Morphogenesis in hydra:II Peduncle and basal disc formation at the distal end of regenerating hydra after exposure to low temperatures

We have previously discussed peduncle and basal disc formation at the distal end in terms of colchicine inhibition of cell division and the possible action of colchicine on the nervous system (Corff & Burnett, 1969). In the present study, we demonstrate that low temperature can initiate the same responses in regenerating hydra as those initiated by colchicine. Evidence provided by recent studies (Burnett, 1961; Burnett & Diehl, 1964; Burnett, Diehl & Diehl, 1964) on the effects of cold on hydra, specifically on the nervous system and growth, permit greater insight into the mechanisms involved in the production of peduncle and basal disc at the distal end of regenerating hydra. In general, the evidence presented here supports our original conclusions regarding the action of colchicine in initiating this response.

Hydra oligactis were cultured according to the method of Loomis & Lenhoff (1956) modified by the use of distilled water in place of tap water. Mass cultures were maintained at 18–20°C. The animals were fed daily on newly hatched Artemia salina.

Hydra were starved for 24 h before each experiment. Depending on the experiment, the hypostome and tentacles were excised from each animal either before or after exposure to cold (8–12°C). After the excision, hydra were placed in the cold or retained at room temperature (18–20°C).

The response of each regenerating hydra to cold treatment was classified within one of the following categories. This same classification had been used in recording the response of hydra to colchicine treatment (Corff & Burnett, 1969).

1. Hydra formed a hypostome and tentacles at the distal end. (Regeneration was frequently delayed. Most of the animals completed normal distal structures; however, a number of animals regenerated only one or two tentacles or abnormally placed tentacles around the hypostome.)

2. Hydra formed a peduncle and basal disc at the distal end.

3. The wound healed over, but no regeneration of distal structures occurred.

4. Disintegration occurred within 24–48 h after excision.

Hydra exposed to cold (12°C) for varying times (6–96 h) before excision of hypostome and tentacles were retained at room temperature (18–20°C) or returned to the cold for 5–8 days following the excision.

Hydra retained at room temperature regenerated as shown in Table 1. None of the animals formed a peduncle and basal disc at the distal end of the body column unless they had been exposed to cold for a minimum of 48 h before excision. These animals responded similarly to colchicine-treated hydra. The animals either regenerated normally, formed fewer tentacles, or a single medial tentacle, did not regenerate or disintegrated (Table 1). In a group of five hydra exposed to cold for 72 h prior to excision, one developed a basal disc at its distal end. This animal also produced a bud with a basal disc at its distal end. This bud was positioned between two other buds, both of which had formed tentacles.

Two other buds from this group of five, exhibited abnormally placed basal discs. One detached bud had two basal discs positioned at its gastric-peduncle junction and one bud still attached to a non-regenerating animal exhibited a basal disc at its gastric-peduncle junction.

None of the hydra returned to the cold directly after excision of hypostome and tentacles regenerated while in the cold. These animals were therefore returned to room temperature after 5 or 8 days in the cold. The results of this transfer to room temperature are shown in Table 2. The frequency of peduncle and basal disc formation at the distal end was higher than in animals exposed to cold only prior to excision. After 24 h at room temperature, two out of five animals previously exposed to cold for 6 h before excision and then returned to the cold for 5 days, regenerated tentacles (one medial tentacle and two small tentacles). After 3 days at room temperature, however, both of these animals exhibited basal discs at the distal end of the animal and the tentacles had disappeared. Twelve hydra exposed to the cold for 6 h prior to excision were returned to the cold for 5 days. No regeneration occurred in the cold and these animals were transferred back to room temperature. Five animals subsequently initiated peduncle and basal disc formation at the distal end of the body column.

Hydra were exposed to cold (8°C) for varying periods of time prior to excision of hypostome and tentacles. One group of animals was retained at room temperature after the excision. The results are shown in Table 3. Hydra exposed for 40 or more h prior to excision formed a high percentage of peduncles and basal discs at the distal end of the body column. However, in the group of 46 hydra exposed for 72 h, no distal basal discs were formed, but a high percentage of animals formed a single medial tentacle. A larger percentage of animals disintegrated after treatment at 8°C.

All hydra returned to 8°C after the excision of hypostome and tentacles disintegrated within 48 h.

Hydra excised below the tentacles were exposed to cold (12°C) for varying periods of time and returned to room temperature. The results of this treatment are shown in Table 4. None of the animals formed a peduncle and basal disc at the distal end of the body column. One hydra in the group of 10, exposed for 7 h, regenerated one tentacle after 48 h at room temperature, but no hypostome. This animal subsequently regenerated normally.

Hydra excised below the tentacles were exposed to cold (8°C) for varying periods of time and returned to room temperature. Three formed a peduncle and basal disc at the distal end of the body column when exposed to the cold for 48–60 h after the excision (Table 4). More animals formed medially placed tentacles after exposure to 8°C than after exposure to 12°C.

Hydra excised and retained at room temperature for varying periods of time subsequently regenerated tentacles as shown in Table 5. Regeneration was considerably delayed (ca. 48 h) when compared with controls at room temperature, regardless of the length of time regeneration had proceeded at room temperature. It is apparent, however, that hydra retained at room temperature for 20–24 h are less affected by cold, since 73 and 95% of the animals, respectively, regenerated at least one tentacle by 72 h after the excision. Hydra retained at room temperature for 4–18 h after the excision, showed progressively less regeneration with decreasing periods of regeneration at room temperature. Regeneration of one or more tentacles in most of these animals did not occur until 96 h after the excision of the tentacles. Hydra placed in the cold almost immediately after the excision of the tentacles were most affected and regeneration did not occur until 144 h after the excision.

Hydra placed in the cold (13–14°C) following regeneration at room temperature subsequently regenerated tentacles at a faster rate than hydra placed in the cold (9–10°C). Regeneration, however, was still delayed by ca. 24 h as compared with controls (Table 6). Hydra placed in the cold (13–14°C) immediately after the excision were delayed 48 h compared with controls.

The various morphological forms observed at the distal end of regenerating hydra following exposure to low temperatures parallel those observed in hydra following exposure to colchicine (Corff & Burnett, 1969). In colchicine-treated hydra, the various morphological forms were arranged in a morphogenetic hierarchy that reflected the effectiveness of colchicine action on individual hydra. This arrangement appears valid for cold-treated animals as well. According to Burnett (1966), polarity, morphogenesis and cell differentiation along the body column of hydra are controlled by quantitative changes in inducer and inhibitor levels along the body column. Inducer is thought to be produced by nerves in the hypostome. The involvement of neurosecretion in regeneration of hypostome and tentacles has been reported (Lesh & Burnett, 1964; Lentz, 1965; Lesh & Burnett, 1966). A gradual reduction of neurosecretion in cold-treated hydra or cold-induced sexual hydra is suggested by Lesh & Burnett (1966) and Burnett & Diehl (1964). In the present study, with increased duration of exposure and decreased temperature, hydra at room temperature subsequently regenerate fewer tentacles, abnormally positioned tentacles, or a single medial tentacle (reflecting lower levels of inducer than normally present at the distal end), no tentacles (reflecting levels of inducer normally present at the midgastric region) or a peduncle and basal disc (reflecting peduncle levels of inducer). At room temperature, neurosecretory droplets usually increase within the nerve cells over a 4 h period in regenerating hydra (Burnett et al. 1964). If hydra are excised and placed in the cold, this build-up of neurosecretory material may be inhibited or may proceed very slowly. When the animals are returned to room temperature, growth and regeneration will resume, but the form regenerated at the distal end will depend on the level of inducer and inhibitor at the cut end.

If the inducer-inhibitor levels at the distal end determine morphological form at the distal end of cold-treated hydra returned to room temperature, why do animals retained in the cold not form peduncles and basal discs at the distal end? Several observations on the effects of low temperature on hydra may help to explain this result. (1) Low temperature inhibits or reduces the rate of budding, suggesting a reduction of growth (mitosis) at least in the epithelio-muscular cells of the epidermis and the digestive cells of the gastrodermis (Burnett, 1961). (2) At room temperature, starving hydra cease budding. (3) Testes on hydra maintained in the cold are stationary. At room temperature, however, they are displaced down the body column suggesting the resumption of growth in the body column. (4) Hydra slowly adapted to low temperatures, will grow and bud normally at temperatures that induce sexuality or inhibit growth in non-adapted hydra. These observations suggest that when growth is slowly reduced, normal morphology and polarity can be maintained. Cold-adapted or starving hydra must therefore maintain their own gradient of inducer and inhibitor levels. When hydra are placed in the cold to regenerate, growth probably occurs slowly enough to permit the establishment of a normal gradient. When cold-treated hydra are returned to room temperature, in some cases, growth resumes before the normal gradient can be established.

Hydra regenerating at room temperature for longer periods of time, regenerate tentacles faster than those placed in the cold immediately after the excision. Since most animals, however, will eventually regenerate in the cold, the temperatures used in this study do not completely prevent growth (cell division) and the ability of nerves to produce neurosecretory material.

Both colchicine and low temperature appear to affect the same processes: cell division and neurosecretion. Although certain concentrations of colchicine (0·015–0·025%) inhibit spindle formation in dividing cells, long exposure to colchicine for three or more hours at these concentrations frequently disrupts hydra at the cellular level (Corff & Burnett, 1969). Preliminary experiments with low concentrations of colchicine (0·005%) and low temperature suggest that the two act synergistically. Hydra placed in 0·005% colchicine in the cold are less affected than those exposed to the same concentration at room temperature. This may be due to the subsequent decrease in growth at cold temperatures. The use of both colchicine and cold treatment, however, may provide a method of retaining c-metaphase cells in hydra over a longer period of time without disorganization at the cellular levels, thereby providing a valuable technique for an approach to the determination of cell cycles in hydra.

1. Hydra can form a peduncle and basal disc at the distal end of the body column when they are exposed to cold for various periods of time either before or immediately after the excision of hypostome and tentacles. The response is not ‘all or none’ and hydra frequently regenerate a smaller number of tentacles, abnormally placed tentacles, or a single medial tentacle. Some hydra do not regenerate.

2. The frequency of peduncle and basal disc formation at the distal end increases with lower temperatures and longer duration of exposure to cold. It decreases with longer duration of regeneration at room temperature prior to cold treatment.

3. Hydra do not form a peduncle and basal disc at the distal end when they are kept in the cold after excision. Normal regeneration is delayed and the length of delay depends upon the decrease in temperature and the length of time regeneration proceeded at room temperature before transfer to the cold.

4. Colchicine and low temperature treatment appear to influence basal disc formation at the distal end in a similar manner. The role of cell division and the nervous system in regeneration, in relation to the effects of cold treatment, is discussed.

1. Les hydres peuvent former un pédoncule et un disque basal à l’extrémité distale de la colonne somatique quand elles sont exposées au froid pendant des périodes variées, soit avant, soit immédiatement après l’excision de l’hypostome et des tentacules. La réponse n’est pas ‘tout ou rien’ et l’hydre régénère fréquemment un plus petit nombre de tentacules, des tentacules en position anormale, ou un seul tentacule médian. Quelques hydres ne régénèrent pas.

2. La fréquence de formation du pédoncule et du disque basal à l’extrémité distale, augmente avec des températures plus basses et une durée accrue d’exposition au froid. Elle diminue avec une durée plus longue de régénération à la température de la pièce avant le traitement par le froid.

3. Les hydres ne forment pas de pédoncule et de disque basal à l’extrémité distale quand on les maintient au froid après excision. La régénération normale est retardée et la longueur du retard dépend de l’abaissement de température et de la durée au cours de laquelle la régénération a eu lieu avant le transfert au froid.

4. La colchicine et le traitement par une basse température apparaissait influencer de la même manière la formation du disque basal à l’extrémité distale. On discute le rôle de la division cellulaire et du système nerveux dans la régénération, en relation avec les effets du traitement par le froid.

This paper was prepared from a thesis submitted in partial fulfilment for the degree of Doctor of Philosophy. This work was supported by the U.S. Public Health Service training grant no. HD 20 (Corff), and by the National Science Foundation grant no. GB-7345 (Burnett).