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Patterns of protein synthesis in livers of Xenopus laevis during metamorphosis: Effects of estrogen in normal and thyrostatic animals
DEVELOPMENTAL
BIOLOGY
82, 158-167 (1981)
Patterns of Protein Synthesis in Livers of Xenopus lawis during Metamorphosis: Effects of Estrogen in Normal and Thyrostatic Animals FELICITY E. B. MAY~,~ANDJOHNKNOWLAND Department of Biochemistry, South Parks Rood, Oeord OX1 SQU, United Kingdom Received April 15, 1980; accepted in revised
form July 21, 1980
Two-dimensional gel electrophoresis has been used to analyse protein synthesis in the livers of Xenqpus luevti larvae during metamorphosis. The patterns found at different developmental stages have been characterised and compared to those found in developmentally static tadpoles and estrogen-treated tadpoles. The results suggest that the majority of proteins synthesized by the larval liver during metamorphosis can be divided equally into three main categories: those which are synthesized continuously, those whose synthesis is lost, and those whose synthesis is gained during development. The synthesis of proteins tends to be lost earlier in metamorphosis than it is gained. The pattern of liver protein synthesis in thyrostatic animals is not characteristic of any single stage of normal development, and displays features characteristic of many different stages. About half the changes in protein synthesis which occur during normal metamorphosis are dependent upon it. All the stages examined are responsive to estrogen, and each has a characteristic response. Half of the estrogen-induced changes in protein synthesis are independent of metamorphosis, while the other half require metamorphosis.
1961), and increases in hydrolases and other enzymes (Frieden, 1968). However, in all these studies the synAmphibians undergo metamorphosis during which thesis of only one or a few proteins has been examined. morphological, physiological, behavioural, and bioIn this report we have extended the range by using twochemical changes occur. The morphological changes are dimensional gel electrophoresis of newly-synthesized of three main types: regression of larval-restricted proteins (O’Farrell, 1975; O’Farrell et al., 19’77), which structures and functions, transformation of larval allows more proteins to be studied simultaneously than structures into an adult form, and de nova development any other method available. We show that the majority of adult structures and functions. They occur in an orof liver proteins synthesized during metamorphosis of derly sequence and at a precise time in development Xenopus laevis can be divided into three categories: (Dodd and Dodd, 1976). Larval development has been those which are always synthesized, those whose syndivided into three stages: premetamorphosis, the truly thesis is lost, and those whose synthesis is gained durlarval period, characterised by growth and development ing metamorphosis; and that the synthesis of twoof larval structures but no metamorphic changes; prometamorphosis, during which growth continues and a thirds of the proteins studied alters, predominantly during metamorphic climax. few minor metamorphic transformations are initiated; By preventing metamorphosis, we have been able to and metamorphic climax, when all the radical changes occur (Etkin, 1964). Amphibian metamorphosis is me- study the dependence of the changes on metamorphosis. diated directly by thyroid hormones, and cannot occur It is possible to halt the development of larvae in late in their absence, but the mechanism of thyroid hormone premetamorphosis by placing premetamorphic animals (Hughes and action is not understood, and the number of changes in compounds such as propyithiouracil Astwood, 1944), which antagonize the action of thyroid under its control is unknown. Much of the biochemical work on amphibian meta- hormone, and thus to alter the normal correlation bemorphosis has been concerned with the changes that tween developmental stage and chronological age. In take place in liver metabolism. Among these are: in- this way it has been shown that the transition from creases in the production of urea cycle enzymes, in- foetal to adult globin synthesis does not require metacreased production of albumin (Herner and Frieden, morphosis (Maclean and Turner, 1976; Just et al, 1977). We show here that this is also true of a much wider range of proteins synthesized by the liver, and that as ’ Present address: Zoologisches Institut, Universitlt Bern, Sahlimany as 50% of the alterations in protein synthesis do strasse 8, CH-3012, Switzerland. ’ To whom reprint requests should be addressed. not require metamorphosis. INTRODUCTION
0012-1606/81/030158-10$02.00/O Copyright All rights
0 1981 by Academic Press, Inc. of reproduction in any form reserved.
158
MAY AND KNOWLAND
Protein
Synthesis
in Xenopus laevis Livers
Finally, we have examined the response of metamorphosing livers to an external stimulus, namely estrogen applied to living tadpoles. It has previously been shown that estrogen, which induces vitellogenin synthesis in adult frog liver, does not do so in living tadpoles before stage 62 (Huber et al., 1979; May and Know-
during
159
Metamorphosis
land, 1980), and that the normal transition from uninducibility to inducibility requires metamorphosis. We show here that while this is also true of some other estrogen-induced proteins, it is not true of them all, suggesting that the transition of the vitellogenin gene to the inducible state is controlled by specific rather
PH
%
L 0
stage54
stage66 FIG. 1. Acidic and basic liver protein Methods.
synthesis
in stage 54 and stage 66 Xmopus.
Proteins
were labelled
as described
under Materials
and
160
DEVELOPMENTAL BIOLOGY
than general factors. The livers of thyrostatic animals are not characteristic of any single stage in normal liver development with respect to either the general pattern of protein synthesis or the response to estrogen, suggesting that many of the changes which occur during metamorphosis may be individually controlled. MATERIALS
AND METHODS
(1) Animals All the animals used in this work were laboratory bred and fed on a suspension of blended, filtered spinach, and dried yeast. Larval stages were identified by the criteria of Nieuwkoop and Faber (1967). To halt metamorphosis tadpoles were transferred to water containing 10 pg/ml of propylthiouracil (0.58 mM) which was changed every 2 weeks. This arrested development at stage 53, but did not inhibit growth, so that at the time of use, which was always after at least 6 months in propylthiouracil, the resulting thyrostatic tadpoles were considerably larger than normal animals of the same developmental stage. To study the effect of exogenous estrogen on liver protein synthesis, tadpoles were transferred to 1.0 &Z estrogen which was changed daily. After 3 days in estrogen the animals were staged and their livers were labelled in vitro with [35S]methionine.
VOLUME 82. 1981 TABLE 1 NUMBER OF PROTEINS CLASSIFIED AT EACH STAGE
Stage 54 57 59 62 66 Thyrostatic, stage 53
Acidic
Basic
Total
58 56 54 62 75
49 42 33 31 30
107 98 87 93 105
77
30
107
electric focussing gels (O’Farrell, 1975) or on nonequilibrium pH gradient gels (pH 7-10; O’Farrell et al., 1977), and in the second dimension on 15% SDS-gels (Knowland, 1974). Gels were fixed and radioactive proteins located by fluorography (Laskey and Mills, 1975). RESULTS
The patterns of [35S]methionine-labelled proteins extracted from livers of stage 54 and stage 66 Xenopus larvae are shown in Fig. 1. There are clearly major differences in the proteins synthesised at these two stages. To assist description of the changing pattern of protein synthesis, we have numbered all the proteins which are reproducibly synthesised and behave consistently at these two stages. The numbering system scans from left to right and from top to bottom, starting at (2) Labelling and Extraction of Livers the top left. Proteins detected after isoelectric focussing Tadpoles were anaesthetised with Tricaine. Their liv- in the first dimension (pH gradient 5-7) will collectively ers were removed under sterile conditions and washed be termed the acidic fraction in the figures and tables in methionine-free medium. Up to 10 mg of each liver and individually prefixed by the letter A in the text, was transferred to 50 ~1 of radioactive medium con- while those separated by nonequilibrium pH gradient sisting of 8 vol of 70% methionine-free Eagle’s medium, electrophoresis in the first dimension (pH gradient 72 vol of 70% complete Eagle’s medium, and 200-300 10) will be referred to as the basic fraction and desig&i/ml of high specific activity [35S]methionine (600-900 nated by the letter B. The number of acidic and basic Ci/mmole; The Radiochemical Centre, Amersham). The proteins whose synthesis was clearly detected at all livers were labelled for 6 hr at 25°C removed from the stages is given in Table 1. Combination of these two radioactive medium, washed, and homogenised at 0°C fractions shows that the total number of proteins dein 100 ~1 of 100 mlM NH4HC03, 5 mlM NaHS03, 20 mM tected is very similar at stages 54 and 66. NazEDTA, 0.1% 2-mercaptoethanol, 150 pg/ml of phenThere are however major differences in the pattern ylmethylsulphonlyl fluoride, and 25 pg/ml each of of protein synthesis between livers from premetaRNAse A and DNAse I, pH 6.8. After aliquots had been morphic tadpoles and newly-metamorphosed frogs. To taken to measure incorporation of isotope, the homog- study the transition from the larval to the adult patenates were lyophilised and frozen at -20°C until an- tern, we first examined protein synthesis during normal alysed. metamorphosis, using three intervening stages, representative of late prometamorphosis (stage 57), onset of (3) Two-Dimensional Gel Electrophoresis metamorphic climax (stage 59), and metamorphic cliThe lyophilised samples were dissolved in lysis buffer max (stage 62). The patterns obtained for the acidic and basic liver proteins synthesised at these three stages (O’Farrell, 1975) and 20 ~1 of each sample containing up to 200,000acid-insoluble cpm was used for each anal- are shown in Figs. 2a and b, and both acidic and basic ysis. All samples were centrifuged for 10 min on a Beck- proteins synthesised at each stage are listed in Table man microfuge. Supernatants were run first on iso- 2. The number of acidic and basic proteins detected at
MAY AND KNOWLAND
Protein
Synthesis
in Xenopus laevis Livers
during
Metamorphosis
161
PH
stage57
L 0
5 20 P 0 z
FIG. 2. Liver proteins synthesized in stage 57,59, and 62 Xenopus larvae. Proteins (a) Acidic proteins; (b) basic proteins.
each stage is summarised in Table 1. The total number decreases during early metamorphosis, and by stage 59 has fallen by 19%) but increases again during the completion of metamorphosis,
were labelled
as described under Materials
and Methods.
The majority of the proteins can be divided into three categories: those synthesised by all larval stages from 54 up to and including 66; those synthesised at stage 54 but whose synthesis ceases as metamorphosis pro-
162
DEVELOPMENTALBIOLOGY
VOLUME82, 1981
TABLE 2 STABLE CHANGES IN PROTEINS SYNTHESIZED AT A GIVEN STAGE RELATIVE TO THE IMMEDIATELY PRECEDING STAGE
Acidic
Basic
Losses
Gains
Stage
Individual
Total
Individual
54
-
0
-
Losses Total
Individual
Gains Total
0
-
0
Individual -
Total 0
57
7, 8, 51, 74
4
23, 77
2
8, 13, 22, 23, 42
5
59
33, 37, 39, 47, 49, 52
6
12, 13, 19, 36, 60, 71, 97
7
12, 14, 25, 26, 31, 35, 48, 55
8
38
1
62
9, 10, 38, 41
4
11, 18, 22, 76, 78, 83, 85-87, 95, 96
11
7, 18, 20, 30, 34, 49
6
59, 60
2
66
20, 21, 26, 30, 62, 63, 75, 90
8
14, 17, 34, 35, 42, 53-55, 65-67, 69, 70, 72, 79-81, 84, 94
20
1-5, 10, 15, 24, 33
9
27-29, 37, 44, 57, 58
7
Stage 53 thyrostatic tadpole compared to normal stage 54
7, 8, 20, 51, 52, 75
6
11, 13, 19, 22, 23, 34-36, 42, 53, 54, 65-67, 70, 71, 76, 77, 79, 81, 83-85, 94, 97
25
3-5, 13, 18, 20, 21-26, 30, 31, 35, 42, 47-49
19
37, 38
2
Proteins synthesized at all normal stages examined
l-6, 15, 16, 24, 25, 27-29, 31, 32, 40, 43-46, 50, 56-59, 61, 64, 68, 88, 89, 91-93
Total
15
48, 73, 82,
9, 11, 36, 40, 51, 52
ceeds; and those whose synthesis is first detected only after stage 54, but are then continuously synthesised at all stages up to and including stage 66. Only 9% of the proteins detected did not follow this simple pattern. Amongst the acidic proteins A82, A48, and A73 are temporarily lost during metamorphosis, and there are three stage-specific acidic proteins synthesised at stage 62 (marked + in Fig. 2a). Amongst the basic fraction, syn-
TABLE 3 NUMBER OF PROTEINS IN EACH OF THE MAIN CATEGORIES DEFINED UNDER RESULTS
Proteins Behaviour during development Always synthesized Synthesis lost Synthesis gained Stage-specific or fluctuating Total
6, 16, 17, 19, 21, 32, 39, 41, 43, 46, 47, 50, 53, 54, 56
33
Proteins whose synthesis fluctuates
0
Acidic
Basic
Total
33 22 40 6
15 28 10 8
48 50 50 14
101
61
162
thesis of proteins B9 and Bll is lost, regained, and finally lost, synthesis of proteins B36, B51, and B52 is temporarily lost, and again there are two stage-specific basic proteins made at stage 57 (marked + in Fig. 2b.). Fluctuating and stage-specific protein synthesis has been observed in much earlier embryonic stages of Xenopus development (Bravo and Knowland, 1979), but not previously during the development of a particular organ. The number of proteins in each of the categories is shown in Table 3. Of the 162 proteins whose synthesis we have followed during metamorphosis 60% were from the acidic and 40% from the basic fraction. When the two fractions are combined, there are approximately the same number of proteins, about 50, in each of the three main categories defined above. Table 4 shows the numbers and percentages of proteins whose synthesis is permanently lost or gained at each stage. This shows that losses in protein synthesis tend to precede gains during metamorphosis, and that almost 50% of the total changes occur during late metamorphic climax, as only 56% have occurred by stage 62.
MAY AND KNOWLAND
Protein
Synthesis
in Xenopus laevis Livers
163
during Metamorphosis
TABLE 4 CUMULATIVECHANGESIN THE NUMBER OF PROTEINS WHOSE SYNTHESIS IS PERMANENTLY LOST OR GAINED BY EACH STAGE
Stage 54 57
Basic
Acidic 0 4
0 5
59
10
62 66
14 22
13 19 28
Total changes
Synthesis gained
Synthesis lost No.
%
Acidic
Basic
No.
%
0 9 23 33 50
0 18 46 66
0 2 9 20 40
0 0
0 2
0 4 20 46
100
Thus we have shown that protein synthesis in larval Xenops liver alters dramatically during metamorphosis and that the majority of changes (67%) occur after stage 59, during metamorphic climax. To test whether the changes observed are under the control of factors altering during metamorphosis and therefore stage-dependent, or due only to the chronological age of the animals, we examined the proteins synthesized by thyrostatic tadpoles. The animals used had been kept for 6 months in 0.58 mM propylthiouracil, and were thus in terms of age equivalent to metamorphosed frogs, but were halted at stage 53 of morphological development. The patterns of proteins synthesized by the livers of these animals are shown in Fig. 3, and should be compared with the results found for normal animals in Figs. 1 and 2. A detailed comparison of the range of proteins synthesized by liver from a thyrostatic tadpole at stage 53 and those synthesized during normal development is given in Table 2, which shows that a thyrostatic tadpole is not equivalent to any single stage of normal development. For example, a thyrostatic tadpole continues to synthesize protein A’74, which is normally lost at stage 57, but does not synthesize proteins A20 and A75, which normally do not disappear before stage 66, nor B21 and B47, which are normally synthesized at all
1
10
3
23 50
10
No.
100
%
0
0
11
11
33 57
33 56
100
100
stages. Changes in protein synthesis which occur during normal development and are also found in thyrostatic tadpoles must depend on age, whereas those which occur during normal development but not in thyrostatic tadpoles must be controlled by factors which alter during metamorphosis. The number of proteins whose synthesis is either lost or gained in thyrostatic tadpole liver is compared in Table 5 with the changes that normally occur between stages 54 and 66. This shows that about half the alterations in liver protein synthesis which occur during normal metamorphosis also take place in thyrostatic tadpole liver, and are therefore independent of metamorphosis, while half are dependent on it. Response of Tadpole Liver to Estrogen To study the response of the larval liver to estrogen we exposed the animals to the hormone by immersion in 10m6M estradiol-170 for 3 days prior to dissection. As we have previously shown that only 12 hr of such exposure to estrogen is necessary to produce an alteration in protein synthesis (May and Knowland, 1980), the animals were staged at the end of the treatment. The resulting patterns for the acidic and basic fraction of tissue proteins are shown in Fig. 4. Most of the estrogen-induced changes in protein synthesis were detected amongst the acidic fraction, but all the changes PH I7
I’0
80,
FIG. 3. Acidic and basic liver proteins synthesized in thyrostatic tadpoles. Proteins were labelled as described under Materials and Methods.
164
DEVELOPMENTALBIOLOGY
VOLUME82, 1981
PH
FIG. 4. Liver proteins synthesized in Xenopus larvae after estrogen treatment. Proteins were labelled as described under Materials and Methods. Some proteins are arrowed to provide a reference for comparison with other figures. Proteins whose synthesis is repressed are marked (-I). (a) Acidic proteins; (b) basic proteins.
observed are listed in Table 6. In order to identify collectively and unambiguously on the figures the proteins whose synthesis is affected by estrogen, they are all pre-
fixed by the letter E, but, as indicated in Table 6,60% are proteins which are clearly synthesized in the absence of exogenous estrogen, and also appear in Table 2.
MAY AND KNOWLAND
165
Protein Synthesis in Xenopus laevis Livers during Metamorphosis PH
110
I7
I’0
I7
SO60do-
zo,:_-, /’
_I.
sMge54
---
_
FIG. 4. (Continued)
DISCUSSION
The extraction methods used mean that not all proteins synthesized by the liver are included in the analyses. Secreted, membrane-bound, and insoluble proteins
are excluded, so that the proteins analysed are mainly the soluble, cytoplasmic proteins. The results also show that there is an enormous difference between the rate of synthesis of the easily detectable proteins and the minor proteins, which are not included in the tables.
166
DEVELOPMENTALBIOLOGYV0~~~~82,1981
TABLE5 LOSSES ANDGAINSIN PROTEINS SYNTHESIZED BY A THYROSTATIC morphic climax (Huber et al., 1979; May and Knowland, TADPOLEORANEWLYMETAMORPHOSEDFROG COMPAREDTOANOR- 1980), suggesting that in Xenopus, tadpole liver acquires all the characteristics of adult liver at this time, even MALSTAGE 54 TADPOLE Gains
Losses
Basic
Acidic
Basic
Total changes
6
19
25
2
52
22
28
40
10
100
Acidic Thyrostatic tadpole, stage 53 Normal frog, stage 66
It is impossible to say whether the minor proteins behave in the same way as the major ones, or indeed how many minor proteins there are, but it is clear that the behaviour described below for the various classes of protein applies to a large proportion of proteins synthesized by the liver. In the two-dimensional protein separations, we have used both isoelectric focussing and nonequilibrium pH gradient electrophoresis in the first dimension, so that we can study both acidic and basic proteins. This not only increases the range of proteins studied; it also reduces the chances of making false generalisations about their behaviour. For example, Table 3 shows that the proportions in each of the three main categories are different if either of the two protein fractions are examined separately, but very similar if they are combined. The proportion of proteins whose synthesis is lost during development is 22% of the acidic fraction and 46% of the basic fraction, but 31% of the total. Figure 1 shows that the pattern of proteins synthesized before and after metamorphosis differs considerably. We have divided the proteins synthesized into the three main categories described under Results because fewer than 10% behaved differently. Of the total number detected at any one stage, which varies from a minimum of 8’7to a maximum of 107 (Table l), about half are accounted for by the 48 continuously-synthesized proteins (Table 4), but over the whole period of metamorphosis the synthesis of nearly 70% of the proteins detected changes in a stable way (50 losses and 50 gains among the 148 proteins whose synthesis behaves consistently and does not fluctuate; Table 3). These changing proteins represent functions which are strongly associated with metamorphosis, and may include some which govern the changes in liver function. Only 30% of the changes had occurred by the onset of metamorphic climax (stage 59), and almost 50% did not occur until late climax, showing that most of the alterations occur after cessation of larval growth and over a short time span. The ability of estrogen to induce vitellogenin synthesis also appears during late meta-
though they may not be required immediately. Table 4 shows that losses in liver protein synthesis characteristic of metamorphosis precede the gains. Eighteen percent of the losses in protein synthesis occur by stage 57, but only 4% of the gains. This pattern continues throughout metamorphosis, so that of the total alterations, there are 39% more losses than gains by stage 59, and 18% more by stage 62. There seems therefore to be a fall in gene activity in tadpole liver during metamorphic climax which is only restored during the completion of metamorphosis, coinciding with the physiological changes that accompany the conversion of a tadpole into a frog. In order to study the relation of these changes to chronological age and developmental stage we used developmentally static tadpoles. It has previously been shown that the transition from foetal to adult globin chain synthesis can occur in the absence of complete metamorphosis (Maclean and Turner, 1976; Just et al., 1977). Figure 3 shows the gel profiles presented by and Table 2 lists the proteins synthesized by thyrostatic tadpole liver. Half the total changes in synthesis of acidic and basic proteins which normally occur during metamorphosis also occur in thyrostatic tadpoles. This proportion of the alterations in liver protein synthesis must depend on age, showing that adult globin is only one representative of a large number of adult proteins whose synthesis does not require metamorphosis, while the remaining 50% do require metamorphosis. Because the total changes that occur in the thyrostatic animals would normally occur separately at very different developmental stages, their livers are not characteristic of any single stage of normally-metamorphosing tadpole liver. It is therefore unlikely that the changes that do occur are caused by residual thyroxine, which would be expected to bring about partial metamorphosis in an orderly manner reflecting the early stages of normal metamorphosis. The effects of estrogen treatment on normal liver protein synthesis are summarised in Table 6. As expected, there was very little repression of protein synthesis by estrogen. All the stages studied were responsive to estrogen, which presumably means that even premetamorphic larval liver contains a functional estrogen receptor. They all exhibited a different estrogen response, and as with general liver protein synthesis the estrogen response of thyrostatic tadpole liver was not characteristic of any single stage of liver development. About half of the proteins whose synthesis becomes inducible by estrogen during normal development become inducible in thyrostatic tadpole liver,
Protein
MAY AND KNOWLAND
Synthesis
in Xenopus laevis Livers
167
during Metamorphosis
TABLE 6 PROTEINS WHOSE SYNTHESIS IS AFFECTED BY ESTROGEN Repressed
Induced or increased
Acidic
Basic
Acidic
Stage 54
El 37
E2 38
E3 39
E4
E5
E6 43
El 27
E2 28
-
57
El 37
E2 38
E4
E5
E7
ES
E2 28
E3 49
-
59
E4
E5
E7
E8
E9 56
El0 73
E3 49
El1 40
El2
El3
El4
El5 91
62
El6 87
66
El7
El8
El9 47
E3 39
E4
E5
E6 43
E7
E9 56
El0 73
El2
El3 41
El5 91
Stage 53 Thyrostatic
tadpole
E8
Note. A number in italics below a protein indicates that it is also synthesized that used in Table 2. No basic proteins are repressed by estrogen.
while the other half, like vitellogenin, remain uninducible, showing that vitellogenin is characteristic of a wider range of proteins whose transition from the uninducible to the inducible state requires metamorphosis. Of the 11 proteins whose synthesis is stimulated by estrogen in normal tadpole liver at or after stage 62, the synthesis of only one (basic protein E6) is stimulated by estrogen in thyrostatic tadpoles. It may be that the induction of these proteins by estrogen requires increased nuclear estrogen receptor, the induction of which by estrogen is stage dependent and does not occur before stage 62 (May and Knowland, unpublished). F.E.B.M. thanks the Medical Research Council for a Scholarship for training in research methods. This work was supported by the MRC.
REFERENCES BRAVO, R., and KNOWLAND, J. (1979). Classes of proteins synthesized in oocytes, eggs, embryos, and differentiated tissues of Xenopus laevis. Differentiation 13,101-108. DODD, M. H. I., and DODD, J. M. (1976). The biology of metamorphosis. In “Physiology of the Amphibia” (B. Lofts, ed.), Vol. III, pp. 467599. Academic Press, New York. ETKIN, W. (1964). Metamorphosis. Zn “Physiology of the Amphibia” (J. A. Moore, ed.), pp. 427-468. Academic Press, New York. FRIEDEN, E. (1968). Biochemistry of amphibian metamorphosis. In
71
E4 20
E5
E4 20
E5
3 Stage-specific E6 19
E7
ES
E6 19
in the absence of exogenous estrogen,
-
the number
being
“Metamorphosis: A Problem in Developmental Biology” (W. Etkin and L. I. Gilbert, eds.), pp. 349-398. Appleton, New York. HERNER, A. E., and FRIEDEN, E. (1961). Biochemistry of anuran metamorphosis VII. Changes in serum proteins during spontaneous and induced metamorphosis. J. Biol. Chem. 235,2845-2851. HUBER, S., RYFFEL, G. LJ., and WEBER, R. (1979). Thyroid hormone induces competence for oestrogen dependent vitellogenin synthesis in developing Xenopus laevis. Nature (London) 278, 65-67. HUGHES, A. M., and ASTWOOD, E. B. (1944). Inhibition of metamorphosis in tadpoles by thiouracil. Endocrinology 34,138-139. JUST, J. J., SCHWAGER, J., and WEBER, R. (1977). Haemoglobin transition in relation to metamorphosis in normal and isogenic Xenopus. Wilhelm Roux’ Archiv. 183, 307-323. KNOWLAND, J. (1974). Protein synthesis induced by the RNA from a plant virus in a normal animal cell. Genetics 78, 383-394. LASKEY, R. A., and MILLS, A. D. (1975). Quantitative film detection of 3H and 14C in polyacrylamide gels by fluorography. Eur. J. Biochem. 56,335-341. MACLEAN, N., and TURNER, S. (1976). Adult haemoglobin in developmentally retarded tadpoles of Xenopus laevis. J. Embryol. Exp. Morphol. 35,261-266. MAY, F. E. B., and KNOWLAND, J. (1980). The role of thyroxine in the transition of vitellogenin synthesis from non-inducibility to inducibility during metamorphosis in Xenqwus laevis. Develop. Biol. 77, 419-430. NIEUWKOOP, P. D., and FABER, J. (1967). “Normal Table of Xenopus la&s (Daudin),” 2nd ed. North-Holland, Amsterdam. O’FARRELL, P. H. (1975). High resolution two-dimension electrophoresis of proteins. J. Biol. Chem. 250,4007-4021. O’FARRELL, P. Z., GOODMAN, H. M., and O’FARRELL, P. H. (1977). High resolution two-dimensional electrophoresis of basic as well as acidic proteins. Cell 12,1133-1142.
BIOLOGY
82, 158-167 (1981)
Patterns of Protein Synthesis in Livers of Xenopus lawis during Metamorphosis: Effects of Estrogen in Normal and Thyrostatic Animals FELICITY E. B. MAY~,~ANDJOHNKNOWLAND Department of Biochemistry, South Parks Rood, Oeord OX1 SQU, United Kingdom Received April 15, 1980; accepted in revised
form July 21, 1980
Two-dimensional gel electrophoresis has been used to analyse protein synthesis in the livers of Xenqpus luevti larvae during metamorphosis. The patterns found at different developmental stages have been characterised and compared to those found in developmentally static tadpoles and estrogen-treated tadpoles. The results suggest that the majority of proteins synthesized by the larval liver during metamorphosis can be divided equally into three main categories: those which are synthesized continuously, those whose synthesis is lost, and those whose synthesis is gained during development. The synthesis of proteins tends to be lost earlier in metamorphosis than it is gained. The pattern of liver protein synthesis in thyrostatic animals is not characteristic of any single stage of normal development, and displays features characteristic of many different stages. About half the changes in protein synthesis which occur during normal metamorphosis are dependent upon it. All the stages examined are responsive to estrogen, and each has a characteristic response. Half of the estrogen-induced changes in protein synthesis are independent of metamorphosis, while the other half require metamorphosis.
1961), and increases in hydrolases and other enzymes (Frieden, 1968). However, in all these studies the synAmphibians undergo metamorphosis during which thesis of only one or a few proteins has been examined. morphological, physiological, behavioural, and bioIn this report we have extended the range by using twochemical changes occur. The morphological changes are dimensional gel electrophoresis of newly-synthesized of three main types: regression of larval-restricted proteins (O’Farrell, 1975; O’Farrell et al., 19’77), which structures and functions, transformation of larval allows more proteins to be studied simultaneously than structures into an adult form, and de nova development any other method available. We show that the majority of adult structures and functions. They occur in an orof liver proteins synthesized during metamorphosis of derly sequence and at a precise time in development Xenopus laevis can be divided into three categories: (Dodd and Dodd, 1976). Larval development has been those which are always synthesized, those whose syndivided into three stages: premetamorphosis, the truly thesis is lost, and those whose synthesis is gained durlarval period, characterised by growth and development ing metamorphosis; and that the synthesis of twoof larval structures but no metamorphic changes; prometamorphosis, during which growth continues and a thirds of the proteins studied alters, predominantly during metamorphic climax. few minor metamorphic transformations are initiated; By preventing metamorphosis, we have been able to and metamorphic climax, when all the radical changes occur (Etkin, 1964). Amphibian metamorphosis is me- study the dependence of the changes on metamorphosis. diated directly by thyroid hormones, and cannot occur It is possible to halt the development of larvae in late in their absence, but the mechanism of thyroid hormone premetamorphosis by placing premetamorphic animals (Hughes and action is not understood, and the number of changes in compounds such as propyithiouracil Astwood, 1944), which antagonize the action of thyroid under its control is unknown. Much of the biochemical work on amphibian meta- hormone, and thus to alter the normal correlation bemorphosis has been concerned with the changes that tween developmental stage and chronological age. In take place in liver metabolism. Among these are: in- this way it has been shown that the transition from creases in the production of urea cycle enzymes, in- foetal to adult globin synthesis does not require metacreased production of albumin (Herner and Frieden, morphosis (Maclean and Turner, 1976; Just et al, 1977). We show here that this is also true of a much wider range of proteins synthesized by the liver, and that as ’ Present address: Zoologisches Institut, Universitlt Bern, Sahlimany as 50% of the alterations in protein synthesis do strasse 8, CH-3012, Switzerland. ’ To whom reprint requests should be addressed. not require metamorphosis. INTRODUCTION
0012-1606/81/030158-10$02.00/O Copyright All rights
0 1981 by Academic Press, Inc. of reproduction in any form reserved.
158
MAY AND KNOWLAND
Protein
Synthesis
in Xenopus laevis Livers
Finally, we have examined the response of metamorphosing livers to an external stimulus, namely estrogen applied to living tadpoles. It has previously been shown that estrogen, which induces vitellogenin synthesis in adult frog liver, does not do so in living tadpoles before stage 62 (Huber et al., 1979; May and Know-
during
159
Metamorphosis
land, 1980), and that the normal transition from uninducibility to inducibility requires metamorphosis. We show here that while this is also true of some other estrogen-induced proteins, it is not true of them all, suggesting that the transition of the vitellogenin gene to the inducible state is controlled by specific rather
PH
%
L 0
stage54
stage66 FIG. 1. Acidic and basic liver protein Methods.
synthesis
in stage 54 and stage 66 Xmopus.
Proteins
were labelled
as described
under Materials
and
160
DEVELOPMENTAL BIOLOGY
than general factors. The livers of thyrostatic animals are not characteristic of any single stage in normal liver development with respect to either the general pattern of protein synthesis or the response to estrogen, suggesting that many of the changes which occur during metamorphosis may be individually controlled. MATERIALS
AND METHODS
(1) Animals All the animals used in this work were laboratory bred and fed on a suspension of blended, filtered spinach, and dried yeast. Larval stages were identified by the criteria of Nieuwkoop and Faber (1967). To halt metamorphosis tadpoles were transferred to water containing 10 pg/ml of propylthiouracil (0.58 mM) which was changed every 2 weeks. This arrested development at stage 53, but did not inhibit growth, so that at the time of use, which was always after at least 6 months in propylthiouracil, the resulting thyrostatic tadpoles were considerably larger than normal animals of the same developmental stage. To study the effect of exogenous estrogen on liver protein synthesis, tadpoles were transferred to 1.0 &Z estrogen which was changed daily. After 3 days in estrogen the animals were staged and their livers were labelled in vitro with [35S]methionine.
VOLUME 82. 1981 TABLE 1 NUMBER OF PROTEINS CLASSIFIED AT EACH STAGE
Stage 54 57 59 62 66 Thyrostatic, stage 53
Acidic
Basic
Total
58 56 54 62 75
49 42 33 31 30
107 98 87 93 105
77
30
107
electric focussing gels (O’Farrell, 1975) or on nonequilibrium pH gradient gels (pH 7-10; O’Farrell et al., 1977), and in the second dimension on 15% SDS-gels (Knowland, 1974). Gels were fixed and radioactive proteins located by fluorography (Laskey and Mills, 1975). RESULTS
The patterns of [35S]methionine-labelled proteins extracted from livers of stage 54 and stage 66 Xenopus larvae are shown in Fig. 1. There are clearly major differences in the proteins synthesised at these two stages. To assist description of the changing pattern of protein synthesis, we have numbered all the proteins which are reproducibly synthesised and behave consistently at these two stages. The numbering system scans from left to right and from top to bottom, starting at (2) Labelling and Extraction of Livers the top left. Proteins detected after isoelectric focussing Tadpoles were anaesthetised with Tricaine. Their liv- in the first dimension (pH gradient 5-7) will collectively ers were removed under sterile conditions and washed be termed the acidic fraction in the figures and tables in methionine-free medium. Up to 10 mg of each liver and individually prefixed by the letter A in the text, was transferred to 50 ~1 of radioactive medium con- while those separated by nonequilibrium pH gradient sisting of 8 vol of 70% methionine-free Eagle’s medium, electrophoresis in the first dimension (pH gradient 72 vol of 70% complete Eagle’s medium, and 200-300 10) will be referred to as the basic fraction and desig&i/ml of high specific activity [35S]methionine (600-900 nated by the letter B. The number of acidic and basic Ci/mmole; The Radiochemical Centre, Amersham). The proteins whose synthesis was clearly detected at all livers were labelled for 6 hr at 25°C removed from the stages is given in Table 1. Combination of these two radioactive medium, washed, and homogenised at 0°C fractions shows that the total number of proteins dein 100 ~1 of 100 mlM NH4HC03, 5 mlM NaHS03, 20 mM tected is very similar at stages 54 and 66. NazEDTA, 0.1% 2-mercaptoethanol, 150 pg/ml of phenThere are however major differences in the pattern ylmethylsulphonlyl fluoride, and 25 pg/ml each of of protein synthesis between livers from premetaRNAse A and DNAse I, pH 6.8. After aliquots had been morphic tadpoles and newly-metamorphosed frogs. To taken to measure incorporation of isotope, the homog- study the transition from the larval to the adult patenates were lyophilised and frozen at -20°C until an- tern, we first examined protein synthesis during normal alysed. metamorphosis, using three intervening stages, representative of late prometamorphosis (stage 57), onset of (3) Two-Dimensional Gel Electrophoresis metamorphic climax (stage 59), and metamorphic cliThe lyophilised samples were dissolved in lysis buffer max (stage 62). The patterns obtained for the acidic and basic liver proteins synthesised at these three stages (O’Farrell, 1975) and 20 ~1 of each sample containing up to 200,000acid-insoluble cpm was used for each anal- are shown in Figs. 2a and b, and both acidic and basic ysis. All samples were centrifuged for 10 min on a Beck- proteins synthesised at each stage are listed in Table man microfuge. Supernatants were run first on iso- 2. The number of acidic and basic proteins detected at
MAY AND KNOWLAND
Protein
Synthesis
in Xenopus laevis Livers
during
Metamorphosis
161
PH
stage57
L 0
5 20 P 0 z
FIG. 2. Liver proteins synthesized in stage 57,59, and 62 Xenopus larvae. Proteins (a) Acidic proteins; (b) basic proteins.
each stage is summarised in Table 1. The total number decreases during early metamorphosis, and by stage 59 has fallen by 19%) but increases again during the completion of metamorphosis,
were labelled
as described under Materials
and Methods.
The majority of the proteins can be divided into three categories: those synthesised by all larval stages from 54 up to and including 66; those synthesised at stage 54 but whose synthesis ceases as metamorphosis pro-
162
DEVELOPMENTALBIOLOGY
VOLUME82, 1981
TABLE 2 STABLE CHANGES IN PROTEINS SYNTHESIZED AT A GIVEN STAGE RELATIVE TO THE IMMEDIATELY PRECEDING STAGE
Acidic
Basic
Losses
Gains
Stage
Individual
Total
Individual
54
-
0
-
Losses Total
Individual
Gains Total
0
-
0
Individual -
Total 0
57
7, 8, 51, 74
4
23, 77
2
8, 13, 22, 23, 42
5
59
33, 37, 39, 47, 49, 52
6
12, 13, 19, 36, 60, 71, 97
7
12, 14, 25, 26, 31, 35, 48, 55
8
38
1
62
9, 10, 38, 41
4
11, 18, 22, 76, 78, 83, 85-87, 95, 96
11
7, 18, 20, 30, 34, 49
6
59, 60
2
66
20, 21, 26, 30, 62, 63, 75, 90
8
14, 17, 34, 35, 42, 53-55, 65-67, 69, 70, 72, 79-81, 84, 94
20
1-5, 10, 15, 24, 33
9
27-29, 37, 44, 57, 58
7
Stage 53 thyrostatic tadpole compared to normal stage 54
7, 8, 20, 51, 52, 75
6
11, 13, 19, 22, 23, 34-36, 42, 53, 54, 65-67, 70, 71, 76, 77, 79, 81, 83-85, 94, 97
25
3-5, 13, 18, 20, 21-26, 30, 31, 35, 42, 47-49
19
37, 38
2
Proteins synthesized at all normal stages examined
l-6, 15, 16, 24, 25, 27-29, 31, 32, 40, 43-46, 50, 56-59, 61, 64, 68, 88, 89, 91-93
Total
15
48, 73, 82,
9, 11, 36, 40, 51, 52
ceeds; and those whose synthesis is first detected only after stage 54, but are then continuously synthesised at all stages up to and including stage 66. Only 9% of the proteins detected did not follow this simple pattern. Amongst the acidic proteins A82, A48, and A73 are temporarily lost during metamorphosis, and there are three stage-specific acidic proteins synthesised at stage 62 (marked + in Fig. 2a). Amongst the basic fraction, syn-
TABLE 3 NUMBER OF PROTEINS IN EACH OF THE MAIN CATEGORIES DEFINED UNDER RESULTS
Proteins Behaviour during development Always synthesized Synthesis lost Synthesis gained Stage-specific or fluctuating Total
6, 16, 17, 19, 21, 32, 39, 41, 43, 46, 47, 50, 53, 54, 56
33
Proteins whose synthesis fluctuates
0
Acidic
Basic
Total
33 22 40 6
15 28 10 8
48 50 50 14
101
61
162
thesis of proteins B9 and Bll is lost, regained, and finally lost, synthesis of proteins B36, B51, and B52 is temporarily lost, and again there are two stage-specific basic proteins made at stage 57 (marked + in Fig. 2b.). Fluctuating and stage-specific protein synthesis has been observed in much earlier embryonic stages of Xenopus development (Bravo and Knowland, 1979), but not previously during the development of a particular organ. The number of proteins in each of the categories is shown in Table 3. Of the 162 proteins whose synthesis we have followed during metamorphosis 60% were from the acidic and 40% from the basic fraction. When the two fractions are combined, there are approximately the same number of proteins, about 50, in each of the three main categories defined above. Table 4 shows the numbers and percentages of proteins whose synthesis is permanently lost or gained at each stage. This shows that losses in protein synthesis tend to precede gains during metamorphosis, and that almost 50% of the total changes occur during late metamorphic climax, as only 56% have occurred by stage 62.
MAY AND KNOWLAND
Protein
Synthesis
in Xenopus laevis Livers
163
during Metamorphosis
TABLE 4 CUMULATIVECHANGESIN THE NUMBER OF PROTEINS WHOSE SYNTHESIS IS PERMANENTLY LOST OR GAINED BY EACH STAGE
Stage 54 57
Basic
Acidic 0 4
0 5
59
10
62 66
14 22
13 19 28
Total changes
Synthesis gained
Synthesis lost No.
%
Acidic
Basic
No.
%
0 9 23 33 50
0 18 46 66
0 2 9 20 40
0 0
0 2
0 4 20 46
100
Thus we have shown that protein synthesis in larval Xenops liver alters dramatically during metamorphosis and that the majority of changes (67%) occur after stage 59, during metamorphic climax. To test whether the changes observed are under the control of factors altering during metamorphosis and therefore stage-dependent, or due only to the chronological age of the animals, we examined the proteins synthesized by thyrostatic tadpoles. The animals used had been kept for 6 months in 0.58 mM propylthiouracil, and were thus in terms of age equivalent to metamorphosed frogs, but were halted at stage 53 of morphological development. The patterns of proteins synthesized by the livers of these animals are shown in Fig. 3, and should be compared with the results found for normal animals in Figs. 1 and 2. A detailed comparison of the range of proteins synthesized by liver from a thyrostatic tadpole at stage 53 and those synthesized during normal development is given in Table 2, which shows that a thyrostatic tadpole is not equivalent to any single stage of normal development. For example, a thyrostatic tadpole continues to synthesize protein A’74, which is normally lost at stage 57, but does not synthesize proteins A20 and A75, which normally do not disappear before stage 66, nor B21 and B47, which are normally synthesized at all
1
10
3
23 50
10
No.
100
%
0
0
11
11
33 57
33 56
100
100
stages. Changes in protein synthesis which occur during normal development and are also found in thyrostatic tadpoles must depend on age, whereas those which occur during normal development but not in thyrostatic tadpoles must be controlled by factors which alter during metamorphosis. The number of proteins whose synthesis is either lost or gained in thyrostatic tadpole liver is compared in Table 5 with the changes that normally occur between stages 54 and 66. This shows that about half the alterations in liver protein synthesis which occur during normal metamorphosis also take place in thyrostatic tadpole liver, and are therefore independent of metamorphosis, while half are dependent on it. Response of Tadpole Liver to Estrogen To study the response of the larval liver to estrogen we exposed the animals to the hormone by immersion in 10m6M estradiol-170 for 3 days prior to dissection. As we have previously shown that only 12 hr of such exposure to estrogen is necessary to produce an alteration in protein synthesis (May and Knowland, 1980), the animals were staged at the end of the treatment. The resulting patterns for the acidic and basic fraction of tissue proteins are shown in Fig. 4. Most of the estrogen-induced changes in protein synthesis were detected amongst the acidic fraction, but all the changes PH I7
I’0
80,
FIG. 3. Acidic and basic liver proteins synthesized in thyrostatic tadpoles. Proteins were labelled as described under Materials and Methods.
164
DEVELOPMENTALBIOLOGY
VOLUME82, 1981
PH
FIG. 4. Liver proteins synthesized in Xenopus larvae after estrogen treatment. Proteins were labelled as described under Materials and Methods. Some proteins are arrowed to provide a reference for comparison with other figures. Proteins whose synthesis is repressed are marked (-I). (a) Acidic proteins; (b) basic proteins.
observed are listed in Table 6. In order to identify collectively and unambiguously on the figures the proteins whose synthesis is affected by estrogen, they are all pre-
fixed by the letter E, but, as indicated in Table 6,60% are proteins which are clearly synthesized in the absence of exogenous estrogen, and also appear in Table 2.
MAY AND KNOWLAND
165
Protein Synthesis in Xenopus laevis Livers during Metamorphosis PH
110
I7
I’0
I7
SO60do-
zo,:_-, /’
_I.
sMge54
---
_
FIG. 4. (Continued)
DISCUSSION
The extraction methods used mean that not all proteins synthesized by the liver are included in the analyses. Secreted, membrane-bound, and insoluble proteins
are excluded, so that the proteins analysed are mainly the soluble, cytoplasmic proteins. The results also show that there is an enormous difference between the rate of synthesis of the easily detectable proteins and the minor proteins, which are not included in the tables.
166
DEVELOPMENTALBIOLOGYV0~~~~82,1981
TABLE5 LOSSES ANDGAINSIN PROTEINS SYNTHESIZED BY A THYROSTATIC morphic climax (Huber et al., 1979; May and Knowland, TADPOLEORANEWLYMETAMORPHOSEDFROG COMPAREDTOANOR- 1980), suggesting that in Xenopus, tadpole liver acquires all the characteristics of adult liver at this time, even MALSTAGE 54 TADPOLE Gains
Losses
Basic
Acidic
Basic
Total changes
6
19
25
2
52
22
28
40
10
100
Acidic Thyrostatic tadpole, stage 53 Normal frog, stage 66
It is impossible to say whether the minor proteins behave in the same way as the major ones, or indeed how many minor proteins there are, but it is clear that the behaviour described below for the various classes of protein applies to a large proportion of proteins synthesized by the liver. In the two-dimensional protein separations, we have used both isoelectric focussing and nonequilibrium pH gradient electrophoresis in the first dimension, so that we can study both acidic and basic proteins. This not only increases the range of proteins studied; it also reduces the chances of making false generalisations about their behaviour. For example, Table 3 shows that the proportions in each of the three main categories are different if either of the two protein fractions are examined separately, but very similar if they are combined. The proportion of proteins whose synthesis is lost during development is 22% of the acidic fraction and 46% of the basic fraction, but 31% of the total. Figure 1 shows that the pattern of proteins synthesized before and after metamorphosis differs considerably. We have divided the proteins synthesized into the three main categories described under Results because fewer than 10% behaved differently. Of the total number detected at any one stage, which varies from a minimum of 8’7to a maximum of 107 (Table l), about half are accounted for by the 48 continuously-synthesized proteins (Table 4), but over the whole period of metamorphosis the synthesis of nearly 70% of the proteins detected changes in a stable way (50 losses and 50 gains among the 148 proteins whose synthesis behaves consistently and does not fluctuate; Table 3). These changing proteins represent functions which are strongly associated with metamorphosis, and may include some which govern the changes in liver function. Only 30% of the changes had occurred by the onset of metamorphic climax (stage 59), and almost 50% did not occur until late climax, showing that most of the alterations occur after cessation of larval growth and over a short time span. The ability of estrogen to induce vitellogenin synthesis also appears during late meta-
though they may not be required immediately. Table 4 shows that losses in liver protein synthesis characteristic of metamorphosis precede the gains. Eighteen percent of the losses in protein synthesis occur by stage 57, but only 4% of the gains. This pattern continues throughout metamorphosis, so that of the total alterations, there are 39% more losses than gains by stage 59, and 18% more by stage 62. There seems therefore to be a fall in gene activity in tadpole liver during metamorphic climax which is only restored during the completion of metamorphosis, coinciding with the physiological changes that accompany the conversion of a tadpole into a frog. In order to study the relation of these changes to chronological age and developmental stage we used developmentally static tadpoles. It has previously been shown that the transition from foetal to adult globin chain synthesis can occur in the absence of complete metamorphosis (Maclean and Turner, 1976; Just et al., 1977). Figure 3 shows the gel profiles presented by and Table 2 lists the proteins synthesized by thyrostatic tadpole liver. Half the total changes in synthesis of acidic and basic proteins which normally occur during metamorphosis also occur in thyrostatic tadpoles. This proportion of the alterations in liver protein synthesis must depend on age, showing that adult globin is only one representative of a large number of adult proteins whose synthesis does not require metamorphosis, while the remaining 50% do require metamorphosis. Because the total changes that occur in the thyrostatic animals would normally occur separately at very different developmental stages, their livers are not characteristic of any single stage of normally-metamorphosing tadpole liver. It is therefore unlikely that the changes that do occur are caused by residual thyroxine, which would be expected to bring about partial metamorphosis in an orderly manner reflecting the early stages of normal metamorphosis. The effects of estrogen treatment on normal liver protein synthesis are summarised in Table 6. As expected, there was very little repression of protein synthesis by estrogen. All the stages studied were responsive to estrogen, which presumably means that even premetamorphic larval liver contains a functional estrogen receptor. They all exhibited a different estrogen response, and as with general liver protein synthesis the estrogen response of thyrostatic tadpole liver was not characteristic of any single stage of liver development. About half of the proteins whose synthesis becomes inducible by estrogen during normal development become inducible in thyrostatic tadpole liver,
Protein
MAY AND KNOWLAND
Synthesis
in Xenopus laevis Livers
167
during Metamorphosis
TABLE 6 PROTEINS WHOSE SYNTHESIS IS AFFECTED BY ESTROGEN Repressed
Induced or increased
Acidic
Basic
Acidic
Stage 54
El 37
E2 38
E3 39
E4
E5
E6 43
El 27
E2 28
-
57
El 37
E2 38
E4
E5
E7
ES
E2 28
E3 49
-
59
E4
E5
E7
E8
E9 56
El0 73
E3 49
El1 40
El2
El3
El4
El5 91
62
El6 87
66
El7
El8
El9 47
E3 39
E4
E5
E6 43
E7
E9 56
El0 73
El2
El3 41
El5 91
Stage 53 Thyrostatic
tadpole
E8
Note. A number in italics below a protein indicates that it is also synthesized that used in Table 2. No basic proteins are repressed by estrogen.
while the other half, like vitellogenin, remain uninducible, showing that vitellogenin is characteristic of a wider range of proteins whose transition from the uninducible to the inducible state requires metamorphosis. Of the 11 proteins whose synthesis is stimulated by estrogen in normal tadpole liver at or after stage 62, the synthesis of only one (basic protein E6) is stimulated by estrogen in thyrostatic tadpoles. It may be that the induction of these proteins by estrogen requires increased nuclear estrogen receptor, the induction of which by estrogen is stage dependent and does not occur before stage 62 (May and Knowland, unpublished). F.E.B.M. thanks the Medical Research Council for a Scholarship for training in research methods. This work was supported by the MRC.
REFERENCES BRAVO, R., and KNOWLAND, J. (1979). Classes of proteins synthesized in oocytes, eggs, embryos, and differentiated tissues of Xenopus laevis. Differentiation 13,101-108. DODD, M. H. I., and DODD, J. M. (1976). The biology of metamorphosis. In “Physiology of the Amphibia” (B. Lofts, ed.), Vol. III, pp. 467599. Academic Press, New York. ETKIN, W. (1964). Metamorphosis. Zn “Physiology of the Amphibia” (J. A. Moore, ed.), pp. 427-468. Academic Press, New York. FRIEDEN, E. (1968). Biochemistry of amphibian metamorphosis. In
71
E4 20
E5
E4 20
E5
3 Stage-specific E6 19
E7
ES
E6 19
in the absence of exogenous estrogen,
-
the number
being
“Metamorphosis: A Problem in Developmental Biology” (W. Etkin and L. I. Gilbert, eds.), pp. 349-398. Appleton, New York. HERNER, A. E., and FRIEDEN, E. (1961). Biochemistry of anuran metamorphosis VII. Changes in serum proteins during spontaneous and induced metamorphosis. J. Biol. Chem. 235,2845-2851. HUBER, S., RYFFEL, G. LJ., and WEBER, R. (1979). Thyroid hormone induces competence for oestrogen dependent vitellogenin synthesis in developing Xenopus laevis. Nature (London) 278, 65-67. HUGHES, A. M., and ASTWOOD, E. B. (1944). Inhibition of metamorphosis in tadpoles by thiouracil. Endocrinology 34,138-139. JUST, J. J., SCHWAGER, J., and WEBER, R. (1977). Haemoglobin transition in relation to metamorphosis in normal and isogenic Xenopus. Wilhelm Roux’ Archiv. 183, 307-323. KNOWLAND, J. (1974). Protein synthesis induced by the RNA from a plant virus in a normal animal cell. Genetics 78, 383-394. LASKEY, R. A., and MILLS, A. D. (1975). Quantitative film detection of 3H and 14C in polyacrylamide gels by fluorography. Eur. J. Biochem. 56,335-341. MACLEAN, N., and TURNER, S. (1976). Adult haemoglobin in developmentally retarded tadpoles of Xenopus laevis. J. Embryol. Exp. Morphol. 35,261-266. MAY, F. E. B., and KNOWLAND, J. (1980). The role of thyroxine in the transition of vitellogenin synthesis from non-inducibility to inducibility during metamorphosis in Xenqwus laevis. Develop. Biol. 77, 419-430. NIEUWKOOP, P. D., and FABER, J. (1967). “Normal Table of Xenopus la&s (Daudin),” 2nd ed. North-Holland, Amsterdam. O’FARRELL, P. H. (1975). High resolution two-dimension electrophoresis of proteins. J. Biol. Chem. 250,4007-4021. O’FARRELL, P. Z., GOODMAN, H. M., and O’FARRELL, P. H. (1977). High resolution two-dimensional electrophoresis of basic as well as acidic proteins. Cell 12,1133-1142.