Transcription and Translation Inhibitors Permit Metamorphosis up to Radiole Formation in the Serpulid Polychaete Hydroides elegans Haswell

12 Pages·2003·5.1 MB·English

by  Eugenio Carpizo-Ituarte

Reference: Biol. Bull. 204: 1 14-125. (April 2110?)
2003 Marine Biological Laboratory
Transcription and Translation Inhibitors Permit
Metamorphosis up to Radiole Formation in the
Haswell
Serpulid Polychaete Hydroides elegans
EUGENIO CARPIZO-ITUARTE1 2 AND MICHAEL G. HADFIELD
Kewalo Marine Laboratory and Department ofZoology. University ofHawaii.
41 Almi St.. Honolulu. Hawaii 96813
Abstract. Settlement and metamorphosis in most well- tion. and differentiating the collar: once thecollaris formed,
studied marine invertebrates are rapid processes, triggered they begin secreting the secondary, calcified tube. During
by external cues. How this initial environmentally mediated the second phase, the small worm develops branchial radi-
response is transduced into morphogenetic events that cul- oles and begins to grow, requiring new mRNA and protein
minate in the formation of a functional juvenile is still not syntheses.
well understood for any marine invertebrate. The response
of larvae of the serpulid polychaete Hydroides elegans to Introduction
inhibitors ofmRNA and protein synthesis was examined to
determine if metamorphosis requires these molecular pro- Complex life histories in which a larval stage undergoes
cesses. Competent larvae of H. elegant were induced to metamorphosis to achieve a juvenile form are common
metamorphose by exposing them to a bacterial film or a 3-h throughout the animal kingdom, including at least 1? phyla
mM
pulse of 10 CsCl in the presence ofthe gene-transcrip- of marine invertebrates (Strathmann. 1493). Well-known
tion inhibitor DRB (5,6-dichloro-l-/3-D-ribofuranosylben- examples ofthis postembryonic transformation are amphib-
zimida/ole) or the translation inhibitor emetine. When in- ians and insects, which havebeen usedas model systems for
duced to metamorphose in the presence of either inhibitor, vertebrates and invertebrates fordecades (see: Gilbert et <//.,
larvae of H. elegans progressed through metamorphosis to 1996; Nijhout, 1999; Rose, 1999). In both groups, the
the point at which branchial radicles start to develop. DRB metamorphic process is driven by the action of stage-spe-
and emetine inhibited the incorporation ofradiolabeled uri- cific hormones that activate gene expression. In amphibians,
dine into RNA and radiolabeled methionine into peptides, thyroid hormones act directly on target tissues by interact-
respectively, indicating that they were effective in blocking ing with specific nuclear thyroid-hormone receptors that
the appropriate syntheses. Taken together, these results in- regulate transcription of target genes containing thyroid-
dicate that the induction of metamorphosis in H. elegans hormone response elements (Evans, 1988: Tata. 1996.
does not require de novo transcription or translation, and 1999). Similarly, metamorphosis in insects is regulated by a
that the form of the juvenile worm is achieved in two complex interaction between a unique group ofcompounds,
phases. During the first phase, larvae respond to the inducer thejuvenile hormones and ecdysteroids. which also activate
by attaching to the substratum, secreting a primary tube, transcription factors (see Gilbert et a!.. 1996).
resorbing the prototroch cilia, undergoing caudal elonga- In contrast, metamorphosis in most well-studied marine
invertebrates is set in motion by environmental cues that
triggerthe developmental events that culminate in the trans-
Received 16 April 2002; accepted 4 December 20(12 formation of a larva into a functional juvenile (Hadtield.
1 Present address: Institute de Investigaciones Oceanologicas. UABC. 2000). This complex set ofinteractions, from the perception
Apdo. Postal 453, Ensenada. B.C. Mexico 22800.
To whom correspondence should be addressed. E-mail of an inducer signal to the acquisition ofthejuvenile form,
[email protected] is poorly understood. Some insights into the signal-trans-
Abbreviations: DRB. 5,6-dichloro-1-/3-D-nboturanosylbenzimida/ole. duction mechanisms have been gained for different groups.
14
1
METAMORPHOSIS AND GENE EXPRESSION
including coelenterates (Freeman and Ridgway, 1990; mechanisms that regulate metamorphosis had not been re-
Schneider and Leitz. 1994; Leitz. 1997: D. W. McCauley. ported for a polychaete.
1997; Thomas et ui. 1997: Berking. 1998), polychaetes The major questions addressed in this study are. can
(Jensen and Morse. 1990: Pawlik. 1990; Ilan ct ai, 1993: metamorphosis be triggered in Hydmides elegans, and how
Holm et til., 1998; Biggers and Laufer. 1999), molluscs far can it progress without de novo synthesis of mRNA or
(Trapido-Rosenthal and Morse. I985a. h; Baxter and proteins? Hydmides elegans is an excellent model for stud-
Morse. 1987; Hadtield, 1998; Hadfield et ai, 2000: Pires et ies ofmetamorphosis in marine invertebrates because it has
til.. 2000), and crustaceans (Clare et ai, 1995; Clare. 1996; a short generation time (about 21 days), it reproduces year-
Knight et nl.. 2000). Recent data on the tropical nudihranch round in Hawaii, and its metamorphosis may be manipu-
Phestillti sihogae show that the apical sensory organ in the latedby natural and artificial inducers (Hadfieldetai, 1994;
veliger larvae bears the receptors for the inducer of settle- Carpizo-Ituarte and Hadfield. 1998; Holm et ai, 1998;
ment and metamorphosis (Hadfield el a!.. 2000). However, Unabia and Hadfield, 1999). The results ofthe present study
little is yet known about how initial inducer-signal trans- show the extent to which syntheses ofRNA and protein are
duction mechanisms are coupled with the morphogenetic involved in the progression ofmetamorphosis in Hvdroides
changes ofmetamorphosis orto what extent metamorphosis elegans.
in marine invertebrates requires de uovo gene expression.
To date, the molecular mechanisms that shape the mor- Materials and Methods
phogenetic changes during metamorphosis have been doc- Collection ofadults and culture oflan'ae
umented in only two aquatic invertebrate phyla. In coelen-
terates, pattern formation has been analyzed for H\dra spp. Adults of Hydmides elegans were collected from Pearl
(summarized by Steele. 2002), and the molecular events Harbor. Hawaii. Gametes were collected, and competent
during metamorphosis have been documented for Hvdrac- larvae were reared by methods described previously (Had-
tiniti echinata (summarized by Berking. 1998; Leitz. 1998). field etal., 1994; Carpizo-Ituarte and Hadfield. 1998). Com-
Recently, many new signaling molecules that have morpho- petence is defined for this species as the ability oflarvae to
genetic activity and genes that participate in head formation settle and metamorphose when exposed to a well-developed
have been characterized in Hydra spp. (see: Broun et ai. marine biofilm. Larvae attained metamorphic competence
2000; Takahashi et ai, 2000; Yan et ai, 2000a. b; Leon- as three-segmented nectochaetes about 4-5 days after fer-
tovich et ui.. 2000). Intensive efforts to understand the tilization (at 25-26 C).
molecular events during metamorphosis in molluscs have
been made in the nudibranch Phestilla sibogae (B. J. Mc- Effects of5,6-dichloro-l-^-D-ribofuranosylbenzimidazole
Cauley. 1997; Hadtield. 1998; Del Carmen and Hadfield. IDRB) and emetine
1999) and the red abalone Haliotis rufescens (Cariolou and
Assays formetamorphosis, using 5-6-day-old competent
Morse, 1988: Degnan and Morse. 1993. 1995; Fenteany and larvae, were conducted in 5-ml petri dishes containing nat-
Morse, 1993; Degnan et ai. 1995).
ural seawater that had been passed through a 0.22-/xm filter
In the serpulid polychaete Hydmides elegans. most ofthe (FSW). Four replicates per treatment were used in all ex-
morphogenetic changes of metamorphosis are completed
periments.
during the first 3.5 h after contact with a metamorphic cue To examine the effects of inhibitors of transcription
contained in bacterial biofilms (Carpizo-Ituarte and Had- (DRB) or translation (emetine) on the ability of larvae to
field. 1998; Unabia and Hadtield, 1999). Within 5 min of complete metamorphosis. 30-90 competent larvae were
being exposed to a biofilmed surface, larvae of H. elegans added to each dish containing one of the inhibitors. To
begin tocrawl upon it and then quickly tether themselves to induce metamorphosis, the larvae were exposed to a marine
the surface by a thin mucous thread secreted from their biofilm ora 3-h pulse of 1 mA/CsCl in the presence ofone
posterior segments. They next lie prone upon the surface ofthe inhibitors. Two typesofexperiments were performed.
and rapidly secrete a primary membranous tube. Within the In one, larvae were exposed to the inducer (marine biofilm
primary tube, the prototroch is resorbed. the larval body or CsCl) and the inhibitor (DRB or emetine) simulta-
elongates, and the collar region becomes functional. When neously. In the second type, larvae were preincubated in
processes are complete, the small worm begins to secrete DRB for 1-12 h or in emetine for 1-2 h before inducing
the calcified secondary tube, starting from the aperture of them to metamorphose in the presence ofthe inhibitor. The
the primary tube. Two to three hours after the initiation of number of larvae that had metamorphosed was determined
settlement, the lobes that will form the branchial radioles between 4 and 24 h afterinitial induction by observing them
become apparent on the head. At this point metamorphosis with a dissecting microscope. Larvae were considered to
iscomplete (Carpizo-Ituarte and Hadtield. 1998). Before the have metamorphosed when they hadelongated, resorbed the
present investigation, attempts to characterize the molecular prototroch. and secreted the primary and secondary (calcar-
116 E. CARPIZO-ITUARTE AND M.G. HADFIELD
eous) tubes. At higher concentrations of inhibitors, the fluid (ScintiSafe, Econo 2, Fisher Scientific), and radioac-
branchial radicles did not differentiate, and the newly meta- tivity was measured in a Beckman LS 7000 liquid scintil-
morphosed worms were transferred from the solution with lation system or a Packard liquid scintillation analyzer 1900
the inhibitor to fresh FSW and observed for an additional TR. The remaining 200 jid from the initial samples was used
16-24 h. to measure protein concentrations by the Bradford method
In all experiments, either pieces of plastic mesh or petri (Ausubel et <//., 1992).
dishes that had accumulated a marine biofilm while soaking Four experiments were carried out to measure the effect
in the laboratory seawater tables for several days were used of emetine on metamorphosis. In each of the experiments,
as a positive control to verify that the larvae were metamor- 400-1000 competent larvae were used. In the first experi-
phically competent. A negative control, included to measure ment, larvae were incubated for4h in 0. 250, 500, and 1000
spontaneous metamorphosis, consisted of placing larvae in nM emetine, and 1 ju.1 (0.01 mCi) of ^S-methionine (Am-
FSW in clean plastic petri dishes during the experimental ersham, 10 mCi/ml) was added during the last 1.5 h of
period. incubation in the inhibitor. In a second experiment, larvae
Stock solutions of emetine (Sigma Chemical Co.) and were incubated in 250 nM emetine for 0, 6, and 9 h before
DRB (Calbiochem) were prepared in 100% ethanol (ETOH) adding 1 ;ul (0.01 mCi) of ^S-methionine (Amersham. 10
and added to filtered seawater to desired concentrations. mCi/ml) during the last 2 h of incubation. That is. for the
Controls were included in each experiment to detect possi- treatment of h. emetine was added simultaneously with
ble effects of ETOH on metamorphosis. ^S-methionine and incubated for2 h. In a third experiment,
the effect ofdifferent periodsofincubation in 1 ju,Memetine
Labeling ofRNA andprotein synthesis "in vivo" w2a50snmeMaseumreetdin.e,Laarnvdae1 w/^elr(e0.i0n1cumbCait)edoffo^rS1-,m2e,th4,ioannidn6ehwaisn
To confirm that DRB and emetine were effectively in- added to the emetine-FSW solution for an additional 45
hibiting RNA and protein synthesis, respectively, the syn- min. Larvae in the treatment of 1-h emetine remained atotal
thesis ofthese macromolecules was measured in vivo in the of 1 h and 45 min in emetine, but were only in the presence
presence and absence of the inhibitors. All of the experi- of both emetine and 35S-methionine for the last 45 min.
ments were conducted in 1.5-ml microcentrifuge tubes (Ep- During the fourth experiment, the effect of250 nM emetine
pendorf) using artificial seawater (ASW) (Cavanaugh. was measured when larvae were induced to metamorphose
mM
1956) containing antibiotics (0.1 mg/1 penicillin; 0.1 mg/1 with a 3-h pulse of 10 CsCl. During this experiment,
streptomycin). larvae were placed for 1 or 2 h in 250 nM emetine before
Two experiments were carried out to measure the effects being exposed to a 3-h pulse of CsCl (10 mM) in the
of DRB. In the first, 400-1000 larvae were incubated for emetine-FSW solution. One microliter (0.01 mCi) of ~S-
2.25 h with 10. 30, 50. or 70 /nA-/ DRB, and then 0.3 /ul (0.3 methionine was added during the last 1.5 h oftheCs+ pulse.
juCi) of [5,6-3H]uridine (Amersham, 38 Ci/mmol) was After the period of incubation in 3SS-methionine. the larvae
added for the last 45 min of incubation in the inhibitor. In were centrifuged (15,120 X i>, 2 min). and a 5()-/il sample
the second experiment, 400-1000 larvae were exposed to of the supernatant fluid was recovered to measure the ra-
10 ixM DRB for 3. 6. 12, or 24 h. and then 3 jul (0.3 juCi) dioactivity remaining in it. The remaining supernate was
of [5,6-3H]uridine was added during the last 45 min of removed from the tube, and the larvae were washed once
incubation in DRB. The labeling was stopped by eentrifug- with ASW and resuspended in 500 /id of 50 mM sodium
ing the tubes (15.120 X #, 5 min) at room temperature phosphate buffer. pH 7.2. The larval tissue was lysed by
(RT = 25 C) and then removing the supernatant fluid and sonication for 30 s in a Branson Sonifier (model 450.
immersing the pelleted larvae, still in the tubes, in liquid Branson Ultrasonics Corporation). The sample was re-
nitrogen. The pellets were homogenized on ice and resus- moved from the sonicatorevery 10 s and put on ice for 20 s
pended in 1 ml of SSC buffer (sodium chloride/sodium to prevent overheating ofthe tissue. TCA precipitation was
citrate. pH 7.0) prior to trichloroacetic acid (TCA) precip- carried out according to the protocol described by Ausubel
itation and protein-concentration analysis. TCA precipita- ct ul. (1987) to measure incorporation of "S-methionine
tion of RNA was carried out according to the protocol into newly synthesized proteins. The sonicated sample was
described by Fenteany and Morse (1993). Absolute TCA centrifuged ( 15.120 x #, 2 min) at RT, and the supernatant
was added to 800 ;al of resuspended sample (to a final fluid was transferred to a clean 1.5-ml microtube (Eppen-
concentration of 10% TCA). Immediately afterward, the dorf). To a 50-/U.1 sample ofthis supernate was added 0.5 ml
sample was vortexed and allowed to precipitate (in ice for of bovine serum albumin (0.1 mg/ml) containing 0.02%
30 min. The precipitate was collected on a microfiber filter NaN, followed by 0.5 ml of ice-cold 20% TCA, and the
(Gelman, Type E), washed three times with 5% TCA and suspension was incubated on ice for 30 min. After incuba-
twice with 100% ETOH, and dried for an hour at RT. The tion, the suspension was filtered under vacuum onto a mi-
filters were introduced to vials with 5 ml of scintillation crofiberfilter(Gelman, Type E), washed twice with ice-cold
METAMORPHOSIS AND GENE EXPRESSION 117
10% TCA and twice with 100% ETOH. and the filters were metamorphosed was observed (Bonferroni's method, P <
allowed to dry for 30-45 min at RT. To quantify the 0.05, Fig. 5).
incorporation of 35S-methionine. radioactivity was mea-
sured by introducing the fiberglass filters with the TCA Effects ofthe protein-synthesis inhibitor emetine on
precipitate into vials containing 5 ml of scintillation fluid
metamorphosis
(ScintiSafe, Econo 2; Fisher Scientific). Radioactivity in the
vials was measured in a Beckman LS 7000 liquid scintilla- Competent larvae of H. elegans metamorphosed when
tion system or a Packard liquid scintillation analyzer 1900 exposed to a biofilm in the presence of 1 ju,A/ emetine (Fig.
TR. Results were expressed as DPM/fj,g of protein. The 6), with no significant reduction in the proportion of larvae
protein concentration in the remaining sample (250-450 that metamorphosed after 3 h compared with induction by a
jul) was measured using the spectrophotometric method biofilm alone. The larvae progressed in metamorphosis to
described by Whitaker and Granum (1980). the point at which branchial radioles begin to develop:
concentrations ofemetine as low as 250 nM were effective
in preventing the formation of these structures. Preincuba-
Statistical analysis ofmetamorphosis
tion for 1 h in 1 p.M emetine prior to exposure to a biofilm
The proportion of larvae that underwent metamorphosis did not curtail metamorphosis (Fig. 7), but the proportion of
was determined, and these values were arcsine-transformed larvae that had metamorphosed after 3 h was slightly re-
to estimate statistical differences among treatments using duced.
one-way ANOVAs or Kniskal-Wallis ANOVAs of ranks Results similar to the ones observed when a biofilm was
when equal-variance tests failed. Pairwise multiple compar- used as inducer were obtained when larvae were stimulated
mM
isons were tested using the Student-Newman-Keuls test or to metamorphose with a 3-h pulse of 10 CsCl in the
Dunnett's method when all treatments were compared to presence of emetine (Fig. 8). The proportion of larvae that
each other, or Bonferroni's method when treatments were metamorphosed was similar in the presence and absence of
compared to a control. All statistical tests were conducted 1 iJiM emetine, and the proportions that metamorphosed in
with the aid of SigmaStat software (SPSS Inc.). these treatments were similar to those obtained in response
to a bacterial biofilm.
Results
Labeling RNA andproteins in vivo
Effects ofthe transcription inhibitor DRB on
metamorphosis Incorporation of [5,6 3H]uridine into RNA by competent
larvae of H. elegans in the presence of DRB varied with
When competent larvae of Hydroides elegans were in- concentration and period of exposure. In comparison to
duced to metamorphose with a bacterial biofilm or a 3-h incorporation without an RNA-synthesis inhibitor, [5.6
pulse of CsCl in the presence of DRB. they completed H]uridine incorporation was reduced up to 80% after an
metamorphosis of the larval body after 3-4 h but did not incubation period of2.25 h in 70 /u.M DRB (Fig. 9A). Even
develop branchial radioles (Fig. 1 ). The minimum effective with the lowest concentration of DRB tested (10 ;uM),
concentration of DRB to prevent branchial radicle forma- incorporation of [5,6 3H]uridine was reduced by 60% (Fig.
tion in all the larvae tested with either inducer was 10 ^M 9A).
(Figs. 2, 3; Bonferroni test, P < 0.05). Lower concentra- When larvae were exposed to 10 fj.M DRB for 24 h
tions ofDRB tested eitherhad noeffect (0.1 ;u,M, Figs. 2. 3) before addition of [5,6 3H]uridine, incorporation of [5.6
or showed only a slight, but significant (Bonferroni test, 'Hjuridine was reduced up to 88% in comparison to that in
P < 0.05), reduction in the proportion of larvae that com- larvae incubated in the absence of DRB (Fig. 9B). A sub-
pleted formation of branchial radioles (1 juA/, Fig. 2). stantial decrease in incorporation of [5.6 ^Hjuridine, up to
Preincubation of competent larvae of H. elegans in 10 42%, was evident after the first 3 h of exposure to DRB
H.MDRB forupto9 h before additionofan inducer(biofilm (Fig. 9B). Spontaneous metamorphosis was 3.2% and6% in
orCsCl) did not inhibit metamorphosis (Figs. 4, 5). In fact, the two experiments in control larvae kept in conditions
larvae responded significantly faster (Student-Newman- equivalent to those of larvae incubated in the presence of
Keuls test. P < 0.05)tobiofilminthe presence ofDRB than DRB.
in its absence (Fig. 4A). a difference that was reduced after Synthesisofnew proteins in larvaeofH. elegansexposed
14 h of incubation when the experiment was stopped (Fig. to emetine was dependent on the concentration and length
4B). of exposure to the inhibitor. When larvae were exposed to
Preincubation in 10 p.M DRB for up to 9 h did not stop different concentrations of emetine for 4 h, a decrease in
the larvae from metamorphosing in response to a pulse of ^S-methionine incorporation was observed. A reduction of
CsCl, but a significant reduction in the percentage that 41% in incorporation of 35S-methionine into proteins
118 E. CARPIZO-ITUARTE AND M.G. HADFIELD
R
I"
iM^-'iP
C/
Figure1. MetamorphosisinHydroideselegansintheabsenceorpresenceofaninhibitorofmacromolecular
synthesis. (A) A worm newly metamorphosed in response to a bacteria] biotilm. (B) A worm in which
metamorphosiswasinducedin thepresenceofthetranslation inhibitoremetine. (C)Theposteriorsectionofthe
tube ofthe worm shown in B. Newly metamorphosed wormsexposed to induction cues in the presenceofthe
trancription inhibitor DRB have the morphology shown in B. br. branchial radicles; c,collar, pt. primary tube;
t, secondary calcareous tube. Scale bar = 20 fj.m for all micrographs.
occurred with exposure to 250 nM emetine, and a reduction mum reduction of81% in 35S-methionine incorporation was
of up to 87% occurred in response to 1 jj.M emetine (Fig. detected after 6 h ofexposure to 1 ^M emetine (Fig. 10B).
10A). Exposure of larvae to 1 /j.M emetine for 1 h was An increase in the length ofexposure oflarvae to 250 nM
sufficient to reduce incorporation of 35S-methionine into emetine resulted in lower levels of incorporation of 35S-
newly synthesized protein by 79%. An increase in the length methionine. Periods ofexposure to the inhibitorof6and 9 h
of exposure to 1 juA/ emetine did not substantially reduce reduced '^S-methionine incorporation by 68% and 74%
the incorporation of the radiolabeled umino acid. A maxi- respectively, in comparison to controls where no emetine
METAMORPHOSIS AND GENE EXPRESSION 119
ox
120 E. CARPIZO-ITUARTE AND M.G. HADFIELD
METAMORPHOSIS AND GENE EXPRESSION 121
100 cles, is dependent on transcription, because a broad spec-
trum ofnew tissues including nerves, muscles, and secre-
.3oac5 80 ntoerwy eppriottehienlsi.umExpmeursitmenbetsgeonnertahteedh,yderacohzoraenquHiyrdirnagctmianniya
echinata revealed that morphogenesis ofthe adult polypcan
60
fe be influenced by pulse applications of a-amanitin or
mRNA
cordycepin. These compounds, which block tran-
| 40 scription at different levels, inhibit the formation of tenta-
cles and stolons in H. ec/iinata when applied during late
^ 20 gastrulation or 3 h after induction ofmetamorphosis (Eiben,
1982).
When larvae ofHyilroides elegans were induced to meta-
Cs+emetine Cs emetine BIO FSW
A.
Treatment
Figure 8. Effects ofthe translation inhibitor emetine on induction ot 1.4e+6
m\eMtaemmoertpihnoesiisn fiinltHeyrdednrsiedaewsateelreg(aFnsS.WlLatrhvaatealwseoreconitnuciunbtaetded1f0orm3MhCisnCl1 1.2e+6
toinduce metamorphosis. Solutionswerereplacedwith FSWafter3h.and '5 l.Oe+6
thelarvaewereallowedtocompletemetamorphosisforanother 16h. Bars
indicate percentages oflamrvMae that metamorphosed I SE (;i = 4 repli- Q. 8-0e+5
cates/treatment). Cs, 10 CsCl/3 h: emetine. 1 /xM emetine; BIO, 6i1
substratumcoatedwithamarinebiofilm;FSW,seawaterfilteredthrougha 6.0e+5
S
0.22-ju.A/ filter. OQ. 4.0e+5
2.0c+5
of DRB, transcription and translation were drastically re-
duced without inhibiting induction of metamorphosis in 0.0
competent larvae of P. sibogae. Further experiments II 10 30 50 70
showed that activation of the receptor for the metamorphie DRB(|aM)
signal is not affected by reduction in transcription, but new
B.
transcription appears to be necessary for the final, elonga-
tion phase ofmetamorphosis in P. sibogae (Del Carmen and
Hadfield. 2000).
The observation that metamorphosis in H. elegans
progresses in the presence of DRB, together with the dem-
DRB RNA
onstration that effectively inhibits synthesis, 4e+6
indicates that the initiation and early phases of metamor-
phosis in this species are independent ofnew RNA synthe- o 3e-f6 -
sis. However, the fact that we were neverable tocompletely EL
inhibit RNA synthesis leaves open the possibility that syn- SI 2e+6
thesis of some mRNAs was resistant to the action of DRB, E
and thus that new transcripts may play an essential role QO.
le+6
during metamorphosis for the first hours after induction.
Transcriptional resistance to DRB in HeLa cells was re-
036
ported by Zandomeni el al. (1982).
12 24
The observation that development in the presence of
DRB stops at the point when branchial radicle development Incubation (h)
begins indicates that new transcripts are necessary for the Figure 9. Incorporation of [5-6 'H|uridine into newly synthezised
growth of the radicles and that these transcripts are among RNA by larvae ofHydroides elegans. In (A), larvae were incubated for
the more than 80% of RNA syntheses inhibited by DRB. 2.15 h in the presenceofthedifferentconcentrationsofDRB indicatedon
Even a 40% reduction in synthesis of RNA, measured by the .v axis. In (B), larvae were incubated in 10 p.M DRB in FSW forthe
incorporation of [5,6 3H]uridine, was sufficient to prevent dliafrfveareenwterpeerailoldsowoefdetxopionscuorrepoirnadtieca[t5e-d6o3nH]tuhrei.dvianxeisd.uIrninbgotthhe(lAa)sta4n5dm(iBn)..
the formation of branchial radicles. It is not surprising that Points are means of uridine incorporation 1 SE (n = 2 replicates/
active growth of new structures, such as the branchial radi- treatment).
122 E. CARPIZO-ITUARTE AND M.G. HADFIELD
A. Larvae of the archaeogastropod Haliotis ntfescens
(Fenteany and Morse, 1993) and the nudibranch Pliestilla
80x10' sibogae (B. J. McCauley, 1997; Del Carmen and Hadfield,
1999) initiate metamorphosis in the presence of translation
inhibitors. When competent larvae of H. nifescens were
60.x103 - induced to metamorphose in the presence ofconcentrations
ofemetine or anisomycin sufficient to block protein synthe-
D,
40x1 3 sis, settlement and plantigrade attachment still occurred,
2 indicating that these premetamorphic processes do not
a. require de novo protein synthesis (Fenteany and Morse,
20.x101 - 1993). The authors suggest that the induction of settlement
A.
250 500 1000
Concentration ofemetine(nM) 140\10'
B.
'3 lOOxlO3
p
XO.xlO3
.
WO
.9- 60x1 3
SOxlO E
40.x10'
5. 60x1O3 - noemetine 20x10'
emetine 1 uMolar
E 40x10'
-na. 9h 6h noemetine
20x10-' - O O Periodofincubation
inemetine
Treatment
B.
1 4 6
Period ofincubation (h)
Figure 111. Incorporation of 33S-methionine into newly synthesized
proteins by larvae ofHyJmitles elegans in the presence ofthe translation
inhibitoremetine. In (A), larvae were incubated in varying concentrations
of emetine, shown on the _v axis, for 4 h with incorporation of "S-
methionine duringthe last 1.5 h. Pointsare means ofmethionine incorpo-
ration 1 SE (H = 2 replicates/treatment). In (B). larvae were incubated
in 1 fj.M emetine for the intervals indicated on the .v axis and allowed to
incorporate "S-methionine for an additional 45 min. Points are mean
methionine incorporation 1 SE.
morphose with either a bacterial biotilm or a pulse ofCsCl incubationinemetine
in the presence of the translation inhibitor emetine, just as beforeinductionwithCsCl
Treatment
with DRB, they completed metamorphosis and stopped
developing before the outgrowth of the branchial radioles. Figure II. Incorporation of 35S-methionine into newly synthesized
nNoeirthaenretxhteenhdigehdesptrecionnccuebnattriaotniopneroifodemeintienmeetuisneed s(t1ojpupMe)d ctpioroonntceeiinnntsr2ab5ty0iolnnasMrviaenemdeiotcfiatnHeeyd.droIonnid(teAh)se,evllaeragvxaainesswaaenfrtdeeraidlinlfcofuweberaedtnettdopeiinrniecoomdresptooirfnaetienactu3b5taSh--e
initiation of mctamorphic morphogenesis in response to a methionine during the last 2 h. Points are means of methionine incorpo-
bacterial biofilm or cesium as inducers. These results are ration 1 SE(n = 2replicates/treatment,exceptfor6h.whichrepresents
consistent with the hypothesis that initiation of metamor- 1 sample). In (B). larvae were first incubated in 1 fj.Memetine in filtered
phosis in H. elegim* is also highly independent ofsynthesis seawater(FSW)beforebeinginducedtometamorphosewitha3-hpulseof
of new proteins, a result that is concordant with previous CafstCelr t(h1e0 CmsAC/)l apnudlseallaonwdinignltahrevaeprteoseinncceorpoofraetmeetmienteh.ioPnoiinnetsfoarre45memainn
studies of other species, especially certain molluscs (Fen- methionine incorporation I SE(H = 4 replicates/treatment for2, 1. and
teany and Morse, 1993; Del Carmen and Hadtield. 1999). h, and 2 replicates/treatment forCsCl and not induced (no CsCl)).
METAMORPHOSIS AND GENE EXPRESSION 123
and plantigrade attachment in the presence of protein-syn- metamorphosis and begun earlyjuvenile development (B. J.
thesis inhibitors is consistent with the notion that these McCauley, 1997). In embryos of Hydractinia echinuta. the
behavioral responses are controlled by chemosensory mech- index of cell proliferation measured by the BrdU method
anisms mediated by the nervous system. Similar results decreased to 4% by 78 h after fertilization. In larvae of H.
were reported forthe larvae ofP. sibogae: experiments with echinata kept at low temperatures, the cell proliferation
emetine showed that metamorphosis can be initiated in the index went to zero after60 to 70 days without affecting the
presence of the translation inhibitor and that development ability of the larvae to undergo metamorphosis (Plickert el
stopsjust before final elongation ofthe body (B. J. McCau- ul, 1988). Preliminary observations on Hydroides elegans
ley, 1997). using PCNA indicated that cell proliferation decreases
It is particularly interesting that protein synthesis in lar- sharply when larvae become competent and increases nota-
vae of Hvdroides elegans was reduced even further when bly in the developing branchial radioles of newly settled
CsCl was added to induce metamorphosis (Fig. 11B). juveniles (Carpizo-Ituarte. unpubl. results).
Kroiher el at. (1991) also reported decreased incorporation Metamorphosis in most well-studied insects and amphib-
of amino acids into proteins when larvae of the hydrozoan ians is a relatively slow process wherein hormonal tran-
H\dractinia echinata were exposed to CsCl, attributing it to scription factors mediate essential cascades of "de novo"
a reduction in the Na+ concentration of the medium when transcription and translation to regulate metamorphic mor-
CsCl is added. Transport of amino acids across the cell phogenesis (Gilbert era!., 1996;Tata. 1996, 1999; Shimizu-
membrane is known to be a function of a Na+-transport in Nishikawael /., 2002). In contrast, the experimental results
marine invertebrates (Stephens, 1988). obtained with the polychaete Hydroides elegans support a
Inhibition of development of the branchial radioles by generalization noted by Hadfield et al. (2001): for most
emetine at concentrations at which protein synthesis was well-studied marine invertebrates, the competent larva car-
reduced by only 40% is consistent with the data obtained ries within it a well-formedjuvenile body, which metamor-
with DRB. Thus, unsurprisingly, both new mRNA tran- phosis need only liberate from larval structures. In this case,
scripts and their translation products are necessary for pro- metamorphosis should require little or no de novo transcrip-
duction of the branchial radioles. tion or translation, processes that will be necessitated when
Taken together, the data on RNA and protein synthesis in juvenile growth begins. This may be the result ofadaptation
metamorphosing Hvdroides elegans strongly support the to undergo rapid metamorphosis and make an extremely
conclusion that metamorphosis and juvenile development vulnerable period that of a planktonically adapted larva
occur in two phases one independent of new gene tran- residing on the benthos as brief as possible (Hadfield.
scription and translation, and a second in which these two 2000).
molecular processes are necessary for development and The more precisely we define the molecular events of
growth to continue. During the first phase, larvae respond metamorphosis in Hydroides elegans and other marine in-
rapidly to the bacterial inducer, change their swimming vertebrates, the betterweare able to makecomparisons with
behavior, attach, secrete primary and secondary tubes, lose model systems among insects and amphibians. So far. the
the prototrochcilia, differentiate the collar, andelongate the capacity for metamorphosis to proceed almost indepen-
caudal region (Carpizo-Ituarte and Hadfield, 1998). During dently ofnew geneexpression in many marine invertebrates
the second phase, the metamorphosed worm develops points toward different mechanisms to initiate postlarval
branchial radioles, a process that requires new RNA and development. To what extent these mechanisms are differ-
protein synthesis. Results reported for larvae of other ma- ent from those known in insects and amphibians awaits
rine invertebrates reinforce the idea that metamorphic com- further investigation. Knowledge of the signal-transduction
petence in many marine invertebrate larvae is a develop- pathways that are activated during induction of metamor-
mental stage primed to metamorphose and in which the phosis and how the initial triggering event orchestrates the
activation and near completion of metamorphosis requires genetic machinery to turn a larva into ajuvenile in a wide
neither de novo synthesis of mRNA nor proteins (Hadfield, variety of marine invertebrates will help us to understand
2000). how the widespread phenomenon of metamorphosis in ma-
Since larvae ofH. elegans survive through metamorpho- rine invertebrates evolved.
sis with very reduced synthesis ofnew mRNAs or proteins,
cell proliferation, which requires both processes, probably
does not play an essential role during metamorphosis. Pre- Acknowledgments
vious studies in other invertebrate species are consistent
with this hypothesis. In Phestilla sibogae, labeling ofdivid- Contributions of colleagues in the Hadfield lab at the
ingcells with an antibodyagainstaproliferatingcell nuclear University of Hawaii's Kewalo Marine Laboratory were
antigen (PCNA) decreases when larvae reach competency many, andwe are grateful forall ofthem. This research was
and is detected again only after the larvae have completed supported by ONR grants NOOO14-95-1015 and N00014-