The accumulation of δ-crystallin mRNA in transdetermination and transdifferentiation of neural retina cells into lens

Cell Differentiation, 12 (1983) 177-183 Elsevier Scientific Publishers Ireland, Ltd.

177

The accumulation of -crystallin mRNA in transdetermination and transdifferentiation of neural retina cells into lens K. Y a s u d a , K. O k u y a m a a n d T.S. O k a d a Institute for Biophysics, Faculty of Science, University of Kyoto, Kyoto 606, Japan

(Accepted 14 September 1982)

By means of hybridization with DNA complementary to 8-crystallin mRNA (8-mRNA) sequences (8-cDNA), the levels of 8-mRNA in three different culture systems of 8-day-oldchick embryonicneural retina were determined. After 30 days of culturing in vitro, the level of 6-mRNA in cells cultured under conditions of spreading cultures (SpC) was 50 times higher than in the cells maintained in aggregatecultures (AgC) throughout. When the cells pre-cultivated in SpC for an initial 10 days were transferred into AgC, the 8-mRNA level in 30-daycultures was 40 times higher than that in 3-day SpC. The level of ~-mRNA in neural retina in situ was negligible, but it became detectable in 10-daySpC. The initial appearance of detectable 8-mRNA in 10-day SpC coincides with the timing of ' transdetermination' of neural retina cells into lens cells. transdifferentiation neuralretina lens differentiation tion cDNA(complementaryto 8-crystallin mRNA)

1. Introduction Neural retina (NR) cells of vertebrate embryos often change their usual pathways of differentiation and transdifferentiate into lens and pigment cells in in vitro cultures (see reviews by Okada, 1976, 1980; Clayton 1978, 1982). Our recent work has demonstrated that the results of aggregate culture (AgC) with constant gyration of freshly dissociated N R cells contrasts sharply with those of stationary cultures (spreading cultures, SpC) of sister cell populations, only the latter system being permissive of transdifferentiation (Okada et al., 1982, 1983; see also Moscona and Degestein, 1981). However, when N R ceils which have been precultivated in SpC for an initial period are transferred into AgC (SpC ~ AgC), lentoidogenesis occurs within aggregates with the accumulation of a large amount of crystallin proteins. Thus, N R cells are transdetermined into lens ceils in SpC, prior to detectable expression of the lens phenotypes.

8-crystallin mRNA

transcriptional control

transdetermina-

It has been revealed recently that crystallin synthesis in the transdifferentiating system of SpC is primarily under transcriptional control. The level of expression of crystallin genes in this system was determined by hybridization to D N A which is complementary to most m R N A present in chick lenses (Thomson et al., 1979, 1981). We were interested in assaying the level of lens-specific m R N A in three different culture systems of AgC, SpC and SpC ~ AgC. It seemed to be particularly important to know the level at the time when N R cells are 'transdetermined' in SpC so as to differentiate into lens after being transferred to AgC (Okada et al., 1982, 1983). In the present study, the hybridization assay was achieved by using c D N A (8-cDNA) which is complementary to specific m R N A of 8-crystallin (8-mRNA). The levels of D N A synthesis of the different culture systems were also determined to be correlated with the transcriptional level of 8-crystallin genes.

0045-6039/83/0000-0000/$03.00 © 1983 Elsevier Scientific Publishers Ireland, Ltd.

178

2. Materials and methods

2.1. Cultures Dissociated cells from NR of 8-day-old chick embryos were cultured in three different systems as described previously (Okada et al., 1982, 1983): (1) AgC of freshly dissociated NR cells on a gyratory shaker (70 rpm; 3 / 4 inch diameter of rotation) throughout; (2) SpC of freshly dissociated cells throughout; (3) AgC of cells that were pre-cultivated for an initial 10 days in SpC. All cultures were maintained for a total of 30 days and samples to determine the levels of 8-mRNA and of DNA synthesis as well as of 8-crystallin content were harvested at appropriate intervals. For culturing, Eagle's minimum essential medium supplemented with 10% fetal calf serum was used.

E

5

2.2. Preparation of 8-crystallin mRNA (8-mRNA) Lens fiber masses were removed from the eyes of 1-day-old white Leghorn chicks. From about 400 fiber masses, 8-mRNA was isolated, according to the procedure given by Yasuda et al. (1982), as follows: (1) extraction of crude RNA by a mixture of buffer-saturated phenol : chloroform : isoamylalcohol (25 : 24 : 1) ; (2) isolation of poly(A) ÷-RNA by applying the crude RNA samples to oligo(dT)cellulose column (P.-L. Biochemicals, Type III); (3) centrifugation of poly(A)+-RNA samples layered on a 5-20% linear sucrose density gradient; (4) fractionation of the gradient into 30 fractions followed by ethanol precipitation and dissolving RNA in distilled water. To identify 3m R N A fractions a translation assay of an aliquot of each fraction was performed using a reticulocyte lysate (New England Nuclear). [H3]Leucinelabelled translation products were subjected to electrophoreses on a SDS/15% polyacrylamide slab gel (Laemmli, 1970). The gel was fluorographed by the method of Bonner and Laskey (1974). The radioactivity profile of the gel was determined by slicing the gel into 2 mm sections with a razor, depolymerizing the slices with 0.4 ml of 15% (w/v) H202, and scintillation counting with 10 ml of Aquasol (New England Nuclear). The cpm from the gel slices were positioned accu-

50

100

Migration distance(mm) Fig. 1. Top, a fluorograph of SDS-polyacrylamide gel electrophoresis of the in vitro translation products of 8-crystallin mRNA used for the synthesis of c D N A . Bottom, a radioactivity profile of each 2 mm section of the gel depolymerized by H202.

rately on the fluorogram by using two thin reference lines of Idia ink which were injected into each gel before fluorographing and slicing. 95% of the translation products corresponded to the band of 8-crystallin of which 92% was immuno-precipitated with anti-8-crystallin serum (Fig. 1). Fractions containing 8-mRNA were pooled and stored as ethanol-precipitate.

2.3. Preparation of 3H-labelled DNA complementary to 8-crystallin mRNA 3H-labelled cDNA was synthesized in a 25 /,1 reaction mixture using reverse transcriptase (Chang et al., 1978). The reaction mixture contained 100 /~g/ml oligo(dT)~2_18, 50 # g / m l actinomycin D, 30 mM mercaptoethanol, 50 mM Tris-HC1 (pH 8.3 at 43°C), 70 mM KC1, 10 mM MgC12, 2 mM

179 each of dATP, dCTP, dTTP, 500/~M of [3H]dGTP (27 Ci/mmol), 30 U of avian myeloblastosis virus reverse transcriptase (a gift from Dr. Beard) and 2 /~g of purified 8-crystallin mRNA (Yasuda et al., 1982). After incubation at 43°C for 90 min, 12.5 mM EDTA, 0.05% SDS and 100 btg/ml of E. coli tRNA were added and the mixture was extracted with an equal volume of phenol saturated with 0.1 M Tris-HC1 (pH 8.0). To the aqueous phase 0.3 M sodium acetate (pH 7.0) was added and the R N A was precipitated with 2.5 vol of ethanol. The pellet was dissolved in 0.1 N N a O H and incubated for 30 min at 68°C. The solution was neutralized with HC1 and applied to a Sephadex G 150 column (0.8 x 20 cm) equilibrated with 10 mM Tris-HC1 (pH 7.4), 10 mM NaC1 and 1 mM EDTA. The materials eluted in the void volume were precipitated. The cDNA had a specific activity of 4.2 x 10 7 d p m / ~ g . The yield of the cDNA was 20.4% of the weight of the mRNA. The cDNA denatured by glyoxal was electrophoresed on a 1.5% agarose gel in 10 mM sodium phosphate buffer (pH 7.0) (McMaster and Carmichael, 1977). rRNAs of chick cells and E. coli were used as size markers. The size of the cDNA was from 800 to 1600 nucleotides.

2.4. Preparation of RNA from cultured NR cells Cultures at appropriate stages were washed three times with Hanks' saline and were pooled to store at - 8 0 ° C before obtaining sufficient amounts of samples. Cells were dissolved in l0 mM Tris-HC1 (pH 7.5) and 5 mM EDTA, homogenized and centrifuged for 10 min at 4°C. The supernatant was incubated in the presence of 50 /~g/ml of proteinase K (Sigma) and 1% SDS for 1 h at 37°C and total cytoplasmic RNA was prepared by extraction with phenol-chloroform.

overlaid with the buffer-saturated paraffin liquid (Merck) and the reaction was carried out at 75°C for various periods, to give the appropriate Rot values. Hybridization was stopped by diluting the mixtures in 1 ml of cold 50 mM sodium acetate (pH 4.5), 2 mM ZnSO 4, 0.1 M NaCl and l0 /~g/ml of heat denatured-sonicated salmon sperm D N A (Sigma, Type III). Each sample was divided into two aliquots. One of them was incubated for 1 h at 37°C in the presence of 20 U of S 1 nuclease (Sigma, Type III), and the [3H]cDNA precipitable with cold 10% TCA was collected on Whatman G F / C glass-fiber filters. The filters thus obtained were counted in l0 ml of Toluene Scintillation solution. The percentage of [3H]cDNA hybridized was calculated from the ratio of [3H]cDNA precipitated in a tube containing S~ nuclease to that in a tube without the enzyme. The hybridization data provide information about the purity of 8-mRNA. We can estimate the Rotl/2 value of pure ~ - m R N A - c D N A hybridization kinetics by comparing it with that of an mRNA standard of known complexity, provided that all other parameters of the hybridization reaction are the same. We have chosen as a standard rabbit globin mRNA, which has a complexity of 1200 and a Rot1~ 2 of 3.7 X 10 - 4 S" M • 1- l (Honjo et al., 1974). Northern blotting of 8-mRNA showed that & m R N A is approximately 2000 nucleotides long (Yasuda et al., unpubl, data). From these values, the estimated Rot~/2 value of 3m R N A - c D N A hybridization kinetics is 6.17 x 10 -4 s . M . 1 - 1 . The results of our experiments gave a Rotl/2 value of 7 . 0 X l 0 - 4 s . M . 1 -~ Therefore, the purity of 8-mRNA of our preparation is about 90% and this value is nearly equal to that calculated from the in vitro translation assay (95%).

2.5. Conditions for R N A - D N A hybridization

2.6. Incorporation of [3H]deoxythymidine ([3H]TdR) into DNA

The hybridization reaction was performed in 20-50/~l of the reaction mixture containing 0.6 M NaC1, 0.2 mM EDTA, 20 mM Tris-HC1 (pH 7.4), 0.1 ng of [3H]cDNA (1500 cpm) and various amounts of RNA (150 n g / m l - 1 4 /~g/ml). The mixture in an Eppendorf polyethylene tube was

Cell cultures at various periods were labelled for 4 h in fresh medium containing 20/~Ci/ml of [3H]TdR (6.7 Ci/mmol). Labelled cells were washed three times with Hanks' saline to which cold 5% PCA was added, vortexed and cooled to 0°C for 15 min. The PCA soluble fraction was

180 removed from the mixture by centrifugation. P C A insoluble materials were washed twice, and resuspended in 5% P C A and boiled for 30 min. After centrifugation, an aliquot of the supernatant was used for determining the D N A content according to the method of Giles and Mayers (1965). To the P C A soluble and insoluble fractions 0.5 M K O H was added, and the mixture centrifuged to remove the insoluble potassium perchlorate. The radioactivities of the supernatants were counted in 10 ml of Aquasol scintilation solution (Amersham).

100

A °f°

B0

60

/

20

o/i/,// -2

-3

' -i

0

1

2

.,

3

4

log Rot

2.7. Determination of 8-crystallin content The level of 8-crystallin in aliquots of the supernatant of culture homogenates used to prepare the culture R N A was determined by quantitative imm u n o e l e c t r o p h o r e s i s (Laurell, 1966) and by hemagglutination inhibition assays (De Pomerai et al., 1977) using antiserum specific to 3-crystallin (Araki et al., 1979).

100

"15

°/

80

~ .< 40 20

./ -5

3. Results

3.1. Accumulation of 8-mRNA in different culture systems The accumulation of 8 - m R N A in N R cultures of the three different systdms (SpC throughout, A g C throughout, and SpC ~ AgC) was assayed by hybridization of total cytoplasmic R N A to 8c D N A . As controls, assays were also made of cytoplasmic R N A of freshly prepared N R (0-day culture) and of headless embryos. The results are shown in Figs. 2a and b. In both controls, small a m o u n t s of hybridization were detected at a Rot up to 4 x 103. In A g C the accumulation of 8m R N A occurred during 30 days of culturing. However, its level was about 1 / 5 0 of that attained in SpC in the same period (see also Table I). A measurable a m o u n t of hybridizable 8 - m R N A was detected already in 10-day SpC, and then it increased about 100 times for a further 20 days. The accumulation was, however, particularly striking, when cells of 10-day SpC were transferred into A g C (10 SpC---, AgC). The level of 8 - m R N A attained in such cultures after a total of 30 days of

/

60

/

o -Z.

-3

-2

-1

y 0

1

2

3

log Rot

Fig. 2. Kinetics of hybridization of cytoplasmic RNAs of various samples to 3H-labelled 8-cDNA. With each RNA sample 2000 cpm of cDNA synthesized from 8-crystallin mRNA was hybridized. The aliquot was assayed by single-strand specific nuclease digestion as described in section 2. The percentage of hybridization is plotted against Rot, the product of concentration of RNA, in moles of nucleotide per liter x time (seconds). The line represents a best fit to the data derived from a computer analysis and fitted by a nonlinear least-squares regression method. A, the cytoplasmic RNAs were extracted from lens (D D), 30-day SpC (O O), 15-day SpC (O O), 10-day SpC (zx zx), 5-day SpC (n . . . . . . (pz), 8-day headless embryos (11. . . . . . II), 8-day NR (I t) and E. coli (A A); B, the cytoplasmic RNAs were extracted from lens (12 D), 30-day 10 SpC---, AgC (11 II), 30-day SpC (O O) and 30-day AgC (t O). The line (zx zx) shows kinetics of hybridization of purified 8-mRNA to the 8-cDNA.

cultivation was about 40 times higher than in 30-day SpC and the increase was more than 4000 times during 20 days of culturing in AgC of cells pre-cultivated for 10 days in SpC.

181 TABLE 1 Dosages of 8 - m R N A and 8-crystallin protein Sample

Roll~2 a

Dosage b

% of 8-crystallin in ¢ total soluble proteins

8-day-old embryonic N R 8-day-old headless embryos 10-day SpC 30-day AgC 30-day SpC 30-day (10 SpC ~ AgC) l-day-old chick lens (cytoplasmic RNA) 8-mRNA

1.05 x 5.0 x 1.07 x 2.8 x 7.5 x

0.00007 0.00015 0.0070 0.2679 1.0

not detectable not detectable not detectable < 0.1 l.l, 1.8 23.3, 24.6 62.3

l03 l02 l01 10-i 10 -2

7.0 x 10 -4

a R 0t 1/'2 values were calculated using a least-squares program which provided a single component curve which was the best fit for the data points in Fig. 2. R N A samples were cytoplasmic R N A s isolated from tissues or cultures in various conditions and were hybridized with 3H-labelled 8-cDNA. b Dosage of m R N A is calculated relative to the levels present in 1-day post-hatch chick lens, which have been given values of 1. c The amount of 8-crystallin in each sample was measured using the method of Laurell (1966). The percentage of 8-crystallin was calculated from the ratio of 8-crystallin to the total soluble protein in each sample.

3.2. Accumulation of g-crystal~in in different culture systems The accumulation of 8-crystallin, a specific marker protein of lens differentiation, in culture homogenates is shown in Table I together with ~-mRNA dosages (see also Fig. 3). The level of

xlO-4

5•C

J

~s o

83 I

SpC--AgC 1

0

~n, i

5

Fig. 3. An immunofluorescent photograph of a histological section of two aggregates of l0 SpC ~ AgC cultured for a total of 30 days, after indirect staining with anti-8-crystallin serum (see Okada et al., 1983). ( × 90).

.o AgC I

i

I

10 15 20 Days in cutture

I

25

Fig. 4. Changes in the incorporation of [3H]TdR into D N A in cultures of N R cells under different conditions. The radioactivity and D N A content in cold PCA-insoluble fractions were measured at the time points indicated in the figure. See text for SpC, AgC and SpC --* AgC.

182

8-crystallin content was well in parallel with that of 8-mRNA. In aggregates of N R cells pre-cultivated in SpC, both levels were much higher than in cultures maintained in SpC throughout. It should be noted that no translation products of 8-mRNA were detected in SpC at l0 days, when considerable accumulation of 8-mRNA was detectable.

3.3. DNA synthesis Incorporation of [3H]TdR into DNA was determined in cultures at several different stages of their maintenance in vitro. As shown in Fig. 4, little incorporation occurred in AgC of the freshly dissociated cells. In AgC of cells pre-cultivated in SpC, some incorporation was detected, but its level was much lower than in SpC at comparable times of culturing. In SpC, incorporation was still low in 10-day cultures and becomes higher thereafter with culture time.

4. Discussion

We have previously demonstrated (Okada et al., 1982, 1983; see also Moscona and Degenstein, 1981), that the two different culture systems, SpC and AgC, provide conditions permissive and nonpermissive, respectively, of transdifferentiation of N R cells into lens is under transcriptional control (Thomson et al., 1979, 1981). The present results seem to support this, by showing a considerable difference between the levels of & m R N A accumulation by the cells cultured in the two different culture systems. In order to obtain unambiguous evidence of transcriptional control of transdifferentiation, however, further studies are needed, since the present studies were done only with cytoplasmic RNAs using the 8-cDNA probe. The previous results revealed that N R cells pre-cultivated in SpC for an initial l0 days are transdetermined and can differentiate into lens cells when transferred into AgC, which is a nonpermissive condition for transdifferentiation of the freshly dissociated N R cells (Okada et al., 1982, 1983). The accumulation of 8-mRNA in AgC of transdetermined cells after l0 days of pre-cultivation in SpC was particularly conspicuous, reaching

about 40 times more than that in cells cultured in SpC throughout. This result parallels well with the accumulation of 8-crystallin. Thus, the conditions of AgC, though non-permissive of transdetermination, must be favorable for the expression of lens phenotypes of transdetermined cells. At l0 days of SpC, which is the critical timing for the transdetermination, low levels of accumulation of 8-mRNA are detected, although 8-crystallin is not yet detected. Thus, there is an interesting coincidence in the timing of transdetermination and of the first detectable accumulation of 8mRNA. It is not known, however, whether this initiation of 8-mRNA accumulation is necessarily related to the transdetermination of N R cells into lens or not. It has been reported that an appreciable level of crystallin messages can be detected in both neural and pigmented retina of chick embryos in situ. Clayton et al. (1979; see also Clayton, 1982) assumed that such an initial level of transcription is a necessary prerequisite for the ability of transdifferentiation of these tissue cells into lens, and proposed an interesting idea of 'molecular overlap between different types of tissue cells' to explain such a unique ability of retinal cells in their remarkable plasticity in the future pathways of differentiation. In the present work, 8-mRNA accumulation was hardly detected in N R of 8-day embryos in situ or in headless embryos. One explanation for this discrepancy in the results may be a difference in cDNAs used as the probe for hybridization assays of the crystallin messenger. Clayton et al. used cDNA which is complementary to the most abundant mRNAs isolated from 1day-old chick lenses, whereas our cDNA was prepared to be complementary to the isolated 8mRNA to code 8-crystallin specifically (Yasuda et al., 1982). Another explanation could be the use of total cytoplasmic RNA, not poly(A)+-RNA, of N R to be hybridized in the present study. Careful examination of these points is now in progress.

References A r a b , M. and T.S. Okada: Dev. Growth Differ. 20, 71-78

(1978).

183 Bonner, W.M. and R.A. Laskey: Eur. J. Biochem. 46, 83-88 (1974). Chang, A.C.Y., J.H. Nunberg, R.J. Kaufman, H.A. Erlich, R.T. Schimke, and S.N. Cohen: Nature (London) 275, 617-624 (1978). Clayton, R.M.: In: Stem cells and Tissue Homeostasis, eds. B.I. Lord, C.S. Potten and R.J. Cole (Cambridge University Press, London)pp. 115-138 (1978). Clayton, R.M.: In: Differentiation in vitro, eds. M.M. Yeoman and D.E.S. Truman (Cambridge University Press, London) pp. 83-120 (1982). Clayton, R.M., I. Thomson and D.I. de Pomerai: Nature (London) 282, 628 (1979). De Pomerai, D.I., D.J. Pritchard and R.M. Clayton: Develop. Biol. 60, 416-427 (1977). Giles, K.W. and A. Mayers: Nature (London) 206, 93 (1965). Honjo, T., S. Packman, D. Swan, M. Nau and P. Leder: Proc. Natl. Acad. Sci. U.S.A. 71, 3659-3663 (1974). Laemmli, U.K.: Nature (London) 227, 680-685 (1970). Laurell, C-B.: Anal. Biochem. 15, 45-52 (1966). McMaster, G.K. and G.G. Carmichael: Prec. Natl. Acad. Sci. U.S.A. 74, 4835-4838 (1977).

Moscona, A.A. and L. Degenstein: Cell Differ. 10, 39-46 (1980). Okada, T.S.: In: Tests of Teratogeneicity In Vitro, eds. J.D. Ebert and M. Marois (North-Holland, Amsterdam) pp. 91-105 (1976). Okada, T.S.: In: Current Topics in Developmental Biology, eds. A.A. Moscona, A. Monroy and H.K. Hunt (Academic Press, New York and London) Vol. 16, pp. 349-380 (1980). Okada, T.S., K. Yasuda, H. Kondoh, K. Nomura, S. Takagi and K. Okuyama: In: 'Embryonic Development', Prec. 1X Congr. Int. Sec. Develop. Biol., ed. M. Burger (Alan R. Liss Inc., New York) pp. 249-255 (1982). Okada, T.S., K. Nomura and K. Yasuda: Cell Differ. 12, 85-92 (1983). Thomson, I., D.I. de Pomerai, J.F. Jackson and R.M. Clayton: Exptl. Cell Res. 122, 73-81 (1979). Thomson, 1., K. Yasuda, D.I. de Pomerai, R.M. Clayton and T.S. Okada: Exp. Cell Res. 135, 445-449 (1981). Yasuda, K., H. Kondoh, T.S. Okada, N. Nakajima and Y. Shimura: Nucleic Acids Res. 10, 2879-2891 (1982).