VITAL MARKING OF SINGLE CELLS IN DEVELOPING TISSUES: INDIA INK INJECTION TO TRACE TISSUE MOVEMENTS IN HYDRA

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1 J. Cell Set. 13, ! (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 Biology University of California, Irvine, California, U.S.A and Max-Planck-Institut fur Virusforschung Molektddrbiologische Abteilung Tubingen, Germany SUMMARY A new vital marking procedure was devised, studied, and applied to the phenomenon of hydra column tissue movements. The method involves injecting colloidal carbon (India ink) into the ectodermal epithelium of hydra. The carbon is taken up specifically by epithelial cells and packed into lysosome residual bodies. Each marked cell is then identifiable by its aggregated carbon particle, which appears as a black dot; this is passed to a daughter cell during 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 relative movements of ectoderm and endoderm along the body column were analysed. Ectoderm and endoderm move in different directions or at different rates in the top quarter of the gastric column, but move together in the lower part of the column. This indicates that some epithelial cell movements involve cells migrating relative to the mesoglea; other movements involve translocation of the entire body wall, including ectoderm, mesoglea and endoderm. INTRODUCTION Vital marking of cells in developing organisms is one of the major techniques of experimental and descriptive embryologists, and a considerable number of methods have been devised. The most common techniques as applied to tissue movements in hydra involve local application of vital dyes, such as methylene blue and neutral red. Serious disadvantages of such staining methods include the progressive loss of sharpness and boundary locations of the marks due to dye diffusion, and the inability to stain single cells. Another useful vital marker in hydra is the symbiotic algal population inside the digestive cells (Browne, 1909); when portions of a green hydra are grafted to algal-less tissue of a bleached hydra strain, the graft junction may be followed for a number of days before algae spread into adjacent tissue. However, these and other vital staining methods in hydra (see Kanaev, 1952) stain mainly endodermal 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.

2 652 R. D. Campbell difficulties. It involves injecting India ink (a suspension of bacteria-sized carbon particles) into the tissue where it enters the epithelial cells through phagocytosis and becomes concentrated in a lysozome. In the ectoderm the ink remains in the cells without diffusion, and is passed to daughter cells during mitosis. Using this method the continual movements of tissues along the body of hydra have been reexamined. A variety of marking methods indicate that hydra column tissues expand and are thus continually displacing themselves towards the ends of the polyp; upwards into the tentacles and downwards towards the budding region and basal disk (Campbell, 1967a). It has been deduced from radioautographic studies (Shostak, Patel & Burnett, 1965; Campbell, 19676) that the ectoderm and endoderm move at different rates although the 2 epithelia rest on the same basement membrane (mesolamella). However, this differential epithelial movement has not been observed in live animals because of the limitations in vital marking techniques. This report describes the differential epithelial movement using carbon to label ectoderm 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 in individual 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 diameter of about 5 /mi. This was attached to a mouth pipette. Injections may be done in 2 ways. To obtain a small spot of ink, the hydra, attached by its base to the dish, is held still using forceps to grasp a tentacle. Then a micropipette tip is inserted into the ectoderm by slipping the tip through 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 the ink goes into the gastric cavity. Only a very small volume of ink can be injected into the ectoderm; excess fluid oozes out around the micropipette shaft or through small ruptures in the ectodermal surface. To mark a large area of ectoderm, the micropipette is pressed perpendicularly against, so as to indent slightly, the ectodermal surface. Then ink is expelled with as much force as possible. This results in a large area (up to one quarter of the entire hydra; see example in Fig. 4) being almost instantly labelled with India ink. Apparently a narrow jet of ink has the force to penetrate the ectodermal surface, and from there to spread into the intercellular spaces almost instantaneously. Histological studies were made using io-//m-thick paraffin sections stained with Mallory's Trichrome (Humason, 1967), i-/im-thickand ultrathin Epon sections. Maceration preparations were made according to the method of David (1973): hydra were placed in glycerine: acetic acid:water = 1:1:13, agitated 10 min later to separate cells, and fixed by addition of formalin to final concentration of 04 % and OsO 4 to 02 %. The resulting suspension was spread on a gelatin-coated microscope slide, allowed to dry overnight, and stained by the Feulgen method or in haematoxylin. For electron microscopy, hydra were fixed for 2 h in hydra growth medium containing 1% glutaraldehyde and o-4%oso 4. The hydra were dehydrated, stained 24 h in 15% uranyl acetate in ethanol at 50 C C (Locke, Krishnan & McMahon, 1971) and embedded in epoxy 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 large spots of India ink 2 days before grafting. Grafts were made by selecting one green and one white animal of similar sizes, one of which was ink labelled, and bisecting them transversely at 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

3 Carbon marking of hydra cells 653 medium surface and then removed from the hair. Column positions of the edge of the green and 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, ink within small marks appears uniform and diffuse (Fig. 3). Large marks appear feathery at the edges (Fig. 4). In all cases the ink pattern appears reticulate when viewed at higher magnification (Fig. 5); this appearance is due to the ink being distributed in the intercellular spaces, thus outlining the uncoloured interstitial cells and epithelial cell bases. Subsequently, the spot becomes granular as seen under low magnification (Figs. 7, 8). High-magnification observation on whole hydra (Fig. 6) indicates that the carbon particles become collected in aggregates 2-10 /tin in diameter, within epithelio-muscular cells, in a vesicle just under the ectodermal surface. 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 to coalescence of multiple spots within single cells, spots gradually decrease in number over periods of days and weeks. Also, the labelled areas become more diffuse with time due to growth of the tissue. Observations on histologically prepared marked hydra are also consistent with this time course of marking. The appearances of tissue 10 s, 10 min, and 2 days after marking are shown in Figs The initial site of carbon particles is in the intercellular 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 rapidly removed from the intercellular spaces. Then during the next several days the carbon becomes compacted into aggregates in the apical region of the ectoderm epithelial cells. Figs. 13 and 14 show the electron-micrographic appearance of injected ink 10 s and 2 days after injection. When the broad marking method (see Materials and Methods) is used, some carbon may be forced into and remain in the mesolamella (see Fig. 12). Some also may 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 the minute marking method is used, carbon is restricted to the ectoderm. The location of carbon aggregates in the epithelio-muscular cells is best seen in isolated cells obtained by maceration (Fig. 15). The vesicle containing the carbon aggregates is retained through cell division and passed to one of the daughter cells (Fig. 15). The appearance and position of this vesicle is also seen in the electron micrograph of Fig. 14. Interstitial cells, nematoblasts, nematocytes, and nerve cells never acquire carbon. 42 CEL 13

4 654 R. D. Campbell Column tissue movements Column tissue movements observed in this study were similar to those described for hydra generally (Brien & Reniers-Decoen, 1949; Burnett, 1961; Campbell, 19676); there is a region in the upper portion of the column where movement is slow or not apparent ('stationary region', Campbell, 19676), from which tissue moves upwards and downwards. Ectodermal and endodermal marker displacements of 4 representative grafts are shown as curves in Fig. 1; the shaded area in each case represents the degree to which the ectodermal and endodermal markers separated. Tentacles 20- so Budding zone Fig. 1. Movement of ectoderm (open circles) and endoderm (closed circles) after grafting unmarked Chlorohydra viridissima top portions to ink- and algal-marked lower portions, as illustrated in Figs. 8 and 9. These 4 grafts (A-D) are representative of 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 time after grafting (abscissa). Ectoderm and endoderm marker boundaries always started at 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. Both tissues move distally, with the ectoderm moving faster, leading to marker separation (shading). B, graft site about 10% of distance from tentacles to budding region. Ectoderm moves distally while endoderm moves proximally. c, graft site about 30 % of distance between tentacles and budding region. Both tissues move proximally, but initially the ectoderm moves more tlowly leading to marker separation. D, graft site about 50% of distance between tentacles and budding region. Ectoderm and endoderm move proximally at same rate. Days In the lower column regions the 2 epithelia are displaced downward at similar rates; this is illustrated by curves c and D. In the upper column regions there is rapid separation of markers in the 2 epithelia. This is due to two factors: the stationary region for endoderm is higher on the column (about 10-15% f tne distance from the tentacles to the budding region) than that of the ectoderm (about 25 % of the

5 Carbon marking of hydra cells 655 distance down the column); and distal (upward) movement of ectoderm appears to be faster than that of endoderm. Thus in almost all grafts made in the upper quarter of 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 position of the stationary region is not possible to specify, and is variable from one hydra to the next (compare Fig. 1 A with B). In all grafts, there was no separation of markers during grafting due to irregular healing. DISCUSSION Carbon labelling by phagocytosis offers a vital method by which individual cells or tissue regions may be followed for much longer periods than is possible using vital dyes. The carbon is aggregated in a vesicle whose inert contents are quite permanent. This vesicle is probably a lysozome, since (1) it is a general property of lysozomes to accumulate inert materials in those cells which do not have a means of 'emptying' them (de Duve & Wattiaux, 1966); in these cases they are termed residual bodies; (2) these vesicles in hydra contain acid phosphatase activity and have the appearance of lysozomes (Lentz, 1966); and (3) extracellular substances in the ectoderm, such as injected materials or externally applied vital dyes, are all quickly moved 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 placing larger grains of carbon on to or into tissues. A problem which has inevitably raised questions with these uses is the possible movement of carbon particles relative to the cells. Some other investigators, particularly those working with tissue-cultured cells (see Rabinowitz & Sachs, 1968), however, have made successful use of ingested colloids to mark vitally cells. The present method of carbon marking is being profitably applied to a variety of problems in the cytology and development of coelenterates (Moore & Campbell, 1973; Campbell, 1973). The great rapidity with which epithelial cells take up injected carbon particles indicates that one function of these cells is to scour continually the meagre intercellular spaces in hydra. A few other epithelial tissues have also been found to clear materials rapidly from the intercellular spaces (see Wolff & Konrad, 1972), and it may be that this activity is general for epithelial cells. While this function has never before been recognized in hydra, it may be of extreme importance in physiological and developmental regulation. Transfer of nutrients from the digestive layer to the ectoderm could depend on the efficient uptake of materials by the ectodermal cells (of glycogen, for example; Gauthier, 1963). Developmentally, if the epithelial cells can rapidly remove materials from the intercellular spaces, the interstitial cells' responsiveness to environmental parameters may thus be completely controlled by the surrounding epithelial cells rather than by materials liberated from nerve cells or interstitial cell derivatives. This may also explain why vital dyes do not seem to stain interstitial cells; the epithelial cells may prevent any foreign substances from entering the intercellular spaces which surround the interstitial cells. 42-2

6 656 R. D. Campbell Differential movement of the 2 layers, whose occurrence was deduced but not vitally demonstrated by Shostak et al. (1965) and Campbell (1967a) is of interest because it implies that epithelial cell movement can occur independently of basement membrane movement. Thus the mesolamella of hydra may act more as a substratum for, than as a 'glue' between, the 2 cell layers. Hausman & Burnett (1971 and earlier papers) propose that the mesolamella functions as a patterning substratum, with which differential cell displacement is consistent. However, the fact that in much of the gastric region the 2 epithelia remain stationary relative to one another indicates that many of the displacements called 'tissue movements' involve the entire body wall of hydra, not independent cell or single tissue movements. Epithelial cell displacements must be correlated with an imbalance between tissue growth and utilization (Campbell, 19676, 1973) and thus the endoderm and ectoderm of C. viridissima apparently have different patterns of formation or utilization. The low column position of the ectodermal stationary region indicates that either the growth in this epithelium is predominantly in the lower region of the column, or that the hydranth recruits ectodermal cells faster than endodermal cells from the upper column. In Hydra attenuata there are 2-3 times as many ectodermal epithelial cells as endodermal cells in the tentacles (Bode et al. 1973); since the tentacles are the primary site of column cell disappearance in the distal regions of hydra (Campbell, 1967a), it seems likely that differential cell utilization in tentacle formation is responsible for differential epithelial movement patterns on the column. While this report has dealt with carbon granules, a number of different materials have successfully been injected intraectodermally using the' minute method' described in Materials and Methods; these materials include other colloids (Thorotrast, ferritin, and gold), oil droplets, latex beads, and cells. Thus the injection method probably has broader applications in developmental and histological studies on this simple animal. This work was supported by NIH Research Career Development Award 5-KO4-GM 2595 and NSF Research Grant GB 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, BRIEN, P. & RENIERS-DECOEN, M. (1949). La croissance, la blastogenese, l'ovogenese chez Hydra fusca (Pallas). Bull. biol. Fr. Belg. 83, BROWNE, E. (1909). The production of new hydranths in hydra by the insertion of small grafts. J. exp. Zool. 7, BURNETT, A. L. (1961). The growth process in hydra. J. exp. Zool. 146, CAMPBELL, R. D. (1967a). Tissue dynamics of steady state growth in Hydra littoralis. II. Patterns of tissue movement. J. Morph., 121, 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, 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,

7 Carbon marking of hydra cells 657 DE DUVE, C. & WATTIAUX, R. (1966). Functions of lysozomes. A. Rev. Physiol. 28, GAUTHIER, G. F. (1963). Cytological studies on the gastroderm of hydra. J. exp. Zool. 152, HAUSMAN, R. E. & BURNETT, A. L. (1971). The mesoglea of hydra. IV. A qualitative radioautographic study of the protein component. J. exp. Zool. 177, 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 of Sciences, Publ. by the Editor, 452 pp. LENHOFF, H. & BROWN, R. (1970). Mass culture of hydra: an improved method and its application to other aquatic invertebrates. Laboratory Animals 4, 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, 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, ROMEIS, B. (1954). Mikroskopische Technick. Miinchen: Libniz Verlag. RUDNICK, D. (1944). Early history and mechanics of the chick blastoderm. Q. Rev. Biol. 19, SHOSTAK, S., PATEL, N. G. & BURNETT, A. L. (1965). The role of mesoglea in mass cell movement in hydra. Devi Biol. 12, SPRATT, N. T. (1946). Formation of the primitive streak in the explanted chick blastoderm marked with carbon particles. J. exp. Zool. 103, SPURR, A. R. (1969). A low-viscosity epoxy resin embedding medium for electron microscopy. y. Ultrastruct. Res. 26, WESTON, JAMES A. (1967). Cell marking. In Methods in Developmental Biology (ed. F. Wilt & N. Wessells), pp New York: Crowell. WOLFF, K. & KONRAD, K. (1972). Phagocytosis of latex beads by epidermal keratinocytes in vivo. J. Ultrastruct. Res. 39, (Received 24 April 1973)

8 658 R. D. Campbell Fig. 2. Injection of small ink spots into ectoderm of Hydra attenuata. The micropipette (mp) tip has been, slid along the body within the ectoderm. Small amounts of ink are being injected at intervals as the micropipette is being withdrawn. One can apply only a single mark. The hydra is attached to the glass dish by its basal disk (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 injection; 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 is forcefully applied. Ink intercalates through the ectoderm, between the cells, in featherlike patterns. This photograph was made using flash illumination at the instant of injection; within a second the area becomes obscured with a black cloud of free ink (just beginning in the centre of the top edge of the ink mark). The feather-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. The ink outlines the cells which are colourless. The upper part of the photograph is focused on the ectoderm near the mesoglea. Fig. 6. Same large ink spot after 2 days. The carbon has been aggregated into discrete patches, about one in each epithelial cell. These are mainly near the ectodermal surface. Magnification as in Fig. 5. Fig. 7. Grafted Chlorohydra viridissima, 2 h old, composed of the bottom half of a 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 inkmarked tissue. The surface illumination largely obscures the green colour of the lower portion. Fig. 8. Thirty-six-hour graft illustrating extensive distal ectodermal movement with slight proximal endodermal displacement. The grafted animal consisted of the top of an albino C. viridissima with the bottom of a green, ink-marked animal. The ectodermal junction (ect) indicated by the highest black granule, is more 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 endodermal junction. The focus is on the horizon of the hydra, so the ink marks in the ectoderm are seen in optical median section. Fig 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 the body column. The edge of ink marking (arrows point to several black ink spots) and green tissue are together.

9 Carbon marking of hydra cells 659

10 66o R. D. Campbell Figs Histological cross-sections through gastric region of Hydra attenuate marked with India ink. The ectoderm is in upper part, and the mesolamella runs horizontally across the centre of eachfigure.all 3figuresare 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 into Hydra attenuate ectoderm. The mesolamella is at the bottom of each figure. Scale for both figures is in Fig. 14. Fig. 13. Hydra fixed 10 s after injection. Ink carbon, appearing granular, is found in the intercellular spaces at arrows and elsewhere. In the centre are 2 large interstitial cells; 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 membranebound vesicles in the distal part of the epithelial cells. Fig. 15. Retention of carbon marks during mitosis of epithelial cells in Hydra attenuata. 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 which began 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 stained by the Feulgen method. The scale on ana, whose magnification is 1-4 times greater than that of the other cells, would be equivalent to approximately lofim in the other micrographs of this figure.

11 Carbon marking of hydra cells //m f inter mcta ana 15

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