Objective
In this lab, you will observe the development of a freshwater killifish, the Japanese
Medaka (Oryzias latipes) from freshly fertilized eggs to hatching and beyond. You will
notice basic patterns of embryonic vertebrate development.
Introduction
Medaka are native to Japan, Taiwan, and southeast Asia. They are most commonly found in rice
paddies and other slow moving bodies of water. Their reproductive cycle is closely tied to day length
and they become reproductive in the summer when the days are long. Shortly after dawn, the female
will release clusters of 10 to 30 eggs. Each egg is enclosed within a clear membrane called the chorion
that has many fine curly hairs on it. These hairs cause the egg cluster to adhere to the vent region of the
female. After a very brief courtship, the male releases sperm onto the egg cluster, and fertilization
quickly follows.
In nature, the eggs are then brushed off onto the vegetation in the water and they complete their
development in the protective environment. In the laboratory, the egg clusters are collected from the
females and placed into small dishes containing a rearing solution. The rearing solution is isotonic to
the egg salinity and contains a blue dye (methylene blue) to prevent fungal infections.
Embryo Rearing Medium
To make 1 L: NaCl
KCl
CaCl2.2H2O
MgSO4.7H2O
Methylene blue 0.01 %
Distilled Water
1.0 g
0.03 g
0.04 g
0.163 g
10 ml
To 1 L
The eggs are from 1 to 1.5 mm in diameter. The chorion with numerous filaments is transparent and
encloses the egg cell. The egg cell itself has a yellowish tint due to the presence of the yolk. The yolk
is stored food material that consists of primarily lipids and proteins. Additionally, there are several
small bubble-like lipid droplets evident in the cytoplasm. Do not confuse these with the newly
developing embryo. These small oil droplets eventually coalesce into a single large oil droplet. It is
thought that this droplet maintains the buoyancy of the developing embryo and holds it in the water
column at the correct depth.
The fertilized egg or zygote begins to divide by mitosis to produce the multicellular embryo. These
early divisions are called cleavages. The dense yolk inhibits cell division, and since it is unequally
distributed within the egg, the cleavage takes place more readily in the yolk- free cytoplasm of the
animal pole of the embryo. Development requires two weeks and is dependent upon temperature.
DEVELOPMENT
Optic vesicles (rudimentary eyes) can be observed at the head end of the embryo by looking for two
tiny swellings. Within two days (46 hours), a primitive heartbeat can be observed just ventral to the
head region with a pulse rate of 40 to 60 beats per minute. At the moment there is little or no blood
circulating, but by 75 hours, pink can be obviously seen, especially in the heart. The retina of eyes
begins to become pigmented by 60 hours and the heart rate is about 100 to 120 beats per minute.
Movement, especially of the pectoral fins, can be most readily seen at 120 hours. The twitches are very
infrequent and weak at first, but become very strong and regular after several more hours. The fish
begins to become golden colored by 170 hours, but it is quite difficult to see. The mouth is one of the
last structures to form, and by 200 hours it begins to open and close quite regularly. Hatching occurs at
about 264 hours (11 days) at 25°C. Most of the fry immediately begin to swim and breath regularly,
but a few lie at the bottom of the container for several hours until motor function develops fully. They
are 4 to 4.5 mm at hatching. Sexual maturity occurs within 2 to 6 months. Adults have a life span of 4
or more years.
Live Medaka Embryo Observation Table
Date and time
Estimated Stage or
age in hours
Diagrams for eggs in
A&B
Notes on Observations
(pumping of heart,
presence of eyes)
TEACHER BACKGROUND:
Japanese Medaka are oviparous freshwater killifish native to the rice paddies of Japan,
Taiwan, and southeast Asia. The wild strain is brownish black in color, but through
artificial selection several other color variants are available through biological supply
companies. The adults are 2 to 4 cm in length and exhibit sexual dimorphism; the male
has a larger anal fin and a dorsal fin that is deeply notched between the two rays closest
to the body. Breeding females are distinguished by their distended abdomens.
Reproduction is dependent upon photoperiod with spawning occurring soon
after dawn. Courtship is brief and the males spread milt concurrently with oviposition.
The chorion of the egg is transparent with many filaments. These filaments cause the
eggs to cluster together on the vent of the female until they are brushed off onto a plant
(or removed by people to study them). The yolk is golden in color with several small oil
droplets that eventually coalesce into one large single globule. This oil droplet eventually
migrates to the vegetal pole of the embryo.
Medaka are teleosts members of the class osteichthyes. The teleosts are modern bony
fishes characterized by bony skeletons, pelvic fins nearer the head and thorax region
(compared to non-teleosts), the transformation of the lungs of primitive fishes into a swim bladder with
hydrostatic functions, and the development of stout spines in the pectoral, dorsal, and anal fins.
All bony fishes have eggs in which a large amount of yolk, up to 90%, is concentrated in
the vegetal pole of the embryo where metabolism is lower. The nucleus and cytoplasm is
concentrated in the animal pole of the embryo where metabolism is higher. Cell division
(cleavage) in the Medaka follows a pattern similar to that found in reptiles and birds. In
organisms that store large amounts of yolk, cleavage is typically meroblastic or
incomplete. Cleavage is restricted to a small disk at the animal pole of the cell. The first
cleavage division occurs approximately 1.5 hours after fertilization; the second cleavage
occurs at right angels to the first and follows about 30 minutes after the first cleavage
division. The third cleavage division occurs parallel to the first division and occurs 30
minutes after the third division. As cleavage continues, a blastodisk is formed and rests
on the top of the large, undifferentiated yolk mass. After four hours, movement of cells
begins and the blastodisk is now two layers of cells surrounded by 16 peripheral cells.
Cleavage becomes asynchronous, with the central cells being slightly smaller than the
peripheral cells.
As cell division continues, the blastoderm flattens out and moves down the yolk toward
the vegetal pole. By 13 hours post- fertilization, the blastoderm covers about 50% of the
yolk and gastrulation begins. After 24 hours, the embryo covers nearly 80% of the yolk
and a large yolk plug protrudes from the blastopore. Neural tube formation quickly
follows and is evidenced by a slight ‘‘streak’’ of cells at the animal pole of the embryo. By 30 hours,
the blastopore is completely closed and the yolk is ‘‘inside’’ the embryo.
NOTES FROM THE WEB
Lecture 27: Animal Embryonic Development:
Fetilization to Gastrulation
In Fertilization 2 Cells Fuse to Become One Zygote
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In sexual reproduction a haploid sperm and egg fuse to form a diploid zygote
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Any particular sperm has a very small chance of fertilizing an egg
o Statistics for humans:
 About 300 million sperm deposited in vagina
 About 300 thousand reach the upper end of the uterus
 About 300 make it to the uper end of the oviduct (Fallopian tube) where
fertilization takes place
 Even if an egg has been released, only 1 sperm fertilizes it
Sperm must burrow through several layers to reach the surface of the egg
o Layer of follicle cells in mammals (not found in simpler species)
o Jelly coat (vitelline layer in sea urchins & chickens; zona pellucida in mammals)
o Contact between sperm and jelly coat causes release of enzymes (acrosome reaction)
that help it to burrow through the coat
At surface sperm binds to membrane protein
o Sets off reactions causing fusion of sperm and egg membranes
o This allows sperm nucleus to enter egg
Prevention of polyspermy:
o Polyspermy = fertilization by more than one sperm; egg will not develop in polyspermy
o Within a few seconds after sperm contact the egg cell membrane depolarizes (the
membrane voltage becomes more positive inside)
o Within ~20 seconds a cortical reaction occurs which produces a fertilization membrane;
sperm cannot penetrate this membrane
Fertilization triggers the egg to finish meiosis II (see meiotic arrest)
Sperm and egg nuclei fuse at around 20 min
DNA duplication (S phase of mitosis) starts at about 40 min
First cell division at ~90 min (time figures are typical for frog and sea urchin fertilization)
The sperm provides the centrosome for cell division
Eggs and Zygotes have Animal and Vegetal Poles
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Egg cells are very large:
o Sea urchin: ~70 to 150 microns
o Human ~100 microns
o Frogs & fishes, some insect eggs: 1000 to 2000 microns(1-2 mm)
o Birds & reptiles: millions of microns (many cm)
Eggs store materials needed for development of the embryo
o Yolk: lipids, carbohydrates and proteins organized into granules
Yolk settles to bottom of egg, producing a gradient of stored material
o Top of egg, with little yolk, is called the animal pole
o Bottom of egg, rich in yolk, is called the vegetal pole
o Polar axis goes from animal to vegetal pole
Eggs have different amounts of yolk
o Large animals developing outside mothers body (birds, reptiles) have large eggs with
lots of yolk
o Large animals developing within mother's body (mammals) have small eggs with very
little yolk; they get their food from the mother through the placenta
o Animals which develop into small feeding larvae (sea urchins, sea stars) also have small,
simple eggs
o Frogs and fish are intermediate in egg size and yolk content
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Almost all of the zygote volume comes from the egg, giving the zygote an animal & vegetal
pole
The Embryo Develops Through a Series of Mitotic Divisions (Cleavage)
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To go from a single-cell to an organism the embryo must repeatedly divide by mitosis
o The early set of rapid cell divisions is called cleavage
Cleavage follows different patterns depending upon the amount of yolk in the egg
o Egg with small amount of yolk (sea urchin): entire egg divides (holoblastic cleavage)
 Produces cells of the same size
o Egg with intermediate amount of yolk (frog egg): entire egg divides, but aninal pole
cells divide faster than vegetal pole cells
 Yolk slows cell division
 Result: large number of small cells at animal pole, small number of large cells at
vegetal pole
o Egg with large amount of yolk (bird egg): only the animal pole divides (meroblastic
cleavage)
 Produces plate of embryonic cells (blastodisc) over a large yolk container
In a Series of Mitotic Divisions the Zygote Becomes a Hollow Ball (Blastula)
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The first set of cleavage divisions are synchronized and there is no cell growth between
divisions
o The size of the embryo does not change, but the egg material is partitioned into more
and more cells
o DNA synthesis does occur between divisions since each new cell needs a nucleus
In frogs the first 12 divisions occur without growth, producing 4096 cells much smaller that the
original zygote
o During this set of divisions the embryo stays within its fertilization membrane
The cells arrange themselves into a ball (blastula; called blastocyst in mammals) with the cell
layer surrounding the fluid-filled interior (blastocoel)
o In the bird cleavage occurs in the blastodisc to produce 2 layers of cells, the epiblast and
hypoblast which split apart to form the blastocoel (see figure p. 975 of text)
The Blastula Invaginates to Form the Primitive Gut (Archenteron)
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After the blastula is finished the wall folds inward at one point
Forms a tube, the archenteron or primitive gut
The opening to the archenteron is called the blastopore
Cells at the animal pole grow and spread over outer surface, forcing other cells inward through
the blastopore
o In bird & mammal embryos there is a long furrow, the primitive streak instead of a
blastopore
Three Germ Layers are Formed in the Gastrula
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Three germ cell layers develop in the gastrula:
o Outer lining of gastrula becomes ectoderm
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Lining of archenteron becomes endoderm
Cells in between ectoderm and endoderm become mesoderm
These 3 layers produce all of the tissues of the body:
o Ectoderm produces: skin (outer layer), nervous system, cornea & lens, etc
o Mesoderm produces: notochord, skeleton, muscles, circulatory system, etc.
o Endoderm produces: digestive tract, lung epithelium, liver, many endocrine glands, etc.
Review of Early Embryonic Development
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These pictures depict development as you might see it in a simple egg with little yolk (sea
urchin)
For pictures of development in other species see text, p. 970-972, 975.
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