Stages of Larval Development and Stem Cell Population Changes During Metamorphosis of a Hydrozoan Planula

12 Pages·1997·4.8 MB·English

by  V J Martin

Reference: liiol Hull 192:41-52.(February, 1447)
Stages of Larval Development and Stem Cell
Population Changes During Metamorphosis
of a Hydrozoan Planula
VICKI J. MARTIN AND WILLIAM E. ARCHER
Department ofBiologicalSciences, UniversityofNotreDame, NotreDame, Indiana 46556
Abstract. Scanning electron microscopy and light his- Archer, 1986; Lumsden, 1988; Bronner-Eraserand Era-
tology were used to reveal the changes in overall mor- ser. 1988;PottenandLoeffler, 1990;Weston, 1991: Mar-
phology and in stem cell differentiation and distribution tin, 1991;Medvinskyf/rt/.. 1993).
that occur as a free-swimming, solid hydrozoan planula The study of stem cells in evolutionarily primitive
larva is transformed into a sessile, hollow adult polyp. metazoans may reveal some ofthe earliest mechanisms
Eight stages of development are described: young 10- used forsetting uppatternsofcell distribution and over-
hour planula, mature 48-hour planula, attaching plan- all morphology. Certain marine cnidarians have charac-
ula. disc, pawn, crown, immature polyp, and primary teristicsthat make them ideal forsuch a study. Embryo-
adult polyp. The larval interstitial stem cell population genesis and metamorphosis are relatively rapid and,
(interstitial cells, nematocytes, ganglion cells) undergoes more importantly, both embryos and adults contain a
dramatic changes during metamorphosis: ( 1 ) distribu- population ofmigratory stem cells, makingit possible to
tion patternschange, (2) certain larval derivativesdisap- compare stem cell behavior during embryogenesis with
pear, (3) new types of derivatives differentiate, and (4) behavior in theadult.
migration patterns become morecomplex. This study is To date, studies on cnidarian metamorphosis are few
the first to examine how a stem cell system develops in and virtually nothingisknown aboutthebehaviorofthe
an organism thatgoes from embryo to larvato adult. interstitial cell system during metamorphosis (Martin et
a/., 1983;Berking, 1984; Thomas et a/., 1987; Weis and
Introduction Buss, 1987; Plickert el a/., 1988; Schwoerer-Bohning et
The stem cell, found in metazoans from sponges to ai. 1990; Sommer, 1990). Thusit is not known how the
interstitial stem cell lineagedevelopsinanorganism that
vertebrates, is an intriguing but little understood cell
type. Stem cells, by definition, are not terminally differ- goes from an embryotoalarvatoan adult.
The embryonic interstitial cell system ofthe marine
aenttiinagtemdo:rtehesytehmavceeltlhse(asbeillfirtyenteowadliv)idbeu,tnaoltsoonyliyelgdeinnegr-a hydrozoan Pennana tiarella has been characterized by
variety ofdifferentiated cell types. The mechanisms by Martinand associates(Martin and Thomas, 1977, 1980,
which stem cells differentiate into different phenotypes 1981a, b; Martin and Archer, 1986; Martin, 1988a,
and arrive at the appropriate location are some ofthe 1990, 1991 ). Embryos possessawell-defined population
most important questions in developmental biology and of migratory interstitial stem cells that either divide to
metazoan evolution. Three major migratory stem cell replenish the population or differentiate into ganglion
systems have been extensively studied: vertebrate neural cells or nematocytes. The adult stem cell system ofthis
crest cells, vertebrate hematopoietic cells, and inverte- hydro/oan hasalso been examined, though in lessdetail
brate cnidarian interstitial cells (Bode and David, 1978; (Martin, 1988b). Because embryosand adults ofPenna-
LeDouarin, 1979; Heimfeldand Bode, 1986; Martinand ! : tiarella are easy to obtain and manipulate, develop
quickly, and are small and transparent, the interstitial
stem cell system can be examined continuously starting
Received 13October 1995;accepted 15 November 1996. from the moment it arises in the embryo, progressing
41
42 V. J. MARTIN AND W. E. ARCHER
through embryogenesis and formation and metamor- JEOL JSM T-300 scanning electron microscope oper-
phosisofthe planula larva, and continuingin the adult. atedat 25 kV.
In this study we used both scanning electron micro-
scopyand lighthistologytoexaminethechangesinover- Light microscopy
all morphology and in stem cell differentiation and dis-
tribution that occuras the free-swimming, solid planula Control planulae (various ages) and stages of meta-
larva ofPennaria tiarella is transformed into the sessile, morphosiscomparable to those processed for SEM were
hollow adult polyp. Our results show that the larval in- fixed for 1 h in 10% formalin in seawater. Samples were
terstitialcell system isextensively modifiedduringmeta- dehydrated for 15 min each through an ascending alco-
morphosisto producetheadult pattern. hol series (25%-100% ethanol), followed by a 20-min
rinse in 100% ethanol: 100% tertiary butyl alcohol (1:
Materialsand Methods 1), and an overnight incubation in 100% tertiary butyl
alcohol. After being infiltrated and embedded in Para-
Cultureo/'Pennariaadultsandembryos plast Plus paraffin, animals were serially sectioned at
8 ^m. The sections were mounted on glass slides and
Mature colonies of Pennaria tiarella were collected
stained with azure B, which specifically stains the inter-
from pier pilings in Wilmington and Morehead City, stitial cells and their derivatives (Martin, 1991). Slides
North Carolina. Fronds from male and female colonies
were viewed and photographed using a Zeiss standard
were mixed together in large finger bowls offiltered sea- research microscope.
water;thesebowlswereplaced in thedarkat 1800 hours,
and returned to the light at 2100 hours. Within 1 h after Results
exposure to light, early cleavage stages were observed in
the bottoms of the dishes. These embryos were trans- Planularmorphologyandstagesofmetamorphosis:
ferred to small dishes of filtered seawater and reared at Generalobservations
23Ctothedesired planularstage.
Forty-eight-hour planulae were incubated in cesium Between 8 and 10 h postfertilization the gastrula of
chloridetoinitiate metamorphosis(ArcherandThomas, Pennaria tiarella elongates in an anterior-posterior di-
1983). A stock solution containing 9.76 g CsCl/100 ml rection to producea fat, ciliated, free-swimming planula
distilled water was mixed with Millipore-filtered seawa- larva (Fig. 1 ). This 10-h larva has a distinct enlarged an-
ter at a ratio of 1 (stock):10 (seawater). Planulae were terior apical pole and a narrower posterior basal pole.
placed in 4 ml ofthis mixture for 3 h; some ofthese ani- The surface cells are numerous, small, and uniform in
malswerefixed forscanningelectron microscopy (SEM) size. By 24 h postfertilization 10-h planulae narrow and
immediatelyafterthecesium treatment. Planulaeplaced elongate to form mature, metamorphosis-competent
in cesium for 3 h were also returned to dishes of Milli- planulae. Although planula larvae are competent to
pore-filtered seawater and allowed to continue their de- metamorphose at 24 h, as shown by induction with ce-
velopment. Treated planulae were fixed for SEM aftera sium chloride(Archerand Thomas, 1983), many planu-
recovery period of 2 (disc stage), 6 (pawn stage), 12 lae swim in the water column for 2 to 3 days before at-
(crown stage), 14 (immature polyp), or 18 (primary tachingand metamorphosing. Duringthisswimmingpe-
polyp) h. riodthelarvaecontinuetoelongate.The48-h hydrozoan
planula is solid, elliptical, and moves in the water col-
umn with its enlarged apical end directed forward
Scanningelectron microscopy(SEM)
(Fig. 2).
For SEM, 10- and 48-h control planulae. cesium- During metamorphosis the solid, nonfeeding. motile
treated planulae immediately after treatment, and ce- planula is transformed into a hollow, sessile, feeding
sium-treated planulae allowed to recover for varioMus adult polyp(Figs. 3-9);thisprocesstakesabout 18-20 h.
timeswere fixed for 1 h in 2.5% glutaraldehyde in 0.2 Theapicalend oftheplanulaformsthebaseofthepolyp,
Millonig's phosphate buffer, pH 7.4. Samples were the middle region forms the stalk, and the basal end
rinsed three times in the phosphate buffer, postfixed for forms the hypostome and tentacles. As planulae meta-
1 h in 2% osmium tetroxide in 1.25% sodium bicarbon- morphose their morphology changes dramatically, in
ate buffer, pH 7.2. then rinsed three times in the sodium distinct stages known as shorteningplanula (Fig. 3), disc
bicarbonate buffer. Animals were dehydrated through a (Fig. 4), pawn (Fig. 5), crown (Fig. 6). immature polyp
graded seriesofethanolsto 100%', critical-point dried us- (Fig. 7), and primary polyp(Figs. 8 and 9).
ing CO;, mounted on metal stubs, and sputter coated MatureplanulaeofPennariaattach tosubstrates, usu-
with gold palladium for 1 min in a Denton sputter ally pier pilings in the wild, via their anterior, apical
coater. Samples were viewed and photographed with a poles; shortly thereafter metamorphosisbegins.
STEM CELL DEVELOPMENT 43
Figure I. Ten-hourplanula.Theyounglarvais350urn longand 170fimwide. A,apicalend; B,basal
end. Bar= 50/jm.
Figure2. Forty-eight-hourpre-metamorphicplanula.Theciliatedlarvamoveswithitsenlargedapical
end(A)directedforward. Itis900pm long, 1.10^mwideintheapicalregion,70^m wideinthemidarea,
and 50/xmwideatthebasalend. B.basal. Bar= 100^m.
Figure3. Attachedmetamorphosingplanula.Theoriginalanterior,apicalend(A)ofthelarvaattaches
to the substrate and flattens over it while the original basal end (B) contracts towardsthe attached end.
Thustheanimalbecomesshortand fat. measuring320/im long, 160fim widein theapex,93^mwidein
themiddle,and45urnwideinthebasalregion. Bar = 50jim.
Figure4. Discstage.Thediscisaflattenedball, 180timindiameter,onthesubstrate. Bar= 50^m.
Figure5. Pawnstage. Thebase(B)ofthepawn,nowconsideredtheposteriorendoftheanimal,arises
from the apical end ofthe planula. The anterior, apical region (A) ofthe pawn, derived from the basal
regionoftheplanula. formstheheadandtentaclesoftheprimary polyp.Smallamountsofperisarcmate-
rial (arrow)aredeposited at the base. Thepawn is350^m tall. 160^m wide in theanteriorhead, 73^m
wideinthemid-stalk,and 106^mwideattheposteriorbase. Bar= 50^m.
44 V. J. MARTIN AND W. E. ARCHER
Figure6. Crownstage.Adistinctheadregion(HE),stalk(Si.andbase(Blareevident. Perisarcmaterial
(arrows)coversthesurface.Thecrownis427^mtall, 125pmwideintheapicalcrownregion,SO^mwide
inthestalkregion,and 166pmwideatthebase. Bar= 50^m
Figure7. Immaturepolyp.Theheadregionconsistsofaconical mound,thehypostome(H),amouth
(arrow),andaringofformingfiliformtentacles(F).Astalk(S)connectstheheadtothebase(B).Thepolyp
is 340/jm tall, 120/jni wide in the anterior hypostome region, 73jim wide in the mid-stalk area, and
206nmwideintheposteriorbase. Bar = 50^m.
Figures. Primarypolyp.Theheadiscomposedofthehypostorne(H),shortcapitatetentacles(C),and
thelongerfiliformtentacles(F). A narrowstalk(S),coveredby perisarc(arrow),extendsfrom theheadto
thebase(B)oftheadult.Stolons(T)emergefromthebase.Theprimarypolypis50(1^mtall.200^mwide
inthecrown area, 70fitnwideinthemid-stalkarea,and200^mwideinthebasalarea. Bar= 50/urn.
Figure9. Enlarged head region ofaprimary polypshowinghypostome(H)withcapitate(C)and fili-
form(F)u-i vThesetentaclesarearmedwith nematocxtes(arrows). Bar= 50^m.
Shorteningplanulu (Fig. 3). Once attached the apical Disc (Fig. 4). Within 2 h ofattachment the basal end
planulaend flattensandexpandso\erthesubstratewhile ofthe larva has completely contracted and a round disc
the basal end contractsdown towards the expanded api- shapeisformed. Theanimal appearsasasmall, flattened
cal pole. Thusthe attached larva becomesshort and fat. ball on the surfaceofthe substrate: apical and basal ends
STEM CELL DEVELOPMENT 45
t
are not discernible. Cilia are absent and the surface is - "-"" * 1 *' '''' "
smooth. <"*
relatively -V<
Pawn (Fig. 5). Six h after attachment a tiny bleb ap- /& -^H^^M
pears in the center ofthe disc and begins to elongate in
an upright direction to form a shape that resembles the
pawn of a chess set. The formation of the pawn from
thedisc stage requires4 h. The original apical end ofthe
planula formsthebaseofthepawn andtheoriginal basal
epnadwnofistshmeopoltahnualnadftohremfsirsittsbehgeiandn.inTghseofsaurpfearciesaorcf,tahne iiS't;A^*alii>i"^A *A;'&sTV*,w . IS^^*
outCemrwnonnc(eFlilgu.la6r).prDoutrecitnigvethcoeatnienxgt,6arhesteheenpaatwthneeblaosen.- ".-* . -v ^ _
gates; the anterior head widens, forming a crown; and a
sharp demarcation appears between the head and the
stalk. This crown stage is formed 12 h after attachment,
anditisduringthisstagethatgeneral featuresoftheadult
polyp begin to take shape: crown (future hypostome).
stalk, base. The surface at this stage is smooth and cov-
ered with perisarc material.
Immaturepolyp (Fig. 7). After 2 h, 14 h after attach-
ment,an immaturepolypisformed. Itshead region con-
sistsofahypostome,aconical moundbearingthe mouth
at its tip, and a ring offorming filiform tentacles. These
tentacles arise as tiny evaginations of the body wall at
the base ofthe crown and lengthen to achieve the adult
tentacle morphology. A cleardivision between the polyp
head and the narrowing stalk is evident. The stalk con-
nects the head to an enlarging base. The surface ofthe
immature polyp below the region ofthe head is covered Figure 10. Interstitial cells (arrows) in the central endoderm ofa
wtiatcPhhrmipemenraitrs,yaracp.oplryipma(rFyigsp.ol8y.p9).isWfiotrhmeidn.4Ah.ro1w8 ohfalfotnegr,afti-- wcmaiapttFshiuugralueedrsaeprl(ka1anl1r.uyrlosawt.sNa)eEi,mnaeacodthroncbluealclrlalgesheotalscsulsawe.ailrtiMhg.chtallmpaysergusselotegadslianer(eakSd.Tcc)Byaaptirsonup=ltleha1ses0m(euDnrp)nld..uossdmaearnllumcdloaefruksa
liform tentaclesanda new rowofshort, evaginatingcap- matureplanula. Bar= 10jim.
itate tentacles, just above the filiform tentacles, charac- Figure12. Nematoblastwithabullet-shapedcapsule(arrow)inthe
terize the crown region, constituting the fully formed endoderm ofa mature planula. Developing desmonemes (large dark
atidpulotfthhyephoysptoosmteo(mFeigj.us9)t.aAbomvoeutthhewihsoprrlesoefnctaaptittahteevteerny- cc=aapp1ss0uullMeemss.))aarnedalmsiocrsoeebna.siEc.ehcetteordoetrrmi;chEoNu.sebn-mdaosdteirgmo;phMo.remses(osgmlaella.daBrakr
tacles, a perisarc covers the stalk and basal region ofthe Figure 13. Bipolarganglion cell (arrow) in the ectoderm ofa ma-
polyp, and stolon formation has begun in the basal re- tureplanula. Theendoderm lacks neurons. EN.endoderm; M, meso-
gion ofthe polyp (Fig. 8). These stolons produce addi- glea. Bar= 10/jm.
tional polyps that remain attached to the original pri-
mary polyp, thuscreatingacolony.
croscopiclevel(Figs. 10-13). Inthefollowingsectionswe
Interstitialstemcellsystem describe the behaviors ofthe interstitial cells, the nema-
toblasts and nematocytes, and the neuroblasts and gan-
Planulae ofPcnnaria tiarel/a contain a population of glion cellsduringembryogenesis, in the metamorphosis-
migratory stem cells, interstitial cells, that either divide competent planula. during metamorphosis, and in the
to replenish the population or differentiate into two adultpolyp.
classes ofsomatic products: nematocytes (stingingcells)
organglioncells(neurons). Earlydifferentiatinginterme- Interstitialcells
diatesofthe nematocyte lineagearecalled nematoblasts,
and intermediates ofthe neural lineage are referred to Interstitial cells are small round cells measuring 7.5
as neuroblasts. Interstitial cells and their derivatives are urn in diameter (Fig. 10). They contain a centrally lo-
easily identified in larval and adult tissueat the light mi- cated nucleus with one or more darkly stained nucleoli.
46 V. J. MARTIN AND W. E. ARCHER
These cells arise during gastrulation (8-10 h postfertil- Nematoblastsandncmatocytcs
ization), in the central core ofthe endoderm along the
entire length ofthe youngplanula(Table I). They divide Nematoblasts, immature nematocytes, range from 10
in the endoderm and by 13-14 h postfertilization begin to 12.5 /um in diameter and form distinctive dark-stain-
to emigrate to the ectoderm, migrating as single cells ingor light-staining capsules (Figs. 10-12); each capsule
through the interstitial spaces of the endoderm and containsanematocystthreadthatmaypossessbarbsand
through the mesoglea. By 15 h postfertilization, the in- spines. Nematoblasts are found in both the ectoderm
terstitial cells reach the base ofthe ectoderm. Migration and the endoderm throughout embryogenesis, meta-
occurs along the entire apical-basal axis of the young morphosis, and in the adult polyp. Once nematoblasts
planula. Asplanulaeage, the numbersofinterstitial cells movetotheoutersurfaceoftheectoderm orprojectinto
in both the ectoderm and endoderm increase, and mi- a forming gastric cavity, they complete theirdifferentia-
gration from the endoderm to the ectoderm continues tion and are considered functional nematocytes. A few
alongthe entire planularaxis. Thus the metamorphosis- nematoblasts with dark capsules are first detected in the
competent planula has many interstitial cellsat the base apicalendoderm oftheyoung 10-hplanula(TableI). Mi-
ofitsectodermandintheendodermalongitsentirebody gration ofthe nematoblasts begins by 13 h postfertiliza-
axis(Table I). tion, andthesecellsarethe firstofthe interstitial cell sys-
During metamorphosis, asthe apical pole ofthe plan- tem toappearin theectoderm (by 14 h postfertilization)
ula attaches to a substrate and the basal end contracts ofthe planula. Nematoblastsin theapical endoderm mi-
towards the attached end, the interstitial cells located in grate assinglecells into theapical ectoderm; theydo not
the mid to basal regions ofthe larva move into the ecto- divide and syncytial clusters ofnematoblastsare not ob-
derm and endoderm ofthe attachment region (Figs. 14- served at any stage ofthe life cycle. As planulae mature
16;TableI). Theirmechanism ofmovementisunknown the nematoblasts increase in number in both the ecto-
but probably involves active cellular migration. The ec- derm and the endoderm and are largely confined to the
toderm and the endoderm of the remaining, still con- apical two-thirds ofthe planular axis (Table I). At least
tracting basal portion ofthe attached larva become de- fourtypes ofcapsules have been observed in the mature
void ofinterstitial cells (Fig. 15: Table I). Once the disc planula(Figs. 10-12): a largeclearcapsule(stenoteles), a
stage is formed, interstitial cells fill both the ectoderm large dark capsule (desmonemes), a small dark capsule
and the endoderm (Table I). As the center of the disc (microbasicheterotrichousb-mastigophores),andamet-
elongates in an upright direction, a pawn is produced achromatic bullet-shaped capsule (microbasic heterotri-
(Fig. 17). Thegrowing, apical upright tissue ofthe pawn, chous b-mastigophores with inclusions). Stenoteles and
destined to form the head and stalk oftheadult polyp, is desmonemes predominate; only a few microbasic heter-
devoid ofinterstitial cells (Fig. 17; Table I). These cells otrichousb-mastigophoreswith inclusionsare seen.
remain in the attached, now basal, end ofthe metamor- Fully differentiated nematocytesare found only at the
phosing animal (Fig. 18); where the upright portion of surface ofthe mature planula, the majority in an area
the pawn connects to the basal disc is a sharp demarca- extending from the apical end ofthe planula to the mid
tion between presence ofinterstitial cells in the base and planula (Table I); only a few nematocytes are found at
absence of these cells above the base. The distribution the surface in the basal (posterior) region. Fully differ-
pattern of interstitial cells in the base of the pawn re- entiated nematocytes of planulae contain either a large
mains unchanged from that ofthedisc. clear capsule (stenoteles), a large dark capsule (desmo-
Bythetimethecrown stage hasformed, theinterstitial nemes) ora bullet-shaped capsule (microbasic heterotri-
cells have migrated out from thebasal attachment siteto chous b-mastigophores with inclusions); no fully differ-
populate the entire body axis ofthe animal (Fig. 19; Ta- entiated nematocytes housing the small dark capsules
bleI). Intheattachmentarea(basaldisc)a fewinterstitial (microbasic heterotrichous b-mastigophores) are found
cellsare found in both the ectoderm and the endoderm. in the planula.
Along the body stalk, the region ofthe animal that con- Asplanulaeattach tosubstrates,all nematoblasts move
nectsthe basal discto the head, are scattered ectodermal intotheectoderm andendoderm oftheapicalattachment
interstitial cells and a few endodermal interstitial cells region (Figs. 14and 15). Nematocytesareconfined tothe
(TableI). Theheadofthecrownstagehasinterstitialcells outer surface of the ectoderm of the attachment area.
in both the ectoderm and the endoderm (Fig. 19; Table Hence, thecontractingbasal portion oftheattached larva
I). This same distribution pattern of interstitial cells is is devoid of nematoblasts and nematocytes in both the
maintained in the immature polyp and in the adult pri- ectoderm and the endoderm (Table I). All four types of
mary polyp (Table I). In the primary polyp many inter- nematoblastcapsulesaredetectedintheectodermanden-
stitial cells are seen at the base ofeach filiform tentacle, doderm oftheattachmentarea. Thebulkofthesecellsare
but noneareseen within thetentacles. differentiating stenoteles, desmonemes, and microbasic
STEM CELL DEVELOPMENT 47
TableI
Distribution /theinterstitialcellsystemduringdevelopmentol Pennariatiarella
Stage Interstitialcells Nematoblasts Nematocytes Ganglioncells
10-HourPlanula
Apical Ectoderm
MidEctoderm
BasalEctoderm
ApicalEndoderm + +
Mid Endoderm +
Basal Endoderm +
48-HourPlanula
Apical Ectoderm ++ ++ ++ ++
MidEctoderm ++ ++ ++ ++
Basal Ectoderm ++ + + ++
Apical Endoderm ++ ++
Mid Endoderm ++ ++
Basal Endoderm ++ +
AttachingPlanula
Apical Ectoderm(Attachmentsite) ++ ++ +
Mid Ectoderm
Basal Ectoderm
Apical Endoderm ++ ++
MidEndoderm
Basal Endoderm
DiscStage
Ectoderm ++ ++ +
Endoderm ++ ++
PawnStage
Apical Ectoderm(HeadandStalk)
Basal Ectoderm(Foot) ++ ++ + +
Apical Endoderm(HeadandStalk)
Basal Endoderm(Foot) ++ ++
CrownStage
Apical Ectoderm(Head) + + +
Mid Ectoderm(Stalk) + + + +
Basal Ectoderm(Foot) + ++ + ++
Apical Endoderm(Head) + +
MidEndoderm(Stalk) + +
Basal Endoderm(Foot) + +
ImmaturePolyp
Apical Ectoderm(Head) + + + ++
Apical Ectoderm(Tentacle) + ++ ++
MidEctoderm(Stalk) + + + +
Basal Ectoderm(Foot) + ++ + ++
Apical Endoderm(Head) + +
Apical Endoderm(Tentacle)
Mid Endoderm(Stalk) + +
BasalEndoderm(Foot) + +
PrimaryPolyp
ApicalEctoderm(Head) + + + ++
Apical Ectoderm(Tentacle) + ++ ++
MidEctoderm(Stalk) + + + +
Basal Ectoderm(Foot) + ++ + ++
Apical Endoderm(Head) + +
Apical Endoderm(Tentacle)
Mid Endoderm(Stalk) + +
Basal Endoderm(Foot) + +
TableKey: ++ =Abundanttomoderatein number. + = Afewpresent. =Absent.
48 V. J. MARTIN AND W. E. ARCHER
time the crown stage has formed, the interstitial cell sys-
tem has migrated from thebasal attachment siteto popu-
late the entire body axis ofthe animal. Nematoblasts are
the first ofthe line to appear apically (Figs. 22-24), and
distinct patternsofnematoblastand nematocytedistribu-
EN tion areobserved. In theectoderm oftheattachmentarea
M are all fourtypesofnematoblastsand twotypes ofnema-
M tocytes(stenotelesanddesmonemes). Oneithersideofthe
basal disc just above the substrate attachment zone, the
perisarc isconnected tothebasal discand lowerbodycol-
umn. In these regions ofperisarc attachment to the ecto-
_i derm, the ectoderm has an abundance of nematoblasts
ir
(desmonemes and stenoteles) (Fig. 22). Along the body
14 15
stalk ofthe crown in the ectoderm are the four types of
HE
tit-
EN EN
B
*
A. .ta
16 >*'' ' . -'' M
Figure 14. Apical region ofan attaching planula. The interstitial
cellsystem hasmovedintotheattachmentarea;notethelargenumber
ofdark nematoblast capsules in this region. E. ectoderm: EN, cndo-
derm;M, mesoglea. Bar= 50urn.
Figure 15. Mid to basal region ofan attaching planula. Note the
absence ofthe interstitial cell system in the basal portion (B) ofthe
animal. E.ectoderm,EN,endoderm:M,mesoglea. Bar= 50Mm.
Figure 16. Interstitial cells(arrows) in the attachment region ofa
metamorphosingplanula. E,ectoderm;EN,endoderm. Bar = 10Mm.
Figure 17. Pawn. Theapical head region (HE) isdevoidofthein-
terstitial cell system. These cells remain in the basal region (B)ofthe
pawn. Dark nematoblast capsules are abundant in the base. Bar =
50Mm. EN
heterotrichous b-mastigophores: only a few developing
microbasic heterotrichous b-mastigophores with inclu-
sions are found. Once the disc stage is reached, the four
types of nematoblasts fill the ectoderm and endoderm
(Fig. 20);afewnematocytes(stenotelesanddesmonemes) theFipgauwrne.18T.hesIentceerlsltsitiaarlecmeillgsra(tarirnogwass)iinndtihceateenddboydetrhme patretsheencbeasoefoaf
are observed around the ectodermal surface of the disc singlehlopodial-likeextension. Bar= 10Mm.
(Table I). As the center ofthe disc elongates to form the Figure19. Headregionofthecrownstage.Interstitialcells(arrows)
pawn, the nematoblasts and nematocytes remain in the are detected; however, ganglion cells are absent in this area. E, ecto-
aintgtatcihsmseuentisdidsecv(oFiigds.o1f7naenmdat2o1b)l.aTshtuss,atnhdeunpermiagthotcgyrtoews.- deenrdFmiog;dueErreNm,;20eg.nadnogDldiiesorcnms;ctealMgle,s.amNreeesmoaagbtlsoeenabt.l.aBsEat.rse=c(ta1or0rdoe^wrmsm).:aErNe,abeunnddoadnetrmi.nBtahre
These cells are confined to the substrate-attached basal = 10nm.
disc region ofthe metamorphosing animal and resemble Figure 21. Nematoblasts (arrows) in the endoderm at the base of
the pattern described for the disc stage (Table I). By the thepawn. Bar = 10Mm.
STEM CELL DEVELOPMENT 49
As the crown stage transforms into the immature
polyp, the ectoderm ofthe forming filiform tentaclesbe-
comes filled with three typesofnematoblastsand nema-
tocytes: Stenoteles, desmonemes, and microbasic hetero-
trichous b-mastigophores with inclusions. Other than
thischange, the distribution pattern ofthe nematoblasts
and nematocytes isthesameasseen in thecrown stage.
PE Asimmature polyps form primaryadult polyps,a sec-
ond group ofshort tentacles, the capitate tentacles, ap-
pearsjust above the whorl offiliform tentacles (Fig. 25).
These capitate tentacles are populated with the same
three types of nematoblasts and nematocytes that oc-
cupy the filiform tentacles (Fig. 25). Alongthe body col-
umn mature nematocytes with small dark capsules (mi-
crobasic heterotrichous b-mastigophores) are visible.
Otherthan these changes, the nematoblast and nemato-
cyte system ofthe primary polyp resembles that ofthe
immature polyp. Thus in the primary polyptheconcen-
tration of nematoblasts and nematocytes is high in the
EN head and in the foot and scattered in the stalk. The dis-
tribution pattern ofnematocytes is specific: in the head
are Stenoteles, desmonemes, and microbasic heterotri-
chous b-mastigophores with inclusions; along the body
column are Stenoteles, desmonemes, and microbasic
heterotrichous b-mastigophores; and in the foot aredes-
monemesand Stenoteles.
24
Neuroblastsandganglioncells
Figure 22. Crown stage. The perisarc(PE) isattached totheecto- Differentiatingneuroblastsaredetectedasearlyas 16-
derm(E)justabovethefootregionoftheformingpolyp.Thisregion is 20 h postfertilization in both the ectoderm and endo-
richinganglioncells(arrows)andnematoblasts. Bar= 10jim. dermoftheplanula(Brumwelland Martin, 1996). These
gliFoingcuerlels2(3.arroSwtsa)lkanrdeginoenmaotfobthleasctrso.wEnNs.taegnedsohdoewrimn.gBmaurlt=ip1o0la^rm.gan- first neuroblasts arise in the apical region ofthe larva;
Figure 24. Stenoteles(arrows)and a nematoblast with adark bul- shortly thereafter they are found along the entire length
let-shapedcapsule in the head regionofthecrown stage. E.ectoderm; ofthe planula. Neuroblastsaresmall roundcells, similar
EN.endoderm. Bar = 10^m. in size to interstitial cells, that contain cytoplasm rich
Figure 25. Head region ofa primary pohp showing nematoblasts in neurosecretory vesicles(Brumwelland Martin, 1996).
(1a0rrMomw.s) in a capitate tentacle (C) and a filiform tentacle (F). Bar = Thesedifferentiatingintermediatesmigrateassinglecells
from the endoderm to the base ofthe ectoderm; neuro-
blasts are positioned closerto the mesoglea than are the
interstitial cells ofthe ectoderm. Neuroblasts seemingly
nematoblasts and at the surface a few nematocytes (sten- emigrate in a straight path from the endoderm to the ec-
otelesanddesmonemes). Theendoderm ofthestalk hasa toderm; there is no evidence that they migrate in an api-
few nematoblasts (desmonemes and Stenoteles) (Fig. 23). calorbasaldirection inthelarva.Onceneuroblastsreach
In the head ofthe crown in both the ectoderm and the thebasal ectoderm they stop movingandcompletetheir
endoderm are three types ofnematoblasts: desmonemes, differentiation byextendingneural processes. These pro-
Stenoteles, and microbasic heterotrichous b-mastigo- cesses are filled with neural vesicles and form an exten-
phores with inclusions (Fig. 24). Three kinds ofnemato- sive neural plexusoftransversely and longitudinally ori-
cytes, thesame varietiesasthehead nematoblasts, project ented processes throughout the length ofthe planula. As
from the ectodermal surface of the head. Prior to the the planula ages additional ganglion cells differentiate
crownstage,thenematoblastswith bullet-shapedcapsules and incorporate into the larval network. Fully differen-
(microbasic heterotrichous b-mastigophores with inclu- tiated larval ganglion cellsare 5 ^m in diameter, bipolar,
sions)areseen sparinglyalongthewholebodyaxisofmet- spindle-shaped, and positioned in the ectoderm just
amorphosinganimals; however, bythecrown stage many above the mesoglea along the entire apical, basal axis of
nematoblastsofthistype haveaccumulated in the head. the planula(Fig. 13;Table I).
50 V. J. MARTIN AND W. E. ARCHER
glion cells: triangular-, star- or spindle-shaped (Fig. 23).
Ganglion cells are not detected in the head ofthe crown
* T stage. As the crown stage develops into the immature
polyp, many ganglion cells (bipolar, multipolar) appear
in the head ectoderm and tentacles(Table I). By the pri-
mary polyp stage, the head ofthe animal is enriched in
ganglion cells, especially around the mouth (Fig. 27).
Ganglion cellsare found at the base ofeach filiform ten-
tacle and in the ectoderm along the lengths ofthe tenta-
cles (Fig. 28). In the ectoderm ofthe body stalk are scat-
teredganglioncells(Fig. 29),andthedistribution pattern
in thebasal disc mimicsthat ofthe immature polyp(Ta-
ble I). Thus in the primary polyp there is a high concen-
tration ofganglion cells in the ectoderm ofthe head and
basal disc and some scattered ganglion cells in the ecto-
derm ofthe bodycolumn (Table I). Theganglion cellsof
the polyp are three types: spindle-shaped bipolar, star-
shaped multipolar, and triangular-shaped multipolar.
Discussion
Thetransformation ofthecnidarian planulalarvainto
the adult phenotype is rapid, taking only 18-20 h in the
hydrozoan Pcnnuria tiarella, and is characterized by
general body reorganization and modification of the
stem cell system. During metamorphosis the hydrozoan
planula ceases swimming, loses its cilia, and attaches to
the substrate by its apical (aboral) pole. Both glandular
Figure 26. Neurons(arrows)and ncmatohlaslswith dark capsules secretions and nematocytes may be used for securing
in the hasal disc ofthe crown stage. E, ectoderm; EN, endoderm; M. planulaetoasubstrate(Martin elai, 1983). Shortlyafter
mesoglea. Bar= 10^m. attachment the basal (oral) end of the larva contracts
Figure 27. Ganglion cells(arrows)in the head regionofaprimary down towards the apical pole until it disappears into the
polFyipg.urBear28=.10Fijilmi.form tentacle ofa primary polyp. Note the abun- attached pole. Atinycirculardisc isformed. Next, atiny
dance of neurons (arrows) at the base ofthe tentacle and along its bleb appears in the centerofthedisc and begins to elon-
length. Bar= 10^m. gateinan uprightdirection formingapawn shape.Three
Figure 29. Stalk ofa primary polyp. Note the ganglion cells (ar- distinct regions ofthe pawn are evident: an apical head,
rows)intheectoderm. PE,perisarc. Bar= 10^m. a mid-stalkregion, and abasaldisc. Thepawngrowsand
reshapesto produce a crown stage, in which general fea-
tures ofthe adult polyp begin to take shape: head, stalk,
During attachment and the early stages of metamor- and base, with a clear separation between the head and
phosis, the larval ganglion cells disappear, by the disc the stalk. Tentacles evaginate from the head region and
stage they aregone (Table I). In the pawn a fewganglion a mouth breaksthrough at the tipofthe head, producing
cells differentiate in the basal disc (Table I). These neu- an immature polyp. Thisstagebecomesa primary polyp
ronsare found in the ectodermjust above the mesoglea: when a row oflong filiform tentacles and a row ofshort
they are triangular or star-shaped and are multipolar. capitate tentacles adorn the head. A mouth is present at
These neurons have smallercell bodiesand thinner pro- the very tip ofthe headjust above the whorl ofcapitate
cessesthan did the planularganglion cells. By the crown tentacles, andaperisarccoversthestalkand basal region
stageganglioncellsarefound intheectoderm ofthebasal ofthepolyp. Toform acolony,thepolypextendsstolons
discand thebody stalk (Table I). Thebasal disccontains from the base and asexually buds additional polyps, all
a large number of ectodermal bipolar and multipolar ofwhich remain connected together.
ganglion cells(Fig. 26), and multipolarganglion cellsare Fourmajorchangesoccurin theinterstitialcellsystem
abundant in the region of perisarc attachment to the duringmetamorphosis: ( 1 )thedistributionpatternofthe
basal disc and lower body column (Fig. 22). Along the cellsalongthebodyaxischanges,(2)certain larvalderiv-
bodystalkintheectoderm areat least threetypesofgan- atives disappear, (3) new types ofderivatives different!-