Distribution of the head-activating substance in hydra and its localization in membranous particles in nerve cells

Transcription

1 /. Embryol. exp. Morph. Vol. 29, 1, pp , Printed in Great Britain Distribution of the head-activating substance in hydra and its localization in membranous particles in nerve cells By H. SCHALLER 1 AND A. GIERER From the Max-Planck-Institut fur Virusforschung Molekularbiologische Abteilung, Tubingen SUMMARY The low-molecular-weight substance activating head and bud formation in hydra is shown to occur in the animal as a gradient decreasing from the hypostomal to the basal region. The concentration of head-activating substance increases during head regeneration and during bud initiation. Most of the low-molecular-weight head-activating substance is present in the animal in a structure-bound form. More than 90% was sedimentable; 70% was recovered in a highly purified fraction consisting of membranous particles of ~ 1200 A diameter. This implies that in the animal only a minor portion of the total activating activity is freely diffusible, i.e. present in the low-molecular-weight form. The head-activating substance is mainly produced by and/or stored in nerve cells or a subgroup of the nerve cells. Nerve cells were enriched tenfold in a fraction containing most of the head-activating substance in a more than 10 times higher specific activity than in the animal. In addition, it is shown that only the nerve cells are positively correlated with the distribution of head-activating activity both with regard to localization within the animal as to time sequence of appearance during head regeneration and bud formation. INTRODUCTION In the previous paper (Schaller, 1973), it was shown that from crude extracts of hydra a low-molecular-weight substance, probably an oligopeptide, can be isolated which activates head and bud formation in hydra. Preliminary evidence suggested that the head-activating substance is present in the animal in a structure-bound form. The aim of this study was (1) to measure the distribution of the head-activating activity in different regions of the animal and during different states of morphogenesis, and (2) to show to which cell type and to which cell structure the activating substance is bound. 1 Author's address: Max-Planck-Institut fur Virusforschung, Molekularbiologische Abteilung, 74 Tubingen, Spemannstr. 35, Germany.

2 40 H. SCHALLER AND A. GIERER MATERIALS AND METHODS Biological assay. Mass cultured Hydra attenuata was used for all experiments. As described in the previous paper (Schaller, 1973), the activation (A) was assayed by incubating regenerating gastric pieces with or without the extracts to be tested and measuring after 2 days the percentage increase in tentacle number of the treated sample (T) over that of the control (C): A = 100 x(r-c)/c. Serial dilutions were assayed for each extract. The amount of substance necessary to achieve a 5-8 % increase in an assay sample containing 10 ml medium and regenerating pieces was arbitrarily defined as one biological unit (BU). The specific activity is expressed as BU/o.D.280. The significance of an increase or a difference in tentacle number was ascertained by comparing the mean tentacle numbers obtained in different dishes of one sample with those of the control or of other samples by means of the t test of significance or-at higher levels of activation - by comparing the tentacle numbers directly by means of the ^-test. Maceration of hydra tissue. As described by David (1972), pieces of hydra or whole animals were dissociated into cells by incubation and shaking in 7 % glycerol, 7 % acetic acid. The total number of cells was determined in a Neubauer cell counter, depth 0-1 mm; the distribution of cell types was counted with phase-contrast optics according to the criteria described by David (1972). Separation of cell types. The macerated cells were collected by centrifugation at looog for 5 min and layered on discontinuous glycerol (50, 30, 25, 20 and 15 % glycerol in 7 % acetic acid) or sucrose gradients (60, 40, 20, 10 and 5 % sucrose in 1 % acetic acid). These gradients were centrifuged at 500 g for 3-4 min and the fractions collected from the top. After determining the cell distribution for each fraction the cells were washed in 15 % sucrose, and resedimented at 3000 g for 10 min. The sediment was dissolved in distilled H 2 O, sonicated for 5 min with an MSE sonicator and assayed for head activation. Nitrogen-mustard treatment. Hydra from the mass culture were incubated in 0-01 % nitrogen mustard for 10 min (Diehl & Burnett, 1964), washed several times in fresh hydra medium, and subsequently treated as normal animals. Isolation of particles containing head-activating activity. The following buffers were used-buffer A: 0-4M sucrose, 10" 2 M Tris-HCl, ph 7-4, 10-3 M-MgCl 2, 4x 10~ 3 M-CaCl 2 ; buffer B: 0-2M-NaCl instead of 0-4M sucrose, the other constituents as in buffer A. Since sucrose inhibits head regeneration (rate and tentacle number) in concentrations above 10~ 3 M, dilution in buffer B and recentrifugation was always used as a last step to facilitate the biological assay. To release the low-molecular-weight activating substance, particulate fractions were freeze-thawed and shocked osmotically by dilution in hydra medium before testing. Homogenization: Concentrated hydra (approximately 500 hydra/ml) were mixed with an equal volume of 2 x buffer A or B. This mixture was

3 Characterization of activating substance 41 homogenized gently at 5 C in a glass or teflon homogenizer until complete disintegration of tissue and cells was achieved (as checked under the microscope). Centrifugation: The homogenate was centrifuged at 5 C in a Spinco (Model L) ultracentrifuge using a No. 30 rotor for 10 min at rev/min and the supernatant for 60 min at rev/min (35000 g SEDIMENT). The respective pellets were resuspended in buffer A for further purification or dissolved in buffer B to be stored frozen for the bioassay. Equilibrium sedimentation in discontinuous sucrose density gradient: Sucrose solutions contained 10-2 M-Tris-HCl, ph 7-4, 10-3 M-MgCl 2, 4x 10~ 3 M- CaCl 2. On to 10 ml of 50 % (w/w) sucrose were layered 10 ml of 40 %, 5 ml of 30 % sucrose+ 5 ml of the 35000g SEDIMENT in buffer A (0-4 M sucrose). The gradient was centrifuged at 5 C in a SW 25/1 rotor at rev/min for 2 h. Fractions were collected from the bottom of the tube, the sucrose density determined with a refractometer, each fraction diluted with buffer B, and concentrated by recentrifugation at g for 60 min (FRACTION A). Equilibrium sedimentation in continuous sucrose gradient: A continuous % sucrose gradient was layered on a 50 % sucrose cushion (0-2 ml). 1-5 ml of FRACTION A was added and centrifuged in a SW 25/3 rotor at 5 C and rev/min for 6-9 h. Fractions were collected from the bottom of the tube, diluted with buffer B and concentrated by centrifugation at g for 60 min (FRACTION B). Sedimentation velocity centrifugation: 1 ml of FRACTION B was layered on to a preformed linear gradient of 5-20 % sucrose and centrifuged in a SW25/3 rotor at 5 C and rev/min for 70 min. All fractions were washed in buffer B, recentrifuged and stored frozen (FRACTION C). RESULTS l(tf). Distribution of head-activating activity in hydra To determine the distribution of the substance activating head and bud formation in hydra, animals without buds were divided into four segments and the concentration of activating activity measured for each. As Table 1 shows, the activating substance is present in hydra as a gradient from the hypostomal to the basal region. The differences between each region are significant (t test, P < 0-05). The head region, including hypostome and tentacles, contains almost 50 % of the total activity. In it the activating substance is more concentrated than in any other region. The concentration of activating activity in the tentacles (2-5 BU/O.D.280) is lower than in the total head region, indicating that the activating substance may be even more concentrated in the hypostome. The remaining 50 % of the activity are found in the body column with an approximately twofold higher specific activity in the upper than in the lower half. This is in agreement with the finding presented in the previous paper,

4 SCH i Table 1. Distribution of the activating activity and nerve cells in freshly dropped buds Region Head region Upper gastric region Lower gastric region Basal region Mass/region (O.D.280) Concentration of extract for 1 BU (O.D.280) 0-3 ± ± ± Serial dilutions were assayed for each extract number (1 BU). The distribution of nerve cells is Specific activity (BU/O.D.280) ±0-4 l-2± ±0-2 Distribution of activating activity Total no. of cells to determine ithe concentration leading taken from Bode et al. (1972)i. No. of nerve cells Distribution of nerve cells > r tn > to a 5 % increase in tentacle O > o tn tn

5 Characterization of activating substance 43 Extract from 3-5 pieces/ml Time of regeneration (hours) Extract from 2 pieces/ml Time of regeneration (hours) Fig. 1. Activity of crude extracts of pieces regenerating a head at different hours after removal of the original head. (a) The extracts from equal-sized gastric pieces (approximately 0-1 o.d.280/piece) were assayed at two concentrations: x x, extract corresponding to 0-35 o.d.280/ml or 3-5 regenerating pieces/ml; A A, extract corresponding to 0-2 O.D. 280/ml or 2 pieces/ml (two independent experiments). (b) O O, Extract from lower gastric regions, the concentration corresponded to 01 o.d.280/ml or 2 pieces/ml (two independent experiments). --, Extract from basal regions, the concentration corresponded to 004O.D.280/ml or 2 pieces/ml. where the upper gastric region of animals starved for 3 days contained approximately 2-5 times more of the activating activity than the lower gastric region. The specific activities of these animals were for all regions approximately 2 times higher than for freshly dropped buds which is in agreement with a higher percentage of nerve cells (as shown by Bode et al. 1972), and as will be discussed later. The continuous decrease in concentration of activating activity from hypostomal to basal region is only found in freshly dropped buds or in animals without buds starved at least for 3 days. If well-fed animals without buds are used which would produce buds during the next 1 or 2 days, the specific activity

6 44 H. SCHALLER AND A. GIERER Table 2. Activity of crude extracts derived from basal regions regenerating a head Source of tissue for crude extract Regenerate at 0 h Regenerate at 24 h Regenerating surface at 24 h Tissue below regenerating surface at24h Concentration of extract (o.d.280/ml) Activation 6±2 0±2 10 ± Specific activity (BU/O.D. 280) 0-7 >2-5 >2-5 <2-5 of the lower gastric region rises significantly from 1-1 ±0-4 in animals without bud Anlage to 2-5 ±0-8 BU/o.D.280 in animals with bud Anlage. The activity of this future budding region is higher than that of the body region above it (t test, P < 005). 1 (b). Changes in concentration of head-activating activity during regeneration To observe small differences in content of activating activity the concentration of extract should be in the quasi-linear part of the activation curve (see fig. 1, Schaller, 1973), where small changes in concentration cause relatively large changes in tentacle number. Fig. 1 (a) (this paper) shows how the appropriate concentration was determined for the first 24 h of head regeneration for relatively large pieces from the body column. In the course of head regeneration two types of changes in the concentration of the activating activity were observed. (1) There is a slight, but measurable drop in activity 4-8 h after the onset of head regeneration {t test, P < 0-05). To determine the extent of this reduction in activating activity, serial dilutions were assayed of pieces 6 h after removal of the original head. It was found that the extract from four regenerating gastric pieces or 0-11 o.d.280/ml were needed at 6 h to achieve the same effect as from three pieces or 0-08 o.d.280/ml at 0 h. Activating substance is released from the regenerating surface into the surrounding medium. This was measured either by incubating other regenerates in the medium in which regeneration had occurred or by determining the minimal volume by which a regenerating piece influenced itself. Incubating a regenerating piece in less than 0-2 ml medium lead to an activation corresponding to a 5-8 % increase in tentacle number. This indicates that quite a considerable amount of the substance present in the regenerate is released during the first hours of head regeneration (approximately %). Pieces of tissue regenerating a basal region did not show such a drop in head activating activity. (2) h after cutting, the concentration of activating activity increases. This increase in activity at 24 h was found to be independent of the size or the origin of the regenerating tissue, i.e. does not seem to be dependent on the time

7 Characterization of activating substance 45 Table 3. Procedure for the purification of particles containing activating activity.1. Homogenization in buffer A (04 M sucrose, 10~ 2 M Tris-HCI, ph 7-4, 10 3 M-MgCl 2, 4x 10^3 M-CaCl 2 ), centrifugation at at 5 C for 10 min; resuspension of sediment in buffer A containing 10~ 2 M EDTA, homogenization, centrifugation at 10000g for 10 min; centrifugation of the two supernatants at g for 60 min to give g SEDIMENT. 2. Centrifugation of the g SEDIMENT in a discontinuous 30-50% (w/w) sucrose gradient at g for 2 h (5 C) to give FRACTION A (reddish band at interphase to 30 % sucrose). 3. Equilibrium sedimentation of FRACTION A in a continuous % (w/w) sucrose-density gradient at 50000g (5 C) for ca. 8 h (overnight) to give FRACTION B (corresponding to % sucrose). 4. Sedimentation of FRACTION B (after suspension in 02 M-NaCl and resedimentation) in a 5-20% (w/w) sucrose-density gradient (SW 25/3, 13000g rev/min, 75 min, 5 C) to give FRACTION C (migration of activity from cm radial distance corresponding to % sucrose). Fig. 2 shows an electron micrograph of FRACTION C. Crude extract (10000 hydra) g sediment Fraction A Fraction B Fraction C Low-molecular-weight activator after G-10 Table 4. Purification of particles containing the head-activating activity Mass (o.d.280) <1 <001 BU Specific activity (BU/O.D.280) > Yield Purification (x -fold) > From fraction C the low-molecular-weight head-activating substance was released by osmotic shock (dist. H 2 O) and subsequent sonication. After centrifugation at g for 60 min the supernatant was chromatographed on a Sephadex G-10 column (as described by Schaller, 1973). required for head regeneration. Whereas the gastric pieces used for Fig. l(a) regenerated a head in 2 days, the pieces used in Fig. l(b) were sections (as diagrammed) from the lower part of the body column, which have a slower rate of head regeneration, i.e. they needed 3 and 4-5 days, respectively, to regenerate a head. The relative increase in specific activity is especially high for a piece with an originally low level of activating activity, e.g. a piece from the basal region showed a more than threefold increase in concentration of activity at 24 h as compared to 0 h (Table 2). This increase in concentration of activity is mainly

8 46 H. SCHALLER AND A. GIERER 2001) A Fig. 2. Electron micrograph of particles containing the head-activating substance. A drop of FRACTION C was placed on a grid and negatively stained in I % aqueous uranyl acetate. due to a higher specific activity of the regenerating surface. In tissues regenerating basal structures no increase in concentration of head-activating activity was observed at 24 h, indicating that the changes measured in the course of head regeneration are specific for head regeneration. 2. Localization of the low-molecular-weight head-activating substance in membranous particles In the previous paper (Schaller, 1973) it was shown that the head-activating substance stimulates head formation in a concentration which is far below that present in the regenerating tissue itself. It acts in a concentration corresponding to approximately 1/1000 of that in the whole animal. This is only explicable by

9 Characterization of activating substance 47 assuming that large amounts of the low-molecular-weight substance are not freely diffusible in the animal, but are present in a stored form, i.e. probably structure-bound. Furthermore, Lentz (1965) recovered head-activating activity from a particulate fraction. To show that most of the low-molecular-weight head-activating substance is structure-bound, hydra were homogenized gently (glass or teflon homogenizer, iso-osmolarity, 4 C), centrifuged at 35000g for 1 h (0-2M-NaCl, 5 C), and sediment and supernatant assayed separately for activity. Under these conditions only 5 10 % of the activating substance were found in the supernatant, i.e. in a low-molecular-weight form, whereas more than % were structure-bound. Since some breakage is unavoidable even under mild homogenization conditions, in the animal the actual percentage of structure-bound head-activating substance is probably higher. To further purify and characterize this particulate fraction, the procedure outlined in Table 3 was developed. By equilibrium density and velocity centrifugation the activity was enriched about 1000-fold in a fraction containing 70 % of the original activity (Table 4). As shown in the electron micrograph in Fig. 2, this fraction consists mainly of particles of approximately 1200 A diameter. This particle size is consistent with a sedimentation coefficient of approximately 800 S calculated from the sedimentation velocity in the sucrose gradient and a density of 1-09 as determined by the density equilibrium centrifugation. The density of 1-09 is characteristic for membranous structures with a high lipid content, especially for constituents of the smooth endoplasmic reticulum, e.g. products of the Golgi complex. From these membranous particles the lowmolecular-weight substance is released quantitatively (Table 4) by osmotic shock, ultrasonication, or repeated freeze-thawing. 3. Localization of the head-activating substance in nerve cells Lentz (1965) and Lesh & Burnett (1966) discussed nerve cells as possible sources for substances responsible for the polarity of hydra. Muller & Spindler (1971) postulated that the head-inducing substance may be a product of the nematocytes. To exclude nematocytes as sources for the head-activating activity, mustardtreated animals (Diehl & Burnett, 1964) - which, as Table 5 shows, contain less than 2 % of the normal complement of nematoblasts and nematocytes, very few interstitial cells, but quite normal numbers of nerve cells - were assayed for their content of head-activating activity. It was found that they still contained 70 % of the activity of normal animals: the extract from 1-2 mustard-treated animals was as active as the extract from 0-8 normal animals. Therefore nematocytes and also interstitial cells are excluded as major sources for the headactivating activity. To prove that nerve cells produce and/or store the head-activating substance the attempt was made to isolate or enrich them. A suspension of macerated cells

10 Table 5. Distribution of cell types and activity in normal and nitrogen mustard-treated animals Normal animals Animals 9 days after treatment Total Epithelial Big Little Nematocytes Specific cell and inter- inter- and activity no. digestive stitial stitial nematoblasts Nerve Gland Mucous (BU/O.D. 280) /o No. /o No } } 1-25 ± Table 6. Distribution of activating activity and cell types after maceration and separation in a glycerol gradient Origin of cell mixture Unseparated cells Fraction 1 Fraction 2 Fraction 3 Fraction 4 Fraction 5 Distribution of cell types in c Nerve cells <5 <5 5 A Small cells* each fraction Large cellsf Distribution of total cell mass (O.D.280) Specific activity (BU/O.D. 280) TV f ih trm X-/lolllUU LIUI1 of activity (7o) The cells accumulate at the interphase of the different glycerol concentrations. Fraction 1 contains the cells collected from the uppermost interphase, i.e. between 10 and 20 % glycerol (see Materials and Methods). Fraction 5 was rich in tissue clumps. * Including interstitial cells, nematoblasts, and nematocytes. t Including epithelial, mucous, and gland cells. oo. c/a n tn > w w

11 Characterization of activating substance 49 was used as starting material. As described in detail by David (1972), all cell types, including nerve cells, are easily and quantitatively recognizable in such preparations. The macerated cell mixture still contained % of the headactivating activity present in the living animal, and was therefore suitable for cell separation experiments. The macerated cell mixture was separated by density centrifugation in discontinuous glycerol or sucrose gradients. The distribution of cell types in the different fractions after centrifugation and the specific activity of each fraction is given in Table 6. Whereas the unseparated cell mixture consisted to only 5 % of nerve cells, fraction 1 consisted to 50 % of nerve cells, fraction 2 to 25 %, i.e. the separation led to a tenfold enrichment of nerve cells in fraction 1, fivefold in fraction 2. Concomitantly, fraction 1 containing 60 % of the total activity showed a more than 10 times higher specific activity than the unseparated cells or a times higher specific activity than the other fractions containing either enriched concentrations of large or small cells. It seems therefore very unlikely that cell types other than nerve cells contain major quantities of the head-activating substance. The only objection to this conclusion is the possibility that the activity may not be bound to cells in the macerate, but to some larger structures (e.g. pieces of cell debris) that band in the same region as the nerve cells and are sedimentable in 5 min at 1000 g. However, since fraction 2 contained practically no debris, but still increased amounts of activating activity in correlation with the increased percentage of nerve cells, it is more likely that the activity is bound to nerve cells (or a subgroup of the nerve cells). This is supported by the finding that of all the cell types only the nerve cells show a similar distribution (Bode et al. 1972) to the head-activating activity both with regard to time sequence of appearance during head regeneration and bud initiation as to localization within the animal. During bud initiation the first detectable change in the distribution of cell types is a local increase in density of nerve cells at the site where the bud tip becomes visible. In head regeneration the density of nerve cells doubles at the regenerating surface h after removal of the head. As shown in Table 1 in the animal the concentration of nerve cells is highest in the head region, with a maximal density in the hypostomal region, from where it decreases as a gradient down the body column and into the tentacles. The density of nerve cells increases again in the basal region. No other cell type follows such a distribution. Interstitial cells, nematoblasts and gland cells have a low density in the hypostomal region; their density is higher in the gastric region. Nematocytes occur almost exclusively in the tentacles. Mucous cells of the hypostome are more or less confined to the head region, and epithelial cells are relatively evenly distributed over all body regions of hydra. EMB 29

12 50 H. SCHALLER AND A. GIERER DISCUSSION The low-molecular-weight substance which as shown in the previous paper (Schaller, 1973) activates head and bud formation in hydra is present in the animal as a gradient from the hypostomal to the basal region. The graded distribution of the activating substance is found only in animals without visible or developing buds. In animals with a developing bud the specific activity of the budding region is almost as high as that of the hypostomal region, i.e. the gradient from top to bottom becomes interrupted in the future budding region by the establishment of a second area of high activator concentration. Together with the fact that the purified substance stimulates bud formation this suggests that the head-activating substance plays a role in bud initiation as well. The concentration of activating activity changes with time in a piece of tissue regenerating a head. During the first 4-8 h there is a slight, but significant decrease in the concentration of activating activity, at 16-24h there is a considerable increase. Neither of these changes are observed during regeneration of basal structures and are therefore probably specific for regeneration of head structures. The head-activating substance stimulates head formation in a concentration which is far below that present in the regenerating tissue itself. This is only explicable by assuming that large amounts of the low-molecular-weight substance are not freely diffusible in the animal, but are present in a stored form, i.e. probably structure-bound. It could be shown that under mild homogenization conditions % of the total activity are sedimentable. This was already indicated by Lentz's finding (1965) that head-activating activity can be recovered from a particulate fraction. By various centrifugation methods 70 % of the total activity were enriched 1000-fold in a fraction consisting of membranous particles of ~ 1200 A diameter. From these particles the low-molecular-weight substance was released quantitatively by osmotic shock or ultrasonication. The high percentage of total activating activity bound to these particles together with the high specific activity of this fraction makes it very probable that the activating substance is located in these structures in the animal also. Lentz (1966) presented evidence that nerve cells play a role in regulating the polarity of hydra. Furthermore he showed that one group of nerve cells contains neurosecretory granules which resemble the particles isolated in size and properties. To correlate activating activity with nerve cells the distribution of activity was compared with the distribution of cell types (as measured by Bode et al 1972). Of all the cell types only the nerve cells show a positive correlation with the activating activity both with regard to localization within the animal as to time sequence of appearance during different states of growth and morphogenesis. Activity as well as nerve cells are most concentrated in the hypostomal region, decreasing in density in the tentacles and down the body column. During head

13 Characterization of activating substance 51 regeneration and bud initiation increase in specific activity and increase in nerve cell density again coincide. The high nerve cell density in the basal disc as well as the increase in nerve cell density during foot regeneration are not accompanied by an increase in activity, indicating that the nerve cells of the basal disc do not contain major amounts of the head-activating substance and/or that they serve a different function. Since these findings hinted at nerve cells the attempt was made to isolate them. They could be enriched tenfold in one fraction, fivefold in another. Together these two fractions contained only 12 % of all the cells but 80 % of the total activity. The three lines of evidence, namely (1) the similarity of the isolated particles with neurosecretory granula, (2) the isolation or enrichment of nerve cells in a cell fraction containing most of the activity, and (3) the positive correlation in time and localization of nerve cells and activity, make it very probable that the nerve cells or a subgroup of the nerve cells are the main site of production and/or storage of the head-activating substance. All experiments presented in this and the previous paper (Schaller, 1973) are consistent with the assumption that the substance activating head and bud formation is a true morphogen which influences or regulates hydra morphogenesis, probably in conjunction with other such substances. For the mode of action of the head-activating substance the following tentative model is suggested. The low-molecular-weight head-activating substance is mainly or exclusively produced by nerve cells and is stored there in particles resembling neurosecretory granules. From the nerve cells the low-molecular-weight substance is released steadily or dependent on certain stimuli in minute concentrations. Since the granules containing head-activating activity are present in hydra as a gradient decreasing from the hypostomal to the basal region, this release would suffice to build up and maintain a gradient of freely diffusible head-activating substance. To explain regeneration and budding the release rate must be assumed to depend not only on granula concentration, but also on other effects controlling or affecting the release. The experiments have shown that during the first hours of head as opposed to foot regeneration increased amounts of the stored substance are released. This increased release can only be caused by the removal of the head. Probably the normal balance of morphogenetically active substances is disturbed or the release may be affected by some other mechanism. At the cellular level the concentration of head-activating substance together with the presence or absence of other such morphogens probably determines what types of differentiations occur. Since during head regeneration the level of head-activating activity rises due to increased release, a gastric region is reprogrammed to form head structures, i.e. differentiate into those cell types characteristic for head as opposed to gastric regions. The cell distribution data (Bode et al. 1972) indicate that at the regenerating surface, for example, more 4-2

14 52 H. SCHALLER AND A. GIERER interstitial cells become determined to differentiate into nerve cells, whereas the differentiation into nematoblasts seems to be turned down. The concentration of head-activating substance probably also influences the other cell types: epithelial cells differentiate into the battery cells of the tentacles, the production of gland cells is reduced, that of the mucous cells enhanced etc. Since other morphogens may be involved in this process a detailed analysis of the effects of the isolated substance on the differentiation of cell types is necessary. We thank H. Schwarz and Dr H. Frank for taking electron micrographs. REFERENCES BODE, H., BERKING, S., DAVID, C. N., GIERER, A., SCHALLER, H. & TRENKNER, E. (1972). Quantitative analysis of cell types during growth and morphogenesis in hydra. Wilhelm Roux Arch. EntwMech. Org. Ill, DAVID, C. N. (1972). Quantitative method for maceration of hydra tissue. Wilhelm Roux Arch. EntwMech. Org. Ill, DIEHL, F. & BURNETT, A. L. (1964). The role of interstitial cells in maintenance of hydra. I. Specific destruction of interstitial cells in normal, asexual, non-budding animals. /. exp. Zool. 155, LENTZ, T. L. (1965). Induction of supernumerary heads by isolated neurosecretory granules. Science, N. Y. 150, LENTZ, T. L. (1966). The Cell Biology of Hydra. Amsterdam: North-Holland Publishing Co. LESH, G. E. & BURNETT, A. L. (1966). An analysis of the chemical control of the polarized form in hydra. /. exp. Zool. 163, MULLER, W. A. & SPINDLER, K. (1971). The 'polarizing inducer' in Hydra: a reexamination ofit s properties and its origin. Wilhelm Roux Arch. EntwMech. Org. 167, SCHALLER, H. (1973). Isolation and characterization of a low-molecular-weight substance activating head and bud formation in hydra. J. Embryol. exp. Morph. 29, {Manuscript received 24 April 1972, revised 23 June 1972)