Development 104 Supplement, 161-168 (1988)Printed in Great Britain @ The Company of Biologists Limited 1988

Segmentation in leech development

DAVID A. WEISBLAT, DAVID J. PRICE and CATHY J. WEDEEN

Department of Zoology, (Jniversity of California, Berkeley, California 94720, USA

Summary

Segments in glossiphoniid leeches, such as Helobdellatriserialis, are the products of stereotyped cell lineages

that yield identifiable cells from first cleavage. Celllines generating segmental tissues are separated fromthose generating prostomial tissues early in develop-ment. Segments arise from five bilateral pairs oflongitudinal columns of primary blast cells that aregenerated by five bilateral pairs of embryonic stemcells called teloblasfs. There are four ectodermal celllines (N, O, P and Q) and one mesodermal cell line (M)on each side of the embryo. In normal development,each cell line generates a segmentally iterated set ofidentified definitive progeny comprising a mixture ofcell types. In the M, O and P cell lines, each blast cellgenerates one segment's worth of definitive progeny(segmental complement). But the clones of blast cells

in each of these three cell lines interdigitate longitudi-nally with cells of the adjacent clones from the sameline, so that the clone of an individual hr o and p blastcell is distributed across more than one segment.Thus, there is no simple clonal basis for morphologi-cally defined segments. In the N and Q cell lines, twoblast cells are required to produce one segmentalcomplement of definitive progeny; in each of these twocell lines, two classes of blast cells (nf and ns, qf andqs) are produced in exact alternation. Primary n and qblast cells are about the same size and are produced at

L6T

the same rate as blast cells for the o and p bandlets,but the longitudinal extent of their clones is roughlyhalf that of the o and p blast cells' clones. Duringdivision of the blast cells, the n and q bandlets becomecompressed relative to the o and p bandlets, so that thesegmental complements of the different cell lines cancome into register. This compression movement ismanifest as a movement of n and q bandlets relative too and p bandlets in the posterior portion of thegerminal band. The number of true segments in leechis fixed at 321' the counting mechanism is not known,but several hypotheses have been disproved. Segmen-tation in annelids and arthropods differs extensively atthe cellular level, yet these phyla are presumed toshare a common segmented ancestor. One strategy toidentify homologous processes in annelid and arthro-pod segmentation is to compare the patterns of ex-pression of evolutionarily conserved, developmentallyimportant genes. Preliminary observations using a

cross-reacting antibody that is thought to recognize ahighly conserved region of a Drosophilq segmentationgene, engrailed, labels nuclei of some blast cells earlyin development and, later, some neurones in thedifferentiating suboesophageal ganglion.

Key words: leech, segmentation, cell lineage, primaryblast cell, teloblast.

Introduction

Segmentation in annelid development is of interestbecause the segmented body plan is such a prominentfeature of this phylum. In addition, the fact thatsegmentation occurs in several phyla raises questionsabout the phylogenetic origins of this developmentalprocess and how it has changed during evolution. It isgenerally held that the most recent common ancestorof chordates and arthropods was not segmented and

that segmentation has therefore arisen separatelyduring the evolution of these two groups (Dobson &Dobson, 1985). On the other hand, annelids andarthropods, along with Onychophora and relatedphyla ) are thought to share a common segmentedancestor, so segmentation in these phyla should behomologous. If true, these assumptions suggest thatwe are more likely to find common elements whencomparing the segmentation processes of annelidsand arthropods than when comparing the segmen-

162 D. A. Weisblat, D. f . Price and C. J. Wedeen

tation processes in either of those groups with chor-dates. But first, it is essential to describe segmen-tation in the organisms of interest in sufficient detailso that meaningful comparisons can be made. Here,we describe segmentation in leech development.Most of the observations are from Helobdella triseria-lis, a glossiphoniid species.

Segmental tissues arise by stereotypedlineages from five bilaterally paired cell lines

Development of glossiphoniid leeches, first describedover 100 years ago by C. O. Whitman (1878), pro-ceeds through a series of cell divisions that are largelyinvariant from embryo to embryo. A series ofunequal cleavages (stages 0-6) generates flve bilat-eral pairs of embryonic stem cells called M, N , O lP,O lP and O telobla^r/,s. Throughout these stages ofdevelopment, there is no evidence of segmentation.The first manifestations of segmentation come about

Ventralmidline

Rostral

Caudal

in stag a 7 , as each teloblast makes a series of severaldozen highly unequal divisions, producing a coherentcolumn of segmental founder cells called primaryblast cells (Fig. 1). Within each column, or bandlet,the birthranking of the primary blast cells is main-tained; older cells lie more distal (future anterior) tothe teloblast (future posterior). The five bandlets oneach side, designated ffi, tr, o, p and e, are arranged instereotyped order into a curving ridge of cells knownas the germinal band. The left and right germinalbands conne ct via their distal ends just ventral to theanimal pole of the embryo. As more primary blastcells are produced, the germinal bands lengthen andmove across the surface of the embryo, eventuallycoalescing rostrocaudally along the ventral midlineduring stage 8, like the two halves of a zipper,forming the germinal plate.

The proliferation of blast cells within the germinalplate (stages 9-10) gives rise to the definitive segmen-tal tissues of the leech. During this process, thegerminal plate lengthens and expands laterally.

Germinal plate

Germinal band

Bandlet

Blast cell

Teloblast

o/P

Stage 8 (early)

Fig. L. Schematic representation of late stages of development in Helobdella triserialis.Left: Hemilateral arrangementof teloblasts and their primary blast cell bandlets within the germinal band and germinal plate. Near right: Dorsal viewsof three embryos: early stage-7 embryo in which the teloblasts have begun to produce blast cell bandlets; mid stage-7embryo in which the bandlets have merged to form germinal bands; and early stage-8 embryo showing the heart-shapedgerminal bands that have begun to coalesce into the germinal plate. Far right: Ventral views of two embryos: latestage-8 embryo showing the germinal plate on the ventral midline, with the nascent ventral nerve cord and its gangliaand ganglionic primordia indicated by filling; stage-L0 embryo in which the chain of ganglia, shown by filling, alreadyclosely resembles the adult nerve cord.

Stage 7 (early)Stage 8 (late)

\\.):'. l'. -.-- \tr.- --^ \. \

Stage 7 (middle)

Stage L0

Eventually, its outer edges zipper together along thedorsal midline, which closes the body tube of theleech.

Additional details of this developmental processhave been obtained in the last decade, using micro-injected cell lineage tracers in conjunction with a

variety of other techniques (e.g. see Weisblat et al.1984). This work builds on anatomical and neurobio-logical studies of leeches begun by Retzius (1891) andcontinued by Nicholls and others since the 1960s

(reviewed in Nicholls , 1987 and Muller et al. 1981).

One result of this large body of work, which has

concentrated primarily on the hirudinid species Hir-udo medicinalis, is that many cells, especiallyneurones and glia ) are known to be individuallyidentifiable from segment to segment and animal toanimal in various leech species.

Lineage-tracing techniques have shown that innormal development, although cell lines are notspecialized for the exclusive production of a particu-Iar cell type, individual, identified cells arise invari-antly from a particular cell line (M, N, O, P or Q).Moreover, although the complete lineages for defini-

Segmentation in leech development 163

tive progeny are not yet known, it seems likely thatthey arise from stereotyped cell lineages, as in thenematode Caenorhabditis elegans (Zacksor, 1984;Shankland , 1987).

When a teloblast is injected with lineage tracerafter it has already begun producing primary blastcells (stage 7) and the embryo is examined at stage L0,

a boundary is seen between anterior, unstained de-finitive progeny derived from blast cells producedprior to injection and posterior, stained cells derivedfrom blast cells produced after the injection. Byexamining the position of the boundaries in suchembryos, it is possible to infer the number of blastcells in each bandlet needed to generate one hemiseg-mental complement of progeny and also the spatialdistribution of the clone descended from an indi-vidual blast cell (Weisblat et al. 1984; Weisblat &Shankland, 1985). These inferences, summartzed inFig. 2, have been confirmed by other procedures(Zacksotr, 1984; Shankland , 1987). Several featuresof the segmentation process in Helobdella can bediscerned from this lineage analysis and associatedexperiments.

nf

qs

C o.,iii,js,

I

IItlIIf '::.:t',t'.'.i.9aI

Fig. 2. Schematic representation of the seven primary blast cell clones in the stage-L0 Helobdella embryo. Not all cellsare shown for the m clone; cross-hatching indicates the extent but not the disposition of muscle cells; the diagonal linerepresents the dorsoventral muscles; spindle-shapes between the first and second ganglia represent muscles associatedwith the interganglionic nerve; in the third ganglion, the filled contour represents M-derived neurones; the small filledcontour outside the ganglion represents presumptive gonoblasts; the large filled contour represents the nephridium. Foreach ectodermal clone (o, p, trs, flf, qs,qf) central and,peripheral neurones are represented as unlabelled filled circles(individual neurones) or multilobed contours (clusters of neurones), glia are represented as filled stars, and epidermalcells by stippling and by unfilled contours. In the o clone , &n O-derived cell at the distal tip of the nephridial tubule isshown just anterior to the medial patch of epidermis. For each clone, anterior is up; ganglia are shown in outline;dashed lines through the centres of ganglia and to their right indicate ventral and dorsal midlines, respectively.(Adapted from Weisblat & Shankland, 1985.)

: f l

II

164 D. A. Weisblat, D. J. Price and C. J. Wedeen

(A) There are seven basic classes of primary blastcells

The boundary analyses show that, in the ffi, o and pbandlets, each primary blast cell generates a completesegmental complement, i.e. one set of the definitiveprogeny from the cell line in question is found in a leftor right half segment. And accordingly, each m, o orp blast cell normally undergoes a stereotyped set offurther mitoses that is characteristic of its bandlet(Zackson , 1984).

For the N and O cell lines, by contrast, two blastcells are needed to generate one segmental comp-lement of progeny. In the N cell line, for example,one primary blast cell generates ganglionic neuronesand epidermal cells primarily in the anterior portionof the segment, while the primary blast cell justbehind it in the bandlet generates a small set ofperipheral neurones, a glial cell and posterior gangli-onic neurones (Weisblat 8. Shankland, L985; Bissen& Weisblat, I9S7). The distinction between theprogeny generated by the two primary q blast cellsneeded to make one segmental complement of defini-tive progeny is similarly clear. In addition to thesedifferences in definitive fates of the two n and two qblast cells, alternate blast cells in the n and q bandletsexhibit differences in the timing and symmetry oftheir first mitoses (Zackson , 1984); and these differ-ences serve to predict the two different fates (Bissen& Weisblat, 1987). Ablation experiments have so farfailed to reveal any plasticity in the fates of cells in then and q bandlets. So, in contrast to the m, o, and pbandlets, each of which comprises a single class ofblast cell, the n and q bandlets each comprise twodistinct classes of blast cells (called nf and ns, qf andqs) that arise in exact alternation. Thus, a total ofseven classes of prim ary blast cells can be defined bytheir intermediate lineages (e.g. the timing and sym-metry of their first mitosis) and by their definitivefates (the phenotype and spatial distribution of theirdefinitive progeny). This analysis ignores the import-ant issue of segment-specific differences in the pheno-type and occurrence of definitive progeny (Macagno,1980; Glover & Mason , 1986; Jellies et al. 1987).

(B) Segmental boundaries are not coincident withclonal boundaries

In the simplest case, it could be imagined, from theresults outlined above, that each left or right halfsegment of the leech would consist of no more and noless than all the progeny of seven primary blast cells,one of each class. This would be consonant with thetheory that the segments or parts of segments arepolyclones comprising all the surviving progeny of asmall set of founder cells (Garcia-Bellido et al. L973;

Crick & Lawrence , 1975). But the situation is morecomplex, because there is no simple relationship

between morphologically defined segments and pri-mary blast cell clones. In particular , each individualclone derived from an o, p and m prim ary blast cell inthe midbody of the leech extends longitudinallyacross parts of two or more segments; thus, longitudi-nally adjacent clones interdigitate so that it is imposs-ible to draw segment boundaries that include all theprogeny of just one set of primary blast cells, even ifwe restrict our analysis to the ectodermal cell lines. Ingeneral, any given midbody segment will containsome of the progeny of two o, two p and three m blastcells and all of the progeny of two n and two q blastcells. From the foregoing, and since leeches are nottoroidal, the details of the processes by which seg-

ments form at the ends of the animal must differ fromthose by which midbody segments form. Forexample, there may be cell deaths or missingbranches in blast cell sublineages otherwise destinedfor non-existent segments, andf or there may bemodifications of the differentiation or migration pat-terns of cells so that they remain in the terminalsegments.

(C) Segment founder cells do not come into properregister until after they have already begun dividingFrom various lines of evidence it is known thatprimary ectodermal blast cells are all about the samesize and are all produced at the same rate (Worde-man, 1982). Yet two blast cells from each N and Oteloblast are required to generate one hemisegmentalcomplement, whereas only one ffi, o or p blast cell isneeded per hemisegmental complement. Temporally,this discrepancy is resolved by the simple fact that theN and Q teloblasts continue making blast cells afterthe other teloblasts have stopped. The spatial aspectof this discrepancy is resolved by compression of theprogeny of the n and q bandlets into half of theiroriginal longitudinal extent, relative to those of the oand p bandlets. Since the distal (anterior) ends of thebandlets are all fixed, this requires that blast cells inthe proximal segments of the n and q bandlets moveforwards relative to their neighbours in the adjacent oand p bandlets (Weisblat & Shankland, 1985). In fact,these movements are going on well after the primaryblast cells have begun the divisions leading to theirdefinitive progeny. An apparent corollary of thesenormal movements of ectodermal bandlets relative toone another is the discovery by Shankland (1984)

that, if any of the ectodermal bandlets is lesionedwithin the posterior portion of the germinal band, theblast cells posterior to the break slip backwardsrelative to their neighbours in adjacent bandlets andassume ectopic positions in more posterior segments.These morphogenetic movements constitute a fasci-nating, and as yet unstudied, aspect of leech segmen-tation.

Little is known about how segments arecounted out and limited to a fixed number

One difference between segmentation in leeches and

in other annelids is that the number of segments inleeches is fixed and constant throughout life.

[Another difference is that leeches are apparentlyunable to regenerate segments (see Sawyer, 1986).]

Although the constancy of segment number has been

accepted for a long time, there has been dispute over

exactly what the number is, because of uncertaintyover just what constitutes a segment. Taking the

nervous system aS a convenient marker for Segmen-

tation in leech, there are 2l obvious segments in themidbody, each innervated by one ganglion of theventral nerve cord. At the ends of the animal'specializations associated with the two suckers com-

plicate matters, but embryological evidence and the

occurrence of identifiable neurones homologous tothose of the midbody ganglia indicate that the tailbrain represents the fusion (more properly, the fail-ure to separate during development) of seven ganglia.

From similar evidence, the suboesophageal ganglion

of the head brain comprises four segments (Fernan-

dez & Olea, 1982; Yau , 1976).

Analysis of the supraoesophageal ganglion was

more difficult because it resembles the typical mid-

body ganglia neither in gross morphology nor in the

phenotype of individual neurones, and because its

embryological origins were also obscure. Then, fromcell lineage studies it became apparent that the

supraoesophageal ganglion and associated epidermisarises not from the five teloblast-derived cells lines at

all, but rather from micromere-derived cell lines thatare separated from the teloblast lineages as early as

the third cleavage (Weisblat et al.1984). Thus, we can

finesse the question of how many segments are in the

most anterior part of the leech by excluding thisregion from the discussion of segmentation altogetherand restricting our analysis to the 7 +2I*4:32 true

segments derived from primary blast cells.

These results should be useful in refining the

question of how segments afe counted during leech

development, but little progress has been made inunderstanding the counting mechanism. However'several key observations have served to eliminateotherwise attractive hypotheses.

(1) Blast cell nuclei are made one at a time, &s blast

cells are produced (Zackson , 1982). This eliminatesthe notion that the teloblasts undergo five (M and

O lP teloblasts) or six (N and Q teloblasts) rounds ofkaryokinesis without cytokinesis to generate the pre-

cise number of nuclei needed for the subsequent

production of the segmental precursor cells.(2) Ablation of various teloblasts eliminates

progeny from segments, but does not affect the

Segmentation in leech development 165

number of segments produced (Blair, 1982; Blair &Weisblat, 1982). Such findings eliminate the extreme

holistic hypothesis that some interaction between allthe teloblasts or all the bandlets is necess ary toterminate segmentation normally. But not all possible

experiments of this sort have been carried out and

there is evidence that interactions between mesoderm

and ectoderm are necess ary for normal pattern for-mation (Blair, 1982). The effects of bilateral ablationof mesodermal cell lines should be carefully exam-

ined. For example, if the posterior mesodermalprecursors are missing, will a normal caudal sucker

and tail brain form, truncating the midbody of the

leech?(3) Every cell line, both ectodermal and mesoder-

mal, produces supernumerary blast cells that die

without contributing to definitive segments (Zackson,1982). This result eliminates the possibility that the

number of segments is limited simply by the numberof blast cells produced, although it remains to be

proven that the supernumerary blast cells are ofnormal viability.

(4) Duplicated ectodermal cell lines make twicethe normal number of viable blast cells. It was

discovered that microinjecting polyadenylic acid(polyA) into newly formed teloblasts or their precur-sor blastomeres alters the normal cleavage pattern inHelobdella so that supernumerary teloblasts, blast

cell bandlets and definitive progeny are producedthroughout the length of the leech (Ho & Weisblat,I9S7). This result runs counter to the hypothesis thatsome factor that is passively distributed among the

teloblasts serves to limit the number of viableprogeny and hence the number of segments; if thiswere the case, then dividing the limiting factor among

supernumerary teloblasts should result in its exhaus-

tion after each has made only its anterior complementof prim ary blast cells. Of course, &s with the ablationexperiments, it will be important to confirm this resultwith all cell lines, especially the mesodermal line.

Fernan dez & Stent (1982) reported an importantobservation relevant to the termination of the seg-

mentation process in the hirudinid leech Hirudomedicinalis. According to these authors, the develop-

ing germinal plate in Hirudo can be divided into an

anterior part, within which segmentation is evidentfrom the massing of cells into somites and ganglionicprimordia, and a posterior 'ribbon' pafi, within whichthe columns of blast cells are still largely in parallelarrays. As development proceeds, the anterior partincreases in length and the bound ary between it and

the ribbon part moves posteriorly. Termination ofsegmentation is evident as a gap in the mesodermalbandlet within the ribbon part, just behind the cells

that will form the last definitive segment. Ectodermalcells deprived of mesodermal contact and cells pos-

166 D. A. Weisblat, D. I. Price and C. J. Wedeen

terior to the gap soon degenerate, while cells anteriorto the gap continue dividing to generate the posteriorsegments.

This observation suggests that the initial signal forthe termination of segmentation arises from themesodermal cell line or that the mesodermal cells areresponding to a signal received from the ectodermallineages or from some other source in the embryo.Support for the notion that the termination signal isunlikely to arise in the ectodermal bandlets comesfrom similar observations reported for Helobdella. fnHelobdella, the gap in the mesodermal bandlet isseen before it merges with the ectodermal bandletsinto the germinal ban d (Zackson , 1982). The resultsfrom Hirudo and Helobdella are not strictly compar-able, of course. Moreover, the possibility remainsthat the ectodermal bandlets in Helobdella mayfragment independently as well (Marty Shankland,personal communication).

lnterphyletic comparisons of segmentationmust distinguish between homologous andoperationally equivalent processes

Segmentation is unarguably an important aspect ofthe development of many higher eukaryotic phyla.But in analysing segmentation processes, it is import-ant to remember that simply using the same word,'segmefrt' , to describe the subunits of the body plan intwo groups does not guarantee that these structuresarise by homologous processes. If segmentation aroseindependently in different groups, then the observedsegmentation in, say, frogs and worms may be merelyoperationally equivalent processes, with no underly-ing mechanistic similarity.

With this caveat in mind, and also the views ofphylogeny stated in the introduction, a search forinterphyletic common ground might entail compari-sons of segmentation in annelids and arthropods. Buteven here the search for homology is far from trivial,especially since, for historical and technical reasons,we are driven to compare highly derived representa-tives of the two phyla, such as insects and leeches,rather than more primitive species or apparentlyintermediate phyla, such as Onychophora. Evenignoring this problem, yet another is that the view ofphylogeny stated in the introduction is not universallyshared and may not be true. Field et al. (1988), forexample, conclude on the basis of a molecular phylo-genetic analysis that arthropods, chordates and anne-lids may in fact all have arisen from a commonsegmented ancestor, and that the arthropods andannelids are not closely related at all. Given thisuncertainty, the introduction of molecular phylogen-etics and the analysis of conserved gene families like

the homeobox-containing genes is a welcome comp-lement to more classical cellular and morphologicalapproaches to analysing the processes we lumptogether under the rubric of segmentation.

ln Helobdella, a putative homologue to theDrosophila gene engrailed appears to beexpressed early in development and indifferentiating neurones

Unlike early development in leech, which is charac-terized by holoblastic cleavages, early developmentin the insect Drosophila melanogaster proceeds via asyncytial blastoderm, in which the zygote nucleusundergoes 13 rounds of karyokinesis without cytokin-esis (Foe & Alberts, 1983). By the last rounds, mostof the nuclei migrate to the periphety, where cellular-izatton and then gastrulation occur. Much work hasshown that in the final syncytial stages and aftercellularization, nuclei differentially express certainmembers of a class of at least 35 genes that regulatethe number, polarity and identity of segments inDrosophila (reviewed by Akam , 1987). A number ofthese genes have been shown to contain similarsequences of about 180 base pairs, called homeo-boxes, which encode protein domains of about 60amino acids thought to mediate DNA binding. Workin Drosophila has demonstrated complex regulatoryrelationships among the various members of this genefamily.

It appears that the homeobox sequence has beenhighly conserved throughout evolution, becausegenes containing homeoboxes have been identified inmost higher eukaryotic phyla, including echino-derms, chordates and annelids (e.g. Carrasco et Al.1984; Levine et al.1984; McGinnis et Al.1984; Doleckiet al. 1986), oS well as in arthropods. In Helobdella,we estimate that there are at least 18 putative genesthat cross-hybridize with various homeobox probes(unpublished results). Given the likelihood thatannelids and arthropods evolved from a segmentedcommon ancestor, an analysis of homeobox geneexpression in Helobdella may bring us closer tounderstanding evolutionary homologies underlyingthe radically different cellular processes of segmen-tation in insects and annelids.

One gene that has been especially highly conservedin evolution is engrailed (tr). In Drosophila, €fr isexpressed in the posterior portion (compartment) ofevery segment and is required for cells in the pos-terior compartment to assume their normal identities(Kornberg, 1981; Kornberg et a|.1985; DiNardo et al.1985). We have identified an en homologue in Helob-della (unpublished data). In addition, using a broadlycross-reactive mouse monoclonal antibody (N. Patel,

K. Colemdn, T. Kornberg &" C. Goodmln, unpub-lished data) that appears to recognize a highly con-

served portion of the enpfotein (S. Toole, C. Lehner,personal communications), we have carried out pre-liminary studies on the expression in Helobdella ofthe epitope reco gntzed by the antibody (in collabor-ation with Patel and Goodman). Using horseradishperoxidase staining, we found immunoreactivity at

three different times of development. In the stage-7

embryo, a small number of nuclei stain, all of whichare apparently within a single bandlet (Fig. 3A). Inthe late stage-B embryo, a segmentally repeatingpattern, apparently a single large cell in the middle ofthe segment, is observed in the central midbody

Fig. 3. Photomicrographs of Helobdella embryos stained

with a monoclonal antibody that was raised to part of the

Drosophila invected gene product and that recognizes a

highly conserved epitope of the engrailed gene (N. Patel,

K. Coleman, T. Kornberg & C. Goodman, unpublisheddata). (A) Dorsal view of a late stage-7 embryo. The leftgerminal band makes a c-shaped curve in this panel, withthe proximal portions of the bandlets at the bottomcentre and distal portions at upper left. Three blast cell

nuclei are labelled in a single bandlet. (B) Lateral view ofa stage-9 to -10 embryo; ventral is down, anterior to the

left. Nuclear staining is evident in neurones of the

suboesophageal ganglion (left arrow) but not in midbodyganglia (right arrows). Scale bar, ca. 50prm in A; ca.

L50 prm in B.

Segmentation in leech development 167

segments (not shown). And in the stage-9 to -10

embryo, the antibody stains nuclei of many neurones,primarily in the second of the four fused ganglia in thesuboesophageal ganglion (Fig. 38).

Given the preliminary nature of these results, it isinappropriate to try to make much of a comparisonbetwe en Drosophila and Helobdella. But it seems fairto say that the staining patterns we observe are atleast vaguely reminiscent of those rn Drosophila, inthat expression is observed early in development in a

subset of segmental founder cells, and later in devel-opment in a subset of differentiating neurones. Moreimportantly, these results indicate that analysingexpression patterns of evolutionarily conserved genes

that are putative regulators of development will beuseful in studying leech development. By combining a

variety of experimen(al approaches to the analysis ofthe segmentation problem, it is possible that answers

to questions about how segmentation works, how andhow often it arose, and how it has been modifiedduring evolution may soon be forthcoming.

We thank Nipam Patel and Corey Goodman for anti-invected antibody and for their generous collaboration on

the preliminary staining experiments, Shirley Bissen,

Robert Ho and Stephanie Astrow for helpful discussions.

This work was supported by NIH First Grant8R9HD23328A-01, to D.A.W. C.J.W. is supported byDamon Runyon-Walter Winchell Fellowship DRG 925;

D.J.P. was supported in part by an MRC (UK) TravellingFellowship.

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