ZEBRAFISH

Zebrafishes
Bandonil

Comia

Lego

Malit

Versoza

Fertilization

Overview of Zebrafish fertilization

Sperm attachment
Fast block to polyspermy
Slow block to polyspermy
Ooplasmic segregation
Pronuclear Fusion

The zebrafish egg

Relatively small
Macrolecithal
Two layers
Vitelline envelope
Chorion
External fertilization

External fertilization
ZEBRAFISH

Increase in motility upon release

to sea water
Do not release any
chemoattractant
May possess micropylar sperm
attractants
Sperms do not have an acrosome

SEA URCHIN

Increase in motility upon release

to sea water
Egg jelly releases
sperm-activating peptides
(chemoattractant)
Sperms have an acrosome

Blocks to polyspermy
ZEBRAFISH
Fast block: plugged micropyle
Slow block: cortical granule reaction

SEA URCHINS

Fast block: depolarization of the

cellular membrane
Slow block: cortical granule
reaction

Cortical granule reaction

Formation of the perivitelline space

Lifting of the chorion from the membrane (increase in cell diameter)

Ooplasmic segregation

Separation of the yolk from other cytoplasmic components

Formation of the blastodisc

Pronuclear fusion

Oocytes are arrested at the second meiotic division prior to fertilization

Upon sperm entry, egg activates and completes meiosis formation of the
second polar body
Sperm nuclear envelope is degraded while chromatin disperses
Fusion of the egg and sperm pronucleus forms the zygote

Fertilized vs. Unfertilized Zebrafish eggs

Morphology
Fertilized egg = relatively larger due to lifted chorion
Cell division
Survival
Unfertilized egg can only survive for 8 hours, with deterioration of cytoplasmic
contents starting at 3 hours after shedding (cytoplasmic darkening)

Early development

CLEAVAGE
* 1-cell to 64-cell stages; 45 minutes post-fertilization
* discoidal meroblastic - only the cytoplasm of blastodisc
becomes embryo
* synchronous (up to 6th cleavage) and metasynchronous (7th
cleavage onwards)

CLEAVAGE
* initially, all cells have an open connection to the yolky
cytoplasm
* body axes are not yet specified

Fertilization

CLEAVAGE | 1-cell stage | 15 minutes

CLEAVAGE | 2-cell stage | 45 minutes

CLEAVAGE | 4-cell stage | 1 hour

CLEAVAGE | 8-cell stage | 1.25 hours

CLEAVAGE | 16-cell stage | 1.5 hours

CLEAVAGE | 32-cell stage | 1.75 hours

CLEAVAGE | 64-cell stage | 2 hours

BLASTULA
* yolk syncytial layer (YSL), enveloping layer (EVL), deep cells,
periderm formed
* epiboly occurs; mediated by microtubules
* zygotic genes are turned on
* mid-blastula transition at the tenth cell division

BLASTULA | 128-cell stage | 2.25 hours

BLASTULA | 256 to 512-cell stage | 2.5 to 2.75 hours

BLASTULA | High stage | 3.3 hours

BLASTULA | Oblong to Sphere stage | 3.6 to 4 hours

BLASTULA | Dome stage | 4.3 hours

BLASTULA | 30% Epiboly | 4.7 hours

GASTRULATION
* involution begins, forming the epiblast and hypoblast
* germ ring and embryonic shield are formed
* ends in the bud stage (100% epiboly)
* brain region forms anteriorly; tail bud forms posteriorly

GASTRULA | 50% Epiboly | 5.25 hours

GASTRULA | Germ ring to Shield stage | 5.6 to 6 hours

GASTRULA | 90% Epiboly | 9 hours

Bud | 10 hours

Zebrafish Organogenesis

Stages of Zebrafish Organogenesis

Segmentation (10 -22 hours)
Pharyngula (24-42 hours)
Hatching (42-72 hours)

Segmentation
(10 -22 hours)

Basically the development

of the somites and the
rudiments of primary
organs, and the elongation
of the embryo

1-somite (10 hours)

Parts: first somite
Somites are mesoderm blocks,
specifically the sclerotome,
myotome and dermatome

A new pair of somites

develops every half
hour.

5-somite (11 hours)

Parts: optic vessel (eye)

10-somite (14 hours)

Parts: otic placode,
eye, somites
Placodes are sensory
flat, round plate of
cells

14-somite (16 hours)

Present: brain neuromeres;
V-shaped trunk somites
Neuromeres are regions of swellings
of the neural tube. First three
correspond to telencephalon,
diencephalon, and mesencephalon,
remaining seven correspond to
rhombencephalon.

18-somite (18 hours)

Parts: olfactory,
rhombomeres, somites and
lens placode

20-somite (19 hours)

Present: otic vesicle, and
hindbrain neuromeres

Tail well extended, twitches

are observed

26-somite (22 hours)

Parts: otoliths
Otoliths are inner ear structures that
function in gravity, balance, movement
and directional indicators.

Pharyngula
(24-42 hours)

Presence of the primordia of the

pharyngeal arches. Embryo possess
the classic vertebrate body plan.
Embryo also respond to touch and
pigmentation is pronounced.

Prim-5 (24 hours)

Parts: optic vesicle (lens and optic cup)
skin, retinal pigments, RBCs in yolk cup,
fin fold, olfactory placode, otic placode
heart, and opening of pronephric duct
Fin fold is the median epidermal fold that give rise to
dorsal, caudal and anal (tail) fins .

Heart starts beating

yolk

Prim-15 (30 hours)

Present: caudal artery, fin buds
Embryo responds to touch,
spontaneous movements
reduced.

Fin buds are mesenchyme outgrowths that give rise to pectoral and
pelvic fins

Prim-25 (36 hours)

Present: pigmented tail, single aortic
arch
Blood circulation is strong. Caudal
artery extends three-quarters of the
way to the end of the tail.

High-pec (42 hours)

Present: dorsal stripe, xanthophores in
head, iridiphores in retina, mandibular
and hyoid arches and lateral line.
Dechorionated embryo rests on its
side after swimming.

Lateral line is asystem of canals/grooves in the skin containing

neuromasts, which are sensory structures that detect low-frequency
motions in water that help inform the fish of prey and predators

Hatching
(48-72 hours)

Cartilaginous skeleton of
pharyngeal region is being laid
down. Larva is colored yellow due to
iridiphores.

Long-pec (48 hours)

Present: Dorsal and ventral stripes, more iridophores in
retina, 24 aortic arches with circulation and pectoral fin.

Optic cup

Otic placode
somites

Olfactory
placode
lens
Opening of pronephric
duct

Pec-fin (60 hours)

Present: flatenned pectoral fin,
early jaw cartilages, 5-6 aortic
arches with circulation.
Movements are very rapid.
Iridophores are seen in the
dorsal stripe.

Protruding mouth
(72 hours)
Present: mouth anterior to
the eye, gill slits and cartilage
in branchial arches.
The dorsal body is as yellow as
the head.

Gill slits and branchial cartilages.

Teratogenesis

Lithium
Exposure at cleavage affects
pattern formation

Lithium
Exposure at later stages affect
anterior development

Lithium
Two teratogenic stages:

16 cell stage
2000 cell midblastula stage

Lithium affects the Wnt/beta catenin pathway through GSK3B

Ethanol
Mechanism is unknown

Affects nervous system and skeletal development

Major effect on development of visual function

Retinoic acid/retinoids
Affects circulatory and central nervous system structures

Teratogenicity from excessive amounts

Retinoic acid/retinoids
Unknown mechanism and pathogenesis

Proposed mechanism centered on a paradoxical approach

Endocrine disruptors
Several methods of interaction with biological processes

Usually endogenous sex hormone mimics

Temperature
Development achieved between 23-34 C

Developmental plasticity may also occur

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