner plexiform layer; NBL, neuroblastic layer; NFL, nerve fiber layer; ON, optic nerve. Scale bars (A and C) 30 mm, (B and D) 10 mm.
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Fig. 2. Photomicrographs of P7 (A and B), P14 (C and D), P21 (E and F) and adult (G and H) retinas. The development of DCC (left column) and ChAT (right column) immunoreactivities is illustrated, as detected by double-labeled sections. (A–F) DCC and ChAT immunoreactivities are localized to the cholinergic strata in the IPL during postnatal stages (P7–P21). (G) The cholinergic strata of adult rats (Ad) are devoid of DCC immunoreactivity. Scale bar 15 mm.
axons begins at their distal parts after finishing their targeting phase and it continues proximally to their axons within the retina [7]. This may explain our observation of continued immunohistochemical expression of DCC in the optic nerve and nerve fiber layer after birth. Second, DCC protein is present in the IPL before birth, but persists in diminishing amounts from the end of the second postnatal week. In the mature rat retina, no DCC immunoreactivity was observed in this layer. The expression of DCC immunolabeling in the IPL coincides with the period in postnatal development when the amacrine cell populations
migrate and extend neurites into the IPL [11,14]. This feature is supported by previous observations of distinct DCC protein expression in differentiating amacrine cells [8,20]. Since DCC expression correlates with cell migration and the active guidance of neuronal processes [10], it may also govern the migration of neuroblasts and the navigation of neurites within the inner plexiform layer of the postnatal retina. Our data revealed a specific and transient expression of DCC on cholinergic amacrine cell processes during postnatal development. This DCC expression is an early
K. Johansson et al. / Developmental Brain Research 130 (2001) 133 – 138
137
Committee for the Blind, as well as the Knut and Alice Wallenberg, the Crafoord, and the Clas Groschinsky Foundations.
References
Fig. 3. Confocal images of double-labeled P10 retina. (A and B) Localization of DCC (red) and VAChT (green) immunoreactivities to the cholinergic strata. (C) Micrographs of the DCC and VAChT positive strata shown merged, visualizing the coexpression of these markers. Scale bar 10 mm.
phenomenon that also temporally coincides when the cholinergic system emerges and establishes its connectivity [11,12]. In addition, cholinergic amacrine cells display spontaneous activity during early postnatal development and have been suggested to be crucial for the formation of appropriate connections in the immature IPL [6]. Whether or not DCC is involved in the formation of the cholinergic plexus in the developing rat retina remains to be established.
Acknowledgements This study was supported by the Swedish Medical Research Council (13012-01A and 14X-2321), the Foundation Fighting Blindness, Crown Princess Margrets
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