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Interdisciplinary Studies on Environmental Chemistry—Environmental Pollution and Ecotoxicology,
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© 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
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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).
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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).
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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
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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. Key (1999): Novel guidance cue during neuronal pathfinding in the early
scaffold 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 early
scaffold 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 exposed
Japanese 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 cat
shark, Scyliorhinus torazame, with special reference to rhombomeres, cephalic mesoderm, and
distribution patterns of cephalic crest cells. Zool. Sci., 17, 893–909.
Murakami, Y. and A. Watanabe (2009): Development of the central and peripheral nervous systems
in the lamprey. Dev. Growth Differ., 51, 197–205.
Okuda, N., S. Ito and H. Iwao (2002): Female spawning strategy in Rhinogobius sp. OR: how do
females 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 water
currents. 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: [email protected])