t /. Embryol. exp. Morph. Vol. 30, l,pp. 1-19, 1973 Printed in Great Britain h Size determination in Hydra: The roles K of growth and budding By JOHN W. BISBEE1 ^ From the Department of Biology, University of Pittsburgh r P SUMMARY \~ Hydra pseudoligactis cultured at 9 °C for 3-4 weeks are one-and-a-half times larger than ^ those cultured at 18 °C. The size of Hydra is correlated with the numbers of epithelio- muscular and digestive cells in the distal portion of the animal and with the diameters of the k- epithelio-muscular cells in the peduncle. [ Counts of mitotic figures and tritiated-thymidine-labeled nuclei and determinations of T increase in mass of Hydra populations suggest that the difference caused by these tempera- y. tures does not affect mitosis. At 9 °C buds are initiated at a lower rate and take longer to develop than at 18 °C. The surface-areas of buds raised at the two temperatures are similar. T Because Hydra raised at the two temperatures have similar growth dynamics, the differences u in sizes of the animals cannot be due to growth rate. The observed effect of temperature on bud initiation and development is probably relevant to the increased size of animals raised at 9 "C, since these larger animals may be accumulating more cells while losing fewer to buds. INTRODUCTION The shape and size of Hydra seems to be a consequence of several dynamic processes. Growth, budding, cell sloughing, cell migration, and mesogleal metabolism have possible roles in Hydra morphogenesis (Burnett, 1961, 1966; Burnett & Hausman, 1969; Brien & Reniers-Decoen, 1949; Campbell, 1965, 1967 a, b, c, 1968; Shostak, Patel & Burnett, 1965; Shostak & Globus, 1966; Shostak, 1968). This paper deal with the roles of growth and budding in deter- mining the dimensions of Hydra pseudoligactis. Hydra is essentially a cylinder made up of two cell layers, the epidermis and the gastrodermis, with an acellular mesoglea between them. The cylinder has a ring of tentacles and a mouth at its distal end, and an adhesive 'foot' at its proximal end. The animal is a cellular system with 'input' by cell division, and 'output' primarily by budding; Campbell (1965) and Shostak (1968) have estimated that 60-85 % of cell loss occurs in buds. Additional cell loss occurs via cell sloughing at both ends of the animal, and possibly along its length. Both Stiven (1965) and Park & Ortmeyer (1972) observed that lowering the ambient temperature increased the size of Hydra. This paper confirms Stiven's observation for Hydra pseudoligactis, and asks the following questions: Is the 1 Author's address: Department of Biology, Mundelein College, Chicago, 111. 60660, U.S.A. I E M B 30 J. W. BISBEE change in size due to differential increase in one body region or is it uniform throughout the body column ? Is it due to differential cell size or rate of cell division ? Is'the change in Hydra size due to differential cell loss ? The approach taken was to measure body column dimensions and cell numbers and dimensions on serial cross-sections. Since Hydra size was correlated with cell number, an effort to understand the mechanism through which temperature alters cell j numbers was made, by determinations of increase in mass of Hydra populations and counts of mitotic figures and tritiated-thymidine-labeled nuclei. Finally, J budding, as the main form of cell loss, was studied. MATERIALS AND METHODS ^ A. Culture methods A clone was identified by species as Hydra pseudoligactis, according to A Forrest's (1963) key. The tentacles on buds arose successively in a fixed pattern as pictured in fig. 10 of Forrest (1963); adults had tentacles approximately three ^ times column length. The holotrichous isorhizas were narrowly oval with trans- A verse coils. As in Hyman's (1931) original description of Hydra pseudoligactis, i an individual's body column was differentiated into stalk and body (Fig. 1). A Also some animals raised at 9 °C in the fall were observed to be sexual, having rather stout testes with nipples. Stocks of animals were maintained in Pyrex-brand baking dishes kept in incubators at 9 ± 1 and 18 ± 1°C. They were fed to repletion three times a week on freshly hatched Artemia sp. nauplii at room temperature. The culture solution (Shostak et al. 1965) was poured off daily (after feeding if they were fed) and replaced with fresh solution already at the appropriate temperature. Animals were transferred to clean dishes every 4-10 days, with the density kept below one Hydra per 0-5 ml of solution. The Hydra used in experiments, drawn from stocks, were raised in finger bowls. Animals raised for dry-weight determinations and those to be injected with tritiated thymidine were maintained at a density of one Hydra per 10 ml of culture solution; all other experimental animals were raised at a density of one per 20 ml of solution. All groups were incubated for 3 or 4 weeks at the appropriate temperature and starved 48 h before use. With one exception (a Hydra raised at 9 °C and used for cell and size determinations), all the Hydra used were asexual budding animals. B. Histology Hydra were placed in 50 x 15 mm Petri dishes with 2-3 ml of approximately 25 °C culture solution at 16.00 h, 52 h after feeding. Within 15 min, having extended to the approximate proportions of Fig. 1, they were quickly flooded with hot Bouin's fluid (Pearse, 1960), which prevented them from contracting. After fixation of all animals for 16-18 h the picric acid was removed by placing Size determination in Hydra 3 the animals in LiCO3 in 70 % alcohol. They were dehydrated in an alcohol series, cleared in xylol and embedded in 56 °C paraplast. Serial sections 10 /im. thick were cut perpendicular to the long axis of the Hydra. Slides were de- paraffinized, hydrated, stained in toluidine blue, dehydrated, and mounted in permount. The number of epidermal epithelio-muscular cells and gastrodermal digestive cells in each section were estimated from counts of their nuclei. These cells were identified as having cytoplasm extending from the mesogleal surface of the respective epithelium to the surface of the layer, and as having nuclei with prominent nucleoli. A cell was considered to be undergoing mitosis if part of •* the mitotic figure was visible on the section. Mitotic counts of all cells (as listed || in Campbell, 1967a) were made. C. Autoradiography Tritiated thymidine, 0-5 /i\ (Schwartz Bioresearch, Inc., 6-0 Ci/mmole, 1-0 r mCi/ml), was injected through the mouth into the Hydra enteron (Campbell, 1965). The animals were returned to culture solution at the appropriate in- cubation temperature and fixed 6 h later. Serial cross-sections on slides were dipped in Kodak NTB-3 nuclear track emulsion, stored in the freezer for 5 days, and developed in D-19. After being stained with toluidine blue, the slides were mounted and examined at x 200. A nucleus was considered labeled if one half or more of it was uniformly blackened by silver grains. D. Mass determinations The dry weight of Hydra was determined on groups of animals that had been starved for 48 h. The animals were lyophilized and weighed on an Oertling R20 analytical balance, which can be read to 0-1 mg. E. Observations of buds Data on budding were collected by observation often adults at room tempera- ture with a dissecting microscope at x 12, noting the number of buds attached and detached daily. Newly detached buds were discarded. RESULTS A. Size of Hydra Observations of Hydra cultured at 18 and 9 °C (Figs. 1, 2) show that animals raised at the lower temperature were larger than those at the higher temperature. The lengths and diameters of the animals were measured and the circumferences calculated. The average dimensions at each temperature, their standard devia- tions, and the locations of the measurements on the body column (axial position) are shown in Table 1 and Fig. 4. J. W. BISBEE Fig. 1. Representative Hydra pseudoligactis raised at 18 °C for 3 weeks, x 10. Fig. 2. Representative Hydra pseudoligactis raised at 9 °C for 3 weeks, x 10. Fig. 3. Photomicrograph of a portion of a Hydra pseudoligactis cross-section in the gastric region. Epidermis is the cell layer on the right; gastrodermis, on the left. E, Epithelio-muscular cell; /, interstitial cell; D, digestive cell; g, gland cell, x 825. K Size determination in Hydra 5 % Table 1. Linear dimensions of the Hydra body column^ Region and its [position]:}: on the body column h Temperature Peduncle [1-3] Budding [4-5] Gastric [6-10] Total y 9 C 89(17) 30(14) 135(46) 253(47) L 18 X 58 (5) 21(19) 84(16) 162(17) t Averages and (standard deviations) of the number of cross-sections, based on four T animals at each temperatuie. Y % Numbers refer to positions on Hydra body column of Fig. 4. | Table 2. Analysis of variance for the body column circumferences at the mesoglea accumulated for corresponding axial positions at both temperatures Y Source of Degrees of Sum of Mean variation freedom squares squares F f Treatments 19 38-26 — — Temperature 1 010 010 186 I Axial position 9 37-92 4-21 84-20* Temperature v. axial position 9 0-24 003 0-50 •" Error 80 4-28 005 — Total 99 42-54 — — * Probability less than 1 % that the difference is due to random error. 1. Length The total lengths and the lengths of regions of Hydra raised at the two temperatures were determined by counting the serial cross-sections 10 /im thick.