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 RNAon amphibian histogenesis

By CYRIL V. FINNEGAN1 & WILLIAM P. BIGGIN2

Department of Zoology, University of British Columbia

INTRODUCTION

In the cell-free inductor demonstrated by Niu & Twitty (1953) the effectivematerial was later characterized by Niu (1956) as ribonucleoprotein with thesuggestion that the ribonucleic acid (RNA) fraction was the active component.Subsequently Niu (1958a, b) reported tissue-specific (thymus-like) induction inamphibian material exposed to calf thymus RNA, though Yamada (1961), usingidentical 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 toensure removal of contaminants from the isolate prior to testing for inductivecapacity with competent amphibian ectoderm and embryos. Sedimentationanalysis with the ultracentrifuge indicated that the isolated spleen ribosomalRNA was undegraded when introduced into the culture medium. In a limitedseries of test cases neither embryos nor ectodermal isolates of Xenopus laevisdemonstrated any tissue-specific inductive response (i.e. erythropoiesis), thougha general enhancement of epithelial development was obvious. Further informa-tion on such enhancement of epithelial histogenesis was obtained when post-gastrula stages of Taricha torosa and Ambystoma gracile were cultured in acommercial 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 spleentissue was sliced into pieces approximately 1 x 2 cm and then homogenized, in2-5 ml. of 001 5M naphthalene-1,5-disulfonate and 2-5 ml. of 88-90 % phenolper gram weight of tissue, for 2 minutes in a Waring Blendor at room tempera-

1 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 J E E M 15

2 C. V. FINNEGAN & W. P. BIGGIN

ture (Kirby, 1956, 1962). The cloudy supernatant was carefully sucked off andsubjected to a total of three extractions with phenol (Biggin, 1964). Glasswareand equipment were washed with lN-NaOH and with versene to remove RNaseand heavy metal contaminants. The purity of the isolate was indicated with theBiuret 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 & David-son, 1955) to estimate RNA. Following extraction of the isolate with 2-methoxyethanol, no polysaccharide contamination was demonstrable eitherwith the anthrone reagent or by chromatography.

Spectrophotometric analysis was made of the isolates in 0-lM-NaCl solutionwith the Unicam SP500, and from these data the ratio of absorption values for260/230 mfi and for 260/280 m/* were calculated to indicate, according toMorton (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 conduc-ted with a Spinco model E analytical ultracentrifuge, employing Schleiren opticswith photographs taken at 4 min intervals at 44,770 r.p.m. The sedimentationcoefficients of the isolated RNA were determined according to Markham (1960,1962).

The method of Brown & Littna (1964) was employed to obtain eggs of Xenopuslaevis which were removed for use at late blastula to early gastrula stages (stages9-9 + , 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 observa-tions. 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 immediatelybefore use to minimize possible RNA degradation effects. All embryos (sixteen)and a few randomly selected control and experimental explants (four) were fixedin Carnoy's acetic-alcohol, paraffin-embedded, sectioned at 5-8/tm and stainedwith haematoxylin-eosin or Chromotrope 2R for histological examination.

Late gastrula of Taricha torosa (stage 13), from California, and Ambystomagracile (stage 12), obtained locally, were freed from the egg jelly and (themajority) from vitelline membranes before being placed in a medium producedby adding Reagent Grade Beef liver RNA (Nutritional Biochemical Company)at a concentration of 25 mg/ml. to Niu-Twitty solution. Ten animals of eachspecies 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 whichthey were fixed in Carnoy's and stained with the modified Mallory's (Liisberg,1962).

The spectrophotometric data for the commercial-liver s-RNA were obtainedas were the calf spleen RNA data and are presented with the latter in Table 1 andFig. 1. In the sedimentation analysis with the Spinco model E analytical ultra-

Isolated RNA and histogenesis 3centrifuge, UV optics were used and photographs were taken at 4 min intervalsafter the speed of 55,770 r.p.m. was attained. The commercial liver RNA, dis-solved in 0-lM-NaCl, was observed in the ultracentrifuge for 20 min.

RESULTS

When calf spleen RNA isolated with a single phenol extraction was examinedspectrophotometrically (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 contami-nation of the isolate (see Table 1). On the other hand, after three phenol extrac-tions, these same ratios were 2-23 and 2-20, respectively, indicating a moreeffective 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

Wave-length (m/*)300 320

Fig. 1. The ultraviolet absorption spectra of isolated calf-spleen RNA, after one andafter three phenol extractions, and of reagent-grade beef-liver RNA. # — # , Onephenol 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 at265 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 spleenisolation (see Table 1).

A spectrophotometrically measurable positive response was obtained with thediphenylamine test for DNA with the single phenol extract which was not detect-able in the isolate after three phenol extractions. It is possible that the smallquantity of DNA observed in the first case is the result of the nuclei demon-strated by Georgiev, Mantirva & Zbarsky (1960) to be present in the intermediatelayer after centrifugation.

Table 1. Spectrophotometric analysis of isolated calf-spleen RNA andreagent-grade liver RNA

One phenol extractionThree phenol extractionsReagent-grade liver RNA

Optical

230

0-6900-5500-266

densities

260

1-2401-2300-588

(m/i)

280

0-6800-5600-421

Ratios

260/230

1-802-232-20

260/280

1-822-201-82

Polysaccharide contamination, which could be demonstrated chromato-graphically and with the anthrone reagent even after three phenol extractions,and previously shown by Kirby (1956,1962) and by Ralph & Bellamy (1964), waseffectively removed by methoxyethanol. Thus, the isolate from calf spleen con-tained RNA in the apparent absence of peptide, protein, DNA and carbo-hydrate. However, these data gave no clue as to the physical condition of theisolated RNA molecule and Laskov, Margoliash, Littauer & Eisenberg (1959)and Ralph*& Bellamy (1964) have reported the RNA molecule to be degradedwhen isolated with the original Kirby (1956) method.

Information on the size and condition of the isolated RNA molecules wasobtained with the analytical ultracentrifuge and the resulting sedimentationpatterns are shown in Figs. 2 and 3. Three definite molecular species of RNAwere present in the calf-spleen isolate, whose sedimentation coefficients (inSvedberg units) were calculated to be 27 S, 18 S and 7-8 S (S2Olw)> and with thefastest moving component, the 27 S fraction, being present in the highest con-centration. These results indicate that the isolated molecules were not degradedby the procedure (Darnell, Penman, Scherrer & Becker, 1963; Ralph & Bellamy,1964) and it is this RNA which was used in the induction experiments withXenopus tissues. With the commercial liver RNA in the ultracentrifuge there wasno apparent movement of the molecules, which may be taken to indicate thatthese s-RNA molecules are quite small; an explanation consistent with presentknowledge 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

6-1

60

5-9

8S

1 2 3 4 5 6

Exposure

Fig. 2. Representation of the data obtained for the isolated calf-spleen RNA withthe analytical ultracentrifuge. The slope of the curves is a measure of the sedimenta-tion coefficients.

yyyyyyyyyyyy

y

7-8 Si

M

I 18 S

yyyyyyyyyy

yy

Fig. 3. A drawing made from a Schlieren photograph of a 0-1 M-NaCl solution ofspleen 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, and27 S.

6 C. V. FINNEGAN & W. P. BIGGIN

and rolled into a ball in approximately 30min in both control and RNA-containing media. Daily examination during the culture period, in which con-trol Xenopus embryos reached swimming larval stages, showed no tissue-specificinduction (i.e. erythropoiesis) or, indeed, any histogenesis in either control orexperimental cases. In preliminary experiments it was found that the controlexplants would not survive much beyond a 5-day culture period whereas theexplants in RNA medium would survive well into the second week. At the sametime, in all experiments, the explants in RNA-medium were less susceptible todisintegration 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 liverRNA medium, which shows the dorsal mesencephalic chaotic hyperplasia. (Tracedfrom a projection.)

Fig. 5. A section of a Taricha torosa (animal no. 7) grown in a commercial liver RNAmedium and showing a hyperplasia in the dorsal preotic rhombencephalon but noincrease in the ventral neural fiber material (white matter). (Traced from a pro-jection.)

Thus, it was apparent that the maintenance and the survival of the explants wereenhanced in the RNA-enriched medium as compared with the control medium.Since the daily observations for evidence of erythropoiesis in the explants showednone, only two of each series, randomly selected from the experimental andcontrol explants, were sectioned for histological examination. Again no erythro-poiesis was seen, but in the similar-sized explants the single layered squamous-cuboidal 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-spleenRNA showed what appeared to be an accelerated histogenesis as compared withthe controls, for when sectioned after 5 days' culture it could be seen that an

Isolated RNA and histogenesis 1increase in epithelial cell number and/or size was present which was non-specificsince it involved such diverse epithelial systems as that of the otic vesicles, theintestine and some glandular epithelia (see also Taricha torosa results below).

In the commercial liver RNA medium the T. torosa embryos all developedposterior dorsal head lesions and excessive cranial flexion during the cultureperiod. It was apparent in the sectioned material that a very definite hyperplasiahad occurred in the mesencephalon roof, i.e. the tectum (see Fig. 4), whichcontinued posteriorly in the rhombencephalon (see Figs. 5, 6). This neuralhyperplasia was rather unorganized in the mesencephalon and anteriorrhombencephalon but became less chaotic posteriorly in the hind brain (seeFig. 7). Although the magnitude of the chaotic histogenesis varied it waspresent in all experimental animals (see Fig. 8).

Table 2. Auditory vesicle size in Taricha torosa embryos grown incommercial liver-RNA medium

ControlfExp. 1

2345

LengthGO210259287245231217

Cross-sectionaldiameters*

< •

21-324-333-825023-823-8

K

273043-827-528-830

No. of cellsper section*

rMin.

848595959196

A

Max.

12488

11811610598

* The vesicle diameters and the number of nuclei per section were measured at severalidentical anterior-posterior levels in all animals, namely the endolymphatic duct, the auditoryganglion and the largest, or a larger, section. The diameter values for this table are those of theendolymphatic duct level.

f A single control animal, which approximated the average control length, was chosen forthis comparison since all control measurements were less than the experimentals.

Auditory vesicles of these T. torosa embryos were measured as to totalanterior-posterior length, as to both dorsal-ventral and medial-lateral widthand as to cell population (number of nuclei per section at various levels along theanterior-posterior axis). An example of the data obtained is given in Table 2.All experimental auditory vesicles were larger than the controls in length andwidth 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 rhombence-phalon indicated an increase in this population which is probably of neural crestorigin, 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 dorsalsomite tips and the increase in size of the basal portion of the dorsal fin. Thishyperplasia did not continue into the posterior trunk though the base of the finappeared to remain large. In the posterior trunk the cloacal endodermal epi-thelium was enlarged in thickness, apparently because individual cells werecolumnar and not the normal cuboidal shape, for no increase in cell numbercould be shown in this tissue.

No external evidence of an effect of the commercial-liver RNA was shown bythe 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 liverRNA medium which shows hyperplasia in the dorsal postotic rhombencephalon.Again, no increase in the ventral neural fiber material was demonstrated. (Tracedfrom a projection.)

Fig. 7. (A) A section of a Taricha torosa (animal no. 2) grown in a commercial liverRNA medium and showing the reduced neural hyperplasia found in these animals atthe anterior trunk level of the neural tube. (Traced from a projection.) (B) A sec-tion of a control Taricha torosa animal, grown in a salt solution, at approximatelythe 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 lesspronounced than it had been in Taricha torosa. There was some indication ofchaotic development in the anterior rhombencephalon roof in only two of theexperimental animals. No increase in the auditory vesicles could be demonstra-ted 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 RNAmedium which show less extensive hyperplasia in the dorsal postotic rhombencepha-lon than that seen in Fig. 6. A, Animal no. 7; B, animal no. 9. (Traced from aprojection.)

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 hind-brain-anterior trunk region of the experimental animals resulting in a broadbase to the dorsal fin. This increase in fin size was continued into the posteriortrunk, in the absence of any mesenchyme increase, and in the presence of a simi-lar, though more loose, fiber content: this is taken to indicate a probable increasein ground substance, particularly hyaluronic acid which is so highly concentratedin the dorsal fin (C. V. Finnegan, unpublished). The cloacal endodermal epi-thelium 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 wasdeleted to reduce this degradation (Niu et al. 1961), it would seem that the poly-saccharide contamination might have become significant, particularly since liverand muscle tissues were often the source material used. With the single phenolextraction used it is possible that the protein and/or peptide contamination alsomight have been significant, since Huppert & Pelmont (1962) were able to demon-strate 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 diffi-cult 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 theKirby-isolated RNA. However, after the additional phenol washings such con-tamination would appear to be reduced to a concentration which does not actinductively in the experimental system used in this investigation.

Analysis with the ultracentrifuge demonstrated that undegraded ribosomalRNA (Spirin, 1963), namely the 27 S and the 18 S fractions, and possible mes-senger RNA (Spiegelman & Hayashi, 1963; Darnell et ah 1963), namely the 8Sfraction, were present initially in the isolate, but the instability of isolated RNAmolecules at room temperature has been shown by Huppert & Pelmont (1962),Kubinski & Koch (1963) and Amos & Moore (1963). Degradation is initiatedbefore 130 min and is drastic within 24 h. However, Amos & Moore (1963) andAmos, Askonas & Soeiro (1964) reported that the biological activity of isolatedRNA is retained over a period of several weeks if stored at - 20° C and thus theRNA of the present study was either employed immediately following isolationor after being stored at - 20° C for no more than 7 days. Once exposed in theculture medium it is probable that the rate of RNA degradation in the presentinvestigation would have been less than that reported by Huppert & Pelmont(1962) since the embryos and explants were cultured at 12° C rather than at roomtemperature (20 + °C). It would seem, then, that a large concentration of intactribosomal and possible messenger RNA molecules would have been present inthe conditioned medium during the early hours of exposure of the competentamphibian embryos and ectodermal isolates.

It is probable that any induction of the Xenopus tissue, under the present testconditions, 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 solutionof nucleotides and nucleosides rather than intact RNA molecules. But it is pre-cisely during this period when any inductive activity would have occurred, sinceYamada (1962) has shown that a 55 % inductive response was achieved inamphibian ectoderm exposed for 30 min to a protein medium and that thiscould 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 develop-ment (namely an increase in epithelial cell number and/or size) in the RNA-conditioned medium. This effect appears to be similar to results described byAmbellan (1955, 1958) and Ambellan & Webster (1962) in which solutions ofvarious nucleotides did not induce, but did accelerate, the formation of neuraltubes in amphibian embryos.

In the test series using commercial-liver RNA it was initially considered thatwhile the surface coat (particularly in Ambystoma gracile) would probablyfunction to prevent most, if not all, RNA uptake by the superficial cells, suffi-cient uptake would take place in the various invaginated epithelia (after their

Isolated RNA and histogenesis 11invagination) to demonstrate an effect if one were to occur. The obvious en-hancement of anterior neural histogenesis observed in both T. torosa andA. gracile would probably have been the result of tissue exposure, immediatelyfollowing neurulation, to both intact RNA and nucleotides. Butros (1965) notedan enhancement of neural fiber histogenesis (which was not seen in the presentwork) when chick-heart RNA was applied to explanted segments of chickblastoderm, which he attributed to the versatility of the responding cells in usingthe foreign RNA for their own purposes (i.e. the synthesis of neuron*brils). Hehad previously called attention to epidermal hyperplasia which resulted whenchick tissues were exposed to specific foreign RNA, namely brain, heart andpancreas (Butros, 1963), and it would seem probable that the suggestion ofButros (1965), that in these test systems the added RNA is depolymerized andthe nucleotides then used in the synthesis of homologous RNA, best conforms tothe reported observations. The suggestion has the added merit of emphasizingthat the resulting histogenesis in the experimental tissue is that which theassimilating 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 isolatedRNA in experimental procedures of this type may be due to a failure to obtain abiologically 'active' preparation.

SUMMARY

1. Ribonucleic acid (RNA) was isolated from calf spleen tissue by the newKirby procedure followed by three phenol washings to reduce the peptide-protein contamination. Ultraviolet absorption spectra'and colorimetric analysesindicated the reduction or absence of protein, peptide, DNA and, after methoxy-ethanol purification, polysaccharide from the isolated RNA.

2. Ultracentrifuge sedimentation analysis showed three components in theundegraded RNA isolate: a 27S fraction, an 18S fraction and a 7-8S fraction.This ribosomal-messenger RNA was added to non-nutrient medium forinduction testing.

3. Neither embryos nor competent gastrula ectoderm of Xenopus laevisdemonstrated, in a limited series, any tissue-specific inductive response, but bothexperimental systems in the RNA-conditioned medium did demonstrate en-hanced epithelial development as compared with the control systems.

4. Postgastrula stages of Taricha torosa and Ambystoma gracile exposedduring development to reagent-grade beef liver s-RNA demonstrated hyper-plasia in the dorsal mesencephalon-rhombencephalon and the anterior neuralcrest mesenchyme population along with an enhancement of auditory andposterior entodermal epithelial development.

12 C. V. FINNEGAN & W. P. BIGGIN

RESUME

Etude de Vinfluence d'ARN isole de la rate du veau sur Vhistogenese desAmphibiens

1. On a isole de l'acide ribonucleique a partir de tissu splenique de veau al'aide du nouveau procede de Kirby suivi de trois lavages au phenol pourreduire la contamination par les peptides et les proteines. Les spectres d'absorp-tion dans l'ultraviolet et les analyses colorimetriques ont indique la diminutionou l'absence de proteines, de peptides, d'ADN et, apres purification au methoxy-ethanol, de polysaccharides, dans l'ARN isole.

2. L'analyse par sedimentation et ultracentrifugation a decele trois com-posants dans l'ARN isole non degrade: une fraction 27 S, une fraction 18 S et unefraction 7-8 S. Ces ARN ribosomique et messager ont ete ajoutes a un milieunon nutritif pour des essais d'induction.

3. Dans une serie limitee, ni les embryons ni l'ectoderme competent degastrula de Xenopus laevis n'ont manifeste de reponse inductive specifique dutissu, mais les deux systemes experimentaux, dans le milieu conditionne parl'ARN, ont presente un developpement epithelial accru par rapport auxsystemes 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, ontpresente une hyperplasie du mesencephale et du rhombencephale dorsaux, et dela population mesenchymateuse de la crete neurale anterieure, en meme tempsqu'un accroissement du developpement des epitheliums auditif et entodermiqueposterieur.

It is a distinct pleasure to acknowledge the assistance, both in discussion and the provisionof facilities, of Dr M. E. Reichmann of the Agricultural Research Station of the Governmentof Canada, Vancouver, B.C. The assistance of Mrs Maureen Douglas in preparing the illustra-tions is gratefully acknowledged. This research was supported by grants from the NationalResearch Council of Canada.

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{Manuscript received 20 February 1965, revised 20 July 1965)