phuket mar. biol. cent. res. bull. 66: embryonic brain ...€¦ · after collection. the embryonic...

Phuket mar. biol. Cent. Res. Bull. 66: 155–165 (2005)

EMBRYONIC BRAIN DEVELOPMENT OF LOLIGINIDS: AXONAL SCAFFOLD ANDNEUROPIL FORMATION RELATED TO EARLY LIFE STYLES

Shuichi Shigeno1 and Masamichi Yamamoto2

1 Evolutionary Regeneration Biology Group, RIKEN Center for Developmental Biology,Kobe, Hyogo 678-0047 Japan

2 Ushimado Marine Laboratory, Okayama University, Ushimado, Okayama, 701-4303 Japan

ABSTRACT: To understand the neural basis for a variety of early life modes in cephalopods, weexamined morphological processes of the brain development in Loliolus japonica, Sepioteuthislessoniana, and Loligo edulis by immunostaining with a neuron specific marker, anti-acetylated á-tubulin antibody. In the early embryonic brain of Loliolus japonica, a simple axonal “scaffold” appearedprior to neuropil formation. A ladder-like axonal scaffold, consisting of longitudinal connectives andcommissural tracts, was formed first in the subesophageal mass, and the neuropils differentiated onthe basis of the pre-existing axonal scaffold. The development of brain lobes began with thesubesophageal and periesophageal masses; the basal lobes then appeared in the supraesophagealmass, and finally the vertical lobe system and the subpedunculate lobe appeared in the dorsal regionof the supraesophageal mass. The developmental sequences were basically very similar in the threeloliginids, but the vertical lobe system in the hatchling brain of S. lessoniana was more highly developedthan those in the other two species. The subvertical lobe in the hatchlings of S. lessoniana showed anespecially complicated domain structure, which may reflect the active predatory life of the hatchlingsof these species.

INTRODUCTION

The cephalopod brain is a complex of simpleganglionic units typical of protostomal invertebrates(Young, 1971). In cephalopods, the embryonicdevelopment (Naef, 1928; Boletzky, 1989 forreviews) and organization of the brain (Young,1971; Budelmann, 1995 for reviews) have beendescribed in many species, but little is known aboutthe early ontogeny of the axonal and neuropilpatterns during their embryonic and hatchlingstages, partly because of the absence of suitablemarkers applicable to early differentiating neurons(Martin, 1977). We have recently found that anti-acetylated á -tubulin antibody is a convenientmarker for the study of neural development incephalopods (Shigeno et al., 2001c). Previoushistological and immunohistochemical studies haveshown that the early neural patterns are basicallyconserved among various cephalopod groups.There are fundamental similarities in thedevelopment of the brain in ommastrephids(Shigeno et al., 2001a, b), loliginids (Meister, 1972;

Shigeno et al., 2001c), sepioids (Yamamoto et al.,2003) and octopods (Marquis, 1989; Yamazaki etal., 2002; Shigeno et al., in prep.), although thereare some differences characteristic to each group(Frösch, 1971). To understand the neural basis ofdifferent life modes in cephalopod hatchlings andjuveniles, knowledge of details of the braindevelopment is important. Though there is nodetailed comparative study for paralarval behaviorin loliginids, the hatchlings of S. lessoniana arepowerful swimmers with many body patternscompared to those of any other loliginids (e.g.Segawa, 1987; Watanabe, 1997). We here comparethe development of the axonal tracts and neuropilsin the embryonic brains of three loliginid species,Loligo edulis, Loliolus japonica and Sepioteuthislessoniana.

MATERIALS AND METHODS

SpecimensLoliolus (Nipponololigo) japonica (Hoyle,

1885): fertilized egg strands were collected from

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the shallow waters off Haneda in Tokyo Bay. Theembryos were transported to the laboratory of theTokyo University of Fisheries and kept in smallplastic vessels at 20–22°C (Yoshioka et al.,unpublished). At 22°C, hatching began 18 daysafter collection. The embryonic development wasdescribed in Watanabe (1997) only for the laterstages, but the normal embryonic stages of Arnold(1965) for Loligo pealei were applicable. Theembryos of blastoderm stage were 2.5 mm long(animal and vegetal axis) and the mantle length ofhatchling was 3.4 mm (Watanabe, 1997).

Sepioteuthis lessoniana Lesson, 1830:embryos and hatchlings were collected from theshallow waters near the Banda Marine BiologicalLaboratory of the Tokyo University of Fisherieson the west coast of the Boso Peninsula facing thePacific Ocean. The staging criteria of Segawa(1987) were used. All embryos and hatchlings weremaintained in small tanks with running freshseawater at a temperature of about 25°C (Shigenoet al., 2001c). The embryos of blastoderm stagewere 5.8 mm long and the mantle length ofhatchlings was 7 mm (Segawa, 1987).

Loligo (Photololigo) edulis (Hoyle, 1885):fertilized egg strands were collected from theshallow waters near the Banda Marine BiologicalLaboratory of the Tokyo University of Fisheries.The species were identified by the egg numbers inthe strand, the small egg-size, and the samplinglocality (Watanabe, 1997). The hatchlings werekept in small glass dishes at room temperature.The embryos of blastoderm stage were 1.8 mmlong and the mantle length of hatchlings was 1.7mm (Watanabe, 1997).

HistologyAll specimens were anesthetized in 2–3%

ethanol/seawater and fixed in calcium carbonatebuffered 10% formalin/seawater as previouslydescribed by Shigeno et al. (2001c). Wholemountembryos and hatchlings were stained by a modifiedCajal silver method (Stephens, 1971).

Wholemount immunohistochemistryWholemount immunohistochemistry was

performed as previously described by Shigeno andYamamoto (2002a, b), with some modifications.

The embryos and hatchlings were mechanicallydechorionated, fixed in Bouin’s solution/seawateror 10% formalin/seawater, washed well and storedin 70% ethanol at 4°C. The stored specimens werewashed in PBS containing 0.1% Tween20 (PBT)and incubated in 5µg/ml proteinaseK/PBT at 37°C.The incubation times were 5–30 min for largerspecimens, such as Sepioteuthis lessoniana andthe late embryonic stages of Loliolus japonica,and less than 5 min for smaller specimens. Afterwashing with PBT, the specimens were placed inglycine (2mg/ml, Sigma) to stop the reaction. Forimmunostaining, samples were placed whole, orafter dissection into a few pieces, in ice-cold 50%dimethyl sulfoxide (DMSO)/methanol with 10%hydrogen peroxide for 5 min. They were incubatedfor 30 min at 4°C in the DMSO solution with 1%Triton X-100, washed with Tris-buffered saline(TST; 20mM Tris-HCl, pH 8.0, 150mM NaCl,0.1% Triton X-100) containing 5% DMSO, andblocked with 5% non-fat dry milk (TSTM)overnight at 4°C. The specimens were incubatedwith anti-acetylated á-tubulin antibody (Sigma)diluted 1:1000 in TSTM for 2 days at 4°C. Afterwashing, the samples were incubated in pre-dilutedgoat anti-mouse antibody conjugated to peroxidase(Envision+, DAKO) for 12–24 hrs at roomtemperature, immersed in ice-cold 3,3’-diaminobenzidine (DAB) (1mg/ml TST) for 1 hrand reacted by adding hydrogen peroxide (0.01%)for 5–20 min in the dark.

RESULTS AND DISCUSSION

The previous results on the developmentalprocesses of the loliginid brain, obtained in Loligovulgaris (Meister 1972) and Sepioteuthis lessoniana(Shigeno et al., 2001c), are schematicallysummarized in Figure 1. In the first step, threepairs of ectodermal ganglionic placodes form acircumesophageal brain consisting of thesubesophageal, periesophageal, and supraesophagealmasses. Then, the development of neuropilsproceeds in the masses in the following order: (i)the subesophageal and periesophageal masses (e.g.the palliovisceral and magnocellular lobes), (ii) thebasal region of the supraesophageal mass (the basallobes), and (iii) the dorsal region of the

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(vmC) and paired brachio-palliovisceralconnectives (bvC) are prominent (Fig. 2B). Thepallial nerves (pN) from the stellate ganglion rundirectly into the posterior subesophageal mass (Fig.2C). Two distinct axonal bundles are observed inthe anterior and posterior region of the posteriorsubesophageal mass. One is diffused tracts at theanterior region of the posterior subesophageal massand another component can be identified asposterior magnocellular commissure (pmC); thesetwo axonal components also connect to the middlesubesophageal mass (Fig. 2A). These patterns ofthe axonal scaffold were very similar to those foundin the embryonic brain of Todarodes pacificus(Shigeno et al., 2001a, b). A major difference inthe scaffold pattern of these two squids (L.japonica and T. pacificus) is the presence of athick commissural tract in the anteriorsubesophageal mass of L. japonica, suggesting thatthis difference may be related to the heterochronicretardation (late appearance according to a commonstaging criteria) of the arm crown development inT. pacificus: there are only six arms with nascentsuckers that are observed at the hatchling stage(Hamabe, 1962).

In the subesophageal mass, neuropils beginto accumulate after stage 23 along the pre-existingaxonal scaffold. At stage 27, the posterior pedal(ppL) and palliovisceral lobes (pvL) have largeneuropil regions (Fig. 2D). The brachio-palliovisceral connectives (bvC) are particularlywell immunostained. Many tracts enter from theposterior pedal lobe (ppL) into the posteriorsubesophageal mass, where the tracts appear tobe included in the neuropils of the fin and posteriorchromatophore lobes (Fig. 2D). The neuropils ofthe palliovisceral (pvL) and posterior magnocellularlobes (pmL) are continuous but most of the axonsare commissural tracts (Fig. 2E). The distinctmiddle pedal commissure (mpC) divides the middlesubesophageal mass into two domains: the anteriorpedal lobe (apL) and the posterior pedal lobes (ppL)(Fig. 2E). The anterior end of the middlesubesophageal mass is still very immature at stage27. At the hatching stage (stage 30), thesubesophageal mass has longitudinally elongatedand almost all the brain nerves are identifiable there(Fig. 2F). The anterior subesophageal mass is

Figure 1. Schematic depiction of brain developmentin the loliginid embryo. Left-side view; top, dorsal.The black and white regions within the black areasrepresent masses of neuronal cells and neuropilsdeveloping in them. bm, buccal mass; cG, cerebralganglion; m, mantle; pG, pedal ganglion; pvG,palliovisceral ganglion; oy, outer yolk sac; sc, axonalscaffold; SEM, subesophageal mass; SPM,supraesophageal mass.

supraesophageal mass (the frontal lobes and verticallobe system) (Shigeno et al., 2001c). In thepresent study, we identified the primary structuresof the neural networks prior to the neuropildifferentiation. We could obtain three-dimensionalimages of the primary neural tracts and earlyneuropils by whole mount immunostainingtechnique using a monoclonal antibody againstacetylated á-tubulin.

The primary neural network in thesubesophageal mass appears as a very simple“scaffold” (Fig. 2A for a schematic summary).Primary tracts of the subesophageal mass can beseen as characteristic connective and commissuralpathways at stage 23. In the middle subesophagealmass, the thick ventral magnocellular commissure

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clearly separated from the middle subesophagealmass at this stage.

Axonal scaffold formation in thesupraesophageal mass is observable at stage 27.The early lobes in the supraesophageal mass areapparently along some certain specific axonalcomponents: (1) commissural tracts connectingthe right and left dorso-lateral lobes (the dorso-lateral commissure of Shigeno and Yamamoto,2002b), (2) the peduncle commissure partlyincluding tracts from the olfactory commissure,and (3) the optic commissure. In longitudinalsections, the cerebral connectives (cC), which runfrom the precommissural lobe (prL) to the superiorbuccal lobe are conspicuous (Fig. 3A). In the basalregion of the supraesophageal mass, the neuropilsof the anterior basal lobes (abL) and the dorsalbasal lobes (dbL) are also prominent (Fig. 3B).The vertical lobe (vtL) and the subpedunculate lobe(spL) are much smaller than other lobes in thesupraesophageal mass (Fig. 3B).

At hatching (stage 30), the neuropils of thevertical lobe (vtL), superior frontal lobe (sfL), andsubpedunculate lobe (spL) are well defined in themost dorsal region of the supraesophageal mass(Fig. 4A, B). A pair of neuropils in the inferior frontallobe (ifL) fuse into one mass (Fig. 4C). Thecerebral connectives (cC) are the largest tracts inthe supraesophageal mass. The subvertical lobe(svL) is characterized by its large paired neuropils,large axonal bundles from the superior frontal lobe,and very fine tracts connecting the paired neuropils(Fig. 4B). The vertical lobe (vtL) is dome-shapedabove the subvertical lobe (svL) and showed manyneural connections with the subpedunculate lobe(spL) (Fig. 4D). Young (1979) divided the spL into3 domains (spL1, 2, 3) in some loliginids. In L.japonica, we found 4 domains in thesubpedunculate lobe 3, two of which are shownin figure 4E. Many fine tracts connect the 4 neuropildomains with each other (Fig. 4E).

In Sepioteuthis lessoniana, Shigeno et al.(2001c) have described distinctive axons, stainablewith the Cajal silver technique (Stephens, 1971) inthe late embryonic stages, but little is known aboutthe neural network formation in the supraesophagealmass. The embryos and hatchlings of S. lessonianaare relatively large in size among loliginids. From a

Figure 2. The axonal scaffold and neuropils in thesubesophageal mass of Loliolus japonica.Immunostaining with anti-acetylated á -tubulinantibody. (A-C, E, F) Ventral view; top, anterior. (D)Left-side view; top, dorsal. A: Schematic figure of theladder-like axonal framework in the subesophagealmass at stage 23. Brain nerves are omitted forsimplification. B: Left part of the anterior and middlesubesophageal mass at stage 23. C: Left part of theposterior subesophageal mass at stage 23. D: Wholeview of the subesophageal mass at stage 27, lateralview. E: Whole view of the subesophageal mass atstage 27, ventral view. F: Anterior and middlesubesophageal mass at stage 30. ASM, anteriorsubesophageal mass; apL, anterior pedal lobe; bvC,brachio-palliovisceral connective; mpC, middle pedalcommissure; MSM, middle subesophageal mass;pmC, posterior magnocellular commissure; pmL,posterior magnocellular lobe; pN, pallial nerve; ppL,posterior pedal lobe; PSM, posterior subesophagealmass; pvL, palliovisceral lobe; st, statocyst; vmC,ventral magnocellular commissure; vmL, ventralmagnocellular lobe; vsN, visceral nerve. Bars, 0.3mm.

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Figure 3. The axonal scaffold and neuropils in the supraesophageal mass of Loliolus japonica atstage 27. Immunostaining with anti-acetylated á-tubulin antibody. A: Dorsal view of the wholesupraesophageal mass. Top, anterior. B: Left-side view of the whole supraesophageal mass. Top,dorsal. abL, anterior basal lobe; cC, cerebral connective; dbL, dorsal basal lobe; dlL, dorsolateral lobe;oC, optic commissure; pdC, peduncle commissure; prL, precommissural lobe; sfL, superior frontallobe; spL, subpedunculate lobe; svL, subvertical lobe; vtL, vertical lobe. Bar, 0.3mm.

comparative point of view, we examined the higherintegration centers in the supraesophageal massof the late embryonic brains of S. lessoniana byimmunohistochemical technique. At stage 28,tubulin positive axons were clearly visible in thesupraesophageal mass (Fig. 5A). A massiveconnective can be seen running from theprecommissural lobe to the superior frontal lobe(Fig. 5B). In the subvertical lobe (svL), large pairedneuropils grow to a posterior direction, and manyaxons enter it mainly from the superior frontal lobe(sfL) and the precommissural lobe (prL) (Fig. 5C).In the optic tract zone of the hatchling brain, thedorso-lateral lobe (dlL) grows to a posteriordirection (Fig. 5D, E). Many axons run out fromthe peduncle lobe (pdL) (Fig. 5E). The subverticallobe is subdivided into several domains: anteriorsubvertical lobe (asvL), posterior subvertical lobe(psvL), posterior protrusion (pp), and proliferationzone (pz) (Fig. 5F). The posterior subvertical lobeextends broadly in the posterior region of thesubvertical lobe, and receives dense axonal tractsfrom many lobes, particularly from the superiorfrontal lobe (sfL) and the precommissural lobe(prL) as described by Young (1979). Theproliferation zone is situated at the posterior to theposterior subvertical lobe and consists of many

small clusters of neuropils that are mutuallyinterconnected (Fig. 5F). The vertical lobe (vtL)is a thin and U-shaped plate at earlier stages (Fig.6A), markedly extends both in anterior andposterior directions at late embryonic stages (Fig.6B), and eventually forms a large dome in the mostdorsal zone of the supraesophageal mass (stage30) (Fig. 6C). Numerous nerve fibers arise fromthe superior frontal lobe and subpedunculate lobe,spreading broadly over the neuropil of the verticallobe (Fig. 5D; 6C). These structures describedabove in the developing brain of S. lessoniana arebasically similar to those of L. japonica, but ahigher degree of development of the neuropil inthe vertical lobe (Fig. 5D) and the complicatedfeature of the subvertical lobe (svL) (Fig. 5F) wereunique features of S. lessoniana.

To confirm whether the differences betweenthe supraesophageal mass of the early brains inloliginids are related to the embryonic size, weexamined the supraesophageal mass of the hatchlingbrains (stage 30) of Loligo edulis, whoseparalarvae are characteristically small amongstloliginids (Watanabe, 1997). The developing brainof the embryos and hatchlings of L. edulis closelyresembles that of L. japonica, but some lobes,especially the subvertical lobe, shows characteristic

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Figure 4. Neuropils in the supraesophageal mass of Loliolus japonica at hatching stage (stage 30).Immunostaining with anti-acetylated á-tubulin antibody. (A) Left-side view; top, dorsal. (B-E) Dorsalview; top, anterior. A: Whole view of the supraesophageal mass. B: The supraesophageal mass at thelevel of the subvertical lobe. C: Anterior part of the supraesophageal mass. D: Middle part of thesupraesophageal mass. Arrowhead indicates connectives radiating from the vertical (vtL) and thesubvertical lobe. E: Posterior part of the supraesophageal mass. Two domains of the subpedunculatelobe are shown by 1 and 2, and small fibers running over the precommissural lobe (prL) are shown bya white arrowhead. Magnification is equal to D. abL, anterior basal lobe; cC, cerebral connective; dbL,dorsal basal lobe; dlL, dorsolateral lobe; ibL, inferior buccal lobe; ifL, inferior frontal lobe; ofL, olfactorylobe; pdL, peduncle lobe; prL, precommissural lobe; sbL, superior buccal lobe; sfL, superior frontallobe; spL, subpedunculate lobe; svL, subvertical lobe; vtL, vertical lobe. Bars, 0.3mm.

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Figure 5. Neuropils in the supraesophageal mass of Sepioteuthis lessoniana at late embryonic stages.Immunostaining with anti-acetylated á-tubulin antibody. A: Whole lateral view at stage 28; top, dorsal.B: Lateral view of the supraesophageal mass at stage 28+. C: Subvertical lobe (svL) at stage 28. D:Whole dorsal view of the supraesophageal mass at stage 30. White arrowheads indicate the superiorfrontal to vertical lobe tracts. E: Right optic tract region at stage 30. Dorsal view. F: Largely developedsubvertical lobe to show the complex domains at hatching stage (stage 30). Dorsal view. The proliferationzone (pz) is enclosed by a white dotted line. asvL, anterior subvertical lobe; cC, cerebral connectives;dbL, dorsal basal lobe; dlL, dorsolateral lobe; ifL, inferior frontal lobe; iy, inner yolk; ofL, olfactorylobe; pdL, peduncle lobe; pp, posterior protrusion; ppL, posterior pedal lobe; prL, precommissurallobe; psvL, posterior subvertical lobe; sfL, superior frontal lobe; spL, subpedunculate lobe; svL,subvertical lobe; vtL, vertical lobe. Bars, 0.3mm (A-E), 0.4mm (F).

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differences from those in the developing brain ofS. lessoniana. The mid-sagittal section reveals thatthe neuropils of the precommissural lobe and thesuperior frontal lobe are large in the central zoneof the supraesophageal mass of the late embryonicbrain of both L. edulis and S. lessoniana (Fig. 7A–C). Small neuropils of the subpedunculate lobe(spL), similar in size in both L. edulis and S.lessoniana, are visible in the posterior area of thesupraesophageal mass (arrowheads in Fig. 7A–C).In the hatchling brain of L. edulis, the subverticallobe (svL) is simple in outline of the neuropiloccupied only the region just behind the superiorfrontal lobe (sfL) (Fig. 7A). In contrast, in theembryonic brain of S. lessoniana, the neuropil ofthe subvertical lobe is already large and complexin outline and grows posteriorly (Fig. 7B, C). Inthe hatchling of L. edulis, as in the hatchling of L.japonica (Fig. 4B), the posterior domains of thesubvertical lobe (asvL, psvL, pp, and pz) are notso prominent compared to the hatchling of S.lessoniana (Fig. 8 for a schematic figure). In mid-sagittal sections of the embryonic brains, the

neuropil of the vertical lobe (vtL) shows a slenderprofile. In S. lessoniana, the profile is short at stage27 (Fig. 7B) but elongated both in anterior andposterior directions at stage 28 (Fig. 7C). In S.lessoniana, the dorsal perikaryal layer of the verticallobe is two or three cells thick at the midsagittalsections of stage 27, and becomes at least fourcells after stage 28. In contrast, the vertical lobe(vtL) in the hatchling of L. edulis, as in thehatchlings of L. japonica (Fig. 4A), is relativelyundeveloped (Fig. 8); the mid-sagittal profile ofthe vertical lobe is short (Fig. 7A) and the dorsalperikaryal layer is two cells thick.

The developing brains of L. japonica and L.edulis resemble each other but differences wererecognized in the vertical lobe system between S.lessoniana and these two species (Fig. 8). Thecontrast is especially prominent in the neuropilpattern of the subvertical lobe (svL) (Fig. 8).Frösch (1971) and Nixon and Mangold (1996)have suggested that the volume of lobes indeveloping brains reflects the specific mode of lifein various cephalopod species. We have reported

Figure 6. Dorsal views of the supraesophageal mass in the developing brain of Sepioteuthis lessoniana.Immunostaining with anti-acetylated á-tubulin antibody. Top, anterior. A: U-shaped nascent neuropilof the vertical lobe (vtL) (enclosed by dotted line) at stage 27. The lateral lines in the dorsal surface ofthe embryo are innervated. B: Neuropil of the vertical lobe (vtL) extending in anterior and posteriordirections at stage 28. C: Vertical lobe (vtL) and many fine tubulinergic tracts at stage 30. dbL, dorsalbasal lobe; dl, dorsal lateral line; sfL, superior frontal lobe; spL, subpedunculate lobe. vtL, verticallobe. Bars, 0.4mm (A, B), 0.3mm (C).

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that development of the vertical lobe system inTodarodes pacificus shows a prominentheterochronic delay (Shigeno et al., 2001a, b) incomparison with that in S. lessoniana (Shigeno etal., 2001c) and in Idiosepius paradoxus(Yamamoto et al., 2003; Shigeno and Yamamoto,in prep.). We have suggested that the heterochronyis a reflection of the mode of paralarval life “ theparalarvae of Todarodes pacificus are presumedto be suspension feeders (O’Dor et al., 1985) butthe paralarvae of S. lessoniana (Shigeno et al.,2001d) and Idiosepius paradoxus are activepredators. Among the three loliginid speciesexamined in the present study, the paralarva of S.lessoniana is much larger in size and shows moreactive and sophisticated predatory behaviors thanthose of the other two loliginid species. In S.lessoniana, heterochronic shifts have beenreported in the development of various organsagainst conserved external structures (Shigeno etal., 2001d). The structural differences in thedeveloping brain among the three loliginids studiedmay be a result of the heterochronic shift: thecomplex structure of the subvertical lobe in S.lessoniana may appear at postembryonic stagesof L. japonica and L. edulis. It is necessary toinvestigate more detailed comparative anatomy forthe subvertical lobe with relation to the paralarvalbehavior.

ACKNOWLEDGEMENTS

We thank S. Segawa, K. Fuzita, M. Yoshioka,T. Izuka, K. Watanabe, and members of the BandaMarine Biological Laboratory of the TokyoUniversity of Fisheries for collecting andmaintaining materials. This work was supportedby a grant from the Research Institute of MarineInvertebrates to S.S.

Figure 7. Midsagittal section of the wholesupraesophageal mass. Cajal silver staining. Top,dorsal; left, anterior. A: Loligo edulis. Stage 30. B:Sepioteuthis lessoniana. Stage 27. C: Sepioteuthislessoniana. Stage 28. The black and white arrowheadsindicate the subpedunculate lobe 1 and 3, respectively.abL, anterior basal lobe; dbL, dorsal basal lobe; prL,precommissural lobe; sfL, superior frontal lobe; svL,subvertical lobe; vtL, vertical lobe. Bars, 0.5mm (A)and 0.4mm (B, C).

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Figure 8. Schematic figures to compare the arrangement of neuropils in the supraesophageal mass(above) and the arrangement of neuropils in the subvertical lobe (below) between the hatchlings ofLoliolus japonica and Sepioteuthis lessoniana. Dorsal view; top anterior. asvL, anterior subverticallobe; dlL, dorsolateral lobe; ifL, inferior frontal lobe; ofL, olfactory lobe; pdL, peduncle lobe; pp,posterior protrusion; psvL, posterior subvertical lobe; pz, proliferation zone; sfL, superior frontallobe; spL, subpedunculate lobe; vtL, vertical lobe.

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