haploid embryos can be produced by fertilizing eggs with sperm that have been irradiated with ultra violet light (UV sperm) destroying the sperm DNA so that it cannot contribute genetically to the embryo but leaving the sperm intact and able to activate the eggs. Haploid embryos develop from the female genetic complement only. Any mutation carried by the female will be seen in her haploid offspring. Additionally it is possible to make these haploid embryos diploid by application of pressure or heat shock during early developmental stages. This section provides an overview of these genetic methods. Detailed procedures follow in a later section.UV SpermSperm is collected and pooled into ice cold Hank's solution as described inEmbryo Production By In Vitro Fertilization. It is important to keep the sperm solution relatively dilute because of possible shielding of the UV.Ice is put in the bottom half of a 10 cm glass petri plate. Sperm is pipetted onto a watch glass sitting on top of the ice taking care not to introduce bubbles. Removing the glass lid under a UV lamp exposes the sperm to UV. Sperm is irradiated by a Sylvania 18 inch 15W germicidal lamp at 38 cm or 15 inches from the watch glass for 2 minutes with gentle mixing. Wear latex gloves and glass safety glasses for your own protection from the UV. Use a clean pipette to pipette the sperm into a clean glass tube and kept on ice. It will fertilize eggs efficiently for up to 90 minutes (just as non-irradiated sperm) producing haploid embryos with only 25 chromosomes in each cell.Haploid EmbryosHaploid embryos have a characteristic syndrome. The body is shorter and thicker than a diploid; the brain is less clearly sculptured; the ears are variable in number; and the heart beats in a swollen pericardial cavity. Haploid cells are smaller than diploid cells and there are problems with organogenesis. For example, blood cells seem to be too large for the blood vessels and most haploid embryos have circulation problems. Haploid embryos live only about five days.For all their faults, haploids are consistent enough to be useful for identifying changes in early development caused by mutations. The haploid mutant phenotype sometimes differs from the mutant phenotype observed in diploids (often the haploid phenotype is more severe), but one is alerted to the mutation and can do the appropriate crosses to produce a diploid.Haploid embryos are used to identify new mutations in mutagenized females as well as to screen and identify mutation-bearing heterozygous females.Production of Gynogenetic Diploid EmbryosThere are two times during the first cell cycle of a UV-fertilized egg when there is a diploid complement of chromosomes from the female. One is during the second meiotic division and the other is during the first mitosis. Preventing chromosome separation at either of these times will restore the diploid number of chromosomes.1. Early PressureWhen eggs are first laid, the chromosomes are condensed, ready to separate in the second meiotic division. A pulse of hydrostatic pressure (EP) at the right time will break down the spindle, preventing chromosome separation and the extrusion of the polar body. The then diploid set of chromosomes replicates and separates in the first mitotic division, producing a viable gynogenetic diploid embryo.Eggs are squeezed from the female and fertilized with UV-irradiated sperm as described in Embryo Production By In Vitro Fertilization. The timer is started when the first water is added to the egg-sperm mixture. Eggs are quickly put into a pressure vial and the vial filled totally with egg water. The vial is capped with a rubber sheet and plastic snap cap being careful not to leave any air bubbles in the vial. The vial is put into the water-filled French pressure chamber and the chamber is put on the French Press. At 1.4 min after fertilization, pressure is applied until the gauge reads 1800 (or 8,000 lbs per square inch). At 6 min, the pressure is slowly released over a period of 1 min. The vial is removed and the eggs left undisturbed in the vial at least an hour. Then fertile eggs are sorted from infertile or dead eggs.T = 0 Fertilize eggs with UV-irradiated spermT = 1.4 min Apply pressure quicklyT = 6.0 min Slowly relieve pressureIt is easy to stop the second meiotic division and one can anticipate 50%-80% of the offspring to be viable in an EP experiment. The males and females which grow from an EP stock are fertile, but not quite as hardy as normal diploid fish.Even though the genes from EP diploids all come from the female, they are not homozygous at all loci. This is because sister chromatids in the 2nd meiosis have already undergone crossing over in the first meiosis. Genes close to the centromere are likely to be homozygous; those far from the centromere are not. In fact, EP is used to construct a crude genetic map of linkage of genetic loci to the centromere.The vials used for EP experiments are 5 ml milk dilution vials covered with a rubber sheet and a plastic snap cap. A hole is cut in the cap exposing the rubber sheet to the pressure. Two vials fit comfortably in the French pressure chamber so two batches of eggs fertilized simultaneously can be pressure treated together.2. Heat ShockThe aim of heat shock (HS) is to prevent chromosome separation and cytokinesis of the first mitotic division. It is different from EP in that meiosis has occurred and the haploid set of chromosomes of the female have replicated themselves prior to the 1st cell division. They are, therefore, homozygous for every gene. Heat shock prevents the first cell division by some unknown mechanism, thus restoring diploidy in some embryos. The second cell division becomes the "first" and so on.Eggs are squeezed from the female and fertilized with UV-irradiated sperm. After 5 min the eggs are transferred to heat shock cylinders (plastic cylinders with a mesh bottom described below) and put into a large beaker filled with egg water sitting in a 28.5•C water bath. At 13 min after fertilization, the vial is moved quickly to a large beaker of water at 41.4•C transferring as little of the 28.5•C water as possible. After two minutes, the cylinder is moved back to the 28.5•C water. After sitting in the 28.5•C beaker for 45 min, the eggs are transferred to a glass beaker and eventually the surviving embryos are sorted from the infertile or dead eggs.T = 0 Fertilize eggs with UV-irradiated spermT = 05 min Transfer embryos to 28.5CT = 13 min Move cylinder from 28.5 to 41CT = 15 min Move cylinder from 41 to 28.5CIt is very difficult to stop the first mitotic division. Only 10%-20% of the embryos survive as diploids to grow to adulthood. Homozygous diploids can be fertile males or females.We make HS cylinders from Beckman Ultraclear centrifuge tubes by cutting off the bottom of the tube and attaching a nylon mesh bottom using methylene chloride as the "glue" (done in a hood). The thin plastic of the cylinders transfers very little heat or cold.The temperature of the 41C bath is held very accurately. Too low a temperature produces haploids and too high a temperature kills embryos. Different strains may require a slightly different temperature. An immersible stirrer drives a magnetic stirring bar in the 41C beaker to keep the temperature uniform.
Insertional mutagenesis strategies used in vertebrates. In each case, a schematized endogenous locus is represented by exons (E) and an endogenous regulatory element. A nonintegrated vector is also shown above, with integrated vector below. Transcriptional start sites are shown as an arrow above each diagram. (a) Integration of DNA into a coding exon can mutate the locus, resulting in a truncated gene product. (b) Retroviral insertional mutagenesis alters the tagged locus using multiple methods, including the loss of the encoding transcript.(c) 5' gene trapping in mouse embryonic stem cells. Shown is one approach whereby the resulting fusion transcript encodes a truncated gene product fused to the selectable marker protein. (d) Insertional mutagenesis in zebrafish using transposons. Based on 3' exon or poly(A) trapping methods, this approach uses two components: a transcriptional termination cassette to truncate the integrated locus and a separate 3' exon trap gene finding cassette. See text for details. pA, polyadenlyation signal; SA, splice acceptor; SD, splice donor.Sivasubbu et al. Genome Biology 2007 8(Suppl 1):S9 doi:10.1186/gb-2007-8-s1-s9The mechanism of mutation using a retroviral gene-trap cassette. If the retrovirus inserts in an intron in the correct orientation, the splice donor from the previous exon can splice to the gene trap cassette (gtc) and then splice out to the next endogenous exon, thus creating a frameshift. The exons are shown in purple; the provirus is depicted by the white box (flanked by long terminal repeats shown in black); the gtc is shown in yellow; the exon with a frameshift mutation is shown in violet. Abbreviations: SD, splice donor; SA, splice acceptor.
one of the advantages of the zebrafish embryo is its translucency, which enables the investigator to examine its development readily with the dissecting microscope. Figure 1A gives an overview of development during the first 24 hours (Haffter et al., 1996), and development from 1 to 5 days is shown in Figure 1B.
We are using a variety of techniques to investigate the possible importance of the three alpha crystallins in the development of the zebrafish embryo. The zebrafish has become a powerful model species for studying vertebrate development because females lay transparent eggs, allowing direct examination of the embryo in real time as it grows. In one set of experiments we are cloning the promoter regions for zebrafish alpha crystallins, the portions of DNA that determine where each gene is used to make protein. By connecting a gene for green fluorescent protein (GFP) to each promoter we can visualize where and when that promoter is active. We can also test the promoter regions from other species. The images above show a 2-day old zebrafish embryo under brightfield microscopy (A) and under fluorescent microscopy (B) indicating that the promoter region for mouse alpha A-crystallin can drive the expression of the attached GFP in the lens. We are using similar techniques to detail the function of the zebrafish promoters. By using a synthetic anti-sense RNA called a morpholino we can also silence the production of individual alpha crystallin proteins in zebrafish embryos. We inject small quantities of each morpholino into zebrafish embryos when they contain only one to four cells (seen at the right with a red tracer dye in an injected embryo) using a picoinjector. The growing embryos can then be examined to see if the lack of specific alpha crystallins cause developmental abnormalities, suggesting possible roles for each.
The first recessive mutation studied in zebrafish (Daniorerio), golden (golb1), causes hypopigmentation of skin melanophores (Fig. 1) and retinal pigment epithelium (Fig. 2) Rescue and morpholino knockdown establishslc24a5 as the goldengene. Lateral views of 48-hpf (A) wild-type and (B)golb1 zebrafish larvae. (C) 48-hpf wild-type larva injected with morpholino targeted to the translational start site ofslc24a5 phenocopies thegolb1 mutation. Lateral view of eye (D) and dorsal view of head (E) of 72-hpf wild-type embryos. (F andG) golb1 pigmentation pattern at 72 hpf, showing lightly pigmented cells. (Hand I) 72 hpf golb1 larva injected with PAC215f11 show mosaic rescue; arrow identifies a heavily pigmented melanophore. (J and K) 72-hpf golb1 larva injected with full-length zebrafish slc24a5 RNA. (L and M) 72-hpf golb1 larvae injected with full-length human European (Thr111)SLC24A5 RNA. Rescue with the ancestral human allele (Ala111) is shown in fig. S4. Rescue in RNA-injected embryos is more apparent in melanophores (K) and (M) than in RPE. Scale bars in [(A) to (C)], 300 μm; in (D), (F), (H), (J), and (L), 100 μm; in (E), (G), (I), (K), and (M), 200 μm.
http://www.nature.com/nrc/journal/v13/n9/fig_tab/nrc3589_F1.htmlDifferently aged animals each offers distinct advantages for cancer-relevant phenotypes. Embryos can be used to identify phenotypes that are highly relevant to cancer biology, such as defects in the cell cycle or genomic instability. Other embryo phenotypes may include stem or progenitor cells that act as cell of origin of the tumour or changes in embryo morphology on transplantation of human cells. Any of these phenotypes can then be used as the basis of chemical or genetic screens to find modifiers, which can be tested for their relevance to human cancer. Juvenile fish have the capacity for modelling early tumorigenesis and remain optically fairly translucent, lending themselves to detailed in vivo imaging. These cancers can be either from transgenic models or can arise via transplantation of tumour cells, and confocal imaging can be used to assess the tumour–stroma interaction at single-cell resolution. Adult fish develop fully penetrant and advanced cancers, both through transgenic techniques and through the transplantation of either zebrafish or human tumour cells. These animals are ideally suited to cross-species oncogenomics, either by directly testing candidate human genomic changes in the fish (by rapid transgenesis) or by comparing the profiles (DNA or RNA) of the mature tumour in the fish to that of the human to look for evolutionarily conserved events. Both the wild-type fish and the transparent casper model add improved capacities compared to mouse models for in vivo imaging and analysis of tumour stem cells and tumour progression and metastasis.
Fig. 3. Fgfsignaling is required for blastemaformation, including cell proliferation and expression of the blastemalmarkermsxb. A: Fin from untreated fish at 4 days postamputation (dpa), showing normal regrowth and new segmentation. Arrows demarcate the amputation plane in each photograph. The distal, regenerating end is positioned toward the top in each image. B: Fin from fish treated with SU5402 (Ri) for 4 days immediately after amputation. These fins showed no new growth. Here, the amputated edge appears saw-toothed due to the retraction of tissue between rays. C: Hematoxylinstain of 1 dpa fin regenerate section from untreated fish (asterisk denotes new blastema). D: Fin regenerate section from fish treated with Ri for 24 hr. Note the lack of blastema. However, Ri-treated fin regenerates showed mesenchymal disorganization (arrowheads mark boundary between organized and disorganized tissue), as well as longitudinal arrangement suggestive of migration. E: Whole-mount in situ hybridization of msxb expression at 1 dpa, indicating strong blastemal expression. F: Expression of msxb is reduced or absent in fins treated with Ri for 24 hr after amputation. G: An untreated bromodeoxyuridine(BrdU) incorporation control. Animals were incubated with BrdU from 42–48 hpa. H: Animals were treated with SU5402 from 40–48 hpa, and BrdU from 42–48 hpa. BrdU incorporation (brown) in proximal blastemal cells was dramatically reduced by drug treatment. Adapted with permission from Developmental Biology (Poss et al., 2000b).
Fast-track zebrafish embryo toxicity (ZET) testing is a much more robust and sensitive model for evaluating vertebrate development pathways and toxicity than small-animal models. Genotoxicity and developmental toxicity are readily viewable through the transparent embryo during development. Microtest's zebrafish embryo assay has better sensitivity and generates more scientific data than the small-animal tests currently recommended by the U.S. Food and Drug Administration (FDA). It also saves time and money because the embryos develop in 72 hours.Newly fertilised zebrafish eggs are exposed to the test chemical for a period of 96 hrs. Every 24 hrs. Twenty embryos (one embryo per well) are exposed to the chemical tested at each concentration level. The test includes five increasing concentrations of the chemical tested and a control. Every 24 hours, four apical observations are recorded as indicators of lethality: (i) coagulation of fertilised eggs, (ii) lack of somite formation, (iii) lack of detachment of the tail-bud from the yolk sac, and (iv) lack of heartbeat. At the end of the exposure period, acute toxicity is determined based on a positive outcome in any of the four apical observations recorded, and the LC50 is calculated. The test report also includes a number of other important information elements related to the conduct of the test, in particular: the concentration of dissolved oxygen, pH, total hardness, temperature et conductivity of solutions, measured concentrations of the chemical tested, and whether the validity criteria of the test were met.1) High fertility: Hundreds embryos can be obtained in one morning, so researchers can start quickly their study. It gives the possibility to test more than 10 compounds a week, and to develop high throughput screening (HTS) assays.2) Very small size: As they are very small, the embryos can be kept in small chambers (as 96-well plates) for a few days. Very small quantity of test compounds, which are simply dissolved in the medium, is needed; therefore, drugs can be tested in earlier discovery stages than with classical assays.3) High predictivity for humans: Zebrafish is a highly predictive tool for toxicity or efficacy endpoints, based on its genetic homology with humans (>80% of genes in common), and on its similarities in cellular physiology, disease pathways, and behavioral phenotypes (learning, sleep, drug addiction, etc) with other vertebrates.4) Embryo transparency and fast development: the larvae are transparent during the first two days, and have all organ buds after 72 hours, allowing easy visual analysis of organ development. Moreover, several transgenic lines that express fluorescent proteins in a specific organ (pancreas, eyes, CNS, blood vessels,…) are available, so that the morphological evolution of this organ can be assessed at a glance. Automated image analysis is also under development.
General Features Benefits
-Dimension ~4 cm
-Salient distinguishable features of
male and female
-Often transparent adult bodies
Large number can be kept easily and
cheaply in lab
Good model for visualization of cellular
-Fresh water fish
- Tropical fish
-Omnivorous Low cost of maintenance
-Female spawns every 2-3 days
-Breeds all year round
-Several hundreds of eggs produced
in single clutch
Large number of offspring- good for
batch variation studies
Easy availability of eggs
• Total life span: 42-66 months
• Good model for
because of transparency
at early stages
• Largest set of vertebrate genome so far sequenced
• Unique features of chromosomal DNA
– Unique repeat content
– Scarcity of pseudo-genes
– Chromosome 4 is enriched with zebra fish specific genes and sex-
• Mitochondrial genome completely sequenced
• According to a paper published in Nature, 70 per cent of protein-coding human
genes are related to genes found in the zebrafish (Danio rerio), and 84 per cent of
genes known to be associated with human disease have a zebrafish counterpart.
• Model for First mutagenesis studies
• Homozygous strain production by using genetically inactivated sperm
• Easy visualization of tumor
development (in heart, brain,
lateral vessels, and intestinal
tube), cell migration, fluorescent
probes, pigmented cells
• One can monitor a disease
without harming the organisms
Homozygous recessive mutants of mitfa gene (associated to melanophores) & an
unnamed gene (associated to iridophores)
- Easy to manipulate: easy mRNA, DNA,
fluorescent tag micro-injection and their
-Egg size ~ (0.7mm)
- Optically transparent : easy to study
embryonic development (epiboly,
gastrulation, segmentation), growth
delay, malformations or morphological
Pigmentation and melanocyte development studies
- required for melanin
- orthologous gene is
present in humans also
• Shares most of their organs with
• Differently aged animals each offers
distinct advantages for cancer-relevant
• Regeneration of
– fins, skin, heart in adult
– brain in larvae
– photoreceptor cells and retinal neurons
Developmental Dynamics 226:202–210, 2003
Why using Zebrafish?
-Very small size
- High fertility
- High predictivity for Human
- Embryo transparency and fast
Zebrafish Embryo Toxicity Test (ZEFT) : Robust and sensitive model
• Typical chemical induced malformations in Zebrafish
• Several mammalian organs are not present in the
zebrafish, including breast tissue, lungs, and prostrate.
Skin lacks some specific cellular components found in
• ectothermic (cold-blooded): physiology not identical to
• Fish are greatly influenced by their environment and
many environment variables, including temperature,
lighting, population density, water quality and nutrition,
must be tightly controlled in order to accurately interpret
• A known problem with gene knockdowns is
that, because the genome underwent a
duplication after the divergence of toleost
It is not always easy to
silence the activity of
one of the two gene
paralogs reliable due
to complementation by
• In 2009, researchers at the Institute of Genomics and
Integrative Biology in Delhi, India, announced the sequencing
of the genome of a wild zebrafish strain, Containing 1.7 billion
Comparative analysis with the
zebrafish reference genome
revealed over 5 million single
nucleotide variations and over
1.6 million insertion deletion
• It is difficult to draw evolutionary
conclusions because it is difficult to
determine whether base pair changes
have adaptive significance via comparisons
with other vertebrates' nucleotide
Zebrafish Model for Toxicology and
Toxicologic Pathology Research
• Shortage of Basic Data on Zebrafish Pathology.
There are scant baseline data on zebrafish
pathologic lesions in infectious and noninfectious
• Many lines of knockout mice for which the inactive
genes are not related directly to the immune
system still are immunodeficient and at high risk
for opportunistic infection
Infectious Diseases of Zebrafish
• Two infectious diseases that commonly occur in
well- managed zebrafish colonies are
microsporidiosis ( infects the central nervous
system, cranial and spinal nerves, and skeletal
muscle of zebrafish) and mycobacteriosis.
• We are uncertain whether vertical transmission of
microsporidia can occur in zebrafish.
Unfortunately, these parasites are very difficult to
inactivate and remove from recirculating
• Piscine mycobacteriosis remains a tenacious problem
in aquarium fish colonies. It most often occurs as an
opportunistic infection in fish over 1 year of age. The
sources of infection and epizootiology are not well
defined. Currently no effective chemopreventative or
therapeutic regimens are defined for fish.
• Because mycobacterial antigens are potent immune
adjuvants, these agents can seriously confound
research in disease resistance or immune responses.
Animal models of human disease
zebrafish swim into view
• First, human pathogens cause disease at 37°C,
whereas zebrafish are maintained at 28°C; it might not
be possible, therefore, to study some pathogens at this
lower temperature. So, most host–pathogen
interactions that could be studied using this approach
might be fish-specific.
• Finally, crucial gaps remain in the comparative
physiology of the zebrafish immune system, which
might impinge on the validity and usefulness of
modelling human infections in zebrafish.
Zebrafish as a model to study the
neurodevelopmental causes of autism?
• First, the genetic and neuroanatomical defects that
cause autistic disorders in humans are largely
unknown. Thus, it is not completely clear what
aspects of zebrafish brain development should be
investigated in a model of autism.
• Second, the constellation of behaviors that define
autism may not be suitably represented in the
repertoire of non-human behaviors.
Transgenic zebrafish model for blood-
brain barrier development
• The BBB, initially regarded as static and rigid, have
now been proven to be dynamic structures with both
paracellular and transcellular pathways capable of
rapid modulation in response to physiological or
• One of the major limitations in this regard has been
the absence of an easily regulated in vivo system
that allows alterations of these barriers.
• In 2012, Australian scientists published a study
revealing that zebrafish use a specialised protein,
known as fibroblast growth factor, to ensure their
spinal cords heal without glial scarring after injury.
• If similar processes occur , absence of the glial scar
has been associated with impairments in the repair
of the blood brain barrier???????
Influence of Diet and Husbandry
Systems on Spontaneous Neoplasia
• Zebrafish colonies fed commercial diets and
maintained in standard recirculating systems have
different patterns of neoplasia and spontaneous
pathologic lesions than those observed in the Core
Fish Facility of the Marine/Freshwater Center at
Oregon State University.
• But systemic mechanism of this spontaneous
neoplasia, its molecular basis and gene expression
variance has not been characterized, challenging
its suitability to cancer model or toxicity research.
The Mystery of Hepatic
• In diagnostic cases from around the world and in about 50% of
groups of broodstock from standard husbandry systems feeding
commercial diets, show mild to moderate hepatocyte megalocytosis
• The toxicant sources causing megalocytosis are uncertain.
• Problems: Reducing early life stage survival, longevity, reproductive
potential, immune competence, and disease resistance.
Zebrafish in hematology: sushi or science
• Different morphology of blood cells, eg, erythrocytes and
thrombocytes are nucleated.
• Different gross anatomy, eg, what is the equivalent of the marrow
• Lack of cell markers/antibodies
• Lack of hematopoietic cell lines
• Lack of biochemical reagents, eg, purified cytokines
• Lack of in vitro differentiation system
(hematopoietic cell culture assays)
• Lack of inbred strains, eg, for transplantation
studies; to facilitate gene mapping.
The Problem of Skewed Sex Ratios in
Cohorts of Zebrafish
• Problems with skewed sex ratios in cohorts of zebrafish can
interfere with natural breeding, and can complicate studies such
as carcinogen or other toxicant bioassays where balanced sex
ratios in control groups are desired.
• In contrast to mammalian species, sex determination in fish is
more flexible, often reversible, and less dictated by genetic factors.
• The relative influences of and interactions between environment,
genetics, and toxicants in sex determination in zebrafish are not
yet clearly defined.
Zebrafish-Based Small Molecule
• Lack of phenotypic fluidity. Only extremes phenotypes
(subtle or distorted are seen in general).
• Therefore, despite a few notable successes, the
benefits of whole-organism, phenotype-based small
molecule discovery have been overshadowed by the
practical limitations of the approach.
Ethanol Disorder of embryo
• Several human ethanol disorders are difficult or impossible to
model in this species (e.g. cardiac septation defects)
• Different scales of phenotypic response to ethanol in case of
humans and zebrafish.
• A further challenge will be to maintain the ongoing development of
community-based resources for zebrafish research. (ZFIN)
• The main pressure points will be stock centers and the sequencing of
the zebrafish genome, both of which will require continued ongoing
investment to provide the necessary quality of material and sufficient
access to it.