ZEBRAFISH
Zebrafishes Bandonil
Comia
Lego
Malit
Versoza
Fertilization
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 ● ●
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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 à 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 ⅓ -22 hours) Pharyngula (24-42 hours) Hatching (42-72 hours)
Segmentation (10 ⅓ -22 hours)
Basically the development of the somites and the rudiments of primary organs, and the elongation of the embryo
1-somite (10 ⅓ 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 ⅔ 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, 2–4 aortic arches with circulation and pectoral fin.
Optic cup
Otic placode somites
Olfactory placode lens Opening of pronephric
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
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Affects nervous system and skeletal development
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Major effect on development of visual function
Retinoic acid/retinoids ●
Affects circulatory and central nervous system structures
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Teratogenicity from excessive amounts
Retinoic acid/retinoids ●
Unknown mechanism and pathogenesis
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Proposed mechanism centered on a paradoxical approach
Endocrine disruptors ●
Several methods of interaction with biological processes
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Usually endogenous sex hormone mimics
Temperature ●
Development achieved between 23-34 C
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Developmental plasticity may also occur
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Mattei, X., & Thiaw, O. (1993). Acrosome-like structures in the spermatozoa of teleost fishes. Can. J. Zool., 883-888. Negron, J., & Lockshin, R. (2004). Activation of Apoptosis and Caspase -3 in Zebrafish Early Gastrulae. Developmental Dynamics, 161-170. Parinov S. Parinov, I. Kondrichin, V. Korzh, A. Emelyanov. (2004). Tol2 transposon-mediated enhancer trap to identify developmentally regulated zebrafish genes in vivo Dev. Dyn., 231 (2004), pp. 449–459 Tyler, M. (2010). Developmental Biology: A Guide for Experimental Study. Sunderland, Massachusetts: Sinauer Associates. Westerfield, M. (2000). The zebrafish book. A guide for the laboratory use of zebrafish (Danio rerio). 4th ed., Univ. of Oregon Press, Eugene. Yanagimachi, R., Cherr, G., Pillai, M., & Baldwin, J. (1992). Factors controlling sperm entry into the micropyles of salmonid and herring eggs. Develop. Growth. Differ., 447-461.
Stachel, S.E., Grunwald, D.J. & Myers, P.Z. (1993). Lithium perturbation and goosecoid expression identify a dorsal specification pathway in the pregastrula zebrafish. Development, 117, 1261-74. Giles, J.J. & Bannigan, J.G. (2006). Teratogenic and developmental effects of lithium. Current Pharmaceutical Design, 12, 1531-41. Carvan, M.J. III, et. al. (2004). Ethanol effects on the developing zebrafish: neurobehavior and skeletal morphogenesis. Neurotoxicology and Teratology, 26, 757-68. Matsui, J.I. et. al. (2006). Effects of ethanol on photoreceptors and visual function in developing zebrafish. Retinal Cell Biology, 47 (10), 4589-97. Collins, M.D. & Mao, G.E. (1999). Teratology of retinoids. Annu. Rev. Pharmacol. Toxicol., 39, 399-430. Herrman, K. (1995). Teratogenic effects of retinoic acid and related substances on the early development of the zebrafish (Brachydanio rerio) as assessed by a novel scoring system. Toxic. In Vitro, 9(3), 267-83
