Embryonic development of the hirudinid leech Hirudo medicinalis: structure, development and segmentation of the germinal plate

Most tissues and organs of clitellate annelids, including leeches, arise from the bandlets of stem cells that join to form the paired germinal bands (Schleip, 1936; Dawydoff, 1959). Right and left germinal bands coalesce on the future ventral midline of the embryo and give rise to the germinal plate. Histo- and organogenesis then proceed by proliferation of stem cells and their blast-cell descendants in the expanding germinal plate. The germinal plate becomes fragmented into a rostrocaudal series of distinct tissue blocks. Out of these blocks arise the metameric segmental structures, such as somites and nerve ganglia, of which each is founded by a discrete number of stem cells (Devries, 1973; Fernández & Stent, 1980; Weisblat, Harper, Stent & Sawyer, 1980). The germinal plate of clitellate embryos presents important structural and developmental similarities with the germ band and germinal disk of arthropod embryos (see Green, 1971; Turner & Mahowald, 1977, 1979).

Most of the information regarding the early stages of leech development has been obtained from embryos of the family of Glossiphonidae (order Rhychobdellidae). By contrast, very little is known about embryogenesis in the Hirudinidae (order Gnathobdellidae), despite the fact that leeches belonging to that family, especially Hirudo medicinalis, have been the favourite working material for the analysis of the structure and function of the relatively simple leech nervous system. This lack of developmental studies on the Hirudinidae is probably attributable to their embryos being too small for convenient experimental manipulation. Furthermore, early development of Hirudinidae is more complicated than that of the Glossiphonidae, since the low yolk content of the hirudinid egg requires the initial formation of a cryptolarva that feeds on the nutrient cocoon fluid to nourish the embryo. Since the composition of that fluid has not yet been defined, early hirudinid embryos cannot be cultured in artificial media. However, the absence of yolk presents also some experimental advantages: hirudinid embryos are sufficiently transparent to allow detailed microscopic viewing in whole mounts of the structure and arrangement of the different types of cells in the germinal plate.

What is known about hirudinid leech ontogeny is derived mainly from the classical works of Leuckart (1863), Bergh (1885) and Bürger (1894) and from more recent work by Shumkina (1951 a–d and 1953). According to these studies, a gravid Hirudo usually lays several cocoons during the breeding season. Each cocoon includes about 5–25 eggs, about 100 μm in diameter, that are bathed in the viscous cocoon fluid referred to as albumen. During the first 2 days of development of Hirudo at 25 °C, cleavage leads to the formation of a 30- to 40-cell embryo. The majority of the cells in this embryo are small and lie at the top of three large cells, or macromeres, designated as cells A, B and C. The macromeres no longer divide and are destined to disintegrate in later stages of development. The small cells fall into three groups. One group, lying in the anterior dorsal region of the embryo, forms the micromere cap. Cells of the micromere cap give rise to the larval mouth and envelope. A second group of cells, lying in the middle dorsal region, include the five pairs of teloblasts, which are descended from a fourth large cell, the D macromere. Iterated divisions of the teloblasts produce the bandlets of stem cells that give rise to the germinal plate. Four teloblast pairs designated as N, O, P and Q [to match the designation used for their homologs in glossiphoniid embryos (Fernández, 1980; Weisblat et al. 1980)] are the precursors of ectoderm. One teloblast pair, designated as M, is the precursor of mesoderm. A third group of small cells lying in the posterior dorsal region have been reported to give rise to endoderm. During days 2μ4 of development, the size of the embryo increases considerably, due to ingestion of albumen. By the end of day 4, the embryo has grown to about 450 μm in diameter, and the early larva has formed. That larva consists of a membranous envelope, a primordial mouth, paired germinal bands and the degenerating macromeres. A germinal band extends along either side of the larva, from the primordial mouth to the teloblasts. Right and left germinal bands are associated at their caudal region. Each germinal band consists of five bandlets of stem cells formed by division of the teloblasts lying at the caudal end of the germinal bands. The stem cell bandlets are designated as m, n, o, p and q, with each lower case letter corresponding to the upper case letter of the teloblast of origin. Lateral outgrowths of the germinal bands form the primordial protonephridia of the larva. During days 5–9 of development, larval structures become fully developed and albumen is ingested intensively via the larval mouth. Coalescence of the germinal bands on the ventral midline, and thus formation of the germinal plate, is completed by day 5 of development. Segmentation of the germinal plate tissue is initiated by day 6 of development and is completed by day 11. Metamorphosis of the larva into a juvenile leech begins at about day 9, when active swallowing of albumen is terminated, and lasts until day 18–20, when the lateral edges of the expanding germinal plate join along the dorsal midline. Larval structures, such as the larval envelope and protonephridia, appear to disintegrate and not to contribute to the formation of adult tissues. The last 8–10 days of intracocoon development are devoted mostly to morphogenesis of the gut epithelium. Juvenile leeches perforate the cocoon wall and hatch with their gut filled with albumen at about day 30. The albumen is gradually digested and nourishes the juvenile until its first meal is taken from a suitable prey.

This paper presents studies of two hitherto largely unexplored aspects of the germinal plate of leech embryos, using 5- to 12-day-old larvae of Hirudo. One aspect of the present studies concerns the origin of the orderly arrangement of cells within the germinal plate tissue. Another aspect concerns the mechanisms involved in the formation of the fixed number of 32 body segments.

Cocoons were obtained from a breeding population of Hirudo medicinalis maintained in the laboratory since 1976. Leeches are kept in aerated aquaria (about 25 specimens per 20 1 aquarium) containing artificial spring water at 23–25 °C. Leeches are fed every 30–45 days by allowing them to suck blood from adult unanaesthetized rabbits having their ventral skin shaved. To avoid regurgitation of the blood meal, fed leeches are kept for about a week at lower temperatures. They are then isolated in individual glass jars for 2-3 weeks. Isolation appears to promote copulation when leeches are put back together in the aquarium. After a few weeks, gravid leeches are detected in the aquaria. The sign of gravidity is the swelling and change of colour of the clitellum. This appears as a yellowish region of the ventral skin, extending between the sexual orifices of the leech. Gravid leeches are transferred to terraria at about 25 °C containing humid peat. Under these conditions, leeches lay 1–3 cocoons over a period of a few weeks. Daily inspection of terraria allowed determination of the approximate time cocoons were laid. Since cocoon laying may be interrupted when leeches are disturbed, searching for cocoons must be performed carefully. Cocoons are ovoid bodies 1–3 cm in length. Their wall consists of an outer spongy layer that retains humidity, and an inner compact layer, that completely seals the cocoon chamber. Cocoons were cleaned of peat by a jet of air, lightly wrapped in wet paper towels and placed in shallow glass containers. Embryonic development within the cocoon was allowed to proceed in an incubator at 23–25 °C. To maintain a population of breeding leeches, some cocoons were allowed to develop until the middle of the fourth week. These cocoons were opened and the juveniles were released into spring water. One-month-old juveniles were allowed to feed on mice. For further feedings rabbits were used. Leeches 9–12 months old become sexually mature. More details on breeding and raising of Hirudo can be found in Sine va (1944, 1949).

It also proved possible to culture embryos and early larvae, up to 7 days of development, outside their cocoons. For that purpose, cocoons were opened under sterile conditions soon after laying. Glassware was autoclaved and dissection tools were soaked in 100% acetone. The spongy outer layer of the cocoon was removed with a razor blade, embryos or larvae and their surrounding albumen were forced out of the cocoon (by gently pressing its surface) on to a watch glass maintained in a moist chamber. Older larvae were cultured in Millipore-filtered spring water.

To allow larval muscle fibres to relax prior to fixation, 5- to 12-day-old embryos were anaesthetized in 8 % ethanol in spring water. When movements of the larval mouth and envelope ceased, embryos were transferred to spring water. This procedure allowed further relaxation of the muscle fibres and reduced distortion of the germinal plate. Five- to seven-day-old embryos were then fixed for 2 h at 4 °C in 50% Karnovsky solution, or in a 1:1 solution of 10% formaldehyde and 5 % glutaraldehyde in double-distilled water. Older embryos were fixed for 2–4 h in cold 1:1 solution of 17% formaldehyde and 12% glutaraldehyde in double-distilled water. After fixation the embryos were rinsed for 2–4 h in 3 changes of cold 0-1 M cacodylate buffer (for larvae fixed in Karnovsky solution) or in double-distilled water. The embryos were then postfixed for 1–2 h in 1 % OsO₄ in cacodylate buffer or in double-distilled water at room temperature in the dark. After several rinses in the appropriate solution, the germinal plate and part of the surrounding larval envelope were dissected out. Tissues were dehydrated in graded ethanol, cleared for several hours in xylene-phenol and whole mounted with or without coverslips, using Permount resin, in such a manner that the inner surface of the germinal plate faced up. Whole mounts were examined under the phase-contrast microscope.

Embryos were fixed for 2 h in cold 50 % Karnovsky solution, as described above. After dehydration in graded ethanol, germinal plates were embedded in Epon 812 and then sectioned at 1 μm in a Porter Blum ultramicrotome. Serial sections of the germinal plate were stained with 1 % toluidine blue in borate buffer.

In embryos of H. medicinalis formation of body segments is initiated on day 5–6 of development at 23–25 °C, and completed on day 10–11. The structure of the germinal plate during that phase of development is described in the following.

On day 6 the germinal plate is 0·9–1·5 mm long and consists of two morphologically distinct sectors, one anterior and the other posterior, of roughly equal lengths. Of these, the posterior sector appears as a ribbon of uniform width of about 60 /un. This sector will be referred to as the ribbon part. At the caudal tip of the ribbon part lie the teloblasts, visible as five pairs of large cells (Figs. 1c and 18–19). The anterior sector of the germinal plate gradually widens towards the front, forming a fan-shaped structure that reaches a width of about 150 μm at the larval mouth (Fig. 1a).

The germinal plate consists of two cell layers: an outer ectodermal layer, formed by the n, o, p and q bandlet pairs, and an inner mesodermal layer formed by the m bandlet pair. In the ribbon part, the ectodermal layer is one cell thick and eight cells wide. The n bandlet pair provides the most medial two and the q bandlet pair the most lateral two of these eight cells, with the o and p bandlet pairs providing the other four cells. The n bandlet cell pair straddles the embryonic midline and lies more dorsally than the o, p and q bandlet cell pairs, which are in contact with the ventral region of the larval envelope. Hence, the eight cells form a bilateral pair of ectodermal arches whose concavity is orientated toward the dorsal embryonic surface. The m bandlet lies in the concavity of the ectodermal arches, and most of its cells are larger and usually stain more darkly than those of the n, o, p and q bandlets. Most of the mesoderm of the ribbon part is several cells thick and wide (Figs. 1c, 11, 13).

The ribbon part ends at that longitudinal position of the germinal plate to the front of which the ectodermal arches become progressively wider and thicker (Figs. 2, 3). In that anterior sector the ectodermal arches of the ribbon part become five, six, seven or more cells in width and several cells thick (Figs. 16, 12, 14). The widest and thickest ectoderm lies adjacent to the larval mouth. This expansion of the outer ectodermal layer is accompanied by an expansion of the inner mesodermal layer, which similarly thickens and widens. The anterior sector of the germinal plate in front of the ribbon part includes five to eight early ganglionic primordia (or about one quarter of the final number of ganglia). These primordia appear as a longitudinal series of transverse bands of ectodermal tissue separated from one another by rounded or rectangular regions of mesoderm of low cell density called interprimordial tissue (Fig. 3). The set of ganglionic primordia gives the anterior sector of the germinal plate a ladder-like appearance. More developed early ganglionic primordia lie in the frontmost region of the germinal plate and appear as dumb-bell-shaped structures that consist of a median part and two lateral parts. Less developed early ganglionic primordia lie to the rear, just forward of the ribbon part, and mostly consist of a median part (Fig. 14). Concomitant with the formation and development of ganglionic primordia, proliferation of cells of the mesoderm leads to the formation of somites. In whole-mounted embryos stretched out prior to fixation, paired somites and ganglionic primordia lie in register (Fig. 3).

On day 7 of development the germinal plate is 2–2·5 mm in length, of which only the caudal tenth is made up by a ribbon part (whose structure is similar to that seen in 6-day-old embryos). The rest of the germinal plate is thicker and wider than the plate of 6-day-old embryos, reaching a width of 250–400 μm at the larval mouth. The germinal plate now contains 14–20 ganglionic primordia, or about half of the final number of ganglia, of which the rostral half to two-thirds are late primordia. Such late primordia present three important characteristics: (a) their lateral parts have moved medially and thus they appear as figure-of-eight structures; (6) they are linked via three short nerve tracts that correspond to developing connectives ; and (c) they are separated by narrower interprimordial tissue due to medialward displacement of the nerve bundles of the connectives (Fig. 8). The structure of early primordia is shown in Figs. 15 and 16. The germinal plate contains 12–18 pairs of somites that are intercalated between ganglionic primordia. Thus somites and ganglionic primordia lie no longer in register (Fig. 8). Formation of nephridial primordia has begun in the somite pairs lying between the 4th and 5th ganglionic primordia.

On day 9 of development the germinal plate is 3·5–4 mm in length, of which only the caudal twentieth is made up of a ribbon part. The cellular components of the residual ribbon part show signs of disorganization and disintegration. The shape of the germinal plate has changed from fan to ellipse, in that its widest part now lies at some distance from the larval mouth. The mesodermal tissues have formed about 25 paired somites, 17 pairs of nephridial primordia and the primordia for the male and female genitalia. The frontmost 10–15 late ganglionic primordia have matured into morphologically intact ganglia and connectives, surrounded by a perineural coelom, thus forming an embryonic nerve cord. The frontmost four ganglia are figure-of-eight-shaped structures linked via very short connectives; they form the suboesophageal ganglion (Fig. 9). The next 6–10 ganglia are globular, linked via longer connectives. Except for the first two or three ganglia, paired intersegmental septa are seen to course circumferentially from the lateral surfaces of each ganglion to the lateral edges of the germinal plate. The rear part of the anterior sector of the germinal plate contains 10–15 late and early ganglionic primordia (Fig. 17).

On day 11 of development the germinal plate is about 8 mm in length. It can be subdivided into cephalic, abdominal and caudal regions. The cephalic region has grown around the larval mouth, encircling it completely. The larval mouth itself has become significantly smaller. The cephalic region includes the frontmost four ganglia of the nerve cord, which form a conical mass of nerve tissue corresponding to the suboesophageal ganglion. A pair of perioesophageal commissures connect the suboesophageal to the supraoesophageal ganglion. The supraoesophageal ganglion has arisen from a mass of cells that lie in front of the larval mouth (Fig. 2), and thus the developmental origin of that ganglion is not the germinal plate. The abdominal region of the germinal plate includes the 21 abdominal ganglia of the embryonic nerve cord, separated by connectives of different lengths, 17 pairs of embryonic nephridia, and the primordia of the male and of the female genitalia that lie respectively in front of and behind the sixth abdominal ganglion. The paired deferential ducts have begun to grow rearward from the primordium of the male genitalia. The caudal region of the germinal plate appears as a disk-like structure from which the caudal sucker will arise. The caudal region includes the last seven ganglia of the nerve cord, which form an elongated mass of nerve tissue corresponding to the caudal ganglion. The cells of the residual ribbon part have degenerated, and their debris is found scattered over the caudal region (Fig. 31).

In the abdominal region of the germinal plate the body segments are organized in the manner characteristic of late embryos. Paired intersegmental septa course at right angles to the longitudinal axis but are slanted with respect to the dorsoventral axis. The anterior (dorsal) edge of each septum lies in register with the ganglion, and the septum extends posteriorly (and ventrally) to about the interganglionic midpoint of the connective (Figs. 9, 10). Thus, this arrangement of the slanted septa gives rise to a rostrocaudal succession of bilaterally symmetric, pocket-like compartments. This septal slanting has the following consequences: (a) the position of the ganglia becomes intersegmental because their rostral and caudal halves come to lie on opposite sides of the septum; (b) successive body segments overlap for about half of their length; (c) most of the components of nephridia lie in the septal tissue since they arise from the rostral mesoderm of each body segment. This arrangement of the septa may be explained as the result of a rostrocaudal expansion of somite tissue due to differential growth.

The teloblasts are globular or pear-shaped cells, 15–30 μm in diameter. They have a globular nucleus, about 10 μm in diameter, which encloses a prominent nucleolus, about 4 μm in diameter. The M and N teloblast pairs lie dorsally, whereas the O, P and Q teloblast pairs lie ventrally. The M teloblast pair has a nearly bilaterally symmetrical disposition in the rearmost region of the germinal plate, usually overlying the O and P teloblasts. The M teloblast pair is not in mutual contact (Figs. 18, 19). The N teloblast pair is the most rostrally situated pair. Usually it is asymmetrically disposed, with one N teloblast - either the right or the left - lying more caudally than its contralateral homolog. In most embryos, the N teloblast pair is in mutual contact, and the more caudal member of the pair is usually in contact also with its ipsilateral O teloblast (Figs. 18–19, 21–23). The O, P and Q teloblasts are nearly symmetrically disposed, with the O pair being in mutual contact at the embryonic midline and the Q pair having the most lateral and slightly dorsal position. As shown in Fig. 18, the paired teloblasts form an arch-like arrangement.

Although the pattern of stem-cell production by teloblast division has not yet been studied in great detail, in all embryos so far examined only one or the other of the teloblasts of a given pair was seen to be dividing. Furthermore, concurrently dividing members of different teloblast pairs were seen to lie either on the same side or on opposite sides of the embryo. Therefore, it appears that teloblast division is asynchronous (Figs. 19, 21).

The manner in which teloblasts divide to form stem cells may be judged from the position of the last-formed stem cell of any given bandlet relative to the rostrocaudal axis of its progenitor teloblast. In the case of the M teloblast, the last-formed stem cell may lie either to the right or to the left of rostrocaudal teloblast axis, giving rise to a zig-zag cell pattern in the M bandlet proximal to its teloblast (Figs. 21–23). This finding suggests that the M teloblasts divide according to a spiral cleavage pattern. By contrast, there is little evidence for a spiral division pattern of the N, O, P and Q teloblasts. In most of the embryos, the last-formed stem cells of the n, o, p and q bandlets could be seen to lie on the same side of the rostrocaudal teloblast axis. Therefore, the N, O, P and Q teloblasts probably divide unidirectionally.

In the ribbon part of the germinal plate, the spatial arrangement of the band-lets corresponds to the relative disposition of their progenitor teloblasts. Cells of the m bandlet are globular in shape, measure 8–20 μm in diameter and usually stain more darkly than the cells of the other bandlets (Figs. 21–23). Cells of the n, o, p and q bandlets are globular or cubical, or oblong in shape and measure 5–12 μm in width (Figs. 13, 18, 19). The bandlet cell nuclei include one nucleolus, and sometimes two nucleoli, whose size and shape varies considerably (Figs. 11–14, 18–26).

In the ribbon part of the germinal plate mitotic cells are found scattered along the bandlets. Mitotic bandlet cells lying close to their teloblasts of origin probably correspond to dividing stem cells, whereas those distant from their teloblasts may be dividing blast cells. Since both the distribution and the number of mitotic cells differ in a given bandlet pair, it would appear that bilaterally homologous stem and blast cells divide asynchronously. In the n, o, p and q bandlets of the ribbon part, the planes of cell division are perpendicular to the longitudinal axis of the embryo. Hence, these bandlets grow in length, without becoming either wider or thicker. This unidirectional, interstitial mode of cell division leads to an increase in the number of 4-cell-wide, bilaterally paired, ectodermal arches characteristic of the ribbon part (Fig. 20). By contrast, division of the cells of the m bandlet in the ribbon part follows a spiral division pattern, and this gradually obscures the initial zig-zag pattern of arrangement of the stem cells (Figs. 21–23). In addition, spiral divisions in the m bandlets lead to the formation of cell clusters (Figs. 24 and 25). Figure 27 shows how the observed cell arrangement pattern’in the m bandlets and lengthening of the mesoderm in the ribbon part of the germinal plate can be accounted for by successive spiral division of teloblasts and of stem and blast cells.

Forward of the ribbon part, changes in the mode of division of mesodermal and ectodermal bandlet cells result in an increase in width and the thickness of the germinal plate. The mesodermal bandlets become segregated into solid cell clusters that constitute primordial somites. There is evidence that cells of the somites continue to divide in a spiral pattern (Fig. 26). Widening and thickening of the ectoderm is the result of division of the n, o, p and q bandlet cells in planes parallel (rather than perpendicular, as was the case in the ribbon part) to the longitudinal embryonic axis. Division in vertical planes leads to an increase in the width of the ectodermal arches (Figs. 1b, 12, 14). In regions where ganglionic primordia are about to be formed, mitotic cells are more abundant in the medial than in the lateral portion of the ectodermal arches (Figs. 14, 16). These medial mitotic cells are probably blast cells derived from the n bandlet and founders of the median part of the ganglionic primordia. Division in horizontal planes leads to thickening of the ectodermal arches. This expansion process is accompanied by formation in the lateral portion of the ectodermal arches of two layers of cell clusters, one dorsal and the other ventral. These clusters include cells of the o, p and q bandlets because these bandlets constitute the lateral portion of the ectodermal arches. The dorsal cluster, which remains associated with the median part of the ganglionic primordium, includes founder cells for the lateral parts of the ganglion. There-fore, ganglionic primordia consist of cells derived from the n, o, p and q bandlets. The ventral cluster of cells presently splits into two cell layers. Of these, the outer layer, lying next to the larval envelope, forms the epidermis and the inner layer seems to develop into the circular muscles of the body wall (Fig. 15). Therefore, the epidermis and possibly the circular muscles are derived from cells of the o, p and q bandlets.

Formation of further body segments ceases in 9-day-old embryos, as manifest by an interruption in the m bandlets just to the fore of the residual ribbon part of the germinal plate. This interruption consists of a mesoderm-free gap about 50 μm in length on either side of the germinal plate, within which the outer ectoderm is deprived of contact with inner mesoderm. As shown in Figure 32, the mesoderm-free gap is about the size of one adjacent cluster of mesodermal cells. The mesoderm-deprived ectoderm appears normal (Fig. 33), and mitotic cells can be seen in its bandlets (Fig. 32). Anterior to the ribbon part, the germinal plate of 9-day-old embryos contains 25 discernible somites, and in addition, 7 bilateral pairs of mesodermal cell clusters, precursors of somites 26–32 (Figs. 6, 30). Therefore, the interruption of the m bandlets occurs just at that position of the germinal plate, anterior to which there is just sufficient mesoderm to form 32 body segments. In the residual ribbon part, posterior to the gap, several paired clusters of mesodermal cells are present that resemble somite precursors (Fig. 32).

Interruption of the m bandlets is soon followed by degeneration of the residual ribbon part of the germinal plate, as manifested by progressive disintegration of the bandlets and teloblasts, and the appearance of abundant cell debris (Figs. 29–31, 34–35). The first signs of degeneration are usually seen at the front of the residual ribbon part, where ectodermal bandlets have been deprived of contact with mesoderm. Cell degeneration then proceeds rearward until the bandlets and, finally the teloblasts, have disintegrated. However, some of the mesodermal cells of the residual ribbon part do not degenerate; they detach from the bandlets, enter mitosis, and migrate from the degenerating ribbon part into the larval membranes (Fig. 34).

Comparison of the disposition of the teloblasts of 6- to 9-day-old embryos with the disposition of their precursor cells in earlier embryos (see Shumkina, 1951 a) leads to the conclusion that the arch-like arrangement of the teloblasts is the consequence of an orderly procession of spiral cleavages. This is also the case for the formation of teloblasts in the glossiphoniid Theromyzon rude (Fernández, 1980). However, the relative position of the teloblasts in the embryo of Hirudo is the inverse of that found in glossiphoniid embryos (Fernández, 1980): in Theromyzon the lateromedial sequence of ectoteloblasts is N, O, P, Q, whereas in Hirudo it is Q, P, O, N. Accordingly, in Hirudo embryos it is the right and left members of the O and N ectoteloblast pairs that are in contact rather than the right and left members of the Q ectoteloblast pair, as is the case in Theromyzon embryos. Furthermore, the M mesoteloblast pair is in mutual contact in Theromyzon embryos but not in Hirudo embryos. The Hirudo-type of arrangement of the teloblasts is also seen in embryos of other clitellate annelids, such as those of Erpobdelliid and Piscicoliid leeches and of some oligochaetes (see Schleip, 1936; Dawydoff, 1959, and Devries, 1973).

This difference in the relative position of teloblasts in the two types of leeches appears to reflect a difference in the early cleavage pattern leading up to teloblast formation. In Hirudo, the ectodermal proteloblast NOPQ cleaves to yield a caudomedial cell NO and a rostrolateral cell PQ (Shumkina, 1951 a). Cleavage of cell NO thereupon yields the rostromedial teloblast N and the caudolateral teloblast O, whereas cleavage of PQ yields the caudomedial teloblast P and the rostrolateral teloblast Q, resulting in the teloblast arch in mediolateral order N, O, P, Q. In glossiphoniids by contrast, the ectodermal proteloblast NOPQ cleaves to yield the caudomedial cell OPQ and the rostrolateral N teloblast. In two further cleavages, OPQ yields the rostromedial teloblast Q and the caudolateral cell OP, and cell OP yields the caudomedial teloblast P and the rostrolateral teloblast O, resulting in the teloblast arch in mediolateral order Q, P, O, N (Fernández, 1980). Despite this apparent difference in cell lineage of origin and relative position in the early embryo, the four ectoteloblast pairs of Hirudo and of glossiphoniid leeches have similar presumptive fates and hence can be considered as homologous blastomeres.

Unlike the teloblasts of glossiphoniid embryos, which make rotational and translational movements during the early phase of embryogenesis (Fernández & Stent, 1980), the teloblasts of Hirudo embryos do not appear to make such movements. This difference in teloblast movement is undoubtedly related to the fact that the germinal bands of glossiphoniid embryos do and those of Hirudo embryos do not migrate circumferentially prior to their coalescence to form the germinal plate.

Growth of the germinal plate proceeds concomitantly with the expansion of the embryo as it ingests albumen from the cocoon fluid. Ingestion of albumen ceases at about the 9th day of development, after which time the growing germinal plate gradually spreads over the larval surface. The available evidence indicates that the larval surface membrane degenerates and does not form part of the later embryo (Schleip, 1936; Shumkina, 1951 b, 1953).

Lengthening, widening and thickening of the germinal plate is clearly the result of an orderly accumulation of ectodermal and mesodermal stem and blast cells. Stem cells are produced for about 6 days, their formation by teloblasts being initiated on day 3 (Shumkina, 1951 b–d) and terminated on day 9. If the rate at which stem cells are produced by teloblast cleavage is assumed to be about the same as the rate of blastomere cleavage, namely about once every 2h (Shumkina, 1951 a), each teloblast would form a total of about 70 stem cells. As shown here, the last of these 70 stem cells to be produced disintegrate upon the degeneration of the residual ribbon part of the germinal plate. Thus, in view of the total number of available stem cells, no more than two stem cells from each bandlet are likely to contribute to the foundation of each of the 32 segments. This estimate of the total number of stem cells produced is in good agreement with that previously reported for embryos of Theromyzon (Fernández & Stent, 1980).

The division pattern of the mesoteloblasts differs from that of the ectoteloblasts : the former divide spirally and the latter unidirectionally. Thus, the early spiral cleavage pattern of the blastomeres that gave rise to the five teloblast pairs is maintained in subsequent divisions of the mesoteloblast pair but not of the ectoteloblast pairs. It seems likely that the switch from spiral to unidirectional cleavage in the ectoteloblast cell line reflects a developmental change in a determinant of the orientation of the mitotic spindle in successive cell divisions. This change would originate in, and be passed on to the descendants of, the ectodermal precursor blastomere arising from cleavage of the D blastomere. By contrast, the other daughter of that cleavage, the mesodermal precursor blastomere, would preserve the determinant for the spiral cleavage pattern and pass it on to the M teloblasts and their daughter cells of the m bandlets. These differences in the division pattern of ecto- and mesoteloblasts, and of their descendant bandlet cells, are likely to play an important role in the differential morphogenesis of ectodermal and mesodermal components of body segments.

As a consequence of the degeneration of the residual ribbon part of the germinal plate, an as yet undetermined number of stem cells and their progeny disintegrate or fail to participate in further development of the germinal plate. It would follow, therefore, that the exact number 32 of segments formed is not determined by the total number of stem cells produced by any of the teloblasts. However, the fact that the 32nd segment arises from tissues lying immediately in front of the gap in the m bandlets suggests that the mesoderm limits the number of body segments produced. One possibility to account for the gap is that after having produced the number of m stem cells needed to found 32 somites, the M teloblasts temporarily cease dividing. Meanwhile, formation of ectodermal stem cells and their division into blast cells, and hence elongation of the ectodermal bandlets, would continue. Upon resumption of division of the M teloblasts, a bilateral, mesoderm-free gap would appear in the germinal plate. An alternative explanation for the appearance of such a gap would be that the m stem cells produced in excess of the number needed to found 32 somites receive some signal that causes them to degenerate in a rostrocaudal sequence, beginning with the frontmost of the surplus cells. It should be noted that the dimensions of the gap are similar to the dimensions of adjacent bilateral clusters of mesodermal cells which give rise to paired somites and that in leech (as well as oligochaete embryos) each half somite appears to be founded by one m stem cell (Devries, 1973 ; Fernández & Stent, 1980). It would follow, therefore, that the gap arises as a consequence of the absence of the descendants of a right and a left mesodermal stem cell. In any case, there are good reasons to think that the gap in the m bandlets induces the degeneration of the residual ribbon part of the germinal plate. First, in all embryos so far examined degeneration of the residual ribbon part is always preceded by a gap in the m bandlets. Second, the first cells of the residual ribbon part to show signs of degeneration are those of the ectodermal bandlets not in contact with mesoderm. Since in Theromyzon embryos many of the m stem cells produced last fail to be incorporated into the germinal plate, the mechanism of limiting segment formation is probably similar in glossiphoniids to that obtaining in Hirudo.

The present results confirm previous findings concerning the dual developmental origin of the central nervous system of Hirudinea. The supraoesophageal ganglion arises from a cluster of cells lying in front of the larval mouth. This cluster is separate from the germinal plate (which gives rise to the remaining 32 ganglia) and corresponds to the cephalic plate of hirudinid leeches (Bergh, 1885; Shumkina, 1951c; Dawydoff, 1959). The origin of the cephalic plate has not yet been clearly established. Shumkina (1951c) reported that the cephalic plate of Hirudo originates from cells migrating out of the germinal bands of 3-day-old embryos, whereas Weisblat et al. (1980), using horseradish peroxidase as a cell lineage marker, have demonstrated that the supraoesophageal ganglion of the glossiphoniid Helobdella triserialis is founded by cells derived from the micromere cap.

We thank Ildina Cerda, Georgia Harper, Margery Hoogs, Nora Loyarte, Victor Monasterio, Nancy Olea and Lilio Yáñez for technical assistance and Serena Mann for preparing the illustrations. We also thank Dr. R. T. Sawyer for many valuable suggestions on how to breed H. medicinalis. This investigation was supported by grant B 257–803, from Servicio de Desarrollo Científico, Artístico y de Cooperación Internacional, Universidad de Chile, NSF grant BMS 74–24637, from the National Foundation – March of Dimes, and grant RLA 78-024-11, from PNUD/UNESCO.