Table Of ContentReference: Biol. Bull. 201: 394-404. (December 2001)
NO/cGMP and HSP90
Signaling Activity Represses
Metamorphosis in the Sea Urchin Lytechinus pictus
CORY D. BISHOP AND BRUCE P. BRANDHORST*
Department ofMolecular Biology ami Biochemistry, Simon Fraser University;
Burnaby, British Columbia V5A 1S6. Canada
Abstract. Nitric oxide (NO) signaling repressively regu- Introduction
lates metamorphosis in two solitary ascidians and a gastro-
pod. We present evidence for a similarrole in the sea urchin Many species of sea urchin undergo maximal indirect
Lytechinus pictus. NO commonly signals via soluble gua- development (Davidson, 1991). Embryonic development
snyolmyelcmyacmlamsael(isaGnC)c.elNlist,riicncolxuiddiengsynnetuhraosnes,(NdOeSp)enadcstiovintythien gtheanterbaetaerss anobirlaetseermalbllyanscyemmteotraincaaldulfteesdeiangurcphliunt.eusAftlearrvaa
molecular chaperone heat shock protein 90 (HSP90); this openritohdeolfefgtrsoiwdtehoifn tthhee pllaarvnak,tonw,ithainnadtuhlet vreusdtiibmuelnet. fOonrcmes
may be so in echinoid larvae as well. Pluteus larvae con-
competence is reached, and in response to appropriate cues,
tL-aionrinDg-njiutvreonairlgeirniundei-mmeentthsylw-eersetetrr(eaLt-edNwAiMtEh eaintdheor-rNaAdiMcEic)o,l tmhaetipolnutienutso alaprevnatasreatdtileasllaynsdymumnedterrigcoaelsjuavernaidlicealseatruarncshfionr.-
or IH-[l,2,4]oxadiazolo[4,3-a]quinoxalin-l-one (ODQ). in- The initial events of this transformation, as described for
hibitors of HSP90, NOS, and sGC. respectively. In all Lytechinus pictus, are completed within an hour (Cameron
qinusetnacncyeso,f mdertuagmotrrpehaotmseinst. SsiNgAniPf,icaantNlyOidnocnroera,sesduptphreesfsreed- aBrnidefHliy,netghaerdlanrevra,l a1r97m4s,b1e9n7d8;awPaeyarsferoamndthCeavmeestriobnu,le,19f9r1o)m.
the inductive properties of L-NAME and biofilm, a natural which the tube feet ofthe rudiment extend, allowing podial
inducer of metamorphosis. NADPH diaphorase histochem- attachment to the substratum. The rudiment becomes ex-
istry indicated NOS activity in cells in the lower lip of the posed to the exterior and then everts. The larval epithelium,
larval mouth, the preoral hood, the gut, and in the tube feet including that of the arms, contracts and collapses onto the
of the echinus rudiment. Histochemical staining coincided aboral surface of the juvenile, involving drastic changes in
with NOS immunostaining. Microsurgical removal of the cell shape. The vestibular epithelium extends to cover the
oral hood or the pre-oral hood did not induce metamorpho- aboral surface, forming the epithelium of the juvenile and
sis, but larvae lacking these structures retained the capacity enclosing degenerating larval cells. Extensive remodeling
to metamorphose in response to ODQ. We propose that the ofinternal structures such as the digestive tract continue for
production of NO repressively regulates the initiation of several days as thejuvenile begins the reproductive stage of
metamorphosis and that a sensory response to environmen- its life cycle as a benthic feeder.
tal cues reduces the production of NO, and consequently Competent echinoid larvae will settle and initiate meta-
cGMP, to initiate metamorphosis. morphosis if provided with a hard surface covered with an
appropriate organic film, particularly a microbial film (re-
viewed by Strathmann. 1987; Pearse and Cameron. 1991;
Received 17 November 2000; accepted 7 September 2001. also see Discussion). In the absence of such cues, some
*Towhomcorrespondenceshouldbeaddressed. Email:[email protected] species delay metamorphosis (Caldwell, 1972; Cameron
Abbreviations:D-NAME,D-nitroarginine-methyl-ester;GA,geldanamy- and Hinegardner. 1974). When placed in clean glass or
cscLioy-nln;;NthAGsaMGBsCEeD;,,,OsLgo-DlenQuilb,tdlraeonlaHgar-umg[aiylnnc,iyi2nlne,-4cbm]yieocntxlhdaayidslnei-g;aezsSdotolNeomrA[a;P4i.,Nn3O;-S,a-H]nnSqiiPuttirr9ino0cos.xooa-xhNlie-idaanetc-;elsNt-hyoOolnSpce,ek;ninRpicrDtior,ltilecraiaomndxiiin9cde0i.e;- ptmlheaetsatiimncodrudcpithsiohosense.owfiTmthheitsafrmaeolsrlhpohwsosesaiwesax.tpeerTr,himeLe.nmtepaiclchtiaiinsnvieslsamtrivbgaayetiwroahnrieclohyt
394
NO REPRESSES ECHINOID METAMORPHOSIS 395
external cues are detected and transduced into the initiation pictus, that it is downstream from a natural inductive cue,
of metamorphosis remains poorly understood, but appar- and that this regulation may be dependent upon HSP90
ently involves a neurosensory response. Further, it is not function. NOS was detected in both larval and juvenile
clearwhether larval orjuvenile sensory perception (orboth) organs; such organs may be involved in sensing or trans-
is responsible for transducing external signals under natural ducing the response to natural inductive cues.
conditions. Evidence for the involvement of neural re-
sponses from both the larva and the juvenile has been Materials and Methods
reported. Electrical stimulation of the oral ganglion or the
apical neuropile of Dendraster excentricus larvae induced Obtaining and culturing lan-ae
metamorphosis (Burke. 1983). In contrast, observation of Specimens of Lytechinus pictus were purchased from
settling behaviors and the prevention of settling (and con- Marinus (Long Beach, CA) and held in recirculating sea-
sequently of metamorphosis) in the presence of inducers water tanks. Eggs were induced to shed by intracoelomic
clearly demonstrates a role for thejuvenile sensory appara- injection of 0.5 mol KC1, then washed and fertilized. Em-
tus in L. pictus (Cameron and Hinegardner. 1974; Burke. bryos in Millipore (0.45 /im) filtered seawater (MFSW) at
1980: ourobservations). Investigations ofthe molecularand 16 Ccontaining 50 /ng/ml penicillin and streptomycin were
anatomical basis of signaling events that regulate echinoid continuously stirred at 20 or 60 rpm using plastic paddles.
metamorphosis can thus be placed in this historical context. After hatching, the embryos were collected by filtration on
Nitric oxide synthase (NOS) catalyzes the conversion of 93-;um-mesh Nitex and resuspended in fresh MFSW; this
oL-xairdgein(iNnOe)t.o LN-cOiStruellxipnreeswistihonthaenpdroNdOuctfiuonnctoifotnhehagvasenbietreinc wvaalshgirnogwtphr,ocaenddurtehewacosncreenpteraatteidonfroefquleanrtvlayetwhraosugghroaudtuallalry-
documented in both nervous and non-nervous tissues alike reduced to I/ml or less. Algae were obtained from the
across a range of eukaryotic organisms, indicating their Northeast Pacific Culture Collection (NEPCC) at the Uni-
antiquity and importance in regulating many cellular pro- versity of British Columbia. Vancouver. Either a mix of
cesses (Schulte elai. 1998; Cueto elal, 1996: Kuzin elal. Pyrenomonas salina (NEPCC strain 076; CenterforCulture
1996; Czar et al.. 1997). That NO is diffusible through ofMarine Phytoplankton (CCMP) strain 3C)andDunaliella
biological membranes suggests that it may have served as a tertiolecta (NEPCC strain 001: CCMP strain 1320) or only
primitive signaling system between cells before more elab- the latterwere fed to plutei every 2-3 days in quantities such
orate mechanisms of cell adhesion and receptor-based sig- that most algae had been ingested as of the next feeding.
naling evolved. In mammalian cells, NOS activity in vivo
requires interaction with heat shock protein 90 (HSP90)
(Garcia-Cardena et al.. 1998: Bender et al., 1999). We Pharmacological inhibition
recently reported that metamorphosis of two species of L-NAME (L-nitroarginine-methyl-ester) and its enantio-
ascidian tadpole larvae is induced by drugs that inhibit the mero-NAME, radicicol (RD), and ODQ (lH-[l,2.4]oxadia-
activity of the protein chaperone HSP90, NOS. or soluble zolo[4,3-a]quinoxalin-l-one) were obtained from Sigma
guanylyl cyclase (sGC) (Bishop etal.. 2001). Among larval Chemical Corp. (St. Louis. MO). Because there is variation
tissues, NOS activity is concentrated in the tail muscle cells in the rate of development of the juvenile rudiment, indi-
of the ascidian tadpole Cnemidocarpa finmarkiensis. Re- vidual L. pictus plutei were selected by examination under
moval of the tail stimulates metamorphosis of the head, a stereomicroscope and transferred to wells of24-well plas-
consistent with there being a signal, probably NO. from the tic culture dishes (Flow Labs, McLean. VA). Larvae were
tail that represses metamorphosis. NOS produces NO. a selected for experiments based on the presence of a large,
gaseous signaling molecule whose most common effectoris pigmented rudiment having well developed spines and tube
sGC (Garthwaite et al., 1995; Salter et al.. 1996: Hebeiss feet. Each well contained about 10 larvae in 2 ml MFSW or
and Kilbinger. 1998). Thus, inhibition of NOS often results experimental solutions in MFSW. To quadruplicate sets of
in a corresponding reduction of cGMP (McDonald and these selected larvae were added L-NAME, o-NAME. RD.
MFSW
Murad. 1996. for review). Metamorphosis of the marine ODQ. or in 1- or 2-ml final volumes. Metamorpho-
gastropod llyanassa obsolete! was also reported to be in- sis was monitored using astereomicroscope: itwas scored if
duced by inhibition of NOS activity (Froggett and Leise, the larval epithelium had collapsed on top of an everted
1999). indicating that NO may repress metamorphosis in a juvenile. The activity of tube feet was used as an indicator
variety of animals. of larval vitality. The concentrations of L-NAME. RD, and
To further test the idea that NO-mediated repression of ODQ used in the experiments reported here were chosen
metamorphosis occurs widely within the bilaterian clade. because they elicited a metamorphic response in ascidian
we used the sea urchin L. pictus. We report that NO/cGMP larvae (Bishop et al.. 2001). L-NAME and D-NAME were
M
signaling is an important regulatoroftheevents surrounding prepared as 1 stocks in water and diluted to a final
the transition of form from the larva to the juvenile in L concentration of 1-10 mM with MFSW. ODQ was prepared
396 C. D. BISHOP AND B. P. BRANDHORST
as a 100 mM stock in DMSO and diluted into MFSW to 50 Sectioned larvae were examined using an Olympus Vanox
ju,M. RD was prepared as a 5 mM stock in DMSO and microscope, and images were captured using a Sony DXC-
diluted into MFSW to 5/iAf. SNAP (S-nitroso-N- 950 3CCD camera.
mM
acetylpenicillamine) was prepared as a 100 stock in Universal anti-NOS (Affinity Bioreagents, Golden, CO)
DMSO and diluted to 0.1 mM in MFSW. For RD, ODQ. polyclonal rabbit antibody was used todetect NOS in grow-
and SNAP treatments, experimental and control wells all ing and mature larvae. Larvae were fixed for 2 h at room
contained a final concentration of 0.1% DMSO; this con- temperature in 4% formaldehyde (prepared as outlined
centration of DMSO did not have any inductive properties. above). Fixed larvae were blocked with PB saline contain-
Unless significant metamorphosis was observed sooner, ex- ing 5% bovine serum albumin and 0.1% Triton-X-100 and
periments were scored at 24,48, and sometimes 72 h. A low then incubated in 1:100 anti-NOS overnight at 4 C. Larvae
frequency of spontaneous metamorphosis was observed for were incubated in secondary antibody (goat anti-rabbit-
larvae placed in plastic dishes; this response tends to occur Alexa 568, Molecular Probes, Eugene OR) for 2 h at room
shortly after the assessment ofa larvae and its transfer into temperature and then rinsed, mounted, and viewed on a
a well. If such a response was observed before the addition Zeiss LSM 410 confocal microscope. Images were pro-
of drugs, juveniles were removed. cessed using Adobe Photoshop 5.5 or 6.0.
To create a natural inductive cue, glass Syracuse dishes
were submerged for several days in recirculating tanks Results
containing naMtuFrSalWseawater. Ten larvae were exposed to the Inhibitors ofnitric oxide synthase, guanylyl cyclase, and
smubMstrSaNtAePi.n Resultsesihthoewrninartehefprroemseancseinoglreaebxspeenrciemeonft0..1 HSP90 induce metamorphosis
Microsurgical removal of oral hoods and pre-oral hoods Treatment of larvae with the NOS inhibitor L-NAME in-
was accomplished using a fine-edged stainless steel pin ducedasignificant increase in the frequencyofmetamorphosis
(Fine Science Tools, Vancouver, BC) fused to a glass pi- in comparison with larvae treated with seawater (Fig. 1A) or
pette. Dissected oral and pre-oral hoods retained their ca- D-NAME (Fig. !B). In a time-course experiment, the fre-
pacity to swim. Quadruplicate sets of5 larvae or hoods per quency ofmetamorphosis was scored every hour for6 h (Fig.
well (a total of 20 operations) were used for each experi- IB). Some larvae responded rapidly (within 2 h) toeitherL- or
ment. D-NAMEbutothers requiredseveral hours. Because D-NAME
All experiments were tested for statistical significance by is used as an inactive enantiomer of L-NAME, the observed
performing a one-tailed Student's / test with the assumption inductive property of D-NAME was unexpected and substan-
of hoinoscedastic variance. In all graphs (made using Mi- tial,althoughlesssothanforL-NAME(Fig. 1).Toconfirmthat
crosoft Excel 97), the asterisks denote statistical signifi- the inductive properties ofL-NAME or D-NAME were due to
cance; P values are provided in the figure legends. Specific a reduction in NO levels, larvae were co-incubated with L-
statistical comparisons are described in the figure legends. NAME or D-NAME and the NO donor SNAP. At a 10-fold
lower concentration than i.-NAME or D-NAME, SNAP com-
NADPHd histochemistry and NOS immunohistochemistry pletely suppressed their inductive properties (Fig. IA). In a
variation of that experiment. SNAP was added 4 h after L-
The NADPH diaphorase staining protocol described by NAME had been added. This also resulted in the suppression
Weinherg et nl. ( 1996) was used with modifications. Larvae ofmetamorphosis (Fig. 2).
were fixed in 2% glutaraldehyde and 1% formaldehyde in Marine biotilms consisting of bacteria and other micro-
sodium phosphate bufferfor 1 h at room temperature. Form- organisms have previously been shown to induce metamor-
aldehyde was freshly prepared by dissolving paraformalde- phosis in L. pictiis larvae (Cameron and Hindgardner,
hyde (EM grade, Ted Pella, CA) in MFSW, adjusting the 1974). This is considered to be a cue that approximates that
pH to 7.4, and then diluting in PB to 1%. After rinsing with of a natural benthic environment. We exposed larvae to a
PB, fixed larvae were incubated in 0.4 mg/ml nitrotetrazo- biofilm grown in recirculating tanks (containing natural
lium blue substrate with 2 mg/ml NADPH from 2 to 16 h at seawater from local sources) in the presence or absence of
37 C. As a negative control, specimens were incubated in SNAP to test whether NO signaling was downstream of a
50% ethanol for 2 h and then incubated in nitrotetrazolium sensory pathway that is responsive to a natural cue. SNAP
blue in the absence of NADPH; no staining was observed suppressed the inductive properties of the biofilm in a
under these conditions. Under the fixation conditions used, reversible manner(Fig. 3). SNAP waseffective at suppress-
NOS is the only diaphorase expected tobe active (Weinberg ing metamorphosis among larvae that had been exposed to
c/ /.. 1996). Stained larvae were examined as whole biofilm for several hours, but had not yet metamorphosed
mounts b\ microscopy or were dehydrated in a graded (Fig. 3).
ethanol series, embedded in polyester wax (BDH Labora- Soluble guanylyl cyclase (sGC) is the most common
tory Supplies. Poole, England), and sectioned at 8 /xm. downstream effector of NO signaling (Salter ct ai, 1996;
NO REPRESSES ECHINOID METAMORPHOSIS 397
(A) DOhourspost-SNAP D7hourspost-SNAP 124hourspost-SNAP
1.2-
- 1
|
o 0.8
E
D8 D24
I
E 0.6 I
0.4
O
D
f
0.2H
X
(B)
0.8
398 C. D. BISHOP AND B, P. BRANDHORST
geal lumen. The number of immunoreactive cells in the
lower lip was variable from larva to larva. It is not clear if
this perceived variation was due to variation in the actual
D3 hours number of NOS-positive cells in this region or in the sen-
o D24 hours sitivity of immunostaining. Since the variation was ob-
I '6 48 hours served among larvae in individual immunostaining experi-
OJ ments, the former possibility is more likely. To gain a
0.4 three-dimensional perspective on the organization of NOS-
positive cells in the lower lip of the mouth, serial optical
0.2- sections were captured and projected as three-dimensional
images (Fig. 5L, M). In agreement with histochemically
stained sections, these cells extend processes toward the
s.w. ODQ apical surface ofthe oral epithelium. We also saw a general
correspondence between histochemical and immunohisto-
wfinrederFiqeicuagettunrereceayate4o.dsfiwmgineIittnfhhaiimc0baoi.nrt1top%rhdsoiDsfoifMfesSrHewSOnaPcse9(c0moionnontrritothlsoe)Gr.eCf5driepanqfdMuteuercnRec3D.ym.2eo4to.framam5one0rdtpfa4hmi8ooMshri.OpsAh.DsotQsIeiarsiiT\shakoeesf sciinhtgeemstihocafatlNsisOtteSasinoeifxnpgNreiAsnsDoiPtohnHe.rdtiiaspshueosra(sdeataactniovtitsyhocwonr)r,esipnodnidcatt-o
larvae treated with RD or ODQ compared to controls. (PRD24 0.01; Dendraster excentricus pluteus larvae contain cells that
PRD4S s 0.02: n = 4). ODQ caused a significant increase within 3 h express catecholamines in the lower lip of the mouth; this
(POD(j, 0.0004; n = 4), with no further significant increase. region was thus termed an oral ganglion (Burke, 1983).
Removal of the oral hood (OH), which includes the oral
ganglion, induced metamorphosis (Burke 1983). That ob-
tahnedfirtedqiuden(cFyig.of4)m.etWaemohrapvheosniostomveearstuhreeduntdirreeactteldycwohntertohlesr. servation and the expression of NOS in the oral ganglion
RD reduces the activity of NOS. creelplrsesosfmL.etpiacmtoursphlaorsviase lveida ttohetihrephryopdoutchteisoinsotfhaNtOt.hesTeocetlelsst
NOS activity is present in neurons ofliin'al tissues anil othristhiedepar,e-worealmihcoroodsu(rPgHi)cafllryomremamtouvreedleairtvhaeeratnhde senctoirreedOthHe
tube feet ofthe rudiment
frequency of metamorphosis. This operation did not induce
The NADPH diaphorase histochemical assay was used metamorphosis of L. /rictus after 6 h (not shown), so L-
under conditions specific for vertebrate NOS enzymes. NAME was added to see iflarvae lacking the OH or the PH
Whole larvae were stained and observed; some were then had retained their capacity to undergo metamorphosis. Nei-
fixed and sectioned before examination by microscopy. ther postoperative larvae nor the OH and PH were respon-
Feeding larvae stained for diaphorase activity in the lower sive to i -NAME at concentrations that induced metamor-
lip of the mouth, mid- and hindgut, at the tips of postural phosis in intact control larvae (Fig. 6A). To further test if
arms, and in cells within the lobe between the anterolateral the postoperative larvae and the dissected tissues had re-
arms. These sitesofNADPH diaphorase activity most likely tained the capacity to metamorphose, we added 50 ^M
represent sites of NOS activity. Larvae having large rudi- ODQ after 14 h of incubation in L-NAME. This resulted in
ments resembling those used for the inhibitor assays were a very rapid metamorphic response (Fig. 6A). The OH and
also sectioned and stained. As shown in Figure 5, diapho- PH did not initially undergo epithelial collapse typical of
rase activity was found in a variety of structures. Stained intact metamorphosing larvae, although they did so within
cells were found within the larval gut epithelium (Fig. 24 h (data not shown). Microscopic analysis indicated that
5A-C). The basioepithelial nerve plexus of juvenile tube the OH and PH were not necrotic, but rather they had
feet was intensely stained and appeared to extend processes undergone genuine cellular rearrangements characteristic of
to the outer surfaces of the tube foot (Fig. 5D, E). Stained the epithelium ofmetamorphosing larvae. Therefore, micro-
cells were observed at the tips of larval arms (Fig. 5F| and surgical removal of the OH or the PH did not lead to
in the pre-oral hood (Fig. 5A). Stained cells, often having a metamorphosis of postoperative larvae, and apparently de-
neuronal appearance, were observed in epaulettes (Fig. 5G) creased their capacity to respond to NOS inhibition but not
and the lower lip of the larval mouth (Fig. 5H-J). sGC inhibition. Dissected OH and PH tissues underwent
We stained larvae with anti-NOS antibodies to see rearrangements typical of metamorphosing larvae, but only
whether sites of NADPH activity were coincident with the after a protracted period in drug.
location of NOS. Prominent staining was observed in the
lower lip of the mouth (Fig. 5K) and in some cells situated Discussion
in the pre-oral hood (not shown). Stained cells in the lower Independent pharmacological inhibition ofNOS, HSP90,
lip are roughly symmetrically arranged around the pharyn- and sGC led to a significant increase in the frequency of
NO REPRESSES ECH1NOID METAMORPHOSIS 399
r-
W
'<
\-&*
-*
*&
/:'"'~
/
Figure 5. NOS expression in larvae was analyzed by NADPH diaphorase histochemistry and NOS immuno-
histochemistry. (A)Sectionofa26-daylarvashowingdarkbluestainingintheforeandmid-gut(M)andinacellin
thepre-oralhood(arrowhead).(B)Obliquelongitudinalsectionshowingthearrangementofstainedmid-gutepithelial
cells. (C) Highermagnificationcrosssectionshowingstaininginthebasalportionofcolumnarepithelialcellslining
the mid-gut. The inset showsa low magnification whole-mount view ofastained larval mid-gut. (D) Longitudinal
sectionofatubefootfromajuvenile rudimentcontainedinalarva. Stainedcellsofthebasioepithelial nerveplexus
aretightlyapposedtotheectodermalepithelium.(E)Slightlyobliquetransversesectionofatubefootshowingstained
nerveplexuswithpossibleprojectionstotheoutersurfaceoftheepithelium.(F)Whole-mountstainingofapost-oral
larval armfroma26-day-oldlarva. (G)Sectionofanepauletteshowingstainingincellsatthedistal tip. (H)Frontal
section of the larval mouth showing stained cells. (I) Lateral section of the oral hood and mouth. (J) Higher
magnificationviewoftheoralepitheliumshowingbasal positionofstainedcell bodies.O = outsideandI = inside.
A\on-likeprojectionshavingbulboustermini (arrowhead)extendtotheciliatedapicalsurface. Scalebars:40/urnin
AinsetofC;20jim inB. F. H.andI:8/im inC-E.G.J. (K-M)NOS immunostaining. (K)Mouthofalarva.Only
cellsinthelowerlipareimmunoreactive.(L)3-DprojectionofNOS-positiveoralcells. (M)Sameprojectionas(L)
butrotatedtoshow cell polarity. Apical sideoftheoral epithelium (outsideoflarva) istothe left. Sharpline atthe
rightdemarcatestheendoftheconfocal stack. Scale barsare not available forK-M.
metamorphosis of L. pictus larvae. These results are con- guanylyl cyclase to produce cGMP that inhibits metamor-
sistent with our model for the signaling system that regu- phosis (Bishop et ai, 2001 ). This proposal is also based, in
NO
lates the initiation of metamorphosis in ascidians: cells part, on evidence that represses metamorphosis in a
having NOS activity (probably dependent upon interaction gastropod (Froggett and Leise. 1999; Leise et al. 2001).
NO
with HSP90) release that stimulates the activity of Natural inductive cues may be operating via receptor-based
400 C. D. BISHOP AND B. P. BRANDHORST
(A) (B)
Dctrls Dlarva-OH llarvae-PH
L-NAME ODQ
Figure 6. (A) Removal of the entire oral hood or the pre-oral hood does not induce metamorphosis and
diminishes the response to L-NAME. but not ODQ. L-NAME was added to postoperative larvae (POL) and the
dissectedfragments6haftersurgery.After 14hinl.-NAME,themetamorphicresponsewassignificantlyreducedin
comparisonwithintactcontrollarvaeinL-NAME.Asterisksindicatesignificantdifferencesbetweenthefrequencyof
amdedteadmotropbhootshisPOofLinatnadctdaisnsdecptoesdtofpreargamteinvtesl,aarvnadet(hPeOfLr)eq(uPeLnjcrly4<o,fmm,neut..aOmHor<ph0o.s0i2;s^wuar>s-asccomrmeud.,PAfHt<er02.0h.02O).DOQDrQapiwdalsy
induced metamorphosis. Asterisks indicate a significant difference between POL before and after ODQ treatment
(PL ^ ^ ^ ^<0.006:/>lanlon,lnu,thepreol_l|holx)<0.0003).(B)Aschematicdrawingofai,yrer/'Hip/fn larva
indicating the position ofNOS-positive cells found in the lower lip ofthe mouth (arrow) and the point at which
portionsofthe larvae were surgically removed. Dashed lines indicate the plane ofthecuts. NOS-positiveoralcells
areremoved along withtheentireoral but not with the pre-oral hood. Arms are virtuallyabsent in well-fed, stirred
larvae. PH = pre-oral hood; OH = oral hood; M = mouth; G = gut; E = epaulettes.
sensory perception that is upstream ofNO/cGMP signaling. describe a specific biological state in any marine inverte-
A low-molecular-weight, water-soluble compound isolated brate having planktotrophic larvae and benthic adults. Lar-
from biofilm has inductive properties in L pictus (Cameron vae with no rudiments or abnormal rudiments do not re-
and Hinegardner. 1974). spond to inducers of metamorphosis (Cameron and
Frequently, some ofthe treated larvae did not respond by Hinegardner, 1978; CDB, unpubl. obs.), so competence in
initiating metamorphosis, even after longer incubations (72 urchins represents a discrete change in the physiological
h in some cases). In similarexperiments, we have found that state of the larva that is related to the growth and develop-
a fraction ofselected larvae do not respond to dishes coated ment of the juvenile. Competence is a phenomenon that
with microbial film. The fraction ofresistant larvae in such requires further investigation and should be considered in
experiments was variable (not shown), but similar to the all studies on the regulation of metamorphosis. The acqui-
fraction resistant to potent drug treatments such as ODQ sition of competence coincides with the initiation of rneta-
(Fig. 4). Variation in response to inducers. whether natural morphosis in some animals, but not in others (Birkeland el
or otherwise, may represent variation in sensitivity of sen- nl.. 1971; Degnan el til., 1997; Bishop el ai. 2001). This
sory perception, levels of NO repression, or response to a indicates that the fitness consequences associated with the
reduction ofNO signaling (oracombination thereof) among timing of, and substrate choice during, settling and meta-
larvae ofa clutch and among clutches. Perhaps the resistant morphosis vary. What other signaling systems may be con-
larvae had not achieved competence to undergo metamor- tributing to the timing events surrounding life cycle trans-
phosis, despite the morphological similarity of their rudi- formations? Studies on thyroxine in echinoids suggest its
ments to those that did metamorphose. In fact, in some involvement in the evolutionary loss of larval feeding. The
cases, larvae containing less well developed rudiments were addition of exogenous thyroxine leads to a reduction of
responsive to drugs, whereas those with large, highly pig- larval structures and the time to metamorphosis in D. ex-
mented rudiments were not. It is clearthat competence does centricus (}. Hodin, Friday Harbor Laboratories, and A.
not strictly correspond to the presence of a fully formed Heyland, University of Florida; pers. comm.l. It will be
rudiment within the larva. Assessing competence is prob- interesting to know if and how NO/cGMP and hormonal
lematic in that one does not know whether a lack of re- signals interact to regulate the timing of life cycle transfor-
sponse is due to lack ofcompetence or failure to respond to mations in echinoids.
an inductive cue. Between different clutches, we have observed a striking
To our knowledge, the concept of competence does not difference in the response of larvae to NOS inhibition.
NO REPRESSES ECHINOID METAMORPHOSIS 401
Increased sensitivity is manifested as a more rapid response However, a direct demonstration of interaction between
given that identical concentrations of L-NAME and HSP90 and NOS in urchins is warranted.
o-NAME were used. We cannot rule out other variations in All of the biochemical characterizations concerning the
culturing conditions, such as larval densities and food. The inhibitory properties of anti-HSP90 drugs have been con-
dataon NO signaling presented here are from the clutch that ducted with vertebrate cells. It is relevant to assess whether
was the most sensitive to NOS inhibition. This clutch often RD is likely to have the same effect on L. pictits HSP90 as
responded to NOS inhibition within 2 h. whereas another it does on vertebrate HSP90. The crystal structure of a
clutch often took 24-48 h to show a significant effect. The geldanamycin-HSP90 complex has been determined (Steb-
source of this variation is not clear. bins et al., 1997). The geldanamycin binding domain
We have shown that D-NAME has inductive properties (GBD) is 43% conserved at the amino acid level between
that are suppressed by SNAP, indicating that application of vertebrates and E. coli; the aspartic acid residue (Asp93) is
D-NAME also leads to a decrease in NO. Although D- absolutely conserved among all HSP90 homologs from 35
NAME is often used as an inactive negative control for species. A hydrogen bond network between Asp93 and the
GA
L-NAME treatment, we propose that it does inhibit NOS, carbamate group of has been suggested by structural
bthuits leascstiveiftfyect(iBvaelbyaltheatn uLi-,NA2M00E0;).otThheerrsefhoarvee,alDs-oNnAotMeEd abinnddifnugncotifonHaSlP9st0udtioesGtAo p(lSacyhntuhre emtoGsaAlt..cr1i9t9i5ca;lSrtoelbebiinnstheet
should be used as a less active enantiomerofL-NAME, not al.. 1997). Thus, it is probable that has similar inhibi-
tauhsneefisunelancsatisitviaevicteoynnaotnfrtoitlohemfeoerrx.pLeT-rhNiemAeMnextEtaeltnrtseyatstotmeewmnhtitcowhiNllDO-derNepAdeuMncdEtiooinns tGsohArayrefpornroopbeirsttnriduGeicsnBtguoDrnatloHsStihPmei9l0Nar-itfterireomsm,inabalulltoprRogrDatniiocsnamnso.fcoRHmDSpePat9ne0dwtGGihtaAAht
andThtehreecownacsentaraltaigoninofthderurgesupsoends.e to radicicol after the ctorenattamiennst,theRD depl(eStcehsultceellestaol.f,k1n9o98w)n. MHoSrPe9ov0erc,lielnitkepro-
beginning of treatment. Radicicol competes with ATP for teins (Schulte et al., 1998). It is reasonable then to expect a
set ofhighly conserved intermolecular interactions between
binding to HSP90, thereby inhibiting its function in binding the GBD of HSP90 of different organisms and RD and
a1n99d8)f.olAdsinga pprrootteeiinnsch(aSpcehruolntee.eHtSaPl.9,01i9n9t8e;raScthsarwimtahemteumi-, hHeSnPce9,0 bayhRiDg.hlCyoncsoinstseenrtvewdithmethcihsacnoinsclmusoifon,inGhiAbiatinodnRoDf
bers of several signal transduction pathways (reviewed by had similar effects on the initiation of ascidian metamor-
Pprraottm,ote19s98t)h.e fIonldcionngcaerntd mwaiitnhteancacnescseoorfytphreotaecitnisv,e sHtSatPe9o0f pdhuorsiinsg (sBeiashuorpcheitnal.e,m2b0r0y1on)iacnddmeovreplhoopgmeennetti(cCmBo.veunmpeunbtls.
several known client proteins (Aligue etal., 1994; Whitesell
obs.).
et al.. 1994: Nathan and Lindquist, 1995; reviewed by Under natural circumstances, the initiation of metamor-
Caplan, 1999). NOS activity in some mammalian cells, phosis by competent L. pictus larvae results from a sensory
tihnrceleudvienrgtenberuartoensi,sofreoqrumisreosfaNnOiSntaerreacdtieognrawdietdh iHnStPh9e0p;reasl-l risesapobnisoecthoemaipcparolprciuaet,eaelntvhioruognhmeantahlarcduessu.rfMaicneimiaslluys,uatlhliys
ence of geldanamycin (GA), another HSP90 inhibitor (Joly required (Cameron and Hinegardner, 1974). It is not clear
et al.. 1997; Garcia-Cardena et al., 1998; Bender et al.. what cells ororgans are involved in transducing thischemo-
1999). Like RD, this agent inhibits HSP90 function by and mechanosensory perception into a metamorphic re-
competing with ATP for binding (Promrodou et al.. 1997). sponse. The rate of biphasic potentials recorded from the
When the folding function of HSP90 is impaired by inhib- larval body or near the rudiment increases more in response
itory drugs such as RD and GA, its client proteins (which to a substrate "conditioned" with a microbial film than to an
are often in complexes including HSP90) may be caught in unconditioned substrate (Satterlie andCameron, 1985). This
a partially folded state that is then recognized by the ubi- suggests that both the larval andjuvenile neural systems are
quitin-proteasome protein degradation machinery (reviewed responsive to environmental stimuli. We have not tested
by Pratt, 1998; Caplan, 1999). Thus, some client proteins whetherthe drugs used herein can induce metamorphosis in
are expected to be degraded or lose activity after HSP90 the absence ofcontact with a hard surface, but the suppres-
activity is inhibited. In this circumstance, a response to sion by SNAP of the inductive properties of biofilm dem-
inhibition ofHSP90 would not be expected until its activity onstrate that NO signaling is downstream of sensory per-
had become limiting and its critical client proteins had lost ception leading to metamorphic events.
activity ordecayed. Such a lag in response was observed for Various experiments have attempted to address how
three HSP90 inhibitors that induced metamorphosis when metamorphosis is initiated and coordinated. The results can
applied to ascidian larvae (Bishop et al., 2001). Thus, we differ among echinoid species. Although many species re-
consider this lag to be a consequence of the mechanism by quire a hard surface for settlement before metamorphosis,
which RD probably leads to a decline in NOS activity. larvae of the sand dollar D. excentricus suspended in sea-
402 C. D. BISHOP AND B. P. BRANDHORST
water can be induced to metamorphose by a heat-labile, larva; in the latter, they were removed (see Fig. 6B). In
low-molecular-weight compound extracted from the sand of direct contrast toD. excentricus, L. pictus did not metamor-
a bed of adults (Highsmith. 1982; Burke. 1983, 1984). phose in response to the removal of the oral hood, a basic
Low-voltage electrical stimulation of the oral ganglion on distinction between these two species. Moreover, both
the lower lip of the larval mouth or the apical neuropile classes ofL. pictus postoperative larvae were less sensitive
between the preoral and anterolateral arms on the preoral to NOS inhibition than were the intact controls, but they
hood region of the D. excentricus larva induced metamor- apparently retained their sensitivity to inhibition of sGC. In
phosis (Burke. 1983). These sensitive larval areas have Figure 6A, ODQ was added directly to wells containing
axonal connections (Burke, 1983). and there is a ciliary postoperative larvae that had been treated with L-NAME for
patch on the pre-oral hood that may have a sensory function 14 h; there may have been an additive effect of the two
(Nakajima. 1986). Electrical stimulation of the oral gan- drugs. Accordingly, when tested separately, a five-fold ex-
glion has been reported to induce metamorphosis in several cess of t-NAME or ODQ is required to induce metamor-
echinoids, including L. pictus (Burke and Gibson, 1986), phosis of larvae lacking the oral hood over concentrations
although Cameron and Hinegardner (1974) reported other- that induce metamorphosis ofcontrol larvae (CDB, unpubl.
wise for L. pictus. The difference in these results may be obs.). These experiments are difficult to interpret with re-
methodological. Recently, Beer et al. (2001) reported that spect to Burke's model of mutual inhibition, but they do
cells in the lower lip of the larval mouth of the sea urchin suggest the involvementofcells in the oral hood ofL. pictus
NO/cGMP
Psammechinus miliaris develop immunoreactivity to a se- in a pathway that regulates metamorphosis by
rotonin antibody. We found staining for NOS protein and signaling.
NOS activity in cells that appear to be neurons in the lower The regulatory role of these and other NOS-expressing
lip of the mouth, corresponding to the region of the oral cells in larvae orjuveniles may be additive. In L. pictus, the
ganglion (Burke, 1983), and in cells of the preoral hood, tube feet of the rudiment appear to have sensory receptors
perhaps corresponding to the apical neuropile (Burke, that may be involved in inducing metamorphosis (Burke.
1983). When Burke (1983) excised the oral hood of D. 1980). We found intense staining for NOS activity in the
excentricus including the oral ganglion and apical neuro- nerve plexus lining the outerepithelial layerofthe tube feet
pile both fragments of the larva rapidly began metamor- ofthe rudiment. NO has been implicated in the relaxation of
phosis, but this did not occur when only the preoral hood adult tube feet (Billack et al., 1998). The presence of NOS
lacking the oral ganglion or larval arms lacking both in cells associated with other structures that may have a
sites were excised. The excised preoral hood and remain- sensory role (the pre-oral hood, the tips ofthe anterolateral
ing larva were able to respond to a chemical cue for meta- arms and epaulettes) suggests that the drugs we used act on
morphosis, but isolated larval arms did not (Burke. 1983). one or more ofthese groups ofcells to inhibit their produc-
Isolated larval arms ofsome species, including D. excentri- tion of NO. Indeed, microsurgical and expression data in-
cus, can be induced to contract by treatment with divalent dicate that multiple larval structures and perhaps juvenile
NO/cGMP
ionophores or the neural transmitters adrenalin, noradrena- structures transduce sensory information, by sig-
lin. and dopamine (Burke, 1982. 1983). Dopamine induced naling, which leads to the initiation of metamorphosis. The
only a few whole D. excentricus larvae to initiate metamor- frequency of metamorphosis of larvae ofL. variegatus was
phosis, suggesting the local response ofarms can be inhib- increased by excess potassium orcalcium ions (Cameron et
ited centrally. til., 1989). Metamorphosis of Strongylocentrotus purpura-
On the basis of his experiments. Burke (1983) proposed tus larvae was induced by treatment with calcium ionophore
that there is a mutually inhibitory control ofmetamorphosis A23187 or quercetin, an inhibitor of a [Ca.Mgj-ATPase
between the oral hood and remainder of the D. excentricus (Klein et al., 1985). Ionic fluxes may play a role, perhaps in
larva that is switched off in response to an appropriate cue coordinating cellular responses (Burke. 1983; Pearse and
(or electrical stimulation). The inhibitory region ofthe oral Cameron. 1991). Some mammalian isoforms of NOS (en-
hood appears to be localized to the larval mouth (Burke. dothelial and neuronal) are dependent on Ca:+ for their
1983), while the preoral and remaining regions ofthe larva activation (Mayer et al., 1998). The inductive properties of
must have sensory receptors for the chemical cue that in- Ca2+ flux may relate to the role ofCa2+ in the regulation of
duces metamorphosis. Data from histological sectioning NOS activity.
and optical reconstructions of the L. pictus oral epithelium With this report, there is now evidence that NO plays a
stained for NOS suggest that nitrergic neurons may reside repressive role in regulating the initiation ofmetamorphosis
within this epithelium, possibly performing a sensory role in a protostome (llvanassa) and three deuterostomes (two
related to feeding or metamorphosis. These NOS-express- ascidians and an echinoid) (Froggett and Leise, 1999;
ing cells were considered as candidate NO-signaling cen- Bishop et al., 2001). NO is involved in metamorphosis of
ters. We removed the pre-oral hood or the entire oral hood. larvae that do not grow before metamorphosis and retain
In the former case, most ofthe oral cells remained with the much of the larval tissue (ascidians), larvae that grow as
NO REPRESSES ECHINOID METAMORPHOSIS 403
swimming veliger larvae but do not undergo profound Burke, R. D., and A. \V. Gibson. 1986. Cytological studies for the
changes upon metamorphosis (Ilyunussa), and larvae that studies of larval echinoids with notes on methods for inducing meta-
undergo extensive growth and catastrophic metamorphosis morphosis. Methods Cell Biol. 27: 295-308.
Caldwell. J. \V. 1972. Development, metamorphosis, and substrate se-
in which most larval tissues are degraded and replaced by a lection of the larvae of the sand dollar. Mellila quinques perforate.
radically differentjuvenile (echinoids). NO, a universal and M.Sc. Thesis. University ofFlorida. Gainesville.
ancient signaling molecule in eukaryotes. may have arole in Cameron. R. A.,and R. T. Hinegardner. 1974. Initiation ofmetamor-
regulating metamorphosis in a wide diversity of animals. phosis in laboratory-cultured sea urchins. Biol. Bull. 146: 335-342.
Sea urchin larvae are optically clear and can easily be Cameron, R. A., and R. T. Hinegardner. 1978. Early events ofmeta-
cultured in large numbers. This fact, and arichexperimental morphosis in sea urchins; description and analysis. J. Morphol. 157:
21-32.
literature on settling and metamorphosis, make echinoids a Cameron,R.A.,T.R.Tosteson,andV. Hensley. 1989. Thecontrolof
useful system with which toinvestigate the neuroanatomical sea urchin metamorphosis: ionic effects. Dev. Growth. Differ. 31:
basis for the regulation of metamorphosis. These features 589-594.
and our findings provide a basis for a more focused exper- Caplan,A.J. 1999. Hsp90'ssecretsunfold: newinsightsfromstructural
imental effort to identify which cells or organs repress and functional studies. Trends Cell Biol. 9: 262-268.
NO Cueto. M., O. Hernandez-Perera, R. Martin. M. L. Bentura, J. Ro-
metamorphosis by production in L. piclits. drigo,S.Lamas,andM. P.Golvano. 1996. Presenceofnitricoxide
synthase activity in roots and nodules of Lupinus albus. FEBS Lett.
Acknowledgments 398: 59-164.
Czar,M.J.,M.D.Galigniana,A.M.Silverstein,andW.B.Pratt. 1997.
This research was funded by a Research Grant from the Geldanamycin, heat shock protein 90-binding benzoquinone ansamy-
Natural Sciences and Engineering ResearchCouncil ofCan- cin. inhibits steroid-dependent translocation of the glucocorticoid re-
ada. We thank Robert Burke. Andrew Cameron, and Victor ceptor from the cytoplasm to the nucleus. Biochemistry 36: 7776-
7785.
Vacquier for helpful discussions, and two anonymous re- Davidson, E. H. 1991. Spatial mechanisms ofgene regulation in meta-
viewers for useful comments.
zoan embryos. Development 113: 1-26.
Degnan, B. M., D. Souter, S. M. Degnan, and S. C. Long. 1997.
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