Post-Hatching Development of Circular Mantle Muscles in the Squid Loligo opalescens

13 Pages·1997·6 MB·English

by  T Preuss

Reference:Bio/, Bull. 192:375-387.(June, 1997)
Post-Hatching Development of Circular Mantle
Muscles in the Squid Loligo opalescens
THOMAS PREUSS, ZORA N. LEBARIC, AND WILLIAM F. GILLY
Hopkins MarineStation. Department ofBiologicalSciences, Stanford Universitv,
PacificGrove. California 93950
Abstract. Post-hatching development of the circular tion with twolayersofstiff, collagenoustunicsanda net-
muscles in the mantle ofsquid was studied morphomet- work of intermuscular connective tissue fibers (Ward
rically to identify structural changes and to quantify hy- and Wainwright, 1972: Bone el at.. 1981; Gosline et a/.,
perplasia and hypertrophy ofthe muscle fibers. Superfi- 1983; Kier, 1988; Wells, 1988). The circular muscle
cial, mitochondria-rich (SMR) fibers and central, mito- mass, responsible forproducingthrustduringjet propul-
chondria-poor (CMP) fibers are present at hatching. sion, is divided into three layers, largely on the basis of
Although both fibertypesincreaseinsizeand,even more the relative mitochondrial content ofindividual muscle
so, in number during post-hatching development, CMP fiber types. Two thin layers ofsuperficial mitochondria-
fibersincreaseata much higherratethandoSMR fibers. rich, oxidative fibers on the outerand inner mantle sur-
Asaresult,therelativeproportionofSMRtoCMPfibers faceenclosea much thicker layerofmitochondria-poor,
shifts from about 1:1 in a hatchlingtoabout 1:6 in an 8- glycolytic fibers in the central zone (Bone el a/.. 1981;
week-old animal; it then apparently remains constant to Mommsen et a/.. 1981 ). In light ofthese structural and
adulthood. These structural changes are consistent with metabolic differences, it has been proposed that the su-
developmental changes in muscular activity. During perficial layersareactiveduringrespiration and slowjet-
slow,jet-propelled swimming, 1-week-old animalsshow propelled swimming, whereas the central circular fibers,
mantle contractions that have twice the relative ampli- presumed to be innervated by the giant axon system,
tude and frequency ofthose in adults. The presence of power thejet-escape and rapid locomotion (Bone et a/..
Na-channel protein in mantle muscle was detected bio- 1995). Ingeneralagreementwith these ideas, recentelec-
chemically by using site-directed antibodies: the protein trophysiological studies have revealed that at least two
was found to be preferentially expressed in CMP fibers. classes ofcircular muscle fibers can be distinguished on
These results suggest that SMR fibers are an important the basis oftheir electrical properties and that some of
source of locomotory power at hatching, but become thecircular fibers, presumably those innervated bygiant
progressively less important during the first 8 weeks of axons, display Na-channel-based excitability (Gilly et
CMP
developmentas fibersassumethedominant role in at.. 1996).
jet locomotion. Jet propulsion, mantle mechanics, and mantle struc-
ture have been well studied in adult squid, but relevant
Introduction work on hatchlings and developing juvenile squid is
comparatively limited (Zuev, 1966; v. Boletzky, 1982,
Jet propulsion in squids is caused by the antagonistic 1987;O'Dorrta/., 1986; Moltschaniwskyj. 1994, 1995;
action ofcircular and radial mantle muscles in connec- Matsuno, 1987). Although squid begin actively swim-
ming by jet propulsion as soon as they hatch (Packard.
ARbebcreeivvieadti2onDse:ceCmMbPer=199c6e:ntarcaclepmtietdoc3hAopnrdirlia1-99p7o.or muscle fibers; c1o96m9o)t,otrhesyfsultlemfleisxilbaicliktiyngan(dGilflinyeelcoaoir,di1n9a91t;ioCnhoefnthetealio.-
DML = dorsal mantlelength;GFL = giantfiberlobe;SMR = superfi- 1996). The aim ofthe present study was to analyze the
cial mitochondria-rich musclefibers;SR = sarcoplasmicreticulum. maturation of the mantle musculature during post-
375
376 T. PREUSS ET AL.
hatching development and to identify structural and day after collection. Blocks of mantle muscle were cut
functional characteristicsofthedifferenttypesofmuscle parallel tothe main bodyaxisandtaken from an antero-
fiberassociated v -propelledlocomotion. Inpursuit dorsal region close to the stellate ganglion offive adults
ofthat goal, v ned three approaches. First, ana- with dorsal mantle lengths (DML) between 80 and
tomical an.i .^metric methods at light- and elec- 140 mm. Forstudiesofhatchlingandjuvenile squid, six
tron-mienK ievels were used to reveal structural healthy looking animals that displayed vigorous swim-
changes as squu mature from hatchlings to adults. Sec- ming capability were collected for fixation weekly over
ond,thekinematicsofmantlecontractionsinfree-swim- a 10-week period. These animals were fixed whole, and
ming animals were investigated to shed light on func- smaller tissue samples for ultrastructural analysis ofthe
tionalcharacteristicsoflocomotion atdifferent maturity mantle muscles were taken after fixation. In all animals
stages. Third, biochemicalanalysisofthepresenceofNa- (except hatchlings), the skin was carefully removed be-
channel protein in superficial and central fibersprovided fore fixation.
atool to characterize the location and abundance ofpu- For LM, the tissue was fixed in 4% paraformaldehyde
tative fast-twitch fibers in the developing mantle muscu- in filtered seawater for 3-5 days at 4C, dehydrated in
lature. graded ethanol, andeitherinfiltrated with paraffin under
Our results indicate that superficial, mitochondria- vacuumorembeddedin plastic(JB4; Polysciences, Inc.).
rich(SMR) fibersarean importantsourceoflocomotory Thick sections (6-10 ^m) and semithick sections (2-
power at hatching but become progressively less impor- 4 ^m) were cut and stained with conventional histologi-
tant during the first 8 weeks after hatching. Over this cal stains and viewed with an Olympus BH-2 micro-
same period, central, mitochondria-poor (CMP) fibers scope.
increase in number until they contribute most of the For TEM, the tissue samples were fixed in 0.065 A/
overall mantle muscle mass and play the dominant role sodium phosphate buffer (pH 7.4) with 3% glutaralde-
injet locomotion. hyde, 0.5% tannic acid, and 6% sucrose for 8 h aMt 4C
(first 15 minatroom temperature), rinsed in0.065 so-
Materialsand Methods dium phosphate bufferwithout sucrose, and postfixed in
a 1:1 mix of0.13 Mcacodylate buffer (pH 7.2) with 2%
Experimentalanimals potassium ferrocyanide and 2% osmium tetroxide for
L/;#0o/w//rotwi.vwascollected in Monterey Bay,Cal- 40 minMat 4C. Thereafter, the tissue was rinsed in
ifornia, between August and November of 1995. Ani- 0.065 cacodylate buffer, dehydrated in graded etha-
mals were maintained at Hopkins Marine Station in cir- nol, and infiltrated overnight (10-12 h) in resin (LR
cular tanks (2.5 m diameter: 1 m deep) plumbed with White: Sigma). For sectioning, the blocks were oriented
flow-through natural seawater. Spawning typically oc- to obtain transverse, sagittal, and tangential sections of
curred in these tanks within 3-5 days ofcollecting the the circular mantle muscles. Thin (0.8-1 fim) and ultra-
squid. Clusters ofegg cases were removed and cultured thin (gray-silver) sections were obtained in alternating
at the Monterey Bay Aquarium, Monterey, California, section series, thereby collecting ultrathin sectionsevery
in flow-through circular 320-1 tanks (temperature range 4-6 /jin in hatchlings andjuvenilesand every 50-60 ^m
13-16C) until natural hatching occurred. During the in adults. Up to five consecutive section series were col-
first 10 weeks, squid received a diet, adlibitum, ofbrine lected in this manner. Thin sections were stained with
shrimp nauplii (Anemia salina)enriched with algae and toluidineblueandexaminedwithan Olympus BH-2 mi-
SuperSelco(anutrient medium rich in lipids, fattyacids, croscope. Ultrathin sections were collected onto Form-
and vitamins; produced by Artemia Systems N. V., Bel- var-coated meshandslotgrids(Electron MicroscopySci-
gium). Copepods (Acartia sp.) and mysids (Acanmo- ences),stainedeitherwithasaturatedaqueousuranylac-
tmwyisciespsepr.)weweekr)e.added to the diet when available (about epthaotsephsootluuntigosntiacnadcidR,eyannodldesx'amleiandedcitwriatthe PohrilwliiptshE2M%
EM
201 or 401 electron microscopes. The magnification
Mantleanatomy stops were calibrated with a diffraction grating replica
(Ted Pella. Inc.).
The anatomy of the mantle musculature was exam- For morphometric analysis of the muscle fibers, a
inedbylight microscopy(LM)andtransmissionelectron computer-aided image analysis program (NIH-Image
microscopy (TEM). Priorto dissection, all animalswere 1.60) was used on a Power Macintosh 7100/80 com-
anesthetized for20 min in 7.5% MgCl; diluted 1:1 in ox- puter. TEM micrographs ofmuscle fibers were digitized
ygenated seawater and then killed by decapitation. Tis- at 600-dpi resolution using a scanner (Microtek Scan-
sue for fixation was removed from adult squid within 1 MakerHHR),andstored onamagneto-opticaldrive(Pin-
SQUID MANTLE MUSCLE DEVELOPMENT 377
nacle Tahoe 230 MB). In addition. LM video-images affinity-purified using the soluble fraction ofthe fusion
were obtained using an Olympus BH-2 microscope protein coupled to an affinity column (AminoLink,
equipped with a CCD B/W camera (Sony SSC-M374). Pierce)accordingto manufacturer'sprotocol.
and selected framesweredigitized with a high-resolution A monoclonal antibody (mAb|i0?.:i) was produced in
video capturecard (Scion LR-3). collaboration with J. Burkhard and S. L. Feng. Univer-
Measurementsweretaken on longitudinal mantlesec- sity ofCalifornia, San Francisco. This antibody was di-
tions (i.e.. on a cross section of the circular muscle fi- rected against a synthetic peptide (synthesized by the
bers). Two measurements ofeach muscle fiber visible in Protein and Nucleic Acid Facility, Stanford University.
a given electron micrograph were taken: (i) the total Stanford, CA) corresponding to aa 1305-1321 of the
cross-sectional area ofa single muscle fiber and (ii) the GFLN1 sequence. These residues make up the part of
area occupied by its mitochondria! core. The difference the cytoplasmic linker between domains III and IV that
between these values approximates the remaining myo- is well conserved in most sodium channels (Gordon el
filamentarea;thisvaluealso includescytoplasm andsar- a/.. 1988).
coplasmic reticulum (SR). Additional measurements Preparation ofprotein samples. Specificity ofthe anti-
were taken in selected micrographs to determine the bodieswastestedwith tissuesamplesofcleanedgiantax-
cross-sectional area(and thecalculateddiameter)ofsyn- ons and giant fiber lobes (GFL) taken from adult squid.
aptic vesicles in nerve processes. Animalswererapidlydecapitatedbeforetissuesweredis-
sected incold,Ca-freeartificial seawater(480 mAINaCl,
mM mM mM
Mantlekinematics 10 MgCl:, 5 EGTA, 10 HEPES, pH 7.4)
oilndgA)tadanondrkssaaldwualvstiesfwqiuloimfdedfirnweitethleyhirsawrheiismgphme-cirtneigsvoeljhuuotvliedonniinlCegCa(1nDd-wBreee/akrW-- lftnoihergeravbtgeieidoafcnaihtbneedmrasixccoluanenladaanenenraddlymbsiiyticssr.mSoacSsnhecuwogaapmlnielncny-tcossebtlsorlefirpsvsptaehiltenligaaotronhfnfeurrtnevthmieealsiswnomeenardll.ely
video camera (Sony SSC-M374) mounted above the Cleaned axons were cut at both ends and immediately
tanks on a remote controlled panning motor (Prinz
placed in ice-cold lysis buffer containing proteinase in-
Power-Fanner430-62), and recorded on a Sony Hi-8 re- hibitors (Knudson et ai. 1989). Several axons were
corder (EVO-9700). Single video-frame analysis ofdigi- pooled, homogenizedinthesamebuffer,andcentrifuged
tainzaeldysvisidseyostseemqudeensccersibweadsacbaorvrei.edTohuetmuasnitnlgethdeiaimmeatgeer autse1d50f0orXpgroftoeri1n-cmoincnenattr4aCt.ioTnhaenasluypseirsna(tbaincitnwcahosntihneinc
wasmeasuredat itswidestpoint(theanteriormantleend acid assay. Pierce, Rockford, 1L) and for immunoblot-
injuvenilesandabout '/3 from theanteriormantleend in ting. Giant fiberlobe, brain, and corneatissuesweredis-
abdyuldtosr)sailn msaucnctelsesilveen,gtehn)l.arTgoedcovmipdeaoreimmaagnetsle(cdailiabmreatteerd saebcotveed.aTnhdehormeosugletniingzesdupienrtnhaetalnystiswbausffecrenatsrdifeusgcerdibeadt
dimensions in animals of different size, the fractional 00.000 Xgfor30 minat4Ctoformacrudemembrane
mantle diameter was calculated by defining the largest p1ellet. This pellet was resuspended in lysis buffer for de-
diameter in agiven measurementsequenceas 100%. termination of protein concentration and for immu-
noblotting.
Biochemistry Muscle samples were obtained from animals anesthe-
Production oj antibodies. Two antibodies (one poly- tizedin0.5% urethaneinartificial seawateranddissected
clonal and one monoclonal) directed against distinct re- in cold, Ca-freeartificial seawater. Thereafter, all muscle
gionsofaputativesquid sodium channelencodedbythe tissuewasprocessedasdescribed forgiantfiberlobes(see
cDNA GFLN1 (Rosenthal and Gilly, 1993) were used. above). Totest forNa-channel protein within individual
mRNA corresponding to this gene is expressed widely layersofcircularmantle muscle, tissue samples from su-
throughout the squid nervous system (Liu and Gilly, perficial and central layerswere dissected and processed
1995). separately (outer and inner superficial layers were
Polyclonal antisera (produced in collaboration with pooled). To test for the presence ofNa-channel protein
Dr. S.R. Levinson. University ofColorado) were raised in the mantle muscle mass during post-hatching devel-
against a bacterial fusion protein that contained amino opment, samples were taken from skinned mantles of
acids (aa) 483-576 of the predicted GFLN1 sequence. hatchling and juvenile squid at four maturity stages (3
These residues compose the C-terminal halfofthe cyto- days, 2 weeks, 3 weeks, and 14 weeks)and from adults.
plasmic linker between domains I and II. Construction Immunoblotling. Protein samples (10 ^g oftotal pro-
of the fusion protein is described in detail elsewhere tein per lane) were separated by standard SDS-PAGE
(Rosenthal. 1996). Polyclonal antisera (Ab4K3-57<,) were electrophoresisusing 5% gelsand transferredto nitrocel-
378 T. PREUSS KT AL.
lulose. Nonspecific binding ofthe antibodies was mini- ring around the central mitochondrial core (Fig. 2B).
mized by pretreating the nitrocellulose witMh 10% nonfat ThisdifferencebetweenCMPand SMR fiberswasfound
dry milk in PBS ( / Na:HPO4, 0.02 NaH:PO4, in cross sections examined at all maturity stages from
0.1 A/NaCl) . UTinity-purified antibodieswere week 1 on.
diluted ;)v n the figure legends. Undiluted hy- Longitudinal sections confirm the difference in width
bridoma1 ; . ni was usedasasourceofmonoclonal ofthemyofilamentzone in relationtothe mitochondrial
antibody. Goal anti-rabbitorgoatanti-mousesecondary coreofthetwocircularfibertypes(Fig. 3).Thesesections
antibodiesconjugatedto horseradish peroxidase(Sigma) also reveal differences in myofilament staggering in the
CMP
were used at 1:5000 dilution in conjunction with an en- two fibertypes. In fibers, the myofilaments and Z-
hanced chemiluminescence detection system (Renais- bodies form an oblique pattern across the fiber, which
sance. Du Pont NEN Research Products). For control characterizesthem as regular, obliquely striated muscles
experiments with blocked Ab^.svh. purified antiserum similar to those in other cephalopods (Gonzalez-San-
4C
was incubated overnight at with the fusion protein tanderandGarcia-Blanco, 1972; Amsellem and Nicaise,
antigen at aconcentration of0.1 mg/ml. 1980, Kier. 1985). In contrast, the myofilaments and Z-
SMR
bodiesof fibersarelined upnearly in registeracross
SMR
Results the fiber (Fig. 3). Although the appearance ofthe
Structuralaspectsofmusclefibermaturation fibers in cross section and longitudinal section is sugges-
tive ofcross striation rather than oblique striation, fur-
Squid hatchlings (1-2 days old) already possess the ther morphological studies are necessary to clarify this
overall organization of radial and circular muscle fibers point. The degree of myofilament staggering in a given
shown by adults (Bone el at., 1995). Profiles oftwo dis- section depends on the sectioning angle and on the de-
tinct types ofcircular muscle fibers are visible in a longi- gree of contraction of the muscle fiber (Rosenbluth,
tudinal mantle section (Fig. 1A). Layers of superficial, 1972; Kier 1985).
large-diameter fibers on the outer and inner mantle sur- In all maturity stages, numerous profiles ofneuronal
face enclose a central layercomposed offibers ofsmaller processes containing round, clear vesicles were found
diameter.Thesuperficial fibersandthecentral fibersdiffer (Fig. 4). These processescontact CMPand SMR muscle
in theirmitochondrial content. Inanygivencrosssection, fibersatpresumptivechemicalsynapseswithcleftsabout
superficial mitochondria-rich (SMR) fibers often contain 20 nm wide(Fig. 4C). A rich, ramifying networkofthese
CMP
several large mitochondria, whereas central mitochon- processesexistswithin the zoneof fibers, and indi-
dria-poor (CMP) fibers rarely display more than a single vidual processes appear to contact multiple fibers (Fig.
small one (Fig. 1 ). In both fiber types, the mitochondria 4A, B). Within the SMR fiber zone, nerve processes are
are surrounded by loosely packed myofilaments (Fig. 1B, less prominent and appearto run in groovesofthe mus-
C). Myofilamentsareratherpoorlyorganizedatthistime, cle fibers(Fig. 4D). The cross-sectional areasofsynaptic
and sizable areas ofcytoplasm without myofilaments are vesicleswere measured at a magnification of240.000 on
common. Similarly, the SR network is poorly developed scanned micrographs, and vesicle diameters were calcu-
and organized at this stage, with only a few SR tubules lated by assuming a spherical shape. Vesicles from pro-
SMR
scattered among the myofilaments (Fig. 1B, C). Fibers of cesses associated with fibers (mean diameter of
both typesdisplay very large nuclei (Fig. 1A). 39 nm 0.7 SEM) are significantly smaller than those
CMP
By 8 weeksafterhatching, several oftheabovecharac- from processesassociated with fibers (mean diam-
teristics show signs of substantial maturation. In addi- eter of 45.8 nm 0.6 SEM). These values are signifi-
tiontotheirdifferencein sizeand mitochondrialcontent cantlydifferentby Student'sttest(df= 225, P< 0.005).
(see also below), SMR and CMP fibers now clearly dis-
play differences in the organization of their myofila- Quantitativeaspectsofmusclefibermaturation
ments and SR (Fig. 2). The myofilament density in-
CMP
creases considerably in both fiber types, but fibers In longitudinal sections, the circular mantle muscle
showa muchthickermyofilamentzone, both inabsolute mass isdivided by bandsofradial fibers into rectangular
size and in relation to the mitochondrial core (Figs. 2B, muscle segments (Figs. 1A and 2A). The dimensions of
2C, and 3). Moreover, myofilaments in each fiber type such a muscle segment, in a given section, can be de-
are now organized in a characteristic manner. In cross scribed by (i) its thickness (i.e., the distance between the
CMP
sectionsof fibers, myofilaments are divided by the outerand innertunics)and(ii)itswidth(i.e.. thedistance
SR and the Z-bodies into radially oriented, trapezoidal between two successive radial-fiber bands). Horizontal
bloci Fig. 2C). In the same sections, SMR fibers lack sections through the entire body of a juvenile showed
such u ,MIIS, and the myofilaments form acontinuous that the thickness and the width ofthe muscle segments
SQUID MANTLE MUSCLE DEVELOPMENT 379
Figure 1. Transmissionelectronmicrographsofsuperficial(outer)andcentrallayersofmantlemuscle
from asquid hatchling(1-2daysold). (A)Crosssectionofcircularmuscle fibersshowingtheouterlayer
composed of 1-2 superficial mitochondria-rich fibers (SMR) and halfofthe central zone composed of
mitochondria-poorfibers(CMP). Radial fibers(RF;cutlongitudinally)dividemantlemuscleintorectan-
gularsegments.N = nucleus;oT=outertunics.Scalebar= 2^m.(B)CrosssectionofasingleSMRfiber.
Many largemitochondria(Mil)arelocated in thecentralcore. Mf= myofilaments. Scalebar= I nm.(C)
Crosssection ofseveral CMPfibers. Usually one mitochondrion islocated in thecentral coreofan indi-
vidual CMPfiber. Notethe irregularshapeofCMPfibersandthelackofany myofilamentorganization.
Scalebar= 2^m.
vary considerably along the mantle. The thickest and A muscle segment in a hatchling (2 mm DML) en-
widest segments are found about one-third of the way closes the profiles ofabout 20-22 circular muscle fibers,
CMP
fromtheanteriormantleend,whereasmoreanteriorand ofwhich 12-14 are fibers (i.e., 60%). During
posterior segments become progressively thinner and growth, the number offibersenclosed in a single muscle
narrower. Transverse mantle sections, on the other segment increases considerably, but SMR and CMP fi-
hand, show almost no variation in the thickness ofthe bers increase at different rates (Fig. 5). By 8 weeks
muscle segments in a given section, although segments (12 mm DML). the numberofCMP fibershas increased
become very narrow dorsally at the location ofthe pen. about 10-fold, whereas the number of SMR fibers in-
In light ofthese results, the mantle region that contains creasesonly 2.5-fold (Fig. 5A). Thus,therelativepropor-
the thickest muscle segments was chosen for analyzing tionofCMPfibersincreasesduringgrowth(Fig. 5B),and
the growth of the mantle muscle, and two animals of by week 8, about 84% ofthe fibers in a muscle segment
each agegroupwith similardorsal mantle length (DML) are CMP fibers. This proportion is comparable to that
were studied. derivedfromanalysisofasinglemusclesegmentinama-
In adults, muscle segments from this same area ofthe ture animal (110mm DML; muscle segment 2.8 X
mantlewereanalyzed. Although adultanimalswerepart 0.12 mm), which revealed a total ofabout 12.000 circu-
ofa spawning population, only very robust males that larfibers, 86% ofwhich wereCMPfibers(Fig. 5A, B).
showed no sign ofskin damage or senescence were stud- Morphometric measurements on the ultrastructural
ied. We therefore consider it unlikely that any selective level fordeveloping CMP fibers are summarized in Fig-
"deconstruction" ofthe mantle muscle mass took place ure6A. Mean cross-sectional areaofCMPfibersdoubles
priorto fixation (Giese, 1969; O'Dorand Wells, 1978). between 1 week and 8 weeks ofage and increases by a
380 T. PREUSS 7 AL.
Figure2. Transmissionelectronmicrographsofsuperficial(outer)andcentrallayersofmantlemuscles
from ajuvenile squid (8 weeks old). (A) Cross section ofcircular muscle fibers showing the outer layer
composedofseveralSMR fibersand partofthecentral zonecomposedofCMPfibers. Radial fibers(RF)
arecutlongitudinally.CMPfibersshowawell-developedsarcoplasmicreticulum(SR).oT = outertunics.
Scale bar = 2^m. (B)Cross section ofa single SMR fiber. A continuous ringofmyofilaments(Mf) sur-
roundsa massivecoreoflarge mitochondria(Mit). Scale bar = 1 ^m. (C)Crosssection ofasingleCMP
fiber.ThemyofilamentareaisdividedintotrapezoidalblocksbySRandZ-bodies.Onlyasmallproportion
ofthefiberisoccupiedby mitochondria(Mit).Scalebar = I ^m.
total of3- to 4-fold in mature animals. This increase in Functionalmaturation olcircularmusclefibers in
fiber cross-sectional area is almost exclusively due to an locomotion
increase in the filamentand SR area. The mitochondrial
core contributes relatively little to the absolute fiber size Theanatomical datadescribed above indicatethat the
and, moreover, does not grow in proportion to the rest circular muscle of the mantle changes in composition
SMR
ofthe fiber. Asaconsequence, the relativeareaoccupied duringpost-hatchingmaturation. Athatching, and
by the mitochondrial core in CMP fibers decreases dur- CMP fibers contribute almost equally to the circular
ing growth from 16.3% 0.6% SEM in 1-week-old ani- mantle mass. Subsequent growth is mostly due to an in-
malsto6% 0.3% SEM in adults. creasein thecontribution ofCMPfibers, andthe relative
SMR
In Figure 6B, resultsofthesame ultrastructural analy- contribution of fibers to the mantle mass in an
sis are presented for SMR fibers. The mean cross-sec- adultisonlyabout 14%. Thesestructuralchangessuggest
tionalareaofSMRfibersshowsapatternofincreasesim- that the mantles of hatchlings and adults may have
ilarto thatdescribed above forCMPfibers, although the different contractile and endurance properties. For ex-
absolute fiberarea is much largerin SMR fibers(Fig. 6A, ample, the endurance capabilities ofSMR fibers should
B left). Growth in SMR fibers, however, reflects a sub- be manifested in respiration-related slow swimming
stantial increase in both the filament and SR area and (Bone el ai. 1995) or hovering (Zuev. 1966), and their
the mitochondrialcore. Asaresult, theproportional area relativecontribution to jet-propelled locomotion should
occupied by the mitochondrial core in SMR fibers re- be most apparent at theearliest stagesofmaturation. To
mains more-or-lessconstant at about40%. test this idea, we compared the kinematics of mantle
SQUID MANTLE MUSCLE DEVELOPMENT 381
tive squid Na channel encoded by the cDNA GFLN1
mRNA
(Rosenthal and Gilly, 1993). corresponding to
GFLN1 is expressed throughout the squid nervous sys-
tem, particularly in neurons, includingthoseoftheGFL,
whose axons are large, long, or both (Liu and Gilly,
1995). These tissueswere therefore used to test the spec-
ificity ofthe Na-channel antibodies.
Results of the monoclonal antibody mAb^i^, with
GFLand cleaned axon samplesare shown in Figure 8A.
A prominentband withan apparent molecularweightof
about 250 kD is present in both lanes. Control experi-
mentswith secondaryantibodyalonegavenosignal(not
illustrated). Similarresultswere obtained usingthe poly-
clonal antibody Ab4X.i-576with GFL samples(Fig. 8B). In
this case, a control experiment employing blocked-
Ab483-57(, (see Methods) demonstrates specificity. The
specificity ofthese antibodies for Na-channel protein is
also supported by the fact that both antibodiesgive sim-
ilarresults,even thoughtheyaredirectedagainsttwodis-
tinct portionsofthe protein encoded byGFLN1.
Asdetected by the polyclonal antibody, aspecific(i.e.,
blockable) Na-channel band centered around 210-
220 kD is prominent in protein samples derived from
mantle muscle (Fig. 8C). The monoclonal antibody also
recognizesacomparableband in muscletissue(not illus-
trated). Thus, it appears that Na-channel protein is rela-
Figure 3. Transmission electron micrograph ofmantle muscle fi- tivelyabundant in mantle muscletissue.
blaerrsmufsrcolmeafjiubveersnislheoswqiunigdp(8orwteieonkssoolfdt).woLoSngMiRtudfiinbearlssaencdtiotnhroefecCirMcuP- Results obtained with a control tissue expected to
fibers. Note the differences in mitochondrial content and in myofila- show minimal Na-channel proteinarealsoshown in Fig-
ment(MF)staggeringanglebetween SMRandCMPfibers. Mil = mi- ure 8C. The cornea ofthe eye isasimplearrangement of
tochondria;iT = innertunics.Scalebar= 2/JPI alayerofepithelial cellssupported byatransparentlayer
ofmuscle fibers (unpubl. obs.). Whole-cell patch clamp
contractions in freely swimming 1-week-old and adult recordingsmade, usingestablished methods(Gilly et til..
squid duringhoveringbehavior. 1990, 1996), from enzymatically dissociated cells from
Adults and juveniles show quite different locomotor both layers failed to reveal the presence ofany voltage-
behaviorduring hovering. Adults move slowlyback and gated Na currents (unpubl. results); the immunoblot re-
forth, eitherin aslightly head-down orhead-upposition, sults also fail to reveal a strong Na-channel band (Fig.
due to gentlejets and undulatory fin movements. Juve- 8C). The weak bands in the cornea lane may arise from
niles, on the other hand, continuously bob upand down axonal membrane, because the muscle fiber layer ofthe
in a definite head-down position. Although juveniles cornea presumably is innervated. These bands are
beat their very small fins rapidly (up to 16 Hz), locomo- blocked in the control experiment (Fig. 8C).
tionatthisstageisapparentlydrivenprimarilybyjetting. Electrophysiological recordings ofNa currents in cir-
Cthoamtpahroivseroinngofjtuhveenmialnetsle(2k.i5nmemmatiDcsMiLn)Fipgurroedu7csehojewtss acunldaritmwuassclperofpiboesresdofthsaqtuitdhemmaunstclleehfaivbeerbseweinthreNpaortceudr,-
about twice as frequently (2.7 Hz) as do adults ( 1.3 Hz). rents were small-diameter, CMP fibers (Gilly el ai,
jIunveadndiilteison(,=t3h2e%f)racitsioanbaolutchathnrgeeeintimmaenstltehadtiaimneatdeurltisn a1n99a6d)u.lTtosqtuesitdt(hsieseidMeeat,hmoadnst)letotpisrsouveiwdeassadimspsleecsteodffpruorme
(=12%; Fig. 7). CMP circular fibers(plus radial fibers)and samplescon-
Identification ofNa-channelprotein indeveloping taining SMR fibers (plus contaminating CMP fibers as
mantlemuscle well as radial fibers). The results ofan immunoblot with
Both Na-channel antibodies described in this study these samples are shown in Figure 9. The Na-channel
were produced against predicted sequence for the puta- band is most prominent in the central-zone sample.
382 T. PREUSS KT AL
j"-v['>
?i.*.-
^;^i|if^,
$ ' -: '
.-
L^
Figure 4. Transmission electron micrographs ofsynaptic profiles contactingcircular mantle muscle
fibers(juvenile: 8 weeksold). (A and B) Longitudinal mantle section (circular fibers in cross section). A
nerveprocess(NP)runswithinthecentralmusclelayerandformsputativesynapticcontacts(arrows)onto
severalCMPfibers. Notetherelativelylargedimensionsofthesynapticprofilesinrelationtothesizeofthe
musclefiber.(C)NeuromuscularjunctionontoaCMPfiber.Thesynapticprofileisfilledwithround,clear
vesicles. (D) Synaptic profile runningwithin a groove ofan SMR fiber. Scale bars = 0.5Aim forA. and
0.2Aim forB-D.
muchweakerinthesuperficial(inner/outer)sample,and sults in Figure 10 confirm the predicted pattern. The
quite strongin thewhole-mantle sample. muscle-type Na-channel band oflow apparent molecu-
IfNa channelsare preferentially expressed in CMP fi- lar weight (relative to the neuronal form detected in
bers, the relative abundance of Na-channel protein in brain; see also Fig. 8A) increases steadily in intensity be-
whole-mantle samples should increase during the post- tween days 3 and 100 post-hatching. At this latter time,
hatching period of maturation described in this study. the band is comparable to that in the adult squid. Be-
Mantlesampleswerethereforecollected from squiddur- cause each lane in Figure 10 was loaded with the same
ing tl: id and processed for immunoblotting. Re- amount ofprotein, these results strongly suggest that the
SQUID MANTLE MUSCLE DEVELOPMENT 383
relative abundance of Na-channel protein in mantle
muscle isincreasingduring maturation. C"ME 40p CMP fibers
^35^
Discussion 1 week old N=2, n=158
0) 30r 8 weeks old N=1, n=141
Comparison ofthe histology ofthe circular muscle of _03 " adult N=2, n=231
squid mantle during development from hatchling to 1
adult reveals large changes in the size and numberofthe 20
muscle fibers. Moreover, clear changes in the relative CD 15 II
proportions ofthe different types offiber are associated
with maturation;these, in turn, affect mantle kinematics CoO 10
b
andjet-propelled locomotion. 5
0^
E total fiber filament mitochondria
105
- D CMP fibers
io4 SMR
I fibers
i_
CD
1000
SMR
fibers
D 1 week old N=2, n=91
8 weeks old N=1, n=44
adult N=1, n=61
hatchling
I^^HIB^^H
total fiber filament mitochondria
Figure6. Characteristics ofmuscle fiber growth in CMP (A) and
B SMR(B|fibers. Individual histogramsaregivenforthetotalcross-sec-
100 D CSMMRP fibers ttihoenamlitfoicbehronadrreai,althceorfeilfaomretnhtreaendmaStRuriatryea,staagnedst(hmeeaarnesa ocScEuMpi;edNb=y
fibers numberofanimals;n = numberofmeasurements).
80 -
.2 60 Mantlegrowth in squidisduetoan increaseinthesize
of existing muscle fibers (hypertrophy) and to recruit-
2 40 ment ofnew fibers(hyperplasia). The extensive increase
in fiber number in a single muscle segment, however,
20 1 1 suggeststhat thelattermechanism isdominant inoverall
LI L. growth, and may also be responsible for the rapid so-
1 matic growth rates reported in squid (Forsythe and Van
Heukelem. 1987). Similar growth mechanisms have
hatchling 1 week 8 weeks adult been found in other squid (Moltschaniwskyj, 1994) and
Figure 5. Recruitment of CMP and SMR fibers during mantle in teleost fish (Weatherley et ai. 1988).
growth.(A)Absolutefibercountsarefromindividual musclesegments On an ultrastructural level, hypertrophy in both SMR
gfrroammohfatrcehllaitnigv,eplr-owpeoerkt-ioolnd.of8S-wMeeRk-aonldd.CaMnPd afidbuelrtsastqudiidf.fer(eBn|tHmiasttuo-- andCMPfibersisbased on asteady increSasMeRin the num-
ber ofmyonlaments and, in the case of fibers, the
ritystages.
384 T. PREUSS ET AL.
1 week old adult quiring rapid excitation-contraction coupling in the ab-
sence ofa transverse tubular system need to be small in
diameter(Boneand Ryan, 1973; Boneel at., 1995). This
constraint would especially affect the presumptive fast-
CMP
twitch fibers with their high myofilament volume-
to-surface ratio(Fig. 2). Such limitationson fiberdiame-
ter would indeed necessitate extensive recruitment of
new CMP fibers (Fig. 5) to maintain rapid muscular re-
sponsesasan animalgrowsthroughoutdevelopment.
Two biochemical results described in this paper sup-
port the idea that at least some CMP fibers display Na-
channel-basedexcitability. Suchexcitabilityisconsistent
r with the hypothesis that these are fast-twitch fibers re-
~i i 1i 2i1i8i2i4i3i0i3i6i4i2i48i 5\ 4i 60 sponsible for all-or-none mantle contractions (Young,
6
video frames 1938; Gilly et a/., 1996). The first supportingevidence is
[30/s] that Na-channel protein is primarily expressed in the
CMP
Figure 7. Manlle kinematics analyzed from video recordings of fiberlayer. Second, theincreaseofNa-channel pro-
hoveringorslow-swimmingadultandjuvenilesquid. Mantlediameter tein seen in developing mantle is comparable to the se-
wasmeasuredatthewidestpoint(dorsal view),andthefractionalman- lective hypertrophy and hyperplasia ofCMP muscle fi-
tmleeadsiuarmeemteerntwasseqcuaelncculeaatsed10b0y''!de.fTiinminegrtehseolluatrigoenstisdi3a0mfertaemresi/ns.agiven bers as shown by histology. The Na-channel band in
muscletissue isoflowerapparent molecularweightthan
that detected in neuronal tissue (Figs. 8-10), and this
size of the mitochondrial core. CMP fibers, however, suggeststhatadistinctNa-channel isoform existsin mus-
showaselective hypertrophyofthe myofilamentand SR cle. The exact relationship of this Na channel to the
area and thus add a disproportional amount of force- GFLNl-derived channels, which have a predicted core-
generatingcapability at the individual fiberlevel. Never- polypeptide massof203 kD, iscurrently unknown.
theless, hypertrophy of individual muscle fibers is lim- Nerveterminalshave been previously described in the
ited by physiological constraints. Forexample, fibers re- mantleofothersquid(Boneeta/., 1981, 1982, 1995)and
B
0)
I
o I
oLL X Oo 5ro
- 213
201 201-
mAb
1305-21 Ab483-576 Blocked 483-576 Blocked
KigureS. SpecificityofNa-channelantibodiesdeterminedwith immunoblotsofsamplesofgiantfiber
lobe(GFL),cleanedgiantaxon.cornea,and mantlemuscle.(A)mAbm,5.2i wasusedasundiluted hybrid-
oma supernatant. Secondary goat anti-mouse antibody dilution was 1:5000. (B) AbJKM76was used at 1:
1000dilution, whiledilution ofsecondarygoatanti-rabbitantibody was 1:10.000.(C)Ab48v!7t, usedat I:
2000.dilutionofsecondarygoatanti-rabbitantibodywas 1:5000.Sampleswerepreparedasdescribedand
separatedon 5't SDS-PAGEgels. Eachlanewasloadedwith lO^goftotalprotein. Followingtransferand
antibodyincubation,bandsweredetectedbychemiluminescencereagent.A260-280kDbandisdetected
inGFLandcleanaxon by monoclonal (A)and polyclonal (B)antibodies. Aspecific210-220kDband is
present in mantle muscle, but not in cornea tissue (C). Blockingcontrol wascarried out asdescribed in
Methods.