Poly A (+) mRNA metabolism in wing imaginal discs during normal development and diapause in Pieris brassicae

BIOCHIMIE, 1983, 65, 105-114.

Poly A (+) mRNA metabolism in wing imaginal discs during normal development and diapause in Pieris brassicae. Philippe T A R R O U X <> and Paul B E R R E U R *

(Re9u le 21-6-1982, accept~ apr~s rdvision le 8-12-1982).

Laboratoire de Zoo!ogie, Ecole Normale Supkrieure, 46, rue d'Ulm, 75230 Paris C6dex 05. * E.R.A. 229, C.N.R.S., 91190 GiJ-sur-Yvette, France.

R6sum6.

Summary.

La synthdse de mRNA poly A ( + ) a dtd analysde pendant Ia vie nymphale et la diapause, dans les d&ques alaires du Ldpidopt~re Pieris brassicae. Les principaux dvdnements de la di[fdrenciation terminale du disque (ddpOt de cuticule, formation des dcailles, ~laboration des pigments) out lieu durant cette p~riode. Nous avons d~tectd seulement une phase de synthbse des m R N A poly A (+), de 48 d 72 heures aprds la mue nymphale. La synthkse semble li~e ?z celle des protdines tardives (120-144 heures). L"examen du m~tabolisme des m R N A de disques traitds avec de la cordycdpine (3' dA) montre une ddcroissance de la stabilit~ de ce~ molecules pendant la phase pr~coce. Cette ddcroissance correspond ~ une phase de renouvellement suivie d'une p~riode de stabilisation qui prdckde la traduction des mRNA.

Poly A ( + ) m R N A synthesis was analyzed during the nymphal stage and the diapause in the wing discs o] the Lepidopteran Pieris brassicae. The main events of differentiation, i.e. scale [ormation, adult cuticle elaboration and pigment deposition, occurred during the period studied. We only detected one phase o[ synthesis [or poly A ( + ) m R N A molecules, 48-72 hours after the nymphal moult. This synthesis was found to be related to that of late proteins at 120-140 hours. Examination o] m R N A metabolism in disc~ treated with cordycepin (3" dA) showed a decline in m R N A stability. This decline corresponded to a turn-over phase ]ollowed by a period o[ stabilization which preceded mRNA translation.

Dans les animaux en diapause, le m~tabolisme des mRNA poly A ( + ) est dIevd et de nombreux messagers sont synthdtis& et rapidement ddtruits. Ces messagers sont ldgkrement plus lourds que ceux produits durant le ddveloppement normal, ce qui sugg&e un blocage gz l'une des dtapes de leur maturation. Nous avons ddveloppd un modkle mathdmatique du mdtabolisme des mRNA qui nous permet de calculer les eflets des paramP.tres de synthbse et de ddgradation sur la quantitO de m R N A et donc la demi-vie du pool de ces moldcules. Dans la mesure oh, chez Pieris, les phases de d~termination assocides aux phOnombnes embryonnaires sont distinctes des phases de diff&enciation terminales, il para~t possible d'dtudier sur ce matdriel les ~vdnements lids aux engagements qui prennent place fi la fin de la pdriode larvaire et au ddbut de la pdriode nymphale.

<> To whom all correspondence should be addressed.

In diapausing animals, poly A ( + )mRNA metabolism was unexpectedly high, and many messengers were synthesized and rapidly destroyed. These messengers were [ound to be slightly heavier than the m R N A s produced during normal development, suggesting a blocking at some step in their maturation. We developed a mathematical model for m R N A metabol&m which enabled us to calculate the effects of synthesis and degradation on the quantity of mRNAs, from the poly A ( + ) m R N A concentration and the turnover time in the mRNA pool. In addition determination phases associated with embryological events and terminal dff[erentiation are clearly distinguished. This feature offers opportunities to investigate the commitment events which take place during the end of the larval stage and the beginning of the nymphal stage.

106

P. Tarroux and P. Berreur.

can follow two opposite ways, depending on the temperature a n d / o r the photoperiod. L o n g days or high temperatures allow the increase of ecdysteroid level and thus normal development and imaginal differentiation. Short photophase or low temperatures induce diapause characterized by a low level of ecdysteroids. These animals, whose metamorphosis was p r o g r a m m e d so that development is stopped at the beginning of the n y m p h a l stage provide an interesting a p p r o a c h to the problem of regulating cellular activity [5[ [6] [7].

Introduction. T h e imaginal discs of insects m a y offer insight into the regulation of cell differentiation. In Lepidopteran, their relatively large size makes conventional biochemical experiments possible, and their ability to differentiate in vitro has already been clearly demonstrated [1] [2]. The original character of wing disc development offers ideal opportunities to tackle the problem of differentiation, since the morphological events involved (scale formation, cuticle deposition, cell polyploidization) are closely related to the biochemical events (specific protein synthesis, pigment deposition, etc.) [3] [4]. These p h e n o m e n a have been extensively studied in our laboratory on the comm o n Lepidopteran Pieris brassicae, and the in vivo and in vitro observations accumulated permit detailed investigation of mechanisms which control genetic expression.

Material and Methods. Developing animals: Pieris brassicae larvae and nymphae were reared at 22°(2, as previously described [5], and carefully synchronized at each moult by manual sorting. Under these conditions the nymphal stage lasts ten days.

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FIG. 1 . - Long and short-term specific activities o] RNA in developing wing discs. SFC

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Curve a (O--O and ©--O) shows the variations in specific activity in 28S and 18S rRNA species, respectively, for long-term (24 hours) in vivo labeling. Pupae at different stages of development were injected with 10 M of [14Cl urid,ine (CEA, specific activity 370 MBq/ mmol). Total RIgA was analyzed on 5-20 per cent sucrose gradient in 10 mM Na acetate, pH 5.4 ; radioactivity and optical density at 254 nm were then measured on each fraction. Curve b (A---A) shows the variations in specific activity in rR,NA speoies for short in vivo labeling (1 hour). Values for 18S and 28S are not distinguishable. RNA labeling and extraction procedures were the same as for curve a, except that [a4C] uridine was replaced by 5 ~1 of [3H] uridine (CEA, specific activity 740 GBq/mmol). Devel,opmen,t indicated at the top of the figure by schemas showing the slate of differentiation of the epidermal (ec), scale-forming (SFc) and socket-forming (SRFc) ceils at the stage indicated. NM and IM refer to nymphal and imaginal moRs respectively.

(hours)

We centered our investigations on the characterization of certain protein markers which are specifically synthesized at different stages of development. T h e result of the initial experiments led us to examine in greater detail the level of expression of the genetic material, and we conducted a series of studies of m R N A molecule metabolism. I n this paper, we report quantitative observations on the poly A ( + ) m R N A molecules extracted from the imaginal wing discs during the n y m p h a l stage, when most of the biochemical wing differentiation occurs. In addition, we report experiments on diapausing animals. A t the end of the larval stage in Pieris brassicae the development BIOCHIMIE, 1983, 65, n ° 2.

Diapausing animals : These animals were reared in an atmosphere controlled room, with programmed photophase (8 hou~rs of light). Diapausing nymphae were obtained after ten days under these conditions but experiments animals were used only three months later, in order to ensure that diapause was complete. Disc Labeling : In vitro experiments were performed in order to obtain incorporation of the precursors independent of their concentration in the hemolymph and of blood volume. This technique was used except for th~ experiments reported in figure 1 (see legend of this figure). Most imaginal discs were dissected, rinsed in disse~tion medium containing 138 m,M NaC1, 91 mM KCI, 3.7 mM KH~PO,, 7.8 mM NazI-IPO, and then layered on the surface of A22 Landureau culture medium [8] in

Poly ,4 ( + ) m R N A s

metabolism in wing imaginal discs.

a 3 cm plastic Petri d~sh. Discs from four nymphae were incubated with 1.5 ml of medi.um containing [all] uridine (CEA, 740-925 GBq/mmol, 46.2:5 M,Bq/ml. 1 Ci = 3.7 10TM Bq).

The R N A pellet obtained was generally resuspended in H~O and precipitated twice with ethanol in order to remove phenol.

mRNA analyses : Poly A ( + ) m R N A molecules were separated from the bulk of the R N A by affinity techniques.

Petri dishes were stored for 3 hours in an incubator at 24°C and gently shaken. Discs were quickly rinsed and immediately homogenized, or stored at - - 8 0 ° C after rapid freezing under liquid n~trogen for further study. Repeated checking showed that this technique makes it possible to evaluate normal disc activity and also amplifies precursor incorporation. However, this only applies to short term experiments of a few hours and we therefore limited incubation to 3 hours. No change was observed in the wing discs during this period of labeling, either in their morphological and structural aspects, or in their precursor incorporation rate.

FiG. 2. - - Evolution oJ the specific activity and quantities of total RNA during normal development in wing discs. Curve a (A--A) shows the evolution of the quantity of total R N A per imaginal~ disc. R N A labeling was performed by the in vitro procedure described in Material and Methods. Total RN~A was e-valuated by U V absorption at 254 nm, assuming 'that 1 mg of R N A gave 24 units of optical densi~ at 254 nm. Curve b ((3---(3) shows the variation in specific activi,ty in this RIGA. RNAs were extraoted and analyzed as described in Material and Methods.

107

Radioactive mP~NAs were recovered by hybridization on poly U × G F / C filters prepared as described elsewhere [9]. For subsequent studies, poly A ( + ) m R N A s were purified on an oligo dT-cellulose column (0.5 g of dry powder in a 1 × 10 cm column), essentially as described by Aviv and Leder [10]. Type 3 otigo dT-celintose from Collaborative Research was used (up to 20 nucleotides long with 20 mg of nucleo-

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RNA ertraction : Total wing discs were homogenized with a Potter-Etvejhem tissue grinder in one volume of phenol-chloroform (1:1) and one volume of extraction buffer (0.5 per cent SDS, 0.12 M NaCI, 10 mM Tris-C1, pH 7.4), in order to minimize degradation by RNases. After a first centrifugation (10 rain, 7,500 rpm, Sorvall SS34 rotor) the supernatant aqueous phase was carefully recovered ; the protein interphase was extracted with 1/2 volume of extraction buffer and the pooled aqueous phases were extracted twice with phenol-chloroform under the same conditions. The total aqueous phase was precipitated overnigh,t by two volumes of. cold ( - - 2 0 ° C ) NaCl-saturated ethanol.

BIOCHIMIE, 1983, 65, n ° 2.

stage (hours)

tides/g of cellulose). Binding and elution buffers were those described by Aviv and Leder [10] vcith 0.5 per cent SDS. The polyA content of each sample was determined by the technique of Bishop and coll. [11].

Results. D e v e l o p i n g pupae : P r e v i o u s studies in o u r l a b o r a t o r y s h o w e d t w o p h a s e s of p r o t e i n synthesis in t h e i m a g i n a l w i n g disc d u r i n g n y m p h a l differ e n t i a t i o n a n d L a f o n t has d e m o n s t r a t e d the c o r r e -

108

P. Tarroux and P. Berreur.

lation of these phases with the two steps in total RNA elaboration [12]. Many investigations also made it clear that this R N A is essentially ribosomal. To complete these observations, we performed the experiments reported in figure 1. They show the specific activity in the rRNA extracted from discs of animals injected with radioactive uridine at various stages of their development. Two phases of incorporation appeared, extending respectively from 12-24 hours and 120-140 hours after pupariation. After 1 hour of labeling specific activity was higher during the first phase than during the second (dashed curve). After 24 hours of labeling, however, this activity reached the same level for both phases of rRNA synthesis.

For poly A ( + ) m R N A , figure 3 illustrates the same type of experiments as figure 2. The evolution of the amount of mRNA (fig. 3, curve a) showed a large drop in the early stages of nymphal development (10 to 24 hours) followed later by a rise (24 to 96 hours). At this stage~ the mRNA content reached a plateau and then fell from 140 hours on. Specific activity reflected this synthesis and decrease from 24 to 140 hours (fig. 3, curve b). This observation coincides with the accumulation of mRNA molecules by the wing discs. [3H] uridine incorporation into mRNA molecules (fig. 4, curve a) increased during the early stages of nymphal differentiation but the specific activity peak preceded the radioactivity peak

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FIG. 3. - - Evolution of the specific activity and quantity o[ poly A (+) mRNAs during normal development. Curve a (A A) shows the variations of the quantity of poly A ( + ) mRNAs per animal, measured by the hybridization method of Bishop and Coll. [11] using [3H] poly U (see Material~ and Methods). Curve b (
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BIOCHIM1E, 1983, 65, n ° 2.

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In vitro experiments were performed to obtain a more accurate evaluation of the specific activity of the RNA molecules as well as measurements independent of the hemolymph pool. Figure 2 shows the evolution of the quantity and specific activity of total RNA after 3 hours of in vitro labeling. It was not possible, for this short-term labeling, to detect the two phases of synthesis mentioned above. However, the total RNA (fig. 2, curve a) displayed two periods of increase (2448 hours and 120-140 hours after the nymphal moult). In agreement with this result we observed two phases in the diminution of specific activity (curve b).

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(fig. 3, curve b). This result was expected, since simple mathematical calculation, not reported here, showed that specific activity was greater at the beginning of a phase of synthesis than at the end. After the peak of incorporation, we observed a decline in total precursor incorporation as well as in the amount of mRNA and rRNA present (data not replotted). Figure 4, curve b, also shows the variations in the m R N A / r R N A ratio. It increase is correlated to the differential accumulation of these two species of RNA during the 48-140 hour period.

Poly A ( + ) m R N A s

109

metabolism in wing imaginal discs.

later stages (curve b). Evaluations based on the calculations given in the section dealing with the model (see below) show the average half-life of the pools of poly A ( + ) m R N A s (T1/2) to be

These results might indicate variations in m R N A synthesis during the early phases of nymphal development, a hypothesis supported by the degradation process which occurred in m R N A molecules during these phases and was followed by a further synthesis of poly A ( + ) m R N A species. To substantiate the existence of such variations, experiments were performed in order to obtain information about the stability of the poly A ( + ) mRNA molecules (fig. 5). After three hours

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FIG. 5. - - Stability of the poly ,4 (+) mRN.4s in 3'd,4-treated wing discs at diJferent stages of development.

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Fro. 4. - - Relative evolution of poly A (+) mRNAs during wing disc development.

Curve a ( I - - I ) shows the [all] uridine incorporation into poly A (+) mRNAs on poly U GF/C filters during developmen,t. Curve b (n._ _rn) shows the evolution of the ratio of the quantity of poly A (+) mRNAs (measured by [aH] pc)ly U hybridization) to total RNA (measured as in figure 2).

of in vitro labeling in the presence of [3H]uridine, poly A ( + )mRNA synthesis was blocked by 1 I~g/ ml of cordycepine (3' dA) in the presence of 5 mM of cold uridine. The decrease of radioactivity in poly A ( + ) m R N A s was measured at various times after the introduction of 3' dA The m R N A from the discs of Pieris in early developmental stages (curve a) exhibited greater stability than that from the discs of animals at BIOCHIMIE, 1983, 65, n ° 2.

Curve a (D--O): poly A (+) mRNAs extracted from cultured wing discs of pupae 0-hour-old. Curve b (0--0) : poly A (+) mRNAs extracted from cultured wing discs of 24-hour-old pupae. Extraction and measurements were performed as described in Material and Methods. Regression lines were computed by linear regression analysis. Correlation coefficients exceeded 0.98.

57.6 ± 5.3 hours for the 0 hour stage and 25.43 -- 4.2 hours for the 24 hour stage, respectively. Diapausing pupae : The development of diapausing animals was stopped by lack of ecdysone secretion at a stage close to that reached by the 48-hour-old pupae. As already reported for diapausing animals, only the first protein synthesis took place at 24-48 hours and it is possible to observe the differentiation of the scale-forming cells as well as the early phases of polyploidization, but not the later phases. Note that scale formation was completely blocked in the absence of ecdysone (Mauchamp : personal communication). Only a few proteins were expressed in the diapausing animals and cell metabolism essentially concerned cuticle formation and the basic turnover of cell constituents.

110

P. Tarroux and P. Berreur.

Table I summarizes RNA metabolism in the wing discs of diapausing animals. Their relatively low content of total RNA probably corresponds to a regression of part of the wing tissues and to a low level of ribosomes within the cells. In contrast, the mRNA percentage is scarcely lower than in 24-hour-old nymphae. The high RNA

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FIG. 6. - - 15-30 per cent sucrose gradient analysis o] poly A (+) m R N A s extracted # o m developing and diapausing pupae. m R N A s labeled for 3 hours with [SH] uridine were purified by affinity chroma~tography on an oligo d T cellulose column (Collaborative Research T3), precipitated overnight at - - 2 0 ° C with a carrier (100 ~xg of cold Pieris rRNA), centrifuged, dissolved in 0.5 per cent SDS, 0.12 M NaCI' and 10 m M Tris-C1 buffer, p H 7.4, and analyzed on sucrose grad'ients in the same buffer at 20°C (50,000 rpm, 110 min, SW50. 1 rotor of a Spinco L-~o ultracentrifuge). Optical density (Am) was determined and fractions were collected with an ISCO automatic gradient fractionator with a UA5 optical unit (path length of the continuous flow cell : 10 mm).

precursor incorporation indicates synthesis of a large amount of ribosomal RNA and poly A ( + ) mRNAs. Figure 6 shows a sucrose gradient analysis of these poly A ( + ) m R N A molecules purified by affinity chromatography and extracted from the wing discs of diapausing animals. The same figure also gives the level of precursor incorporation ill BIOCH1M!E~ 1983~ 6.5) n ° 2,

the poly A (+)mRNAs extracted from developing pupae. The high level observed for diapausing wing discs (table I) was consequently no due to the predominant radioactivity of a particular species. However, some differences can be noted between extracts from diapausing and developing animals. In diapausing samples, we observed a peak in the 4S region. The absence from the mRNA pattern of a 28S contaminating peak ruled out the possibility of contamination by 4S or 5S RNA. We also measured the contamination by poly A dosage in the fraction retained on oligo dT cellulose by passing this fraction twice on an column. The second recovery and the pattern observed after sucrose gradient analysis were the same as for the first chromatography. This peak therefore only seems to be constituted by a few species of light poly A ( + ) m R N A which are presently under investigation. The mean sedimentation constant of the mRNAs from diapausing animals seemed to be slightly higher than in normal animals. In the 10-16S ,region radioactivity was greater in 24 hour~old than in diapausing pupae. In the latter, it was relatively greater in the 28S region. Although radioactivity distribution along the gradient was largely heterogeneous, the mRNAs extracted from diapausing wings seemed to be more homogeneous around the 18S region. This corresponds to the expression of a small number of different proteins. Modeling : To fully describe our experimental results we constructed a model for RNA synthesis and degradation. This enabled us to integate the different observations and test their internal coherence, thus facilitatin~ the formulation of hypotheses on the system The complete description of the model has already been published, but we report here those features that are essential to clarify particular points in the discussion. The metabolic pathways are summarized by the following scheme :

k2 k3 A ->X ~-M for the non-radioactive material, and kl ka y >-x >-m-

k~ ~k~ >-

for the radioactive molecules, where k~ is the kinetic parameter of the i reaction, A, the molarity in the pool of precursors, X or x and M or m, the molarity or the radioactivity in the nucleotides and mRNA pools, respectively, and y, the radioactivity of the external radioactive precursor. This _model closely corresponds to that of Puckett

Poly A ( + )mRNAs metabolism in wing imaginal discs. a n d D a r n e l l [13]. We chose an 0-order representation for these chemical reactions because the label decrease in 3' d A blocked discs suggests such kinetics (fig. 5). It is noteworthy that the variation in time of R N A synthesis z:nd degradation can be simulated by varying the k!netic parameters, so that the 0-order process is only apparent. T h e radioactivity in m R N A molecules at time t, after blocking of the synthesis is : m = mo - - k4m0t/M0

(1)

where m0 a n d Mo are respectively the radioactivity

111

a n d the q u a n t i t y of the m R N A species at time 0. The t u r n o v e r time of the pool of M is : T1/,~ = M0/2k4

(2)

a n d depends o n the m R N A concentration. F r o m the m a t h e m a t i c a l m o d e l corresponding to the chemical m o d e l presented above and consisting of a set of linear differential equations, we obtained the expression of m = fit) which is in the multiexponential form. If we assume the kinetic parameters to be i n d e p e n d e n t of time, each exponential term is a p p r o x i m a t e d by a T a y l o r series restricted

TABLE I.

Comparison o[ total R N A and m R N A metabolism in wing discs during normal development and diapause. [:~H] uridine incorporation (cpm/disc)

Normal development Diapause

Unbound (a)

bound (b)

Total RNA content 0g/disc)

5712.5 8100.0

478.7 2188.2

265 175

Per cent of poly A (q--) mRNAs in total RNA 0.7 0.55

Normal development : 24-hour-old nymphae. Diapause : induced as described in Material and Methods. Animals were stored for 2 months at 17°C after nymphal molt. Discs were labeled in vitro for 3 hours. Total RNA was extracted and fractionated on oligo dT columns. Unbound fractions (a) were precipitated with ethanol, dissolved in water and then precipitated with 10 per cent cold tricbloracetic acid on GF/C filters and counted. Bound fractions (b) were eluted by low ionic strength buffer and analyzed for poly A content and radioactivity, as described in Material and Meehods.

TABLE II.

Calculation of degradation parameters for m R N A s at two stages of normal development.

Stage

1

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4

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1-k~=1Mo q-- k ~ / 2 M o ": ---- 3 h

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9.743 10-1 9 427 10-I

286 10-~ ~ 0 53 3.65 10 -~' -~- 0 79

(h)

(h)

k4/Mo (1/2Tt/0 (h-1)

0 24

57 6 h 25.4

8.7 ~ 0.75 19.66 ± 2.31

9.74 10-1 9.43 10-t

1'

2'

3'

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2.27 ~- 0 46

1.77 ~ 0 3 4

0.78 -~- 0.31

Column 1 : half-l,ife of mRNA pool (experimental). Column 2: first-order degradation parameter (calculated). Column 3-4 : comparison of the respective degradation term of the equation m = f(x) (column 3) and of its 2nd order Taylor approximation (column 4). The equality of the two terms justifies the use of the approximation. Column 5 : calcafl~ated absolute rate of degradation. Columns 1', 2' and 3' show the respective rations of T,/2, the amount of mRNA (Q) and k~ calculated in 0-hour-old pupae to those calculated in 24-hour-old pupae.

BIOCHIMIE, 1983, 65, n ° 2.

112

P. Tarroux and P. Berreur.

to the second order for simplification. After canceiling the homologous terms we obtain : m ~ klkz Z2/2Xo (3) f o r the expression of the radioactivity in M after a labeling time of ~. This simplification shows that the radioactivity m is independent of the k4 parameter as long as the 1: variable is small in comparison with M0/k4. Equation (2) shows that this condition occurs when z ~ T1/2. It is fulfilled (table lI) for the 0 hour and 24 hour stages. The duration of the experiments (3 hours) is always shorter than the turnover time of the mRNA molecule pool. Comparison of columns 3 and 4 of table 2 shows that the parabolic approximation used to establish equation (3) is entirely justified. The value obtained by the exact calculation (column 3) is close to the approximated one (column 4) and very close to 1. It is important to stress the great importance of this observation in the interpretation of incorporation experiments. The results in table 2 demonstrate that variations in the radioactivity incorporated in poly A (+)mRNAs only correspond to variations in the synthesis parameter k~. We can deduce from equation (2) the value of k4 at different stages if we know the value of M0 at these same stages; the result of such a calculation is given in column 5 of table 2, for 0 and 24-hour-old nymphae. The values used for T1/2 (column 1) are deduced from the experiments reported in figure 5. At this stage of our investigations it is interesting to correlate the variations in turnover time between the two stages considered with one or other of the variables Mo and k4 which determine T1/2. This is why table 2 includes the ratio of T,/o (1'), M,, (2') and k4 (3') calculated in 0-hour-old pupae to these calculated in 24-hour-old pupae. Discussion.

The main purpose of this work was to examine mRNA formation during development and diapause in insect imaginal discs. This apparently simple problem is complicated by the existence of variable pools of precursors whose modifications complicate interpretation of labeling experiments, a difficulty reported in several systems [14] [151. An alternative method of determining the synthesis phases of poly A (+)mRNAs would be an accurate evaluation of these molecules. However, their accumulation need not necessarily be related to an increase in real synthesis but may rather be the result of stabilization. By integrating variaBIOCHIMIE, 1983, 65, n ° 2.

tions of the different biochemical parameters, the construction of a model for mRNA elaboration and degradation provides another me.ans o[ approaching these problems. Lafont and coll. [3] previously showed that the two nymphal phases of ribosomal RNA syntbesis are focused on the 24-28 hour and 120-140 hour stages of nymphal development. The situation is entirely different for messenger RNAs which seem to be produced during a single phase occurring in pupae aged from 48-72 hours. During this period, the total quantity of mRNAs increased by a factor of 5 (fig. 3, curve a) and the ratio of mRNAs/rRNA, by a factor of 2 (fig. 4, curve b) in 24-72-hour-old pupae. The differential phases of accumulation of both species of RNA are reflected in the evolution of this ratio. After sucrose gradient analysis, the polysome zone was compared to the monosome peak and no difference was found in the saturation levels of the ribosomes at 24 and 140 hours. Since in vivo protein synthesis was maximal in the terminal stage, this result suggests an increase in the translation speed of the proteins in the wings of 140-hours-old pupae. As we pointed out in the modeling section, precursor incorporation during the labeling time chosen is not dependent upon the stability of the mRNAs (k4). Moreover, uridine incorporation into poly A ( +)mRNAs was closely related to the synthesis variations determined on the basis of mRNA accumulation (curve a, figs. 3 and 4). These results imply that the variations in precursor pools are not large at the 2 s:ages considered here, although the amount of radioactivity incorporated in mRNAs depends upon the incorporation kinetic constant kl and upon the concentration of the precursor pool Xo (see equation (3)). Degradation : The comparison of the half life of the species of poly A ( + ) m R N A extracted from 0 and 24-hour-old pupae showed relative destabilization of messages in 24-hour-old pupae. This result and the accumulation observed after 72 hours is interpreted as indicating a modification in the protein expressed at the early and late stages. A first set of messages is expressed during the early stages and is replaced by a second one during the late stages. Analyses of protein expressed, by two-dimensional electrophoresis [291 and in vitro translation experiments (data not reported), show that these differences are qualitative. Experiments to test this hypothesis are presently in progress. Although in an 0-order model, variations in mRNA stability depend on M0, comparison of columns 1', 2' and 3' of table 2

Poly A (+)mRNAs metabolism in wing imaginal discs. shows that k4 variations are still partly responsible for message destabilization. By contrast, in the late phase of disc evolution (48-140 hours) the increasing difficulty of labeling mRNAs and the fact that their total quantity forms a plateau suggest that these molecules are subject to a period of stabilization. Such stabilization has been noted in several systems [16], and the half-life values (T1/2) obtained in our case (fig. 5) did not differ from those reported elesewhere. The values most often found range from 5 to 50 hours [17] [18] [13]. However, like Galau and coll. who worked on sea urchin embryos [19], we were unable to confirm by our method that mRNA populations contain a few rapidly decaying or very stable kinds of mRNA in insect wing discs, as previously reported for various other types of tissues [13] [20]. This problem requires further investigation, which the elaboration of a model of mRNA degradation would greatly facilitate. During normal development, most mRNAs seem to be synthetized 48 to 72 hours before translation; however, the increasing difficulty of labeling all RNA species during short pulses could be connected with a defective precursor penetration in the late phases of wing differenciation. This fact is not clearly established but the decrease in radioactivity (see specific activity curves b, figs. 2 and 3) may be associated with the programmed cellular death which usually occurs in wing discs 72-96 hours after synthesis at the same rate as the radioactivity declines [4]. This suggests the existence of an interesting cell designation mechanism for programmed degeneration. To conclude this discussion on mRNA stability, we could like to reexamine the question of the 0-order kinetics of mRNA degradation. Our results for degradation kinetics in 3'dA-treated discs and the close agreement between our data and the model described above led us to deduce that this type of kinetics were involved in mRNA synthesis. Other authors reached a similar conclusion [26] [27] when explaining why such synthesis was independent of nuclear nucleotide pools. As already stated, 0-order kinetics might in fact only be apparent, especially when the kinetic parameters are not time-independent during the differentiation process. Nevertheless, several comments can be made about the significance of such a process : in an 0-order model for mRNA degradation the reaction is not random, unlike a first-order reaction, since, it is independent of the concentration of mRNA molecules. The advantage of such kinetics for the

BIOCHIMIE, 1983, 65, n* 2.

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cell is obvious, since the relative abundance of the message is not directly related to the expression of the biological function o~ these molecules. Different explanations can be suggested for such a mechanism : an attractive one is the possibility that the degradative system is physically related to the mRNA complex in such a way that their interaction may be independent of their relative concentrations. As reported in several papers [28], we suppose that specific RNases are bound either to the mRNA molecules or to the ribosomal system. Whether a message is translated or degraded depends on the program of the message itself (a program written into part of its sequence) as well as on its relative concentration in the cell. This however is not explained by a set of first order reactions. Consequently, the limiting factor in mRNA molecule degradation is not the amount of specific RNases, but rather an information borne by the RNA itself. Such a feature is well explained by a 0-order degradation kinetic. Diapause : A particularly interesting problem is the relation of the diapause to this mRNA regulation. In pupae programmed for diapause, the expression of normal diffentiation mav be blocked for periods as Ions as six months. The results reported here suggest that mRNA metabolism during diapause is continuous and that mRNA half-life is shorter than in normal development. This contradicts the visual assumption that diapause is a sleeping phase in the differentiation of imaginal organs. Nevertheless, certain observations concerning protein metabolism or cuticle elaboration [21], show that the imaginal disc in diapausing animals is active. However, as we have shown, protein synthesis in such cases seems to be reduced and several mRNAs may be not translated. The data replotted in figure 5 suggest that maturation of the majority of the mRNAs synthesized is blocked during diapause, and mRNAs from diapausing pupae are certainly heavier than those extracted from normal animals, although proteins and the poly A tail are of similar molecular weight in both types of animals. Several systems in which a block in normal development occurs have been studied for protein expression, and various explanations have been proposed for the lack of protein synthesis. Sequestered mRNAs were generally assumed to be present in Artemia embryos or dormant seeds [22] [23]. Polyadenylation of these messengers

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has b e e n suggested to be the m o d e of activation in b o t h A r t e m i a and Cotton seeds [24] [25] ; the possibility that such activation occurs in m o s t of the m R N A s of d i a p a u s i n g wings can be discounted, since these messages b e a r a n o r m a l p o l y A tail, b u t we c a n n o t exclude the possible existence of u n t r a n s l a t e d m R N A s n o t b o u n d to oligo d T cellulose. A n o t h e r m o d e of sequestration which does n o t exclude the p r e c e d i n g one was r e p o r t e d by H a m m e t a n d K a t t e r m a n for Cotton seeds ; they observe preferential m R N A a c c u m u l a t i o n in the nucleus of d o r m a n t seeds ; this might corr e s p o n d to o u r results for m R N A centrifugation (fig. 5), a n d to the b l o c k i n g of poly A ( + ) m R N A m a t u r a t i o n , T h e R N A m e t a b o l i c system during n y m p h a l d e v e l o p m e n t and d i a p a u s e in Pieris brassicae is an e x a m p l e of a p r o g r a m m e d process. I n this p a p e r we a t t e m p t e d a general description of certain difficulties e n c o u n t e r e d in the i n t e r p r e t a t i o n of e x p e r i m e n t a l observations caused b y v a r i a b l e p o o l s of precursors. W e s h o w e d that m R N A expression in the insect wing disc system p r o v i d e s a u n i q u e m o d e l of strictly p r o g r a m m e d cell differentiation.

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