electrophoresis at 20 mA/plate and stained with coomassie brilliant blue. Gels were destained for 6 h with several changes ofdestaining solution I (45% ...
Vol. 41, No. 2, February 1997
CHANGES IN' PROTEIN DEVELOPMENT
BIOCHEMISTRYond MOLECULAR BIOLOGY INTERNATIONAL Pages339-347
COMPOSITION
DURING
CHICK
EMBRYO
RETINA
M. Carabillr, E, Gerbino, H. Main, G. Calvaruso and G. Tesoriere Istituto di Chimica Biologica, Universit& of Palermo, Policlinico, via del Vespro 129, 90127 Palermo, Italy. Received December5, 1996 ABSTRACT. Two dimensional gel electrophoresis was employed to analyse the protein composition of chick embryo retina during "in ovo" development in order to individuate some components which are typical of different stages of growth. The study of protein composition of 7-day-old chick embryo retinas revealed the presence of about eighty different soluble components. In four of these proteins the staining intensity decreased during retina development. One of these components, of 3 5 KD with an isoelectric point of about 5.15, was partially purified by means of preparative isoelectric focusing. Other components, which were absent at day 7 appeared during the second week of development and were clearly visualised at day 16. These results were also compared with those obtained by means of 2DGE performed on liver and brain of chick embryo at day 7 and 16 of development. Key words: chick embryo retina, development, two-dimensional gel electrophoresis, preparative IEF During the last few years in our laboratory we have studied the development of chick embryo retina. It has been ascertained that in retina developing in
ovo
three different stages can be
distinguished (1). After a first stage of rapid growth corresponding to the first week of development, cell proliferation declines from day 7 to day 13. During this second stage retinoblasts differentiate into different types of cells while the typical structural organisation appears. Differentiative events continue in the third successive stage until hatching. Recently we have undertaken further research on the development of retinas cultured vitro
in
in serum-free medium and we have demonstrated that under this condition retinas maintain
their vitality for many days, although a great retardation in maturation is noted compared to uncultured controls (2). In addition we have investigated the effects of both insulin (2) and IGF I (3) on retina development in culture. In accordance with other authors (4,5), we have demonstrated (2) that insulin exerts neurotrophic effects in cultured retinas o f chick embryos, in particular by increasing both wet weight and protein content with a marked effect on the nonassembled fraction of tubulin.
1039-9712/97/020339-09505.00/0 339
Copyright 9 1997 by Academic Press Australia. A II rights ~[ reproduction in any form reserved.
Vol. 41, No. 2, 1997
BIOCHEMISTRYand MOLECULAR BIOLOGY INTERNATIONAL
The goal of the present study was to analyse the protein content of chick embryo retina and to demonstrate eventual changes in this composition during the process of development. These experiments were performed in retinas developing in ovo,
using two-dimensional gel
electrophoresis (2DGE). This study permitted us to individuate the presence of some components at day 7 whose content decreased with development, while an increment with age was observed in other components which could thus be clearly seen at day 16. One acidic component, visualised at day 7 of development but completely absent at day 16, has been partially purified by means of preparative IEF. Experiments are now in progress to isolate and characterise these proteins.
METHODS Chemicals were purchased from Sigma Chemical Co. (St. Louis, MO). Fertilized white leghorn chicken eggs were obtained from Centro Avicolo Mediterraneo of Palermo. Eggs of uniform size and weight were placed in an automatic incubator (Victoria) at 38~ and 60% relative humidity.
Retinal explants: Explants of retinas, removed from chick embryo at different days of development, were cultured in agarose-coated dishes (55 mm diameter, three retinas per dish) in 8 ml of culture medium. Agarose allows the retinas to be cultured for a long time without attachment to the wells (2). Culture medium was Dulbecco's modified Eagle's medium (DMEM) containing sodium selenite (5 ng/ml), transferrin (5 ~g/ml) and fatty acid free albumin (1 mg/ml) supplemented by 1% pen-strep. The dishes were incubated in a moist atmosphere of 94% air and 6% CO2 at 37~ The cultures were fed every third day. At the end of incubation retinas were transferred to tubes and centrifuged for 2 min at 2000 rpm. Tissue extracts preparation: Tissues were homogenized (procedure A) in 10 vol of 50 mM Tris-HCl (pH 7.4), 25 mM MgCI2, 0.01 mM EDTA and sonicated twice at 14 microns for 10 sec with Soniprep 150 and then centrifuged at 20,000 rpm for 20 min at 4~ Five ~tl of the supernatant was kept for a protein content assay according to the technique of Lowry (6). Leupeptin and PMSF were then added to the sample to a final concentration of 5 gg/ml and 0.1 mM, respectively. Samples were then dialyzed against 50 mM ammonium acetate for 20 h in a Spectrapore 3 dialysis tubing (molecular weight cut off of 500, Spectrum Med. Ind.) and then lyophilized. The lyophilized samples were resuspended in isoelectric focusing (IEF) lysis buffer (5 ml/mg protein) containing 9 M urea, 2% nonidet P-40 (w/v), 6% ampholines (3% pH range 5-7 and 3% pH range 3.5-10) and 50 mM DTT. in some experiments retinas were homogenized in 10 vol oflEF lysis buffer directly and submitted to 2 DGE (procedure B). Two dimensional gel electrophoresis: 2DGE was carried out by a modification of the method of O'Farrell (7,8), using a Hoefer tube gel adaptor Kit SE 220, and Mighty Small II Unit SE 250. Isoelectric focusing gel buffer contained 6% ampholine (3% pH range 5-7 and 3% pH range 3.5-10). Prefocusing was carried out for l0 min at 200V, 10 min at 300V and 10 min at 400V, then 50 ~tg of protein was loaded and the first dimension run (IEF) was carried out for 30 min at 100 V, l h at 200 V, 1 h at 300 V and overnight at 400 V. After the run, the gels were equilibrated with 50 mM Tris-HC1 (pH 6.7) containing 2% SDS, 10% glycerol and 10 mM DTT.
340
Vol. 41, No. 2, 1997
BIOCHEMISTRYond MOLECULAR BIOLOGY INTERNATIONAL
For the run in the second dimension a discontinuous SDS gel system was used as described by Laemmli (9). The resolving gel contained 12% acrylamide/bisacrylamide (from a 30% stock solution composed of 29.2% acrylamide and 0.8% bisacrylamide). The gel was subjected to electrophoresis at 20 mA/plate and stained with coomassie brilliant blue. Gels were destained for 6 h with several changes ofdestaining solution I (45% methanol, 10% acetic acid) and left overnight in destaining solution II (7% acetic acid, 5% methanol). Gels were then treated for 2 h in 35% ethanol, 1% glycerol and dried in Easy Breeze Gel Dryer (Hoefer). Reproducible electrophoretic patterns were obtained either from multiple two dimensional separations of the same tissue preparation or from different preparations of the same tissue. Molecular weights were estimated on SDS ,gels using 13-galactosidase (l16K), fructose-6-phosphate-kinase (85K), glutamate dehydrogenase (55K), aldolase (39K), triosephosphate isomerase (26K), trypsin inhibitor (20.1K) as standards. Furthermore protein standards at indicated isoelectric points were employed: trypsin inhibitor (4.6 pI), carbonic anhydrase II (5.9 pI) and lactic dehydrogenase (8.6 pI).
Preparative isoelectric focusing: Retinas were homogenized in 5 vol of IEF lysis buffer and centrifuged at 20,000 rpm. Aliquots of supernatants, corresponding to 50 mg of protein, were made up to 32 ml with a final concentration of : 7 M urea, 10% glycerol, 1% ampholine pH range 5-7 and 1% ampholine pH range 3.5-10. This solution was loaded in a Miniphor apparatus (Rainin Instruments Co., Inc). Preparative isoelectric focusing was performed first at 1,000 V for 1.30 h to produce a stable pH gradient and then at 500 V for a further 15 min. During the run, proteins were separated into 20 fractions according to their isoelectric point. The pH of the collected fractions ranged from 12.6 for fraction 1 to 3.1 for fraction 20. The fractions 13-18 (pH range 6-4.7) were dialyzed for 48 h against 20 mM Tris-HCl (pH 7.4), lyophilized and resuspended in IEF lysis buffer. About 50 ~tg of proteins were then submitted to 2 DGE as described above.
~S~TS
We have analysed, using two-dimensional gel electrophoresis, the protein composition of chick embryo retina from day 7 to day 16 of embryonic life. The electrophoretic procedure employed was highly reproducible and although a large number of spots were visualised on two dimensional gels after staining with coomassie brilliant blue, it was found that when two identical samples were separated on two different 2D gels, each spot corresponded to a single spot on the other gel The protein composition of chick embryo retina was studied at different days of "in ovo" development. About eighty different spots were visualized on the electrophoretograms shown in Fig. 1. Among these spots some proteins, such as tubulin and actin, were identified by comigration with protein standards. For four proteins shown in retinas of 7-day-old chick embryos (Fig. 1A) staining intensity decreased with development of the retina and they were absent on the electrophoretograms performed for retinas at day 12 (Fig. IB) and day 16 (Fig. 1C) of development. These four proteins, typical of the early stages of development, were indicated as group I and were numbered in Fig. 1A from 1 to 4 in relationship to the decreasing value of their
341
Vol. 41, No. 2, 1997
BIOCHEMISTRYond MOLECULAR BIOLOGY INTERNATIONAL
IEF 116 k 85 k 55 k 39 k
26 k 20 k
IEF 116 k 85 k 55 k 39 k
26 k , 20 k -
# : ::
i?
B IEF 116 k 85 k 55 k 39k-
26 k -
20 k -
(: Fig. 1. Two dimensional analysis of soluble proteins of chick embryo retinas. Comparison of retinas from chick embryos at day 7 (A), 12 (B) and 16 (C) of development. Samples of soluble proteins were prepared from chick embryo retinas as described in "Methods" (procedure A). Focusing was performed with 6% ampholines (3% pH 5-7 and 3% pH 3.5-10). The second dimension was on 12% SDS-Polyacrylamide gel. A and T represent actin and tubulin respectively. The electrophoretogram was stained with coomassie brilliant blue and photographed. The position of those spots which changed their staining intensities during development are indicated by arrows. Spots (I-4) shown in A decreased during development; spots (1-6) shown in B and C increased during development. 342
BIOCHEMISTRYond MOLECULAR
Vol. 4 1 , No. 2, 1 9 9 7
BIOLOGY INTERNATIONAL
isoelectric point. The components of group 1 were not specific to retinal tissue, as they were also found at day 7 of development in brain (Fig. 2A) and liver (Fig. 3A). Two of the components of group 1 (components 3 and 4) were acidic proteins with isoelectric points of about 5 and molecular weights of about 35 KD. Component 3, in particular, was detected in all gels performed with retinas at day 7 and was already completely absent at day 10 of tissue development (data not shown). Furthermore, although component 3 was also present at day 7 in brain and liver, its concentration was much higher in retina than in the former tissues. Figs. 1B and 1C show proteins of embryo retina at day 12 and day 16 of development respectively. It was noted that six new protein components were present on the 2DGE performed with retinas at day 16 which were not visualized at day 7 of development. These proteins, which represent components of group 2, are indicated in Fig. 1C numbered from 1 to 6 in relationship to their decreasing isoelectric point. Among proteins of group 2, components 5 and 6 were acidic proteins. Components 2, 4, 5 and 6 were clearly distinguishable also on 2DGE from retinas at day 12 of development (Fig. 1B, see IB=
116 k 85 k - ~ 55 k - ~ 39 k 26 k 2Ok-
~
A
IEF
116 k 85 k -
"
55 k - w 39 k
;,, 9
"~--
~
Ep,
-
=
~
,41, - -
o ,n
9
26 k 20 k -
~'
B Fig. 2. Two dimensional analysis of soluble proteins from chick embryo brain. Samples of soluble proteins were prepared from brains of chick embryo at day 7 (A) and 16 (B) of development as described in "Methods" (procedure A). 2DGE was performed as reported in the legend of Fig. 1. Spots which seem to correspond to retinal proteins of group 1 are indicated in (A) by arrows.
343
Vol. 41, No. 2, | 997
BIOCHEMISTRY and MOLECULAR BIOLOGY INTERNATIONAL IEF 116 k 85 k 55
k-
39 k
et
-
b
26 k -
2Ok-
9
i
A
IEF 116 k85 k55 39k
~&
k-
,~ ~
,~ ~
-
,,
26 k - ~
%
"
~,
~"
-
20 k -
B Fig. 3. Two dimensional analysis of soluble proteins from chick embryo liver Samples of soluble proteins were prepared from liver of chick embryos at day 7 (A) and 16 (B) of development as described in "Methods" (procedure A). 2DGE was performed as reported in the legend of Fig. 1. Spots which seem to correspond to retinal proteins of group 1 are indicated in (A) by arrows.
arrows), although they were less strong than the corresponding spots found at day 16. Components 3, 4, 5 and 6 seem to be specific to the retina as they were not visualised in 2DGE of brain (Fig. 2B), liver (Fig. 3B) and whole body (data not shown). Component 3 of group 1 was partially purified from retinas at day 7 of development. To this purpose, samples containing 50 mg of soluble retinal protein in 32 ml of preparative IEF solution were submitted to preparative isoelectricfocusing as described in "Methods". Fractions 13-18, collected in the range of pH 6-4.7, were dialyzed, lyophilized and then submitted to 2DGE It was observed that the electropboretogram obtained from fraction 17, eluted from preparative IEF at pH 5.15, contained as a predominant component a protein indicated in Fig. 4 by an arrow, which corresponded to component 3 of group 1. In some experiments extracts were prepared following procedure B, which allowed a complete solubilization of cellular proteins. Also in this case proteins of group 1 were found to be present in the gels from retinas at day 7 of development, while proteins of group 2 were visualised
344
Vol. 41, No. 2, 1997
BIOCHEMISTRY and MOLECULAR BIOLOGY INTERNATIONAL
IEF 55 k 39 k 26 k -
Fig. 4. Two dimensional analysis of acidic proteins of chick embryo retinas. Retinas from 7-day-old chick embryo were homogenized in lysis buffer and submitted to preparative isoelectric focusing as reported in "Methods". Fraction 17 (pH 5.15) was dialyzed and lyophilized. The residue, resuspended in buffer, was employed for 2DGE. Focusing was performed with 6% ampholines (3% pH 5-7 and 3% pH 3.5-10). An arrow indicates the presence of component 3 with a molecular weight of 35 KD.
on the gels from retinas at day 16. Furthermore, when procedure B was employed, many proteins appeared on the electrophoretogram (Fig. 5) which were totally absent when proteins were extracted following procedure A. Among these proteins, the components indicated by arrows increased their staining intensity during development (Fig. 5B).
DISCUSSION
One approach to studying the molecular basis of development is to individuate the proteins which show a specific behaviour during development, purify these molecules and identify their functions (10). Two useful tools for protein separation are isoelectric focusing and SDSpolyacrylamide gel electrophoresis. The combination of both techniques permits high resolution analysis of individual proteins from complex mixtures (I1-14). An analysis of developmental changes in protein composition by means of two dimensional electrophoresis was performed by Shirao and Obata in chick optic tectum (10). Protein composition "of chick embryo retina has been analysed by Neukirchen et al. (13), but changes in protein composition during development were not studied. Our present paper analyses the protein composition of soluble extracts obtained from chick embryo retina between day 7 and day 16 of embryonic life. In particular our study aims to individuate the protein components whose abundance increases or decreases during development. Another aim was to ascertain the presence of components specific to the retina which are absent in 2D gel electrophoresis performed on other tissues. The results permitted us to conclude that among the proteins detected in retina at day 7 of embryonic life, four components disappear at
345
Vol. 41, No. 2, 1997
BIOCHEMISTRYond MOLECULAR BIOLOGY INTERNATIONAL IEF
IEF
116 k
116 k
55 k
55 k
26 k-,':'
26 k
O
A
B
Fig. 5. Two dimensional analysis of total proteins of chick embryo retinas. Comparison of retinas from chick embryos at day 7 (A) and 16 (B) of development. Samples of total proteins were prepared as described in "Methods" (Procedure B). The position of those spots which increased their staining intensities during development are indicated in (B) by arrows.
more advanced stages of development. Because these components were also found in brain and liver and their abundance also decreased during development of these tissues, we conclude that these proteins are involved in some developmental function and are not specific to retinal tissues. One of these proteins, indicated as component 3 of group I, has been studied by us with particular interest. This acidic protein, which has been partially purified by means of preparative IEF, disappeared in retina at day 10 of development and was represented in the retina at day 7 much more than in other tissues. Some other spots increased their staining intensities
markedly during embryonic
development (group 2). Among these, component 2 could be recognized as calretinin, a 29 KD neuronal protein with a pI of 5.3 which belongs to the calmodulin family and has been found to be present in chick retina (15). Furthermore, component 3 seems to correspond to a soluble protein designated by Kuo et al. (16) as MEKA protein. This component, which is present in chick embryo retina as a 33 KD protein with a pl of 5.35, accumulates in the same manner as opsin in the inner segment of photoreceptor cells. Component 3 was totally absent in 2DGE performed on brain, liver and total body at day 16. The other components are unknown proteins specific to the retina. Because their concentration increases in the retina during the third week of embryonic life, it is possible that they have some relationship to neuronal differentiation and network formation. In the future we intend to purify by means of preparative IEF and HPLC both the acidic proteins which are present in the first stages of development (components 3 and 4 of group 1) and the unknown proteins o f group 2 (component 4,5 and 6). We aim to ascertain their amino acid sequences and their developmental or differentiative function. At this moment studies are in
346
Vol. 41, No. 2, 1997
BIOCHEMISTRYond MOLECULAR BIOLOGY INTERNATIONAL
progress using 2D gel electrophoresis on the protein composition of different layers of retina after differentiation, in particular the layers of photoreceptor cells and those of ganglion cells. In conclusion, the 2 D gel electrophoresis technique presented in this paper seems to be a useful procedure for studying
chick embryo retina development in vivo. Furthermore this
procedure permits the individuation of several protein components whose abundance changes during embryonic life. Studies on the characters and functions of these proteins are now in progress.
ACKNOWLEDGEMENT This research was supported by "Ministero della Universit~i e della Ricerca Scientifica" (MLrRST, Italy). The authors wish to express their gratitude also to Associazione Italiana per la Ricerca sul Cancro (AIRC, Milan) for its financial support.
REFERENCES 1 Tesoriere, G., Vento, R., Taibi, G., Calvaruso, G. and Schiavo, M. R. (1989) J. Neurochem. 52, 1487-1494. 2 Tesoriere, G., Vento, R., Morello,V.,Tomasino, R. M., Carabill6, M. and Lauricella, M. (1995)Neurochem Res. 20, 803-813. 3 Calvaruso, G., Vento, R., Giuliano, M., Lauricella, M., Gerbino, E. and Tesoriere, G. (1996) Regul. Pept. 61, 19-25. 4 Hausman, R. E., Vivek Sagar, G. D. and Shah, B. H. (1991) Dev. Brain Res. 59, 31-37. 5 Shah, B. H. and Hausmann R. (1993) Dev. Brain Res. 72, 151-158. 6 Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. (1951) J. Biol. Chem. 193, 265-275. 7 O'Farrell, P. H. (1975) J. Biol. Chem. 250, 4007-4021. 8 O'Farrell, P. Z. Goodman, H. M. and O'Farrell, P. H. (1977) Cell 12, 1133-1142. 9 Laemmli, U. K. (1970). Nature, 227, 680-685. 10 Shirao, T. and Obata, T. (1985) J. Neurochem. 44, 1210-1216. 11 Czosnek, H., Soifer, D. and Wisniewski H. M. (1980) J. Cell. Biol. 85, 726-734. 12 Strocchi, P., Gilbert M. J., Benowitz L. I., Dahl, D. and Lewis, R. E. (1984) J. Neurochem 43,349-357. 13 Neukirchen, R. O., Schlosshauer, B., Baars, S., Jackle, H. and Schwarz, U. (1982) J .Biol. Chem. 24, 15229-15234. 14 Walsh, M. J. and Kuruk, N. (1992) J. Neurochem. 59, 667-678. 15 Rogers, J. H. (1987) J. Celt Biol. 105, 1343-1353. 16 Kuo, C. H., Akiyama, M. and Miki, N. (1989) Brain Res. Mol. Brain Res. 6, 1-10.
347
