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Figure 1. Forward Screen Strategy
Following ENU mutagenesis of postmeiotic sperm and fertilization of wild-type eggs, a founder F1 generation was raised. Males were used in a reverse genetic strategy (see Figure 7). F1 females were used to generate gynogenetic embryos that were screened for embryonic defects. F1 females carrying defects were outcrossed and the resulting F2 embryos screened for carriers, then sibling intercrossed. Color code indicates status of specific mutations (see Figure 2 and Tables 2 and 3): red for phenotypes confirmed in the progeny of a conventional F2 sibling intercross, orange for phenotypes confirmed heritable by backcross or F2 gynogenesis, green for phenotypes observed twice from gynogenesis of an individual F1 female, and blue for phenotypes observed once and not yet retested.
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Figure 2. Phenotypes Detected
Defects were sorted into ten broad categories (shown with representative images): eye (zatoichi), inner ear and otolith (komimi), neural crest/pigment (cyd vicious), dwarf (issunboushi), axial (bulldog), circulation (desert tad), gut (haggis), cardiovascular system and motility, and head (troll). Color code (green, orange, red) is described in Figure 1 and used in Tables 2 and 3 to denote current confirmation status of individual mutations in the pipeline. Provisional alleles (�prov,� green) have not yet been assayed for heritability.
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Figure 3. Axis Extension Mutations
The dwarf phenotypes tansoku, yodaa, and issunboushi show relatively normal head and trunk structures, but are defective in tail extension. Anti-laminin immunohistochemistry reveals discrete defects in axial structures, with tansoku ([C] and [D]) displaying a reduced number of relatively well-ordered somites, issunboushi ([G] and [H]) showing highly disordered intersomitic boundaries, and yodaa ([E] and [F]) displaying an intermediate phenotype.
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Figure 4. The mlo Mutation Exhibits Paralysis and Motor Neuron Defects
Neural tissue of mlo and diploid control was stained with the HNK-1 antibody. In wild-type tadpoles ([A] and [C]), motor neuron axons (white arrow) travel down the intermyotomal cleft from the neural tube; in mlo ([B] and [D]) the axonal tracts (black arrow) wander away from the intermyotomal space. In situ hybridization with cardiac actin ([E] and [F]) shows that somite structure is relatively unaffected.
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Figure 5. wha Embryos Show Defects in Hematopoeisis
Whole mount in situ hybridization with α-globin suggests that wha blood distribution is aberrant ([A] and [C]), with reduced globin staining pooled ventrally (black arrows) rather than distributed throughout the circulatory system as in wild-type tadpoles. Comparison of ventral views of wha (C) globin staining with that of muzak (B), a mutant which is impaired in heart function but not hematopoiesis, leading to ventral pooling of normal levels of blood (white arrows), confirms that wha is quantitatively defective in blood formation rather than circulation. See also Tables 6 and 7 for microarray analysis of wha.
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Figure 6. The Mutation cyd vicious [Xtra.supt5h] Displays Neural Crest and Eye Defects
Brightfield images of stage ~38 outcrossed sibling wild-type embryo (A) and gynogenetic cyd embryo (B). Likely neural crest-derived pigmented cells (arrows, [C] and [D]) fail to migrate in cyd, and instead populate the lumen of the neural tube. St. 40 wild-type eye (E) displays laminar organization surrounded by prominent pigmented epithelium. cyd eyes form a poorly laminated ball of neural retina surrounding a central mass of pigmented tissue, and no lens tissue is visible (F).
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Figure 7. Reverse Screen Strategy
ENU-treated sperm (G0) was used to fertilize wild-type eggs (in vitro fertilization), and the resulting F1 families raised to adulthood. F1 males were killed and their testes dissociated, with a portion used to generate F2 tadpoles and the remainder frozen in several aliquots per individual (F1 library). F1 females were used in the gynogenetic forward screens (see Figure 1). F2 genomic DNA was isolated from the tadpoles for reverse genetic (TILLING) screens. Known genomic sequences were used to design nested PCR primers, and then individual F2 tadpole amplicons were sequenced to detect induced mutations. Mutations are then recovered from frozen testes by in vitro fertilization for subsequent phenotypic analysis.
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Figure 8. Mutation Detection
All sequences generated by TILLING (see Figure 7) are examined and compared to a reference sequence by a Mutation Finder program. Any disparities with reference sequences are recorded for view in a Mutation Display window (A). Reference DNA and amino acid sequence is displayed above the trace and the TILLING trace below. Boxes around reference sequence nucleotides denote alterations in one or more TILLING traces; box color indicates number of traces altered. The asterisk above the reference amino acid sequence designates a position at which a mutation has been visually confirmed and recorded. Clicking on a box or asterisk will recover the trace(s) containing the change. Traces that are not confirmed are dismissed. All processed traces are accessible via the pull down trace lists. Examples of mutations are displayed for silent, missense, and nonsense alongside wild-type traces for comparison (B).
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Xtr.mlo tadpole, also known as 'mrs lot' phenotype. ENU-mutants exhibits paralysis and motor neuron defects. Neural tissue of mlo and diploid control was stained with the HNK-1 antibody. In wild-type tadpoles motor neuron axons travel down the intermyotomal cleft from the neural tube; in mlo ([B] and [D]) the axonal tracts (black arrow) wander away from the intermyotomal space.
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WISH with α-globin suggests that the blood distribution in mutant Xtr.smad4.1 tadpoles (on right, also known as whitehart or wha) is aberrant, with reduced globin staining pooled ventrally (black arrows) rather than distributed throughout the circulatory system as in wild-type tadpoles (left).
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hba1 (hemoglobin subunit alpha 1) gene expression in Xtr.smad4.1 mutant at tadpole stage, ( also known as whitehart or wha), asayyed bu WISH, ventral view, anterior left. This assays confirms phenotype as defects in blood formation with normal circulation.
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hba1 (hemoglobin subunit alpha 1) gene expression in Xtr.myh6 mutant tadpole, ventral view, anterior left. This mutant has impaired in heart function but normal hematopoiesis, leading to ventral pooling and normal levels of blood (white arrows)
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