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The fine structure of epidermal glands of regenerating and mature globiferous pedicellariae of a sea urchin (Lytechinus pictus)
TISSUE & CELL 1975 7 (4) 723-737 Published by Longman Group Ltd. Printed in Great Britain
LINDA Z. H O L L A N D and N I C H O L A S D. H O L L A N D
THE FINE STRUCTURE OF EPIDERMAL GLANDS OF REGENERATING AND MATURE GLOBIFEROUS PEDICELLARIAE OF A SEA URCHIN (LYTECHINUS PICTUS) ABSTRACT. After a globiferous pedicellaria is lost from a sea urchin, a new appendage of the same kind is usually regenerated in the weeks that follow. During the latter part of regeneration, head glands and stalk glands, both of epidermal origin, develop from undifferentiated cells. Head gland cells begin morphological differentiation in the epidermis and then delaminate into the underlying dermis. In the formation of the stalk gland, by contrast, undifferentiated cells delaminate from the epidermis and then begin morphological differentiation in the dermis. During late regeneration, ceils in the head and stalk glands are characterized by extensive rough endoplasmic reticulum distended with intracisternal material; moreover, the Golgi complex is closely associated with some of the large cytoplasmic vacuoles. The accumulating secretions of the two glands differ both in fine structure and in site of storage. Head gland secretions are stored intracellularly in the cytoplasmic vacuoles, while stalk gland secretions leave the gland ceils in an apocrine fashion and are stored in an extracellular lumen. After regeneration, the mature cells of the head glands and stalk glands contain relatively little distended endoplasmic reticulum, although a Golgi complex is still present. Presumably, mature gland cells, in comparison to regenerating gland cells, produce relatively little secretion; instead, the glandular products elaborated during regeneration are probably stored in the mature glands with little augmentation or turnover.
Introduction
PEDICELLARIAE o f sea u r c h i n s are small a p p e n d a g e s arising f r o m t h e external surface of the b o d y wall. T h e m a i n parts o f a pedicellaria are t h e apical head, w h i c h is typically divided into t h r e e m o v e a b l e jaws, a n d the b a s a l stalk. Pedicellariae, o f w h i c h there are four chief types, p r o b a b l y f u n c t i o n in g r o o m i n g , in d e f e n d i n g against p r e d a t o r s a n d in c a p t u r i n g f o o d ( H y m a n , 1955). T h e globiferous pedicellariae h a v e been studied m o s t intensively, since they have epidermal glands, at least s o m e o f w h i c h c o n t a i n toxins (Alender a n d Russell, 1966). After a pedicellaria bites a n object a n d the g l a n d u l a r secretions are discharged, t h e h e a d ( a n d often From the Division of Marine Biology, Scripps Institution of Oceanography, University of California at San Diego, La Jolla, California 92037. Received 21 July 1975.
the stalk as well) is lost; in the s u b s e q u e n t weeks, a new globiferous pedicellaria is usually regenerated to replace the lost o n e (Chia, 1970). It is r e a s o n a b l e to assume t h a t the g l a n d cells o f a globiferous pedicellaria pass t h r o u g h a very active secretory p h a s e d u r i n g the course of regeneration. T h e p u r p o s e o f the present investigation is to describe the fine structure o f these g l a n d cells d u r i n g a n d after their active secretory phase. W e h a v e studied the stalk glands as well as the h e a d glands of Lytechinus pietus, a species h a v i n g globiferous pedicellariae with two k i n d s o f e p i d e r m a l glands. T h e fine s t r u c t u r e o f the general epidermis a n d of the muscles investing the glands will also b e described; however, the o t h e r tissues of the pedicellaria are b e y o n d t h e scope o f this study. Previous electron microscopic w o r k o n e p i d e r m a l glands of globiferous pedicellariae 723
724
HOLLAND AND HOLLAND
(O'Connell et al., 1974) dealt with mature head glands of Strongylocentrotus purpuratus, a species lacking stalk glands. Those authors recognized the importance of studying regeneration stages, but found that the dense spination of Strongylocentrotus prevented such a study. In Lytechinus, by contrast, the spines are relatively short and sparsely distributed; thus we were able to make repeated observations on individual pedicellariae throughout the course of their regeneration. Our anatomical description of regeneration of globiferous pedicellariae provides a frame of reference for our electron microscopy. Materials and Methods
Adult specimens of Lytechinus pictus, each weighing about 15 g, were collected in a few meters of water in Mission Bay near San Diego, California. In the laboratory, animals were held in tanks of running sea water at 16+ 2°C and fed ad libitum on brown algae heavily encrusted with ectoprocts. Regeneration was initiated by inducing each of 149 globiferous pedicellariae to bite on to a piece of meat (clam adductor muscle) held by forceps; the meat, with the head of the pedicellaria embedded in it, was then pulled away from the body of the sea urchin. Subsequently, the stalk always regressed completely, and, in two-thirds of the experiments, an entirely new globiferous pedicellaria regenerated at the same location. Relocation of individual globiferous pedicellariae was possible, since each sea urchin bore only about a score of such appendages. It was sometimes necessary to remove the distal ends of spines blocking a view of the regeneration site. For electron microscopy, several dozen regenerating and mature pedicellariae were fixed by one of the following two methods. By the procedure of O'Connell et al. (1974), we fixed some specimens in 2% glutaraldehyde in sea water at pH 7'6; fixation was started at 3°C and then continued at room temperature for two more hours. After several changes of chilled sea water, the specimens were postfixed in 1.33% osmic acid in sea water at 3°C for 2 hr. Following rapid dehydration in a chilled ethanol series, specimens were transferred through propylene oxide and then embedded in Epon 812
(Luft, 1961). The only deviation from the method of O'Connell et al. (1974) was our substitution of Epon for Araldite. By the procedure of Holland (1970), we fixed other specimens in 3% glutaraldehyde in 0.1 M phosphate buffer (pH 7.3) with 0.45 M sucrose for 90 min at room temperature. After several changes of 0'45 M sucrose in 0"1 M phosphate buffer (pH 7'3) at room temperature, the specimens were postfixed in 1 ~o osmic acid in 0.1 M phosphate buffer (pH 7.3) with 0.45 M sucrose for 6 0 m i n at 3°C. Following rapid dehydration in an ethanol series at room temperature, specimens were transferred through propylene oxide and embedded in Epon 812. Silver sections of pedicellariae fixed by the foregoing methods were cut, and contrast was enhanced with uranyl acetate and lead citrate. Results
Light microscopic observations A longitudinal section through a mature globiferous pedicellaria is shown in Fig. la. The figure is diagrammatic, since the plane of section passes through two of the three head glands and two of the three stalk glands; actually, since there are three glands of each type and since the head glands alternate with stalk glands, all four of the glands shown could not be demonstrated in a median longitudinal section. Each head gland (Fig. la, HG) is divided into a right and left portion along much of its length (this division is not evident in the figure); the two portions become continuous both at the base and at the apex of the jaw. The mature head gland, in spite of its epidermal origin, is sunk deeply into the dermis except at the jaw apex. Each mature stalk gland (Fig. la, SG) is approximately spherical. In comparison to a head gland, a stalk gland is much smaller and less deeply sunk into the dermis. The epidermal glands are largely invested with muscle layers (hatched areas in Fig. la) that are probably of dermal origin. The only other tissues shown in the figure are the general epidermis and the dermal skeleton. Comprehensive light microscopic descriptions and references to the older literature on globiferous pedicellariae can be found in the works of Cannone (1970) and Chia (1970). Fig. 2 shows a living, mature globiferous
SEA URCHIN GLOBIFEROUS PEDICELLARIAE
725
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a Fig. la. Mature globiferous pedicellaria in longitudinal section. Coarse stippling indicates the head gland (HG) and stalk gland (SG), while hatching indicates the gland musculature. Fig. lb. Regenerating globiferous pedicellaria at stage e. Coarse stippling indicates regions that will give rise to the head glands (HG) and stalk glands (SG), whilehatching indicates the location of dermal cells that will probably give rise to the gland musculature. In Figs. a and b the generalepidermisis indicatedby fine stippling and the dermal skeleton by solid black.
pedicellaria in approximate side view. Basally, the stalk articulates with the body wall of the sea urchin; the skeleton of the stalk is visible through the soft tissues. Apically, the stalk bears a head consisting of three closed jaws. Each jaw appears swollen, since it contains a relatively massive head gland. By contrast, the small stalk glands are inconspicuous swellings near the apex of the stalk; one of them is indicated by the arrow in Fig. 2. The same pedicellaria just described is shown with wide open jaws in Fig. 3. The skeleton of each jaw is visible through the soft tissues. After a mature pedicellaria bites a piece of meat and loses its head, the subsequent events are broadly divisible into (1) a stalk regression phase, (2) a lag phase, and (3) a regeneration phase. The time course of the foregoing phases is shown in Table 1 for a typical globiferous pedicellaria of Lytechinus; for the same pedicellaria, the gross anatomi-
cal changes at the regression-regeneration site are depicted in Figs. 4-16. The duration of the stalk regression phase (Figs. 4-6) varied little from one globiferous pedicellaria to the next, averaging about 9 days (Table l). It was not determined if the regression was caused by the old stalk tissues being resorbed, sloughed off, or both. The lag phase (Fig. 7) started with the complete disappearance of the old stalk and ended with the start of regeneration. From one pedicellaria to the next, the lag phase varied greatly in duration, ranging from no lag phase at all to one of at least 50 days (at which time observations were discontinued; this happened in about one-third of the instances studied). The regeneration phase (Figs. 8-16) began with the appearance of a minute protuberance at the regeneration site and continued until a mature globiferous pedicellaria finished developing there. From start to finish, regeneration took 6 or 7 weeks,
HOLLAND AND HOLLAND
726 with little v a r i a t i o n f r o m o n e pedicellaria to the next ( T a b l e 1). O n the a p p r o x i m a t e l y 100 r e g e n e r a t i o n sites w h e r e a n a p p e n d a g e grew, it i n v a r i a b l y t u r n e d o u t to be a globiferous pedicellaria. I n T a b l e 1, the r e g e n e r a t i o n p h a s e is subdivided i n t o nine a r b i t r a r y stages design a t e d a-i. F o r each stage, the table also gives the size a n d definitive m o r p h o l o g y o f the regenerate. Stages a - d (Figs. 8-11) occur l o n g before epidermal gland cells first a p p e a r a n d are b e y o n d the scope o f the present paper. T h e earliest regenerating globiferous pedicellaria described by electron microscopy was in stage e (Fig. 12). A d i a g r a m m a t i c l o n g i t u d i n a l section t h r o u g h such a regenerate is s h o w n in Fig. l b . Coarse stippling indicates the e p i d e r m a l regions t h a t will give rise to the h e a d glands ( H G ) a n d stalk glands (SG). H a t c h i n g indicates the l o c a t i o n o f undifferentiated d e r m a l cells p r e s u m a b l y destined to b e c o m e
t h e m u s c u l a t u r e o f the h e a d glands a n d o f the stalk glands.
Electron microscopic observations F o r m o s t cells, the two different fixation p r o c e d u r e s gave similar results. H o w e v e r the 1974 m e t h o d o f O ' C o n n e l l et al. (Fig. 20), in c o m p a r i s o n to the 1970 p r o c e d u r e o f H o l l a n d (Fig. 21), preserved m o r e of the secretion in the m a t u r e h e a d glands. U n f o r tunately, neither m e t h o d preserved m u c h o f the secretion in regenerating h e a d glands. E x c e p t i n g Fig. 20, all electron m i c r o g r a p h s in the present p a p e r are o f pedicellaria fixed a c c o r d i n g to H o l l a n d (1970). By r e g e n e r a t i o n stage e (Fig. 12), the globiferous pedicellaria consists o f a core o f dermis covered b y a n epidermis. A t this stage, b o t h the dermis a n d epidermis include m a n y undifferentiated cells. Such cells are j u d g e d to be undifferentiated by m o r p h o logical criteria a l o n e ; they m i g h t n o t
Fig. 2. Approximate side view of a mature globiferous pedicellaria with closed jaws. The arrow points to one of the stalk glands, x 12. Fig. 3. Looking into the wide open jaws o f a globiferous pedicellaria, x 12. Fig. 4. The stalk of a globiferous pedicellaria an hour after head loss. In this figure and in the subsequent 12 figures, the arrow points to the same regression-regeneration site. × 12. Fig. 5. Regressing stalk 5 days after head loss, x 12. Fig. 6. Regressing stalk 7 days after head loss. × 12. Fig. 7. Lag phase; stalk regression is complete, but regeneration has not started. x12. Fig. 8. Regeneration stage a. The regenerate is at the tip of the arrow and should not be confused with ophiocephalous pedicellariae regenerating nearby. × 12. Fig. 9. Regeneration stage b. x 12. Fig. 10. Regeneration stage c. × 12. Fig. 11. Regeneration stage d. × 12. Fig. 12. Regeneration stage e. × 12. Fig. 13. Regeneration stage f. × 12. Fig. 14. Regeneration stage g. x 12. Fig. 15. Regeneration stage h. × 12. Fig. 16. Regeneration stage i. x 12.
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H O L L A N D AND H O L L A N D
Table 1. The thne course o f regression and regeneration following head loss from a typical glob~ferous pedicel/aria o f Lytechinus Regeneration phase Mm total Figure
Days after head loss
Stalk regression phase
4 5 6
0 5 7
Lag phase
7
9
8 9 10 11 12 13 14 15 16 2, 3
27 31 34 37 39 41 44 48 60 72
Regeneration phase
Days after start
Stage
Length
Definitive features
0 4 7 10 12 14 17 21 33 45
a b c d e f g h i Mature
0.1 0.3 0.5 0' 8 1 •0 1.3 1 '5 1.9 2.9 3.5
Skeletal ossicles absent Skeletal ossicles present Jaws start diverging Jaws widely divergent Jaws start converging Jaws convergent Head base wider than apex Jaws can open and close Head as wide as long Maximum size attained
necessarily be undifferentiated by behavioral, chemical or developmental criteria (as defined by G r o b s t e i n , 1959). Undifferentiated cells o f the dermis and epidermis (Fig. 17, arrow) are morphologically identical, E a c h cell is spherical to oval and usually lacks c o n s p i c u o u s cytoplasmic processes. The nucleus contains one or two nucleoli. The c o n d e n s e d c h r o m a t i n is distributed in a
thin layer adjacent to the inner nuclear m e m b r a n e and is scattered in patches elsew h e r e in the nucleus. The sparse cytoplasm c o n t a i n s a b u n d a n t free r i b o s o m e s and an occasional flattened cisterna o f r o u g h e n d o plasmic reticulum. Also present are s o m e mitocilondria, lipid droplets a n d a l p h a rosettes o f glycogen. A small Golgi complex and a pair o f centrioles m a y also be encoun-
Fig. 17. A section through the general epidermis of a globiferous pedicellaria at regeneration stage e. In addition to several columnar epithelial cells, the epidermis includes undifferentiated cells (arrow) and a colorless spherule cell (at the left). The external surface is at the top of the figure, and the inconspicuous basal lamina between epidermis and dermis is at the lower right, x 4000. Fig. 18. A section through the epidermal region wherein head gland cells are beginning to differentiate at regeneration stage f. The external surface is at the top and the dermis is at the bottom, x 3800. Fig. 19. The juxtanuclear portion of a head gland cell at regeneration stage f. Prominent structures from top to bottom are a part of the nucleus, the Golgi complex and part of a cytoplasmic vacuole. × 16,000. Fig. 20. A section through the periphery of a mature head gland fixed by the method of O'Connell et al. (1974). Conspicuous structures in the gland cell are the cytoplasmic vacuoles of secretion and the nucleus. The inconspicuous Golgi complex is indicated by the arrow. Part of the musculature investing the head gland shows at the bottom left. x 6000.
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730 tered. Presumably, some of the undifferentiated cells of the epidermis are destined to become gland cells, while some undifferentiated cells of the dermis are destined to become the muscles investing the glands. The epidermis and dermis of all stages included some electron-lucent cells, a few red spherule cells and a few colorless spherule cells (like the one at the left in Fig. 17). The spherule cells, at least, are doubtlessly related to the coelomocytes which circulate in the perivisceral coelom. We do not know what role, if any, such cell types play during regeneration, and their fine structure will not be described here. By regeneration stage e, the epidermis is separated from the dermis by a basal lamina (Fig. 17, bottom right). At this stage, most of the regenerate is covered with a general epidermis, which includes the regions where the head glands and neck glands will develop. However, on the inner surface of each jaw, the epidermis is already differentiating into two sensory hillocks, one basal and one apical. The fine structure of the sensory hillocks has been described by Cobb (1968) and will not be included here. At stage e, the general epidermis of the head and stalk is composed of numerous columnhr epithelial cells (Fig. 17) in addition to undifferentiated cells; by the stage of the mature pedicellaria, the epithelial ceils have become cuboidal to squamous. The apical end of each columnar cell lacks cilia, but bears numerous microvilli that are associated with a fibrous glycocalyx (the glycocalyx was Well preserved by the method of O'Connell et al., 1974, but not by the method of Holland, 1970). Apically, the columnar epithelial cells are associated with one another by intermediary junctions (in the terminology of Satir and Gilula, 1970). The apical cytoplasm contains a pair of centrioles, a Golgi complex and vacuoles containing material ranging from electron lucent to very dense. Throughout the cytoplasm, free ribosomes and profiles of rough endoplasmic reticulum are abundant; also present are mitochondria, lipid droplets and glycogen rosettes. The nucleus resembles that already described for an undifferentiated cell. Basally, some of the columnar epithelial cells give off a cytoplasmic process that might possibly be an axon. That is, some of the epithelial cells of the general epidermis
HOLLAND AND HOLLAND may actually be neuroepithelial cells (as was first proposed by Kawaguti and Kamishima, 1964).
Differentiation of the head gland cells and their musculature. In stage f, head gland cells first appear among the cells of the general epidermis. Although a few undifferentiated cells still occur in the epidermis at stage f, they disappear during subsequent stages, presumably by differentiating into gland cells. Each young head gland cell is elongated, but its apical end does not reach the outer surface of the epidermis (Fig. 18). The most conspicuous cytoplasmic inclusions are membrane-bound vacuoles ranging from 0"2 t, to about 5 t~ in diameter. Some of the vacuoles appear empty, while others are filled with a flocculent material of low electron density. Some vacuoles also contain small patches of relatively dense granular material. In general, the contents of the vacuoles appear to have been partially removed by both of the fixation procedures that we used. The juxtanuclear cytoplasm contains a Golgi complex with dilated cisternae (Fig. 19), which are probably involved in packaging secretory products into the vacuoles. The rough endoplasmic reticulum of the youngest head gland cells is not distended with intracisternal material. The cytoplasm also contains free ribosomes, mitochondria, lipid droplets and glycogen rosettes. The nucleus resembles that already described for an undifferentiated cell. During stage g, the population of head gland cells delaminates from the general epidermis and sinks down into the dermal layer. The delamination procedes from the base to the apex of the jaw, and the gland cells retain a close association with overlying epidermal cells only at the apex of the jaw. The solid cord of delaminated gland cells is invested with a basal lamina. Within each gland cell, the nucleus is located at the end nearest the basal lamina, while the vacuolated cytoplasm extends toward the center of the lumenless gland. Each head gland cell enlarges during stage g, as the cytoplasmic vacuoles increase in number and size. The rough endoplasmic reticulum becomes more extensive and its intracisternal spaces become dilated with a finely granular material of low electron density. N o conspicuous changes occur in the other cytoplasmic organelles or
SEA URCHIN GLOBIFEROUS P E D I C E L L A R I A E in the nucleus. During stage h, gland cell vacuoles continue to enlarge in size and number, and the intracisternal spaces of the rough endoplasmic reticulum remain distended with granular contents. By stage i, the rough endoplasmic reticulum is less distended and the Golgi complex is less conspicuous. By the time of the mature stage, the few profiles of each endoplasmic reticulum remaining are not distended at all. Moreover, the Golgi complex consists mainly of a stack of flattened saccules (Fig. 20, arrow). The cytoplasm of a mature head gland cell is dominated by the vacuoles, some of which have reached diameters of nearly 30ix (Fig. 21, at right). The less dense flocculent contents of the vacuoles are preserved better by the 1974 method of O'Connell et al. (Fig. 20) than by the 1970 method of Holland (Fig. 21). Much of the cytoplasm is compressed into thin sheets which intervene between the vacuoles. In addition to the organelles just mentioned, the cytoplasm contains free ribosomes, mitochondria, lipid droplets, glycogen rosettes, myelin figures, and probable lysosomes. These latter organelles may contain dense material, compacted membranous material, or numerous small vesicles. In the mature stage, the nucleus is located near the end of the cell that is closest to the periphery of the head gland (Figs. 20, 21). As O'Connell et al. (1974) demonstrated, the mature head gland has no lumen; many light microscopists had previously mistaken the highly vacuolated cytoplasm for a lumen. When a mature globiferous pedicellaria bites an object, the vacuoles of the head gland cells rupture, releasing their secretions. These secretions exit from the gland where it is closely associated with the epidermis at the apex of the jaw. A head gland that had bitten an object was fixed shortly thereafter for electron microscopy (Fig. 22). The discharged gland appeared as a packed mass of cytoplasmic profiles. It could not be determined whether these profiles were largely interconnected or were small fragments of the gland cells. As the head gland cells begin to delaminate from the epidermis in stage g, some of the surrounding undifferentiated cells begin to develop into smooth muscle cells. Because the developing muscle cells are separated from the gland by a basal lamina, it is
731
probable that they are of dermal origin; however, an epidermal origin for some of the developing muscle cells cannot be ruled out on strictly morphological evidence. As delamination proceeds, the muscle cells almost completely invest the head gland, except near the tip of the jaw where the secretion will be expelled. The developing muscle cells first elongate, and then a few scattered muscle fibrils appear in the cytoplasm among the organelles already described for an undifferentiated cell. During stages h and i (Fig. 23), cell elongation continues, muscle fibrils become abundant, and the centrioles disappear; the other cytoplasmic organelles of the undifferentiated cell are still present, but are inconspicuous. Several muscles of the mature head gland are shown at the bottom left in Fig. 20. Differentiation o f the stalk gland cells' and their musculature. The stalk glands develop from the general epidermis, which has already been described above for stage e; the site of stalk gland development is labelled SG in Fig. 1b. During regeneration stage g, undifferentiated cells delaminate from the columnar epithelial cells of the epidermis to form an approximately spherical mass. The undifferentiated cells in this mass do not begin to develop into gland cells until stage h. Thus, in the stalk gland, delamination precedes differentiation, while, in the head gland, differentiation precedes delamination. As differentiation of the stalk gland cells begins in stage h, a small lumen appears in the center of the gland. This space seems to open up within the mass of differentiating gland cells, which are arranged around it. Each developing stalk gland cell is elongate (Fig. 24). The nucleus is located near the end of the cell farthest from the lumen. The nuclear fine structure resembles that of an undifferentiated cell. At the luminal end, each gland cell is associated with its neighbors by a junctional complex consisting of an intermediary junction and a poorly developed septate desmosome (in the terminology of Satir and Gilula, 1970). The most conspicuous cytoplasmic inclusions are membranebound vacuoles about 1"5 tx in diameter. The contents of each vacuole are divisible into a core of finely granular material
HOLLAND AND HOLLAND
732 a n d a s u r r o u n d i n g cortex o f sparsely distributed flocculent material. A Golgi complex is present, a n d its dilated cisternae pres u m a b l y help package the c o n t e n t s o f the cytoplasmic vacuoles already described. The c y t o p l a s m also c o n t a i n s a b u n d a n t free ribosomes, m i t o c h o n d r i a , lipid droplets a n d glycogen rosettes. The r o u g h e n d o p l a s m i c reticulum consists of a few flattened cisternae t h a t are n o t dilated with intracisternal material. R u n n i n g parallel to the long axis o f the gland cell are a few cytoplasmic filaments a b o u t 3 0 0 , ~ in diameter. These organelles, a l t h o u g h wider t h a n typical microfilaments, might possibly play some role in g l a n d cell e l o n g a t i o n or in cell movem e n t s related to l u m e n formation. The c y t o p l a s m at the luminal end of the cell c o n t a i n s a pair o f centrioles; they are sometimes associated with striated rootlets or with the cytoplasmic filaments described a b o v e (Fig. 24, inset). By r e g e n e r a t i o n stage i, the l u m e n o f the stalk g l a n d is enlarged a n d c o n t a i n s n u m e r o u s secretory granules (Fig. 25, at center a n d
right). T h e l u m e n is s u r r o u n d e d by a single layer o f g l a n d cells, which are n o w low c o l u m n a r in shape (Fig. 25, at left). By this stage, the gland cell cytoplasm rarely includes centrioles a n d the r o u g h e n d o p l a s m i c retic u l u m h a s b e c o m e very p r o m i n e n t . T h e intracisternal spaces o f the r o u g h e n d o p l a s m i c r e t i c u l u m are distended with finely g r a n u l a r m a t e r i a l o f m o d e r a t e electron density (Fig. 26). Parts of the cisternae of the e n d o p l a s m i c reticulum nearest the f o r m i n g face of the G o l g i c o m p l e x lack ribosomes. M o s t of the c y t o p l a s m i c vacuoles are n o w a b o u t 2-3 t~ in diameter, a n d their c o n t e n t s are divisible into regions of finely g r a n u l a r material a n d regions c o n t a i n i n g a loose m e s h w o r k of fibrous material. In m o s t cells, a vacuole is closely associated with the m a t u r e face o f the G o l g i complex. In some places (Fig. 26, arrows), G o l g i vesicles are p r o b a b l y fusing with the m e m b r a n e of the vacuole. In the lumen, m o s t of the secretory granules are a b o u t 2-3 ~ in diameter, a n d their c o n t e n t s resemble those o f the cytoplasmic vacuoles. T h e c o n t e n t s of each secretory granule
Fig. 21. A section through the periphery of a mature head gland fixed by the method of Holland (1970). The gland cell resembles the one in the preceding figure, except for the loss of the flocculent material from the secretory vacuoles, x 4500. Fig. 22. A section through a mature head gland fixed shortly after biting and expulsion of the secretions, x 4500. Fig. 23. Several of the smooth muscles investing a head gland (at left) at regeneration stage i. x 4500. Fig. 24. A section through a stalk gland at regeneration stage h. The lumen is at the top right, and a dermal cell just starting to differentiate into a muscle cell is at the far left. ×5000. The inset (× 14,000) shows the luminal tip of a gland ceil containing a centriole associated with cytoplasmic filaments. Fig. 25. A section through the periphery of a stalk gland at regeneration stage i. The lumen filled with secretory granules is at the center and right, while low columnar gland cells are at the left. × 5000. Fig. 26. A section through a stalk gland cell at stage i. From right to left, conspicuous structures are the distended rough endoplasmic reticulum, the Golgi complex, a cytoplasmic vacuole and a portion of the nucleus. The arrows indicate places where Golgi vesicles appear to be fusing with the periphery of the vacuole, x 26,000. Fig. 27. Secretory granules in the lumen of a stalk gland at regeneration stage i. x 27,000. Fig. 28. Secretory granule in the lumen of a stalk gland of a mature globiferous pedicellaria. × 27,000.
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SEA URCHIN GLOBIFEROUS P E D I C E L L A R I A E are surrounded by two concentric membranes which are separated from one another by a thin layer of cytoplasm (Fig. 27). There can be little doubt that the outermost membrane corresponds to part of the plasma membrane of a gland cell. The cytoplasmic vacuoles are apparently budded off into the lumen along with a small amount of gland cell cytoplasm and plasma membrane around them. In the mature globiferous pedicellariae, the large lumen of each stalk gland is filled with secretory granules. Within the mature granule (Fig. 28), the finely granular component of the preceding regeneration stage has become organized into numerous spherules, each about 700 A~ in diameter. Mature stalk gland cells are cuboidal to squamous. Each mature cell contains the same organelles that were present in the stalk gland cells of regeneration stage i. The Golgi complex and a few cytoplasmic vacoules are conspicuous. However, the rough endoplasmic reticulum is much less extensive than before. The differentiation of the stalk gland musculature begins at regeneration stage h, one stage later than the start of muscle differentiation around the head gland. The stalk gland muscles probably originate from undifferentiated cells in the dermis near the basal lamina covering the gland. A developing muscle cell that is just elongating is shown at stage h (Fig. 25, far left). By stage i, the centrioles have disappeared, and the cytoplasm contains abundant muscle fibrils. The smooth muscles surrounding the mature stalk gland resemble those illustrated for the mature head gland (Fig. 20, bottom left). The muscle cells almost completely envelop the stalk gland except on its epidermal side, where the secretion will be expelled. Discussion
The results of the present study indicate that most of the secretory activity of the head glands and stalk glands of globiferous pedicellariae takes place during a limited period during the later stages of regeneration. In mature head glands and neck glands there is probably little elaboration of secretory products; instead, the secretions produced during regeneration are presumably stored in the mature glands with little augmentation
735
or turnover. O'Connell et al. (1974) have proposed 'a continuous activity in secretory cells of fully developed pedicellariae', since the Golgi complex and related vesicles were demonstrable by electron microscopy. However, we believe that such organelles are relatively inactive and are simply left over from an earlier cell phase of intense synthesis. As suggested by O'Connell et al. (1974), autoradiographic and biochemical studies might resolve this problem. Unfortunately, an autoradiographic study of the head gland will require a better fixation method than now available to preserve the secretory products in situ. Moreover, possible changes in gland cell permeability and in size of precursor pools will have to be taken into account when comparing different regeneration stages. In Lytechinus pictus, the head gland and the stalk gland of a mature globiferous pedicellaria are made up exclusively of highly differentiated cells; no morphologically undifferentiated cells are mixed with them. Thus the gland cell populations are of the static type and not of the expanding or renewing types (in the terminology of Messier and Leblond, 1960). A similar lack of replacement cells was reported for the head gland of Strongylocentrotus purpuratus by O'Connell et al. (1974). The same tissue was classified as a static cell population in an autoradiographic study with tritiated thymidine (Holland, 1964); all other tissues of the adult sea urchin were, by contrast, expanding or renewing cell populations. Conspicuous components of the secretions in the head and stalk glands of Lytechinus pictus are acid mucopolysa.ccharides and mucoproteins (Holland and Holland, unpublished light microscopic histochemistry); similar histochemical results have previously been published for other species of sea urchins (P6r~s, 1950; Chia, 1970; O'Connell et al., 1974). The histochemistry is in agreement with the fine structure of the gland cells, which contain distended endoplasmic reticulum as well as Golgi complexes associated with secretory vacuoles. Such gland cells resemble better studied cells that synthesize mucopolysaccharides and glycoproteins. According to the review of Spiro (1969), peptides are formed on the ribosomes and enter the intracisternal spaces of the endoplasmic reticulum, where some carbo-
736 hydrates may be added. Subsequently, the secretion proceeds to the Golgi complex for further augmentation with carbohydrates. Finally, the Golgi saccules concentrate and package the secretions. In Lytechinus pictus, the chief function of the head gland of the globiferous pedicellaria is probably to elaborate a toxic secretion, as in other sea urchins (see review by Alender and Russell, 1966). Although secretions of the mature head gland are toxic, it is not known if secretions of the regenerating head gland are toxic as soon as they are produced. This question might be answered if extracts of regenerating head glands were tested on a suitable biological assay. The function of the stalk gland secretions of Lytechinus pictus remains enigmatic. P6r~s (1950), in his study of Sphaerechinus gramdaris, presented evidence that the stalk gland secretions were not particularly toxic; he presumed that they acted physically to immobilize small organisms. When a globiferous pedicellaria bites an object, the musculature covering the glands contacts. This contraction apparently causes a massive exocytosis of the contents of the cytoplasmic vacuoles from the head gland cells. F r o m each cell, the secretions probably exit via simultaneous ruptures of the vacuole
HOLLAND AND HOLLAND membranes and plasma membrane at many points on the cell surface. This is an example of holocrine secretion. After entering the intercellular spaces of the head gland, the secretions are rapidly expelled to the exterior near the apex of the jaw. It is not clear whether they exit by a well-defined duct or by a localized rupture of the overlying epidermis; the gland ducts reported by some light microscopists (e.g. Hamann, 1887) might possibly be slight ruptures of epithelia taking place at fixation. Pressure from the musculature covering the stalk glands causes an expulsion of secretory granules from the gland lumen. This massive expulsion, if observed at the organ level might be considered holocrine; however, the production of the secretory granules, if observed at the cellular level, is clearly apocrine. As in the case of the head gland, it is not clear whether the stalk gland secretions exit via a duct or a rupture of the epidermis.
Acknowledgement We are deeply indebted to Mr Fritz G o r o for his invaluable help with the photomicrography of living pedicellariae (Figs. 2-16).
SEA URCHIN
GLOBIFEROUS
PEDICELLARIAE
737
References ALENDER, C. B. and RUSSELL, F. E. 1966. Pharmacology. In Physiology of Eehinodermata (ed. R. A Boolootian), pp. 529 543. Wiley Interscience, New York. CANNONE, A. J. 1970. The anatomy and venom-emitting mechanism of the globiferous pedicellariae of the urchin Parechinus angnlosus (Leske) with notes on their behaviour. Zool. Afrieana, 5, 179-190. Cl-I~A, F. S. 1970. Histology of the globiferous pedicetlariae of Psammecllinus miliaris (Echinodermata: Echinoidea). J. Zool., Lond., 160, 9-16. COBB, J. L. S. 1968. The fine structure of the pedicellariae of Echimts esculenttts (L.). II. The sensory system. J. R. microsc. Sot., 88, 223 233. GROBSTEIN, C. 1959. Differentiation of vertebrate cells. In The Cell (eds. J. Brachet and A. E. Mirsky), Vol. I, pp. 437-496. Academic Press, New York. HAMANN, O. 1887. Beitrfige zur Histologie der Echinodermen. lII. Anatomie und Histologie der Echiniden und Spatangiden, Jena Z. Naturwiss., 21, 87-266. HOLLAND, N. D. 1964. An autoradiographic investigation of the gonads of the purple sea urchin (Strong),locentrotus purpuratus): an autoradiographic study employing tritiated thymidine. Ph.D. Dissertation, Stanford University. HOLLAND, N. D. 1970. The fine structure of the axial organ of the feather star Nemaster r,biginosa (Echinodermata: Crinoidea). Tissue & Cell, 2, 625 636. HYMAN, L. H. 1955. The invertebrates, Vol. IV. Eehinodermata. McGraw-Hill, New York. KAWAGUTI, S. and KAMISmMA, Y, 1964. Electron-microscopic study of the integument of the echinoid, Diadema setosum. Annot. zool. jap., 37, 147 152. LUFT, J. H. 1961. Improvements in epoxy resin embedding methods. J. biophys, bioehem. Cytol., 9, 409-414. MESSIER, B. and LEBLOND, C. P. 1960. Cell proliferation and migration as revealed by autoradiography after injection of thymidine-H 3 into male rats and mice. Am. J. Anat., 106, 247-265. O'CONNELL, M. G., ALENDER,C. B. and WOOD, E. M. 1974. A fine structure study of venom gland cells in globiferous pedicellariae from Strongylocentrotns parpuratns (Stimpson). J. Morph., 142, 41 I 432. P[~RI~S,J. M. 1950. Recherches sur les pddicellaires glandulaires de Sphaerechinus granldaris (Lamarck). Arch. zool. exp. gdn. (Notes et Revue), 86, ll8 136. SATIR, P. and GILULA,N. B. 1970. The cell junction in a lamellibranch gill ciliated epithelium. Localization of pyroantimonate. J. Cell Biol., 47, 468 487. SPiRO, R. G. 1969. Glycoproteins: their biochemistry, biology and role in human disease. New Engl. J. Med., 281, 991-1001.
LINDA Z. H O L L A N D and N I C H O L A S D. H O L L A N D
THE FINE STRUCTURE OF EPIDERMAL GLANDS OF REGENERATING AND MATURE GLOBIFEROUS PEDICELLARIAE OF A SEA URCHIN (LYTECHINUS PICTUS) ABSTRACT. After a globiferous pedicellaria is lost from a sea urchin, a new appendage of the same kind is usually regenerated in the weeks that follow. During the latter part of regeneration, head glands and stalk glands, both of epidermal origin, develop from undifferentiated cells. Head gland cells begin morphological differentiation in the epidermis and then delaminate into the underlying dermis. In the formation of the stalk gland, by contrast, undifferentiated cells delaminate from the epidermis and then begin morphological differentiation in the dermis. During late regeneration, ceils in the head and stalk glands are characterized by extensive rough endoplasmic reticulum distended with intracisternal material; moreover, the Golgi complex is closely associated with some of the large cytoplasmic vacuoles. The accumulating secretions of the two glands differ both in fine structure and in site of storage. Head gland secretions are stored intracellularly in the cytoplasmic vacuoles, while stalk gland secretions leave the gland ceils in an apocrine fashion and are stored in an extracellular lumen. After regeneration, the mature cells of the head glands and stalk glands contain relatively little distended endoplasmic reticulum, although a Golgi complex is still present. Presumably, mature gland cells, in comparison to regenerating gland cells, produce relatively little secretion; instead, the glandular products elaborated during regeneration are probably stored in the mature glands with little augmentation or turnover.
Introduction
PEDICELLARIAE o f sea u r c h i n s are small a p p e n d a g e s arising f r o m t h e external surface of the b o d y wall. T h e m a i n parts o f a pedicellaria are t h e apical head, w h i c h is typically divided into t h r e e m o v e a b l e jaws, a n d the b a s a l stalk. Pedicellariae, o f w h i c h there are four chief types, p r o b a b l y f u n c t i o n in g r o o m i n g , in d e f e n d i n g against p r e d a t o r s a n d in c a p t u r i n g f o o d ( H y m a n , 1955). T h e globiferous pedicellariae h a v e been studied m o s t intensively, since they have epidermal glands, at least s o m e o f w h i c h c o n t a i n toxins (Alender a n d Russell, 1966). After a pedicellaria bites a n object a n d the g l a n d u l a r secretions are discharged, t h e h e a d ( a n d often From the Division of Marine Biology, Scripps Institution of Oceanography, University of California at San Diego, La Jolla, California 92037. Received 21 July 1975.
the stalk as well) is lost; in the s u b s e q u e n t weeks, a new globiferous pedicellaria is usually regenerated to replace the lost o n e (Chia, 1970). It is r e a s o n a b l e to assume t h a t the g l a n d cells o f a globiferous pedicellaria pass t h r o u g h a very active secretory p h a s e d u r i n g the course of regeneration. T h e p u r p o s e o f the present investigation is to describe the fine structure o f these g l a n d cells d u r i n g a n d after their active secretory phase. W e h a v e studied the stalk glands as well as the h e a d glands of Lytechinus pietus, a species h a v i n g globiferous pedicellariae with two k i n d s o f e p i d e r m a l glands. T h e fine s t r u c t u r e o f the general epidermis a n d of the muscles investing the glands will also b e described; however, the o t h e r tissues of the pedicellaria are b e y o n d t h e scope o f this study. Previous electron microscopic w o r k o n e p i d e r m a l glands of globiferous pedicellariae 723
724
HOLLAND AND HOLLAND
(O'Connell et al., 1974) dealt with mature head glands of Strongylocentrotus purpuratus, a species lacking stalk glands. Those authors recognized the importance of studying regeneration stages, but found that the dense spination of Strongylocentrotus prevented such a study. In Lytechinus, by contrast, the spines are relatively short and sparsely distributed; thus we were able to make repeated observations on individual pedicellariae throughout the course of their regeneration. Our anatomical description of regeneration of globiferous pedicellariae provides a frame of reference for our electron microscopy. Materials and Methods
Adult specimens of Lytechinus pictus, each weighing about 15 g, were collected in a few meters of water in Mission Bay near San Diego, California. In the laboratory, animals were held in tanks of running sea water at 16+ 2°C and fed ad libitum on brown algae heavily encrusted with ectoprocts. Regeneration was initiated by inducing each of 149 globiferous pedicellariae to bite on to a piece of meat (clam adductor muscle) held by forceps; the meat, with the head of the pedicellaria embedded in it, was then pulled away from the body of the sea urchin. Subsequently, the stalk always regressed completely, and, in two-thirds of the experiments, an entirely new globiferous pedicellaria regenerated at the same location. Relocation of individual globiferous pedicellariae was possible, since each sea urchin bore only about a score of such appendages. It was sometimes necessary to remove the distal ends of spines blocking a view of the regeneration site. For electron microscopy, several dozen regenerating and mature pedicellariae were fixed by one of the following two methods. By the procedure of O'Connell et al. (1974), we fixed some specimens in 2% glutaraldehyde in sea water at pH 7'6; fixation was started at 3°C and then continued at room temperature for two more hours. After several changes of chilled sea water, the specimens were postfixed in 1.33% osmic acid in sea water at 3°C for 2 hr. Following rapid dehydration in a chilled ethanol series, specimens were transferred through propylene oxide and then embedded in Epon 812
(Luft, 1961). The only deviation from the method of O'Connell et al. (1974) was our substitution of Epon for Araldite. By the procedure of Holland (1970), we fixed other specimens in 3% glutaraldehyde in 0.1 M phosphate buffer (pH 7.3) with 0.45 M sucrose for 90 min at room temperature. After several changes of 0'45 M sucrose in 0"1 M phosphate buffer (pH 7'3) at room temperature, the specimens were postfixed in 1 ~o osmic acid in 0.1 M phosphate buffer (pH 7.3) with 0.45 M sucrose for 6 0 m i n at 3°C. Following rapid dehydration in an ethanol series at room temperature, specimens were transferred through propylene oxide and embedded in Epon 812. Silver sections of pedicellariae fixed by the foregoing methods were cut, and contrast was enhanced with uranyl acetate and lead citrate. Results
Light microscopic observations A longitudinal section through a mature globiferous pedicellaria is shown in Fig. la. The figure is diagrammatic, since the plane of section passes through two of the three head glands and two of the three stalk glands; actually, since there are three glands of each type and since the head glands alternate with stalk glands, all four of the glands shown could not be demonstrated in a median longitudinal section. Each head gland (Fig. la, HG) is divided into a right and left portion along much of its length (this division is not evident in the figure); the two portions become continuous both at the base and at the apex of the jaw. The mature head gland, in spite of its epidermal origin, is sunk deeply into the dermis except at the jaw apex. Each mature stalk gland (Fig. la, SG) is approximately spherical. In comparison to a head gland, a stalk gland is much smaller and less deeply sunk into the dermis. The epidermal glands are largely invested with muscle layers (hatched areas in Fig. la) that are probably of dermal origin. The only other tissues shown in the figure are the general epidermis and the dermal skeleton. Comprehensive light microscopic descriptions and references to the older literature on globiferous pedicellariae can be found in the works of Cannone (1970) and Chia (1970). Fig. 2 shows a living, mature globiferous
SEA URCHIN GLOBIFEROUS PEDICELLARIAE
725
SO
a Fig. la. Mature globiferous pedicellaria in longitudinal section. Coarse stippling indicates the head gland (HG) and stalk gland (SG), while hatching indicates the gland musculature. Fig. lb. Regenerating globiferous pedicellaria at stage e. Coarse stippling indicates regions that will give rise to the head glands (HG) and stalk glands (SG), whilehatching indicates the location of dermal cells that will probably give rise to the gland musculature. In Figs. a and b the generalepidermisis indicatedby fine stippling and the dermal skeleton by solid black.
pedicellaria in approximate side view. Basally, the stalk articulates with the body wall of the sea urchin; the skeleton of the stalk is visible through the soft tissues. Apically, the stalk bears a head consisting of three closed jaws. Each jaw appears swollen, since it contains a relatively massive head gland. By contrast, the small stalk glands are inconspicuous swellings near the apex of the stalk; one of them is indicated by the arrow in Fig. 2. The same pedicellaria just described is shown with wide open jaws in Fig. 3. The skeleton of each jaw is visible through the soft tissues. After a mature pedicellaria bites a piece of meat and loses its head, the subsequent events are broadly divisible into (1) a stalk regression phase, (2) a lag phase, and (3) a regeneration phase. The time course of the foregoing phases is shown in Table 1 for a typical globiferous pedicellaria of Lytechinus; for the same pedicellaria, the gross anatomi-
cal changes at the regression-regeneration site are depicted in Figs. 4-16. The duration of the stalk regression phase (Figs. 4-6) varied little from one globiferous pedicellaria to the next, averaging about 9 days (Table l). It was not determined if the regression was caused by the old stalk tissues being resorbed, sloughed off, or both. The lag phase (Fig. 7) started with the complete disappearance of the old stalk and ended with the start of regeneration. From one pedicellaria to the next, the lag phase varied greatly in duration, ranging from no lag phase at all to one of at least 50 days (at which time observations were discontinued; this happened in about one-third of the instances studied). The regeneration phase (Figs. 8-16) began with the appearance of a minute protuberance at the regeneration site and continued until a mature globiferous pedicellaria finished developing there. From start to finish, regeneration took 6 or 7 weeks,
HOLLAND AND HOLLAND
726 with little v a r i a t i o n f r o m o n e pedicellaria to the next ( T a b l e 1). O n the a p p r o x i m a t e l y 100 r e g e n e r a t i o n sites w h e r e a n a p p e n d a g e grew, it i n v a r i a b l y t u r n e d o u t to be a globiferous pedicellaria. I n T a b l e 1, the r e g e n e r a t i o n p h a s e is subdivided i n t o nine a r b i t r a r y stages design a t e d a-i. F o r each stage, the table also gives the size a n d definitive m o r p h o l o g y o f the regenerate. Stages a - d (Figs. 8-11) occur l o n g before epidermal gland cells first a p p e a r a n d are b e y o n d the scope o f the present paper. T h e earliest regenerating globiferous pedicellaria described by electron microscopy was in stage e (Fig. 12). A d i a g r a m m a t i c l o n g i t u d i n a l section t h r o u g h such a regenerate is s h o w n in Fig. l b . Coarse stippling indicates the e p i d e r m a l regions t h a t will give rise to the h e a d glands ( H G ) a n d stalk glands (SG). H a t c h i n g indicates the l o c a t i o n o f undifferentiated d e r m a l cells p r e s u m a b l y destined to b e c o m e
t h e m u s c u l a t u r e o f the h e a d glands a n d o f the stalk glands.
Electron microscopic observations F o r m o s t cells, the two different fixation p r o c e d u r e s gave similar results. H o w e v e r the 1974 m e t h o d o f O ' C o n n e l l et al. (Fig. 20), in c o m p a r i s o n to the 1970 p r o c e d u r e o f H o l l a n d (Fig. 21), preserved m o r e of the secretion in the m a t u r e h e a d glands. U n f o r tunately, neither m e t h o d preserved m u c h o f the secretion in regenerating h e a d glands. E x c e p t i n g Fig. 20, all electron m i c r o g r a p h s in the present p a p e r are o f pedicellaria fixed a c c o r d i n g to H o l l a n d (1970). By r e g e n e r a t i o n stage e (Fig. 12), the globiferous pedicellaria consists o f a core o f dermis covered b y a n epidermis. A t this stage, b o t h the dermis a n d epidermis include m a n y undifferentiated cells. Such cells are j u d g e d to be undifferentiated by m o r p h o logical criteria a l o n e ; they m i g h t n o t
Fig. 2. Approximate side view of a mature globiferous pedicellaria with closed jaws. The arrow points to one of the stalk glands, x 12. Fig. 3. Looking into the wide open jaws o f a globiferous pedicellaria, x 12. Fig. 4. The stalk of a globiferous pedicellaria an hour after head loss. In this figure and in the subsequent 12 figures, the arrow points to the same regression-regeneration site. × 12. Fig. 5. Regressing stalk 5 days after head loss, x 12. Fig. 6. Regressing stalk 7 days after head loss. × 12. Fig. 7. Lag phase; stalk regression is complete, but regeneration has not started. x12. Fig. 8. Regeneration stage a. The regenerate is at the tip of the arrow and should not be confused with ophiocephalous pedicellariae regenerating nearby. × 12. Fig. 9. Regeneration stage b. x 12. Fig. 10. Regeneration stage c. × 12. Fig. 11. Regeneration stage d. × 12. Fig. 12. Regeneration stage e. × 12. Fig. 13. Regeneration stage f. × 12. Fig. 14. Regeneration stage g. x 12. Fig. 15. Regeneration stage h. × 12. Fig. 16. Regeneration stage i. x 12.
i¸
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728
H O L L A N D AND H O L L A N D
Table 1. The thne course o f regression and regeneration following head loss from a typical glob~ferous pedicel/aria o f Lytechinus Regeneration phase Mm total Figure
Days after head loss
Stalk regression phase
4 5 6
0 5 7
Lag phase
7
9
8 9 10 11 12 13 14 15 16 2, 3
27 31 34 37 39 41 44 48 60 72
Regeneration phase
Days after start
Stage
Length
Definitive features
0 4 7 10 12 14 17 21 33 45
a b c d e f g h i Mature
0.1 0.3 0.5 0' 8 1 •0 1.3 1 '5 1.9 2.9 3.5
Skeletal ossicles absent Skeletal ossicles present Jaws start diverging Jaws widely divergent Jaws start converging Jaws convergent Head base wider than apex Jaws can open and close Head as wide as long Maximum size attained
necessarily be undifferentiated by behavioral, chemical or developmental criteria (as defined by G r o b s t e i n , 1959). Undifferentiated cells o f the dermis and epidermis (Fig. 17, arrow) are morphologically identical, E a c h cell is spherical to oval and usually lacks c o n s p i c u o u s cytoplasmic processes. The nucleus contains one or two nucleoli. The c o n d e n s e d c h r o m a t i n is distributed in a
thin layer adjacent to the inner nuclear m e m b r a n e and is scattered in patches elsew h e r e in the nucleus. The sparse cytoplasm c o n t a i n s a b u n d a n t free r i b o s o m e s and an occasional flattened cisterna o f r o u g h e n d o plasmic reticulum. Also present are s o m e mitocilondria, lipid droplets a n d a l p h a rosettes o f glycogen. A small Golgi complex and a pair o f centrioles m a y also be encoun-
Fig. 17. A section through the general epidermis of a globiferous pedicellaria at regeneration stage e. In addition to several columnar epithelial cells, the epidermis includes undifferentiated cells (arrow) and a colorless spherule cell (at the left). The external surface is at the top of the figure, and the inconspicuous basal lamina between epidermis and dermis is at the lower right, x 4000. Fig. 18. A section through the epidermal region wherein head gland cells are beginning to differentiate at regeneration stage f. The external surface is at the top and the dermis is at the bottom, x 3800. Fig. 19. The juxtanuclear portion of a head gland cell at regeneration stage f. Prominent structures from top to bottom are a part of the nucleus, the Golgi complex and part of a cytoplasmic vacuole. × 16,000. Fig. 20. A section through the periphery of a mature head gland fixed by the method of O'Connell et al. (1974). Conspicuous structures in the gland cell are the cytoplasmic vacuoles of secretion and the nucleus. The inconspicuous Golgi complex is indicated by the arrow. Part of the musculature investing the head gland shows at the bottom left. x 6000.
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730 tered. Presumably, some of the undifferentiated cells of the epidermis are destined to become gland cells, while some undifferentiated cells of the dermis are destined to become the muscles investing the glands. The epidermis and dermis of all stages included some electron-lucent cells, a few red spherule cells and a few colorless spherule cells (like the one at the left in Fig. 17). The spherule cells, at least, are doubtlessly related to the coelomocytes which circulate in the perivisceral coelom. We do not know what role, if any, such cell types play during regeneration, and their fine structure will not be described here. By regeneration stage e, the epidermis is separated from the dermis by a basal lamina (Fig. 17, bottom right). At this stage, most of the regenerate is covered with a general epidermis, which includes the regions where the head glands and neck glands will develop. However, on the inner surface of each jaw, the epidermis is already differentiating into two sensory hillocks, one basal and one apical. The fine structure of the sensory hillocks has been described by Cobb (1968) and will not be included here. At stage e, the general epidermis of the head and stalk is composed of numerous columnhr epithelial cells (Fig. 17) in addition to undifferentiated cells; by the stage of the mature pedicellaria, the epithelial ceils have become cuboidal to squamous. The apical end of each columnar cell lacks cilia, but bears numerous microvilli that are associated with a fibrous glycocalyx (the glycocalyx was Well preserved by the method of O'Connell et al., 1974, but not by the method of Holland, 1970). Apically, the columnar epithelial cells are associated with one another by intermediary junctions (in the terminology of Satir and Gilula, 1970). The apical cytoplasm contains a pair of centrioles, a Golgi complex and vacuoles containing material ranging from electron lucent to very dense. Throughout the cytoplasm, free ribosomes and profiles of rough endoplasmic reticulum are abundant; also present are mitochondria, lipid droplets and glycogen rosettes. The nucleus resembles that already described for an undifferentiated cell. Basally, some of the columnar epithelial cells give off a cytoplasmic process that might possibly be an axon. That is, some of the epithelial cells of the general epidermis
HOLLAND AND HOLLAND may actually be neuroepithelial cells (as was first proposed by Kawaguti and Kamishima, 1964).
Differentiation of the head gland cells and their musculature. In stage f, head gland cells first appear among the cells of the general epidermis. Although a few undifferentiated cells still occur in the epidermis at stage f, they disappear during subsequent stages, presumably by differentiating into gland cells. Each young head gland cell is elongated, but its apical end does not reach the outer surface of the epidermis (Fig. 18). The most conspicuous cytoplasmic inclusions are membrane-bound vacuoles ranging from 0"2 t, to about 5 t~ in diameter. Some of the vacuoles appear empty, while others are filled with a flocculent material of low electron density. Some vacuoles also contain small patches of relatively dense granular material. In general, the contents of the vacuoles appear to have been partially removed by both of the fixation procedures that we used. The juxtanuclear cytoplasm contains a Golgi complex with dilated cisternae (Fig. 19), which are probably involved in packaging secretory products into the vacuoles. The rough endoplasmic reticulum of the youngest head gland cells is not distended with intracisternal material. The cytoplasm also contains free ribosomes, mitochondria, lipid droplets and glycogen rosettes. The nucleus resembles that already described for an undifferentiated cell. During stage g, the population of head gland cells delaminates from the general epidermis and sinks down into the dermal layer. The delamination procedes from the base to the apex of the jaw, and the gland cells retain a close association with overlying epidermal cells only at the apex of the jaw. The solid cord of delaminated gland cells is invested with a basal lamina. Within each gland cell, the nucleus is located at the end nearest the basal lamina, while the vacuolated cytoplasm extends toward the center of the lumenless gland. Each head gland cell enlarges during stage g, as the cytoplasmic vacuoles increase in number and size. The rough endoplasmic reticulum becomes more extensive and its intracisternal spaces become dilated with a finely granular material of low electron density. N o conspicuous changes occur in the other cytoplasmic organelles or
SEA URCHIN GLOBIFEROUS P E D I C E L L A R I A E in the nucleus. During stage h, gland cell vacuoles continue to enlarge in size and number, and the intracisternal spaces of the rough endoplasmic reticulum remain distended with granular contents. By stage i, the rough endoplasmic reticulum is less distended and the Golgi complex is less conspicuous. By the time of the mature stage, the few profiles of each endoplasmic reticulum remaining are not distended at all. Moreover, the Golgi complex consists mainly of a stack of flattened saccules (Fig. 20, arrow). The cytoplasm of a mature head gland cell is dominated by the vacuoles, some of which have reached diameters of nearly 30ix (Fig. 21, at right). The less dense flocculent contents of the vacuoles are preserved better by the 1974 method of O'Connell et al. (Fig. 20) than by the 1970 method of Holland (Fig. 21). Much of the cytoplasm is compressed into thin sheets which intervene between the vacuoles. In addition to the organelles just mentioned, the cytoplasm contains free ribosomes, mitochondria, lipid droplets, glycogen rosettes, myelin figures, and probable lysosomes. These latter organelles may contain dense material, compacted membranous material, or numerous small vesicles. In the mature stage, the nucleus is located near the end of the cell that is closest to the periphery of the head gland (Figs. 20, 21). As O'Connell et al. (1974) demonstrated, the mature head gland has no lumen; many light microscopists had previously mistaken the highly vacuolated cytoplasm for a lumen. When a mature globiferous pedicellaria bites an object, the vacuoles of the head gland cells rupture, releasing their secretions. These secretions exit from the gland where it is closely associated with the epidermis at the apex of the jaw. A head gland that had bitten an object was fixed shortly thereafter for electron microscopy (Fig. 22). The discharged gland appeared as a packed mass of cytoplasmic profiles. It could not be determined whether these profiles were largely interconnected or were small fragments of the gland cells. As the head gland cells begin to delaminate from the epidermis in stage g, some of the surrounding undifferentiated cells begin to develop into smooth muscle cells. Because the developing muscle cells are separated from the gland by a basal lamina, it is
731
probable that they are of dermal origin; however, an epidermal origin for some of the developing muscle cells cannot be ruled out on strictly morphological evidence. As delamination proceeds, the muscle cells almost completely invest the head gland, except near the tip of the jaw where the secretion will be expelled. The developing muscle cells first elongate, and then a few scattered muscle fibrils appear in the cytoplasm among the organelles already described for an undifferentiated cell. During stages h and i (Fig. 23), cell elongation continues, muscle fibrils become abundant, and the centrioles disappear; the other cytoplasmic organelles of the undifferentiated cell are still present, but are inconspicuous. Several muscles of the mature head gland are shown at the bottom left in Fig. 20. Differentiation o f the stalk gland cells' and their musculature. The stalk glands develop from the general epidermis, which has already been described above for stage e; the site of stalk gland development is labelled SG in Fig. 1b. During regeneration stage g, undifferentiated cells delaminate from the columnar epithelial cells of the epidermis to form an approximately spherical mass. The undifferentiated cells in this mass do not begin to develop into gland cells until stage h. Thus, in the stalk gland, delamination precedes differentiation, while, in the head gland, differentiation precedes delamination. As differentiation of the stalk gland cells begins in stage h, a small lumen appears in the center of the gland. This space seems to open up within the mass of differentiating gland cells, which are arranged around it. Each developing stalk gland cell is elongate (Fig. 24). The nucleus is located near the end of the cell farthest from the lumen. The nuclear fine structure resembles that of an undifferentiated cell. At the luminal end, each gland cell is associated with its neighbors by a junctional complex consisting of an intermediary junction and a poorly developed septate desmosome (in the terminology of Satir and Gilula, 1970). The most conspicuous cytoplasmic inclusions are membranebound vacuoles about 1"5 tx in diameter. The contents of each vacuole are divisible into a core of finely granular material
HOLLAND AND HOLLAND
732 a n d a s u r r o u n d i n g cortex o f sparsely distributed flocculent material. A Golgi complex is present, a n d its dilated cisternae pres u m a b l y help package the c o n t e n t s o f the cytoplasmic vacuoles already described. The c y t o p l a s m also c o n t a i n s a b u n d a n t free ribosomes, m i t o c h o n d r i a , lipid droplets a n d glycogen rosettes. The r o u g h e n d o p l a s m i c reticulum consists of a few flattened cisternae t h a t are n o t dilated with intracisternal material. R u n n i n g parallel to the long axis o f the gland cell are a few cytoplasmic filaments a b o u t 3 0 0 , ~ in diameter. These organelles, a l t h o u g h wider t h a n typical microfilaments, might possibly play some role in g l a n d cell e l o n g a t i o n or in cell movem e n t s related to l u m e n formation. The c y t o p l a s m at the luminal end of the cell c o n t a i n s a pair o f centrioles; they are sometimes associated with striated rootlets or with the cytoplasmic filaments described a b o v e (Fig. 24, inset). By r e g e n e r a t i o n stage i, the l u m e n o f the stalk g l a n d is enlarged a n d c o n t a i n s n u m e r o u s secretory granules (Fig. 25, at center a n d
right). T h e l u m e n is s u r r o u n d e d by a single layer o f g l a n d cells, which are n o w low c o l u m n a r in shape (Fig. 25, at left). By this stage, the gland cell cytoplasm rarely includes centrioles a n d the r o u g h e n d o p l a s m i c retic u l u m h a s b e c o m e very p r o m i n e n t . T h e intracisternal spaces o f the r o u g h e n d o p l a s m i c r e t i c u l u m are distended with finely g r a n u l a r m a t e r i a l o f m o d e r a t e electron density (Fig. 26). Parts of the cisternae of the e n d o p l a s m i c reticulum nearest the f o r m i n g face of the G o l g i c o m p l e x lack ribosomes. M o s t of the c y t o p l a s m i c vacuoles are n o w a b o u t 2-3 t~ in diameter, a n d their c o n t e n t s are divisible into regions of finely g r a n u l a r material a n d regions c o n t a i n i n g a loose m e s h w o r k of fibrous material. In m o s t cells, a vacuole is closely associated with the m a t u r e face o f the G o l g i complex. In some places (Fig. 26, arrows), G o l g i vesicles are p r o b a b l y fusing with the m e m b r a n e of the vacuole. In the lumen, m o s t of the secretory granules are a b o u t 2-3 ~ in diameter, a n d their c o n t e n t s resemble those o f the cytoplasmic vacuoles. T h e c o n t e n t s of each secretory granule
Fig. 21. A section through the periphery of a mature head gland fixed by the method of Holland (1970). The gland cell resembles the one in the preceding figure, except for the loss of the flocculent material from the secretory vacuoles, x 4500. Fig. 22. A section through a mature head gland fixed shortly after biting and expulsion of the secretions, x 4500. Fig. 23. Several of the smooth muscles investing a head gland (at left) at regeneration stage i. x 4500. Fig. 24. A section through a stalk gland at regeneration stage h. The lumen is at the top right, and a dermal cell just starting to differentiate into a muscle cell is at the far left. ×5000. The inset (× 14,000) shows the luminal tip of a gland ceil containing a centriole associated with cytoplasmic filaments. Fig. 25. A section through the periphery of a stalk gland at regeneration stage i. The lumen filled with secretory granules is at the center and right, while low columnar gland cells are at the left. × 5000. Fig. 26. A section through a stalk gland cell at stage i. From right to left, conspicuous structures are the distended rough endoplasmic reticulum, the Golgi complex, a cytoplasmic vacuole and a portion of the nucleus. The arrows indicate places where Golgi vesicles appear to be fusing with the periphery of the vacuole, x 26,000. Fig. 27. Secretory granules in the lumen of a stalk gland at regeneration stage i. x 27,000. Fig. 28. Secretory granule in the lumen of a stalk gland of a mature globiferous pedicellaria. × 27,000.
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.
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SEA URCHIN GLOBIFEROUS P E D I C E L L A R I A E are surrounded by two concentric membranes which are separated from one another by a thin layer of cytoplasm (Fig. 27). There can be little doubt that the outermost membrane corresponds to part of the plasma membrane of a gland cell. The cytoplasmic vacuoles are apparently budded off into the lumen along with a small amount of gland cell cytoplasm and plasma membrane around them. In the mature globiferous pedicellariae, the large lumen of each stalk gland is filled with secretory granules. Within the mature granule (Fig. 28), the finely granular component of the preceding regeneration stage has become organized into numerous spherules, each about 700 A~ in diameter. Mature stalk gland cells are cuboidal to squamous. Each mature cell contains the same organelles that were present in the stalk gland cells of regeneration stage i. The Golgi complex and a few cytoplasmic vacoules are conspicuous. However, the rough endoplasmic reticulum is much less extensive than before. The differentiation of the stalk gland musculature begins at regeneration stage h, one stage later than the start of muscle differentiation around the head gland. The stalk gland muscles probably originate from undifferentiated cells in the dermis near the basal lamina covering the gland. A developing muscle cell that is just elongating is shown at stage h (Fig. 25, far left). By stage i, the centrioles have disappeared, and the cytoplasm contains abundant muscle fibrils. The smooth muscles surrounding the mature stalk gland resemble those illustrated for the mature head gland (Fig. 20, bottom left). The muscle cells almost completely envelop the stalk gland except on its epidermal side, where the secretion will be expelled. Discussion
The results of the present study indicate that most of the secretory activity of the head glands and stalk glands of globiferous pedicellariae takes place during a limited period during the later stages of regeneration. In mature head glands and neck glands there is probably little elaboration of secretory products; instead, the secretions produced during regeneration are presumably stored in the mature glands with little augmentation
735
or turnover. O'Connell et al. (1974) have proposed 'a continuous activity in secretory cells of fully developed pedicellariae', since the Golgi complex and related vesicles were demonstrable by electron microscopy. However, we believe that such organelles are relatively inactive and are simply left over from an earlier cell phase of intense synthesis. As suggested by O'Connell et al. (1974), autoradiographic and biochemical studies might resolve this problem. Unfortunately, an autoradiographic study of the head gland will require a better fixation method than now available to preserve the secretory products in situ. Moreover, possible changes in gland cell permeability and in size of precursor pools will have to be taken into account when comparing different regeneration stages. In Lytechinus pictus, the head gland and the stalk gland of a mature globiferous pedicellaria are made up exclusively of highly differentiated cells; no morphologically undifferentiated cells are mixed with them. Thus the gland cell populations are of the static type and not of the expanding or renewing types (in the terminology of Messier and Leblond, 1960). A similar lack of replacement cells was reported for the head gland of Strongylocentrotus purpuratus by O'Connell et al. (1974). The same tissue was classified as a static cell population in an autoradiographic study with tritiated thymidine (Holland, 1964); all other tissues of the adult sea urchin were, by contrast, expanding or renewing cell populations. Conspicuous components of the secretions in the head and stalk glands of Lytechinus pictus are acid mucopolysa.ccharides and mucoproteins (Holland and Holland, unpublished light microscopic histochemistry); similar histochemical results have previously been published for other species of sea urchins (P6r~s, 1950; Chia, 1970; O'Connell et al., 1974). The histochemistry is in agreement with the fine structure of the gland cells, which contain distended endoplasmic reticulum as well as Golgi complexes associated with secretory vacuoles. Such gland cells resemble better studied cells that synthesize mucopolysaccharides and glycoproteins. According to the review of Spiro (1969), peptides are formed on the ribosomes and enter the intracisternal spaces of the endoplasmic reticulum, where some carbo-
736 hydrates may be added. Subsequently, the secretion proceeds to the Golgi complex for further augmentation with carbohydrates. Finally, the Golgi saccules concentrate and package the secretions. In Lytechinus pictus, the chief function of the head gland of the globiferous pedicellaria is probably to elaborate a toxic secretion, as in other sea urchins (see review by Alender and Russell, 1966). Although secretions of the mature head gland are toxic, it is not known if secretions of the regenerating head gland are toxic as soon as they are produced. This question might be answered if extracts of regenerating head glands were tested on a suitable biological assay. The function of the stalk gland secretions of Lytechinus pictus remains enigmatic. P6r~s (1950), in his study of Sphaerechinus gramdaris, presented evidence that the stalk gland secretions were not particularly toxic; he presumed that they acted physically to immobilize small organisms. When a globiferous pedicellaria bites an object, the musculature covering the glands contacts. This contraction apparently causes a massive exocytosis of the contents of the cytoplasmic vacuoles from the head gland cells. F r o m each cell, the secretions probably exit via simultaneous ruptures of the vacuole
HOLLAND AND HOLLAND membranes and plasma membrane at many points on the cell surface. This is an example of holocrine secretion. After entering the intercellular spaces of the head gland, the secretions are rapidly expelled to the exterior near the apex of the jaw. It is not clear whether they exit by a well-defined duct or by a localized rupture of the overlying epidermis; the gland ducts reported by some light microscopists (e.g. Hamann, 1887) might possibly be slight ruptures of epithelia taking place at fixation. Pressure from the musculature covering the stalk glands causes an expulsion of secretory granules from the gland lumen. This massive expulsion, if observed at the organ level might be considered holocrine; however, the production of the secretory granules, if observed at the cellular level, is clearly apocrine. As in the case of the head gland, it is not clear whether the stalk gland secretions exit via a duct or a rupture of the epidermis.
Acknowledgement We are deeply indebted to Mr Fritz G o r o for his invaluable help with the photomicrography of living pedicellariae (Figs. 2-16).
SEA URCHIN
GLOBIFEROUS
PEDICELLARIAE
737
References ALENDER, C. B. and RUSSELL, F. E. 1966. Pharmacology. In Physiology of Eehinodermata (ed. R. A Boolootian), pp. 529 543. Wiley Interscience, New York. CANNONE, A. J. 1970. The anatomy and venom-emitting mechanism of the globiferous pedicellariae of the urchin Parechinus angnlosus (Leske) with notes on their behaviour. Zool. Afrieana, 5, 179-190. Cl-I~A, F. S. 1970. Histology of the globiferous pedicetlariae of Psammecllinus miliaris (Echinodermata: Echinoidea). J. Zool., Lond., 160, 9-16. COBB, J. L. S. 1968. The fine structure of the pedicellariae of Echimts esculenttts (L.). II. The sensory system. J. R. microsc. Sot., 88, 223 233. GROBSTEIN, C. 1959. Differentiation of vertebrate cells. In The Cell (eds. J. Brachet and A. E. Mirsky), Vol. I, pp. 437-496. Academic Press, New York. HAMANN, O. 1887. Beitrfige zur Histologie der Echinodermen. lII. Anatomie und Histologie der Echiniden und Spatangiden, Jena Z. Naturwiss., 21, 87-266. HOLLAND, N. D. 1964. An autoradiographic investigation of the gonads of the purple sea urchin (Strong),locentrotus purpuratus): an autoradiographic study employing tritiated thymidine. Ph.D. Dissertation, Stanford University. HOLLAND, N. D. 1970. The fine structure of the axial organ of the feather star Nemaster r,biginosa (Echinodermata: Crinoidea). Tissue & Cell, 2, 625 636. HYMAN, L. H. 1955. The invertebrates, Vol. IV. Eehinodermata. McGraw-Hill, New York. KAWAGUTI, S. and KAMISmMA, Y, 1964. Electron-microscopic study of the integument of the echinoid, Diadema setosum. Annot. zool. jap., 37, 147 152. LUFT, J. H. 1961. Improvements in epoxy resin embedding methods. J. biophys, bioehem. Cytol., 9, 409-414. MESSIER, B. and LEBLOND, C. P. 1960. Cell proliferation and migration as revealed by autoradiography after injection of thymidine-H 3 into male rats and mice. Am. J. Anat., 106, 247-265. O'CONNELL, M. G., ALENDER,C. B. and WOOD, E. M. 1974. A fine structure study of venom gland cells in globiferous pedicellariae from Strongylocentrotns parpuratns (Stimpson). J. Morph., 142, 41 I 432. P[~RI~S,J. M. 1950. Recherches sur les pddicellaires glandulaires de Sphaerechinus granldaris (Lamarck). Arch. zool. exp. gdn. (Notes et Revue), 86, ll8 136. SATIR, P. and GILULA,N. B. 1970. The cell junction in a lamellibranch gill ciliated epithelium. Localization of pyroantimonate. J. Cell Biol., 47, 468 487. SPiRO, R. G. 1969. Glycoproteins: their biochemistry, biology and role in human disease. New Engl. J. Med., 281, 991-1001.