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Heat shock protein 108 mRNA expression during chicken retina development
Neuroscience Letters 344 (2003) 25–28 www.elsevier.com/locate/neulet
Heat shock protein 108 mRNA expression during chicken retina development Dong Hoon Shina, Hyun Joon Kimb, Jaehyup Kima, Su-ryeon Baea, Sa Sun Choa,* a
Department of Anatomy, Seoul National University College of Medicine, Yongon-Dong 28, Seoul 110-799, South Korea b Department of Anatomy, Gyeongsang National University College of Medicine, Chinju, South Korea Received 27 January 2003; received in revised form 20 March 2003; accepted 26 March 2003
Abstract In a developmental study on the expression of heat shock protein 108 (HSP108) mRNA in the chicken retina, we found different spatial and temporal expressions of HSP108 mRNA in each retinal layer. While intense HSP108 signals were found in the retina neuroblast layer at embryonic day 5 (E5), the ganglion cell population (GC), inner nuclear layer (IN) and pigment epithelium (PE) showed HSP108 expression at E9. At E14, HSP108 signals were reduced versus the previous stages even though signals were still detected in the GC, the IN, the outer nuclear layer and the PE. HSP108 signals were still detectable at the E21 stage, although each retinal layer showed a much differentiated morphology and diminished signal intensity. These results suggest that HSP108 expression might be developmentally regulated throughout eye organogenesis and that it plays a role in ocular development. q 2003 Elsevier Science Ireland Ltd. All rights reserved. Keywords: In situ hybridization; Retina; Heat shock protein; Ganglion cell layer; Inner nuclear layer; Oligodendrocyte
The heat shock proteins (HSPs) are known to include a series of stress proteins, which are induced by several different stresses, such as heat, ischemia, inflammation, oxidative stress, and even behavioral and psychological stresses [8,10]. Although the exact roles of HSPs have not yet been clearly elucidated, several researchers have suggested that HSPs might play a role in the clearing or production of proteins formed in response to the various external stresses [22]. Even in the normal state, HSPs are induced by cell cycle progression, embryonic development, cell differentiation and growth in microorganisms [10,21]. The HSPs are classified into five different groups in accordance with their molecular weights, and HSP90, HSP70 and HSP60 have been studied in greatest detail. It is now known that HSPs act as molecular ‘chaperons’ in protein folding, unfolding, and oligomerization [12]. However, much study remains to be done before the exact roles of HSPs are understood under normal physiological conditions, because they are known to participate in a diverse range of cellular activities. HSP108 or Transferrin binding protein (TfBP), the avian * Corresponding author. Tel.: þ 82-2-740-8204; fax: þ82-2-745-9528. E-mail address: [email protected] (S.S. Cho).
homologue of the mammalian Glucose Regulated Protein 94 (GRP94) family of stress-regulated proteins [3,12], was originally purified from the chicken oviduct [23]. It exhibits transferrin binding activity and shares a number of physical properties with the human transferrin receptor [13]. However, based on peptide map analysis and immunologic studies [14], TfBP is clearly distinct from the avian erythrocyte transferrin receptor, and was identified as a HSP108, which was shown to be highly homologous to yeast HSP90 [3,11]. In our previous studies on the distribution of HSP108 mRNA in adult chicken brain, HSP108 mRNAs were found to be mainly localized in various types of neuroglial cells in the brain [18]. This tendency was also observed in the cerebellum; HSP108 signals are found in Bergmann glial cells and oligodendrocytes, but not in the Purkinje cells. Therefore, the expression pattern of HSP108 mRNA differs from that of its homologous HSP90, which is mainly expressed in neurons [15]. Although our previous studies on HSP108 mRNA suggest that HSP108 might play an important role in the normal metabolism of neuroglial cells in the chicken brain, no studies on the expression of HSP108 mRNA during chicken development have been available until now. In
0304-3940/03/$ - see front matter q 2003 Elsevier Science Ireland Ltd. All rights reserved. doi:10.1016/S0304-3940(03)00409-9
26
D.H. Shin et al. / Neuroscience Letters 344 (2003) 25–28
particular, in the case of the retina, several reports exist on the expressional changes of the other types of HSPs [4,9, 20], but no report is available on the expression of HSP108 mRNA during retinogenesis. Therefore, the present study was designed to determine the expression of HSP108 mRNA by in situ hybridization during retinal development. The animals used in this experiment were treated in accordance with The Guide for the Care and Use of Laboratory Animals (NIH publication No. 86-23, 1985 edition). The retinas of embryonic day 5 (E5), 9 (E9), 14 (E14) and 21 (E21) embryos were used in this study. Fertilized eggs were incubated at 38 8C in a humidified chamber, and subsequently the tissues were sliced to 12 mm on a cryostat (Reichert Jung), fixed in 4% (w/v) paraformaldehyde, and treated with chloroform for protein removal. Total cellular RNA was extracted from white leghorn chickens, aged E18, using the guanidine thiocyanate (GTC) method [16]. HSP108 cDNA was amplified by reversetranscription PCR (RT-PCR) using oligonucleotide primers, namely, 50 HSP108 primer: CCA GTT TGG TGT TGG CTT TT and 30 HSP108 primer: CCT CCT TTG CTT CCT CCT CT. These PCR primers were designed on the basis of previous cloning results [1,6]. The amplified PCR products obtained were cloned into a T easy vector (Promega). A digoxigenin-11-UTP (Boehringer-Mannheim) labeled antisense HSP108 cRNA probe was generated by transcribing NcoI (Boehringer-Mannheim) linearized plasmid with SP6 RNA polymerase (Boehringer-Mannheim). A sense probe was also transcribed with T7 RNA polymerase (BoehringerMannheim). In situ hybridization was performed using a previously described method [19]. Briefly, tissue sections were incubated overnight with 350 ng/ml sense and antisense probes at 42 8C in a humidified chamber. After hybridization, the sections were treated with RNase A (20 mg/ml) in NTE buffer (500 mM NaCl, 10 mM Tris, and 1 mM EDTA, at pH 8.0) to remove the unhybridized probes, immersed in buffer 1 (0.1 M maleic acid, 0.15 M NaCl, pH 7.5) containing 1% blocking reagent (Boehringer-Mannheim)
for 1 h at room temperature, and then incubated with sheep anti-digoxigenin antibody (1.5 units/ml). After incubation in biotinylated anti-sheep IgG (1:500, Vector) for 1 h, the sections were visualized with Cy3-conjugated streptavidin (1:1000, Jackson Immunoresearch) for 1 h. Sections were observed under a fluorescence microscope (Zeiss), and the mRNA expression of HSP108 in each retinal layer during prenatal development was analyzed densitometrically using the NIH image program (Scion Image). Densitometric data were expressed as mean densities, i.e. the sum of the gray values of all the pixels in a selection divided by the number of pixels. In situ hybridization of all sections treated with sense probes showed no specific HSP108 signals in any of the development stages, while sections treated with antisense probes showed meaningful signals. In the case of the sections treated with antisense probes, the HSP108 signals showed different spatial and temporal expressions in each retinal layer. Although the retina is composed almost entirely of a single cell population, i.e. neuroblast cells [17], intense HSP108 signals were found at E5 in the outermost region of the retinal neuroblast layer (NB) and in the pigment epithelium (PE) (Fig. 1A,B). The HSP108 mRNAs were mainly present within the cytoplasm near signal-free nuclear regions (Fig. 1C). At E9, the inner plexiform layer (IP) was discernable between the NB and the newly emerging ganglion cell population (GC) [17]. At this stage, intense HSP108 signals were observed within the GC, the inner region of the inner nuclear layer (IN) and the PE, while much weaker and fewer signals were detected in the optic fiber layer, the IP and the outer regions of the IN (Fig. 1D – F). According to a previous study, the major stratification of the retina is completed by E11, at which stage both plexiform layers are clearly discernable [17]. In the E14 retina, we observed that the general structure was similar to that of the adult stage, except that the photoreceptor layer was not completely differentiated (Fig. 1G). However, signals for HSP108 mRNA at E14 were much weaker than those at E9, though meaningful signals were still detected in the GC, the inner region of the
Table 1 Mean density of heat shock protein (HSP) 108 mRNA in the developing chicken retina E5 NB, inner
NB, outer
PE
0.81 ^ 0.17
137.42 ^ 9.14
19.57 ^ 1.92
NF GC IP IN, inner IN, outer OP ON PR PE
E9
E14
220.22 ^ 14.53 19.81 ^ 3.82 230.17 ^ 11.29 5.66 ^ 1.36
18.53 ^ 5.45 1.67 ^ 0.04 19.67 ^ 5.38 1.51 ^ 0.13 1.94 ^ 0.06 185.98 ^ 16.6
254.96 ^ 15.67
195.11 ^ 11.1
E21 6.84 ^ 1.87 28.93 ^ 2.11 5.76 ^ 1.81 24.94 ^ 3.7 9.49 ^ 2.24 4.60 ^ 1.56 64.63 ^ 6.69 58.37 ^ 5.25 69.48 ^ 7.49
The sum of the gray values of all the pixels in a selection was divided by the number of pixels within the selection. The maximum allowed gray value is 256. NB, neuroblast layer; PE, pigment epithelium; NF, nerve fiber layer; GC, ganglion cell layer; IP, inner plexiform layer; IN, inner nuclear layer; OP, outer plexiform layer; ON, outer nuclear layer; PR, photoreceptor layer.
D.H. Shin et al. / Neuroscience Letters 344 (2003) 25–28
Fig. 1. Heat shock protein 108 (HSP108) mRNA expressions during the development of the chicken retina. (A) Cresyl violet staining at embryonic day 5 (E5). The retina was composed of only neuroblast cells (NBs). PE, pigment epithelium. (B) HSP108 mRNA expression at E5. Only the outer layers of the NBs in the retina produced positive signals. PE also contained HSP108þ cells. (C) Magnified image of (B). HSP108 mRNA signals were mainly localized to the cytoplasm, and not to nuclei (arrowheads). (D) Cresyl violet staining at E9 showed that the inner plexiform layer (IP), which contained some punctuate cells (arrows), was discernable between the ganglion cell layer (GC) and the inner nuclear layer (IN). (E) HSP108 mRNA expression at E9. HSP108 signals were found mainly within the GC, and in the inner regions of the IN and PE, while much weaker signals were found in the nerve fiber layer, the IP and the outer regions of the IN. Cells (arrows) in the IP of (E) also showed HSP108 signals. (F) Magnified image of (E). (G) Cresyl violet staining of the E14 retina showed a similar structure to that of an adult retina, except for the absence of a photoreceptor layer. (H) Also at E14, HSP108 signals were significantly reduced though positive signals were detected in the GC, the inner region of the IN, throughout the outer nuclear layer (ON) and the PE. (I) Magnified image of the ON and the PE in (H). (J) A cresyl violet stained retina at E14. All the retinal layers were remarkable. PR, photoreceptor. (K) HSP108 mRNA expression at E14. HSP108 mRNA signals were detected in the GC, IN, ON and PR. The ON and the PR showed much more intense signals than the GC and the IN. (L) Magnified image of (K). Scale bar, 50 mm.
27
IN, and throughout the outer nuclear layer (ON) and the PE. In addition, we also found that HSP108 signals in the outer retinal layers, i.e. ON and PE, showed much more intense signals than those of the GC and IN (Fig. 1H,I). At E21, the general structure and HSP108 mRNA expression were similar to those at E14. Comparing the E14 and E21 retinas, each retinal layer at E21 showed a markedly more differentiated morphology and diminished signal intensity, though the distribution pattern of HSP108þ signals was relatively unaltered in the GC, IN, ON and in photoreceptors (Fig. 1J– L). At E21, the signal intensities of HSP108 mRNA in the outer retinal layers were still much more intense than those in the outer retinal layers. The densitometric findings of HSP108 mRNA expression in the retina at each stage are summarized in Table 1. Previous studies have reported that spatial and temporal expression changes occur in various HSPs during embryonic development [2,5,7]. According to these studies, some HSPs, which were detected at a high intensity in the early embryonic stages, began to decrease during the late embryonic stages. For example, HSP90s, which are known to be homologous to HSP108 [11], were maximally expressed at the earliest stage analyzed and then gradually decreased as embryogenesis progressed [7]. From these observations, it was concluded that HSPs may be expressed strongly during developmental stages involving cell proliferation rather than cell differentiation. On the other hand, it was also reported that the decreased expression of HSPs during the late embryonic stage might be important for the successful maturation of retinal cells [20]. Tanaka et al. [20] observed that some types of HSPs, i.e. HSC70, HSP84, HSP86 and HSP60, exhibited intense mRNA expression in the mouse retina during E11.5 to E14.5 and a marked decrease during E15.5 to E18.5. In another study on the expression of HSPs in the developing rat retina by Kojima et al. [4], the expression of inducible HSP70 was also found to be transiently decreased at the perinatal stage, while intense mRNA signals re-emerged after P7. The involvement of HSPs in the development of the retina was also observed by Morales et al. [9]; although they only observed the sections during E4 to E12, they found that various kinds of HSPs (HSP40, HSP60, HSP70 and HSP90) were expressed during retinal development in the chicken. Our results on HSP108 mRNA expression during eye development were very similar to those of previous studies. As shown in Table 1, intense HSP108 signals were observed at E9, and these decreased during E14 to E21. Moreover, HSP108 mRNA signal intensity changed in accord with each stage and retinal layer. Although a decrease in HSPs was observed during different embryonic days in the present and previous studies, this is probably explained by the differences in the types of HSPs examined or the species used. The intense expression of HSP108 mRNA in retinal cells during early developmental stages raises the possibility that HSP108 might be associated with cellular proliferation,
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D.H. Shin et al. / Neuroscience Letters 344 (2003) 25–28
because HSP108 signals seem to be correlated with active proliferation, such as in the outermost zone of neuroblast cells. This suggestion is supported by the weaker HSP mRNA signals observed during the later developmental stages, by which time cells had differentiated and proliferation was much reduced. In summary, despite being unable to explain the meaning of HSP108 expression during chicken retinal development, we suggest that HSP108 mRNA expression is developmentally regulated and that it plays an important role in ocular development.
Acknowledgements This study was supported by a grant (2000-1-20700-0063) from the Basic Research Program of the Korea Science and Engineering Foundation and a grant from 2002 BK21 project for medicine, dentistry and pharmacy.
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[8] S. Lindquist, E.A. Craig, The heat-shock proteins, Annu. Rev. Genet. 22 (1988) 631–677. [9] A.V. Morales, M. Hadjiargyrou, B. Diaz, C. Hernandez-Sanchez, F. de Pablo, E.J. de la Rosa, Heat shock proteins in retinal neurogenesis: identification of the PM1 antigen as the chick Hsc70 and its expression in comparison to that of other chaperones, Eur. J. Neurosci. 10 (1988) 3237–3245. [10] R.I. Morimoto, Cells in stress: transcriptional activation of heat shock genes, Science 259 (1993) 1409–1410. [11] I. Poola, An estrogen inducible 104kDa chaperone glycoprotein binds ferric iron containing proteins: a possible role in intracellular iron trafficking, FEBS Lett. 416 (1997) 139–142. [12] I. Poola, J.G. Kiang, The estrogen-inducible transferrin receptor-like membrane glycoprotein is related to stress-regulated proteins, J. Biol. Chem. 269 (1994) 21762–21769. [13] I. Poola, J.J. Lucas, Purification and characterization of an estrogen inducible membrane glycoprotein: evidence that it is a transferring receptor, J. Biol. Chem. 63 (1988) 19137–19146. [14] I. Poola, A.B. Mason, J.J. Lucas, The chicken oviduct and embryonic red blood cell transferrin receptors are distinct molecules, Biochem. Biophys. Res. Commun. 171 (1990) 26–32. [15] H. Quraishi, I.R. Brown, Expression of heat shock protein 90 (hsp90) in neural and nonneural tissues of the control and hyperthermic rabbit, Exp. Cell Res. 219 (1995) 358–363. [16] J. Sambrook, E.F. Fritsch, T. Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989. [17] A.M. Sheppard, M. Konopka, P.L. Jeffrey, Thy-1 expression in the retinotectal system of the chick, Dev. Brain Res. 43 (1988) 49–60. [18] D.H. Shin, H.J. Kim, H.Y. Lee, K.H. Lee, G.S. Jeon, J.H. Seo, S.H. Baik, S.S. Cho, Distribution of heat shock protein 108 mRNA in the chicken central nervous system, Neurosci. Lett. 283 (2000) 181 –184. [19] D.H. Shin, H.Y. Lee, H.W. Lee, H.J. Kim, E. Lee, S.S. Cho, S.H. Baik, K.H. Lee, In situ localization of p53, bcl-2 and bax mRNAs in rat ocular tissue, NeuroReport 10 (1999) 2165– 2167. [20] Y. Tanaka, K. Kobayashi, M. Kita, S. Kinoshita, J. Imanishi, Messenger RNA expression of heat shock proteins (HSPs) during ocular development, Curr. Eye Res. 14 (1995) 1125–1133. [21] D. Walsh, Z. Li, Y. Wu, K. Nagata, Heat shock and the role of the HSPs during neural plate induction in early mammalian CNS and brain development, Cell. Mol. Life Sci. 53 (1997) 198–211. [22] W.J. Welch, Mammalian stress response: cell physiology, structure/ function of stress proteins, and implications for medicine and disease, Physiol. Rev. 72 (1992) 1063–1081. [23] T. Zarucki-Schulz, M.S. Kulomaa, D.R. Headon, N.L. Weigel, M. Baez, D.P. Edwards, W.L. McGuire, W.T. Schrader, B.W. O’Malley, Molecular cloning of a cDNA for the chicken progesterone receptor B antigen, Proc. Natl. Acad. Sci. USA 81 (1984) 6358–6362.
Heat shock protein 108 mRNA expression during chicken retina development Dong Hoon Shina, Hyun Joon Kimb, Jaehyup Kima, Su-ryeon Baea, Sa Sun Choa,* a
Department of Anatomy, Seoul National University College of Medicine, Yongon-Dong 28, Seoul 110-799, South Korea b Department of Anatomy, Gyeongsang National University College of Medicine, Chinju, South Korea Received 27 January 2003; received in revised form 20 March 2003; accepted 26 March 2003
Abstract In a developmental study on the expression of heat shock protein 108 (HSP108) mRNA in the chicken retina, we found different spatial and temporal expressions of HSP108 mRNA in each retinal layer. While intense HSP108 signals were found in the retina neuroblast layer at embryonic day 5 (E5), the ganglion cell population (GC), inner nuclear layer (IN) and pigment epithelium (PE) showed HSP108 expression at E9. At E14, HSP108 signals were reduced versus the previous stages even though signals were still detected in the GC, the IN, the outer nuclear layer and the PE. HSP108 signals were still detectable at the E21 stage, although each retinal layer showed a much differentiated morphology and diminished signal intensity. These results suggest that HSP108 expression might be developmentally regulated throughout eye organogenesis and that it plays a role in ocular development. q 2003 Elsevier Science Ireland Ltd. All rights reserved. Keywords: In situ hybridization; Retina; Heat shock protein; Ganglion cell layer; Inner nuclear layer; Oligodendrocyte
The heat shock proteins (HSPs) are known to include a series of stress proteins, which are induced by several different stresses, such as heat, ischemia, inflammation, oxidative stress, and even behavioral and psychological stresses [8,10]. Although the exact roles of HSPs have not yet been clearly elucidated, several researchers have suggested that HSPs might play a role in the clearing or production of proteins formed in response to the various external stresses [22]. Even in the normal state, HSPs are induced by cell cycle progression, embryonic development, cell differentiation and growth in microorganisms [10,21]. The HSPs are classified into five different groups in accordance with their molecular weights, and HSP90, HSP70 and HSP60 have been studied in greatest detail. It is now known that HSPs act as molecular ‘chaperons’ in protein folding, unfolding, and oligomerization [12]. However, much study remains to be done before the exact roles of HSPs are understood under normal physiological conditions, because they are known to participate in a diverse range of cellular activities. HSP108 or Transferrin binding protein (TfBP), the avian * Corresponding author. Tel.: þ 82-2-740-8204; fax: þ82-2-745-9528. E-mail address: [email protected] (S.S. Cho).
homologue of the mammalian Glucose Regulated Protein 94 (GRP94) family of stress-regulated proteins [3,12], was originally purified from the chicken oviduct [23]. It exhibits transferrin binding activity and shares a number of physical properties with the human transferrin receptor [13]. However, based on peptide map analysis and immunologic studies [14], TfBP is clearly distinct from the avian erythrocyte transferrin receptor, and was identified as a HSP108, which was shown to be highly homologous to yeast HSP90 [3,11]. In our previous studies on the distribution of HSP108 mRNA in adult chicken brain, HSP108 mRNAs were found to be mainly localized in various types of neuroglial cells in the brain [18]. This tendency was also observed in the cerebellum; HSP108 signals are found in Bergmann glial cells and oligodendrocytes, but not in the Purkinje cells. Therefore, the expression pattern of HSP108 mRNA differs from that of its homologous HSP90, which is mainly expressed in neurons [15]. Although our previous studies on HSP108 mRNA suggest that HSP108 might play an important role in the normal metabolism of neuroglial cells in the chicken brain, no studies on the expression of HSP108 mRNA during chicken development have been available until now. In
0304-3940/03/$ - see front matter q 2003 Elsevier Science Ireland Ltd. All rights reserved. doi:10.1016/S0304-3940(03)00409-9
26
D.H. Shin et al. / Neuroscience Letters 344 (2003) 25–28
particular, in the case of the retina, several reports exist on the expressional changes of the other types of HSPs [4,9, 20], but no report is available on the expression of HSP108 mRNA during retinogenesis. Therefore, the present study was designed to determine the expression of HSP108 mRNA by in situ hybridization during retinal development. The animals used in this experiment were treated in accordance with The Guide for the Care and Use of Laboratory Animals (NIH publication No. 86-23, 1985 edition). The retinas of embryonic day 5 (E5), 9 (E9), 14 (E14) and 21 (E21) embryos were used in this study. Fertilized eggs were incubated at 38 8C in a humidified chamber, and subsequently the tissues were sliced to 12 mm on a cryostat (Reichert Jung), fixed in 4% (w/v) paraformaldehyde, and treated with chloroform for protein removal. Total cellular RNA was extracted from white leghorn chickens, aged E18, using the guanidine thiocyanate (GTC) method [16]. HSP108 cDNA was amplified by reversetranscription PCR (RT-PCR) using oligonucleotide primers, namely, 50 HSP108 primer: CCA GTT TGG TGT TGG CTT TT and 30 HSP108 primer: CCT CCT TTG CTT CCT CCT CT. These PCR primers were designed on the basis of previous cloning results [1,6]. The amplified PCR products obtained were cloned into a T easy vector (Promega). A digoxigenin-11-UTP (Boehringer-Mannheim) labeled antisense HSP108 cRNA probe was generated by transcribing NcoI (Boehringer-Mannheim) linearized plasmid with SP6 RNA polymerase (Boehringer-Mannheim). A sense probe was also transcribed with T7 RNA polymerase (BoehringerMannheim). In situ hybridization was performed using a previously described method [19]. Briefly, tissue sections were incubated overnight with 350 ng/ml sense and antisense probes at 42 8C in a humidified chamber. After hybridization, the sections were treated with RNase A (20 mg/ml) in NTE buffer (500 mM NaCl, 10 mM Tris, and 1 mM EDTA, at pH 8.0) to remove the unhybridized probes, immersed in buffer 1 (0.1 M maleic acid, 0.15 M NaCl, pH 7.5) containing 1% blocking reagent (Boehringer-Mannheim)
for 1 h at room temperature, and then incubated with sheep anti-digoxigenin antibody (1.5 units/ml). After incubation in biotinylated anti-sheep IgG (1:500, Vector) for 1 h, the sections were visualized with Cy3-conjugated streptavidin (1:1000, Jackson Immunoresearch) for 1 h. Sections were observed under a fluorescence microscope (Zeiss), and the mRNA expression of HSP108 in each retinal layer during prenatal development was analyzed densitometrically using the NIH image program (Scion Image). Densitometric data were expressed as mean densities, i.e. the sum of the gray values of all the pixels in a selection divided by the number of pixels. In situ hybridization of all sections treated with sense probes showed no specific HSP108 signals in any of the development stages, while sections treated with antisense probes showed meaningful signals. In the case of the sections treated with antisense probes, the HSP108 signals showed different spatial and temporal expressions in each retinal layer. Although the retina is composed almost entirely of a single cell population, i.e. neuroblast cells [17], intense HSP108 signals were found at E5 in the outermost region of the retinal neuroblast layer (NB) and in the pigment epithelium (PE) (Fig. 1A,B). The HSP108 mRNAs were mainly present within the cytoplasm near signal-free nuclear regions (Fig. 1C). At E9, the inner plexiform layer (IP) was discernable between the NB and the newly emerging ganglion cell population (GC) [17]. At this stage, intense HSP108 signals were observed within the GC, the inner region of the inner nuclear layer (IN) and the PE, while much weaker and fewer signals were detected in the optic fiber layer, the IP and the outer regions of the IN (Fig. 1D – F). According to a previous study, the major stratification of the retina is completed by E11, at which stage both plexiform layers are clearly discernable [17]. In the E14 retina, we observed that the general structure was similar to that of the adult stage, except that the photoreceptor layer was not completely differentiated (Fig. 1G). However, signals for HSP108 mRNA at E14 were much weaker than those at E9, though meaningful signals were still detected in the GC, the inner region of the
Table 1 Mean density of heat shock protein (HSP) 108 mRNA in the developing chicken retina E5 NB, inner
NB, outer
PE
0.81 ^ 0.17
137.42 ^ 9.14
19.57 ^ 1.92
NF GC IP IN, inner IN, outer OP ON PR PE
E9
E14
220.22 ^ 14.53 19.81 ^ 3.82 230.17 ^ 11.29 5.66 ^ 1.36
18.53 ^ 5.45 1.67 ^ 0.04 19.67 ^ 5.38 1.51 ^ 0.13 1.94 ^ 0.06 185.98 ^ 16.6
254.96 ^ 15.67
195.11 ^ 11.1
E21 6.84 ^ 1.87 28.93 ^ 2.11 5.76 ^ 1.81 24.94 ^ 3.7 9.49 ^ 2.24 4.60 ^ 1.56 64.63 ^ 6.69 58.37 ^ 5.25 69.48 ^ 7.49
The sum of the gray values of all the pixels in a selection was divided by the number of pixels within the selection. The maximum allowed gray value is 256. NB, neuroblast layer; PE, pigment epithelium; NF, nerve fiber layer; GC, ganglion cell layer; IP, inner plexiform layer; IN, inner nuclear layer; OP, outer plexiform layer; ON, outer nuclear layer; PR, photoreceptor layer.
D.H. Shin et al. / Neuroscience Letters 344 (2003) 25–28
Fig. 1. Heat shock protein 108 (HSP108) mRNA expressions during the development of the chicken retina. (A) Cresyl violet staining at embryonic day 5 (E5). The retina was composed of only neuroblast cells (NBs). PE, pigment epithelium. (B) HSP108 mRNA expression at E5. Only the outer layers of the NBs in the retina produced positive signals. PE also contained HSP108þ cells. (C) Magnified image of (B). HSP108 mRNA signals were mainly localized to the cytoplasm, and not to nuclei (arrowheads). (D) Cresyl violet staining at E9 showed that the inner plexiform layer (IP), which contained some punctuate cells (arrows), was discernable between the ganglion cell layer (GC) and the inner nuclear layer (IN). (E) HSP108 mRNA expression at E9. HSP108 signals were found mainly within the GC, and in the inner regions of the IN and PE, while much weaker signals were found in the nerve fiber layer, the IP and the outer regions of the IN. Cells (arrows) in the IP of (E) also showed HSP108 signals. (F) Magnified image of (E). (G) Cresyl violet staining of the E14 retina showed a similar structure to that of an adult retina, except for the absence of a photoreceptor layer. (H) Also at E14, HSP108 signals were significantly reduced though positive signals were detected in the GC, the inner region of the IN, throughout the outer nuclear layer (ON) and the PE. (I) Magnified image of the ON and the PE in (H). (J) A cresyl violet stained retina at E14. All the retinal layers were remarkable. PR, photoreceptor. (K) HSP108 mRNA expression at E14. HSP108 mRNA signals were detected in the GC, IN, ON and PR. The ON and the PR showed much more intense signals than the GC and the IN. (L) Magnified image of (K). Scale bar, 50 mm.
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IN, and throughout the outer nuclear layer (ON) and the PE. In addition, we also found that HSP108 signals in the outer retinal layers, i.e. ON and PE, showed much more intense signals than those of the GC and IN (Fig. 1H,I). At E21, the general structure and HSP108 mRNA expression were similar to those at E14. Comparing the E14 and E21 retinas, each retinal layer at E21 showed a markedly more differentiated morphology and diminished signal intensity, though the distribution pattern of HSP108þ signals was relatively unaltered in the GC, IN, ON and in photoreceptors (Fig. 1J– L). At E21, the signal intensities of HSP108 mRNA in the outer retinal layers were still much more intense than those in the outer retinal layers. The densitometric findings of HSP108 mRNA expression in the retina at each stage are summarized in Table 1. Previous studies have reported that spatial and temporal expression changes occur in various HSPs during embryonic development [2,5,7]. According to these studies, some HSPs, which were detected at a high intensity in the early embryonic stages, began to decrease during the late embryonic stages. For example, HSP90s, which are known to be homologous to HSP108 [11], were maximally expressed at the earliest stage analyzed and then gradually decreased as embryogenesis progressed [7]. From these observations, it was concluded that HSPs may be expressed strongly during developmental stages involving cell proliferation rather than cell differentiation. On the other hand, it was also reported that the decreased expression of HSPs during the late embryonic stage might be important for the successful maturation of retinal cells [20]. Tanaka et al. [20] observed that some types of HSPs, i.e. HSC70, HSP84, HSP86 and HSP60, exhibited intense mRNA expression in the mouse retina during E11.5 to E14.5 and a marked decrease during E15.5 to E18.5. In another study on the expression of HSPs in the developing rat retina by Kojima et al. [4], the expression of inducible HSP70 was also found to be transiently decreased at the perinatal stage, while intense mRNA signals re-emerged after P7. The involvement of HSPs in the development of the retina was also observed by Morales et al. [9]; although they only observed the sections during E4 to E12, they found that various kinds of HSPs (HSP40, HSP60, HSP70 and HSP90) were expressed during retinal development in the chicken. Our results on HSP108 mRNA expression during eye development were very similar to those of previous studies. As shown in Table 1, intense HSP108 signals were observed at E9, and these decreased during E14 to E21. Moreover, HSP108 mRNA signal intensity changed in accord with each stage and retinal layer. Although a decrease in HSPs was observed during different embryonic days in the present and previous studies, this is probably explained by the differences in the types of HSPs examined or the species used. The intense expression of HSP108 mRNA in retinal cells during early developmental stages raises the possibility that HSP108 might be associated with cellular proliferation,
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because HSP108 signals seem to be correlated with active proliferation, such as in the outermost zone of neuroblast cells. This suggestion is supported by the weaker HSP mRNA signals observed during the later developmental stages, by which time cells had differentiated and proliferation was much reduced. In summary, despite being unable to explain the meaning of HSP108 expression during chicken retinal development, we suggest that HSP108 mRNA expression is developmentally regulated and that it plays an important role in ocular development.
Acknowledgements This study was supported by a grant (2000-1-20700-0063) from the Basic Research Program of the Korea Science and Engineering Foundation and a grant from 2002 BK21 project for medicine, dentistry and pharmacy.
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