The Patterns of Bromodeoxyuridine Incorporation in the Nervous System of a Larval Ascidian, Ciona intestinalis

9 Pages·1993·3.7 MB·English

by  T Bollner

Reference: Biol Bull. 184: 277-285. (June, 1993)
The Patterns of Bromodeoxyuridine Incorporation in
the Nervous System of a Larval Ascidian,
dona
intestinalis
TOMAS BOLLNER1 2* AND I. A. MEINERTZHAGEN2
'Department ofZoology, Stockholm University. 106 91 Stockholm, Sweden, and2Neuroscience
Institute, LifeSciences Centre, Dalhousie University, Halifax, Nova Scotia, Canada B3H 4JI
Abstract. The fates ofcells from the anterior region of Introduction
the ascidian neural plate are described eitheras neural or Ascidiansorsea-squirtshave longattracted interest be-
asmixed neural and non-neural. In Ciona intestinalis. all cause, as urochordates, they are considered to be closely
cellular progeny are accounted for until a time 60% be- relatedtothecommon ancestorofallchordates(Garstang.
tween the onset of embryonic development and larval 1928; Berrill, 1955; Bone, 1972). Itisnotthesessileadult,
hatching. To resolvethe issue oftheir fatesin thisspecies, however, but rather the short-lived larval stage that ex-
wehaveexaminedthelatermitotichistoryofneural-plate hibits some ofthe typical features of a chordate. These
cells. Becausecessation ofcell division in the neural plate include a dorsal tubular central nervous system (CNS),
has been claimed to occur at 70% of embryonic devel- and the fact that this arises from an embryonic neural
opment,weneedtoaccount forcellproduction from 60% plate. On the other hand, the number ofcells within the
onward, to determine whether more cells are produced larval CNS is approximately 370 and is apparently fixed,
than populate the larval CNS, allowing some to adopt at least within narrow limits, in closely related offspring
non-neural fates. The embryonic incorporation of bro- ofthe mutual fertilization oftwo hermaphrodite adults
modeoxyuridine (BrdU), 500 jtMin seawater, was mon- (Nicol and Meinertzhagen, 1991). Such cell constancy,
itored in 1-h larvae byanti-BrdU immunocytochemistry. orcutely, is not an invariably identifiable feature ofchor-
The pattern of incorporations indicates that all larval date nervous systems (Williams and Herrup, 1988), but
neuronsare born before 70% ofembryonicdevelopment, rather is usually associated with invertebrate animals.
but that cell division unexpectedly continues to generate Most ofthe cells constitute the functional larval nervous
ependymal cells until at least 95%. Divisions in the neuro- system, which consists ofan anterior sensory vesicle, the
hypophysiscontinuethroughoutembryonicdevelopment. visceral ganglion in the posterior part ofthe trunk, and
The total numberofcells produced appearssufficient only the nervecord in the tail (Nicol and Meinertzhagen, 1991;
tocomplete the complement oflarval CNS cells, denying Fig. 1). The formation ofthis CNS follows the pattern in
non-neural fatesforanteriorly migrating neural platecells, other chordate embryos (Nicol and Meinertzhagen,
and indicating ageneral absence ofcell death. Consistent 1988a), with neurulation following the formation ofthe
numbersofincorporationsafterthesameexposurein dif- neural plate.
ferent larvae provide evidence for determinacy ofneural Two current ideas exist about the fate of the cells in
plate lineages. The last three conclusions confirm those the embryonic neural plate. Descriptive studies in Ciona
reached previously (Nicol and Meinertzhagen, 1988b). (Nicol and Meinertzhagen, 1988a,b) indicate that all
neural plate progeny contributetotheCNS, and thusoffer
an orthodox viewofthe fate ofthisstructure in ascidians.
On the other hand, injections of horseradish peroxidase
*RePcreeisveendt2adJdruenses:19D9e2p;aractcmeepnttedof26BiFoleobgryu,arRyoy1a9l93H.ollowayand Bed- (HRP) into seventh and eighth generation neural-plate
ford New College (London University), Egham, Surrey TW20 OBX, blastomeres in Halocynthia (Nishida and Satoh, 1985;
UK. Nishida, 1987) implythat someanteriorneural platecells
277
278 T. BOLLNER AND I. A. MEINERTZHAGEN
NC tuted nucleotide, 5-bromodeoxyuridine (BrdLJ). to expose
late cell divisions in the neural tube. Our observations
build on the only precedent in this direction (Reverberi
c/ nl., 1960), a previous study in which thymidine incor-
Oc porations were examined, but only at earlier stages in
neurulation.
Materials and Methods
Adult dona intestinaliswereobtained from the Marine
Resources Department of the Marine Biological Labo-
EC POL
ratory, Woods Hole, Massachusetts, or from Tjarno Ma-
Figure 1. Diagram ofthe entire larva, from the right rostral side. rine Biological Laboratory, Sweden, and maintained in
Abbreviations: Nervoussystem.C:cavityofthesensory-vesicle;n: neck; running seawater. Gametes collected from the gonoducts
NC; nervecord; Nh: neurohypophysis;Oc: ocellus;Ot: otolith; PostSV: oftwo individuals were mixed to produce cross-fertilized
spyosstteermiso:rEsCe:nseonrydovdeseircmlea;lScVa:visteyn;soErp:yveepsiitchlee;liVuGm:: EviSs:ceernaldogdaengrlmiaoln.stOrtahnedr; embryos, which were then raised in plastic petri dishes
Me: mesenchyme; Mu: muscle hand; No: notochord; Pa: papilla; Ph: floating on a water bath at 16C.
pharynx; POL: pre-oral lobe. (From Nicol and Memertzhagen, 1991.) DNA synthesis was monitored by the incorporation of
BrdU (Sigma), and revealed by a monoclonal antibody
directed against BrdU (Gratzner, 1982). Incorporations
occurredwhen embryoswereimmersedin BrdU dissolved
migrate anteriorly, away from the CNS, where they ac- in Millipore-filtered seawater. Initially, three concentra-
quire non-neural fates either in the pharyngeal region or tions, 250nM, 500p.Al, and 1 roA/, weretested.Thelowest
in the epidermal adhesive papillae. Ifmigration ofneural concentration produced less clear immuno-staining. Al-
plate cellswere also to occurin dona, it must, according though the two higher concentrations both gave equally
to the results ofNicol and Meinertzhagen 1988b), occur
later than the 60.5% developmental stage ((where 100% is swtarsoncghiomsemnu.no-Iltabeexlcienegd,edthtehelowdeerteocftitohne ttwhor,esh5o0l0d//fAo/,r
the period ofembryonic development required to attain
larval hatching), because this was the last record ofthese
workers. Ifmigration were to occur, it would further re- 500,
quire that extra cell divisions occur to replace the mi-
grating cells (Nicol and Meinertzhagen, 1988b). The res- embryo
olution ofthis issue hinges on an accurate determination 1T1
ofthe time at which the last cell divisions take place. 100 1T0 neuralplate
When does cell division actually cease? Reliable infor-
50
mation about theactual time forcessation ofcell divisions
in theCNS islacking. Berrill (1935)suggests, but without
clear documentation, that the onset ofpigment differen-
tiation in the ocellus and otolith (70% ofembryonic de-
10
velopment) marks the end of neural proliferation, and
this claim was used to arbitrate the differences between
neural-plate fates in dona and Halocynthia (Nicol and
Meinertzhagen, 1988b). Cell counts in dona, however,
indicate that this is not accurately the case (Fig. 2). At
70%, some 300cellsexist intheCNS(Nicol, 1987), while 40 70 100
at hatchingthereare 371 orso, implyingthat furtherpro- embryonicdevelopment,%
liferation must occur between 70-100%. Previous cell
counts have indicated that 88% ofthe final cell comple- revFeiagluerdef2r.omCceellllcporuonlitfser(aotridoinnatate:disfqfuearreentsystmabgoelss,indatthaepnleoutrtaeld fplraotme
ment in one larva was attained by about 73%- (Balinsky. Nicoland Meinertzhagen, 1988a,b).comparedwithcell numberinthe
1931), and thus confirm this picture. Our study was de- entireembryo(circles,datafromConklin, 1905;NicolandMeinertzhagen
signed to address the question of whether later mitoses 1988a). Arrowheads indicatethegeneration numberofthecellsat that
gdtehoseta.lcIotfcutaathlielosyne,omciicftuonrsoetisntdthoheeofcancteeuu,rr,aolfthtpehlnaetoer,uersausalittamhnetwseapsrfiotggouerinedyse.nstuiTgfo-y scNneteilanclgtoeicl.aolaT,mnhpadelseMsimenheciornnweetnarstoebfzyhitnahtgheteehisner.wnlii1umn9mme9abi1re)nprgornoollgfayrrnevaesatus(riaao1lnt0i0ipm%nle,attpoheirpsoceejpnlellcossttq,eiusdaarrntoeodu,goadhctaltctayuairnefsxlrapttoohem-er
do this, we have exploited the incorporation ofa substi- than 70"; (opensquare,data from Nicol, 1987).
BRDU IN THE LARVAL ASCIDIAN CNS 279
dividing cells in adult dona (Bollner et ai, 1991), but
avoided an unnecessarily high concentration, which could doeveemlborpymoennitc
havedisturbed embryonicdevelopment. Thistypeofper-
turbation has been previously reported for Ciona (Cusi-
mano, 1961) and isalso widely known in otherembryos, Longpulse
such as, for example, insects (Truman and Bate, 1988).
Two strategies for BrdU incorporation were used (Fig.
3). In the first, incorporations resulting from long-pulse
exposures to BrdU which started at 60% of embryonic SOhSohrrtpulse
development or later, and ended when the larva hatched,
were used to reveal all cells that were not already post- Figure 3. Diagrammatic representation ofthe two types of Brdll
mitotic before these times. Long-pulse exposures were exposureused.Long-pulseexposures(heavylines)commenceduringthe
started at 60%, 70%, 72.5%, 75%, and so on, and were final40%ofembryonicdevelopmentandcontinueuntil hatchingofthe
extended in 2.5% increments up to 95%. In the second larva.Short-pulseexposures(heavylines)distributedthroughoutthesame
strategy, short-pulse BrdU incorporations of 30 min period, but in abutting intervalsof30 mm.
(about 2.5%) were also administered, starting at 70% and
then at the same intervals as for the long pulses, but with prior to overnight incubation in anti-BrdU 1:200. The
the embryos transferred to fresh seawater after the pulse. same secondary antiserum and PAP-complex were used
Short-pulseincorporationswereusedtorevealtheregional asforthewholemounts, andpreparationswereincubated
and temporal distribution ofmitoses. In all cases, larvae for 1 h and 30 min, respectively. The DAB reaction was
were fixed 1 h after hatching. enhanced by adding NiCl: to a final concentration of
Fixation for pre-embedding immunocytochemistry was 0.015%.
carriedoutin0.1 A/phosphatebuffer(pH 7.2)containing
4% paraformaldehyde, for 4-12 h at 4C. After fixation, Results
thespecimenswerewashedin thesamebufferandtreated
with 2 N HC1 in phosphate-buffered saline (PBS) for 45 The larval ascidian CNS is divided into three main re-
min, and then rinsed in PBS (pH 7.2) containing 0.1% gions (Fig. 1 ), which, in sequence, consist ofthe anterior
Triton X-100 (Sigma) (PBS-TX). To improve tissue pen- sensory vesicle, with 215 cells, separated by a neck region
etration ofantibodies, the fixed larvae were treated with of6 cells from the visceral ganglion of45 cells, which lies
1% Triton X-100 PBS for 24 h at 4C. After two washes in the posterior part ofthe trunk, and the nerve cord of
4C
in PBS-TX, samples were incubated for 48 h at in 65-66 cells in the tail. The sensory vesicle is situated ros-
anti-BrdU (Becton and Dickinson, Mountain View, Cal- trally in the trunk with its anterior left portion overlain
ifornia)diluted 1:200in PBS-TXwith0.5%bovineserum by the pharynx and the so-called neurohypophysis, the
albumin (BSA). The preparations were incubated over- primordium of the adult neural complex with some 40
night at 4C in rabbit anti-mouse secondary antibody or so cells. These cell numbers derive mostly from one
(Dakopatts) diluted 1:50 in PBS-TX BSA, followed by larva (L4) in Nicol and Meinertzhagen (1991), but are
mouse PAP-complex (Dakopatts) diluted 1:100 in PBS- supported by others reported bythese authorsand by Ni-
TX BSA, before further processing using 0.03% DAB as col (1987). Only some cells are neuronal. Roughly 68%
a chromogen. The preparations were washed in PBS-TX havebeen classifiedasependymal, aspecialized non-neu-
between all steps. Finally, the material was dehydrated ronalcellpeculiartoembryonicandlarvalchordates, from
andembedded, eitherin Historesin (LKB)orin Durcupan theirposition liningthe cavities ofthe neural tube's elab-
ACM (Fluka), viewedeitheraswholemountsoras 1-3-^m orations or from clear similarities in the cytological ap-
sections cut on glass knives, and photographed with dif- pearance to those that do (Nicol and Meinertzhagen,
ferential interference contrast optics. 1991).
Forpost-embedding immunocytochemistry, specimens Initially, long pulse exposures were used to establish
were fixed either in methanol for 20 min at -20C, fol- how late in embryonic development cells ofthe CNS in-
lowed by ethanol under the same conditions, or in cold corporated BrdU. Larvaewereexamined inwholemounts,
Bouin's fluid for a minimum of 3 h to a maximum of after being immersed in BrdU from 70% of embryonic
overnight. All specimenswere stained witheosin dissolved development and allowedtodevelop in this medium until
in 70% alcohol, dehydrated in a routine ethanol series, they hatched as free-swimming larvae (Fig. 3). Embryos
and embedded in paraffin wax. Sections were cut either ofthecorrectdevelopmental stage wereselected according
transversely orsagittally at 4-5 ^m and mounted on poly to the time since their fertilization expressed as a propor-
L-lysine coated slides. The preparations were then de- tion ofthe time until hatching, which for most batches
waxed, rehydrated, and treated with 2 NHC1 for 30 min. was attained after 20 h at 16C. In addition, the devel-
280 T. BOLLNER AND I. A. MEINERTZHAGEN
opment ofa batch ofembryos was calibrated by two fur- beled cells in the posterior part ofthe sensory vesicle and
ther criteria. The first was that 60% ofembryonic devel- others in the anterior visceral ganglion might have been
opmenthasoccurredwhenthelengthofthetailbudequals neck cells, but this could not be ascertained.
that ofthe trunk, and the second was that 70% has oc- Very few cells were labeled from long-pulse BrdU ex-
curred when pigment cells first appear in the sensory ves- posures starting at 85%. ofembryonic development and
icle. extending until hatching (Table I). In one case, seven la-
Long pulse BrdU exposures starting at 70% ofembry- beled cells were found in the CNS, while in the second
onic development produced labeled nuclei in all parts of case, the nine cells in the CNS were found in the sensory
theCNS within the trunk, but not in the nerve cord (Fig. vesicle and none was found in the visceral ganglion. Of
4a). It is thus immediately clear that mitotic activity in thenine, atleast fivecouldbeassignedasependymal cells
the CNS extends beyond 70%. With a single short BrdU lining the nerve cord. The identity ofthe remaining four
pulse administered from 72.5% to 75%., labeled nuclei was unclear, although they were found close to the wall
weredetectedin boththedorsal partofthesensoryvesicle ofthe sensory vesicle (Fig. 6c), an area where most ofthe
and the neurohypophysis (Fig. 4b). A long BrdU pulse cells are also thought to be ependymal (Nicol and Mein-
starting from 85% and continuing to 100%. (Fig. 4c) gave ertzhagen, 1991).
labeled nuclei in the sensory vesicle aswell asin the neu- These results are summarized in Figure 7. In all the
rohypophysis. while a short BrdU pulse starting from the examined groups, BrdU incorporations were detected in
same stage, 85%, produced labeled nuclei in the epen- the neurohypophysis, which thus seemed to continue to
dymal liningofthe nervecordand inthe neurohypophysis proliferate throughout the entire period investigated.
(Fig. 4d). Thus the cells in the sensory vesicle must have
had their S-phase after the period ofthe short pulse, i.e., Discussion
later than 87.5%.
BrdU exposure still produced labeled nuclei in theCNS The primary conclusion ofthis study is that far from
at 95% of embryonic development. At this late stage, a ceasing at the time ofpigment differentiation at 70%, as
few labeled nuclei were found in. or in close association Berrill (1935) has suggested, cell division in the nervous
with, the caudal part ofthe sensory vesicle or the neck system continues throughout the remaining period of
(Figs. 4e. 5). These cells may have been part ofthe epen- embryonic development. In principle, therefore, cells
dymal lining ofthe nerve cord. Mitotic activity was also could arise in excess ofthose surviving in the CNS, and
evident in the neurohypophysis at this stage (Figs. 4e, 5). this excess could allow for some to migrate anteriorly, as
Someideaoftheextentofmitoticactivitycan begained current evidence indicates must happen in Halocynthia
from the numbers oflabeled nuclei. Counts in the CNS (Nishida, 1987). The production ofsurplus cells had pre-
oflong-pulse larvae, which were incubated in BrdU start- viously been thought unlikely because the progressive in-
ing at 60. 70. or 85% and continued until hatching, are crease in theduration ofthecell cycleseemed to preclude
shown in Table I. thepossibility fortheretobesufficienttime foradditional
When the long BrdU pulse started at 60% embryonic rounds ofcell division before 70% (Nicol and Meinertz-
development approximately 50 nuclei (53, 50) showed hagen. 1988b).
staininginthelarvalCNS,excludingtheneurohypophysis. Is there in fact sufficient time to generate an excess of
These included cells in the sensory vesicle, the neck, and cells'?Theobserved patternsofdivisionswithintheneural
thevisceral ganglion. Some ofthe labeled cells in the sen- plate concluded with 84 progeny in the posterior region
sory vesicle were deemed to be neurons from their loca- ofthe neural plate and 85 in the anteriorat 60.5% (Nicol
tion, whereas all other labeled cells at this stage were and Meinertzhagen. 1988b). Nicol (1987) predicted from
thought to be ependymal (Fig. 6a). Most labeled cells in the pattern oftheir previous divisions that these cells will
the visceral ganglion were members of bilateral pairs soon generate a total of 109 llth and 12th generation
forming the ependymal lining ofthe nerve cord. cellsintheposteriorpartoftheneural plate, and 158 such
When the long BrdU pulse started later, at 70% em- cells in the anterior (ofwhich only the pigment cells are
bryonic development, fewer labeled nuclei appeared in still intheir9thgeneration). Shepredictedthatthesewould
theCNS (31, 34 cells: Table I), most ofwhich were in the occur before 70%, although some additional divisions
sensory vesicle (Fig. 6b). The larva with 31 labeled nuclei must also be required to make up the total number of
hadonly 6 thatcould beassigned tothe visceral ganglion. 300 cells counted at that stage (Nicol, 1987). This means
On the other hand, the second larva possibly had eight that 130 divisionsare required between 60% and 70%,an
incorporations in its visceral ganglion, but because the interval corresponding to 2 h at 16C. Most ofthe 170
visceralganglion andsurroundingmesenchymal cellswere neural plate cells recorded at 60.5% (Nicol and Mein-
difficult todistinguish in thislarva, some oftheeight may ertzhagen, 1988b: their 13.3 h) werealready born 48 min
not have been part ofthe CNS. In both larvae, some la- earlier at 57% which, considering a cell cycle time
J
f
Ph
En
f
Figure4. Larval wholemount preparationsshowingthe pattern ofincorporations when theembryo is
exposedtotheBrdLI labelatdifferentages.Incorporationsoccurinnucleiinthesensoryvesicle(arrowheads)
and neurohypophysis(arrows): scale bar: 30/jm. (a) Long-pulse embryoexposed through the period from
70%tohatching. Labelednucleiappearinthedorsalandcaudalpartsofthesensory vesicle,incellsassociated
with the neck between the sensory vesicle and visceral ganglion, and in the neurohypophysis. (b) A short
BrdLIpulseat72.5%(Fig.3B)labelednucleiintheposteriorpartofthesensoryvesicleandtheneurohypophysis
(out offocus), (c) A long BrdU pulse starting at 85% labeled nuclei in the postenor part ofthe sensory
vesicle, alongthe nerve cord, and in the neurohypophysis. (d) A single BrdU pulse at 85% ofembryonic
developmentgavelabeled nucleiintheneurohypophysisandalongthedorsal nervecord,(e)Asinglepulse
at95% incorporated in nuclei ofthedorsal posteriorsensory vesicle, (f) Diagram ofthetrunk shown with
the same orientation as(a-e), identifying the positions ofthe majorcomponents: endoderm (En), neuro-
hypophysis(Nh), pharynx (Ph), and sensory vesicle(SV).
281
282 T. BOLLNER AND I. A MEINERTZHAGEN
**
"
* 9* * / * \
V !*;
'*
4
t,
-
Figure 5. A single BrdU pulse at 95% showing labeled nuclei abutting the cavity (C) ofthe sensory
vesicle, in cells ofthe caudal part ofthe sensory vesicle (arrowheads), as well as in the neurohypophysis
(arrows)ofawholemount section. Bar = 20^m.
of2.5-2.9h(Nicoland Meinertzhagen. 1988b),givessuf- been counted in the larval CNS from long-pulseexposure
ficient time for the 130 divisions needed to reach a total at 60% would seem quiteclearly to indicate not an excess
of300 cells by 70% (15.3 h). ofdivisions, but an insufficiency. On the other hand, if
ThenumbersofcellspredictedbyNicol(1987)compare Nicol's (1987) record of300 CNS cells at 70% is correct,
closely in the posteriorpart ofthe neural plate with those wecanaccount fortheremainingdivisions: approximately
found in the final larva (65-66 cells in the nerve cord 30 cells are born from 70% to hatching (Table I) which
+ 45 cells in the visceral ganglion: Nicol and Meinertz- together with 40 or so cells in the neurohypophysis pro-
hagen, 1991). On the other hand, for the anterior part duces a total of370. The inconsistency between ourdata
where 221 cellsexist in the larva (266 trunk cells-45 cells and the predictions arising from Nicol'sdata(Nicol, 1987)
in the visceral ganglion oflarva L4, in Nicol and Mein- stems, then, from the small number ofBrdU incorpora-
ertzhagen, 1991), 63 cells (=221-158) would have to di- tions seen in our long-pulse animals at 60%.
vide again just to generate the numbers of larval CNS Thereare several possible reasonsforthesesmall num-
cells, without excess. Other larvae may have slightly dif- bers. ( 1) The staging ofourembryos might have differed
fering cell counts. The number ofextra divisions for the from those of Nicol. so that the BrdU pulses actually
fourlarvae in Nicol and Meinertzhagen (1991)would, for commenced later than 60%. Since many of the neural
example, rangefrom 56 to65. These numbers, moreover, plate cells at this stage had been in their 1 1th generation
take no account ofthe neurohypophysis. which requires for some time, it is not impossible that they were about
approximatelyanother40cell divisions, foratotal of 103 to enter their next generation at about the 60% stage.
divisions. Fewer incorporations than expected could then have re-
Are more than 103 BrdU incorporations in fact seen sulted ifthepulsewere, through slightly inconsistent stag-
in the period between 60% and hatching, for any excess ing of embryos in the two studies, administered a little
cells to be created? The 53 or so labeled cells that have laterthan 60%. The fact that incorporationscounted from
larvae exposed at 60% derived from a different batch of
animals than those used to count incorporations in older
Table I larvae (Table I) provides a possible explanation for the
NumberofCNScellslabeledwith BnlU[ internal discrepancy in our study between the 60% batch
and the 70 and 85% batches if, once again, the youngest
Stage Larva I Larva 2 batch had in factactually beensomewhat olderthan 60%.
(2) We also do not know the details ofthe cell-cycle.
60-100% 53 50
70-100% 31 34 It istherefore possiblethatalthough cellswere pre-mitotic,
85-100<rr 7 they had already passed, or were at least in a late part of
theirS-phaseandwereconsequently unabletoincorporate
1 Twolarvaecountedateachstage.Theolderlarvaewereallfromthe sufficient BrdU to be detected.
6s0a%m-ewetrweolaadruvlatesoafntdwowaedrueltpsrwohceiscshedrecteogievtehdearn,widheinlteictalhoBsredlUabterleeadtmfernotm, (3) A third possibility is that embryonic development
but on adifferentdav. was slightly inhibited from the 60% stage by the concen-
BRDU IN THE LARVAL ASCIDIAN CNS 283
in BrdU much shorter than in Cusimano's study, but all
the resultant larvae arising from such treatment also
looked normal. It is nevertheless possible that their cell
division had been slowed or inhibited by long-pulse ex-
posureto BrdU. but thatthiswasundetectableexternally,
and that as a result the number of incorporations was
smallerthan the normal number ofdivisions. In thatcase,
theeffect upon embryosat 70% might be lessthan at60%>
because oftheshortercumulative period ofexposure and
becausethe susceptibility ofembryosstartsatthe neurula
stage (Cusimano, 1961). We were not able to avoid the
possibility ofthis problem by using BrdU at a lowercon-
centration (250 H.M). because that would have diminished
the staining intensity and run the risk of recording too
few incorporations.
* Thus, with theseprovisos, theevidencesupportsamode
of neural plate proliferation which at most attains con-
stancy ofcell number by addition (Williams and Herrup,
.* 1988), possibly by a lineage dependent mechanism. This
proliferation continues, moreover, until the final stage of
embryonic development, because the latest stages inves-
tigated (95%) still revealed incorporations in the CNS.
> The cells produced at this late stage are thought to be
ependymal ratherthan neuronal, asindeed istrueformost
,
** ^
::* ' 4P
Figure 6. Sections through the sensory vesicle oflong-pulse larvae
exposed to BrdU from 60to85%. (a)60%. Incorporationsoccur in the
sensory vesicle wall andagroupposteriortothepigmentcup. (b) 70%.
Incorporationsoccurincpendymalcellsliningthedorsalnervecord,(c)
85%.Incorporationsoccurintwolabelednucleiintheependymallining
ofthenervecord. Positionsoflabeled nucleiareindicatedbyarrows.C:
sensory vesiclecavity; P: pigment cup. Bar = 10^m.
tration of BrdU we used. Cusimano (1961) reports that
afterexposingembryosto 500p.Aldeoxuridine from early Figure7. Schematicrepresentationofthepositionsoflabelednuclei
cdwlaheteaarvetaaogsetehsextspaeogesasurreoenlwataocrki1dn,mg9fMo0r%wBaorsfdcUnl,oerawrmhlayilctholxaiwrcev.aesEuqegugmieevsratgleehdna,ts (c(iacno))mtLLhpooraennregge--cppotuuhullenssitreeepdlloaassrrtivvataagielolsana,bsbemewllaieeddtdheaatmtb6ay80p5s%s%u..po(efbNr)unicemLlupoeroniosgni-aanprlguelncssuahecmolleewairnrav(awNhiilictacohublleealaqednudrdaaaltwMise7ni0izge%nss.-,:
no effect on normal development at 500 y.M. Not only ertzhagen, 1441: their Figs. 16, 17). Ot: otolith; Oc: ocellus: NC: nerve
were the periods for which our embryos were immersed cord.
284 T. BOLLNER AND I. A. MEINERTZHAGEN
BrdU labeled nuclei found in the larval CNS after 60%. and Meinertzhagen, 1988b, 1991) and we find no BrdU
This diagnosis is based on the locations ofthe nuclei rel- incorporationsatthissite, sothatall postmitoticcellsmust
ativeto published maps(Nicol and Meinertzhagen, 1991), survive into the larva. To rule out cell death may seem a
ratherthantheappearanceofthe nuclei themselves, which trivial distinction in the case ofthe nerve cord, however,
did not distinguish ependymal cells from neurons. Our given that its cells will in any case degenerate with the
data thus indicate that all neurons in the larval CNS are onset ofmetamorphosis, soon after larval hatching (Clo-
born before70% ofembryonicdevelopment, and thatonly ney, 1978), and because probably all such cells are epen-
ependymal cell incorporations are revealed thereafter, at dymal not neuronal. Nevertheless, the absence of cell
least outside the neurohypophysis. The neurohypophysis death is a cardinal difference seen when we invoke the
itselfcontinues to proliferate right throughout the period other major example ofchordate neurogenesis, the for-
ofembryonic development, and thus behaves differently mation ofthe CNS in vertebrates (Oppenheim. 1991 ).
from the cells ofthe larval CNS proper. We are therefore confident that our results support all
What happens to the cells produced from divisions be- ofthe following: the absence ofmigration to the papillae
tween 70 and 100% of embryonic development? Three from the neural plate, the birth ofall neurons before 70%
possibilities concerning their fate exist in the light ofour but continued ependymal cell production until at least
new evidence. 95%, the lack ofover-production ofneural plate progeny
The first and by far most likely possibility is that the and the consequent absence of neuronal cell death, and
late divisions of neural plate cells are the final rounds the attainment offinal cell number by the regulated con-
required fortheconstructivegeneration ofthecellsofthe trol ofthe numberofcell divisions. Evidenceforthelatter
CNS by a mechanism ofaddition (Williams and Herrup. comes clearly from consistency among the numbers of
1988), topping up the complement of neural plate cells incorporations seen in larvae afterexposure to BrdU at a
to the final total, which, including roughly 40 cells in the particular embryonic stage. Thus, larvae exposed at the
neurohypophysis, isabout 370(Nicol and Meinertzhagen, same times between 60 and 100% differ by only two to
1991). The only way in which a fixed number of larval three incorporations (Table 1). We take the small range
CNScellscould then be produced would beby regulating ofthe differences between two animals at any one larval
the number ofprogeny ofeach neural platecell, sincethe ageto reflect eitherminordifferences in thestagingofthe
total numberofsuchcellsinthe neural plateisfixed(Nicol larvae or in the timing oftheir mitoses, and to indicate a
and Meinertzhagen, 1988b). If, on theotherhand, asmall determinacy of cell lineage amongst neural plate cells
surplus of cells were produced, if for example we were which hasbeen seenpreviously(Nicol and Meinertzhagen,
unable to record all incorporations successfully, two fur- 1988b). Put another way, ifwe assume the existence of
therpossibilities on the fateofthecellscan be entertained. lineage determinacy, consistency in the number ofBrdU
First, they may migrate away from the neural plate. incorporations provides assurance that developmental
The late migration ofcells to other regions is implied by staging and immunocytochemical detection is also con-
Nishida's (1987) results on I/ulocyntliiu. but because in- sistent between embryos.
corporations were not found in the adhesive papillae in
Ciona, andbecausethiswasaclearsiteoftheirlocalization Acknowledgments
in Halocynthia (Nishida, 1987), these two species must We aregrateful to The Royal Swedish Academy ofSci-
differ in at least this one important respect, reinforcing ences and the Foundation "Lars Hiertas minne" for fi-
the only conclusion reached previously on their compar- nancial support to T.B. We also gratefully acknowledge
ison (Nicol and Meinertzhagen, I988b). Migration to sites the help ofthe staffand the use ofthe excellent facilities
other than the papillae is at least theoretically possible. at the Marine Biological Laboratory, Woods Hole. Mas-
Forexample,weseepharyngeal incorporations, asNishida sachusetts, and the Marine Biological Laboratory at
(1987) found in Halocynthia, but these need not be of Tjarno. Supported by NSERC grant A 0065 (to I.A.M.).
migrating cells. Moreover, from BrdU immunocyto-
chemistry we cannot distinguish them from incorpora- Literature Cited
tions in the neurohypophysis, because the neurohypo-
physis is located close to the pharynx and we lack probes Balinsky,B.I. 1931. UberdenTeilungsrhythmusheiderEntwicklung
des EiesderAscidie Cionti inlestinalis. 11'ilhelni Roux'Arch. Entw.
to its yet undifferentiated cells. Mech. Org. 125: 155-175.
Finally, surplus cells may be later eliminated by cell Berrill, N.J. 1935. VIM. Studiesin tunicatedevelopment. Part III.
death. This has been ruled out fortheearly stagesofneu- Differential retardation andacceleration. Phil. Trans. R Si>c. Limit.
rogenesis (Nicol and Meinertzhagen, 1988b) and is most Ser. B 225: 255-326.
unlikely for later stages. At least in the nerve cord ofthe BBeorlrlinlelr,,N.T.J,.P1.95\5V.. BcTehsleeGyr,ita-nindnMf\.'eCn.ehTrhaloersn.dyCklea.ren1d99o1n.PresBsi,rtOhx-fdoartde.s
tail,celldeathapparentlydoesnotoccur.Cellcomplement and differentiation ofcells during neural complex regeneration in
at60% hasalreadyattained that ofthe mature larva(Nicol theaseidian Ciona inlestinalis. Reg. Peptides35: 227.
BRDU IN THE LARVAL ASCID1AN CNS 285
Bone, Q. 1472. The Origin <>/ Cliordiiiex Oxford University Press, Neural plate morphogenesis and cell lineages during neurulation.
London. DCY Binl 130: 737-766.
Clone},R.A. 1978. Ascidian metamorphosis: reviewandanalysis. Pp. Nicol,D.,andI. A.Meinert/hagen. 1991. Cellcountsand mapsinthe
255-282 in Seiilenieni ami Metamorphosis t Marine Invertebrate larvalcentral nervoussystem oftheascidian Ciniw iiiie\linali\ (I..).
Larvae. F.-S. Chiaand M. E. Rice, eds. Elsevier, New York. ./ Camp Neurnl. 309: 415-429.
Conklin, K.G. 1905. Organizationandcell-lineageoftheascidianegg. Nishida, II. 1987. Cell lineage analysis in ascidian embryos by intra-
J. Acatl .\al Sci Philadelphia 13: 1-114. cellular injection ofa tracerenzyme. III. Up to the tissue restricted
C'usimano, I. 1961. L'azione di alcuni nucleosidi sullo sviluppo stage. Dcv Bml 121: 526-541.
dell'uovodi ascidie. .Ida Knihiyul Murnliul /i".v/> 4:62-69. Mishida, H.,and N.Satoh. 1985. Cell lineageanalysisin ascidianem-
Garstang, \\. 1928. ThemorphologyoftheTunicata.anditsbearings bryos by intraccllular injection ofa tracerenzyme. II. The 16- and
Gratdozonndeetroh,exyIpuIh.r\iGld.oignee1n:9y82ao.fntehMweonrCoehacogrledonanttaalf.oraQndte.it/beocMdtiiyconnto^ocf5-DSbcriN.oAm7o2-:rea5pl1ni-dc1a8t5i7-o.ino.- Oppcs32yn-shcteeelimlm.,.stIaRgn.ens\.VR./cV1\r9.91..HYironlnCnerl1l/10d:e1a4-t44h:04-d54u35r-4i5n.0g1.developmentofthenervous
NNiiccoocSNllic,,odiviDeDa.an.n,c.Sae1cn9(o2d.8t1'7i8lIa..i:.mAn4.17Dn9M4ie4eh-v"i4ipent7pili5.noTt.itplzm/.he\angtePnho..fDIt.9he8t8hlaeas.rivsa.lDDenavelerhvlooouupssimeesnyUtsntioevfm.t,hienHcatelhnietfraaaxsl,- TRervueamArncabaknelir,yisE,Jii.snGh.\onV,f-.i,\t'Vhla.endGMa.osMcriV.pdehirBoalalnyt,ed.ICe:.vx1pe9M8la83on.:ps2mu0ecS2ntp-toa2,t1bia2yal.ntdahneId.ustDee'mAopnfonrraaa.ldi1op9-ai6ts0to.etronpseAson.f
nervous system ofthe larva ofthe ascidian. Cinmi inlevinalis L. I. neurogenesis in the central nervous system ofDnwoplnla nu'lano-
Theearly lineagesofthe neural plate. Dev liinl 130: 721-736. Xmler /)cr Dnil 125: 145-157.
Nicol, D.,and 1. A. Meinertzhagen. 19886. Developmentofthecentral Williams,R.\\'.,andK.llcrrup. 1988. Thecontrolofneuronnumber.
nervoussystem ofthe larvaofthe ascidian, dona imexlimilix L. II. Inn KIT AVimnv;. 11:423-453.