Download NORMAL EMBRYONIC DEVELOPMENT OF ZEBRAFISH, Danio rerio

NORMAL EMBRYONIC DEVELOPMENT OF
ZEBRAFISH, Danio rerio
8.1. INTRODUCTION
The zebrafish, Danio rerio (Hamilton), is one of the most
important vertebrate model organism in developmental biology
(Grunwald and Eisen, 2002). Zebrafish eggs are large relative to other fish
(0.7 mm in diameter at fertilization) and optically transparent, the yolk
being sequestered into a separate cell. Furthermore, fertilization is
external so live embryos are accessible to manipulation and can be
monitored through all developmental stages under a dissecting microscope
(Kimmel et al., 1995). Development is rapid, with precursors to all
major organs developing within 3 hr and larvae displaying food seeking
and active avoidance behaviours within five days post fertilization,
i.e, 2-3 days after hatching (Kimmel et al., 1995).
A popular aquarium species, the zebrafish has been used
in developmental biology for many years (eg, Creaser, 1934). A staging
series is a tool that provides accuracy in developmental studies.
Still, knowledge of early ontogeny is of critical importance in
understanding the biology of a species and the functional trends and
environmental preferences of the different developmental stages
(Koumoundouros et al., 2001; Borcato et al., 2004). A detailed
understanding of the ontogeny is therefore essential to identify
species-specific adaptations and their ecological consequences
(Verreth et al., 1992). An earlier staging series for zebrafish, although
less complete than the present one. The developmental rates of
domesticated strains in the laboratory have been differed from wild
fish (Eaton and Farley, 1974a). This part of the embryo study provide
a detailed illustration of the normal development of zebrafish (D. rerio)
and an integral part of the guideline for the embryo-toxicity tests.
Knowledge of normal early developmental stages of zebrafish as a
important guideline for the eco-toxicological test. Therefore, the
developmental biology of control zebrafish, D. rerio was undertaken as
a part in present work of toxicological testing on zebrafish embryo
with the toxicity of As(III) oxide.
8.2. RESULTS
8.2.1. Normal embryonic development of Danio rerio
The zebrafish (Danio rerio) egg is telolecithal, cleavage is
meroblastic and discoidal. The observed selected major stages of zebrafish
development details are given in Table 18. Entire embryogenesis
was described in D. rerio (from fertilization to the end of hatched out
yolk-sac larvae) with focused on the organogenesis. The cleavage period
(45 min to 1.30 hr), the blastula (4 hr), gastrultion (10 hr),
segmentation (10.30 to 20 hr), pharyngula period (24 to 36 hr) and
hatching (48 to 96 hr) were observed (Table 18 and Plate I).
Cleavage phase
First cleavage occurred at the animal pole (discoidal cleavage)
at 45 min post fertilization (PF), forming two equally sized blatomeres.
The 4 - and 8 - blastomeres stage appeared 1 hr and 1.25 hr post
fertilization (PF), respectively. The 16 - cell blastomeres stage was
observed at 1.5 hr PF followed by the blastula stage at 2-4 hr PF.
Blastula
At 3.45 hr PF, flattening of the cellular materials occurred,
leading the formation of the blastula (Plate I, Fig. A and B).
Gastrula
Cells of the disc spread over the yolk mass towards the
vegetal pole (4 hr PF) replacing the blastoderm margin and initiating
gastrulation. At 5.15 hr PF epiboly covered nearly half of the yolk
(50 % epiboly stage). 10 hr PF the process of epiboly was completed
and the embryonic shield was formed (Plate I, Fig. C and D).
Segmentation
The process of segmentation started at 10.5 hr PF formed
with first somite furrow. At 12 hr PF, somites are developed,
mesodermal component of the early trunk was formed and tail was
segmented. At 20 hr PF, the tail well extended (Plate I, Fig. E).
Hatching
The hatching process started at 48 hr PF and end at 96 hr PF.
The embryo showed twisting movement inside the eggs few hours
before hatching (Plate I, Fig. F and G). 48 hr after fertilization certain
egg membranes was ruptured with caudal region of the embryo and
the tail emerged out followed by the rest of the body.
Newly hatched embryo
The body of the newly hatched embryo remained in a
curved position for few hours after hatching, with the head bend down
over the yolk (Fig. H and I). The newly post hatched (PH) embryo was
transparent, light yellowish in colour. Mouth and anus were not
opened yet and the eyes were still translucent. A thin membranous fin
fold surrounded the caudal region (Fig. I).
24 hr old post hatched larva
The member of myotomes increased, the size of the yolk
sac was reduced and the membranous fin fold expanded.
The tail
curved and barbells appeared. The mouth and eyes were unidentifiable.
The alimentary canal could be seen as a straight tube emerging from
post dorsal part of the yolk sack (Fig. J and K).
48 hr old post hatched larva
The membranous fin fold surrounded the entire area
from behind the head region. Optic and auditory vesicle could be
distinguished. The mouth and jaws began to differentiate and the
barbells become elongated (Fig. L).
72 hr old post hatched larva
The yolk sac was reduced. The barbells became larger
around the well developed mouth. The eyes further differentiated
(Plate I, Fig. M).
96 hr old post hatched larva
The yolk sac was resorbed nearly completely. The mouth
and eyes were well developed. The mouth was terminal position.
The pigmentation increased on the head and snout and extended over
the whole body.
120 old post hatched larva
The yolk sac were gradually replaced by the developing
alimentary canal until the yolk sac was completely resorbed
(Plate I, Fig. N).
Time
(h.min)
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q
Fertilization
0
Zygote period
Cleavage period
0.45
Zygote
Cytoplasm accumulates at the animal pole,
one cell stage
Discoidal partial cleavage division:
Four-cell-stage: (vertical division)
8 cell-stage: (vertical and parallel to the plane of the first division)
1.30
16-cell-stage: (vertical and parallel to the second division)
Blastula period
Start of blastula stage
3
Late cleavage; blastodisc contains more number of blastomers
4
Flat interface between blastoderm and yolk
Gastrula period
50% of epibolic movements; blastoderm thins and blatoderm become
curved
8
75% of epibolic movement
10
Epibolic movement ends
10.30-20
Characterization
1.15
5.15
Two-cell stage: (median, vertical, division)
1
2
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Stage
0
Segmentation period
Somites are developed, undifferentiated mesodermal component of the
early trunk, tail segmented
Muscular twitches; tail well extended
24
Spontaneous movements, tail is detatched from the yolk, early
pigmentation
30
Reduced spontaneous movement; retina pigmented, cellular degeneration
of the tail end
36
Tail pigmentation; heart beating
48-96
Pharyngula period
Hatching period
Heart-beat regular; yolk extension beginning to taper; dorsal and ventral
pigmentation stripes meet at tail; foregut development
!
Plate I. Selected stages of early development of zebrafish,
Danio rerio
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8.3. DISCUSSION
Studies on the normal embryonic development of D. rerio
(zebrafish) are important not only to increase the knowledge about the
developmental process but also to understand the time specific
developmental process in course of particular fish species. The zebra fish
embryo has become major model in neurobiology and developmental
biology (Westerfield, 2000; Wixen, 2000). Previously development of
zebrafish has been described in most detail (Roosen-Runge, 1938;
Thomas and Waterman, 1978; Kimmel et al., 1995). The variation in
egg size and time of developmental stages in zebrafish was recorded
(Thomas and Waterman 1978; Kimmel et al., 1995). This might be
related to the existence of different races in nature (Thakur, 1980).
Alternatively, it may be also depend on the individual parental care,
moderated by the food availability experienced by the female spawner.
At 26qC the incubation period (the period from fertilization of hatching
lasted between 48 hr-96 hr). Zaki and Abdula (1983) and Herath (1988)
reported shorter incubation period for fish embryo at higher temperature.
Therefore, the development of a fish embryo is clearly temperature
dependent as in most fish species (De Graaf and Janssen, 1996).
The zebra fish egg is telolecithal, cleavage in meroblastic and
discoidal. All teleosts show a discoidal meroblastic cleavage pattern,
where the large yolk volume restricts cell division to a small area at
the animal pole close to the micropyle (Hall et al., 2004). The pattern
of cleavage recorded have for D. rerio did not differ from that in
other teleosts such as oryzias latipes, Cichlasoma dimerus and
Gadus morhua (Kimmel et al., 1995; Meijide and Guerrero, 2000) or in
catfish, e.g. P. suchi (Islam, 2005) and M. montanus (Arockiaraj et al., 2003).
However, niter and intra-specific variation exists in the cleavage
pattern (Hall et al., 2004). For example, the first horizontal cleavage
occurs between 32-cell and 64-cell stage in D.rerio and in C. dimerus
(Kimmel et al., 1995; Meijide and Guerrero, 2000), but it is early
observed between 16 and 32-blastomeres in C.gariepinus and medaka
O. latipes (Iwamatsu, 1994). Different timining for the blastula and
morula stage were reported for cyprinid fish Kimmel et al., 1988; 1995;
Thomas and Waterman 1978), but are still congruent with the
observations reported in the present experiment. Gastrulation in
zebrafish starts after the movement of cells over the surface of the yolk
(epiboly) and was observed at the 50% epiboly stage which similar to
the pattern reported by Warga and Kimmel, 1990.
In contrast, the
beginning of gastrulation the 50% epiboly stage was observed in
common carp. Generally, previous observations on the early development
of D. rerio (Kimmel et al., 1995; Warga and Kimmel, 1990) and also on
other teleost fish Cichlasoma dimerus (Meijide and Guerrero, 2000)
agree with the pattern described in the present study, thus confirming the
basic uniformity of teleost development particular in pre-hatching stages.
The newly hatched embryos of D. rerio were characterized
by a large yolk sac which they nourished for 120 hr pH.
At high
temperature (30qC), the yolk sac period lasted within 48 hr pH stage
(Verreth et al., 1992) which indicating the temperature regime might
be influenced the period yolk sac stage. In the present observations,
the terminal mouth, the jaws, the eyes of D. rerio became well
developed at 96 hr pH stage. Active feeding might be initiated as early
as 24 hr before the complete resorption (120 hr pH) of the yolk sac.
In contrast, active feeding of free embryo in assumed inefficient
(Osse, 1990) due to the delayed jaw function. Additional support for
an early onset of external feeding is the morphologic evidence for an
early function of the sensory organs. According to the hypothesis of
Kawamura and Washiyama (1989), vision is the most important sense
of fish larvae used for feeding and prey detection, which is supported
by well developed eyes at 96 hr pH stage in zebrafish. However, one
has to consider that a functional digestive system is completed only
5 days after the onset of exogenous feeding (Verreth et al., 1992) on
the other hand, this might even raise pressure for efficient foraging
compensating for insufficient digestion.