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Interdisciplinary Studies on Environmental Chemistry—Environmental Pollution and Ecotoxicology, Eds., M. Kawaguchi, K. Misaki, H. Sato, T. Yokokawa, T. Itai, T. M. Nguyen, J. Ono and S. Tanabe, pp. 41–48. © by TERRAPUB, 2012. Establishment of the Protocol for Developmental Analysis and Observation of Embryogenesis and Axonogenesis in a Freshwater Goby, Rhinogobius flumineus Masahumi KAWAGUCHI1, Junya SHIBATA2, Ryota KAWANISHI3, Atsushi SOGABE4, Torao KAWANAKA5, Koji MATSUMOTO 5, Koji O MORI1 and Yasunori MURAKAMI3 1 Center for Marine Environmental Studies, Ehime University, 2-5 Bunkyo-cho, Matsuyama 790-8577, Japan 2 Center for Ecological Research, Kyoto University, 2-509-3 Hirano, Otsu 520-2113, Japan 3 Graduate School of Science and Engineering, Ehime University, 2-5 Bunkyo-cho, Matsuyama 790-8577, Japan 4 Graduate School of Biosphere Science, Hiroshima University, 1-4-4 Kagamiyama, Higashi-Hiroshima 739-8528, Japan 5 Ehime 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 the developmental stages of a freshwater goby, Rhinogobius flumineus. The males collected from river showed the same courtship behavior in the test tank as in the field, and the fertilized eggs attached on the plastic film were conveniently acquired. The chronological observation of embryogenesis and axonogenesis revealed that the basic morphological appearance and primitive axonal tract of the freshwater goby embryo have been almost completed until 7th day of incubation. As the experimental procedure is convenient, R. flumineus will be useful as a novel model animal to study neuronal developmental biology and neuroethology. Keywords: embryology, teleost, Gobiidae, nervous system, axonal tract INTRODUCTION Freshwater goby, Rhinogobius species, is a group of Gobiidae and inhabit various freshwater 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 eggs until hatching (Kawanabe and Mizuno, 1989). Additionally, it is reported that the particular courtship behavior and the mating mode are reproducible even in the experimental condition (Takahashi and Kohda, 2001, 2004; Okuda et al., 2002). The ecological investigations have revealed that Gobiidae show the reproductive isolation, suggesting that their visual system recognize the species41 42 M. KAWAGUCHI et al. specific morphological appearance and the combinatorial patterning of courtship behavior (Kawanabe and Mizuno, 1989). Although these observations indicate the sophisticated computation by neural networks, the studies with neuroethological and neuroanatomical approaches have not been performed in Gobiidae. The neural circuits in the brain are so complicated that it is difficult to explore the neural network relating to the visual perception, recognition and decision process in Gobiidae. Therefore, we focused on the embryonic period as a novel approach, because the early scaffold of axonal tract during embryogenesis is conserved in vertebrate and provides a fundamental framework behind the complicated nervous system in adult brain (Easter et al., 1993; Barreiro-Iglesias et al., 2008). Here we established the experimental procedure to observe the embryonic period of a freshwater goby, Rhinogobius flumineus, and determined the 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 subtle regional differences in Japan (Kawanabe and Mizuno, 1989). Therefore, we collected the adult males and females in June, in the upstream of the Shigenobu River (Ehime Prefecture, Japan), with an electrofishing unit (Model LR24 Backpack Electrofisher, Smith-Root Inc.). The collected males and females were maintained 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 caudal fins 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 covered with gravels. As an artificial nest, a short polyvinyl-chloride pipe was halved lengthwise 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 test tank prepared a nest by excavating a canal under the half pipe. The conditioned females developed bright body colors and bulged abdominal regions (Fig. 1B, the back one). As soon as a conditioned female was released into the test tank, the male dramatically changed its body color to black and induced the female to the nest (Fig. 1C). The female that accepted the courtship entered into the nest and the male closed the entrance of nest by gravels (Fig. 1D). After a few days, only the female with flat abdominal region came out from the nest while the entrance of nest was still remained closed. Then, we carefully opened the nest and took out 80–120 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 800 mL 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 Fig. 1. Preparation of fertilized eggs and the developing embryos of freshwater goby. A–B, The adult males (A) and females (B) of freshwater goby rearing in the stock tank. C–D, Mating of the freshwater goby. E, Half pipe attached by a plastic film on the inner side and incubation of the eggs 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. aerated and the bubbles were adjusted to pass through the eggs. The water temperature and light condition were maintained at 21°C and a 12 hours light: 12 hours dark cycle (white fluorescent light), respectively. Dead embryos were removed and ten eggs were sampled everyday from the film with forceps. The collected embryos were observed for their morphological appearance in a stereomicroscope (Carl Zeiss, Thornwood, NY) after removing their egg envelopes physically by forceps. Then, the embryos were fixed and dehydrated with reference to Kawaguchi et al. (2011). Whole mount immunostaining The immunostaining was performed following the method described by Kawaguchi et al. (2011). Three-dimensional images of the embryonic nervous system were visualized on Zeiss LSM 510 inverted laser scan confocal microscope (Carl Zeiss) or Axio Imager.A1 fluorescent microscope (Carl Zeiss). 44 M. KAWAGUCHI et al. RESULTS Morphological appearance of the freshwater goby embryos in chronological order The embryos in a clutch developed almost simultaneously, but the timing of hatching 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% (mean value ± SD, n = 4, data not shown). The time sequential morphology of embryonic freshwater 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 was established. 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 posterior side as the protuberance dissociated from yolk sac. The protrusion in the dorsal head region was observed (Fig. 1G). This stage seems to be similar to medaka Stage 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 of hindbrain. The pectoral fin bud emerged out from the lateral side of anterior trunk region as in medaka Stage 28 (2 days and 16 hours). The heart has appeared on the 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 continuous fin surrounding the tail and dorsal trunk region was established (Fig. 1I). This stage 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 was visible in the ventral trunk region connecting to the remote heart (Fig. 1J). This stage 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 accomplished yet. Mesencephalon was expanded to the posterior side. The caudal region of continuous fin has expanded. Blood vessel formation proceeded (Fig. 1K) as in medaka 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 Fig. 2. Developmental process of the nervous system in freshwater goby embryos. The time sequential axonal scaffolding pattern in the developmental stage of freshwater goby. A, 2nd day from 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 of F. 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 the positioning of cell nuclei. C, D and E were visualized by fluorescent microscope. Bright field and 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, posterior optic commissure; OC, optic chiasm; man, mandibular nerve; nVII, facial nerve; nVIII, vestibulocochlear nerve; nIX, glossopharyngeal nerve; nX, vagus nerve. vessel in the ventral trunk region elongated to the tail edge. The otic vesicle migrated posteriorly and positioned to the lateral side of the ventral hindbrain (Fig. 1L). Developmental process of axonal scaffolding during embryogenesis of the freshwater goby The nervous system of freshwater goby was sequentially constructed as follows. 2nd day of incubation Only a couple of anterior lateral line nerves (nALL) were slightly extended along the antero-posterior axis (Fig. 2A). 46 M. KAWAGUCHI et al. 3rd day of incubation Various developing axons of peripheral and central nervous system were constructed. The olfactory epithelium was established in the anterior region and olfactory nerves (ON) entered into the telencephalion (Fig. 2C). nALL and ophthalmic nerve, a branch of the trigeminal nerves (nV), elongated together above the optic vesicle. Buccal nerve of nALL and maxillary nerve of nV entered into the upper jaw region following similar projection patterns. Posterior lateral line nerve (nPLL) extended toward the trunk region along the hindbrain and spinal cord. The segmental spinal nerves were visible slightly in the trunk region (Fig. 2B). The medial longitudinal fascicle (MLF) was identified in the ventral hindbrain along the antero-posterior axis. The segmental rhombomere has appeared in hindbrain as the transverse pattern. The posterior commissure (PC) was formed in the dorsal side of the brain between diencephalon and mesencephalon or midbrain (Fig. 2C). 4th day of incubation Optic chiasm (OC) was observed in the ventral view. Two types of major ventral commissure, anterior commissure (AC) in telencephalon and posterior optic commissure (POC) in diencephalon were formed (Figs. 2D and E). 5th day of incubation Three pairs of spinal nerves in anterior trunk region entered into the pectoral fin (Fig. 2G, open arrow head). In hindbrain, the segmental pattern of rhombomere has disappeared (Fig. 2F). The axons of optic nerves arrived at the anterior region of dorsal midbrain (Fig. 2F, open arrow head). The commissural tract connecting both midbrain hemispheres was observed (Fig. 2F, closed arrow head). 6th day of incubation The extension of craniofacial peripheral nerves including facial nerve (nVII), vestibulocochlear nerve (nVIII), glossopharyngeal nerve (nIX) and vagus nerve (nX) has almost accomplished. In also nV, the mandibular nerve started to elongate toward the ventral side of mandibular arch, nevertheless the lower jaw has not been formed yet. In addition to optic nerve, the axons emerged from hindbrain entered into midbrain (Fig. 2H). 7th day of incubation The mandibular nerve innervated the lower jaw. The number of axons that emerged from hindbrain and entered into midbrain has increased at the posterior side 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 of freshwater goby in the test tank and easily identified the developmental stage of their embryo, by using of a plastic film. This experimental framework will be useful to observe the courtship behavior and to analyze the embryogenesis of freshwater goby. The important point for maintaining adult fishes is that the density 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 disease or something else. The mating sign of the conditioned male was clear, while it was difficult to define in female. When the egg load was beyond its capacity, the female has spawned some unfertilized eggs in the stock tank. However, the female that has finished spawning once, can be made ready to lay eggs again in a season by the supply of abundant food and stable rearing environment. Therefore, toward preparing the fertilized eggs of freshwater goby efficiently, it is essential to rear fishes with low population density, to supply the comfortable situation 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 and Horigome, 2000; Murakami and Watanabe, 2009). Furthermore, the topological distribution of the identified tracts of longitudinal and transverse axonal bundles in brain was similar as in other vertebrates (Chitnis and Kuwada, 1990; Easter et al., 1993; Anderson and Key, 1999; Barreiro-Iglesias et al., 2008). Therefore, it is suggested that freshwater goby embryo possesses the early axonal scaffold (MLF, PC, AC, POC) that propose the landmark of the complicated neural networks in adult brain. These observations will provide insight into clarification of the neural circuits in adult brain, in order to explore the center for regulating reproductive behavior in freshwater goby. It is important to note that we could clearly visualize the axonal tracts entering into the dorsal midbrain, optic tectum, which is the visual highest center and integrates various sensory perceptions in teleosts (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 of their small size and thick epidermis. The large size and high permeability of freshwater goby embryo enabled us to identify the projection pattern of neural axons in the optic tectum. This advantage will be useful to elucidate the neural circuit 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 of R. 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 film enable us to conduct embryological experiments without removing the eggs from the substrate. The mortality of developing embryo is low, and the body size of larvae is so large that we can rear the early-stage larvae by feeding brine shrimp and artificial diet. In addition, R. flumineus remain at the freshwater for their whole life nevertheless many Rhinogobius species are amphidromous (Kawanabe and Mizuno, 1989). Therefore, it is easy to grow R. flumineus from egg to adult under the experimental condition. In summary, R. flumineus will be a useful model animal to study the effect on adult fishes obtained through the embryonic 48 M. KAWAGUCHI et al. experimental treatment in the physiological, neuroethological and toxicological laboratories. Acknowledgments—We would like to thank Dr. M. Inoue (Graduate School of Science and Engineering, Ehime University, Japan) for his contribution to establish the experimental procedures. We would like to gratefully thank Dr. A. Subramanian (CMES, Ehime University, Japan) for critical reading of the manuscript. This research was partially supported by “Global COE Program” by the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan, awarded to Ehime University and also by the Japan Society for the Promotion of Science (JSPS). REFERENCES Anderson, R. B. and B. 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