ILAR 42 (4)

Introduction. ILAR 42 (4): 271.
One of the reasons why we study fish is because the continuity of biological diversity is the key to wise use of comparative medical investigation. Various political, economic, and scientific pressures are demanding more efficient and less expensive whole animal toxicology screening methods as well as ways to reduce the use of mammals in research. At the same time, we need to protect our aquatic resources. There is much NIH funding available to researchers working with zebrafish (Danio rerio), the current popular candidate for fish-mouse substitute. The zebrafish is an excellent tool for investigating developmental and genetic questions. NIH also supports the zebrafish genome initiative. Fish as research animals present some important challenges for laboratory animal veterinarians and institutional animal care and use committees (IACUCs). There is a need for more information based on good investigative research into the biology and husbandry of laboratory fishes. Using fish for carcinogenicity testing offers advantages and challenges of different exposure designs and delivery routes. The diversity of fishes can also provide necessary tools for the creative research scientist to investigate aspects of physiology that cannot be isolated in mammalian models. Inbred genetic strains of live-bearing platyfish and swordtails have been available and in use for comparative oncology models since the early 1930s. The best known of these models is the Gordon-Kosswig melanoma model. The inducible Xiphophorus tumor models include the controversial UV-induced melanoma models and chemical carcinogen-induced models; these models are also used for studying DNA repair potential. There are several applications of transgenic fish models in environmental toxicology studies. The creation of transgenic medaka with multiple copies of bacteriophage LIZ vector and with lacI and cII bacterial genes as mutational targets reminds us how mechanistically similar the fish model is to the most widely used transgenic rodent mutation assays. One problem with using transgenic fish models of human disease is the challenge of sustaining gene statement in transgenic fish lineages, a challenge rooted in the common use of mosaic integration of transgenes in founder fish into fin and other superficial tissues. Fish developed this way don't often transmit the transgene or do so at very low frequencies. Another concern is that transgenic fish model development cannot approach its potential as long as researchers are the primary care providers for their fish. There is a need to integrate laboratory fish husbandry and facilities into traditional laboratory animal management paradigms. Many advocate increased standards, practices, and facilities for the care and use of fish, consistent with those for mammals, and increased training of laboratory animal caretakers in the care and use of fish in general and transgenic fish in particular.
1. Platyfish-swordtail hybrids are animal models for what disease? Give genus and species for both fish. What are some other animal models for this disease?
2. Which fish (common name, genus and species) is only female and genetically identical?
3. Give the genus and species of the bicolor damselfish. What disease is this fish used as an animal model? What are some other animal models for this disease?
4. Give the genus and species of the fish used as a model for Wilson's Disease. What are some other animal models for this disease?
5. Give the genus and species for medaka.
1. Platyfish-swordtail hybrids are an animal model for malignant melanoma. Platyfish = Xiphophorus maculatus, Swordtail = Xiphophorus helleri. Other animal models for malignant melanoma include Sinclair miniature swine, gray horse, Syrian golden hamster (Mesocricetus auratus), tiger salamander (Ambystoma tigrinum), and Trituris chrisates injected SC with methylcholanthrene.
2. Amazon molly = Poecilia formosa. FYI - these fish reproduce gynogentically (eggs activate after mating with males of related species but sperm doesn't contribute genetically), therefore offspring are female clones.
3. The bicolor damselfish (Pomacentrus partitus), AFIP fascicle #282, is used as an animal model of multiple schwannomas or neurofibromatosis (eponym is von Recklinghausen's disease). Other animal models for this disease includes N-nitroso-N-ethylurea-induced multiple neurofibromas in Syrian golden hamsters (AFIP fascicle #393), and the HTLV 1-tax transgenic mice (AFIP fascicle #397).
4. White perch (Morone americana). Other animal models for Wilson's disease include the Bedlington terrier, LEC rat, and sheep with copper toxicosis.
5. Medaka = Oryzias latipes

Mechanistic Considerations in Small Fish Carcinogenicity Testing. ILAR 42 (4): 274.
Fish species mentioned in this article include; Japanese medaka- Oryzias latipes, Zebra fish- Danio rerio, rainbow trout- Onchorynchus mykiss, platyfish and swordtails- Xiphorus spp., top minnow- Poeciliopsis sp., sheepshead minnow- Cypriodon variegatus, guppy- Poecilia reticulata, and Western mosquitofish- Gambusia affinis. Abstract: Historically small fish have been useful as environmental sentinels and as test animals in toxicity and carcinogenicity studies. They can be bred in large numbers, have low maintenance and bioassay costs, and have a low incidence of background tumors. This paper focuses on mechanistic considerations when using small fish for carcinogenicity testing. The Japanese medaka is the best characterized small fish model for carcinogenicity studies, however the zebrafish is emerging as an important model because it is genetically well characterized. Exposure techniques include introducing compound into the water, dietary exposure and embryo microinjection. Initiating carcinogens such as diethylnitrosamine(DEN) are sometimes used. Differences in xenobiotic metabolism, such as the fact that fish CYP2B is refractory to Phenobarbital induction could be another consideration. The small size of the animal can be limiting for some types of analysis, however improved analytical technologies are making this less of a factor. Immunohistochemistry is an important aspect of these studies and more species specific antibodies need to be developed for fish research. There is also a need for better information on fish cytokines, serum biochemistry, and oncogenes to strengthen the use of these important test models. Small fish in carcinogenicity studies. 80 % of human cancers are caused by environmental exposures. Detection and regulation of carcinogenic compounds is important to prevent cancer in humans. Historically carcinogenicity has been established by using whole animal chronic bioassays. Programs such as the National Toxicology Program were established in the 1960's and 70's. The Clean Air Act Amendments of 1990 directed government agencies to study a large group of priority chemicals in a limited amount of time and for less cost. The standard 2 year rodent carcinogen assay has become too costly so a less expensive animal alternative was sought. Alternative testing such as the short term in vitro Ames test are useful for mass screening. But the validity of these tests is somewhat limited due to false positives and false negatives. There is also an inherent inability to determine target organ-specific carcinogenicity or to detect tumor promoting activity. Accordingly alternative models have been looked at. In 1993 the US Congress instructed the NIH to look for ways to reduce the numbers of animals used and to refine current methods to emphasize relief of pain, maximize information obtained from each animal and utilize animals lower on the phylogenetic tree. In 2000. the National Toxicology Program Interagency Coordinating Committee on the Validation of Alternative Methods was established to assist these efforts. Use of transgenic mouse models can answer some questions but these are expensive sometimes difficult to obtain models. Small fish may but a good model especially in early test stages for screening compounds. They have the advantage of being a whole animal model that is inexpensive and available. They are sensitive to known carcinogens and exhibit a short time to tumorigenesis. Yet they have low spontaneous tumor formation. Early models included zebrafish hepatic neoplasia when exposed to DEN and then cycasin, and rainbow trout hepatic tumors caused by aflotoxins in moldy feed. Hepatocarcinogenicity studies have been carried out in all the species listed previously. Sentinels of environmental degradation Researchers have turned to the use of biomarkers such as measurements of body fluids, cells, or tissues that indicate in biochemical or cellular terms the presence of contaminants and the extent of exposure. Fish have the potential to be used as sentinels to detect the presence of contaminants in the environment, surrogates that indicate the potential human exposure and effects or predictors of long term effects on the population or ecosystems. Cancers occur primary in tissues of epithelial origin such as liver pancreas, skin and GI tract. In 1990 41 areas in North America where clusters of increased cancers in fish were recognized due to environmental exposure to carcinogenic compounds. English sole from Puget Sound have incidence of liver lesions associated with exposure to polycyclic aromatic hydrocarbons (PAH). a novel DNA adduct was found in the neoplastic liver tissue. Cancer epizootic have been noted in both salt water and fresh water species. Notably the English sole, winter flounder, brown bullhead catfish (Ictalurus nebulosus), white sucker and the Atlantic Tomcod (Microgadus tomcod). PAH and PCB's have been implicated in these episodes. Mummichogs (Fundulus heteroclitus) from creosote contaminated water had liver and pancreatic neoplasm. Mechanistic considerations Using animals lower on the phylogenic tree may assist in unlocking the mechanisms of cell damage that lead to neoplastic changes. See figure 1 on page 227 for diagram of stages of hepatocarcinogenesis that should be considered when designing a carcinogenicity bioassay. "Dose" factors to consider are concentration of exposure, uptake and distribution kinetics, solubility of the test compound and physiology of the fish. Water soluble compound are easier to dose. DEN, an alkylating carcinogen which targets the liver is the best characterized carcinogen. 15-100 mg/L for several weeks then clean water for 8 weeks will promote tumor growth. It is important to consider dose response effects on cell injury, cell loss, and regenerative proliferation as too high a dose may overwhelm cellular defense mechanisms such as DNA repair. Route of exposure is also important. The following routes have been used Embryo microinjection into perivitelline space of embryonated eggs. It is necessary to avoid the chorion as it is selectively permeable. After injection eggs are grown out in clean water and fish are checked for tumor growth. This technique is good for poorly soluble compounds. Embryos may be more sensitive than other stages. Early life stage (pulse) exposure. Embryos or fry are exposed to several brief pulse of carcinogen separated by clean water and then grown out in clean water. Exposure is uniform and uptake occurs via gills, gi tract or skin. Dietary exposure. Requires a standardized diet. Good for poorly soluble compounds. a model for biomagnification of toxicants through the food chain. Can require a large amount of compound and uneven dosing of aggressive eaters may occur. Static exposures. Add compound to an aquarium. Constant bath of timed exposure. pH changes in the tank can effect the compound. fish may be exposed to metabolites of the compound too. Problem with infectious disease if the tank is not cleaned. Flow through exposures Ambient water continuously replaced. Compound delivered continuously by computer controlled dosing. Water quality remains good, more "waste" water generated. Initiation carcinogen can be used. A short (48) hour exposure to DEN before exposure to the test compound will increase tumor response. It is easier to pick up subtle carcinogens. Metabolism involving the cytochrome P450's (PYC's) are similar in fish and mammals. CYP1A is the subfamily of the cytochrome P450's that are induced by environmental toxicants in fish. CYP1A has a major impact on the activation or detoxification of carcinogens such as PAH's, PCB's, and polychlorinated dioxins. Pentobarbital normally induces CYP2B in mammals, this does not occur in fish, instead CYP1A is induced. Xenoestrogens which are an important class of aquatic pollutants, may alter the response of fish to carcinogen through the CYPs. CYP1A deficiencies were found in preneoplastic and neoplastic liver lesions in mummichogs. Medakas exposed to trichloroethylene had readily detectable level of CYP1A and low levels of CYP2E1. Other enzymes which have been examined with variable results are gamma glutamyl transpeptidase and glutathione-S transferase. More studies are needed to understand the roles of these enzymes. Immunohistochemistry may prove to be a valuable tool. DNA adducts: Mutagenesis as a mechanism of carcinogenesis Any chemical that forms DNA adducts is a potential mutagen and carcinogen. 1 Most carcinogens are mutagens 2 Mutagenic and carcinogenic properties depend on in vivo conversion of electrophilic derivatives that attack nucleoplilic sites in DNA to form adducts. 3 The degree of DNA adduction correlates with tumorigenic response. 4 Activation of proto-oncogenes has been demonstrated by the interaction of chemical carcinogens with DNA. DNA adduct determinations can provide information on metabolic pathways as well as chemical effects on DNA structure, transcription, synthesis and repair. It is a direct test for somatic mutation and a dosimeter of exposure to chemicals for cancer risk assessment. Analysis of the DNA adducts from fish are essential for mechanistic studies of mutagenesis and carcinogenesis and for biomonitoring populations at risk for environmentally caused cancer. Adducts have been detected using 32P-postlabelling, immunoassays, HPLC(high performance liquid chromatography), GC/MS (gas chromatography/mass spectrometry/ HPLC couples to GC/MS are the most promising methods. Immunohistochemistry May need to use more fish per group in order to get enough tissues for both histopathology and immunohistopathology. In order to preserve epitopes 10% Formalin should be used for no more than 48 hours before sectioning. Following fixation overnight demineralization aids in sectioning. PCNA (proliferating cell nuclear antigen can be used on Formalin fixed embedded tissues to identify replicating cells. BrdU (Bromodeoxyuridine) stain will also identify cells in the active cell cycle, but most be incorporated by live animals into their cells. BrdU is technically more difficult but identifies more replicating cells than PCNA. Oncogenes/tumor Suppressor genes The goldfish ras-oncogene has a high homology (96%) with K-ras human oncogene. point mutations in Ki-ras in rainbow trout correlates with liver tumors induced by various agents. The p53 tumor suppressor gene is another important molecular marker. Over expression of p53 protein products is correlated with liver neoplasm. More work on small fish models is needed to exploit this valuable tool.
1 Match the fish with the proper genus or species. zebrafish
A Onchorhynchus mykiss Japanese makada
B Gambusia affinis rainbow trout
C Poecilia reticulata swordtail
D Danio rerio guppy
E Oryzias latipes western mosquitofish
F Xiphorus
2 Name 3 methods to administer potential toxins to fish.
3 What are biomarkers?
4 Define sentinels, surrogates and predictors as these terms apply to toxicology studies.
5 What is diethyl diethylnitrosamine (DEN) used for ?
6 After exposure of fish to polycyclic aromatic hydrocarbons(PAH's), in what organ are most lesions found?
7 What factors effect the "dose" of toxin received by a fish?
8 Name two methods of exposure that could be used for poorly soluble compounds.
9 What is the relationship between DNA adduction and carcinogenicity?
10 Name 4 techniques used for analysis of DNA adducts.
1 D, E, A, F, C, B
2 In the water, in food and microinjection of embryos
3 Measurements of body fluids, cells or tissues that indicate in biochemical or cellular terms the presence of contaminants or the magnitude of the host response.
4 Sentinels- detect presence of contaminants Surrogates- indicate potential human exposure and effects Predictors- indicate long term effects on populations or ecosystems
5 DEN is a well characterized carcinogen that targets the liver. It is used as an initiation carcinogen. After 48 hour exposure to DEN the test compound is applied. There is an increased tumor response to the test compound, making it easier to pick up subtle carcinogens.
6 liver
7 Dose depends on amount of toxin, uptake by the fish, kinetics, solubility of the test compound, and physiology of the fish
8 Food and microinjection of embryos.
9 DNA adduction is a measure of mutability which is directly related to carcinogenicity.
10 DNA adduct can be analyzed using 32P postlabelling, immunoassays, HPLC or GC/MS.

 A Fish Model of Renal Regeneration and Development. ILAR 42 (4): 285.
Fish, like mammals have the ability to repair injured nephrons following sub lethal toxic injury. This process is called renal regeneration. The response is marked by the recovery from acute renal failure by replacing injured cells with new epithelial cells and restoring tubule integrity. Mammalian kidneys also have the ability to enlarge following unilateral nephrectomy. That is referred to as "compensatory renal hypertrophy." Fish kidneys, however, have the unique ability to respond to renal injury by denovo nephron neogenesis. Some of the species capable of this response include catfish, goldfish, zebrafish, trout, tilapia, and aglomerular toadfish. In mammals, the generation of new nephrons occur primarily during fetal development. A few species (e.g. the rat-up to 3 days after birth) have some neonatal development of new nephrons, but no mammal develops new nephrons after the neonatal period. Some adult fish retain the ability to develop new nephrons throughout life. Because of this fact, they can provide more tissue than can be found in larval or embryonic kidneys. This makes them good models to study both renal regeneration and nephron development.
Renal regeneration in mammals and fish are similar (Insult-> tubular degeneration and death->denudation of basement membrane->basophilic epithelium lines the denuded basement membrane->eventually replaced by cubodal epithelium->normal morphology in a few weeks). Experimantally, some of the nephrotoxicants used to initiate kidney damage in mammals and fish include Mercuric Chloride, Hexachlorobutadiene (HCBD), and Gentamicin (in fish).
Nephrogenesis in fish following toxic injury is similar to that seen during nephrogenesis in developing mammalian kidneys. Toadfish have kidneys that are aglomerular, and are a natural knockout model for studying glomerular structure and function. Toadfish are very sensitive to genatamicin toxicosis. This is probably due to the fact that gentamicin is is excreted primarily by glomerular filtration, and toad fish do not possess glomeruli.
The nephrogenic repair in fish and nephrogenic development in mammals proceed through similar stages indicating that highly conserved gene products may regulate these responses. This model may assist in identifying the genes that restrict nephrogenic responses in mammals, increasing the understanding of nephrogenesis, and ultimately may lead to the utilization of treatments for kidney disease by inducing nephrogenesis in adult mammalian kidneys.
1. Adult mammals have the ability to develop new nephrons. T or F
2. General kidney responses to injury/unilateral removal include:
a. regeneration
b. compensatory hypertrophy
c. nephrogenesis
d. all of the above
3. ___________ is exquisitely sensitive to gentamicin toxicosis.
a. goldfish
b. zebrafish
c. tilapia
d. toadfish
4. All of the following are used to initiate renal injuries in fish and mammals except:
c. Gentamicin
d. Mercuric Chloride
1. F, 2. D, 3. D, 4. A

 Development of Sensory Systems in Zebrafish (Danio rerio). ILAR 42 (4): 292.
The zebrafish is used for analyzing developmental events because of its external fertilization, optically clear chorion, translucent embryo, rapid development, and accessibility of early developmental stages. Zebrafish possess all of the sensory modalities: taste, tactile, smell, balance, vision, and hearing. All but taste has been studied in zebrafish. Zebrafish do not possess any overt inner ear specialization although they do have Weberian ossicles capable of transmitting vibration from the swim bladder to the inner ear. The lateral line and the inner ear are used to detect vibrational stimuli. Most studies of the zebrafish sensory systems involve molecular biology or genetics. More than 1100 genes have been identified and cloned in zebrafish, and more than half has been mapped to a specific linkage group. Dorsal Root Ganglia (DRG) As in all vertebrates, zebrafish DRG are organized segmentally. DRG sensory neurons are derived from neural crest cells that are transient, emybronic, migratory cells originating at the lateral edges of the neural plate. Before DRG formation, zebrafish skin is innervated by Rohon-Beard spinal sensory neurons that die as DRG neurons become established. Rohon-Beard cells and neural crest cells are intermingled in the lateral neural plate. Mutants defective in Delta-Notch signaling have more Rohon-Beard cells and fewer trunk neural crest cells, suggesting that Delta-Notch-mediated lateral inhibition segregates Rohon-Beard cell and trunk neural crest cell fates. Only the earliest migrating trunk neural crest cells generate DRG neurons. Each DRG initially contains only 1-3 cells in zebrafish but by adulthood, there are about 100 neurons per DRG, suggesting some neurons may be capable of division. Examples of zebrafish mutant strains with DRG developmental defects: 1) snail2, forkhead 6 used to identify premigratory neural crest cells via in situ hybridization; 2) alyron significantly reduced # of neural crest cells, lacks DRG at later stages; 3) sdpw15 (sensory deprived) no DRG neurons; 4) narrowminded no DRG and Rohon-Beard cells, suggesting genetic link between formation of neural crest and primary sensory neurons. Olfactory System Zebrafish possess a well-developed sense of smell that governs a variety of behaviors. The # of odorant receptor genes and of glomeruli in the olfactory bulb (OB) are about 1 order of magnitude smaller than those of mammals. The spatial organization of functional properties within the sensory surface and the OB are comparable to mammals. The reduced zebrafish olfactory system makes these animals a good model system to study olfactory development and CNS representation of olfactory information. The olfactory organ is a unique cellular population that arises outside the CNS from the ectodermally derived olfactory placode. As the placode differentiates into olfactory epithelium, adult epithelial stratified cell types (i.e. basal cells, sensory neurons, support cells) appear. Throughout life, basal cells generate olfactory sensory neurons. Neuronal cell bodies remain in the epithelium while axons grow through the olfactory nerve into the CNS. CNS axons segregate to form OB glomeruli. This glomerular organization of olfactory afferents is characteristic of both invertebrates and vertebrates. Each animal species has a stereotyped OB glomerular pattern. Sensory neuron axons expressing the same olfactory receptor converge on the same glomerulus, express receptor transcript in their axon terminals, and respond to the same odorant subset. The pathway that sensory neurons follow into the CNS is established by pioneer neurons; their appearance precedes olfactory neurons. Pioneer neurons establish initial telencephalic contact and are then used as a scaffold by the developing sensory neurons before dying. These pioneer neurons do not express any individual odorant receptors. Odorants are thought to activate specific g-protein-coupled receptors, and each olfactory neuron expresses 1 or a few odorant receptor genes. Neurons with common odorant receptor specificities converge to a small # of OB glomeruli. The logic underlying olfactory coding is a direct consequence of the selectivity of odorant receptor gene regulation and the concomitant targeting of specific OB olfactory neurons. Few zebrafish mutants are known with defects limited to the olfactory system. Lateral Line The lateral line is a rostral to caudal linear arrangement of mechanosensory neuromasts along the lateral aspect of the body. Neuromasts are also found throughout the surface of the head. Each neuromast consists of support and hair cells that resemble the cupula of the inner ear semicircular canals. Each neuromast is a mechanosensory end-organ sensitive to low-frequency vibrations. Mechanosensory information reaches the brain via the rostral and caudal lateral line nerves and is used for prey localization, navigation, schooling behavior, and predator avoidance. The rostral lateral line develops from a placode just rostral to the optic placode and from neural crest cells. The caudal lateral line develops from an embryonic primordium that migrates from its initial postotic position to the tail base. The horizontal myoseptum appears to be necessary for this migration. For example, the no tail mutant lacks the entire horizontal myoseptum and shows erratic primordium migration. Local myoseptal disruptions can also be induced by heat shock during somite formation, leading to abnormal migration pathways. As the fish grows, neuromasts and sensory neurons continue to develop to maintain an even spacing along the lateral aspect of the fish. Lateral line projections appear to be organized in a somatotopic fashion similar to the tonotopic projections of the cochlear hair cells in mammals. Other similarities to mammals, such as a defective myosin isoform linked to hereditary "deafness' in zebrafish, suggest that the zebrafish lateral line might be used as a model for mammalian hearing. Examples of zebrafish mutant strains with lateral line defects: 1) dog reduced neuromast # on trunk and tail; 2) hypersensitive increased neuromast # on trunk and tail. Both mutants have normal # and distribution of neuromasts on the head. Vestibular System The zebrafish inner ear consists of a vestibular end-organ that also serves as an auditory organ. Vertebrate vestibular end-organs are divided into a gravity receptor system and angular acceleration receptor system. The gravity receptor system consists of utricular, saccular, and lagenar maculae, each covered by an otolith. In zebrafish, each otolith has a stereotypic shape, and the maculae have characteristic shapes, patterns of sensory hair cell arrangement, orientations of the ciliary bundles of the hair cells, and characteristic polarization patterns. The angular acceleration receptor system consists of 3 orthogonal semicircular ducts, each with an ampulla containing sensory cristae. Little is known about the central projection of the primary afferent neurons of the zebrafish vestibular end-organs. To date, no genes have been identified as playing a role in guiding the vestibular primary afferent axons to their central targets. Presumably in the zebrafish, the sensory epithelia of the vestibular end-organ receive efferent innervation. Zebrafish inner ear development is probably initiated by release of fibroblast growth factor 3 from the hindbrain. Tether cells (hair cells) usually occur in pairs at the rostral and caudal ends of the ear. The utricular and saccular otoliths develop several days before the lagenar otolith. Development of the first 2 otoliths on each side appears to precede the development of the epithelial swellings destined to become maculae. Mutations in zebrafish have been identified that affect otic placode specification, otolith presence or size, otic vesicle size and shape, and semicircular canal formation. There are also "circling" mutants that have no visible inner ear morphology defects but display swimming behaviors suggestive of vestibular deficits, most likely due to defects of the vestibular primary afferent projection patterns. Visual System Zebrafish eyes have the characteristic vertebrate retina (neural retina + retinal pigmented epithelium). As in other vertebrates, the retinal ganglion cells are the first neurons to be born in the retina. Retinal stratification becomes progressively more distinct at later developmental stages and appears to be directed by pigmented epithelial cells via a pathway involving the mosaic eyes gene. The retinal photoreceptor cell layer contains 5 photoreceptor types: rods, short single cones, long single cones, and long and short members of the double cone pair. A precise retinotectal map is established very early in development and is the result of a 2 step process. First, after leaving the eye, the growth cones of the retinal ganglion cell axons navigate through the brain to find the contralateral tectal lobe. Axons from both eyes cross each other at the ventral diencephalic midline to form the chiasm. Second, after entering the tectum, retinal axons travel farther to their individual target sites within the tectal field where the projection is topographically organized so that neighboring retinal cells connect to neighboring places in the tectum. The retinal image is doubly inverted and topologically mapped onto an area of brain that is concerned with processing visual information. Mutants with retinal development defects include: eye anlage specification, optic cup growth rate, establishment of retinal stratification, differentiation of retinal neurons, and formation of the dorsoventral axis in the developing eye. Mutants with defects in retinotectal projections have also been described. These mutations affect distinct steps in the retinotectal pathway, from pathfinding between eye and tectum to map formation along the dorsal-ventral and the rostral-caudal axes of the tectum. Mutations that disrupt axon pathfinding to the tectum usually don't disrupt retinotopic mapping, and vice versa. In Drosophila melangaster, roundabout genes are members of the immunoglobulin superfamily which play a role in axon pathfinding as a receptor on neuronal growth cones. The astray gene has recently been cloned and is a novel zebrafish roundabout homologue.
1. Mechanical stimuli are detected by the ( ) system, which is composed of the ( ) and ( ).
2. Which cranial nerve innervates the inner ear?
3. Which of the following are disadvantages in using zebrafish to analyze developmental events?
A. External fertilization
B. Optically clear chorion
C. Translucent embryo
D. Rapid development
E. Accessibility of early developmental stages
F. None of the above
4. Match the specific sensory system with the type of neurons or cells found in that sensory system of zebrafish.
A. DRG i. Pioneer neurons
B. Olfactory System ii. Retinal ganglion cells
C. Lateral Line iii. Tether or hair cells
D. Vestibular System iv. Rohon-Beard spinal sensory neurons
E. Visual System v. Mechanosensory neuromasts
5. The fruit fly, Drosophila melanogaster, is used as a model for what? It is also used to study what other things?
1. Acousticolateralis; ear (3 semicircular canals and 3 otolith organs) and lateral line system (lateral line canal organs & superficial neuromasts = small and large pit organs).
2. Eighth cranial nerve innervates the inner ear.
3. F. All are advantages of this animal model for analyzing developmental events.
4. A. iv. B. i. C. v. D. iii. E. ii.
5. The fruit fly is a model for neuroblastoma of genetic origin. Drosophila melanogaster is also used to study genetics, mutagens, developmental biology, and Cu/Zn superoxide dismutase.

Xiphophorus Interspecies Hybrids as Genetic Models of Induced Neoplasia. ILAR 42 (4): 299.
Fishes of the genus Xiphophorus include the southern platyfish (X. maculatus) and the green swordtail (X. helleri). These fish are small, internally fertilizing, livebearing and derived from freshwater habitats. They have been used since the 1920's as a research model to study the genetic components of carcinogenesis. Most of the melanoma studies use interspecies hybrids (both the first generations; F1's, and backcrosses; BC1's) between the southern platyfish and the green swordtail; the hybrids spontaneously develop melanomas, whereas the two parental species rarely develop neoplastic lesions. The majority of the paper deals with the "Gordon-Kosswig Melanoma Model" which is a "two-hit" model for the development of melanoma in the fish hybrids. Loci have been identified which correspond to an oncogene (on the X chromosome of X. maculatus) and a tumor suppressor gene. Therefore, the first prerequisite is overexpression of a copy of a melanoma-determining gene (termed Xiphophorus melanoma receptor tyrosine kinase-2; Xmrk-2). In addition to overexpression of Xmrk-2, the second prerequisite of tumorigenesis is loss of the X. maculatus tumor suppressor locus, which normally regulates growth of macromelanophores and melanocyte precursor cell populations in parental X. maculatus fish.
Recent studies have identified the Xmrk-2 locus and a related proto-oncogene Xrmk-1, which have been mapped within 0.6cM of each other on the X. maculatus X chromosome. They both encode for transmembrane receptor tyrosine kinases, similar in structure to the human epidermal growth factor receptor. Interestingly, the Xmrk-1 transcript is expressed at low levels in all tested fish tissues; however, the Xmrk-2 transcript is virtually undetectable in normal tissues and is distinctly overexpressed in melanomas derived from F1 and BC1 hybrids within the Gordon-Kosswig cross.
It is evident that Xmrk-2 differs from the Xmrk-1 proto-oncogene in two significant ways: (1) through a mutation in the extracellular protein domain, which leads to a potent, constitutive tyrosine kinase receptor activity that exhibits ligand-independent activation, and (2) via constitutive oncogenic RNA/protein overexpression in melanotic tissues derived from hybrid fish. But these phenomena alone clearly do not independently lead to spontaneous melanoma within BC1 hybrids.
The scientific search for the tumor suppressor gene, initially referred to as Diff, has resulted in the cloning of the fish homolog of the cyclin-dependent kinase inhibitor-2 (CDNK2) gene family from the Xiphophorus genome. To distinguish this gene from its mammalian homologs, it is referred to as CDKN2X, wherein the "X" after the gene designation denotes the Xiphophorus form. Analysis of melanoma-bearing BC1 hybrids revealed that the vast majority (136/165) of these fish did not inherit the X. maculatus CDKN2X allele.
Detailed examination of the _expression of CDKN2X has led to the hypothetical model in which CDKN2X _expression levels in melanocytes may influence melanocyte differentiation in hybrid fish. If the CDKN2X gene is inadequately expressed in BC1 hybrids lacking X. maculatus alleles, melanocytes might not fully differentiate into macromelanophores; and this lack of complete differentiation, coupled with Xmrk-2 oncogene activation, could lead to a path of tumorigenesis.
The last part of the paper discusses the inducible Xiphophorus tumor models, which are not associated with the inheritance of CDKN2X. Many of these model crosses require pretreatment of BC1 animals with DNA-damaging agents (ultraviolet light, esp. UVB wavelengths) or N-methyl-N-nitrosurea (MNU) soon after birth to express tumor development. MNU-induced melanomas can arise through genetic mechanisms distinct from those identified for UVB-induced tumorigenesis. In addition, spontaneous melanoma models, that do not involve interpecies hybridization, are seen in Xiphophorus fish, and may be influenced by aging and androgens. Lastly, the paper highlights the research looking at tumor inducibility and DNA repair potential between the various tumor models. Researchers have initiated DNA repair assays in an effort to determine the varying DNA repair capabilities of parental species/strains used in the tumor model crosses.
Nonheritable melanoma is an important public health concern because of an alarming recent increase in worldwide incidence. From 1973-1990, the incidence of cutaneous malignant melanoma in the US increased ~94% - more than that for any other cancer. Therefore, animal models for melanoma, in which genetic components are easily recognized and subject to experimental manipulation and analysis, are needed.
1) What is the mechanism of action of MNU and what types of tumors does it induce in rodents vs. fish?
2) Characterize the different wavelengths of ultraviolet light for UVC, UVB, and UVA; what type of UV radiation is the majority of that which reaches the earth surface?
3) T/F. The web address will link to the Xiphophorus Genetic Stock Center at Southwest Texas State University.
4) Hybrid crosses, with no inducible agents used, between Xiphophorus fishes lead to what type of disease:
a) fibrosarcomas
b) neuroblastomas
c) melanomas
d) rhabdomyosarcomas
1) MNU is an alkylating agent that methylates DNA bases primarily at nucleophilic sites. MNU induces numerous cancers in rodents including mammary carcinomas and thyroid tumors in rats, as well as thymic lymphomas in mice. This carcinogen has also been shown to induce neuroblastomas, melanomas, fibrosarcomas, and rhabdomyosarcomas at high incidence in Xiphophorus hybrids.
2) UVC (230-290 nm), UVB (290-320 nm), UVA (320-400nm). The majority of UV radiation reaching the earth surface is within the UVA range.
3) True.
4) c; note that the other neoplasms are induced with MNU in hybrid fish

 Transgenic Fish as Models of Environmental Toxicology. ILAR 42 (4): 322.
Scientists have generated numerous transgenic fish using a variety of species and trangenes (First trangenic fish were introduced in 1985). Currently the emphasis is on refining fish transgenic technology, developing novel strains of fish with commercial benefits, and also the development of animal models. As an adjunct to environmental testing, it is hoped that soon transgenic fish will carry reporter genes driven by promoters that react to exposure to certain chemicals. The fish would then be removed from the environment and assayed for the chemical of interest. Examples of this include fish carrying metal responsive or heat shock promoters spliced to a green fluorescent or LUC reporter gene. These reporter genes act as indicators for exposure to heavy metals or other pollutants. The major problem associated with this method is the difficulty of sustaining gee statement past the founder generation. Many groups are working to solve that challenge.
Researchers have also developed a transgenic fish model that does not depend on gene statement, but carry prokaryotic vectors containing specific genes that serve as targets for quantifying in vivo mutagenesis. In this case, the fish are exposed to a chemical over time, and then tissues are collected. Genomic DNA is isolated from the tissues and processed to identify target and non-mutant target genes. Transgenic mutation assays are usually very efficient and require small amounts of tissue (1-5mg), and low animal numbers (6-10 animals /treatment) to detect significant mutation induction above a background mutation frequency.
Some of the transgenic mutation models include:
1.The bacteriophage lLIZ vector in which the fish used is the Japanese Medaka. (Oryzias latipes). The fish carry multiple copies of the vector containing lacI and cII bacterial genes (they are mutational targets). The most widely used rodent mutation assay is based on this bacteriophage vector. This increases the potential of these fish being used for comparative mutagenesis studies.
2.The bacteriophage fX174 is based on a bacteriophage vector using the Japanese Medaka and the Mummichog (Fundulus heteroclitus).
Examples of plasmid mutation models include:
1.The pML4 plsmid zebrafish (Danio rerio) model. This model was adapted from a mouse mutation based on the pML4 plasmid. It uses the rspL transgene as a mutational target. Investigators observed that spontaneous mutation frequency in the transgenic zebrafish was similar to that of transgenic mice similarly manipulated.
2.The pUR288 plasmid vector inserted into the medaka or the mummichog. The vector harbors the lacZ gene. The assay has been useful for detecting point mutations, small deletions, insertions, and rearrangements induced by clastogenic agents.
Fish selection for transgenic modeling is based on several factors. These factors include, but are not limited to small size, short generation time, cost effective husbandry, and well described embryology.
Fish have played an important role in assessing the potential risk of chemical contaminants in aquatic environments (sentinels). Like transgenic rodents in biomedicine, transgenic fish can make significant contributions to environmental toxicology. Applications of these fish for environmental studies are in the early stages, but the new models e.g. targeted mutagenesis models, and spontaneous mutagensis (similar to rodents) models have illustrated the value of transgenic fish as a comparative animal model. The challenges that impact the development and application of these models are the need to improve and refine the transgenic technology surrounding the production of the fish, and in establishing proper standards for husbandry and use of these animals.
1.Examples of fish used as transgenic models include:
a.Japanese Medaka
d.a and c
2.What's the gens ad apeces of the Japanese Medaka, Mummichog and the Zebrafish?
2.Medaka (Oryzias latipes), Mummichog (Fundulus heteroclitus), Zebrafish (Danio or Brachydanio rerio).