J. Cell Sci. 5, 699-726 (1969) 699

Printed in Great Britain

DIFFERENTIATION OF NEUROSENSORY

CELLS IN HYDRA

LOWELL E. DAVIS

Department of Zoology, Syracuse University, Syracuse, Neiu York 13210, U.S.A.

SUMMARY

The differentiation of neurosensory cells in Hydra has been studied at the level of the electronmicroscope. These cells arise from interstitial cells (undifferentiated cells) and not from pre-existing nerve cells. Furthermore, there is no evidence to suggest that neurosensory cellsrepresent a stage in the development of other nerve cells, i.e. ganglionic and neurosecretorycells. Major cytoplasmic changes in fine structure during differentiation include development ofa cilium and associated structures (basal body, basal plate, rootlets), development of micro-tubules and at least two neurites, increase in Golgi lamellae and formation of dense dropletstypical of neurosecretory droplets, structural variations in mitochondria and a decrease in thenumber of ribosomes. Granular endoplasmic reticulum is characteristically poorly developed inall stages of differentiation, including the mature neurosensory cell. Nuclear and nucleolarchanges also occur during differentiation but these are less dramatic than the cytoplasmic events.

The possibility of neurosensory cells being bi- or multiciliated and the presence of inter-cellular bridges between these cells are considered. The function of neurosensory cells is dis-cussed briefly in relation to the function of the cilium and neurosecretory droplets.

INTRODUCTION

Histological and ultrastructural studies have shown the existence of at least threetypes of nerve cells in Hydra, namely, neurosensory, ganglionic and neurosecretorycells (Burnett & Diehl, 1964 a, b; Lentz & Barrnett, 1965; Davis, Burnett & Haynes,1968). The morphology of fully differentiated nerve cells has been described previouslyand the ultrastructural criteria for their identification presented (Davis et al. 1968).However, no attempts were made in the latter study to investigate their origins anddevelopmental stages.

It has been shown histologically, however, that interstitial cells differentiate intonerves—presumably all types of nerves—in the oral and budding regions of Hydra(Burnett & Diehl, 1964a). Lentz (1965 a) presented some electron-microscopic evi-dence for the development of nerve cells in general from interstitial cells. Manydevelopmental stages for the various types of nerve cells, however, were lacking, anddifferentiation of nerve cells at the ultrastructural level still requires further study.

Because of certain ultrastructural similarities between different types of nerve cells,it has been suggested, for example, that ganglionic cells and neurosecretory cells mayrepresent the same cell in different stages of development and function (Lentz &Barrnett, 1965). Although this suggestion is entirely plausible, the preliminary evi-dence suggests that each type of nerve cell may arise independently from interstitialcells. An investigation was undertaken therefore to determine the ultrastructural

700 L. E. Davis

changes during differentiation of nerve cells. The present study reveals that neuro-sensory cells are formed independently of neurosecretory and ganglionic cells, and thatthey differentiate directly from interstitial cells. The differentiation of ganglionic andneurosecretory cells will be the subject of future communications.

MATERIALS AND METHODS

The animals used in these studies were normal non-budding Hydra pseiidoligactis. They werecultured by a modified method of Loomis & Lenhoff (1956). All animals were starved for 24 hbefore fixation. They were fixed for 1 h in cold 3 -6% glutaraldehyde buffered with O-IMsodium cacodylate at pH 7-3 (Sabatini, Bensch &Barrnett, 1963). During fixation short segmentswere excised from the hypostome, mid-gastric regions, peduncle and basal disk. The tissueswere washed in buffered sucrose solution (in O-IM sodium cacodylate) and then post-fixed for30-45 min in cold 1 % osmium tetroxide containing sucrose (Caufield, 1957). The fixed tissueswere dehydrated rapidly in alcohol and embedded in Maraglas (Spurlock, Kattine & Freeman,1963)-

Sections were cut with glass or diamond knives on a Huxley or Porter Blum M T 2 ultrami-crotome and mounted on carbon-coated Formvar-filmed grids. The sections were stained withuranyl acetate (Watson, 1958) followed by lead citrate (Reynolds, 1963). Sections were ex-amined in an RCA EMU 3H electron microscope.

OBSERVATIONS AND RESULTS

Interstitial cells are undifferentiated cells which are capable of differentiating intoseveral cell types, including neurosensory cells. Since the ultrastructure of interstitialcells has been well documented by several investigators (Slautterback & Fawcett, 1959;Slautterback, 1961; Hess, 1961; Lentz, 1965 a; Davis, 1968) only a brief descriptionwill be presented here. Interstitial cells are small, round or oval, measuring 5-6 /im indiameter (Fig. 1). The nucleus containing an unusually prominent nucleolus is cen-trally located and occupies most of the cell. The cytoplasm is characterized by thepresence of a few small mitochondria, numerous free ribosomes, few membrane-boundvesicles and the absence of granular endoplasmic reticulum. Occasionally, however,sparse segments of endoplasmic reticulum and an inconspicuous Golgi complex areobserved (Fig. 1).

The structural criteria for the early stages of differentiation are shown in Figs. 1-7.At the outset of nerve cell differentiation, interstitial cells first assume a spindle shape(Fig. 2). Subsequently, the cell elongates, forming what will later be neurites. At thisstage of differentiation, except for the presence of a developing cilium and cytoplasmicextensions, the cell resembles a typical interstitial cell (Figs. 1, 2). However, the deve-loping neurosensory cell is readily distinguishable from other developing cells derivedfrom interstitial cells, e.g. cnidoblasts, by the absence of a conspicuous Golgi complexand extensive endoplasmic reticulum (see Slautterback & Fawcett, 1959; Slautterback,1961).

At this stage of development, also, it is not unusual to observe two neurosensory cellsconnected by intercellular bridges (Fig. 2). Although similar connexions have beenobserved between developing spermatozoa in sexual Hydra (Burnett, Davis & Ruffing,1966), they have been observed previously only between interstitial cells and between

Differentiation of neurosensory cells in Hydra 701

developing cnidoblasts of non-sexual Hydra (Slautterback & Fawcett, 1959; Slautter-back, 1961; Hess, 1961). In the present study, however, both cells differentiate intoneurosensory cells. Since no mature neurosensory cells have been observed with inter-cellular bridges, and since, as will be discussed later, at least some neurosensory cellsmigrate to the surface of the animal, it is assumed that such bridges rupture during orshortly after differentiation.

Development of the ciliary apparatus

The processes of centriole formation and ciliogenesis have been studied in a varietyof cells (Roth & Shigenaka, 1964; Dirksen & Crocker, 1966; Outka & Kluss, 1967;Sorokin, 1968; Steinman, 1968; Boquist, 1968). It appears that some of these pro-cesses occur in a similar manner in neurosensory cells. The first sign of the develop-ment of the ciliary structure is the appearance of several osmiophilic dense dropletssometimes circularly arranged immediately adjacent or in close proximity to the cellmembrane (Fig. 3). These droplets average about 50 nm in diameter and appear to besimilar to the procentriole precursor bodies (30-80 nm in diameter) described bySteinman (1968). Eventually a small centriole is observed (Fig. 4) which apparentlymigrates to the plasma membrane of the cell where two centrioles are later observedoriented at right angles to each other (Figs. 2, 5). It should be noted that when inter-stitial cells divide for the last time prior to differentiation, a single centriole remainsnear the plasma membrane (Slautterback, 1961).

A second type of dense droplet (65-85 nm in diameter) is found at the base of thecentriole which approaches the plasma membrane (Fig. 6). In some instances theyappear fibrous and resemble the structures described by Sorokin (1968) as fibrogranularaggregates, which are one type of precursor material for basal body formation.

A third type of electron-dense granule, 70 nm in diameter, is frequently observed inassociation with the developing cilium. Several of these droplets are arranged linearly,and may extend from the base of the cilium for a distance of up to 3-5 /tm (Fig. 7, seealso Doolin & Birge, 1966). Occasionally, long dense fibres emanating from near thebase of the developing cilium are seen among the linearly arranged granules, indicatingthat these structures may be precursor materials for rootlet formation (Fig. 7). Thedistinction among these three types of granules is based solely on dimensions, arrange-ment and location within the cell.

As differentiation of the neurosensory cell proceeds, the emerging ciliary bud pro-trudes from the cell and is completely surrounded by the plasma membrane (Fig. 9).This structure strongly resembles the young ciliary process of vertebrate neurons(Thornhill, 1967). The developing cilium contains axial microtubules, few small densedroplets dispersed among the microtubules and an amorphous material. As the ciliaryshaft elongates into the extracellular space, internal microtubules extend its entirelength (Figs. 8, 10). At the base of the cilium, rootlets begin to appear but are notstriated at this stage. The electron-dense droplets which previously surrounded orextended linearly from the base of the centriole have disappeared (Fig. 10).

A single well-developed cilium, complete with ciliary shaft, basal plate, basal bodyand rootlets is shown in Fig. 11. The portion of the shaft shown contains microtubules

702 L. E. Davis

throughout the length. The rootlets at this stage reveal typical striations. Cross-sectionsof the cilium at various levels within its cytoplasmic collar are shown in Fig. 8. Theinternal tubules reveal a typical 9 + 2 arrangement (Fig. 8 A) but the number and loca-tion of the central tubules vary from one level to another (see Roth & Shigenaka, 1964;Satir, 1965; Boquist, 1968).

The number of cilia in each neurosensory cell has been suggested as one to several(McConnell, 1932; Burnett & Diehl, 1964a; Bullock & Horridge, 1965; Lentz &Barrnett, 1965; Davis et al. 1968). The present studies lend support to the idea that atleast in some neurosensory cells more than one cilium may begin development, butwhether more than one mature cilium is eventually formed remains questionable.Figure 11 shows a developing cilium with all its associated structures. Close to thiscilium is a pair of centrioles, aligned at right angles to each other. One of these cen-trioles approaches the limits of the so-called ciliary vesicle, but it is uncertain whetherthis gives rise to another mature cilium. Up to the present time, two or more fullydeveloped cilia have not been observed in neurosensory cells.

Golgi complex and formation of droplets

The increase in Golgi lamellae and activity and the ultimate formation of secretorydroplets are closely associated and therefore will be described together. The Golgicomplex of interstitial cells is small (Fig. 1) and sometimes not observed. As differen-tiation of the neurosensory cells proceeds, the lamellar system of the Golgi complexincreases (Fig. 12; see also Fig. 10), and by the time the cilium is completely formedand elaboration of droplets begins, there may be as many as 3 Golgi complexes in anyone cell (Fig. 13). It is interesting to note that there may be as many as 4 Golgi com-plexes in the developing vertebrate sensory neuroblasts (Tennyson, 1965). One ofthese Golgi complexes, however, is located characteristically near the base of the deve-loping cilium (Fig. 14). These associated structures bear a striking resemblance tosimilar structures observed in the developing sensory neuroblasts of rabbit embryos(Tennyson, 1965) and in the biciliated cells of rat adenohypophysis (Wheatley, 1967).It is not known whether this Golgi complex participates in the formation of the ciliaryprecursors, but the close association of these organelles suggests a functional signi-ficance of the Golgi complex.

Once the cilium is formed, the Golgi complexes become extremely active. Materialof relatively high density is observed in the lamellae and in the peripheral vesicleswhich become distended and eventually separate from the Golgi membranes (Figs. 13,15). The droplets (70-130 nm in diameter) in the immediate vicinity of the Golgicomplexes usually occupy the entire vesicle in which they are enclosed (Figs. 13, 15),while the droplets which eventually become scattered throughout the cell body as wellas in the neurites occupy only a portion of the vesicles. In many instances dropletsreveal a low density, and apparently empty vesicles are observed near the plasmamembrane or in the interior of the cell (Figs. 11, 15; see DeRobertis, 1962).

The droplets described above have been reported in previous investigations (Burnett& Diehl, 1964a; Davis et al. 1968) in which the droplets have been shown to beneurosecretory droplets. These neurosecretory droplets are strikingly similar to

Differentiation of neurosensory cells in Hydra 703

comparable droplets in known neurosecretory cells (Bern, Nishioka & Hagadorn,1962; DeRobertis, 1962; Oosaki & Ishii, 1965; Scharrer, 1967, 1968). The mechanismby which the neurosecretory droplets are released from neurosecretory cells hasbeen suggested previously (Davis et al. 1968) and will be the subject of a futurecommunication.

Otlier cytoplasmic structures

Droplets. Another type of electron-dense droplet is frequently observed in deve-loping neurosensory cells. These droplets appear as early as during formation of thecilium. They are highly irregular in shape, size (0-4-1-0/tm), densities and internalstructure (Figs. 13, 16; see also Bern et al. 1962). Their origin is still uncertain, buttentative evidences implicate mitochondria as possible sources.

Ribosomes. Interstitial cells from which neurosensory cells are derived containnumerous free ribosomes (Fig. 1). These ribosomes persist through the early stages ofcilium formation (Figs. 2-7, 9). By the time the cilium is completely formed (Fig. 10),the number of ribosomes diminishes and when the neurosensory cell is fully maturemost ribosomes have disappeared (Figs. 11, 15).

Mitochondria. Mitochondria of interstitial cells undergo several changes duringdifferentiation of neurosensory cells. They increase in number with a correspondingincrease in the density of the matrix (Figs. 1, 6, 7, 14). Certain mitochondria of thematuring neurosensory cells undergo other changes. There may be a decrease in size,but in most cases there is a reduction in the number of cristae and an increase in thedensity of the matrix (Figs. 11, 13, 15, 16). Although the evidence is not conclusive, itappears that these mitochondria may be responsible, at least in part, for the formationof the larger type of droplets described previously.

Endoplasmic reticulum. Endoplasmic reticulum is not a prominent organelle in thedeveloping or fully differentiated neurosensory cell. Occasionally, only short segmentsare observed (Figs. 10, 14). For this reason it has been assumed that this organelleparticipates to a minor degree, if at all, in the formation of neurosecretory droplets.

Microtubules. Microtubules are observed in all stages of neurosensory cell developmentfrom the onset of ciliary development to the mature cell (Figs. 6, 11, 17, 18, 20). Theyare first seen near the developing cilium and as the cell elongates, forming neurites,they extend for long distances, parallel to the long axis of the cell. In some neurites,except for secretory droplets and a few mitochondria, they form the major or solecomponents (Fig. 17).

Nuclear changes. Nuclear changes are usually less dramatic than cytoplasmic changesin differentiating cells. The nucleus of the interstitial cell has been described pre-viously as being round or oval and containing a conspicuous nucleolus. Both structuresreveal granules of various sizes (Fig. 1). As the neurosensory cell begins to differentiatethe nucleus assumes a more oval shape (Figs. 2, 10), and by the completion of differen-tiation, it may be elongated or highly irregular in shape (Figs. 11, 15, 17, 18). Thenucleolus also undergoes certain changes, but this subject will be reported in a futurecommunication.

704 L. E. Davis

The fully differentiated neurosensory cell

Since the fine structure of the fully differentiated neurosensory cell has been des-cribed previously (Davis et al. 1968) only additional information will be presented inthis study. Mature neurosensory cells with their characteristic organelles and inclusionsare shown in Figs. 11, 13, 15 and 17-20. The branching neurite seen in Fig. 20 isespecially interesting. Although numerous neurites have been observed at the electron-microscope level, seldom is a branching neurite seen. As with other neurites, it isdifficult to trace the entire neurite in the electron microscope and accurately identifyterminations. It is emphasized that, to date, no synaptic vesicles or specialized synapticjunctions similar to those of higher invertebrates and vertebrates have been observedin neurites of neurosensory cells or other types of nerve cells.

DISCUSSION

Electron-microscopic studies of the development of neurons in general have beenstudied previously (Lentz, 1965 a, b). However, no complete ultrastructural descrip-tion of the differentiation of the various types of nerve cells in Hydra exists. We havesought in this investigation not only to study the origin of one type of nerve cell—the neurosensory cell—but also to recognize and identify certain crucial developmentalstages of this cell type. Furthermore, the study has given rise to several questions whichwill be considered later.

The fact that interstitial cells give rise to nerve cells—and in this particular case,neurosensory cells—has been established, as well as certain differentiative nuclear andcytoplasmic changes. Although it is generally accepted that nuclear changes tend to beless striking than cytoplasmic modifications during cellular differentiation (Grobstein,1959), the nuclei of neurosensory cells show remarkable structural differences through-out their development. The small oval nucleus of the interstitial cells from which theyarise is finely granular in appearance and contains a conspicuous nucleolus. From thispoint the structural changes become obvious until the mature neurosensory cell isformed in which the nucleus may be oval or highly irregularly shaped, most of thefinely granular material disappears and is replaced by large coarse granules and ac-cumulations of these materials. The nucleolus is reduced in size to such an extentthat only an occasional section may reveal its presence.

The cytoplasm, on the other hand, shows a much greater diversification since it isthe primary area of morphological differentiation (Grobstein, 1959). The formation ofthe cilium and its associated structures (i.e. basal body, basal plate, striated rootlets),the elongation of the cytoplasm in the formation of neurites, the elaboration ofsecretory droplets and the development of or increase in cell organelles are allstructural events in the differentiation of the neurosensory cell.

Neurosensory cells represent another definite cell type derived from interstitial cellsand join such cells as cnidoblasts, germ cells, and digestive cells (Slautterback, 1959,1961; Lentz, 1965a, b; Burnett et al. 1966; Davis, Burnett, Haynes & Mumaw, 1966).Lentz (1965 a) has also included epithelio-muscular cells as belonging to this group,

Differentiation of neurosensory cells in Hydra 705

but the validity of this observation is doubtful (Slautterback, 1967, and unpublishedobservations).

Fully specialized cells which may or may not be derived from interstitial cells inHydra are known to undergo division. These include epithelio-muscular, digestive andmucous cells (Burnett, 1966) and cnidoblasts under special conditions (Davis, 1968).Examination of numerous sections from normal and regenerating animals shows manyneurosensory cells in various stages of differentiation but fails to reveal neurosensorycells or any type of nerve cells in division. These electron-microscopical observationssubstantiate earlier histological findings that nerve cells in Hydra originate from in-terstitial cells and not from pre-existing nerve cells (Burnett & Diehl, 1964a). Furthersupport for this conclusion has been obtained from our studies on the regeneratingisolated gastrodermis of Hydra. In this system, gland cells dedifferentiate into inter-stitial cells, which in turn redifferentiate into cnidoblasts (Davis et al. 1966), sper-matozoa (Burnett et al. 1966) and nerve cells (L. E. Davis, in preparation). Althoughmature nerve cells and many nerve cells in various stages of development have beenobserved, nerve cells in division have not been seen.

It should be noted that all neurosensory cells in various stages of differentiation aresingle cells or two cells held together temporarily by cytoplasmic bridges. In the lattercase the two cells undergo synchronous development. Most if not all developingneurosensory cells are located adjacent to the muscle bases of epithelio-muscular cellsand may be surrounded partly by cells of a similar type, cnidoblasts or other nervecells. Except for cytoplasmic bridges, these cells are 'free'—without specialized at-tachments to other cells—and therefore, presumably, capable of migration. Matureneurosensory cells, however, are observed not only in the area just mentioned but alsoat the periphery of the animal where they are attached firmly by septate desmosomes toepithelio-muscular cells. It would be interesting in this respect to study the formationof septate junctions. Similar junctions have also been observed in the apical region ofperipheral neurosensory cells in Cordylophora (Jha & Mackie, 1967). The above ob-servations, together with the fact that cells in both areas are identical in structure, leadus to suggest that the peripheral neurosensory cells probably migrate from theirlocation near the base of epithelio-muscular cells. In some cases, however, it appearsthat some neurosensory cells extend from the base of the epithelio-muscular cellswhere they originate to the periphery of the body.

The presence of intercellular bridges in developing neurosensory cells is an interes-ting structural phenomenon observed in certain differentiating cells. Similar cyto-plasmic connexions have been observed between developing cnidoblasts, betweenspermatozoa and between interstitial cells in Hydra (Slautterback & Fawcett, 1959;Slautterback, 1961; Hess, 1961; Burnett et al. 1966) as well as between the spermato-cytes and also spermatids of a variety of organisms, including man (Fawcett, Ito &Slautterback, 1959). According to Slautterback & Fawcett (1959), these intercellularbridges between interstitial cells and between cnidoblasts arise by incomplete cyto-kinesis during division of interstitial cells and persist throughout differentiation ofcnidoblasts. It is further suggested that since only one such bridge exists between anytwo cells and these two cells are of the same type and stage of development, the most

45 Cell Sci. 5

706 L. E. Davis

important function of the intercellular bridges is to synchronize differentiation (Slaut-terback, 1961). Developing neurosensory cells represent another cell type in Hydra inwhich intercellular bridges are found and, as in all cases indicated above, neurosensorycells joined by cytoplasmic bridges are precisely synchronized in development. Thesestructures, however, are apparently transitory since no mature neurosensory cells havebeen observed with intercellular bridges. The present observations also lend furthersupport to the fact that neurosensory cells arise from interstitial cells.

The possibility that the neurosensory cell may represent a stage in the developmentof other types of nerve cells (i.e. ganglionic and neurosecretory) should be considered.Lentz &Barrnett (1965) have suggested, for example, that ganglionic and neurosecretorycells may be the same type of nerve cell in different stages of development and function.Although the above possibility is likely, there is no evidence from the present study ofneurosensory cells to warrant such conclusions.

The question regarding the number of cilia in each neurosensory cell remains un-resolved, and unfortunately the present study has not contributed significantly toelucidating this point. McConnell (1932) stated that there may be as many as 5 'hairs'in each of these cells. Burnett & Diehl (1964 a) suggested from their histological studiesthat these cells may contain 1-3 small 'hairs' projecting from the tip of the cell.Bullock & Horridge (1965) stated that neurosensory cells contain one to several 'hairs'.From electron-microscopical studies, Lentz & Barrnett (1965) and Davis et al. (1968)suggested the presence of a single cilium in each neurosensory cell. This apparentdiscrepancy could be explained on the basis that neurosensory cells may differ in thenumber of cilia which they contain, i.e. they may be uni-, bi- or multi-ciliated. Al-though there is slight evidence from the present study that more than one cilium maybegin development, there is still no definite ultrastructural proof of bi- or multi-cilated neurosensory cells.

There are some arguments in favour of neurosensory cells containing a single cilium.Serial sections of numerous neurosensory cells have shown only one cilium. Further-more, in bi- or multi-ciliated sensory or receptor cells of other organisms all cilia arelocated in one region (supranuclear cytoplasm), so that a few adjacent longitudinalsections pass through two or more cilia (Bannister, 1965; Frisch & Reith, 1966;Wheatley, 1967; Thornhill, 1967). It seems likely, therefore, that if neurosensory cellsin Hydra contained two or more cilia, they would be located in one region so that serialsections would certainly reveal them.

The presence of neurosecretory droplets in these and other nerve cells has beendescribed previously (Davis et al. 1968). The droplets in the cell body and neurites areremarkably similar to those known as neurosecretory droplets in higher invertebratesand vertebrates (Oosaki & Ishii, 1965). It has been suggested that the activities of theGolgi complexes and/or granular endoplasmic reticulum are implicated in the originand development of neurosecretory droplets (Bern et al. 1962; Zambrano & De-Robertis, 1966; Zambrano & Mordoh, 1966; Oosaki & Ishii, 1965; Afzelius & Frid-berg, 1963; Scharrer, 1967, 1968). It appears that in neurosensory cells, the Golgicomplexes may be the major site of droplet elaboration.

Although the function of neurosensory cells is not of primary interest in this study,

Differentiation of neurosensory cells in Hydra 707

some comments concerning their possible functions seem appropriate. It has been

suggested earlier that neurosecretory materials may be responsible for promoting

growth and cell differentiation in Hydra (Burnett & Diehl, 19640,6; Davis et al. 1968).

The sensory function of the cilium, however, and the release of secretory materials are

still not clear. Rushforth, Burnett & Maynard (1963) have shown that Hydra contracts

when exposed to light and that the animal is particularly sensitive in the blue range.

Receptors for mechanical stimuli in these animals were also suggested. Lentz &

Barrnett (1965) have alluded to the possibility that neurosensory cells may possess a

photoreceptor function. It may be that the cilium of neurosensory cells represents one

type of receptor, and that in this case, it stimulates autorelease of neurosecretory

materials.

This investigation was supported by the National Science Foundation, Grant no. GB-8384.

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SCHARRER, B. (1968). Neurosecretion. XIV. Ultrastructural study of sites of release of neuro-secretory material in blattarian insects. Z. Zellforsch. mikrosk. Anat. 89, 1—16.

SLAUTTERBACK, D. B. (1961). Nematocyst development. In The Biology of Hydra and of SomeOther Coelenterates (ed. H. Lenhoff &W. Loomis), pp. 77-129. Coral Gables, Florida: Uni-versity of Miami Press.

SLAUTTERBACK, D. B. (1967). The cnidoblast-musculoepithelial cell complex in the tentacles ofHydra. Z. Zellforsch. mikrosk. Anat. 79, 296-318.

SLAUTTERBACK, D. B. & FAWCETT, D. W. (1959). The development of the cnidoblasts ofHydra, an electron microscope study of cell differentiation. J. biophys. biochem. Cytol. 5,441-452.

SOROKIN, S. (1968). Reconstruction of centriole formation and ciliogenesis in mammalian lungs.J. Cell Sci. 3, 207-230.

SPURLOCK, B. O., KATTINE, B. & FREEMAN, J. (1963). Technical modifications in Maraglasembedding. J. Cell Biol. 17, 203-207.

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THORNHILL, R. A. (1967). The ultrastructure of the olfactory epithelium of the lampreyLampetra fluviatilis. J. Cell Sci. 2, 591-602.

WATSON, M. L. (1958). Staining of tissue sections for electron microscopy with heavy metals.J. biophys. biochem. Cytol. 4, 475-478.

WHEATLEY, D. N. (1967). Cells with two cilia in the rat adenohypophysis. J. Anat. 101, 479-485.ZAMBRANO, D. & DEROBERTIS, E. (1966). The secretory cycle of supraoptic neurons in the rat.

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rats. Z. Zellforsch. mikrosk. Anat. 73, 405-413.

{Received 17 February 1969)

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Fig. i. Interstitial cell showing a centrally located nucleus and prominent nucleolus.The cytoplasm contains numerous free ribosomes and few small mitchondria. x 22 200.Inset: small Golgi complex (g) sometimes observed in interstitial cells, x 15400.

Differentiation of neurosensory cells in Hydra 711

Fig. 2. Early stage of differentiating neurosensory cell which has assumed a spindleshape. The nucleus, conspicuous nucleolus and cytoplasmic structures are similar tothose of interstitial cells. The ciliary structures (arrows) have begun to develop nearthe periphery of the cells, x 22200. Inset: early differentiating neurosensory cells withdeveloping cilium (c). Note especially the intercellular bridges (arrows) between the twoneurosensory cells (ns) and between two cnidoblasts (en), x 7965.

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Fig. 3. Small dense droplets (arrows) presumed to be centriolar precursor materials(about 50 nm in diameter) at the periphery of an interstitial cell which has begun todifferentiate into a neurosensory cell, x 29856.Fig. 4. Young centriole (ce) in the cytoplasm of a cell similar to that in Fig. 3. x 22000.Fig. 5. Two centrioles (arrows) oriented at right angles to each other near the peri-phery of a developing neurosensory cell, x 28856.Fig. 6. Developing neurosensory cell showing centriole (ce) aligned beneath the cellmembrane, dense granules (65-68 nm in diameter) at the base of the centriole andcross-section of microtubules (arrows), x 29856.

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Fig. 7. Dense granules (70 nm in diameter) arranged linearly (arrows) extending for adistance of up to 35 fim in a developing neurosensory cell. The nucleus, nucleolus andcytoplasmic structures are similar to those of interstitial cells, x 22000. Inset: centriolesoriented perpendicular to each other at the periphery of developing neurosensory cell.Note the fibres extending from the centrioles into the interior of the cell and smallgranules (similar to those in the larger micrograph) aligned along the fibres, x 15400.

Fig. 8A-D. Cross-sections of the cilium of neurosensory cells at various levels within itscytoplasmic collar. In one instance (Fig. 8 A) the internal tubules reveal a typical9 + 2 arrangement, but the number and location of the fibres vary from one level toanother, A, x 28700; B, X 28700; C, X 30600; D, x 28700.

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Fig. 9. Emerging ciliary bud (c) protruding from the cell contains axial microtubuleswithin an amorphous material. The developing cilium is completely surrounded byplasma membrane. At the base of the cilium is a low electron-dense area in whichsmall dense droplets and rootlet fibres are observed, x 29856.Fig. 10. Two developing neurosensory cells, one of which (top) contains a ciliumextending into the extracellular space. Microtubules appear throughout the entirelength of the cilium. Rootlets are seen at the base of the cilium near a centriole but atthis stage they are not striated. In the cytoplasm are numerous ribosomes, Golgi com-plex (g) and short segments of endoplasmic reticulum (arrows), x 22000.

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Fig. I I . Mature neurosensory cell. The ciliary structures reveal internal microtubulesin the cilium (c) which is surrounded by a cytoplasmic collar, basal plate, basal body andstriated rootlets. Note the small dense membrane-bounded droplets (no—130 nm indiameter) scattered throughout the cell. Most droplets occupy only a portion of thevesicles in which they are enclosed. Microtubules {mi) are parallel to the long axis ofthe cell; mitochondria with dense matrix (m) can be seen; the elongated nucleus (n)reveals dense accumulations and there is a drastic decrease in ribosomes. x 29856.Inset: cilium (c) with its striated rootlets and a pair of centrioles (arrows) one of whichapproaches a vacuole as though to begin the formation of another cilium. x 15400.

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Fig. 12. Differentiation of the neurosensory cell results in an increase in the Golgilamellae (g) but only a few vesicles originate from these membranes at this time. Densefibrous materials (arrows) of the rootlets are observed near the Golgi complex, x 45 600.Fig. 13. Three Golgi complexes (g) in a mature neurosensory cell. Small dense mem-brane-bounded droplets (no-i3onm in diameter) are in close proximity to the Golgimembranes. Larger (o-6 /im) droplets (top right) are also observed in the maturecells. Note the dense matrix of the mitochondria, (c, cilium; en, cnidoblast.) x 22000.Fig. 14. One Golgi complex (g) is characteristically located near the base of the deve-loping cilium (c). These cells contain only short segments of endoplasmic reticulum(arrows), x 28686.

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Fig. 15. Section through the cell body of a mature neurosensory cell. Note the coarsegranular material in the nucleus, cross-section of the cilium (c) with its internal tubules,dense deposits within the membranes of the Golgi complex (g), electron-dense droplets(99-110 nm in diameter), some of which are of low density and appear to be diffusingfrom their membrane-bounded vesicles, empty vesicles (top right), and mitochondria(m) revealing a dense matrix, x 45000.Fig. 16. Mitochondrion (m) with dense matrix and large dense droplets (0-7 /(m) whichmay be derived from mitochondria, x 28686.

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Fig. 17. Mature neurosensory cell showing cell body with oval nucleus containingclumps of nuclear material, and two neurites (top and bottom). Note the Golgi com-plex (g) at the base of the neurite, few mitochondria with dense matrix (m), densedroplets (100 nm in diameter) mostly in the neurite (bottom), and many microtubules(mt) running parallel to the long axis of the neurite. The dense droplets (also in Figs.18—20) occupy only a portion of the vesicles in which they are enclosed, x 14168.

Fig. 18. Mature neurosensory cell with cilium (c) and associated structures, highlyconvoluted nucleus (n), Golgi complex (g), dense droplets (90-110 nm in diameter),larger type droplet (top) and microtubules (mt) aligned along the long axis of the cell.A portion of a flagellum and stereocilia (horizontal view) of a nematocyst is shown(bottom left), x 21424.

Fig. 19. Mature neurosensory cell with cell body containing an oval nucleus and oneneurite (bottom). Small droplets (100 nm in diameter), larger droplets (arrows) about0'4 fim in diameter, Golgi complex (g) at the base of the neurite and part of the ciliaryapparatus (top) are shown, x 14168.

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Fig. 20. Neurite of a mature neurosensory cell showing small dense droplets (90 nmin diameter), large droplets, mitochondria with dense matrix (m) and microtubules(mt) aligned along the long axis of the neurite. A part of a cilium (c) is seen at the baseof the neurite. Note that the neurite is divided into two small branches (arrows) and be-tween the branches are parts of epithelio-muscular cells (ep). x 21 520.