embryonic developmen oft the hirudinid leech hirudo medicinalis: … · raising of hirudo can be...

J. Entbryol. exp. Morph. Vol. 72, pp. 71-96, 1982 7 1

Printed in Great Britain © Company of Biologists Limited 1982

Embryonic development of the hirudinid leechHirudo medicinalis: structure, development

and segmentation of the germinal plate

By JUAN FERNANDEZ1 AND GUNTHER S. STENT2

From the Departamento deBiologia, Facultad de CienciasBdsicas y Farmaceuticas,Universidad de Chile,

and the Department of Molecular Biology, University of California

SUMMARY

The germinal plate of 5- to 12-day-old embryos of the leech Hirudo medicinalis consistsof an anterior and a posterior sector that differ both structurally and developmentally. Theposterior sector includes the five pairs of teloblasts and five paired longitudinal bandlets ofstem cells and their descendant blast cells. The mesoteloblast pair and their descendantcells of the m bandlet divide spirally and give rise to bilaterally paired cell clusters. The fourectoteloblast pairs and their descendant cells of the n, o, p and q bandlet pairs divide uni-directionally and give rise to paired one-cell-thick and four-cell-wide ectodermal arches.The developmentally more advanced anterior sector of the germinal plate consists of differen-tiating ectodermal and mesodermal cells engaged in organogenesis. The mesodermal cellclusters develop into somites, whereas the expanding ectodermal arches develop into nervecord ganglia and epidermis. Rostrocaudal expansion of somite tissue results in the formationof obliquely oriented intersegmental septa, causing the ganglia to take on an intersegmentaldistribution. The first sign that formation of body segments has been completed is theappearance of a bilateral gap in the mesodermal bandlet of the posterior sector of the germinalplate. This gap seems to trigger degeneration of the posterior sector of the germinal plate.

INTRODUCTION

Most tissues and organs of clitellate annelids, including leeches, arise fromthe bandlets of stem cells that join to form the paired germinal bands (Schleip,1936; Dawydoff, 1959). Right and left germinal bands coalesce on the futureventral midline of the embryo and give rise to the germinal plate. Histo- andorganogenesis then proceed by proliferation of stem cells and their blast-celldescendants in the expanding germinal plate. The germinal plate becomesfragmented into a rostrocaudal series of distinct tissue blocks. Out of theseblocks arise the metameric segmental structures, such as somites and nerveganglia, of which each is founded by a discrete number of stem cells (Devries,

1 Author's address: Departamento de Biologia, Facultad de Ciencias Basicas y Farmaceu-ticas, Universidad de Chile, Casilla 653, Santiago, Chile.

2 Author's address: Department of Molecular Biology, University of California, Berkeley,California, 94720, U.S.A.

72 J. FERNANDEZ AND G. S. STENT

1973; Fernandez & Stent, 1980; Weisblat, Harper, Stent & Sawyer, 1980). Thegerminal plate of clitellate embryos presents important structural and develop-mental 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 developmenthas been obtained from embryos of the family of Glossiphonidae (orderRhychobdellidae). By contrast, very little is known about embryogenesis in theHirudinidae (order Gnathobdellidae), despite the fact that leeches belongingto that family, especially Hirudo medicinalis, have been the favourite workingmaterial for the analysis of the structure and function of the relatively simpleleech nervous system. This lack of developmental studies on the Hirudinidaeis probably attributable to their embryos being too small for convenientexperimental manipulation. Furthermore, early development of Hirudinidae ismore complicated than that of the Glossiphonidae, since the low yolk contentof the hirudinid egg requires the initial formation of a cryptolarva that feedson the nutrient cocoon fluid to nourish the embryo. Since the composition ofthat fluid has not yet been defined, early hirudinid embryos cannot be culturedin artificial media. However, the absence of yolk presents also some experi-mental advantages: hirudinid embryos are sufficiently transparent to allowdetailed microscopic viewing in whole mounts of the structure and arrangementof the different types of cells in the germinal plate.

What is known about hirudinid leech ontogeny is derived mainly from theclassical works of Leuckart (1863), Bergh (1885) and Burger (1894) and frommore recent work by Shumkina (1951 a-d and 1953). According to these studies,a gravid Hirudo usually lays several cocoons during the breeding season. Eachcocoon includes about 5-25 eggs, about 100/tm in diameter, that are bathedin the viscous cocoon fluid referred to as albumen. During the first 2 days ofdevelopment of Hirudo at 25 °C, cleavage leads to the formation of a 30- to40-cell embryo. The majority of the cells in this embryo are small and lie at thetop of three large cells, or macromeres, designated as cells A, B and C. Themacromeres no longer divide and are destined to disintegrate in later stages ofdevelopment. The small cells fall into three groups. One group, lying in theanterior dorsal region of the embryo, forms the micromere cap. Cells of themicromere cap give rise to the larval mouth and envelope. A second group ofcells, lying in the middle dorsal region, include the five pairs of teloblasts, whichare descended from a fourth large cell, the D macromere. Iterated divisions ofthe teloblasts produce the bandlets of stem cells that give rise to the germinalplate. Four teloblast pairs designated as N, O, P and Q [to match the designa-tion used for their homologs in glossiphoniid embryos (Fernandez, 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 cellslying in the posterior dorsal region have been reported to give rise to endoderm.During days 2-4 of development, the size of the embryo increases considerably,

Embryonic development of the leech 73

due to ingestion of albumen. By the end of day 4, the embryo has grown toabout 450 [im in diameter, and the early larva has formed. That larva consistsof a membranous envelope, a primordial mouth, paired germinal bands andthe degenerating macromeres. A germinal band extends along either side of thelarva, from the primordial mouth to the teloblasts. Right and left germinalbands are associated at their caudal region. Each germinal band consists offive bandlets of stem cells formed by division of the teloblasts lying at the caudalend of the germinal bands. The stem cell bandlets are designated as m, n, o, pand q, with each lower case letter corresponding to the upper case letter of theteloblast of origin. Lateral outgrowths of the germinal bands form the primordialprotonephridia of the larva. During days 5-9 of development, larval structuresbecome fully developed and albumen is ingested intensively via the larvalmouth. Coalescence of the germinal bands on the ventral midline, and thusformation of the germinal plate, is completed by day 5 of development. Segmen-tation of the germinal plate tissue is initiated by day 6 of development and iscompleted by day 11. Metamorphosis of the larva into a juvenile leech beginsat about day 9, when active swallowing of albumen is terminated, and lastsuntil day 18-20, when the lateral edges of the expanding germinal plate joinalong the dorsal midline. Larval structures, such as the larval envelope andprotonephridia, appear to disintegrate and not to contribute to the formationof adult tissues. The last 8-10 days of intracocoon development are devotedmostly to morphogenesis of the gut epithelium. Juvenile leeches perforate thecocoon wall and hatch with their gut filled with albumen at about day 30. Thealbumen is gradually digested and nourishes the juvenile until its first meal istaken from a suitable prey.

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

MATERIALS AND METHODS

Maintenance and breeding of the medicinal leech

Cocoons were obtained from a breeding population of Hirudo medicinalismaintained in the laboratory since 1976. Leeches are kept in aerated aquaria(about 25 specimens per 20 1 aquarium) containing artificial spring water at23-25 °C. Leeches are fed every 30-45 days by allowing them to suck bloodfrom adult unanaesthetized rabbits having their ventral skin shaved. To avoidregurgitation of the blood meal, fed leeches are kept for about a week at lowertemperatures. 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. Thisappears as a yellowish region of the ventral skin, extending between the sexualorifices of the leech. Gravid leeches are transferred to terraria at about 25 °Ccontaining humid peat. Under these conditions, leeches lay 1-3 cocoons over aperiod of a few weeks. Daily inspection of terraria allowed determination of theapproximate time cocoons were laid. Since cocoon laying may be interruptedwhen leeches are disturbed, searching for cocoons must be performed carefully.Cocoons are ovoid bodies 1-3 cm in length. Their wall consists of an outerspongy layer that retains humidity, and an inner compact layer, that completelyseals the cocoon chamber. Cocoons were cleaned of peat by a jet of air, lightlywrapped in wet paper towels and placed in shallow glass containers. Embryonicdevelopment within the cocoon was allowed to proceed in an incubator at23-25 °C. To maintain a population of breeding leeches, some cocoons wereallowed to develop until the middle of the fourth week. These cocoons wereopened and the juveniles were released into spring water. One-month-oldjuveniles were allowed to feed on mice. For further feedings rabbits were used.Leeches 9-12 months old become sexually mature. More details on breeding andraising of Hirudo can be found in Sineva (1944, 1949).

Culture of embryos and larvae

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

Preparation of wholemounts

To allow larval muscle fibres to relax prior to fixation, 5- to 12-day-oldembryos were anaesthetized in 8 % ethanol in spring water. When movementsof the larval mouth and envelope ceased, embryos were transferred to springwater. This procedure allowed further relaxation of the muscle fibres and reduceddistortion of the germinal plate. Five- to seven-day-old embryos were thenfixed 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 embryoswere 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 rinsedfor 2-^h in 3 changes of cold 0-1 M cacodylate buffer (for larvae fixed inKarnovsky solution) or in double-distilled water. The embryos were thenpostfixed 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 dissectedout. Tissues were dehydrated in graded ethanol, cleared for several hours inxylene-phenol and whole mounted with or without coverslips, using Permountresin, in such a manner that the inner surface of the germinal plate faced up.Whole mounts were examined under the phase-contrast microscope.

Preparation of sections for light microscopy

Embryos were fixed for 2 h in cold 50 % Karnovsky solution, as describedabove. After dehydration in graded ethanol, germinal plates were embedded inEpon 812 and then sectioned at 1 /*m in a Porter Blum ultramicrotome. Serialsections of the germinal plate were stained with 1 % toluidine blue in boratebuffer.

RESULTS

Structure of the germinal plate

In embryos of H. medicinalis formation of body segments is initiated on day5-6 of development at 23-25 °C, and completed on day 10-11. The structureof the germinal plate during that phase of development is described in thefollowing.

(1) Six-day-old embryos (Figs. 1-3). On day 6 the germinal plate is 0-9-1-5 mmlong and consists of two morphologically distinct sectors, one anterior and theother posterior, of roughly equal lengths. Of these, the posterior sector appearsas a ribbon of uniform width of about 60 /*m. This sector will be referred to asthe ribbon part. At the caudal tip of the ribbon part lie the teloblasts, visibleas five pairs of large cells (Figs. \c and 18-19). The anterior sector of thegerminal plate gradually widens towards the front, forming a fan-shaped struc-ture that reaches a width of about 150 [im. at the larval mouth (Fig. 1 a).

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 layerformed by the m bandlet pair. In the ribbon part, the ectodermal layer is onecell thick and eight cells wide. The n bandlet pair provides the most medialtwo and the q bandlet pair the most lateral two of these eight cells, with theo and p bandlet pairs providing the other four cells. The n bandlet cell pairstraddles the embryonic midline and lies more dorsally than the o, p and qbandlet cell pairs, which are in contact with the ventral region of the larvalenvelope. Hence, the eight cells form a bilateral pair of ectodermal archeswhose concavity is orientated toward the dorsal embryonic surface. The mbandlet lies in the concavity of the ectodermal arches, and most of its cells arelarger 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,lc, 11, 13).


le m n o p q

m—

n o p q

1

Fig. 1. Schematic representation of 6-day-old embryo (a) and of the structure of theanterior (b) and posterior (c) sectors of its germinal plate. The oval-shaped larvamay reach up to 1 cm in length and consists of a membranous envelope (le), fourpairs of protonephridia (pn), a larval mouth (mo) and the germinal plate. Themembranous envelope, 2-5 fim in thickness, includes an outer flat epithelium (seealso Fig. 12) and an inner discontinuous muscle layer that is associated with a nerveplexus. Muscle cells (lm) extend radially around the mouth and circumferentiallyaround the rest of the larva. The mouth, which is surrounded by a muscle sphincter,communicates with a short pharynx that opens in the cavity of the primitive gut.The gut contains the ingested albumen. The arrow indicates the anterior limit of theribbon part, i.e. the border between the anterior and posterior sectors of the germi-nal plate. The anterior sector contains ganglionic primordia (gp) and incipientsomites (s). The arrangement of teloblasts and of cells of the bandlets in the anterior(b) and posterior (c) sectors of the germinal plate is shown in dorsal view [upperparts of (b) and (c)] and in transverse section [lower parts of (b) and (c)].

Embryonic development of the leech

Tr^K3Fr • V

Figs. 2 and 3. Whole mounts of an early (2) and of a late (3) 6-day-old embryo thatshow the structure of the germinal plate. The arrows mark the border between theanterior and posterior sectors of the germinal plate. Six segments can be distinguishedin the anterior sector of the late 6-day-old germinal plate, gp, Ganglionic primor-dium; it, interprimordial tissue; lm, larval muscles; In, larval nerve cells; pn, proto-nephridia; s, somite; se, cluster of cells that give rise to the supraoesophagealganglion; T, teloblasts. Phase contrast. Scale bar represents 0-1 mm.


The ribbon part ends at that longitudinal position of the germinal plate tothe front of which the ectodermal arches become progressively wider andthicker (Figs. 2, 3). In that anterior sector the ectodermal arches of the ribbonpart become five, six, seven or more cells in width and several cells thick(Figs. \b, 12, 14). The widest and thickest ectoderm lies adjacent to the larvalmouth. This expansion of the outer ectodermal layer is accompanied by anexpansion of the inner mesodermal layer, which similarly thickens and widens.The anterior sector of the germinal plate in front of the ribbon part includesfive to eight early ganglionic primordia (or about one quarter of the final numberof ganglia). These primordia appear as a longitudinal series of transverse bandsof ectodermal tissue separated from one another by rounded or rectangularregions of mesoderm of low cell density called inter primordial tissue (Fig. 3).The set of ganglionic primordia gives the anterior sector of the germinal platea ladder-like appearance. More developed early ganglionic primordia lie in thefrontmost region of the germinal plate and appear as dumb-bell-shaped structuresthat consist of a median part and two lateral parts. Less developed early ganglionicprimordia lie to the rear, just forward of the ribbon part, and mostly consist ofa median part (Fig. 14). Concomitant with the formation and development ofganglionic primordia, proliferation of cells of the mesoderm leads to theformation of somites. In whole-mounted embryos stretched out prior to fixation,paired somites and ganglionic primordia lie in register (Fig. 3).

(2) Seven-day-old embryo (Figs. 4, 5). On day 7 of development the germinalplate is 2-2-5 mm in length, of which only the caudal tenth is made up by aribbon 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-oldembryos, reaching a width of 250-400 jam at the larval mouth. The germinalplate now contains 14-20 ganglionic primordia, or about half of the final num-ber of ganglia, of which the rostral half to two-thirds are late primordia. Suchlate primordia present three important characteristics: (a) their lateral partshave moved medially and thus they appear as figure-of-eight structures; (b) theyare linked via three short nerve tracts that correspond to developing connectives;and (c) they are separated by narrower interprimordial tissue due to medialwarddisplacement of the nerve bundles of the connectives (Fig. 8). The structure ofearly primordia is shown in Figs. 15 and 16. The germinal plate contains 12-18pairs of somites that are intercalated between ganglionic primordia. Thussomites and ganglionic primordia lie no longer in register (Fig. 8). Formationof nephridial primordia has begun in the somite pairs lying between the 4th and5th ganglionic primordia.

(3) Nine-day-old embryo (Fig. 6). On day 9 of development the germinalplate is 3-5-4 mm in length, of which only the caudal twentieth is made up ofa ribbon part. The cellular components of the residual ribbon part show signsof disorganization and disintegration. The shape of the germinal plate haschanged from fan to ellipse, in that its widest part now lies at some distance


v

JKV

•-. ?U.-A• ; • , •

% • * » " , - JIT • •

Figs. 4, 5. Whole mounts of an early (4) and of a late (5) 7-day-old embryo. Theanterior sector of the germinal plate of the latter includes 20 segments, of which 12have formed nephridial primordia (np). Late ganglionic primordia (lgp) lie to thefront of early ganglionic primordia (egp). The arrows mark the border betweenthe two sectors of the germinal plate, it, Interprimordial tissue; s, somites.Phase contrast. Scale bars represent 0-2 mm.


from the larval mouth. The mesodermal tissues have formed about 25 pairedsomites, 17 pairs of nephridial primordia and the primordia for the male andfemale genitalia. The frontmost 10-15 late ganglionic primordia have maturedinto morphologically intact ganglia and connectives, surrounded by a perineuralcoelom, thus forming an embryonic nerve cord. The frontmost four ganglia arefigure-of-eight-shaped structures linked via very short connectives; they form thesuboesophageal ganglion (Fig. 9). The next 6-10 ganglia are globular, linked vialonger connectives. Except for the first two or three ganglia, paired interseg-mental septa are seen to course circumferentially from the lateral surfaces ofeach ganglion to the lateral edges of the germinal plate. The rear part of theanterior sector of the germinal plate contains 10-15 late and early ganglionicprimordia (Fig. 17).

(4) Eleven-day-old embryo (Fig. 7). On day 11 of development the germinalplate is about 8 mm in length. It can be subdivided into cephalic, abdominaland 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, whichform a conical mass of nerve tissue corresponding to the suboesophagealganglion. A pair of perioesophageal commissures connect the suboesophagealto the supraoesophageal ganglion. The supraoesophageal ganglion has arisenfrom a mass of cells that lie in front of the larval mouth (Fig. 2), and thus thedevelopmental origin of that ganglion is not the germinal plate. The abdominalregion of the germinal plate includes the 21 abdominal ganglia of the embryonicnerve cord, separated by connectives of different lengths, 17 pairs of embryonicnephridia, and the primordia of the male and of the female genitalia that lierespectively in front of and behind the sixth abdominal ganglion. The paireddeferential ducts have begun to grow rearward from the primordium of themale genitalia. The caudal region of the germinal plate appears as a disk-likestructure from which the caudal sucker will arise. The caudal region includes

Fig. 6. Whole mount of a late 9-day-old embryo. The arrow points to the degenera-ting ribbon part of the germinal plate. The anterior sector of the germinal plateincludes 32 body segments (the last 7 are marked by arrow heads) that are atdifferent stages of development. Since development of the germinal plate occurs ina rostrocaudal sequence, the most advanced body segments lie at the front of thegerminal plate. The developing nerve cord consists of 12 ganglia (ga) and 10 late(lgp) and 10 early (egp) ganglionic primordia. The primordia of the female (pf)and of the male (pm) genitalia, as well as the 17 pairs of nephridial primordia (np)are seen. Phase contrast. Scale bar represents 0.4 mm.Fig. 7. Whole mount of an 11-day-old embryo. Intersegmental septa (is) extend fromthe middle of the ganglia to the lateral edge of the germinal plate. Thus, gangliapresent an intersegmental distribution, whereas developing nephridia (ne) andtestes (te) are distributed segmentally. The paired deferential ducts (dd) have begunto grow caudalward from the primordium of the male genitalia. The incipientcaudal sucker (cs) includes 7 ganglia. Scale bar represents 1 mm.


ne

CS


the last seven ganglia of the nerve cord, which form an elongated mass of nervetissue corresponding to the caudal ganglion. The cells of the residual ribbonpart 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 organizedin the manner characteristic of late embryos. Paired intersegmental septacourse at right angles to the longitudinal axis but are slanted with respect to thedorsoventral axis. The anterior (dorsal) edge of each septum lies in register withthe ganglion, and the septum extends posteriorly (and ventrally) to about theinterganglionic midpoint of the connective (Figs. 9, 10). Thus, this arrangementdf the slanted septa gives rise to a rostrocaudal succession of bilaterally

Fig. 8. Whole mount of a late 7-day-old embryo that shows the structure of thefrontmost six body segments. Ganglionic primordia consist of a median (mp) andtwo lateral (lp) parts and are linked to each other by short connectives. Somites(s) and ganglionic primordia are not in register. Darkly stained mesodermal cells(m), that migrated out of somites, are seen scattered, it, Interprimordial tissue.Phase contrast. Scale bar represents 100/*m.Fig. 9. Whole mount of a 9-day-old embryo that shows the structure of the front-most five body segments. Ganglia 1-4 have developed very short connectives (co)and constitute the suboesophageal ganglion. Arrows point to the developingperioesophageal commissure, is, Intersegmental septum; mo, larval mouth. Phasecontrast. Scale bar represents 100/*m.

Fig. 10. Whole mount of an 11-day-old embryo that shows the arrangement ofintersegmental septa. These extend from the emerging root of the posterior nerveto about the interganglionic midpoint of the connective (arrows), cl, Perineuralcoelom; ga, ganglion. Phase contrast. Scale bar represents 100 /*m.Fig. 11. Transverse section through the ribbon part of a 6-day-old embryo. Theectoderm consists of 4-cell-wide arches formed by the n, o, p and q bandlets. Cellsof the mesodermal bandlets (m) lie dorsally. Epon-embedded section stained withtoluidine blue. Scale bar represents 20 /*m.Fig. 12. Transverse section through the rear part of the anterior sector of the ger-minal plate of a 6-day-old embryo. Ectodermal arches have expanded laterally, to awidth of about seven cells. The inner sector of these arches (between arrows)constitute the median part of the ganglionic primordia. The lateral sector of thearches has already begun to separate into two layers and consists of a dorsal (d) anda ventral (v) cell cluster. Paired dorsal cell clusters constitute the lateral parts ofeach ganglionic primordium. The ventral cell clusters give rise to the epidermis,le, Larval envelope; m, migrating mesodermal cell; s, somite. Epon-embeddedsection stained with toluidine blue. Scale bar represents 30 /tm.

Fig. 13. Whole mount of a 6-day-old embryo that shows the structure of the ribbonpart, including the arrangement of the o, p and q bandlets. Cell nuclei include aprominent nucleolus (nu). lm, Larval muscle fibre. Phase contrast. Scale bar repre-sents 20 ftm.Fig. 14. Whole mount of a 6-day-old embryo that shows the structure of the rearregion of the anterior sector of the germinal plate. Ectodermal arches are 6 to 10cells wide and formation of gangl ionic primordia is under way in regions marked withasterisks, mi, Mitotic cell in the n bandlet. Phase contrast. Scale bar represents20 fim.


.mp

s'it

1 v v

.V. *«•%

V

_ri<f*

v\»5

• j

. iJ


symmetric, pocket-like compartments. This septal slanting has the following con-sequences : (a) the position of the ganglia becomes intersegmental because theirrostral and caudal halves come to lie on opposite sides of the septum; (6)successive body segments overlap for about half of their length; (c) most of thecomponents of nephridia lie in the septal tissue since they arise from the rostralmesoderm of each body segment. This arrangement of the septa may be ex-plained as the result of a rostrocaudal expansion of somite tissue due to differen-tial growth.

Structure, distribution and division of teloblasts

The teloblasts are globular or pear-shaped cells, 15-30/mi in diameter. Theyhave a globular nucleus, about 10 /im in diameter, which encloses a prominentnucleolus, about 4 /im 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 anearly bilaterally symmetrical disposition in the rearmost region of the germinalplate, usually overlying the O and P teloblasts. The M teloblast pair is not inmutual contact (Figs. 18, 19). The N teloblast pair is the most rostrally situatedpair. Usually it is asymmetrically disposed, with one N teloblast - either theright or the left - lying more caudally than its contralateral homolog. In mostembryos, the N teloblast pair is in mutual contact, and the more caudal mem-ber 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 Qpair having the most lateral and slightly dorsal position. As shown in Fig. 18,the paired teloblasts form an arch-like arrangement.

Fig. 15. Transverse section across the anterior sector of the germinal plate of a7-day-old embryo that shows the structure of an early ganglionic primordium. ep,Epidermis; cm, differentiating circular muscle cell; lp, lateral parts of the gang-lionic primordium; mp, median part of the ganglionic primordium; s, somites.Epon-embedded section stained with toluidine blue. Scale bar represents 30 /im.Fig. 16. Whole mount of a 7-day-old embryo that shows two nascent ganglionicprimordia (gp). A mitotic cell (mi) is seen in a region of the neuroectoderm thatderives from the n bandlet. Mesodermal cells (m) stain more darkly than ecto-dermal cells, it, Interprimordial tissue. Phase contrast. Scale bar represents 30 /*m.Fig. 17. Whole mount of a 9-day-old embryo that shows the structure of earlyganglionic primordia. it, Interprimordial tissue; lp, lateral parts of primordium;mp, median part of primordium. Scale bar represents 30 /*m.Figs. 18 and 19. Whole mounts of 6-day-old (Fig. 18) and of 7-day-old embryos(Fig. 19) that show the arrangement of teloblasts and of bandlets in the ribbon partof the germinal plate. The N teloblast pair is distributed asymmetrically (see alsoFigs. 21-23). The left N teloblast of Fig. 19 is dividing. Phase contrast. Scale barsrepresent 20 fim.Fig. 20. Whole mount of a 7-day-old embryo that shows mitotic cells (mi) in then and m bandlets of the ribbon part. Phase contrast. Scale bar represents 20/tm.


Although the pattern of stem-cell production by teloblast division has notyet been studied in great detail, in all embryos so far examined only one or theother of the teloblasts of a given pair was seen to be dividing. Furthermore,concurrently dividing members of different teloblast pairs were seen to lieeither on the same side or on opposite sides of the embryo. Therefore, it appearsthat teloblast division is asynchronous (Figs. 19, 21).

The manner in which teloblasts divide to form stem cells may be judged fromthe position of the last-formed stem cell of any given bandlet relative to therostrocaudal axis of its progenitor teloblast. In the case of the M teloblast, thelast-formed stem cell may lie either to the right or to the left of rostrocaudalteloblast axis, giving rise to a zig-zag cell pattern in the M bandlet proximal toits teloblast (Figs. 21-23). This finding suggests that the M teloblasts divideaccording to a spiral cleavage pattern. By contrast, there is little evidence for aspiral 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 onthe same side of the rostrocaudal teloblast axis. Therefore, the N, O, P and Qteloblasts probably divide unidirectionally.

Structure and growth of the bandlets

In the ribbon part of the germinal plate, the spatial arrangement of the band-lets corresponds to the relative disposition of their progenitor teloblasts. Cellsof the m bandlet are globular in shape, measure 8-20 ptm in diameter andusually 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 shapeand measure 5-12 /im in width (Figs. 13, 18, 19). The bandlet cell nuclei includeone nucleolus, and sometimes two nucleoli, whose size and shape varies con-siderably (Figs. 11-14, 18-26).

In the ribbon part of the germinal plate mitotic cells are found scatteredalong the bandlets. Mitotic bandlet cells lying close to their teloblasts of originprobably correspond to dividing stem cells, whereas those distant from theirteloblasts may be dividing blast cells. Since both the distribution and the

Figs. 21-25. Whole mounts of 7-day-old embryos that show the arrangement ofmesodermal cells at the ribbon part. Arrows indicate the direction into which Mteloblasts released the last-formed stem cell. In the proximal region of the m bandletsthe cells lie in a zig-zag arrangement and the metaphase plate of dividing cells(mi) has an oblique orientation. Figs. 24 and 25 show how successive spiral divisionsof m cells lead to the formation of larger and larger cell clusters (cm). These cellclusters eventually become somites. The left Q teloblast of Fig. 21 is dividing. Phasecontrast. Figs. 21, 22 and 24 - scale bars represent 30/*m. Figs. 23 and 25 - scalebars represent 20 fim.

Fig. 26. Whole mount of an 8-day-old embryo that shows part of the anteriorsector of the germinal plate. Mitotic cells (mi) in somites present obliquely orientedmetaphase plates. Phase contrast. Scale bar represents 20 fim.

J. FERNANDEZ AND G. S. STENT

bFig. 27. Schematic representation of the arrangement of cells in the mesodermalbandlets due to spiral divisions of M teloblasts (large white circles), m stem cells(small white circles) and m blast cells (small stippled circles), (a) Zig-zag arrange-ment of stem cells due to spiral division of the teloblast. This cell arrangement issometimes seen in the proximal segment of the m bandlets. (b) Zig-zag arrangementof cells generated after stem cells have undergone one spiral division. Thiscell arrangement is usually seen in the proximal segment of the m bandlets. (c) Cellarrangement generated by repeated spiral divisions of some blast cells. In thismanner, clusters of m cells are formed. Each cell cluster is probably constitutedof the progeny of one stem cell.

number of mitotic cells differ in a given bandlet pair, it would appear thatbilaterally 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 perpen-dicular to the longitudinal axis of the embryo. Hence, these bandlets grow inlength, without becoming either wider or thicker. This unidirectional, inter-stitial mode of cell division leads to an increase in the number of 4-cell-wide,

Fig. 28. Whole mount of an early 9-day-old embryo that shows a bilateral gapin the mesodermal bandlets of the ribbon part (arrows). Phase contrast. Scale barrepresents 100/wn.Fig. 29. Whole mount of a 9-day-old embryo in which the residual ribbon part(between arrows) has started to degenerate, pn, Protonephridia. Phase contrast.Scale bar represents 200 /tm.Fig. 30. Whole mount of a late 9-day-old embryo in which the residual ribbon part(between arrows) is in an advanced state of degeneration. The last seven bodysegments are marked with arrow heads. Phase contrast. Scale bar represents 100 /tm.Fig. 31. Whole mount of an 11-day-old embryo in which remnants of the ribbonpart (arrow) are seen posterior to the developing caudal sucker. The latter includesseven ganglionic masses (ga). Phase contrast. Scale bar represents 200 /*m.


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 followsa spiral division pattern, and this gradually obscures the initial zig-zag patternof arrangement of the stem cells (Figs. 21-23). In addition, spiral divisions inthe m bandlets lead to the formation of cell clusters (Figs. 24 and 25). Figure27 shows how the observed cell arrangement pattern'in the m bandlets andlengthening of the mesoderm in the ribbon part of the germinal plate can beaccounted for by successive spiral division of teloblasts and of stem and blastcells.

Forward of the ribbon part, changes in the mode of division of mesodermaland ectodermal bandlet cells result in an increase in width and the thickness ofthe germinal plate. The mesodermal bandlets become segregated into solid cellclusters that constitute primordial somites. There is evidence that cells of thesomites continue to divide in a spiral pattern (Fig. 26). Widening and thickeningof the ectoderm is the result of division of the n, o, p and q bandlet cells inplanes parallel (rather than perpendicular, as was the case in the ribbon part)to the longitudinal embryonic axis. Division in vertical planes leads to anincrease in the width of the ectodermal arches (Figs. \b, 12, 14). In regionswhere ganglionic primordia are about to be formed, mitotic cells are moreabundant in the medial than in the lateral portion of the ectodermal arches(Figs. 14, 16). These medial mitotic cells are probably blast cells derived fromthe 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 ofthe ectodermal arches of two layers of cell clusters, one dorsal and the otherventral. These clusters include cells of the o, p and q bandlets because thesebandlets constitute the lateral portion of the ectodermal arches. The dorsalcluster, which remains associated with the median part of the ganglionicprimordium, includes founder cells for the lateral parts of the ganglion. There-

Figs. 32-33. High-magnification micrographs of the ribbon part of the germinalplate in the embryo of Fig. 28. Fig. 32 shows that the bilateral gap in the meso-dermal bandlets (bg) is about the size of an adjacent paired cluster of mesodermalcells (cm). A mitotic cell (mi) is seen in the left n bandlet that crosses the gap region.Fig. 33 shows that the gap region (bg) includes normal ectodermal bandlets. The tipof the ribbon part is toward the bottom of the figure. Phase contrast. Scale barsrepresent 40 /*m.Fig. 34. High-magnification micrograph of the ribbon part of the germinal plate inan embryo similar to that of Fig. 29. The ectodermal bandlets (eb) are disorganizedand apparently normal teloblasts (T) are present. Mesodermal cells are seen to bedividing (mi), as well as migrating out the ribbon part (m). Phase contrast. Scalebar represents 40 /«n.Fig. 35. High-magnification micrograph of the ribbon part of the germinal plate ofFig. 30. Although portions of some bandlets remain apparently intact, extensivecell disintegration has occurred. Phase contrast. Scale bar represents 30 [in\.

92 j . F E R N A N D E Z A N D G. S. S T E N T

fore, ganglionic primordia consist of cells derived from the n, o, p and qbandlets. 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 theinner layer seems to develop into the circular muscles of the body wall (Fig. 15).Therefore, the epidermis and possibly the circular muscles are derived fromcells of the o, p and q bandlets.

Termination of body segment formation and degeneration of the residual ribbonpart

Formation of further body segments ceases in 9-day-old embryos, as manifestby an interruption in the m bandlets just to the fore of the residual ribbon partof the germinal plate. This interruption consists of a mesoderm-free gap about50 (im in length on either side of the germinal plate, within which the outerectoderm 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 mesodermalcells. The mesoderm-deprived ectoderm appears normal (Fig. 33), and mitoticcells can be seen in its bandlets (Fig. 32). Anterior to the ribbon part, thegerminal plate of 9-day-old embryos contains 25 discernible somites, and inaddition, 7 bilateral pairs of mesodermal cell clusters, precursors of somites26-32 (Figs. 6, 30). Therefore, the interruption of the m bandlets occurs just atthat position of the germinal plate, anterior to which there is just sufficientmesoderm to form 32 body segments. In the residual ribbon part, posterior tothe gap, several paired clusters of mesodermal cells are present that resemblesomite precursors (Fig. 32).

Interruption of the m bandlets is soon followed by degeneration of theresidual ribbon part of the germinal plate, as manifested by progressive disin-tegration of the bandlets and teloblasts, and the appearance of abundant celldebris (Figs. 29-31, 34-35). The first signs of degeneration are usually seen atthe front of the residual ribbon part, where ectodermal bandlets have beendeprived of contact with mesoderm. Cell degeneration then proceeds rearwarduntil the bandlets and, finally the teloblasts, have disintegrated. However, someof the mesodermal cells of the residual ribbon part do not degenerate; theydetach from the bandlets, enter mitosis, and migrate from the degeneratingribbon part into the larval membranes (Fig. 34).

DISCUSSION

Comparison of the disposition of the teloblasts of 6- to 9-day-old embryoswith the disposition of their precursor cells in earlier embryos (see Shumkina,1951 a) leads to the conclusion that the arch-like arrangement of the teloblastsis the consequence of an orderly procession of spiral cleavages. This is also thecase for the formation of teloblasts in the glossiphoniid Theromyzon rude(Fernandez, 1980). However, the relative position of the teloblasts in the


embryo of Hirudo is the inverse of that found in glossiphoniid embryos (Fer-nandez, 1980): in Theromyzon the lateromedial sequence of ectoteloblasts isN, O, P, Q, whereas in Hirudo it is Q, P, O, N. Accordingly, in Hirudo embryosit is the right and left members of the O and N ectoteloblast pairs that are incontact rather than the right and left members of the Q ectoteloblast pair, as isthe case in Theromyzon embryos. Furthermore, the M mesoteloblast pair is inmutual contact in Theromyzon embryos but not in Hirudo embryos. TheHirudo-typt of arrangement of the teloblasts is also seen in embryos of otherclitellate annelids, such as those of Erpobdelliid and Piscicoliid leeches and ofsome oligochaetes (see Schleip, 1936; Dawydoff, 1959, and Devries, 1973).

This difference in the relative position of teloblasts in the two types of leechesappears to reflect a difference in the early cleavage pattern leading up to teloblastformation. In Hirudo, the ectodermal proteloblast NOPQ cleaves to yield acaudomedial cell NO and a rostrolateral cell PQ (Shumkina, 1951a). Cleavageof cell NO thereupon yields the rostromedial teloblast N and the caudolateralteloblast O, whereas cleavage of PQ yields the caudomedial teloblast P and therostrolateral teloblast Q, resulting in the teloblast arch in mediolateral orderN, O, P, Q. In glossiphoniids by contrast, the ectodermal proteloblast NOPQcleaves to yield the caudomedial cell OPQ and the rostrolateral N teloblast.In two further cleavages, OPQ yields the rostromedial teloblast Q and thecaudolateral cell OP, and cell OP yields the caudomedial teloblast P and therostrolateral teloblast O, resulting in the teloblast arch in mediolateral orderQ, P, O, N (Fernandez, 1980). Despite this apparent difference in cell lineageof origin and relative position in the early embryo, the four ectoteloblast pairsof Hirudo and of glossiphoniid leeches have similar presumptive fates and hencecan be considered as homologous blastomeres.

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

Growth of the germinal plate proceeds concomitantly with the expansion ofthe embryo as it ingests albumen from the cocoon fluid. Ingestion of albumenceases at about the 9th day of development, after which time the growinggerminal plate gradually spreads over the larval surface. The available evidenceindicates that the larval surface membrane degenerates and does not formpart of the later embryo (Schleip, 1936; Shumkina, 19516, 1953).

Lengthening, widening and thickening of the germinal plate is clearly theresult of an orderly accumulation of ectodermal and mesodermal stem andblast cells. Stem cells are produced for about 6 days, their formation by telo-blasts being initiated on day 3 (Shumkina, \95\ b-d) and terminated on day 9.

4 EMB 72


If the rate at which stem cells are produced by teloblast cleavage is assumed tobe about the same as the rate of blastomere cleavage, namely about onceevery 2 h (Shumkina, 1951a), each teloblast would form a total of about 70stem cells. As shown here, the last of these 70 stem cells to be produced disin-tegrate upon the degeneration of the residual ribbon part of the germinalplate. Thus, in view of the total number of available stem cells, no more thantwo stem cells from each bandlet are likely to contribute to the foundation ofeach of the 32 segments. This estimate of the total number of stem cells producedis in good agreement with that previously reported for embryos of Theromyzon(Fernandez & Stent, 1980).

The division pattern of the mesoteloblasts differs from that of the ectotelo-blasts: the former divide spirally and the latter unidirectionally. Thus, the earlyspiral cleavage pattern of the blastomeres that gave rise to the five teloblastpairs is maintained in subsequent divisions of the mesoteloblast pair but notof the ectoteloblast pairs. It seems likely that the switch from spiral to uni-directional cleavage in the ectoteloblast cell line reflects a developmental changein a determinant of the orientation of the mitotic spindle in successive celldivisions. This change would originate in, and be passed on to the descendantsof, the ectodermal precursor blastomere arising from cleavage of the D blasto-mere. By contrast, the other daughter of that cleavage, the mesodermal pre-cursor blastomere, would preserve the determinant for the spiral cleavagepattern and pass it on to the M teloblasts and their daughter cells of the mbandlets. These differences in the division pattern of ecto- and mesoteloblasts,and of their descendant bandlet cells, are likely to play an important role in thedifferential morphogenesis of ectodermal and mesodermal components of bodysegments.

As a consequence of the degeneration of the residual ribbon part of the ger-minal plate, an as yet undetermined number of stem cells and their progenydisintegrate or fail to participate in further development of the germinal plate.It would follow, therefore, that the exact number 32 of segments formed is notdetermined by the total number of stem cells produced by any of the teloblasts.However, the fact that the 32nd segment arises from tissues lying immediatelyin front of the gap in the m bandlets suggests that the mesoderm limits thenumber of body segments produced. One possibility to account for the gap isthat after having produced the number of m stem cells needed to found 32somites, the M teloblasts temporarily cease dividing. Meanwhile, formation ofectodermal stem cells and their division into blast cells, and hence elongationof the ectodermal bandlets, would continue. Upon resumption of division ofthe M teloblasts, a bilateral, mesoderm-free gap would appear in the germinalplate. An alternative explanation for the appearance of such a gap would bethat the m stem cells produced in excess of the number needed to found 32somites receive some signal that causes them to degenerate in a rostrocaudalsequence, beginning with the frontmost of the surplus cells. It should be noted

Embryonic development of the leech 95that the dimensions of the gap are similar to the dimensions of adjacent bilateralclusters 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 onem stem cell (Devries, 1973; Fernandez & Stent, 1980). It would follow, therefore,that the gap arises as a consequence of the absence of the descendants of a rightand a left mesodermal stem cell. In any case, there are good reasons to thinkthat the gap in the m bandlets induces the degeneration of the residual ribbonpart of the germinal plate. First, in all embryos so far examined degenerationof 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 degenerationare those of the ectodermal bandlets not in contact with mesoderm. Since inTheromyzon embryos many of the m stem cells produced last fail to be incor-porated into the germinal plate, the mechanism of limiting segment formationis probably similar in glossiphoniids to that obtaining in Hirudo.

The present results confirm previous findings concerning the dual develop-mental origin of the central nervous system of Hirudinea. The supraoesopha-geal 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 remaining32 ganglia) and corresponds to the cephalic plate of hirudinid leeches (Bergh,1885; Shumkina, 1951c; Dawydoff, 1959). The origin of the cephalic plate hasnot yet been clearly established. Shumkina (1951c) reported that the cephalicplate of Hirudo originates from cells migrating out of the germinal bands of3-day-old embryos, whereas Weisblat et al. (1980), using horseradish peroxidaseas a cell lineage marker, have demonstrated that the supraoesophageal ganglionof the glossiphoniid Helobdella triserialis is founded by cells derived from themicromere cap.

We thank Ildina Cerda, Georgia Harper, Margery Hoogs, Nora Loyarte, Victor Monas-terio, Nancy Olea and Lilio Yanez for technical assistance and Serena Mann for preparingthe illustrations. We also thank Dr. R. T. Sawyer for many valuable suggestions on how tobreed H. medicinalis. This investigation was supported by grant B 257-803, from Servicio deDesarrollo Cientifico, Artistico y de Cooperation Internacional, Universidad de Chile,NSF grant BMS 74-24637, from the National Foundation - March of Dimes, and grantRLA 78-024-11, from PNUD/UNESCO.

REFERENCES

BERGH, R. S. (1885). Die Metamorphose von Aulastoma gulo. Arb. zool. Inst. Wiirzburg 7,231-291.

BURGER, O. (1894). Neue Beitrage zur Entwicklungsgeschichte der Hirudineen. Zur Embryo-logie von Hirudo medicinalis und Aulostomum gulo. Z. wiss. Zool. 58, 440-459.

DAWYDOFF, C. (1959). Ontogenese des Annelides. In Traite de Zoologie vol. 5 (ed. P. P.Grasse), pp. 594-686. Paris: Masson.

DEVRIES, J. (1973). La formation et la destinee des feuillets embryonnaires chez le lombricienEisenia foetida (Annelide Oligochete). Archs Anat. Microsc. Morph. exp. 62, 15-38.

FERNANDEZ, J. (1980). Embryonic development of the Glossiphoniid leech Theromyzon rude:characterization of developmental stages. Devi Biol. 76, 245-262.

4-2


FERNANDEZ, J. & STENT, G. S. (1980). Embryonic development of the glossiphoniid leechTheromyzon rude: structure and development of the germinal bands. Devi Biol. 78, 407-434.

GREEN, J. (1971). Crustaceans. In Experimental Embryology of Marine and FreshwaterInvertebrates (ed. G. Reverberi), pp. 313-362. North-Holland.

LEUCKART, R. (1863). Die menschlichen Parasiten und die von ihnen herriihrenden Krank-heiten, vol. 1. Leipzig und Heidelberg.

SCHLEIP, W. (1936). Ontogenie der Hirudineen. In Klassen und Ordnungen des Tierreichs,vol. 4 (ed. H. G. Bronn), div. 3, book 4, part 2, pp. 2-117. Leipzig: Akad. Verlagsgesell-schaft.

SHUMKINA, O. B. (1951a). Cleavage of the egg in the medicinal leech. (In Russian.) Dokl.Akad. Nauk SSSR11, 353-356.

SHUMKINA, O. B. (19516). Development and metamorphosis of Hirudo medicinalis. (InRussian.) Dokl. Akad. Nauk SSSR 11, 161-164.

SHUMKINA, O. B. (1951C). Germ band and the head primordium of the medicinal leech.(In Russian.) Dokl. Akad. Nauk SSSR 11, 821-824.

SHUMKINA, O. B. (1951 cf). Periods of intracocoon development of the medicinal leech. (InRussian.) Dokl. Akad. Nauk SSSR 11, 1259-1262.

SHUMKINA, O. B. (1953). Embryonic development of Hirudo medicinalis. (In Russian.) Akad.Nauk SSSR 8,216-279.

SINEVA, M. B. (1944). Observations on breeding the medicinal leech. (In Russian.) Zool. Zh.23, 293-303.

SINEVA, M. B. (1949). Biological observations on propagation of Hirudo medicinalis. (InRussian.) Zool. Zh. 28, 213-224.

TURNER, R. F. & MAHOWALD, A. P. (1977). Scanning electron microscopy of Drosophilamelanogaster embryogenesis. II. Gastrulation and segmentation. Devi Biol. 57, 403-416.

TURNER, R. F. & MAHOWALD, A. P. (1979). Scanning electron microscopy of Drosophilamelanogaster embryogenesis. III. Formation of the head and caudal segments. Devi Biol.68, 96-109.

WEISBLAT, D. A., HARPER, G., STENT, G. S. & SAWYER, R. T. (1980). Embryonic celllineages in the nervous system of the glossiphoniid leech Helobdella triserialis. Devi Biol.76, 58-78.

{Received 20 January 1982, revised 15 June 1982)

embryonic developmen oft the hirudinid leech hirudo medicinalis: … · raising of hirudo can be...

Documents