Changes in translatable mRNAs during the larval-pupal transformation of the epidermis of the tobacco hornworm

DEVELOPMENTAL

BIOLOGY

92,330-342

(1982)

Changes in Translatable mRNAs during the Larval-Pupal Transformation of the Epidermis of the Tobacco Hornworm’ LYNN Department

of Zoology,

University

M. RIDDIFORD of Washington,

Seattle, Washington 98195

Received December 7, 1981; accepted in revised fornz

April

5, 1982

At the initiation of metamorphosis when exposed to ecdysteroid in the absence of juvenile hormone (JH), the lepidopteran epidermis changes its commitment from one for larval differentiation to one for pupal differentiation. Changes in mRNA populations during this change both in vivo and in vitro were followed by a one-dimensional SDSgel electrophoretic analysis of translation products made in a mRNA-dependent rabbit reticulocyte lysate system. The larval epidermal cell was found to lose its translatable mRNAs for larval cuticular proteins and the larval-specific pigment insecticyanin during the change in commitment; these never reappeared. For Class I cuticular proteins and for insecticyanin, this loss occurred during the exposure to ecdysteroid, each with a differing time course. By contrast, Class II cuticular mRNAs first increased during this time, then also disappeared by the time the cells were pupally committed. In vitro these mRNAs appeared in only trace amounts in response to 20-hydroxyecdysone (20-HE). The pupally committed cell (late in the wandering stage) contained mRNAs for three low-molecular-weight proteins which were precipitable with the pupal cuticular antiserum. The remainder of the pupal cuticular mRNAs were not translatable until the third day after wandering, a time when pupal cuticle is being deposited in response to a molting surge of ecdysteroid. The pupally committed cell also had at least one new noncuticular mRNA which coded for a 34K protein and which was absent from both larval and pupal epidermal cells making cuticle. Since its appearance in response to 20-HE in vitro is repressed by JH, it is called a pupal commitment-specific protein. Thus, during the change of commitment 20-HE inactivates larval-specific genes irreversibly in a sequential cascade of events. The activation of most pupal-specific genes then requires a subsequent exposure to more ecdysteroid. INTRODUCTION

The sequential polymorphism undergone by many insect cells (Kafatos, 1976) poses important problems for a developmental biologist. How does a differentiated cell change the types of products it produces? Can hormonally regulated genes be turned off so as never to be expressed again even when exposed to the same hormonal milieu? Does the loss of the ability to express certain genes require the acquisition of the ability to express others? The lepidopteran epidermis proves to be a good system in which to study such questions since it produces the cuticle and often pigments, both of which are stage specific. In the tobacco hornworm, Manduca sexta, the epidermis produces the larval cuticle and the blue pigment protein, insecticyanin (Cherbas, 1973), throughout the feeding larval stages under the influence of 20-hydroxyecdysone (20-HE) in the presence of juvenile hormone (JH) at the larval molts (Riddiford, 1980, 1981). During the final (fifth) larval stage the JH titer falls to undetectable levels followed by a small release of i Most of this work was done in the laboratory of Professor Robert Schimke, Department of Biological Sciences, Stanford University, Stanford, Calif., during the author’s sabbatical leave from the University of Washington.

ecdysteroid which initiates metamorphosis that is characterized by the cessation of feeding and the onset of wandering (see Riddiford (1980) for a review). This 20HE acts in the absence of JH to cause the epidermis to lose its ability to make larval products and to become committed to pupal differentiation by late on the day of wandering (Riddiford, 1976,1978). Such pupally committed epidermis cannot produce larval cuticle but only produces pupal cuticle when challenged by the hormonal milieu of a larval molt (ecdysteroid in the presence of JH). Thus epidermal commitment can be conveniently assayed by the assessment of the type of cuticle produced by epidermis implanted into a fourth instar and then recovered after the host molts to the fifth larval stage (Riddiford, 1978). This change of commitment occurs on a cell-by-cell basis over the approximately 24-hr exposure to ecdysteroid in the absence of JH (Riddiford, 1978). It occurs in the absence of DNA synthesis in most cells (Dyer et al., 1981) but requires both mRNA and protein synthesis (Riddiford et al., 1981). In these aspects the change appears similar to that occurring in many undifferentiated cells as they become committed to a certain path of differentiation (Levenson and Housman, 1981). Our preliminary studies of the translatable mRNAs showed that major changes in the mRNA populations 330

0012-1606/82/080330-13$02.00/O Copyright All rights

0 1982 by Academic Press, Inc. of reproduction in any form reserved.

LYNN M. RIDDIFORD

mRNAs

during Larval-Pupal

occurred during the change of commitment (Chen and Riddiford, 1981). To examine in further detail these changes in mRNA populations, I have used antisera to both larval and pupal cuticular proteins as well as to insecticyanin to precipitate translation products of epidermal RNA during the larval-pupal transformation. Both in vivo and in vitro studies showed that the translatable mRNAs for insecticyanin and the larval cuticular proteins disappeared after exposure to 20-HE in the absence of JH and did not reappear when ecdysteroids initiated pupal cuticle synthesis. By contrast, most pupal cuticular mRNAs did not become evident in the pupally committed cells but appeared only later as a prelude to the synthesis of the pupal cuticle. MATERIALS

AND METHODS

Animals Tobacco hornworm (Manduca sexta) larvae were reared according to the techniques of Bell and Joachim (1976) at 255°C 12 hr light:12 hr dark photoperiod, and 60% relative humidity from at least the beginning of the final larval instar. Animals from both the University of Washington and Zoecon Corporation stocks were used for these experiments. No differences in timing of metamorphic events between the two stocks were detected. Animals that ecdysed to the final (fifth) instar at lights off designated 0O:OOAZT (Arbitrary Zeitgeber Time) +-2 hr on Day 0 were used for all experiments, All times are referred to AZT. Prepuration

of’ Epidermis

The larvae or pharate pupae were anesthetized in water or with COz, then surface-sterilized in two changes of 70% alcohol followed by two rinses in sterile distilled water. When RNA was to be isolated from the epidermis directly, the dorsal abdominal integument was removed to ice-cold sterile Manduca saline (Riddiford et al., 1979) and the adhering fat body and muscles were dissected away keeping the dissection dish on ice. As soon as a segment was clean, it was immediately frozen in liquid nitrogen. The elapsed time between the start of the dissection and freezing was lo-15 min. Epidermis that was cultured in vitro was prepared and cultured as previously described (Riddiford, 1978; Riddiford et d., 1979). The cleaned epidermis from one or two fifth instar larvae on Day 2 (14:00-17:00 AZT) was incubated for the requisite time in Grace’s medium (Gibco) either without hormones, with 1 pg/ml 20-HE (Rhoto Pharmaceutical Co.), with 1 pg/ml 20-HE and 3 pg/ml methoprene (Zoecon Corp.) or epoxygeranylsesamole (EGS; Eco-Control) (both JH analogs), or with one of these JH analogs alone. After the requisite time the tissue was rinsed in Manduca saline and most was

331

Transformation

frozen in liquid nitrogen for later RNA extraction. Four to six small pieces were implanted into fourth instar larvae to assay the commitment stage of the epidermis (Riddiford, 1978). The epidermal cyst was recovered after the host molted to the fifth instar, and the type of cuticle secreted by the implant was determined. Larval cuticle indicated that the cells were still larvally committed whereas pupal cuticle indicated that the cells had become pupally committed during the incubation in vitro before implantation. RNA Extraction Total cellular RNA was prepared by the technique of Ross (1976) with the substitution of 6 M urea for 7 M urea. The integumental pieces were homogenized in 10 vol 6 M urea (Schwartz/Mann Ultrapure) and 2% sodium dodecyl sulfate (SDS) in a high salt buffer (0.35 M NaCl, 0.001 M EDTA, 0.01 M Tris, pH 8.0) with a Polytron homogenizer in short 5-see bursts for a total of about 30 set at room temperature. The homogenate was spun at 10,OOOgfor 10 min in a swinging-bucket rotor to precipitate the unhomogenized cuticle. The supernatant was then extracted twice with an equal volume of water-saturated phenol containing 0.1% B-hydroxyquinoline and a half-volume of ehloroform:isoamyl alcohol (24:1), then twice with an equal volume of chloroform:isoamyl alcohol (24:l). The RNA was separated from DNA by CsCl centrifugation (Glisin et al., 1974). The RNA pellets were dissolved in water by heating at 68°C for 30 see, and the rapidly cooled solution was adjusted to 0.1 A4 NaCl. The RNA was reprecipitated by the addition of 2.5 vol cold ethanol overnight at -20°C. After washing with 70% ethanol, the precipitate was lyophilized. All glassware and solutions were autoclaved before use except for the urea and SDS. The Polytron probe was soaked in 0.2% diethylpyrocarbonate in 5% ethanol for 30 min immediately prior to use. Poly(A)‘-RNA was prepared from the total RNA by oligo(dT)-cellulose (Type 3, Collaborative Research) chromatography essentially as described by Alt et al. (1978). The RNA was dissolved in 0.5% SDS, 10 mM Tris, 1 mM EDTA, pH 7.4, heated at 68°C for 3 min, then rapidly cooled in an ethanol-ice bath. This solution was adjusted to 400 mM NaCl and the poly(A)‘-RNA bound by four passes over the column. The bound RNA was eluted with 0.5% SDS, 10 mM Tris, 1 mM EDTA, pH 7.4, adjusted to 400 mM NaCl, and precipitated at -20°C overnight with 2.5 vol ethanol. RNA-Dependent Translations

Rabbit Reticulocyte

Lysate

Micrococcal nuclease-treated rabbit reticulocyte lysate was prepared by a modification of the Pelham and

332

DEVELOPMENTALBIOLOGY

Jackson (1976) procedure outlined by Alt et al. (1978) and used immediately. The translations were run for 1 hr at 25°C with either [3H]leucine or [35S]methionine as described by Alt et al. (1978) and the amount incorporated was determined by trichloracetic acid-precipitable radioactivity in the lysate reaction mixture. Typical stimulation was 15- to 20-fold above the background for a lysate with no RNA added. Recently a freshly prepared lysate has given a 30- to 40-fold stimulation with the same RNA preparations and somewhat increased translation of bands above 60,000 daltons but no major qualitative changes are seen. Usually 50 pg total cellular RNA (assuming that Azso = 1.0 indicates 32 pg/ml RNA (White and DeLucca, 1977)) was used per 90 ~1 lysate incubation. Incorporation was linear in the range tested, 10 to 75 pg, indicating no inhibitory factors in the total RNA preparation. Preliminary experiments showed that heating the aqueous RNA solution at 68°C for 2 to 3 min followed by rapid cooling in ice immediately before adding the treated lysate improved the efficiency of the translation as indicated by the increased total incorporation. More than 5 min heating was deleterious to the translatability of the RNA. Gel Electrophoresis Aliquots of the translation mixture containing an equal number of TCA-precipitable counts were added to an equal volume of 2X SDS-containing sample buffer and run on 0.8-mm vertical SDS-polyacrylamide gel slabs according to the method of Ames (1974). The samples, the molecular weight standards (phosphorylase b, 94,000 daltons (94K), bovine serum albumin 67K, ovalbumin 43K, carbonic anhydrase 30K, soybean trypsin inhibitor 20.1K, and a-lactalbumin 14.4K; Pharmacia), purified rabbit actin (gift of Dr. Helen Blau), and crystalline insecticyanin purified from the hemolymph (gift of Dr. Walter Goodman) were boiled for 3 min in SDS sample buffer, cooled, then centrifuged for 30 set in an Eppendorf microfuge. Twenty to twenty-five microliters was loaded onto each lane. Electrophoresis was performed at room temperature at a constant current of 15 mA through the stacking gel, then at 20 mA. The gels were stained with Coomassie brilliant blue (Weber and Osborn, 1969), then treated with Enhance (New England Nuclear), and dried, and fluorograms were prepared using preflashed Kodak XR-1 or XRP-1 film (Laskey and Mills, 1975). Molecular weights of selected polypeptide bands were determined from the distribution of the weight of standard proteins (Weber and Osborn, 1969). Selected underexposed fluorograms were scanned and the relative areas under the peaks obtained using a Joyce Loebl MKIIIC microdensitometer.

VOLUME92,1982

Antiserum

Preparation

For preparation of antisera against cuticular proteins, the integument was removed from 6.5- to 7-g fifth instar larvae at 15:00 AZT on Day 2 (larval cuticular antiserum) and from pharate pupae 6 to 8 hr before pupal ecdysis (pupal cuticular antiserum) and cleaned of all muscle, epidermis, and fat body under Manduca saline containing crystals of phenylthiourea to prevent darkening. The cleaned cuticle was cut in small pieces and homogenized in 2% SDS in 0.0625 M Tris, pH 6.8, with a motor-driven (ca. 400 rpm) ground-glass homogenizer in an ice bath for 5 min. These conditions had been previously determined in this laboratory to give maximal solubilization of both larval and pharate pupal cuticular proteins (A. C. Chen, unpublished). After centrifugation at 10,000~ for 10 min, the supernatant was adjusted to 0.083 M NaCl and mixed 1:l with complete Freund’s adjuvant; then 2 ml was injected into each of two rabbits for each cuticular preparation. Four and six weeks later second and third injections were given, and the subsequent week the rabbits were bled and tested for antibodies by the Ouchterlochny double-diffusion method. When the titer was sufficiently high, the rabbits were bled, and the antiserum was collected by centrifugation after the clot and red blood cells were removed. Insecticyanin antiserum and partially purified insecticyanin antibody were gifts of Dr. Walter Goodman, University of Wisconsin, and Dr. Peter Cherbas, Harvard University, respectively. Immunoprecipitation Heat-inactivated Staphylococcus aureus Cowan type I (SAC) was used as an adsorbent for the antibodyantigen complexes according to the method of Ivarie and Jones (1979). Briefly, 50 ~1 of the lysate translation mixture was incubated with 10 ~1 freshly washed 10% SAC suspension (IgSorb, New England Enzyme Center) for lo-15 min in ice; then the SAC was removed by centrifugation for 5 min in an Eppendorf microfuge at 2°C. The supernatant was transferred to a clean microfuge tube and incubated with 25 ~1 antiserum in an ice bath for 15-30 min; then 15 ~1 10% SAC suspension was added for an additional 15 min to precipitate the immune complexes. The mixture was centrifuged 2 min in an Eppendorf microfuge at room temperature, and the resultant pellet washed three times with 0.025 M potassium phosphate, pH 7.6, 0.1 M NaCl containing 0.001 M EDTA and 0.25% Nonidet-P40. The washed pellet was then dissolved in 25 yl SDS electrophoresis sample buffer, heated at 100°C for 3 min, and stored at -20°C until electrophoresis. The thawed sample was

LYNN M. RIDDIFORD

TOTAL

POLV

RNA

mRNAs

during Larval-Pupal

333

Transformaticm

guanidine thiocyanate (Chirgwin et al., 1979). Therefore, these procedures were used for all experiments reported in this paper. To determine whether non-poly(A)+-containing RNAs were contributing significantly to the translation products, I prepared poly(A)+-RNA from epidermal RNA from various stages of Manduca larvae. Figure 1 shows that although there were some quantitative differences between the translation products of the total RNA and the poly(A)+-RNA, the same polypeptide bands were found in each. Thus, the major proteins in Manduca epidermis are made on poly(A)+-RNA; furthermore, the presence of ribosomal RNA does not inhibit the in vitro translation of these messengers. Consequently, total RNA was used for the subsequent studies.

A+

$4

34 I!%! MdPd3

WPP

d2

d3

W

-L

FIG. 1. Fluorograms of reticulocyte lysate translation products labeled with [%]methionine of muscle (M) and of epidermal RNA on 11.5% SDS-gel. The epidermis was taken from Manduca larvae on Day 2 at 20:00 (d2), Day 3 at 20:00 (d3) and Day 4 (first day of wandering) at 20:00 (W) and from pharate pupae about 4-6 hr before pupal ecdysis (PP). The poly(A)‘-RNA was prepared from the total RNA by oligo(dT) chromatography. L designates the lysate blank. The numbers indicate the molecular weights in kilodaltons of the standards. INS is the insecticyanin standard (23,000 daltons (Cherbas, 1973)). Arrowheads indicate two protein bands which appear during the change in commitment,

ti

1 a

vortexed and centrifuged 2 min in an Eppendorf microfuge; then the supernatant was loaded into the well. -

RESULTS

Translation Products of Total RNA versus Poly(A)+-RNA Our earlier experiments translating total epidermal RNA isolated either by phenol-chloroform extraction or by CsCl gradient centrifugation in a wheat germ system (Chen and Riddiford, 1981) gave protein products mainly below 40,000 daltons on SDS-gels. The mRNA-dependent rabbit reticulocyte lysate translation system produced proteins up to 100,000 daltons when epidermal RNA was added. The urea-SDS-phenol-chloroform extraction outlined under Materials and Methods gave better yields, higher translation efficiency, and an increased number of protein bands on SDS-gels than did the previous extraction methods or extraction by

3AM 2AM 2PM3AM3PM tram

W

W+I W+ZW+3

PP

FIG. 2. Fluorograms of insecticyanin antibody precipitates of reticulocyte lysate translation products labeled with [%]methionine on a 12.5% SDS-gel. The far left lane shows the translation products from Day 3 (13:OO) epidermal RNA. The RNAs were from epidermis of Day 2 (AM = 13:OO; PM = 21:00), Day 3 (AM = 13:OO; PM = 20:00), and wandering stage (21:00) larvae; from prepupae 1 day (19:00, beginning ocellar retraction) (W + l), 2 days (23:00) (W + 2), and 3 days (23:00) (W + 3) after the onset of wandering; and from pharate pupae 4 hr before ecdysis (PP). Precipitates were from translation products of approximately equal cpm through wandering PM and for pharate pupae; for W + 1 through W + 3 the translation mixtures precipitated contained four times as many cpm. The molecular weight standards in kilodaltons and the crystalline insecticyanin (I) marker (23,000 daltons (Cherbas, 1973)) are shown at the left.

334

DEVELOPMENTAL BIOLOGY

VOLUME 92, 1982

The heavy band at 43,000 daltons seen in Fig. 1 is likely actin as it comigrated with a rabbit actin marker and was the major band found when muscle RNA was translated (Fig. 1). Although muscle was difficult to dissect completely away from the insect integument, a comparison of the relative intensity of the bands in the translation products of muscle RNA and of epidermal RNA (Fig. 1) suggests that little muscle contamination was present and that the epidermal cells contained a significant amount of mRNA for actin, particularly on Day 3.

daltons larger than the crystalline protein obtained from the hemolymph. The disparity in molecular weights suggests the presence of a signal peptide in the translation product that is normally cleaved from the protein before it is secreted into the hemolymph. Large amounts of insecticyanin were present among translation products from epidermal RNA extracted through Day 3 of the fifth instar (Fig. 2). Only traces were observed on the day of wandering, and none were detectable thereafter, even when the antibody precipitation was from translation mixtures containing four times as many counts per minute. Likewise it was not among translation products of RNA taken during the time of pupal Developmental Changes in IdentiJied mRNAs during differentiation, i.e., from 2 days after wandering through the Larval-Pupal Transformation the pharate pupal stage. Thus insecticyanin is made Insecticyanin. To determine which protein band was only by larval cells and its mRNA disappears when the insecticyanin, the insecticyanin antibody was used to cells become pupally committed at the wandering stage. precipitate this protein from the translation products. Larval cuticular proteins. A major product of the epiAs seen in Fig. 2, insecticyanin mRNA was present in dermal cell is cuticle. Amino acid analyses of both larval larval epidermis and produced a protein about 1500 and pupal cuticular proteins of Manduca indicate that

94-

-

Met 01 0

2AMO Trans

I

2

2

3

3

AM

PM

PM

PM

Wander AM PM

wt1w+2

67-

0

PP

PM

I

2

3

Wander

AM

PM

AM

W+I PM

W+2 W+2

W+3

pp

pp-M Tra ns

PP

PP-L Tra ns

b

FIG. 3a. Fluorogram of larval cuticular antiserum precipitates from reticulocyte lysate translation products labeled with [3H]leucine on a 12.5% SDS-gel. RNA was extracted from epidermis of Day 0 (15:00), Day 1 (17:00), Day 2 (AM = 13:00, PM = 21:00), Day 3 (PM = 21:00 and 1700, respectively), and wandering stage (AM = 15:00, PM = 21:00, two different preparations) larvae; from prepupae 20:00, 1 day (W + l), and 2200, 2 days (W + 2), after wandering begins; and from pharate pupae (PP) 4 hr before pupal ecdysis. Precipitates were from equal volumes of lysate (50 fig RNA per 90 ~1 translation mixture), which contained approximately equal cpm (+S%) from Days 0 to 3 and for pharate pupae except for Day 2 PM (ca. 75% of Day 0) and for wandering through W + 2 (ca. 60% of Day 0). The lane on the far left shows the larval cuticular antiserum precipitate from translation products of Day 1 RNA labeled with [?S]methionine. The molecular weight standards in kilodaltons separate this lane from the translation products labeled with [3H] leucine from epidermal RNA. FIG. 3b. Fluorogram of pupal cuticular antiserum precipitates from reticulocyte lysate translation products labeled with [35S]methionine on a 12.5% SDS-gel. RNA was same as used in Fig. 3a except for a second preparation at 19:00, 2 days after wandering (W + 2), and one at 21:00, 3 days after wandering (W + 3). Precipitates were from equal volumes of translation mixtures (50 pg RNA per 90 ~1 translation mixture); hence total counts on the mixtures were about the same (*lo%) except for Day 3 PM (75%), W + 2 (65 and 75%, respectively), and W + 3 and PP (ca. 200%). The two lanes on the far right show the pupal cuticular antiserum precipitate and translation products respectively labeled with [‘Hlleucine using pharate pupal epidermal RNA. The molecular weight standards in kilodaltons are shown at the sides.

LYNN M. RIDDIFORD

mRNAs during Larval-Pupal

335

Transfwmativn

56WANDERING BEGINS

LARVAL ECDYSIS WANDERING BEGINS +

LARVAL ECDYSIS

52

52-

J

t

4040 4444 4040 3636 32-

zs-

I i i

I 1 I I I I

24-

20-

! I i i i :

I

I2

8

0:

I DAYS OF FIFTH

4 INSTAR

a

b

DAYS OF FIFTH

INSTAR

FIG. 4. Diagrams of the relative amounts of Class I (a) and Class II (b) larval cuticular proteins in [3H]leucine-labeled translation products of epidermal RNAs during the fifth larval instar and the larval-pupal transformation. The points on Days 2 through wandering are based on an average density of microdensitometric scans of fluorograms of antiserum-precipitable bands from translations of two different RNA preparations, one of which is shown in Fig. 3a. Those from the pharate fifth instar larva through Day 1 are based on precipitates from two different translations of the same RNA preparation. Since different exposure times and different fluorograms were necessary to obtain all the data, the relative intensities between bands should be considered approximate.

no methionine is present and that leucine comprises about 4% of the total amino acids in both larval and pupal cuticular proteins (Riddiford et al., 1980; Chen and Riddiford, unpublished). When the patterns of translation products obtained with these two amino acids were compared, many quantitative but no major qualitative differences were evident for either larval or pharate pupal RNA (complete data not shown; for an example, see Fig. 3b). Similarly, when cuticular anti-

serum precipitates from the two translations were compared, no qualitative differences were apparent (Figs. 3a, b). Since secreted cuticular proteins lack methionine, the labeling that is seen in the translation system must be due to the initiator methionine plus additional methionines in the signal peptide region (Davis and Tai, 1980). Snyder et al. (1981) have recently shown that at least one of the Drosophila larval cuticular proteins has a signal peptide.

336

DEVELOPMENTALBIOLOGY

Antiserum to Day 2 larval cuticle caused precipitation of 17 protein bands from the translation mixtures from Day 0 epidermal RNA (Fig. 3a). Antiserum from a different rabbit gave identical precipitates except for the 13.4K band (the upper band of the bottom doublet in Fig. 3a) which was missing. Consequently, the first antiserum was used for all subsequent studies. Figure 3a shows that major changes in the larval cuticular proteins found in the translation mixtures occurred on Day 3 when the cells were changing their commitment (Riddiford, 1978). By the afternoon of the wandering stage all but five bands had disappeared, indicating a loss of translatable mRNAs for the major larval cuticular proteins in the pupally committed cells. The larval cuticular proteins found in the translation mixtures appeared to fall into two discrete classes: Class I, those that were present during most or all of the feeding stage; and Class II, those that appeared or increased substantially on Day 3. Figure 4 shows the semiquantitative changes in the protein bands of these two classes as a function of developmental stage based on microdensitometric scans. Most of the mRNAs for Class I cuticular proteins were available for translation on Day 0 to 1, then began declining on Day 2, and disappeared from the pupally committed cells at the onset of the wandering stage (Fig. 4a). The apparently anomalous 14.2K protein band which appeared to increase fourfold in intensity between Days 1 and 2 is likely due to the appearance of a second polypeptide of similar molecular weight which I have been unable to resolve satisfactorily on these first-dimension gels. The Class II proteins appeared first either on Day 2 or on Day 3 (Fig. 4b). All showed the largest increase on Day 3, then most disappeared by the afternoon of wandering when all cells were pupally committed. Pupal cuticular proteins. Antiserum to pharate pupal cuticle primarily precipitated proteins from translation mixtures of RNA taken either the day before (W + 3) or the day of pupal ecdysis (pharate pupa) (Fig. 3b). On the day of pupal ecdysis the pattern of precipitated proteins was similar to that precipitated by the larval cuticular antiserum (Fig. 3a) although the pupal cuticular antiserum did not appear to cross-react with the larval cuticular proteins produced between Days 0 and 3 (Fig. 3b). Importantly, this antiserum precipitated three new low-molecular-weight bands (10.4K, llK, 11.5K) from the pupally committed (wandering stage) epidermal mRNA translation mixtures (Fig. 3b). These were not precipitable by the larval cuticular antiserum (Fig. 3a). By the afternoon of the second day after wandering (just before the beginning of pupal cuticle deposition), these proteins had virtually disappeared from the translation mixtures.

VOLUME 92,1982

Precipitation by the cuticular antisera thus indicates that when the cells become committed for future pupal differentiation, the mRNAs for larval cuticular proteins become untranslatable but the majority of those used for pupal cuticular protein synthesis do not appear until about 2 days later at the time of formation of the new pupal cuticle. Noncuticular proteins. The pupally committed cell has many fewer translatable mRNAs (Fig. 1) due to the loss of the insecticyanin mRNA, the larval cuticular mRNAs, and at least three other noncuticular mRNAs (bands at 39K, 3’7K, and 35.7K). In Fig. 1 only two new bands can be detected (arrows), one about 60K and one about 39.5K. Translations of other preparations of late wandering stage RNA sometimes showed small amounts of a 34K band which was one of the major proteins synthesized in translation mixtures from RNA taken the day after wandering (data not shown). The high-molecular-weight band (60K) was present only on the day of wandering whereas the two lower-molecular-weight bands were still found in small amounts 2 days later (data not shown) but were not present in translations of pharate pupal RNA. Relative to the larval cuticular proteins which were synthesized in large amounts during the feeding stage, these new proteins were very minor. Since they were not precipitable with either the larval or pupal cuticular antisera and appeared only late in the process of pupal commitment, then disappeared either before or just after pupal cuticle formation began, I have termed them “pupal commitment” proteins. Changes in Epidermal mRNAs during Commitment in Vitro

the Change of

The above studies showed that the loss of larval cutitular and insecticyanin mRNAs and the appearance of pupal commitment and three pupal cuticular mRNAs were correlated with the change to pupal commitment which begins on Day 3 and is completed by the afternoon of the wandering stage (Riddiford, 1978). In vitro the change can be effected by the exposure of Day 2 epidermis to 1 pg/ml 20-HE for 24 hr in the absence of JH (Riddiford, 1976, 1978). Therefore, to determine whether all of the changes in mRNAs seen above were the result of ecdysteroid action, Day 2 larval epidermis was exposed to 20-HE for varying lengths of time up to 24 hr by which time all the cells have become pupally committed (Riddiford, 1978). Figure 5 shows that the 20-HE exposure was accompanied by a loss of translatable mRNAs and an appearance of others. The translatable RNAs present in the cells after 24-hr exposure to 20-HE were similar to those seen in Fig. 5a in all seven preparations done, but differed somewhat from

mRNAs

LYNN M. RIDDIFORD

during Larval-Pupal

b

a

67-

--

*

Day2 AM

337

Transformation

3 -Hrs

6

9 20-OH

12 18 Ecdysone-

24

24 3 6 12 -Hrs after Removal20-OH Ecdysone

-

W AM

24E L Cut Ab

24-6

24E-I8 P CutAb

FIG. 5a. Fluorogram of reticulocyte lysate translation products labeled with [?S]methionine on a 12% gel. RNAs were extracted from Day 2 larval epidermis (16:00) cultured with 1.3 pg/ml 20-HE for varying lengths of time up to 24 hr or with 20-HE for 24 hr, then in hormonefree medium for varying lengths of time. Translation products from Day 2 (13:OO) and wandering stage (15:OO) larval epidermal RNA are shown for comparison. The molecular weight standards in kilodaltons are at the left of the gel. (-) Class I larval cuticular proteins; (6) Class II larval cuticular proteins; (a) designates new noncuticular proteins; INS indicates the band that precipitates with the insecticyanin antibody. FIG. 5b. Fluorograms of a 12.5% SDS-gel of larval cuticular antiserum precipitates of translation products labeled with [35S]methionine of three different RNA preparations from epidermis cultured 24 hr with 20-HE (24E) (the middle one corresponds to the 24E translation seen in Fig. 5a) and of epidermis cultured for 24 hr with 20-HE, then in hormone-free medium for 6 hr. Also shown on the far right is a pupal cuticular antiserum precipitate of [35S]methionine-labeled translation products of an RNA preparation from epidermis cultured 24 hr with 20-HE, followed by 18 hr in hormone-free medium (243-18). The molecular weight standards in kilodaltons are at the left of each gel.

the RNAs present in cells pupally committed in viva (wandering AM, Fig. 5a). After subsequent exposure to hormone-free medium, the protein pattern produced by the RNAs was more like that on the afternoon of wandering (cf. Fig. 1). Among the nine major Class I cuticular proteins present in translations of Day 2 RNA, all declined in amount translated by 6-hr exposure to 20-HE, each with its own time course (Figs. 5a, 6a). By 24-hr exposure to 20-HE, these proteins were either absent or considerably reduced (ca. lo- to 20-fold less). Surprisingly, when incubated in hormone-free medium after exposure to 20HE, some of these Class I mRNAs again were translatable (Figs. 5b, 6a), indicating that 24-hr exposure to

20-HE was insufficient to repress these mRNAs permanently. Although implants of tissue after incubation with 20-HE for 24 hr always produced pupal cuticle during a larval molt (confirming that they were pupally committed), after subsequent incubation in hormonefree medium about one-fourth to one-third of the cells produced larval cuticle in the same implantation assay. Of the five Class II cuticular proteins found in vivo all also appeared or increased during the exposure to 20-HE in vitro, then disappeared (Figs. 5a, 6b). The increase, however, was much smaller than that seen in vivo. For example, the 28.5K band increased only 2.5fold in vitro as compared to 16-fold in vivo (Fig. 4b). Trace amounts of the three low-molecular-weight

338

DEVELOPMENTAL BIOLOGY

VOLUME 92, 1982

52

44

40

36

\

/

jl2.8K.13.3K

I---ZO-HYDROXYECDYSONE

z z

32,

Z E Y c 3

-NO

HORMONE-

HOURS

b ISSK

28

24. _j

E

\

20.

16

I z-

8.

4.

+20-HYDROXYECDYSONEL-,36K

I

6

12

18

I

24

‘--20-HYDROXYECDYSONEa

c

--_

0.

6

12

I8

NO

HORMONE4

HOURS

1

24

NO HORMONEHOURS

FIG. 6. Diagrams of the changes in larval cuticular proteins of Class I (a) and Class II (b) and in noncuticular proteins (c) occurring during exposure to 20-HE in vitro for 24 hr and after removal to hormone-free medium. The amounts of cuticular proteins are based on microdensitometer scans of fluorograms of larval cuticular antiserum precipitates of [3H]leucine-labeled translation products after 6, 12, 18, and 24 hr exposure to 20-HE and subsequently 6 and 9 hr in hormone-free medium. Those at 0 and 24 hr exposure to 20-HE are based on averages of scans of two and four different preparations, respectively. The amount of noncuticular proteins (c) is based on microdensitometer scans of the fluorogram in Fig. 5a and a second gel of translation products from different RNA preparations. Insecticyanin was determined by antibody precipitation at the times designated above.

(10.4K-11.5K) protein bands precipitable with the pharate pupal cuticular antiserum were found after 24-hr exposure to 20-HE in three different preparations tested. Increased amounts of these mRNAs were present later after 18-hr incubation in hormone-free medium (Fig. 5b), corresponding to their appearance in vivo in epidermis late on the day of wandering (Fig. 3b). Among the mRNAs for noncuticular proteins, insecticyanin and the ones for the 37K and 39K proteins were

lost (Fig. 6~) in response to ecdysteroid exposure. Only one of the pupal commitment proteins seen in vivo appeared in vitro. This 34K protein (A in Figs. 5a, 6~) was found in translations of four of the seven preparations of epidermal RNA after 24-hr exposure to 20-HE, but never when the epidermis was incubated under conditions in which it retained its larval commitment (see following section and Fig. 7). Therefore, it is truly a pupal commitment-specific protein.

LYNN M. RIDDIFORD

D2

NH JH JHE

E

mRNAs

during Larval-Pupal

D2

I sN H24 J2H4JF4E2Eq FIG. ‘7. Left: Fluorogram of a 12% SDS-gel of [a5S]methionine-containing translation products of RNA from Day 2 epidermis (13:00) (d2) or from explanted Day 2 epidermis (13:OO) incubated 24 hr in hormone-free medium (NH), with 3 rg/ml methoprene (JH), with 3 pg/ml methoprene and 1.3 pg/ml20-hydroxyecdysone (JHE), or with 1.3 pg;/ml 20-hydroxyecdysone alone (E). (*) A new pupal commitment-specific band; (a) another new noncuticular polypeptide; arrow above the insecticyanin (INS) standard marker points to a band which precipitates with the insecticyanin antibody. Right: Fluorograms of larval cuticular antiserum precipitates of the translation products of the above RNAs with the addition of one after incubation in hormonefree medium for only 18 hr (NH18) on a 12.5% SDS-gel. Both sides: Molecular weight standards are on the right of each gel. (-) Class I larval cuticular proteins; (0) Class II larval cuticular proteins.

Inhibition of the Change of Commitment Hormcme

by Juvenile

The change of commitment induced by ecdysteroid in vitro can be inhibited by the presence of JH in the culture medium (Riddiford, 1976, 1978). Also, the epidermis retains its larval commitment when incubated in either hormone-free or JH-containing medium for 24 hr. Therefore, epidermis was cultured under these various hormonal conditions to determine whether all the changes seen in response to ZO-HE in vitro were due to the change of commitment and whether JH could prevent these changes. Figure 7 shows that most of the major Class I cuticular proteins were found in the same or slightly reduced amount when incubated in either hormone-free medium or with JH alone. Only two (21K and 30.5K bands) decreased substantially by 24 hr; these normally begin decreasing in vivo on the afternoon of Day 2 (Figs. 3a,

Transformatirm

339

4a). By contrast, after incubation with both JH and 20HE, most of the major Class I cuticular proteins were decreased as was seen after incubation with ZO-HE alone. Thus, despite the presence of JH and the retention of larval commitment, ZO-HE apparently causes a decrease in translatability of these larval cuticular messengers relative to those seen in Day 2 epidermis or in epidermis incubated with JH alone. Importantly, small amounts of the major Class II cuticular proteins were found under all these treatments in vitro (Fig. 7), appearing as early as 6 hr and reaching maxima at 12-18 hr (data not shown), then declining particularly in those containing ZO-HE by 24 hr (cf. Figs. 5b and 7). Therefore, the increase in at least these major Class II mRNAs which begins in vivo in the afternoon of Day 2 (Fig. 4b) appears not to be initiated by ecdysteroid but their decline at the end of Day 3 apparently is. Traces of the low-molecular-weight bands precipitated by the pupal cuticular antiserum were present at 24 hr in the larvally committed cells, which had been exposed to ZO-HE and JH (data not shown). Consequently, their appearance must be a result of ecdysteroid action unrelated to the change of commitment. Among the noncuticular mRNAs, insecticyanin mRNA declined only slightly in hormone-free or JHcontaining medium but substantially after 24 hr exposure to ZO-HE in the presence of JH (Fig. 7 and confirmed by antibody precipitations not shown). The 38.2K polypeptide band which appeared after incubation with ZO-HE for 12-18 hr (Fig. 6~) also appeared in hormonefree medium or after exposure to ZO-HE and JH but not after exposure to JH alone (4 in Fig. 7). Therefore, its appearance is not related to the change in commitment. By contrast, the 34K noncuticular protein (*, Fig. 7) only appeared after 18-24 hr in vitro with ZO-HE alone (see also Fig. 6a) and not in either JH-containing or hormone-free medium. Consequently, it is truly a pupal commitment-specific protein. DISCUSSION

The changes in the epidermal mRNA populations during the final larval instar described in this study can be correlated with the changes in hormonal milieu at the onset of metamorphosis. As diagrammed in Fig. 8 the JH levels are high to intermediate during the initial part of the final feeding period, then decline to undetectable on Day 2 (see Riddiford (1980) for a detailed review). Subsequently, a small release of ecdysteroid initiates the onset of metamorphosis as signaled by the onset of wandering behavior. The large surge of ecdysteroicl beginning on the second day after wandering causes the pupal molt, leading to the initiation of pupal

340

DEVELOPMENTAL BIOLOGY

insecticyanin

mRNA

Class I Larval

Cuticle

mRNA’s

Larval

Cuticle

mRNA’s

class11

Pupal Commitment Small

Pupal Cuticle

Most Pupal Cuticle I 0 A ecdysis to 5th

I 2

I

-A I

4f wandering

DAYS

I ’

puial’ cuticle deposition

mRNA mRNA’s mRNA’s

1 ’ pup01 ecdysis

FIG. 8. Diagram of hormonal titers in Manduca during the final (fifth) larval stage and the onset of metamorphosis up to pupal ecdysis (from Riddiford, 1981). The changes in selected mRNAs determined in this study are shown schematically below; the pupal commitment mRNA is that for the 34K protein.

cuticle deposition at the beginning of the third day after wandering (W. J. Wolfgang, unpublished studies in this laboratory) and culminating in pupal ecdysis. Since this molting surge of ecdysteroid occurs in the presence of JH just as it does in a larval molt (Riddiford, 1980), a polymorphic cell such as the epidermis must change from its larval to pupal state before this time. This change has been found to occur during Day 3 (Riddiford, 1978) when the cells are exposed to ecdysteroid in the absence of JH for the first time in their postembryonic life. Thus by the afternoon of the wandering stage, the entire epidermis is pupally committed. As summarized in Fig. 8, the loss of translatable larval-specific mRNAs such as that for insecticyanin and larval cuticular proteins coincides with the hormonally induced change of commitment. Even though new mRNA synthesis is required for the ecdysteroid-induced change of commitment (Riddiford et al., 1981), few new mRNAs are found in pupally committed cells: two or three pupal commitment mRNAs and three mRNAs for pupal cuticular proteins. Most of the pupal cuticular mRNAs are not present until pupal cuticle synthesis occurs in response to the second surge of ecdysteroid. Thus the pupally committed cell is not yet capable of making its main differentiated products but requires further hormonal stimulus to do so. Similar losses preceding gains are seen in the changing patterns of liver protein synthesis in metamorphosing Xenopus tadpoles (May and Knowland, 1981). The changes in patterns of proteins synthesized by the epidermis during the fifth larval instar and the on-

VOLUME 92, 1982

set of metamorphosis closely parallel the changes seen here in translatable mRNAs (Riddiford and Kiely, 1981; Kiely, 1982). Therefore, these latter changes likely reflect regulation primarily at the level of transcription and mRNA turnover as also appears to be the case in the developing adult wing of Drosophila (Mitchell and Petersen, 1981). The correctness of this conclusion will have to await the development of probes for the insecticyanin and the cuticular genes. The most striking change accompanying the change of commitment of the larval epidermal cells was the loss of translatable mRNAs for the larval pigment protein, insecticyanin, and for the larval cuticular proteins. Although the data in this paper only show that these larval mRNAs are no longer translatable, it is likely that the larval-specific genes are completely inactivated since they are not expressed again during either pupal or adult development. This lack of expression is not simply a result of the lack of the proper hormonal milieu since the pupal molt occurs in response to ecdysteroid in the presence of JH (Fig. 8) just as does the larval molt (Riddiford, 1980). Insecticyanin is produced by the epidermal cells throughout larval life from the second instar onward; part is released into the hemolymph while the remainder is stored in apical granules (Cherbas, 1973). These granules are lost progressively from the cells beginning a few hours before wandering (Cherbas, 1973; Wolfgang and Riddiford, 1981) until all are gone by about a day before ecdysis. The loss of the translatable mRNA for insecticyanin (Fig. 8) coincides with the beginning of the loss of granules. Both losses are initiated by the action of 20-HE. In vitro the loss of translatable mRNA was complete by 6 hr in hormone-free medium after exposure to 20-HE for 24 hr (Fig. 6~) and visible pigment loss from the cells is well advanced by 18 hr further incubation in hormone-free medium (W. J. Wolfgang, unpublished studies in this laboratory). Importantly, 20-HE must act in the absence of JH to inactivate this gene permanently since the translatable mRNA declines but does not disappear when tissue is incubated in 20-HE in the presence of JH (Fig. 7). Yet no loss of pigment is seen either in vitro (personal observations) or after implantation of this tissue into a penultimate instar larva where it subsequently undergoes a larval molt and continues to make insecticyanin (Riddiford, 1978). The loss of translatable Class I larval cuticular mRNAs in vivo also appeared to be correlated with the rise of ecdysteroid in the absence of JH (Fig. 8). In vitro studies, however, showed the situation to be more complex. After incubation with 20-HE alone for 24 hr, conditions under which all the cells become pupally committed and will produce pupal cuticle under larval molting conditions (Riddiford, 1978), three of the Class I

LYNN M. RIDDIFORD

mRNAs

cuticular mRNAs were no longer translatable, and the remainder were considerably reduced in translatability (Fig. 4a). In contrast to insecticyanin, some of these larval cuticular mRNAs became translatable again when the epidermis was transferred to hormone-free medium, and some of the cells reverted and produced larval cuticle when exposed to the hormonal milieu of the larval molt. Recent data have indicated that slightly longer incubations in low levels of ecdysteroid cause the permanent loss of these mRNAs (Riddiford, unpublished). The inactivation of these larval-specific mRNAs occurred progressively during the incubation with 20-HE; much of it was prevented by cycloheximide (Riddiford, 1981) or actinomycin D or ol-amanitin (unpublished). This system has some similarities to the sequential activation of Drosophila salivary gland chromosome puffing by 20-HE wherein products of early puffs apparently are required for activation of late puffs (Ashburner and Richards, 1976; Richards, 1980; Walker and Ashburner, 1981). In Munduca epidermis the continued presence of ecdysteroid seems necessary until all these larval cuticular genes are completely inactivated. Further experiments are needed to determine the mode of action of 20-HE in this sequential inactivation. The findings that Class 11 mRNAs were not increased to their in viva Day 3 levels by incubation with 20-HE in vitro and that they were present in somewhat larger amounts in Day 2 tissue incubated in hormone-free medium led to the hypothesis that their appearance was caused by the decline of JH. Juvenile hormone application to Day 1 larvae to maintain a high JH level prevented their appearance on Day 3 whereas it had no effect when applied to late Day 2 larvae after the decline in JH (Riddiford, 1982), thereby confirming this hypothesis. Moreover, the prevention of appearance of these mRNAs by JH also prevented the normal change (Wolfgang and Riddiford, 1981) in cuticular morphology from thick (1-p) lamellae to thin (0.1-p) lamellae on Day 3, thus suggesting the participation of the Class II proteins in the new cuticular structure (Wolfgang and Riddiford, in preparation). The pupally committed cell in vivo (late afternoon of wandering) has very few translatable mRNAs for products that are precipitable by either larval or pupal cutitular antisera. The mRNAs for the three low-molecular-weight pupal cuticular proteins (lOK-11.5K) detectable late on the day of wandering and on the following day (Figs. 3b, 8) must be for proteins that are produced before the cuticle actually is deposited since they disappear before the onset of epicuticle deposition at the beginning of the third day after wandering (Fig. 8). Studies of protein synthesis by these cells also indicate that a few pupal cuticular proteins are produced

during Larval-Pupal

Transfwmatimz

341

the day before deposition begins (Kiely, 1982; Kiely and Riddiford, in preparation). Only one (34K) of the new noncuticular proteins that appeared among the translation products can be said to be truly pupal commitment specific. It appears after incubation with 20-HE between 12 and 18 hr and JH blocks this appearance. Its appearance is also prevented by the presence of cycloheximide (band lla in Riddiford, 1981), indicating that the transcription of this mRNA requires the action of at least one primary gene product induced by 20-HE. Several sequential products in the cascade of cellular synthesis initiated and maintained by 20-HE could be required for its turn-on since it does not appear for the first 12 hr. The role of this protein is unknown but its presence is significant since it represents a developmental difference between the “committed” state and either of the cuticle-synthesizing states. Two-dimensional gel electrophoresis of both the translation products and the epidermal proteins has revealed still further changes (Kiely, 1982; Kiely and Riddiford, in preparation) but the basic conclusions remain the same. The primary action of 20-HE in the absence of JH is to inactivate larval-specific genes irreversibly. The time course of the loss of these mRNAs is different, suggesting that all are not coordinately regulated but rather that they respond to different signals in the sequential cascade of gene products elicited and maintained by 20-HE. The activation of most pupalspecific genes then requires a subsequent molting surge of 20-HE. I thank Professor Robert T. Schimke for laboratory space and supplies and for encouragement and advice in the course of these studies, Dr. William Dower for helpful advice, Dr. Gerardus Staal of Zoecon Corporation for use of their Munduca stock and for the methoprene, Drs. Peter Cherbas and Walter Goodman for insecticyanin and its antibody, Dr. Helen Blau for the rabbit actin, Anna Curtis for culturing the epidermis, Garland Bellamy for many of the RNA extractions, Richard Vogt for photography of the gels, and Professor James W. Truman for a critical reading of the manuscript. I was supported by a Guggenheim Foundation fellowship during much of this study. The work was also supported by grants from NIH (A112459) and NSF (PCM 76-14400). REFERENCES ALT, F. W., KELLEMS, R. E., BERTINO, J. R., and SCHIMKE, R. T. (1978). Selective multiplication of dihydrofolate reductase genes in methotrexate-resistant variants of cultured murine cells. J. BioZ. Chem. 253, 1357-1370. AMES, G. F. (1974). Resolution of bacterial proteins by polyacrylamide gel electrophoresis on slabs. J. Biol. Chem. 249, 634-644. ASHBURNER, M., and RICHARDS, G. (1976). The role of ecdysone in the control of gene activity in the polytene chromosomes of Drosophila. In “Insect Development” (P. A. Lawrence, ed.), Symp. Roy. Entomol. Sot. London, Vol. 8, pp. 203-225. Wiley, New York. BELL, R. A., and JOACHIM, F. A. (1976). Techniques for rearing lab-

342

DEVELOPMENTAL BIOLOGY

oratory colonies of tobacco hornworms and pink bollworms. Ann. EntomoL Sot. Amer. 69, 365-373. CHEN, A. C., and RIDDIFORD, L. M. (1981). Messenger RNA in the cellular commitment of Munduca se&u. Gen. Camp. EndocrinoL 43, 315-324. CHERBAS, P. T. (19’73) “Biochemical Studies of Insecticyanin.” Ph.D. thesis, Harvard University, Cambridge. CHIRGWIN, J. M., PRZYBYLA, A. E., MACDONALD, R. J., and RUTTER, W. J. (1979). Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18, 5294-5299. DAVIS, B. D., and TAI, P. C. (1980). The mechanism of protein secretion across membranes. Nature (Landan) 283.433-438. DYER, K. A., THORNHILL, W. A., and RIDDIFORD, L. M. (1981). DNA synthesis during the change to pupal commitment of h4unduca sexta epidermis. Dev. BioL 84, 425-431. GLISIN, V., CRKVENJAKOV, R., and BYUS, C. (1974). Ribonucleic acid isolated by cesium chloride centrifugation. Biochemistry 13, 26332637. IVARIE, R. D., and JONES, P. P. (1979). A rapid sensitive assay for specific protein synthesis in cells and in cell-free translations: Use of Staphylococcus aurens as an absorbent for immune complexes. AnaL B&hem. 97, 24-35. KAFATOS, F. C. (1976). Sequential cell polymorphism: A fundamental concept in developmental biology. Advun. Insect Physiol. 12, 1-15. KIELY, M. (1982). “Temporal Programming of Epidermal Cell Protein Synthesis during the Larval-Pupal Transformation of Munducu sexta.” Ph.D. thesis, University of Washington. LASKEY, R. A., and MILLS, A. D. (1975). Quantitative film detection of ‘H and “‘C in polyacrylamide gels by fluorography. Eur. J. Biu them. 56, 335-341. LEVENSON, R., and HOUSMAN, D. (1981). Commitment: How do cells make the decision to differentiate? Cell 25, 5-6. MAY, F. E. B., and KNOWLAND, J. (1981). Patterns of protein synthesis in livers of Xenopus la&s during metamorphosis: Effects of estrogen in normal and thyrostatic animals. Dev. Biol. 82, 158-167. MITCHELL, H. K., and PETERSEN, N. S. (1981). Rapid changes in gene expression in differentiating tissues of Drosophila. Dev. Biol. 85, 233-242. PELHAM, H. R. B., and JACKSON, R. J. (1976). An efficient mRNAdependent translation system from reticulocyte lysates. Eur. .I B&hem. 67, 247-256. RICHARDS, G. (1980). Ecdysteroids and puffing in Drosophilu melunoguster. In “Progress in Ecdysone Research” (J. A. Hoffmann, ed.), pp. 363-378. Elsevier/North-Holland, Amsterdam. RIDDIFORD, L. M. (1976). Hormonal control of insect epidermal cell commitment in vitro. Nature (London) 259, 115-117. RIDDIFORD, L. M. (1978). Ecdysone-induced change in cellular commitment of the epidermis of the tobacco hornworm, Munducu sexta,

VOLUME 92, 1982

at the initiation of metamorphosis. Gen Camp. Endocrinol 34,438446. RIDDIFOKD, L. M. (1980). Interaction of ecdysteroids and juvenile hormone in the regulation of larval growth and metamorphosis of the tobacco hornworm. In “Progress in Ecdysone Research” (J. A. Hoffmann, ed.), pp. 409-430. Elsevier/North-Holland Press, Amsterdam. RIDDIFORD, L. M. (1981). Hormonal control of epidermal cell development. Amer. ZooL 21, 751-762. RIDDIFORD, L. M. (1982). Hormonal control of epidermal polymorphism. In “Symposium Proceedings of the Ninth International Symposium on Comparative Endocrinology” (B. Lofts, ed.), University Hong Kong Press, Hong Kong, in press. RIDDIFORD, L. M., CHEN, A. C., GRAVES, B. J., and CURTIS, A. T. (1981). RNA and protein synthesis during the change to pupal commitment of Munducu sextu epidermis. Insect Biochem. 11, 121-127. RIDDIFORD, L. M., and CURTIS, A. T., (1978). Hormonal control of epidermal detachment during the final feeding stage of the tobacco hornworm larva. J. Insect. PhysioL 24, 561-568. RIDDIFORD, L. M., CURTIS, A. T., and KIGUCHI, K. (1979). Culture of the epidermis of the tobacco hornworm Munducu sexta. Tissue Cult. Assoc. Manual 5, 975-985. RIDDIFORD, L. M., and KIELY, M. L. (1981). The hormonal control of commitment in the insect epidermis-Cellular and molecular aspects. 1n “Regulation of Insect Development and Behaviour” (F. Sehnal, A. Zabza, J. J., Menn, and B. Cymborowski, eds.), pp. 485496. Wroclaw Tech. Univ. Press, Wroclaw. RIDDIFORD, L. M., KIGUCHI, K., ROSELAND, C. R., CHEN, A. C., and WOLFGANG, W. J. (1980). Cuticle formation and sclerotization in vitro by the epidermis of the tobacco hornworm, Munducu sextu. In “Invertebrate Systems in Vitro” (E. Kurstak, K. Maramorosch, and A. Dubendorfer, eds.), pp. 103-115. Elsevier/North-Holland, Amsterdam. ROSS, J. (1976). A precursor of globin mRNA. J. Mol. Biol., 106,403420. SNYDER, M., HIRSH, J., and DAVIDSON, N. (1981). The cuticle genes of Drosophila: A developmentally regulated gene cluster. Cell 25,165177. WALKER, V. K., and ASHBURNER, M. (1981). The control of ecdysterone-regulated puffs in Drosophila salivary glands. Cell 26,269-277. WEBER, K., and OSBORN, M. (1969). The reliability of molecular weight determination by dodecyl sulfate-polyacrylamide gel electrophoresis. J. BioL Chem. 244, 4406-4412. WHITE, B. N., and DELUCCA, F. C. (1977). Preparation and analysis of RNA. In “Analytical Biochemistry of Insects” (R. B. Turner, ed.), pp. 85-130. Elsevier, Amsterdam. WOLFGANG, W. J., and RIDDIFORD, L. M. (1981). Cuticular morphogenesis during continuous growth of the final instar larva of a moth. Tiss. Cell 13, 757-772.