41

Interdisciplinary Studies on Environmental Chemistry—Environmental Pollution and Ecotoxicology,Eds., M. Kawaguchi, K. Misaki, H. Sato, T. Yokokawa, T. Itai, T. M. Nguyen, J. Onoand S. Tanabe, pp. 41–48.© by TERRAPUB, 2012.

Establishment of the Protocol for Developmental Analysisand Observation of Embryogenesis and Axonogenesis

in a Freshwater Goby, Rhinogobius flumineus

Masahumi KAWAGUCHI1, Junya SHIBATA2, Ryota KAWANISHI3, Atsushi SOGABE4,Torao KAWANAKA5, Koji MATSUMOTO5, Koji OMORI1 and Yasunori MURAKAMI3

1Center for Marine Environmental Studies, Ehime University,2-5 Bunkyo-cho, Matsuyama 790-8577, Japan

2Center for Ecological Research, Kyoto University,2-509-3 Hirano, Otsu 520-2113, Japan

3Graduate School of Science and Engineering, Ehime University,2-5 Bunkyo-cho, Matsuyama 790-8577, Japan

4Graduate School of Biosphere Science, Hiroshima University,1-4-4 Kagamiyama, Higashi-Hiroshima 739-8528, Japan

5Ehime University Senior High School, 3-2-40 Tarumi, Matsuyama 790-8566, Japan

(Received 30 September 2011; accepted 12 December 2011)

Abstract—We established the experimental protocol to analyze thedevelopmental stages of a freshwater goby, Rhinogobius flumineus. The malescollected from river showed the same courtship behavior in the test tank as inthe field, and the fertilized eggs attached on the plastic film were convenientlyacquired. The chronological observation of embryogenesis and axonogenesisrevealed that the basic morphological appearance and primitive axonal tract ofthe freshwater goby embryo have been almost completed until 7th day ofincubation. As the experimental procedure is convenient, R. flumineus will beuseful as a novel model animal to study neuronal developmental biology andneuroethology.

Keywords: embryology, teleost, Gobiidae, nervous system, axonal tract

INTRODUCTION

Freshwater goby, Rhinogobius species, is a group of Gobiidae and inhabit variousfreshwater systems in the temperate zone of Asian countries. In these species,males establish the nest under a big stone and show the unique courtship behavior.The eggs are adhered to the ceiling of the stone and males take care of the eggsuntil hatching (Kawanabe and Mizuno, 1989). Additionally, it is reported that theparticular courtship behavior and the mating mode are reproducible even in theexperimental condition (Takahashi and Kohda, 2001, 2004; Okuda et al., 2002).

The ecological investigations have revealed that Gobiidae show thereproductive isolation, suggesting that their visual system recognize the species-

42 M. KAWAGUCHI et al.

specific morphological appearance and the combinatorial patterning of courtshipbehavior (Kawanabe and Mizuno, 1989). Although these observations indicatethe sophisticated computation by neural networks, the studies withneuroethological and neuroanatomical approaches have not been performed inGobiidae. The neural circuits in the brain are so complicated that it is difficult toexplore the neural network relating to the visual perception, recognition anddecision process in Gobiidae. Therefore, we focused on the embryonic period asa novel approach, because the early scaffold of axonal tract during embryogenesisis conserved in vertebrate and provides a fundamental framework behind thecomplicated nervous system in adult brain (Easter et al., 1993; Barreiro-Iglesiaset al., 2008). Here we established the experimental procedure to observe theembryonic period of a freshwater goby, Rhinogobius flumineus, and determinedthe developmental process of their axonal scaffold.

EXPERIMENTAL PROCEDURE

Preparation of fertilized eggs of the freshwater goby

The mating season of R. flumineus is from May to August with subtleregional differences in Japan (Kawanabe and Mizuno, 1989). Therefore, wecollected the adult males and females in June, in the upstream of the ShigenobuRiver (Ehime Prefecture, Japan), with an electrofishing unit (Model LR24Backpack Electrofisher, Smith-Root Inc.). The collected males and females weremaintained separately at 21°C in the stock tank (600 mm × 300 mm × 350 mm).The males conditioned for mating showed clear red rays in the dorsal and caudalfins and they intimidated by expanding their fins and opening their mouths widely(Fig. 1A). The bottom of test tank (350 mm × 220 mm × 250 mm) was coveredwith gravels. As an artificial nest, a short polyvinyl-chloride pipe was halvedlengthwise and attached a plastic film (Traceter Z-300.S, Somar Co., Ltd, Tokyo)on its inner side (Fig. 1E), in order to adhere the sedentary eggs to the surface(Okuda et al., 2002; Shibata and Kohda, 2006). The male released into the testtank prepared a nest by excavating a canal under the half pipe. The conditionedfemales developed bright body colors and bulged abdominal regions (Fig. 1B, theback one). As soon as a conditioned female was released into the test tank, themale dramatically changed its body color to black and induced the female to thenest (Fig. 1C). The female that accepted the courtship entered into the nest andthe male closed the entrance of nest by gravels (Fig. 1D). After a few days, onlythe female with flat abdominal region came out from the nest while the entranceof nest was still remained closed. Then, we carefully opened the nest and took out80–120 fertilized eggs in a clutch attached on the film (Fig. 1E).

Egg incubation and sampling

The eggs attached on the film were put in a 1 L glass beaker filled with 800mL freshwater, which was filtered through EHEIM classic (EHEIM GmbH & Co.KG) and treated with UV (Fig. 1E). The freshwater in the beaker was continuously

Developmental Biology of Freshwater Goby 43

aerated and the bubbles were adjusted to pass through the eggs. The watertemperature and light condition were maintained at 21°C and a 12 hours light: 12hours dark cycle (white fluorescent light), respectively. Dead embryos wereremoved and ten eggs were sampled everyday from the film with forceps. Thecollected embryos were observed for their morphological appearance in astereomicroscope (Carl Zeiss, Thornwood, NY) after removing their egg envelopesphysically by forceps. Then, the embryos were fixed and dehydrated withreference to Kawaguchi et al. (2011).

Whole mount immunostaining

The immunostaining was performed following the method described byKawaguchi et al. (2011). Three-dimensional images of the embryonic nervoussystem were visualized on Zeiss LSM 510 inverted laser scan confocal microscope(Carl Zeiss) or Axio Imager.A1 fluorescent microscope (Carl Zeiss).

Fig. 1. Preparation of fertilized eggs and the developing embryos of freshwater goby. A–B, The adultmales (A) and females (B) of freshwater goby rearing in the stock tank. C–D, Mating of thefreshwater goby. E, Half pipe attached by a plastic film on the inner side and incubation of theeggs adhered to the film. F–L, Lateral view of the freshwater goby embryos at 1st (F), 2nd (G),3rd (H), 4th (I), 5th (J), 6th (K) and 7th (L) day of incubation.

44 M. KAWAGUCHI et al.

RESULTS

Morphological appearance of the freshwater goby embryos in chronologicalorder

The embryos in a clutch developed almost simultaneously, but the timing ofhatching differed among each embryo. The larvae in a clutch hatched out at 13th–15th day of incubation at 21°C. The hatching rate of larvae was 85.0 ± 8.3% (meanvalue ± SD, n = 4, data not shown). The time sequential morphology of embryonicfreshwater goby in each day of incubation was observed as bellow.

The stage of embryonic shield formation (1st day of incubation)Epiboly proceeded and the embryonic shield was observed at the dorsal side

of embryonic body (Fig. 1F, open arrow head). Antero-posterior axis wasestablished. This stage appears to be corresponding to medaka Stage 13–15 (13–17.5 hours post fertilization; Iwamatsu, 2004).

The stage of segmentation (2nd day of incubation)Optic and otic vesicles have been recognized but unclear yet. The segmental

somites in the trunk region were observed. The tail bud was visible at the posteriorside as the protuberance dissociated from yolk sac. The protrusion in the dorsalhead region was observed (Fig. 1G). This stage seems to be similar to medakaStage 22–23 (1 day and 14–17 hours).

The stage of pharyngula (3rd day of incubation)The pigmentation of eye partially progressed. The rhombencephalic isthmus

was discernible. The otic vesicle was remarkably visible on the ventral side ofhindbrain. The pectoral fin bud emerged out from the lateral side of anterior trunkregion as in medaka Stage 28 (2 days and 16 hours). The heart has appeared onthe dorsal surface of yolk sac, separated from the body axis (Fig. 1H).

The stage of dorsal fin formation (4th day of incubation)A couple of obvious eyespots were visible. The segmental sacromeres have

appeared until the tail edge. The pectoral fin was well defined and the continuousfin surrounding the tail and dorsal trunk region was established (Fig. 1I). Thisstage appears to be similar to medaka Stage 30 (3 days 10 hours).

The stage of blood vessel formation (5th day of incubation)The swelling of mesencephalon was unremarkable because of covering tela

choroidea ventriculi quarti over the hindbrain. The colored blood vessel wasvisible in the ventral trunk region connecting to the remote heart (Fig. 1J). Thisstage seems to be similar to medaka Stage 32 (4 days 5 hours).

The stage of brain expansion (6th day of incubation)The size of the premandibular region increased, following the expansion of

telencephalon. However, the construction of lower jaw has not been accomplishedyet. Mesencephalon was expanded to the posterior side. The caudal region ofcontinuous fin has expanded. Blood vessel formation proceeded (Fig. 1K) as inmedaka Stage 34–35 (5 days 1–12 hours).

The stage of lower jaw formation (7th day of incubation)The lower jaw has clearly appeared and the heart has been stored in the

posterior side of lower jaw as in medaka Stage 36 (6 days). The colored blood

Developmental Biology of Freshwater Goby 45

vessel in the ventral trunk region elongated to the tail edge. The otic vesiclemigrated posteriorly and positioned to the lateral side of the ventral hindbrain(Fig. 1L).

Developmental process of axonal scaffolding during embryogenesis of thefreshwater goby

The nervous system of freshwater goby was sequentially constructed asfollows.

2nd day of incubationOnly a couple of anterior lateral line nerves (nALL) were slightly extended

along the antero-posterior axis (Fig. 2A).

Fig. 2. Developmental process of the nervous system in freshwater goby embryos. The timesequential axonal scaffolding pattern in the developmental stage of freshwater goby. A, 2nd dayfrom lateral view. B–C, 3rd day from lateral (B) and dorsal view (C). D–E, 4th day from dorsal(D) and ventral view (E). F–G, 5th day from dorsal view. G is a higher magnification view ofF. Asterisks show the pectoral fin. H, 6th day from lateral view. I, 7th day from lateral view. A,B, F, G, H and I were observed by the laser scan confocal microscope. Blue signal means thepositioning of cell nuclei. C, D and E were visualized by fluorescent microscope. Bright fieldand fluorescent views were merged. nALL, anterior lateral line nerve; OE, olfactory epithelium;PC, posterior commisure; oph, ophthalmic nerve; buc, buccal nerve; max, maxillary nerve; nV,trigeminal nerve; nPLL, posterior lateral line nerve; nSp, spinal nerve; olf, olfactory nerve;MLF, medial longitudinal fascicle; rho, rhombomere; AC, anterior commissure; POC, posterioroptic commissure; OC, optic chiasm; man, mandibular nerve; nVII, facial nerve; nVIII,vestibulocochlear nerve; nIX, glossopharyngeal nerve; nX, vagus nerve.

46 M. KAWAGUCHI et al.

3rd day of incubationVarious developing axons of peripheral and central nervous system were

constructed. The olfactory epithelium was established in the anterior region andolfactory nerves (ON) entered into the telencephalion (Fig. 2C). nALL andophthalmic nerve, a branch of the trigeminal nerves (nV), elongated togetherabove the optic vesicle. Buccal nerve of nALL and maxillary nerve of nV enteredinto the upper jaw region following similar projection patterns. Posterior lateralline nerve (nPLL) extended toward the trunk region along the hindbrain andspinal cord. The segmental spinal nerves were visible slightly in the trunk region(Fig. 2B). The medial longitudinal fascicle (MLF) was identified in the ventralhindbrain along the antero-posterior axis. The segmental rhombomere has appearedin hindbrain as the transverse pattern. The posterior commissure (PC) was formedin the dorsal side of the brain between diencephalon and mesencephalon ormidbrain (Fig. 2C).

4th day of incubationOptic chiasm (OC) was observed in the ventral view. Two types of major

ventral commissure, anterior commissure (AC) in telencephalon and posterioroptic commissure (POC) in diencephalon were formed (Figs. 2D and E).

5th day of incubationThree pairs of spinal nerves in anterior trunk region entered into the pectoral

fin (Fig. 2G, open arrow head). In hindbrain, the segmental pattern of rhombomerehas disappeared (Fig. 2F). The axons of optic nerves arrived at the anterior regionof dorsal midbrain (Fig. 2F, open arrow head). The commissural tract connectingboth midbrain hemispheres was observed (Fig. 2F, closed arrow head).

6th day of incubationThe extension of craniofacial peripheral nerves including facial nerve

(nVII), vestibulocochlear nerve (nVIII), glossopharyngeal nerve (nIX) and vagusnerve (nX) has almost accomplished. In also nV, the mandibular nerve started toelongate toward the ventral side of mandibular arch, nevertheless the lower jawhas not been formed yet. In addition to optic nerve, the axons emerged fromhindbrain entered into midbrain (Fig. 2H).

7th day of incubationThe mandibular nerve innervated the lower jaw. The number of axons that

emerged from hindbrain and entered into midbrain has increased at the posteriorside of dorsal midbrain (Fig. 2I).

DISCUSSION

Instruction for preparing the fertilized eggs of freshwater goby

In the present study, we successfully induced the courtship behavior offreshwater goby in the test tank and easily identified the developmental stage oftheir embryo, by using of a plastic film. This experimental framework will beuseful to observe the courtship behavior and to analyze the embryogenesis offreshwater goby. The important point for maintaining adult fishes is that thedensity of the goby in stock tanks should be low. The intensive rearing induced

Developmental Biology of Freshwater Goby 47

high mortality because of the fight between fishes, the bacterial infectious diseaseor something else. The mating sign of the conditioned male was clear, while it wasdifficult to define in female. When the egg load was beyond its capacity, thefemale has spawned some unfertilized eggs in the stock tank. However, thefemale that has finished spawning once, can be made ready to lay eggs again ina season by the supply of abundant food and stable rearing environment.Therefore, toward preparing the fertilized eggs of freshwater goby efficiently, itis essential to rear fishes with low population density, to supply the comfortablesituation and to check carefully the condition of individual female.

The early developing nervous system of the freshwater goby

In the freshwater goby embryo, the position of craniofacial peripheral nerves(ON, OC, nV, nVII-X, nALL) was identical to the other vertebrates (Kuratani andHorigome, 2000; Murakami and Watanabe, 2009). Furthermore, the topologicaldistribution of the identified tracts of longitudinal and transverse axonal bundlesin brain was similar as in other vertebrates (Chitnis and Kuwada, 1990; Easter etal., 1993; Anderson and Key, 1999; Barreiro-Iglesias et al., 2008). Therefore, itis suggested that freshwater goby embryo possesses the early axonal scaffold(MLF, PC, AC, POC) that propose the landmark of the complicated neuralnetworks in adult brain. These observations will provide insight into clarificationof the neural circuits in adult brain, in order to explore the center for regulatingreproductive behavior in freshwater goby. It is important to note that we couldclearly visualize the axonal tracts entering into the dorsal midbrain, optic tectum,which is the visual highest center and integrates various sensory perceptions inteleosts (Figs. 2F, H and I). In the major model fish such as zebrafish and medaka,it is difficult to observe the developing neural networks in midbrain, because oftheir small size and thick epidermis. The large size and high permeability offreshwater goby embryo enabled us to identify the projection pattern of neuralaxons in the optic tectum. This advantage will be useful to elucidate the neuralcircuit relating to the information processing of courtship behavior.

Suitability of Rhinogobius flumineus for experimental embryology

Here we designed a convenient framework for the developmental analysis ofR. flumineus. When compared to other Rhinogobius species, the eggs of R.flumineus are so large that it is capable of manipulating the embryo; for instance,microinjection of transgenes or toxicants. The fertilized eggs attached on the filmenable us to conduct embryological experiments without removing the eggs fromthe substrate. The mortality of developing embryo is low, and the body size oflarvae is so large that we can rear the early-stage larvae by feeding brine shrimpand artificial diet. In addition, R. flumineus remain at the freshwater for theirwhole life nevertheless many Rhinogobius species are amphidromous (Kawanabeand Mizuno, 1989). Therefore, it is easy to grow R. flumineus from egg to adultunder the experimental condition. In summary, R. flumineus will be a usefulmodel animal to study the effect on adult fishes obtained through the embryonic

48 M. KAWAGUCHI et al.

experimental treatment in the physiological, neuroethological and toxicologicallaboratories.

Acknowledgments—We would like to thank Dr. M. Inoue (Graduate School of Scienceand Engineering, Ehime University, Japan) for his contribution to establish the experimentalprocedures. We would like to gratefully thank Dr. A. Subramanian (CMES, EhimeUniversity, Japan) for critical reading of the manuscript. This research was partiallysupported by “Global COE Program” by the Ministry of Education, Culture, Sports,Science and Technology (MEXT), Japan, awarded to Ehime University and also by theJapan Society for the Promotion of Science (JSPS).

REFERENCES

Anderson, R. B. and B. Key (1999): Novel guidance cue during neuronal pathfinding in the earlyscaffold of axon tracts in the rostral brain. Development, 126, 1859–1868.

Barreiro-Iglesias, A., B. Villar-Cheda, X. M. Abalo, R. Anadon and M. C. Rodicio (2008): The earlyscaffold of axon tracts in the brain of a primitive vertebrate, the sea lamprey. Brain Res. Bull.,75, 42–52.

Chitnis, A. B. and J. Y. Kuwada (1990): Axonogenesis in the brain of zebrafish embryos. J.Neurosci., 10, 1892–1905.

Easter, S. S., L. S. Ross and A. Frankfurter (1993): Initial tract formation in the mouse brain. J.Neurosci., 13, 285–299.

Iwamatsu, T. (2004): Stages of normal development in the medaka Oryzias latipes. Mech. Dev., 121,605–618.

Kawaguchi, M., J. Y. Song, K. Irie, Y. Murakami, K. Nakayama and S. I. Kitamura (2011):Distribution of Sema3A expression causes abnormal neural projection in heavy oil exposedJapanese flounder larvae. Mar. Pollut. Bull., 63, 356–361.

Kawanabe, H. and N. Mizuno (1989): Freshwater Fishes of Japan. YAMA-KEI Publishers Co., Ltd,Tokyo.

Kuratani, S. and N. Horigome (2000): Developmental morphology of branchiomeric nerves in a catshark, Scyliorhinus torazame, with special reference to rhombomeres, cephalic mesoderm, anddistribution patterns of cephalic crest cells. Zool. Sci., 17, 893–909.

Murakami, Y. and A. Watanabe (2009): Development of the central and peripheral nervous systemsin the lamprey. Dev. Growth Differ., 51, 197–205.

Okuda, N., S. Ito and H. Iwao (2002): Female spawning strategy in Rhinogobius sp. OR: how dofemales deposit their eggs in the nest? Ichthyol. Res., 49, 371–379.

Shibata, J. and M. Kohda (2006): Seasonal sex role changes in the blenniid Pectroscirtes breviceps,a nest brooder with paternal care. J. Fish Biol., 69, 203–214.

Takahashi, D. and M. Kohda (2001): Females of stream goby choose mates that court in fast watercurrents. Behavior, 138, 937–946.

Takahashi, D. and M. Kohda (2004): Courtship in fast water currents by a male strem goby(Rhinogobius brunneus) communicates the paternal quality honestly. Behav. Ecol. Sociobiol.,55, 431–438.

M. Kawaguchi (e-mail: kawa@sci.ehime-u.ac.jp)