J. Cell Set. 13, 651-66! (1973) 651

Printed in Great Britain

VITAL MARKING OF SINGLE CELLS IN

DEVELOPING TISSUES: INDIA INK

INJECTION TO TRACE TISSUE

MOVEMENTS IN HYDRA

R. D. CAMPBELL'Department of Developmental and Cell BiologyUniversity of California, Irvine, California, U.S.A. 92664andMax-Planck-Institut fur VirusforschungMolektddrbiologische Abteilung Tubingen, Germany

SUMMARY

A new vital marking procedure was devised, studied, and applied to the phenomenon ofhydra column tissue movements. The method involves injecting colloidal carbon (India ink)into the ectodermal epithelium of hydra. The carbon is taken up specifically by epithelialcells and packed into lysosome residual bodies. Each marked cell is then identifiable by itsaggregated carbon particle, which appears as a black dot; this is passed to a daughter cellduring cell division. Using this method to stain vitally the ectoderm of the green hydra,Chlorohydra viridissima, whose endodermal cells were marked by symbiotic algae, the relativemovements of ectoderm and endoderm along the body column were analysed. Ectoderm andendoderm move in different directions or at different rates in the top quarter of the gastriccolumn, but move together in the lower part of the column. This indicates that some epithelialcell movements involve cells migrating relative to the mesoglea; other movements involvetranslocation of the entire body wall, including ectoderm, mesoglea and endoderm.

INTRODUCTION

Vital marking of cells in developing organisms is one of the major techniques ofexperimental and descriptive embryologists, and a considerable number of methodshave been devised. The most common techniques as applied to tissue movementsin hydra involve local application of vital dyes, such as methylene blue and neutralred. Serious disadvantages of such staining methods include the progressive loss ofsharpness and boundary locations of the marks due to dye diffusion, and the inabilityto stain single cells. Another useful vital marker in hydra is the symbiotic algalpopulation inside the digestive cells (Browne, 1909); when portions of a green hydraare grafted to algal-less tissue of a bleached hydra strain, the graft junction may befollowed for a number of days before algae spread into adjacent tissue. However,these and other vital staining methods in hydra (see Kanaev, 1952) stain mainlyendodermal cells; the ectoderm is very transparent and binds dyes only transiently.

This report deals with a method for marking hydra cells which gets around these# Address for correspondence.

652 R. D. Campbell

difficulties. It involves injecting India ink (a suspension of bacteria-sized carbonparticles) into the tissue where it enters the epithelial cells through phagocytosis andbecomes concentrated in a lysozome. In the ectoderm the ink remains in the cellswithout diffusion, and is passed to daughter cells during mitosis.

Using this method the continual movements of tissues along the body of hydrahave been reexamined. A variety of marking methods indicate that hydra columntissues expand and are thus continually displacing themselves towards the ends ofthe polyp; upwards into the tentacles and downwards towards the budding regionand basal disk (Campbell, 1967a). It has been deduced from radioautographicstudies (Shostak, Patel & Burnett, 1965; Campbell, 19676) that the ectoderm andendoderm move at different rates although the 2 epithelia rest on the same basementmembrane (mesolamella). However, this differential epithelial movement has notbeen observed in live animals because of the limitations in vital marking techniques.This report describes the differential epithelial movement using carbon to labelectoderm vitally, and symbiotic algae to mark endodermal cells.

MATERIALS AND METHODS

Hydra attenuata and Chlorohydra viridissima (green and albino strains) were grown in' M ' solution according to the methods of Lenhoff & Brown (1970). The animals were kept inindividual 65-mm Petri dishes in an incubator at 22 °C.

Pelikan brand India ink was injected into the tissue using a micropipette with a tip diameterof about 5 /mi. This was attached to a mouth pipette. Injections may be done in 2 ways. Toobtain a small spot of ink, the hydra, attached by its base to the dish, is held still using forcepsto grasp a tentacle. Then a micropipette tip is inserted into the ectoderm by slipping the tipthrough the hydra surface almost parallel to the surface, but well above the mesolamella(Fig. 2). The ink is injected slowly. If the micropipette has punctured the mesolamella theink goes into the gastric cavity. Only a very small volume of ink can be injected into theectoderm; excess fluid oozes out around the micropipette shaft or through small ruptures inthe ectodermal surface. To mark a large area of ectoderm, the micropipette is pressedperpendicularly against, so as to indent slightly, the ectodermal surface. Then ink is expelledwith as much force as possible. This results in a large area (up to one quarter of the entirehydra; see example in Fig. 4) being almost instantly labelled with India ink. Apparently anarrow jet of ink has the force to penetrate the ectodermal surface, and from there to spreadinto the intercellular spaces almost instantaneously.

Histological studies were made using io-//m-thick paraffin sections stained with Mallory'sTrichrome (Humason, 1967), i-/im-thickand ultrathin Epon sections. Maceration preparationswere made according to the method of David (1973): hydra were placed in glycerine: aceticacid:water = 1:1:13, agitated 10 min later to separate cells, and fixed by addition of formalinto final concentration of 04 % and OsO4 to 02 %. The resulting suspension was spread ona gelatin-coated microscope slide, allowed to dry overnight, and stained by the Feulgenmethod or in haematoxylin.

For electron microscopy, hydra were fixed for 2 h in hydra growth medium containing1% glutaraldehyde and o-4%OsO4. The hydra were dehydrated, stained 24 h in 1 5 %uranyl acetate in ethanol at 50 CC (Locke, Krishnan & McMahon, 1971) and embedded inepoxy plastic (Spurr, 1969). Other hydra were fixed in Lavdowsky's fixative (Romeis, 1954)and embedded in paraffin for light microscopy on y-fim sections.

To study column growth movements, green or white C. viridissima were marked with largespots of India ink 2 days before grafting. Grafts were made by selecting one green and onewhite animal of similar sizes, one of which was ink labelled, and bisecting them transverselyat the same column level. The cut was made through the ink region of the marked individual.Complementary tissue pieces were threaded together on a hair, floated for 1 h on the culture

Carbon marking of hydra cells 653

medium surface and then removed from the hair. Column positions of the edge of the greenand black markers were measured using an ocular reticle when the hydra fully extended.Forty-two successful grafts were analysed in this study.

RESULTS

India ink marking method

Injected ink marks change in appearance during the first day or two. Initially, inkwithin small marks appears uniform and diffuse (Fig. 3). Large marks appearfeathery at the edges (Fig. 4). In all cases the ink pattern appears reticulate whenviewed at higher magnification (Fig. 5); this appearance is due to the ink beingdistributed in the intercellular spaces, thus outlining the uncoloured interstitialcells and epithelial cell bases. Subsequently, the spot becomes granular as seen underlow magnification (Figs. 7, 8). High-magnification observation on whole hydra(Fig. 6) indicates that the carbon particles become collected in aggregates 2-10 /tinin diameter, within epithelio-muscular cells, in a vesicle just under the ectodermalsurface.

Individual carbon aggregates apparently remain within particular epithelial cells,for some may be recognized for days or weeks. Some aggregates disappear, however;presumably they are ejected by the cell or the cell dies. In this fashion, and due tocoalescence of multiple spots within single cells, spots gradually decrease in numberover periods of days and weeks. Also, the labelled areas become more diffuse withtime due to growth of the tissue.

Observations on histologically prepared marked hydra are also consistent withthis time course of marking. The appearances of tissue 10 s, 10 min, and 2 daysafter marking are shown in Figs. 10-12. The initial site of carbon particles is in theintercellular spaces; this gives the impression that the cells are outlined with ink(Fig. 10). Within a few minutes this appearance is lost, for the carbon is rapidlyremoved from the intercellular spaces. Then during the next several days the carbonbecomes compacted into aggregates in the apical region of the ectoderm epithelialcells. Figs. 13 and 14 show the electron-micrographic appearance of injected ink10 s and 2 days after injection.

When the broad marking method (see Materials and Methods) is used, somecarbon may be forced into and remain in the mesolamella (see Fig. 12). Some alsomay enter the endoderm, and although this is aggregated in the epithelial (digestive)cells it is not retained as long by the animal as are ectodermal spots. When theminute marking method is used, carbon is restricted to the ectoderm. The locationof carbon aggregates in the epithelio-muscular cells is best seen in isolated cellsobtained by maceration (Fig. 15). The vesicle containing the carbon aggregates isretained through cell division and passed to one of the daughter cells (Fig. 15). Theappearance and position of this vesicle is also seen in the electron micrograph ofFig. 14.

Interstitial cells, nematoblasts, nematocytes, and nerve cells never acquire carbon.

42 C E L 13

654 R. D. Campbell

Column tissue movements

Column tissue movements observed in this study were similar to those describedfor hydra generally (Brien & Reniers-Decoen, 1949; Burnett, 1961; Campbell,19676); there is a region in the upper portion of the column where movement isslow or not apparent ('stationary region', Campbell, 19676), from which tissuemoves upwards and downwards. Ectodermal and endodermal marker displacementsof 4 representative grafts are shown as curves in Fig. 1; the shaded area in each caserepresents the degree to which the ectodermal and endodermal markers separated.

Tentacles

20-

so

Buddingzone

Days

Fig. 1. Movement of ectoderm (open circles) and endoderm (closed circles) aftergrafting unmarked Chlorohydra viridissima top portions to ink- and algal-markedlower portions, as illustrated in Figs. 8 and 9. These 4 grafts (A-D) are representativeof the 4 classes of tissue behaviour seen in 42 cases. The axial positions (ordinate)of the ectoderm and endoderm marker boundaries are indicated as a function of timeafter grafting (abscissa). Ectoderm and endoderm marker boundaries always startedat the same axial position at the graft site. In those cases where they separated (A-C),the separation is represented by shading.

A, graft site is about 15% of distance from tentacles to budding region. Bothtissues move distally, with the ectoderm moving faster, leading to marker separation(shading).

B, graft site about 10% of distance from tentacles to budding region. Ectodermmoves distally while endoderm moves proximally.

c, graft site about 30 % of distance between tentacles and budding region. Bothtissues move proximally, but initially the ectoderm moves more tlowly leading tomarker separation.

D, graft site about 50% of distance between tentacles and budding region.Ectoderm and endoderm move proximally at same rate.

In the lower column regions the 2 epithelia are displaced downward at similarrates; this is illustrated by curves c and D. In the upper column regions there israpid separation of markers in the 2 epithelia. This is due to two factors: the stationaryregion for endoderm is higher on the column (about 10-15% °f t n e distance fromthe tentacles to the budding region) than that of the ectoderm (about 25 % of the

Carbon marking of hydra cells 655

distance down the column); and distal (upward) movement of ectoderm appears tobe faster than that of endoderm. Thus in almost all grafts made in the upper quarterof the column the markers separated; in grafts made in the column regions 10-25%below the tentacles, the markers moved in opposite directions. The exact positionof the stationary region is not possible to specify, and is variable from one hydrato the next (compare Fig. 1 A with B).

In all grafts, there was no separation of markers during grafting due to irregularhealing.

DISCUSSION

Carbon labelling by phagocytosis offers a vital method by which individual cellsor tissue regions may be followed for much longer periods than is possible usingvital dyes. The carbon is aggregated in a vesicle whose inert contents are quitepermanent. This vesicle is probably a lysozome, since (1) it is a general property oflysozomes to accumulate inert materials in those cells which do not have a meansof 'emptying' them (de Duve & Wattiaux, 1966); in these cases they are termedresidual bodies; (2) these vesicles in hydra contain acid phosphatase activity and havethe appearance of lysozomes (Lentz, 1966); and (3) extracellular substances in theectoderm, such as injected materials or externally applied vital dyes, are all quicklymoved into these vesicles.

Carbon marking is a classic and important method in embryology (Rudnick, 1944;Spratt, 1946; Weston, 1967), but it has mainly been used by pushing or placinglarger grains of carbon on to or into tissues. A problem which has inevitably raisedquestions with these uses is the possible movement of carbon particles relative tothe cells. Some other investigators, particularly those working with tissue-culturedcells (see Rabinowitz & Sachs, 1968), however, have made successful use of ingestedcolloids to mark vitally cells. The present method of carbon marking is beingprofitably applied to a variety of problems in the cytology and development ofcoelenterates (Moore & Campbell, 1973; Campbell, 1973).

The great rapidity with which epithelial cells take up injected carbon particlesindicates that one function of these cells is to scour continually the meagre inter-cellular spaces in hydra. A few other epithelial tissues have also been found to clearmaterials rapidly from the intercellular spaces (see Wolff & Konrad, 1972), and itmay be that this activity is general for epithelial cells. While this function has neverbefore been recognized in hydra, it may be of extreme importance in physiologicaland developmental regulation. Transfer of nutrients from the digestive layer to theectoderm could depend on the efficient uptake of materials by the ectodermal cells(of glycogen, for example; Gauthier, 1963). Developmentally, if the epithelial cellscan rapidly remove materials from the intercellular spaces, the interstitial cells'responsiveness to environmental parameters may thus be completely controlled bythe surrounding epithelial cells rather than by materials liberated from nerve cellsor interstitial cell derivatives. This may also explain why vital dyes do not seem tostain interstitial cells; the epithelial cells may prevent any foreign substances fromentering the intercellular spaces which surround the interstitial cells.

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656 R. D. Campbell

Differential movement of the 2 layers, whose occurrence was deduced but notvitally demonstrated by Shostak et al. (1965) and Campbell (1967a) is of interestbecause it implies that epithelial cell movement can occur independently of basementmembrane movement. Thus the mesolamella of hydra may act more as a substratumfor, than as a 'glue' between, the 2 cell layers. Hausman & Burnett (1971 and earlierpapers) propose that the mesolamella functions as a patterning substratum, with whichdifferential cell displacement is consistent. However, the fact that in much of thegastric region the 2 epithelia remain stationary relative to one another indicatesthat many of the displacements called 'tissue movements' involve the entire bodywall of hydra, not independent cell or single tissue movements.

Epithelial cell displacements must be correlated with an imbalance betweentissue growth and utilization (Campbell, 19676, 1973) and thus the endoderm andectoderm of C. viridissima apparently have different patterns of formation or utiliza-tion. The low column position of the ectodermal stationary region indicates that eitherthe growth in this epithelium is predominantly in the lower region of the column, orthat the hydranth recruits ectodermal cells faster than endodermal cells from theupper column. In Hydra attenuata there are 2-3 times as many ectodermal epithelialcells as endodermal cells in the tentacles (Bode et al. 1973); since the tentacles arethe primary site of column cell disappearance in the distal regions of hydra (Campbell,1967a), it seems likely that differential cell utilization in tentacle formation isresponsible for differential epithelial movement patterns on the column.

While this report has dealt with carbon granules, a number of different materialshave successfully been injected intraectodermally using the' minute method' describedin Materials and Methods; these materials include other colloids (Thorotrast, ferritin,and gold), oil droplets, latex beads, and cells. Thus the injection method probablyhas broader applications in developmental and histological studies on this simpleanimal.

This work was supported by NIH Research Career Development Award 5-KO4-GM 2595and NSF Research Grant GB29284.

REFERENCES

BODE, H., BERKING, S., DAVID, C. N., GIERER, A., SCHALLER, H. & TRENKNER, E. (1973).

Quantitative analysis of cell types during growth and morphogenesis in hydra. Arch.EntwMech. Org. 171, 269-285.

BRIEN, P. & RENIERS-DECOEN, M. (1949). La croissance, la blastogenese, l'ovogenese chezHydra fusca (Pallas). Bull. biol. Fr. Belg. 83, 293-386.

BROWNE, E. (1909). The production of new hydranths in hydra by the insertion of smallgrafts. J. exp. Zool. 7, 1-23.

BURNETT, A. L. (1961). The growth process in hydra. J. exp. Zool. 146, 21-83.CAMPBELL, R. D. (1967a). Tissue dynamics of steady state growth in Hydra littoralis. II .

Patterns of tissue movement. J. Morph., 121, 19-28.CAMPBELL, R. D. (19676). Tissue dynamics of steady state growth in Hydra littoralis. III .

Behaviour of specific cell types during tissue movements. J. exp. Zool. 164, 379—391.CAMPBELL, R. D. (1973). Cell movements in hydra. Am. Zool. (in Press).DAVID, C. N. (1973). A quantitative method for maceration of hydra tissue. Arch. EntwMech.

Org. 171, 259—268.

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DE DUVE, C. & WATTIAUX, R. (1966). Functions of lysozomes. A. Rev. Physiol. 28, 435-492.GAUTHIER, G. F. (1963). Cytological studies on the gastroderm of hydra. J. exp. Zool. 152,

13-40-HAUSMAN, R. E. & BURNETT, A. L. (1971). The mesoglea of hydra. IV. A qualitative radio-

autographic study of the protein component. J. exp. Zool. 177, 435-446.HUMASON. G. (1967). Animal Tissue Techniques, 2nd edn. San Francisco: Freeman.KANAEV, I. I. (1952). Hydra. Essays on the Biology of the Fresh Water Polyps. (Transl. by

E. T. Burrows & H. M. Lenhoff, 1966; ed. H. M. Lenhoff). Moscow: Soviet Academy ofSciences, Publ. by the Editor, 452 pp.

LENHOFF, H. & BROWN, R. (1970). Mass culture of hydra: an improved method and itsapplication to other aquatic invertebrates. Laboratory Animals 4, 139-154.

LENTZ, T. L. (1966). The Cell Biology of Hydra. Amsterdam: North Holland Publishing.LOCKE, M., KRISHNAN, N. & MCMAHON, J. T. (1971). A routine method for obtaining high

contrast without staining sections. J. Cell Biol. 50, 540-544.MOORE, L. & CAMPBELL, R. D. (1973). Bud initiation in a non-budding strain of hydra: Role

of interstitial cells. J. exp. Zool. (in Press).RABINOWITZ, A. & SACHS, L. (1968). Reversion of properties in cells transformed by polyoma.

Nature, Lond. 220, 1203-1206.ROMEIS, B. (1954). Mikroskopische Technick. Miinchen: Libniz Verlag.RUDNICK, D. (1944). Early history and mechanics of the chick blastoderm. Q. Rev. Biol.

19, 187-212.SHOSTAK, S., PATEL, N. G. & BURNETT, A. L. (1965). The role of mesoglea in mass cell

movement in hydra. Devi Biol. 12, 434-450.SPRATT, N. T. (1946). Formation of the primitive streak in the explanted chick blastoderm

marked with carbon particles. J. exp. Zool. 103, 259—304.SPURR, A. R. (1969). A low-viscosity epoxy resin embedding medium for electron microscopy.

y. Ultrastruct. Res. 26, 31-43.WESTON, JAMES A. (1967). Cell marking. In Methods in Developmental Biology (ed. F. Wilt &

N. Wessells), pp. 723-736. New York: Crowell.WOLFF, K. & KONRAD, K. (1972). Phagocytosis of latex beads by epidermal keratinocytes

in vivo. J. Ultrastruct. Res. 39, 262-280.(Received 24 April 1973)

658 R. D. Campbell

Fig. 2. Injection of small ink spots into ectoderm of Hydra attenuata. The micro-pipette (mp) tip has been, slid along the body within the ectoderm. Small amountsof ink are being injected at intervals as the micropipette is being withdrawn. Onecan apply only a single mark. The hydra is attached to the glass dish by its basaldisk (lower left) and is stretched into a favourable position for injection by forceps(black in upper right corner) grasping the tentacles.Fig. 3. Appearance of spots made in experiment shown in Fig. 2 shortly after injec-tion; the animal has relaxed into an elongated shape.Fig. 4. Injection of a large ink spot into H. attenuata. The micropipette (mp) tip(not visible) is pushed against the surface of the hydra and a 3udden jet of ink isforcefully applied. Ink intercalates through the ectoderm, between the cells, infeatherlike patterns. This photograph was made using flash illumination at theinstant of injection; within a second the area becomes obscured with a black cloudof free ink (just beginning in the centre of the top edge of the ink mark). Thefeather-like pattern of the mark indicates that the ink is intraectodermal.Fig. 5. Appearance of larger ink spot of Fig. 4, a few seconds after injection. Theink outlines the cells which are colourless. The upper part of the photograph is focusedon the ectoderm near the mesoglea.Fig. 6. Same large ink spot after 2 days. The carbon has been aggregated intodiscrete patches, about one in each epithelial cell. These are mainly near theectodermal surface. Magnification as in Fig. 5.Fig. 7. Grafted Chlorohydra viridissima, 2 h old, composed of the bottom half ofa hydra marked 2 days earlier with India ink, and the top half of an unmarked hydra.The position of the ectodermal graft junction is marked by the edge of the ink-marked tissue. The surface illumination largely obscures the green colour of thelower portion.Fig. 8. Thirty-six-hour graft illustrating extensive distal ectodermal movementwith slight proximal endodermal displacement. The grafted animal consisted ofthe top of an albino C. viridissima with the bottom of a green, ink-markedanimal. The ectodermal junction (ect) indicated by the highest black granule, ismore distal than the endodermal junction (end) which is indicated by the edge of dark(green) tissue. The original graft site was slightly above the present endodermaljunction. The focus is on the horizon of the hydra, so the ink marks in the ectodermare seen in optical median section.Fig. 9. 36-h graft illustrating commensurate displacement of ectoderm and endoderm.The original graft was like that of hydra shown in Fig. 7, but slightly lower on thebody column. The edge of ink marking (arrows point to several black ink spots) andgreen tissue are together.

Carbon marking of hydra cells 659

66o R. D. Campbell

Figs. 10-12. Histological cross-sections through gastric region of Hydra attenuatemarked with India ink. The ectoderm is in upper part, and the mesolamella runshorizontally across the centre of each figure. All 3 figures are at the same magnification.

Fig. 10. Hydra fixed 10 s after injection. Ink is in the intercellular spaces.Fig. 11. Two hours after injection. Ink is largely within the epithelial cells, but

distributed throughout the cells.Fig. 12. Two days after injection. Ink carbon is aggregated into compact masses

fairly high in the epithelial cells. Some carbon remains in the mesolamella.Figs. 13, 14. Electron micrographs showing distribution of ink carbon injected intoHydra attenuate ectoderm. The mesolamella is at the bottom of each figure. Scalefor both figures is in Fig. 14.

Fig. 13. Hydra fixed 10 s after injection. Ink carbon, appearing granular, is foundin the intercellular spaces at arrows and elsewhere. In the centre are 2 large interstitialcells; at lower left, the arrow points to space around migrating nematocyte; at right,the arrow points to intercellular spaces between small, young, clustered nematoblasts.

Fig. 14. Two days after injection. Carbon particles are amassed in membrane-bound vesicles in the distal part of the epithelial cells.Fig. 15. Retention of carbon marks during mitosis of epithelial cells in Hydraattenuata. These cells, in macerated preparations of 3-day-old ink-marked hydra,show the persistence of the carbon aggregates (arrows) during interphase (inter),prophase (pro), metaphase (meta), anaphase (ana) and early post-telophase (telo).The anaphase cell has 2 carbon aggregates; this probably represented a cell whichbegan with 2 aggregates; usually only one daughter cell is marked (e.g. telo).The cells were prepared according to the technique of David (1973) and then stainedby the Feulgen method. The scale on ana, whose magnification is 1-4 times greaterthan that of the other cells, would be equivalent to approximately lofim in the othermicrographs of this figure.

Carbon marking of hydra cells 661

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