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Tail grafts involving anucleolate Xenopus embryos
DEVELOPMENTAL
Tail
Grafts
BIOLOGY,
5, 252-263
involving
( 1962)
Anucleolate
Xenopus
Embryos
H. WALLACE Zoology
Depurtment,
The
Queen’s
Accepted
May
University,
Belfast,
N.
Ireland
14, 1962
INTRODUCTION Direct observations on the nucleolus suggest that it may function in development as an agent in cellular differentiation and growth. The implication of the nucleolus in ribonucleic acid metabolism (Vincent and Baltus, 1960; Woods, 1960) supports this view, as do the demonstrations of abnormal development in embryos lacking nucleoli (Elsdale et al., 1958; Beermann, 1960). Beermann ( 1960) showed that “nucleolusless” (N-) Ch ironomus embryos form germ bands and achieve some cellular differentiation, but die without hatching. He interpreted this lethal mutation in terms of the necessity of the nucleolus for growth. An investigation of anucleolate (On) Xenopus has also revealed a specific lethal syndrome, including neural pycnosis and a general retardation of both cellular differentiation and growth (Wallace, 1960). The growth of the tail of 0~ Xenopus may serve as a model of how the lack of nucleoli affects growth in general. The tails of On embryos grow rapidly at first, at a time when total nucleic acid synthesis has apparently ceased (cf. Wallace, 1962a); their growth is later retarded and finally arrested several days before the larvae die. Further growth of the tail has been detected during the survival of On embryos in parabiosis, but only at a time when cells from the host have migrated into the tail of the On embryo (Wallace, 196217). For this has been undertaken to establish reason a further investigation whether the growing tissues of the tail are affected directly by the mutation, or indirectly as a systemic effect of the mutation, mediated by the blood circulation or by other means. This article describes the results of transplanting tailbuds between On embryos and their viable sibs. The experiments were designed principally to test the 252
TAIL
GRAFTS
OF
XENOPUS
253
MUTANTS
effects of the absence of nucleolar function on an organ whose growth could be measured easily, but the results are also relevant to the control of tail growth in general. AIATERIAL
AND
METHOD
Heterozygous 1 n toads were induced to spawn by hypodermic injections of chorionic gonadotropin. Their progeny consisted of approximately 25% wild-type (2n), 50% heterozygous (In), and 25% anucleolate (On) embryos1 Tailbud stage embryos, at stage 29/30 of Nieuwkoop and Faber (1956), were used for grafting in Niu-Twitty solution on an agar base. The operations were performed on random samples of embryos, as the mutants cannot be distinguished in V~VCJ at tailbud stages. The operation is illustrated in Fig. 1. A tailbud was excised from one embryo (A) and implanted into a flank wound, above and behind the future anal region, of another embryo (B). No pressure was applied to hold the graft in place: it was either shaken off within a few hours or healed permanently in position. Successful combinations ( C ) developed in 5 days to larvae such as D and E, which are extreme examples of the differences observed. Figure 1 shows a moribund On larva (D) bearing a +n graft and a stage 47 +n larva (E) bearing a On graft. The grafts were always attached to the proximal part of the host’s tail, and usually originated close behind the host’s rectum. Out of 74 such operations, involving two series on different spawnings, 50 grafts held. Subsequent losses (abnormal host development and injuries in handling) reduced this number to 42 hosts with grafts, which were kept with control larvae through a lo-day period of observation. As soon as the grafts had healed, the embryos were transferred to one-tenth strength culture solution, then to tap water on the following day. All larvae were kept at 20°C and fed on a suspension of nettle powder after stage 47. Individual records were kept of the heartbeat and of the blood circulation in the tails and grafts. Growth was followed by daily camera lucida drawings of the lateral fin area behind the rectum (marked by broken lines in Fig. 1, D, E) of the hosts and controls, and of the lateral area of the grafts (stippled in Fig. 1, D, E) . The camera lucida drawings, with an area magnifica‘The nucleus.
symbols
denote
the
maximum
number
of nucleoli
present
in
n diploid
254
H.
WALLACE
tion x400, were subsequently measured by planimeter. The genotypes of the hosts, controls, and grafts were finally determined from permanent preparations stained with Mayer’s acid hemalum. It was found that a random combination of host and graft genotypes had
I
5mm.
1
I'
FIG. 1. Diagram of the operation. The grafted tailbud is stippled. Broken lines mark the anterior limits of the measured areas of the hosts’ tails. A full explanation is given in the text.
been achieved: 10 2n, 19 In, and 13 On hosts, bearing I2 2n, 20 In, and 10 On grafts. The controls were selected to provide more than the Mendelian expectation of On tadpoles; there were 8 2n, 17 In, and 11 On controls. A further series of operations provided material for an investigation of cellular migration into the grafts. RESULTS
In this and previous experiments, 2n and In genotypes have given identical results; thus they may be considered together as nucleolate (+n) material. It is convenient to express the results as a function of time after the operation, because that is virtually the time when tail growth begins. For reasons stated below, only the results from the first 5 days of growth are used. The developmental stages reached during successive days after operating on stage 29/30 are: 1, stage 39; 2, stage 42; 3, stage 44; 4, stage 46; 5, stage 47. Heartbeat and Blood Circulation The heart probably begins to beat before stage 35/36 in all genotypes. The heartbeat of On larvae is relatively slow at stage 40 (Wallace, 1960) and is intermittent or has ceased in the moribund
TAIL
CRAFTS
OF
XENOPUS
255
MUTAYR
On larvae at 4 days. The blood circulation in the subcaudal vein is sluggish and intermittent in On larvae, being imperceptible after 4 days. A rapid blood circulation was established in all grafts on +n hosts, beginning at 24 days in +n grafts and rather later, at 3-4 days, in On grafts. No movement of blood corpuscles was detected in any of the grafts on On hosts. Some circulation must have occurred in them, however, for all these grafts gradually accumulated masses of erythrocytes. Tail Growth
in Hosts
and Controls
Analyses of variance on the growth data show no significant differences of the daily size of tails between 2n and In genotypes, nor
op.1
t
+
6-
/"t
x
t +'
-
*(A'
5Ot 4-
op. II
2
3-
2-
III I
I
I
I
I
I
Oo
I
2
3
4
5
OOU
(2) FIG.
Growth of tails of hosts and controls of +n hosts and controls; 0 Fro. 3. Growth of grafts for each series of times 400 square inches; abscissas, age in days show the standard errors of the mean values. +n hosts; e _ @ On grafts carried by +n by On hosts.
n -
2.
n Tails
t’+ /+x x ,-; /t t 4v I
2
3
4
5
(3) from both series of operations. 0 tails of On hosts and controls. operations. Ordinates, lateral area after the operation. Vertical lines + - + fn grafts carried by hosts; X X +n grafts carried
256
H.
WALLACE
between hosts and controls of any genotype-the grafts did not appreciably affect the tail growth of their hosts. It follows that tail growth can be considered in two groups, +n and On, which are shown for both series of operations in Fig. 2. It will be seen from Fig. 2 that +n tails continued to grow rapidly for 5 days (no further growth could be accommodated by the drawing apparatus), whereas the growth of On tails was retarded at 3 days and was virtually arrested after that. The reliability of this result is shown by an analysis of variance between fn and On groups (Table 1). A general T.mLE ANALYSES
OF VARIANCE BETWEEN Operation
Numbers Days
+?I
On
Variance mt.io
1 2 3 4 5
32 32 32 32 32
11 11 11 11 11
0.58 0 .83 33.45 l’L3.51 23s. l!)
ALL
OS fn
T-411, AXI)
1 SIZE ALL
HOSTS
IN On
AND
I
Operation h-nmbers I’
>0.2 >o.a
CONTROLS
MATERIAL
+ 3,
Old
Variance ratio
22 22 a2 22 ,”
IS 1s 13 13 13
2.78 I .38 140 5ti 201.55 425.60
II
P
<0.2 >O.l >0.2 10.001
correspondence may be seen between the inferior blood circulation and retarded tail growth of On larvae, and between their later cessation both of the circulation and of tail growth.
Graft Growth Owing to a variation in the initial size of the grafts, and to the difficulty in keeping them horizontal while drawing, there is more variability in the growth data of the grafts than in that of the host and control tails. It is assumed that this variability would swamp any possible difference between the growth of 2n and In grafts, or any possible differential influence of 212or In hosts on their grafts. Grouping both grafts and hosts into +n and On categories gives four classes of results: +n/+n, On/+n, +n/On, On/On. The single example of the last class showed the least growth of any graft but is ignored as having little comparative value. Results for the other three classesof grafts are shown separately for each series of operations (Fig. 3). The size attained by the grafts implies that they normally form only the distal half of the tail. The grafts of the second
TAIL
GRAFTS
OF
XENOPUS
257
MUTANTS
series of operations were larger than those of the first series throughout the experiment, a result suggesting that they were taken from slightly older and larger tailbuds. The grafts of both series, however, show the same growth characteristics. On hosts were moribund at 5 days; they and their grafts barely survived the following day. Continued observations of the grafts on +n hosts up to 10 days revealed that the difference in size of +n and On grafts at 5 days was maintained. The little further growth of these grafts after 5 days was accompanied by their increased distortion. There are two meaningful comparisons of the data on graft growth, which are analyzed by t tests (Tables 2 and 3). It is concluded that the data show both an autonomous control of growth by the graft itself and an influence of the host on the growth of its graft. 1. Autonomous growth of grafts. When supported by +n hosts, On grafts grew more slowly than +n grafts (Fig. 3). This difference became statistically significant by 3 days (Table 2). For these first 3 days, all fn grafts grew at approximately the same rate, irrespective of whether they were supported by +n or by On hosts (Fig. 3, Table 3). These two results demonstrate that the initial growth of TABLE 2’ TESTS
1
2 3 4 5 10
ON
12 12 12 12 12 12
GRAFT
4 4 4 4 4 4
SIZE
0.569 1.2YL 2.463 2.091 2.340 2.961
BETWEEN
+n
>0.5 >0.2 >0.02 >0.05 >0.02 >O.Ol
2 AND
On GRAFTS,
8 8 8 8 8 8
5 5 5 5 5 5
1.185 1.321 2.572 2.582 4.052 1.878
ALL
ON
<0.3 <0.3 <0.05 <0.05
+n
HOSTS
>0.2 >O.” >0.02 >0.02 >O.OOl >0.05
tailbuds is controlled autonomously; the initial growth of On tailbuds is intrinsically subnormal, as a consequence of the genotype of its own tissues. 2. Host influence. Apart from the requirement of host survival beyond 5 days, no significant effect of the hosts on their grafts can be demonstrated from these results. The size of +n grafts on On hosts shows no statistical deviation from the size of +n grafts on +n hosts (Table 3). Some host influence may be inferred, however, from
258
H.
WALLACE
the following two aspects of the results. First, the growth of +n grafts on On hosts appears to be retarded at 4 or 5 days (Fig. 3), mimicking the virtual arrest of tail growth in On hosts and controls at that time. Secondly, On grafts on +n hosts continued to grow after 2’ TESTS
TABLE 3 ON GRAFT SIZE, ALL +r~ GRAFTS, GROUPELI (fn) OR (On) ACCORDING TO HOSTS
Operation
1
Operation
Numbers nays
(+n)
(On)
1
12 12 12 12 12
6 6 6 6 6
2 3 4 5
II
Numbers
t 0.994 0.724 0.920 1.392 1.016
I’ <0.4 <0.5 <0.4 <0.2 <0.4
(+n) >0.3 >0.4 >0.3 >O.l >0.3
8 8 8 8 8
(On) 6 ti 6 6 6
I 0.505 0.161 1.035 2.051 1.368
P <0.7 <0.9 <0.4
>0.6 >0.8 >0.3 >0.05 >O.l
the tail growth of On larvae was virtually arrested (Figs. 2 and 3). These comparisons suggest that the growth of tailbuds is influenced by systemic factors after 3 or 4 days’ growth; the systemic factors emanating from On larvae then only support a subnormal growth of the tail. Cellular
Migration
An extension of the host epidermis over the basal part of the graft could be detected in all cases where the host and grafts were of different genotype (less than 30% of 2n epidermal cells have fused nucleoli after 4 days). Where the host and graft were either 2n or In genotypes, host epidermis covered usually the basal third of the graft. The greatest epidermal movement was from +n hosts onto On grafts: +n epidermis covered more than half of the graft at 5 days, and all but the distal tip of the graft at 10 days. In the reciprocal combination at 5 days, epidermis from the On host covered only the base of the +n graft. Some mesenchymal migration also occurred between the hosts and grafts. Such migration was always of fn cells invading an area occupied by On tissue, and concerned the mesenchyme of the fin and similar cells which enter the somites and differentiate as muscle cells (cf. Wallace, 1962b). The mesenchyme at the base of On grafts included some fn cells from the hosts at 2 days. Such cells were dis-
TAIL
GRAFTS
OF XENOPUS
hfUTANTS
259
tributed throughout the graft fin and had entered the graft somites at 4 days. They were then more common than On mesenchyme cells derived from the graft and appeared to be partly differentiated, forming much of the fin support of the graft and a minor part of its musculature. In the reciprocal combination at 5 days, no On mesenthyme cells from the host were detected in the fin or somites of the +n graft, but some +n mesenchyme from the graft had entered the fin and neighboring somites of the On host. DISCUSSION
Although Xenopus develops relatively rapidly, the results of this study correspond to the early growth of the tail in investigations on other amphibia. Two such investigations are mentioned here; others may be traced by reference to Bijtel (1958). By interchanging tailbuds between two species of Rana, Harrison ( 1898) demonstrated that about three somites anterior to the tailbud form the base of the tail, the remainder of which develops by the apical growth of the musculature, spinal cord, and notochord. Harrison also showed that trunk epidermis extends to cover the proximal half of the tail. The present results support Harrison’s findings when allowance is made for the differences of material and grafting technique. It is possible that the tailbud contributes less to the tail in Xenopus than in Rana, as the grafts in this experiment grew to only half the normal size of the tail. It is more probable, however, that these grafts were incomplete tailbuds, since they were taken at an earlier stage of development than that used by Harrison. A caudal extension of the epidermis is also found to occur in Xenopus. The epidermal movement is sufficiently powerful to counteract the competitive replacement of On by fn epidermis, which has been described previously (Wallace, 1962b). On the basis of extirpations and transplantations of the prospective tailbud tissues in urodeles, Bijtel (1958, p. 474) ascribes the first growth of the tailbud “to the stretching tendencies of the tail neural tube and especially of the tail somites.” The somites of Xenopus tailbuds both extend and incorporate migratory cells, which consequently could affect the size of the grafts. An influence of the migratory mesenchyme cells on the expansion of the graft fin would also affect the growth data, since the area of the grafts was measured in preference to a linear measurement of their somewhat distorted
260
H.
WALLACE
axes. The observed cellular migration, like the differences in blood circulation, would act systematically to increase in particular the later growth of On grafts carried by +n hosts. It is even conceivable that these migratory cells entirely account for the continued growth of such grafts after the arrest of tail growth in On hosts and controls. The defective blood circulation of On larvae perhaps remains a better explanation of the systemic effect on growth, in that it can also account for the inferredly inferior growth of +n grafts carried by On hosts, and for the death of both these hosts and their grafts. This argument is weakened, however, by the remarkable fact that explanted tails of older Xenopus larvae may survive for more than a month in sterile culture (Hauser and Lehmann, 1962). The demonstration of an initial autonomous factor and the inference of a later systemic factor controlling tail growth do not imply that these factors are mutually exclusive. The initial growth of tailsince explanted tailbuds conbuds probably is strictly autonomous, tinue to grow for several days. The present experiment, however, could only reveal those systemic factors lacking in the homozygous mutants; none were apparent during the first 3 days. Similarly, the continued subnormal growth of On grafts carried by fn hosts, observed up to 10 days, suggests the persistence of an autonomous control where the systemic factors permit normal growth. The relevance of the present results to nucleolar function is based on circumstantial evidence. The nucleolar mutation of Xenopus has been recognized at mitosis as the absence of a secondary constriction, with no other visible loss of the chromosome (Kahn, 19862). It is thus probable that the mutation concerns only the nucleolar organizer although this has not been rigorously region of the chromosome, demonstrated as in N- Chironomus (Beermann, 1960). On Xenopus resembles this and other cases of deleted nucleolar organizers in cytological detail. The On lethal syndrome can also be readily interpreted as a failure of nucleolar function. Probably in Xenopus, the nucleolus is essential for the survival certainly in Chironomus, does not of the organism. The absence of the nucleolus, however, prevent cellular survival superior to that of the organism, as shown by tail grafts. Various tissues of On Xenopus have been kept in parabiosis well beyond their usual life span, without apparent deterioration. Anucleolate cells can divide, differentiate, and function. Their differentiation is retarded and their function may be impaired:
TAIL
GRAFTS
OF
XENOPUS
MUTANTS
261
such abnormalities would have a cumulative effect, leading to the disorganization and death of the organism. It has now been demonstrated that a subnormal growth of the tail is an inherent effect of this mutation, Such a result is in accordance with other evidence of the participation of nucleoli in cellular growth (e.g., Caspersson, 1950). The On syndrome includes a wide range of cell types, and affects some common aspect of their presumably the mutation metabolism. The defective aspects of development in the absence of the nucleolus could then be considered as expressions of a generally slow cellular metabolism, which is sufficient for the life of the cell. That implies the essential function of the nucleolus to be one of maintaining the rate of one or more basic cellular syntheses above a critical level. The variety of mechanisms proposed to account for such a nucleolar function may be divided into two major categories, according to the derivation of the nucleolus. Either the nucleolus is produced by the unique synthetic activity of the nucleolar organizer, or it is also a collection of products from other gene loci (cf. Sirlin, 1960). Mechanisms of the former category have been examined by Beermann ( 1960) ; evidence has also been presented in support of the latter category (Wallace, 1962a). The example of On Xenopus is adaptable to either category, perhaps favoring the latter. The nucleolar organizer is either deleted or inactivated in this mutation, yet bodies with a cytochemical resemblance to nucleoli (blobs) are found in the nuclei of On Xenopus, which develops through stages normally associated with marked nucleolar activity. If the organizer synthesizes all nucleolar material, that material is not essential for basic cellular metabolism but could contribute to the ribosomal nucleoprotein and so facilitate protein synthesis. In this case, blobs are presumably produced by the overactivity of several independent gene loci in “compensation” for the lack of nucleolar synthesis. It is doubtful how real that compensation would be, as genetic overactivity could only supply redundant genetic information. Compensation could occur by synthesis at normally inert gene loci, like the latent organizers of ~riticurn (Longwell and Svihla, 1960). Xenopus does not possess latent organizers, in the sense that the specificity or quantity of blob material is inadequate to allow normal development in the absence of the nucleolus. If the organizer collects gene products, it follows that blobs are accumulations of gene products revealed by the failure of
262
H. WALLACE
the collection system. In this case, typically nucleolar material is still present in the cell and may perform its normal function. A nucleolar collection system can be readily conceived to influence the rate of cellular metabolism. According to the law of mass action, the removal of gene products from their sites of synthesis into a collected nucleolus should accelerate both the synthesis and the possible interaction of the gene products. SUMMARY
A larval lethal mutant of Xenopus which lacks nucleoli suffers a retardation and arrest of tail growth, and a defective blood circulation, several days before death. The factors that control the growth of the tail have been investigated by transplanting tailbuds between these mutants and their viable sibs. The initial growth of the tailbud is autonomous; that of anucleolate tailbuds is subnormal. The autonomous control may persist, subject to the later influence of systemic factors on the survival and probably also on the growth of the tail. A movement of the epidermis from the hosts to the grafts occurs, confirming a previous report of trunk epidermis extending over part of the tail. Cells from viable hosts also migrate into the somites and fin mesenchyme of anucleolate grafts. Such migratory cells, like the blood circulation, could act systemically upon tail growth. The subnormal tail growth of anucleolate embryos is, at least partly, a direct effect of the mutation in the cells of the tailbud. This is suggested to be an instance where a failure of nucleolar function reduces cellular growth, thus implying the necessity of functional nucleoli for normal growth. I am grateful to Professor Sir Alister Hardy, F.R.S., and to Professor R. A. R. Gresson for providing facilities for this research at Oxford and Belfast, respectively. I also thank Professor M. Fischberg for his general supervision of my studies and for his comments on a draft of this article. REFERENCES W. ( 1960). Der Nukleolus als lebenwichtiger Bestandteil des Zellkernes. Chromosoma 11, 263-296. BIJTEL, J. H. ( 1958). The mode of growth of the tail in nrodcle larvae. .I. Embryol. Exptl. Morphol. 6, 466478. CASPERSSON, T. (1950). “Cell Growth and Cell Function.” Norton, New York. ELSDALE, T. R., FISCHBERG, M., and SMITH, S. (1958). A mutation that reduces nucleolar number in Xenopus lazvis. Exptl. Cell Research 14, 642-643. BEERMANN,
TAIL
HARRISON, R. G. (1898).
GRAFTS
OF
XENOPUS
MUTANTS
263
The growth and regeneration of the tail of the frog Arch. EntuUlungsmech. Organ. 7, 430-485. HAUSER, R., and LEHMANN, F. E. (1962). Regeneration in isolated tails of Xenopus larvae. Experientia 18, 83-84. KAHN, J. ( 1962). The nuclcolar organizer in the mitotic chromosome complement of Xenopus laevis. Quurt. J. Microscop. Sci. in press. LONGWELL, A. R. C., and SVIHLA, G. (1960). Specific chromosomal control of the nucleolus and of the cytoplasm in wheat. Exptl. Cell Research 20, 294-312. NIEUWKOOP, P. D., and FABER, J. (lQs6). “Normal Table of Xenopus laevis.” North-Holland, Amsterdam. SIRLIX, J. L. (1960). The nucleolus problem. Nature 186, 275-277. VINCENT, W. S., and BALTUS, E. (1960). TI re ribonucleic acids of nucleoli. In “The Cell Nucleus” (J. S. hlitchell, ed.). pp, 13-23. Academic Press, New York. WALLACE, H. (1960). The development of anucleolate embryos of Xenopus laevis. J. Embryol. Exptl. Morphol. 8, 405-413. WALLACE, H. ( 1962a). Cytological and biochemical studies of anucleolate Xenopus larvae. Quart. J. Microscop. Sci. 103, 25-35. WALLACE, H. (196213). Prolonged life of anucleolate Xenopus tadpoles in parabiosis. J. Embryol. Exptl. ‘Vorphol. 10, 212-223. WOODS, P. S. (1960). Autoradiographic studies of ribonucleic acid metabolism with tritium-labelled cytidine. In “The Cell Nucleus” (J. S. Mitchell, ed.), pp. 127-137. Academic Press, New York. larva.
Tail
Grafts
BIOLOGY,
5, 252-263
involving
( 1962)
Anucleolate
Xenopus
Embryos
H. WALLACE Zoology
Depurtment,
The
Queen’s
Accepted
May
University,
Belfast,
N.
Ireland
14, 1962
INTRODUCTION Direct observations on the nucleolus suggest that it may function in development as an agent in cellular differentiation and growth. The implication of the nucleolus in ribonucleic acid metabolism (Vincent and Baltus, 1960; Woods, 1960) supports this view, as do the demonstrations of abnormal development in embryos lacking nucleoli (Elsdale et al., 1958; Beermann, 1960). Beermann ( 1960) showed that “nucleolusless” (N-) Ch ironomus embryos form germ bands and achieve some cellular differentiation, but die without hatching. He interpreted this lethal mutation in terms of the necessity of the nucleolus for growth. An investigation of anucleolate (On) Xenopus has also revealed a specific lethal syndrome, including neural pycnosis and a general retardation of both cellular differentiation and growth (Wallace, 1960). The growth of the tail of 0~ Xenopus may serve as a model of how the lack of nucleoli affects growth in general. The tails of On embryos grow rapidly at first, at a time when total nucleic acid synthesis has apparently ceased (cf. Wallace, 1962a); their growth is later retarded and finally arrested several days before the larvae die. Further growth of the tail has been detected during the survival of On embryos in parabiosis, but only at a time when cells from the host have migrated into the tail of the On embryo (Wallace, 196217). For this has been undertaken to establish reason a further investigation whether the growing tissues of the tail are affected directly by the mutation, or indirectly as a systemic effect of the mutation, mediated by the blood circulation or by other means. This article describes the results of transplanting tailbuds between On embryos and their viable sibs. The experiments were designed principally to test the 252
TAIL
GRAFTS
OF
XENOPUS
253
MUTANTS
effects of the absence of nucleolar function on an organ whose growth could be measured easily, but the results are also relevant to the control of tail growth in general. AIATERIAL
AND
METHOD
Heterozygous 1 n toads were induced to spawn by hypodermic injections of chorionic gonadotropin. Their progeny consisted of approximately 25% wild-type (2n), 50% heterozygous (In), and 25% anucleolate (On) embryos1 Tailbud stage embryos, at stage 29/30 of Nieuwkoop and Faber (1956), were used for grafting in Niu-Twitty solution on an agar base. The operations were performed on random samples of embryos, as the mutants cannot be distinguished in V~VCJ at tailbud stages. The operation is illustrated in Fig. 1. A tailbud was excised from one embryo (A) and implanted into a flank wound, above and behind the future anal region, of another embryo (B). No pressure was applied to hold the graft in place: it was either shaken off within a few hours or healed permanently in position. Successful combinations ( C ) developed in 5 days to larvae such as D and E, which are extreme examples of the differences observed. Figure 1 shows a moribund On larva (D) bearing a +n graft and a stage 47 +n larva (E) bearing a On graft. The grafts were always attached to the proximal part of the host’s tail, and usually originated close behind the host’s rectum. Out of 74 such operations, involving two series on different spawnings, 50 grafts held. Subsequent losses (abnormal host development and injuries in handling) reduced this number to 42 hosts with grafts, which were kept with control larvae through a lo-day period of observation. As soon as the grafts had healed, the embryos were transferred to one-tenth strength culture solution, then to tap water on the following day. All larvae were kept at 20°C and fed on a suspension of nettle powder after stage 47. Individual records were kept of the heartbeat and of the blood circulation in the tails and grafts. Growth was followed by daily camera lucida drawings of the lateral fin area behind the rectum (marked by broken lines in Fig. 1, D, E) of the hosts and controls, and of the lateral area of the grafts (stippled in Fig. 1, D, E) . The camera lucida drawings, with an area magnifica‘The nucleus.
symbols
denote
the
maximum
number
of nucleoli
present
in
n diploid
254
H.
WALLACE
tion x400, were subsequently measured by planimeter. The genotypes of the hosts, controls, and grafts were finally determined from permanent preparations stained with Mayer’s acid hemalum. It was found that a random combination of host and graft genotypes had
I
5mm.
1
I'
FIG. 1. Diagram of the operation. The grafted tailbud is stippled. Broken lines mark the anterior limits of the measured areas of the hosts’ tails. A full explanation is given in the text.
been achieved: 10 2n, 19 In, and 13 On hosts, bearing I2 2n, 20 In, and 10 On grafts. The controls were selected to provide more than the Mendelian expectation of On tadpoles; there were 8 2n, 17 In, and 11 On controls. A further series of operations provided material for an investigation of cellular migration into the grafts. RESULTS
In this and previous experiments, 2n and In genotypes have given identical results; thus they may be considered together as nucleolate (+n) material. It is convenient to express the results as a function of time after the operation, because that is virtually the time when tail growth begins. For reasons stated below, only the results from the first 5 days of growth are used. The developmental stages reached during successive days after operating on stage 29/30 are: 1, stage 39; 2, stage 42; 3, stage 44; 4, stage 46; 5, stage 47. Heartbeat and Blood Circulation The heart probably begins to beat before stage 35/36 in all genotypes. The heartbeat of On larvae is relatively slow at stage 40 (Wallace, 1960) and is intermittent or has ceased in the moribund
TAIL
CRAFTS
OF
XENOPUS
255
MUTAYR
On larvae at 4 days. The blood circulation in the subcaudal vein is sluggish and intermittent in On larvae, being imperceptible after 4 days. A rapid blood circulation was established in all grafts on +n hosts, beginning at 24 days in +n grafts and rather later, at 3-4 days, in On grafts. No movement of blood corpuscles was detected in any of the grafts on On hosts. Some circulation must have occurred in them, however, for all these grafts gradually accumulated masses of erythrocytes. Tail Growth
in Hosts
and Controls
Analyses of variance on the growth data show no significant differences of the daily size of tails between 2n and In genotypes, nor
op.1
t
+
6-
/"t
x
t +'
-
*(A'
5Ot 4-
op. II
2
3-
2-
III I
I
I
I
I
I
Oo
I
2
3
4
5
OOU
(2) FIG.
Growth of tails of hosts and controls of +n hosts and controls; 0 Fro. 3. Growth of grafts for each series of times 400 square inches; abscissas, age in days show the standard errors of the mean values. +n hosts; e _ @ On grafts carried by +n by On hosts.
n -
2.
n Tails
t’+ /+x x ,-; /t t 4v I
2
3
4
5
(3) from both series of operations. 0 tails of On hosts and controls. operations. Ordinates, lateral area after the operation. Vertical lines + - + fn grafts carried by hosts; X X +n grafts carried
256
H.
WALLACE
between hosts and controls of any genotype-the grafts did not appreciably affect the tail growth of their hosts. It follows that tail growth can be considered in two groups, +n and On, which are shown for both series of operations in Fig. 2. It will be seen from Fig. 2 that +n tails continued to grow rapidly for 5 days (no further growth could be accommodated by the drawing apparatus), whereas the growth of On tails was retarded at 3 days and was virtually arrested after that. The reliability of this result is shown by an analysis of variance between fn and On groups (Table 1). A general T.mLE ANALYSES
OF VARIANCE BETWEEN Operation
Numbers Days
+?I
On
Variance mt.io
1 2 3 4 5
32 32 32 32 32
11 11 11 11 11
0.58 0 .83 33.45 l’L3.51 23s. l!)
ALL
OS fn
T-411, AXI)
1 SIZE ALL
HOSTS
IN On
AND
I
Operation h-nmbers I’
>0.2 >o.a
CONTROLS
MATERIAL
+ 3,
Old
Variance ratio
22 22 a2 22 ,”
IS 1s 13 13 13
2.78 I .38 140 5ti 201.55 425.60
II
P
<0.2 >O.l >0.2 10.001
correspondence may be seen between the inferior blood circulation and retarded tail growth of On larvae, and between their later cessation both of the circulation and of tail growth.
Graft Growth Owing to a variation in the initial size of the grafts, and to the difficulty in keeping them horizontal while drawing, there is more variability in the growth data of the grafts than in that of the host and control tails. It is assumed that this variability would swamp any possible difference between the growth of 2n and In grafts, or any possible differential influence of 212or In hosts on their grafts. Grouping both grafts and hosts into +n and On categories gives four classes of results: +n/+n, On/+n, +n/On, On/On. The single example of the last class showed the least growth of any graft but is ignored as having little comparative value. Results for the other three classesof grafts are shown separately for each series of operations (Fig. 3). The size attained by the grafts implies that they normally form only the distal half of the tail. The grafts of the second
TAIL
GRAFTS
OF
XENOPUS
257
MUTANTS
series of operations were larger than those of the first series throughout the experiment, a result suggesting that they were taken from slightly older and larger tailbuds. The grafts of both series, however, show the same growth characteristics. On hosts were moribund at 5 days; they and their grafts barely survived the following day. Continued observations of the grafts on +n hosts up to 10 days revealed that the difference in size of +n and On grafts at 5 days was maintained. The little further growth of these grafts after 5 days was accompanied by their increased distortion. There are two meaningful comparisons of the data on graft growth, which are analyzed by t tests (Tables 2 and 3). It is concluded that the data show both an autonomous control of growth by the graft itself and an influence of the host on the growth of its graft. 1. Autonomous growth of grafts. When supported by +n hosts, On grafts grew more slowly than +n grafts (Fig. 3). This difference became statistically significant by 3 days (Table 2). For these first 3 days, all fn grafts grew at approximately the same rate, irrespective of whether they were supported by +n or by On hosts (Fig. 3, Table 3). These two results demonstrate that the initial growth of TABLE 2’ TESTS
1
2 3 4 5 10
ON
12 12 12 12 12 12
GRAFT
4 4 4 4 4 4
SIZE
0.569 1.2YL 2.463 2.091 2.340 2.961
BETWEEN
+n
>0.5 >0.2 >0.02 >0.05 >0.02 >O.Ol
2 AND
On GRAFTS,
8 8 8 8 8 8
5 5 5 5 5 5
1.185 1.321 2.572 2.582 4.052 1.878
ALL
ON
<0.3 <0.3 <0.05 <0.05
+n
HOSTS
>0.2 >O.” >0.02 >0.02 >O.OOl >0.05
tailbuds is controlled autonomously; the initial growth of On tailbuds is intrinsically subnormal, as a consequence of the genotype of its own tissues. 2. Host influence. Apart from the requirement of host survival beyond 5 days, no significant effect of the hosts on their grafts can be demonstrated from these results. The size of +n grafts on On hosts shows no statistical deviation from the size of +n grafts on +n hosts (Table 3). Some host influence may be inferred, however, from
258
H.
WALLACE
the following two aspects of the results. First, the growth of +n grafts on On hosts appears to be retarded at 4 or 5 days (Fig. 3), mimicking the virtual arrest of tail growth in On hosts and controls at that time. Secondly, On grafts on +n hosts continued to grow after 2’ TESTS
TABLE 3 ON GRAFT SIZE, ALL +r~ GRAFTS, GROUPELI (fn) OR (On) ACCORDING TO HOSTS
Operation
1
Operation
Numbers nays
(+n)
(On)
1
12 12 12 12 12
6 6 6 6 6
2 3 4 5
II
Numbers
t 0.994 0.724 0.920 1.392 1.016
I’ <0.4 <0.5 <0.4 <0.2 <0.4
(+n) >0.3 >0.4 >0.3 >O.l >0.3
8 8 8 8 8
(On) 6 ti 6 6 6
I 0.505 0.161 1.035 2.051 1.368
P <0.7 <0.9 <0.4
>0.6 >0.8 >0.3 >0.05 >O.l
the tail growth of On larvae was virtually arrested (Figs. 2 and 3). These comparisons suggest that the growth of tailbuds is influenced by systemic factors after 3 or 4 days’ growth; the systemic factors emanating from On larvae then only support a subnormal growth of the tail. Cellular
Migration
An extension of the host epidermis over the basal part of the graft could be detected in all cases where the host and grafts were of different genotype (less than 30% of 2n epidermal cells have fused nucleoli after 4 days). Where the host and graft were either 2n or In genotypes, host epidermis covered usually the basal third of the graft. The greatest epidermal movement was from +n hosts onto On grafts: +n epidermis covered more than half of the graft at 5 days, and all but the distal tip of the graft at 10 days. In the reciprocal combination at 5 days, epidermis from the On host covered only the base of the +n graft. Some mesenchymal migration also occurred between the hosts and grafts. Such migration was always of fn cells invading an area occupied by On tissue, and concerned the mesenchyme of the fin and similar cells which enter the somites and differentiate as muscle cells (cf. Wallace, 1962b). The mesenchyme at the base of On grafts included some fn cells from the hosts at 2 days. Such cells were dis-
TAIL
GRAFTS
OF XENOPUS
hfUTANTS
259
tributed throughout the graft fin and had entered the graft somites at 4 days. They were then more common than On mesenchyme cells derived from the graft and appeared to be partly differentiated, forming much of the fin support of the graft and a minor part of its musculature. In the reciprocal combination at 5 days, no On mesenthyme cells from the host were detected in the fin or somites of the +n graft, but some +n mesenchyme from the graft had entered the fin and neighboring somites of the On host. DISCUSSION
Although Xenopus develops relatively rapidly, the results of this study correspond to the early growth of the tail in investigations on other amphibia. Two such investigations are mentioned here; others may be traced by reference to Bijtel (1958). By interchanging tailbuds between two species of Rana, Harrison ( 1898) demonstrated that about three somites anterior to the tailbud form the base of the tail, the remainder of which develops by the apical growth of the musculature, spinal cord, and notochord. Harrison also showed that trunk epidermis extends to cover the proximal half of the tail. The present results support Harrison’s findings when allowance is made for the differences of material and grafting technique. It is possible that the tailbud contributes less to the tail in Xenopus than in Rana, as the grafts in this experiment grew to only half the normal size of the tail. It is more probable, however, that these grafts were incomplete tailbuds, since they were taken at an earlier stage of development than that used by Harrison. A caudal extension of the epidermis is also found to occur in Xenopus. The epidermal movement is sufficiently powerful to counteract the competitive replacement of On by fn epidermis, which has been described previously (Wallace, 1962b). On the basis of extirpations and transplantations of the prospective tailbud tissues in urodeles, Bijtel (1958, p. 474) ascribes the first growth of the tailbud “to the stretching tendencies of the tail neural tube and especially of the tail somites.” The somites of Xenopus tailbuds both extend and incorporate migratory cells, which consequently could affect the size of the grafts. An influence of the migratory mesenchyme cells on the expansion of the graft fin would also affect the growth data, since the area of the grafts was measured in preference to a linear measurement of their somewhat distorted
260
H.
WALLACE
axes. The observed cellular migration, like the differences in blood circulation, would act systematically to increase in particular the later growth of On grafts carried by +n hosts. It is even conceivable that these migratory cells entirely account for the continued growth of such grafts after the arrest of tail growth in On hosts and controls. The defective blood circulation of On larvae perhaps remains a better explanation of the systemic effect on growth, in that it can also account for the inferredly inferior growth of +n grafts carried by On hosts, and for the death of both these hosts and their grafts. This argument is weakened, however, by the remarkable fact that explanted tails of older Xenopus larvae may survive for more than a month in sterile culture (Hauser and Lehmann, 1962). The demonstration of an initial autonomous factor and the inference of a later systemic factor controlling tail growth do not imply that these factors are mutually exclusive. The initial growth of tailsince explanted tailbuds conbuds probably is strictly autonomous, tinue to grow for several days. The present experiment, however, could only reveal those systemic factors lacking in the homozygous mutants; none were apparent during the first 3 days. Similarly, the continued subnormal growth of On grafts carried by fn hosts, observed up to 10 days, suggests the persistence of an autonomous control where the systemic factors permit normal growth. The relevance of the present results to nucleolar function is based on circumstantial evidence. The nucleolar mutation of Xenopus has been recognized at mitosis as the absence of a secondary constriction, with no other visible loss of the chromosome (Kahn, 19862). It is thus probable that the mutation concerns only the nucleolar organizer although this has not been rigorously region of the chromosome, demonstrated as in N- Chironomus (Beermann, 1960). On Xenopus resembles this and other cases of deleted nucleolar organizers in cytological detail. The On lethal syndrome can also be readily interpreted as a failure of nucleolar function. Probably in Xenopus, the nucleolus is essential for the survival certainly in Chironomus, does not of the organism. The absence of the nucleolus, however, prevent cellular survival superior to that of the organism, as shown by tail grafts. Various tissues of On Xenopus have been kept in parabiosis well beyond their usual life span, without apparent deterioration. Anucleolate cells can divide, differentiate, and function. Their differentiation is retarded and their function may be impaired:
TAIL
GRAFTS
OF
XENOPUS
MUTANTS
261
such abnormalities would have a cumulative effect, leading to the disorganization and death of the organism. It has now been demonstrated that a subnormal growth of the tail is an inherent effect of this mutation, Such a result is in accordance with other evidence of the participation of nucleoli in cellular growth (e.g., Caspersson, 1950). The On syndrome includes a wide range of cell types, and affects some common aspect of their presumably the mutation metabolism. The defective aspects of development in the absence of the nucleolus could then be considered as expressions of a generally slow cellular metabolism, which is sufficient for the life of the cell. That implies the essential function of the nucleolus to be one of maintaining the rate of one or more basic cellular syntheses above a critical level. The variety of mechanisms proposed to account for such a nucleolar function may be divided into two major categories, according to the derivation of the nucleolus. Either the nucleolus is produced by the unique synthetic activity of the nucleolar organizer, or it is also a collection of products from other gene loci (cf. Sirlin, 1960). Mechanisms of the former category have been examined by Beermann ( 1960) ; evidence has also been presented in support of the latter category (Wallace, 1962a). The example of On Xenopus is adaptable to either category, perhaps favoring the latter. The nucleolar organizer is either deleted or inactivated in this mutation, yet bodies with a cytochemical resemblance to nucleoli (blobs) are found in the nuclei of On Xenopus, which develops through stages normally associated with marked nucleolar activity. If the organizer synthesizes all nucleolar material, that material is not essential for basic cellular metabolism but could contribute to the ribosomal nucleoprotein and so facilitate protein synthesis. In this case, blobs are presumably produced by the overactivity of several independent gene loci in “compensation” for the lack of nucleolar synthesis. It is doubtful how real that compensation would be, as genetic overactivity could only supply redundant genetic information. Compensation could occur by synthesis at normally inert gene loci, like the latent organizers of ~riticurn (Longwell and Svihla, 1960). Xenopus does not possess latent organizers, in the sense that the specificity or quantity of blob material is inadequate to allow normal development in the absence of the nucleolus. If the organizer collects gene products, it follows that blobs are accumulations of gene products revealed by the failure of
262
H. WALLACE
the collection system. In this case, typically nucleolar material is still present in the cell and may perform its normal function. A nucleolar collection system can be readily conceived to influence the rate of cellular metabolism. According to the law of mass action, the removal of gene products from their sites of synthesis into a collected nucleolus should accelerate both the synthesis and the possible interaction of the gene products. SUMMARY
A larval lethal mutant of Xenopus which lacks nucleoli suffers a retardation and arrest of tail growth, and a defective blood circulation, several days before death. The factors that control the growth of the tail have been investigated by transplanting tailbuds between these mutants and their viable sibs. The initial growth of the tailbud is autonomous; that of anucleolate tailbuds is subnormal. The autonomous control may persist, subject to the later influence of systemic factors on the survival and probably also on the growth of the tail. A movement of the epidermis from the hosts to the grafts occurs, confirming a previous report of trunk epidermis extending over part of the tail. Cells from viable hosts also migrate into the somites and fin mesenchyme of anucleolate grafts. Such migratory cells, like the blood circulation, could act systemically upon tail growth. The subnormal tail growth of anucleolate embryos is, at least partly, a direct effect of the mutation in the cells of the tailbud. This is suggested to be an instance where a failure of nucleolar function reduces cellular growth, thus implying the necessity of functional nucleoli for normal growth. I am grateful to Professor Sir Alister Hardy, F.R.S., and to Professor R. A. R. Gresson for providing facilities for this research at Oxford and Belfast, respectively. I also thank Professor M. Fischberg for his general supervision of my studies and for his comments on a draft of this article. REFERENCES W. ( 1960). Der Nukleolus als lebenwichtiger Bestandteil des Zellkernes. Chromosoma 11, 263-296. BIJTEL, J. H. ( 1958). The mode of growth of the tail in nrodcle larvae. .I. Embryol. Exptl. Morphol. 6, 466478. CASPERSSON, T. (1950). “Cell Growth and Cell Function.” Norton, New York. ELSDALE, T. R., FISCHBERG, M., and SMITH, S. (1958). A mutation that reduces nucleolar number in Xenopus lazvis. Exptl. Cell Research 14, 642-643. BEERMANN,
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HARRISON, R. G. (1898).
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The growth and regeneration of the tail of the frog Arch. EntuUlungsmech. Organ. 7, 430-485. HAUSER, R., and LEHMANN, F. E. (1962). Regeneration in isolated tails of Xenopus larvae. Experientia 18, 83-84. KAHN, J. ( 1962). The nuclcolar organizer in the mitotic chromosome complement of Xenopus laevis. Quurt. J. Microscop. Sci. in press. LONGWELL, A. R. C., and SVIHLA, G. (1960). Specific chromosomal control of the nucleolus and of the cytoplasm in wheat. Exptl. Cell Research 20, 294-312. NIEUWKOOP, P. D., and FABER, J. (lQs6). “Normal Table of Xenopus laevis.” North-Holland, Amsterdam. SIRLIX, J. L. (1960). The nucleolus problem. Nature 186, 275-277. VINCENT, W. S., and BALTUS, E. (1960). TI re ribonucleic acids of nucleoli. In “The Cell Nucleus” (J. S. hlitchell, ed.). pp, 13-23. Academic Press, New York. WALLACE, H. (1960). The development of anucleolate embryos of Xenopus laevis. J. Embryol. Exptl. Morphol. 8, 405-413. WALLACE, H. ( 1962a). Cytological and biochemical studies of anucleolate Xenopus larvae. Quart. J. Microscop. Sci. 103, 25-35. WALLACE, H. (196213). Prolonged life of anucleolate Xenopus tadpoles in parabiosis. J. Embryol. Exptl. ‘Vorphol. 10, 212-223. WOODS, P. S. (1960). Autoradiographic studies of ribonucleic acid metabolism with tritium-labelled cytidine. In “The Cell Nucleus” (J. S. Mitchell, ed.), pp. 127-137. Academic Press, New York. larva.