J. Embryol. exp. Morph. Vol. 15, 1, pp. 1-14, February 1966
Printed in Great Britain
*
A study of the influence of isolated calf spleen RNA
on amphibian histogenesis
By CYRIL V. FINNEGAN 1 & WILLIAM P. BIGGIN 2
Department of Zoology, University of British Columbia
INTRODUCTION
In the cell-free inductor demonstrated by Niu & Twitty (1953) the effective
material was later characterized by Niu (1956) as ribonucleoprotein with the
suggestion that the ribonucleic acid (RNA) fraction was the active component.
Subsequently Niu (1958a, b) reported tissue-specific (thymus-like) induction in
amphibian material exposed to calf thymus RNA, though Yamada (1961), using
identical procedures, was unable to repeat the results, and Saxen & Toivonen
(1962) considered the thymus-like histogenesis in Niu's results to be poor.
In the present investigation calf spleen ribonucleic acid was isolated so as to
ensure removal of contaminants from the isolate prior to testing for inductive
capacity with competent amphibian ectoderm and embryos. Sedimentation
analysis with the ultracentrifuge indicated that the isolated spleen ribosomal
RNA was undegraded when introduced into the culture medium. In a limited
series of test cases neither embryos nor ectodermal isolates of Xenopus laevis
demonstrated any tissue-specific inductive response (i.e. erythropoiesis), though
a general enhancement of epithelial development was obvious. Further information on such enhancement of epithelial histogenesis was obtained when postgastrula stages of Taricha torosa and Ambystoma gracile were cultured in a
commercial liver s-RNA preparation.
MATERIALS AND METHODS
Calf spleen was used as the source of RNA since it has a high RNA content,
a low connective tissue polysaccharide content and its specific histogenesis
(erythropoiesis) is easy to see in cultured tissues. The freshly frozen spleen
tissue was sliced into pieces approximately 1 x 2 cm and then homogenized, in
2-5 ml. of 001 5M naphthalene-1,5-disulfonate and 2-5 ml. of 88-90 % phenol
per gram weight of tissue, for 2 minutes in a Waring Blendor at room tempera1
Author's address: Department of Zoology, University of British Columbia, Vancouver 8,
Canada.
2
Author's address: Department of Biology, Temple University, Philadelphia, Pennsylvania,
U.S.A.
I
JEEM
15
2
C. V. FINNEGAN & W. P. BIGGIN
ture (Kirby, 1956, 1962). The cloudy supernatant was carefully sucked off and
subjected to a total of three extractions with phenol (Biggin, 1964). Glassware
and equipment were washed with lN-NaOH and with versene to remove RNase
and heavy metal contaminants. The purity of the isolate was indicated with the
Biuret test (Schneider, 1957) for protein, Drey wood's anthrone test (Morris,
1948) for carbohydrate, Dische's diphenylamine test (Chargaff & Davidson,
1955; Schneider, 1957) for DNA and Bial's orcinol method (Chargaff & Davidson, 1955) to estimate RNA. Following extraction of the isolate with 2methoxyethanol, no polysaccharide contamination was demonstrable either
with the anthrone reagent or by chromatography.
Spectrophotometric analysis was made of the isolates in 0-lM-NaCl solution
with the Unicam SP500, and from these data the ratio of absorption values for
260/230 mfi and for 260/280 m/* were calculated to indicate, according to
Morton (1962), the relative amounts of peptide (230 m/i) and protein (280 m/i)
present with the nucleic acid (see Table 1). Sedimentation analysis was conducted with a Spinco model E analytical ultracentrifuge, employing Schleiren optics
with photographs taken at 4 min intervals at 44,770 r.p.m. The sedimentation
coefficients of the isolated RNA were determined according to Markham (1960,
1962).
The method of Brown & Littna (1964) was employed to obtain eggs of Xenopus
laevis which were removed for use at late blastula to early gastrula stages (stages
9-9 + , Nieuwkoop & Faber, 1956). Sterile techniques were used throughout.
Isolated gastrula ectoderm (six cases) and embryos from stage 9 + (eight cases)
were cultured as controls in Niu-Twitty medium for 5 days with daily observations. Similarly, isolated ectoderm (eighteen cases) and embryos (eight cases)
were cultured as experimental cases in Niu-T witty medium to which 50/^g/ml.
(Niu, 19586; Yamada, 1961) of isolated spleen RNA was added immediately
before use to minimize possible RNA degradation effects. All embryos (sixteen)
and a few randomly selected control and experimental explants (four) were fixed
in Carnoy's acetic-alcohol, paraffin-embedded, sectioned at 5-8/tm and stained
with haematoxylin-eosin or Chromotrope 2R for histological examination.
Late gastrula of Taricha torosa (stage 13), from California, and Ambystoma
gracile (stage 12), obtained locally, were freed from the egg jelly and (the
majority) from vitelline membranes before being placed in a medium produced
by adding Reagent Grade Beef liver RNA (Nutritional Biochemical Company)
at a concentration of 25 mg/ml. to Niu-Twitty solution. Ten animals of each
species and five control animals (in salt solution) were grown until stage 39
(2-3 weeks), with the media replaced at least twice per week, after which
they were fixed in Carnoy's and stained with the modified Mallory's (Liisberg,
1962).
The spectrophotometric data for the commercial-liver s-RNA were obtained
as were the calf spleen RNA data and are presented with the latter in Table 1 and
Fig. 1. In the sedimentation analysis with the Spinco model E analytical ultra-
Isolated RNA and histogenesis
3
centrifuge, UV optics were used and photographs were taken at 4 min intervals
after the speed of 55,770 r.p.m. was attained. The commercial liver RNA, dissolved in 0-lM-NaCl, was observed in the ultracentrifuge for 20 min.
RESULTS
When calf spleen RNA isolated with a single phenol extraction was examined
spectrophotometrically (see Fig. 1) the ratio of absorption values 260/230 m/t
(1-80) and the ratio of 260/280 mju, (1-82) indicated protein and peptide contamination of the isolate (see Table 1). On the other hand, after three phenol extractions, these same ratios were 2-23 and 2-20, respectively, indicating a more
effective removal of proteins and/or peptides from the isolates. The Biuret test
1-8
-
1-6
1-4
1-2
10
0-8
0-6
0-4
0-2
220
240
260
280
300
320
Wave-length (m/*)
Fig. 1. The ultraviolet absorption spectra of isolated calf-spleen RNA, after one and
after three phenol extractions, and of reagent-grade beef-liver RNA. # — # , One
phenol extraction; O—O three phenol extractions; A — A , commercial RNA.
4
C. V. FINNEGAN & W. P. BIGGIN
was negative in all cases. The commercial liver RNA had an absorption peak at
265 m/A (see Fig. 1) and, while the peptide removal seemed effective, the protein/
nucleic acid absorption ratio was the same as for the single phenol calf spleen
isolation (see Table 1).
A spectrophotometrically measurable positive response was obtained with the
diphenylamine test for DNA with the single phenol extract which was not detectable in the isolate after three phenol extractions. It is possible that the small
quantity of DNA observed in the first case is the result of the nuclei demonstrated by Georgiev, Mantirva & Zbarsky (1960) to be present in the intermediate
layer after centrifugation.
Table 1. Spectrophotometric analysis of isolated calf-spleen RNA and
reagent-grade liver RNA
Optical densities (m/i)
One phenol extraction
Three phenol extractions
Reagent-grade liver RNA
Ratios
230
260
280
260/230
260/280
0-690
0-550
0-266
1-240
1-230
0-588
0-680
0-560
0-421
1-80
2-23
2-20
1-82
2-20
1-82
Polysaccharide contamination, which could be demonstrated chromatographically and with the anthrone reagent even after three phenol extractions,
and previously shown by Kirby (1956,1962) and by Ralph & Bellamy (1964), was
effectively removed by methoxyethanol. Thus, the isolate from calf spleen contained RNA in the apparent absence of peptide, protein, DNA and carbohydrate. However, these data gave no clue as to the physical condition of the
isolated RNA molecule and Laskov, Margoliash, Littauer & Eisenberg (1959)
and Ralph*& Bellamy (1964) have reported the RNA molecule to be degraded
when isolated with the original Kirby (1956) method.
Information on the size and condition of the isolated RNA molecules was
obtained with the analytical ultracentrifuge and the resulting sedimentation
patterns are shown in Figs. 2 and 3. Three definite molecular species of RNA
were present in the calf-spleen isolate, whose sedimentation coefficients (in
Svedberg units) were calculated to be 27 S, 18 S and 7-8 S (S2Olw)> and with the
fastest moving component, the 27 S fraction, being present in the highest concentration. These results indicate that the isolated molecules were not degraded
by the procedure (Darnell, Penman, Scherrer & Becker, 1963; Ralph & Bellamy,
1964) and it is this RNA which was used in the induction experiments with
Xenopus tissues. With the commercial liver RNA in the ultracentrifuge there was
no apparent movement of the molecules, which may be taken to indicate that
these s-RNA molecules are quite small; an explanation consistent with present
knowledge concerning the size of soluble RNA (Spirin, 1963).
The excised Xenopus gastrula ectoderm remained free of the glass coverslip
Isolated RNA and histogenesis
6-5
6-4
6-3
•E
6-2
8S
6-1
60
5-9
1
2
3
4
5
6
Exposure
Fig. 2. Representation of the data obtained for the isolated calf-spleen RNA with
the analytical ultracentrifuge. The slope of the curves is a measure of the sedimentation coefficients.
y
y
y
y
y
y
y
y
y
y
y
y
7-8 S
i
I 18 S
M
y
y
y
y
y
y
y
y
y
y
y
y
y
Fig. 3. A drawing made from a Schlieren photograph of a 0-1 M-NaCl solution of
spleen RNA after reaching the speed of 44,770 r.p.m. in the analytical ultracentrifuge.
From the meniscus, M, the peaks have sedimentation coefficients of 7-8 S, 18S, and
27 S.
6
C. V. FINNEGAN & W. P. BIGGIN
and rolled into a ball in approximately 30min in both control and RNAcontaining media. Daily examination during the culture period, in which control Xenopus embryos reached swimming larval stages, showed no tissue-specific
induction (i.e. erythropoiesis) or, indeed, any histogenesis in either control or
experimental cases. In preliminary experiments it was found that the control
explants would not survive much beyond a 5-day culture period whereas the
explants in RNA medium would survive well into the second week. At the same
time, in all experiments, the explants in RNA-medium were less susceptible to
disintegration during the 5-day culture period than were the control explants.
Fig. 4
Fig. 5
Fig. 4. A section of a Taricha torosa (animal no. 10) grown in a commercial liver
RNA medium, which shows the dorsal mesencephalic chaotic hyperplasia. (Traced
from a projection.)
Fig. 5. A section of a Taricha torosa (animal no. 7) grown in a commercial liver RNA
medium and showing a hyperplasia in the dorsal preotic rhombencephalon but no
increase in the ventral neural fiber material (white matter). (Traced from a projection.)
Thus, it was apparent that the maintenance and the survival of the explants were
enhanced in the RNA-enriched medium as compared with the control medium.
Since the daily observations for evidence of erythropoiesis in the explants showed
none, only two of each series, randomly selected from the experimental and
control explants, were sectioned for histological examination. Again no erythropoiesis was seen, but in the similar-sized explants the single layered squamouscuboidal ectoderm of the control explants contrasted with the 2- to 4-layered,
frequently columnar, epithelium of the experimental explants.
The intact Xenopus embryos cultured in the medium containing calf-spleen
RNA showed what appeared to be an accelerated histogenesis as compared with
the controls, for when sectioned after 5 days' culture it could be seen that an
Isolated RNA and histogenesis
1
increase in epithelial cell number and/or size was present which was non-specific
since it involved such diverse epithelial systems as that of the otic vesicles, the
intestine and some glandular epithelia (see also Taricha torosa results below).
In the commercial liver RNA medium the T. torosa embryos all developed
posterior dorsal head lesions and excessive cranial flexion during the culture
period. It was apparent in the sectioned material that a very definite hyperplasia
had occurred in the mesencephalon roof, i.e. the tectum (see Fig. 4), which
continued posteriorly in the rhombencephalon (see Figs. 5, 6). This neural
hyperplasia was rather unorganized in the mesencephalon and anterior
rhombencephalon but became less chaotic posteriorly in the hind brain (see
Fig. 7). Although the magnitude of the chaotic histogenesis varied it was
present in all experimental animals (see Fig. 8).
Table 2. Auditory vesicle size in Taricha torosa embryos grown in
commercial liver-RNA medium
No. of cells
per section*
Cross-sectional
diameters*
Length
GO
Controlf
Exp. 1
2
3
4
5
210
259
287
245
231
217
•
21-3
24-3
33-8
250
23-8
23-8
A
r
K
<
27
30
43-8
27-5
28-8
30
Min.
Max.
84
85
95
95
91
96
124
88
118
116
105
98
* The vesicle diameters and the number of nuclei per section were measured at several
identical anterior-posterior levels in all animals, namely the endolymphatic duct, the auditory
ganglion and the largest, or a larger, section. The diameter values for this table are those of the
endolymphatic duct level.
f A single control animal, which approximated the average control length, was chosen for
this comparison since all control measurements were less than the experimentals.
Auditory vesicles of these T. torosa embryos were measured as to total
anterior-posterior length, as to both dorsal-ventral and medial-lateral width
and as to cell population (number of nuclei per section at various levels along the
anterior-posterior axis). An example of the data obtained is given in Table 2.
All experimental auditory vesicles were larger than the controls in length and
width but no variation in cell population could be demonstrated (see Table 2),
which implies that an increase in cell size (not readily apparent histologically)
was responsible for, or accompanied, the increase in volume.
Cell counts made of the mesenchyme cells present dorsal to the rhombencephalon indicated an increase in this population which is probably of neural crest
origin, but the hyperplasia did not involve the ganglia or the melanophores.
The increase in dorsal mesenchyme cells was continued in the anterior trunk, being
8
C. V. FINNEGAN & W. P. BIGGIN
reflected there by the more lateral or ventro-lateral positioning of the dorsal
somite tips and the increase in size of the basal portion of the dorsal fin. This
hyperplasia did not continue into the posterior trunk though the base of the fin
appeared to remain large. In the posterior trunk the cloacal endodermal epithelium was enlarged in thickness, apparently because individual cells were
columnar and not the normal cuboidal shape, for no increase in cell number
could be shown in this tissue.
No external evidence of an effect of the commercial-liver RNA was shown by
the Ambystoma gracile embryos during the culture period but, in the sectioned
Fig. 6. A section of a Taricha torosa (animal no. 4) grown in a commercial liver
RNA medium which shows hyperplasia in the dorsal postotic rhombencephalon.
Again, no increase in the ventral neural fiber material was demonstrated. (Traced
from a projection.)
Fig. 7. (A) A section of a Taricha torosa (animal no. 2) grown in a commercial liver
RNA medium and showing the reduced neural hyperplasia found in these animals at
the anterior trunk level of the neural tube. (Traced from a projection.) (B) A section of a control Taricha torosa animal, grown in a salt solution, at approximately
the same anterior trunk level as in the above (A). (Traced from a projection.)
Isolated RNA and histogenesis
9
material, the dorsal neural hyperplasia was definitely present though much less
pronounced than it had been in Taricha torosa. There was some indication of
chaotic development in the anterior rhombencephalon roof in only two of the
experimental animals. No increase in the auditory vesicles could be demonstrated and it was evident that the more subtle hyperplasia of the neural tissue had
Fig. 8. Sections of Taricha torosa embryos grown in a commercial liver RNA
medium which show less extensive hyperplasia in the dorsal postotic rhombencephalon than that seen in Fig. 6. A, Animal no. 7; B, animal no. 9. (Traced from a
projection.)
not interfered with neural organogenesis in this species as it had in T. torosa.
The dorsal mesenchyme population was increased about one-third in the hindbrain-anterior trunk region of the experimental animals resulting in a broad
base to the dorsal fin. This increase in fin size was continued into the posterior
trunk, in the absence of any mesenchyme increase, and in the presence of a similar, though more loose, fiber content: this is taken to indicate a probable increase
in ground substance, particularly hyaluronic acid which is so highly concentrated
in the dorsal fin (C. V. Finnegan, unpublished). The cloacal endodermal epithelium was enlarged as had been seen in T. torosa.
DISCUSSION
The procedure for the isolation of ribonucleic acid as employed by Niu
(1958a, b); Niu, Cordova & Niu (1961); Hillman & Niu (1963a, b); Yamada
(1961) and Butros (1963) was the Kirby (1956) method reported by Laskov et al.
(1959) to yield a degraded product. When the methoxyethanol purification was
deleted to reduce this degradation (Niu et al. 1961), it would seem that the polysaccharide contamination might have become significant, particularly since liver
and muscle tissues were often the source material used. With the single phenol
extraction used it is possible that the protein and/or peptide contamination also
might have been significant, since Huppert & Pelmont (1962) were able to demonstrate with the sensitive Lowry method the presence of between 20 and 50/*g/ml.
10
C. V. FINNEGAN & W. P. BIGGIN
protein following three phenol extractions of the isolated RNA. Thus it is difficult to consider, as do many workers (Gierer, 1957; Gierer & Schramm, 1956;
Kirby, 1962; Niu, 1958a, b; Niu et at. 1961; Hillman & Niu, 1963a, b; Butros,
1963), that protein and/or peptide contamination is completely absent from the
Kirby-isolated RNA. However, after the additional phenol washings such contamination would appear to be reduced to a concentration which does not act
inductively in the experimental system used in this investigation.
Analysis with the ultracentrifuge demonstrated that undegraded ribosomal
RNA (Spirin, 1963), namely the 27 S and the 18 S fractions, and possible messenger RNA (Spiegelman & Hayashi, 1963; Darnell et ah 1963), namely the 8S
fraction, were present initially in the isolate, but the instability of isolated RNA
molecules at room temperature has been shown by Huppert & Pelmont (1962),
Kubinski & Koch (1963) and Amos & Moore (1963). Degradation is initiated
before 130 min and is drastic within 24 h. However, Amos & Moore (1963) and
Amos, Askonas & Soeiro (1964) reported that the biological activity of isolated
RNA is retained over a period of several weeks if stored at - 20° C and thus the
RNA of the present study was either employed immediately following isolation
or after being stored at - 20° C for no more than 7 days. Once exposed in the
culture medium it is probable that the rate of RNA degradation in the present
investigation would have been less than that reported by Huppert & Pelmont
(1962) since the embryos and explants were cultured at 12° C rather than at room
temperature (20 + °C). It would seem, then, that a large concentration of intact
ribosomal and possible messenger RNA molecules would have been present in
the conditioned medium during the early hours of exposure of the competent
amphibian embryos and ectodermal isolates.
It is probable that any induction of the Xenopus tissue, under the present test
conditions, would have had to occur during the first hour(s) of exposure, if at all,
since some time later, and certainly after 24 h, there would have been a solution
of nucleotides and nucleosides rather than intact RNA molecules. But it is precisely during this period when any inductive activity would have occurred, since
Yamada (1962) has shown that a 55 % inductive response was achieved in
amphibian ectoderm exposed for 30 min to a protein medium and that this
could be increased to a 94 % response with an exposure of 180 min. In the event,
no induction was evidenced but there was observed an enhancement of development (namely an increase in epithelial cell number and/or size) in the RNAconditioned medium. This effect appears to be similar to results described by
Ambellan (1955, 1958) and Ambellan & Webster (1962) in which solutions of
various nucleotides did not induce, but did accelerate, the formation of neural
tubes in amphibian embryos.
In the test series using commercial-liver RNA it was initially considered that
while the surface coat (particularly in Ambystoma gracile) would probably
function to prevent most, if not all, RNA uptake by the superficial cells, sufficient uptake would take place in the various invaginated epithelia (after their
Isolated RNA and histogenesis
11
invagination) to demonstrate an effect if one were to occur. The obvious enhancement of anterior neural histogenesis observed in both T. torosa and
A. gracile would probably have been the result of tissue exposure, immediately
following neurulation, to both intact RNA and nucleotides. Butros (1965) noted
an enhancement of neural fiber histogenesis (which was not seen in the present
work) when chick-heart RNA was applied to explanted segments of chick
blastoderm, which he attributed to the versatility of the responding cells in using
the foreign RNA for their own purposes (i.e. the synthesis of neuron*brils). He
had previously called attention to epidermal hyperplasia which resulted when
chick tissues were exposed to specific foreign RNA, namely brain, heart and
pancreas (Butros, 1963), and it would seem probable that the suggestion of
Butros (1965), that in these test systems the added RNA is depolymerized and
the nucleotides then used in the synthesis of homologous RNA, best conforms to
the reported observations. The suggestion has the added merit of emphasizing
that the resulting histogenesis in the experimental tissue is that which the
assimilating tissue is already competent to express.
Finally, it must be noted that Amos & Moore (1963) and Amos et al. (1964)
have suggested that the absence of any specific inductive effect with isolated
RNA in experimental procedures of this type may be due to a failure to obtain a
biologically 'active' preparation.
SUMMARY
1. Ribonucleic acid (RNA) was isolated from calf spleen tissue by the new
Kirby procedure followed by three phenol washings to reduce the peptideprotein contamination. Ultraviolet absorption spectra'and colorimetric analyses
indicated the reduction or absence of protein, peptide, DNA and, after methoxyethanol purification, polysaccharide from the isolated RNA.
2. Ultracentrifuge sedimentation analysis showed three components in the
undegraded RNA isolate: a 27S fraction, an 18S fraction and a 7-8S fraction.
This ribosomal-messenger RNA was added to non-nutrient medium for
induction testing.
3. Neither embryos nor competent gastrula ectoderm of Xenopus laevis
demonstrated, in a limited series, any tissue-specific inductive response, but both
experimental systems in the RNA-conditioned medium did demonstrate enhanced epithelial development as compared with the control systems.
4. Postgastrula stages of Taricha torosa and Ambystoma gracile exposed
during development to reagent-grade beef liver s-RNA demonstrated hyperplasia in the dorsal mesencephalon-rhombencephalon and the anterior neural
crest mesenchyme population along with an enhancement of auditory and
posterior entodermal epithelial development.
12
C. V. FINNEGAN & W. P. BIGGIN
RESUME
Etude de Vinfluence d'ARN isole de la rate du veau sur Vhistogenese des
Amphibiens
1. On a isole de l'acide ribonucleique a partir de tissu splenique de veau a
l'aide du nouveau procede de Kirby suivi de trois lavages au phenol pour
reduire la contamination par les peptides et les proteines. Les spectres d'absorption dans l'ultraviolet et les analyses colorimetriques ont indique la diminution
ou l'absence de proteines, de peptides, d'ADN et, apres purification au methoxyethanol, de polysaccharides, dans l'ARN isole.
2. L'analyse par sedimentation et ultracentrifugation a decele trois composants dans l'ARN isole non degrade: une fraction 27 S, une fraction 18 S et une
fraction 7-8 S. Ces ARN ribosomique et messager ont ete ajoutes a un milieu
non nutritif pour des essais d'induction.
3. Dans une serie limitee, ni les embryons ni l'ectoderme competent de
gastrula de Xenopus laevis n'ont manifeste de reponse inductive specifique du
tissu, mais les deux systemes experimentaux, dans le milieu conditionne par
l'ARN, ont presente un developpement epithelial accru par rapport aux
systemes temoins.
4. Des stades post-gastruleens de Taricha torosa et d'Ambystoma gracile,
exposes au cours de leur developpement a de l'ARN-S de foie de bceuf, ont
presente une hyperplasie du mesencephale et du rhombencephale dorsaux, et de
la population mesenchymateuse de la crete neurale anterieure, en meme temps
qu'un accroissement du developpement des epitheliums auditif et entodermique
posterieur.
It is a distinct pleasure to acknowledge the assistance, both in discussion and the provision
of facilities, of Dr M. E. Reichmann of the Agricultural Research Station of the Government
of Canada, Vancouver, B.C. The assistance of Mrs Maureen Douglas in preparing the illustrations is gratefully acknowledged. This research was supported by grants from the National
Research Council of Canada.
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{Manuscript received 20 February 1965, revised 20 July 1965)