Goda T et al. (2006), Genetic screens for mutations affecting develop...

XB-ART-203

PLoS Genet 2006 Jun 01;26:e91. doi: 10.1371/journal.pgen.0020091.

Show Gene links Show Anatomy links

Genetic screens for mutations affecting development of Xenopus tropicalis.

Goda T , Abu-Daya A , Carruthers S , Clark MD , Stemple DL , Zimmerman LB .

???displayArticle.abstract???
We present here the results of forward and reverse genetic screens for chemically-induced mutations in Xenopus tropicalis. In our forward genetic screen, we have uncovered 77 candidate phenotypes in diverse organogenesis and differentiation processes. Using a gynogenetic screen design, which minimizes time and husbandry space expenditures, we find that if a phenotype is detected in the gynogenetic F2 of a given F1 female twice, it is highly likely to be a heritable abnormality (29/29 cases). We have also demonstrated the feasibility of reverse genetic approaches for obtaining carriers of mutations in specific genes, and have directly determined an induced mutation rate by sequencing specific exons from a mutagenized population. The Xenopus system, with its well-understood embryology, fate map, and gain-of-function approaches, can now be coupled with efficient loss-of-function genetic strategies for vertebrate functional genomics and developmental genetics.

???displayArticle.pubmedLink??? 16789825
???displayArticle.pmcLink??? PMC1475704
???displayArticle.link??? PLoS Genet
???displayArticle.grants??? [+]

Species referenced: Xenopus tropicalis
Genes referenced: actc1 actl6a alas2 atp12a b3gat1l btg3 cldn6.1 fgfr4 glul hba1 hba4 krt18.1 lama1 myh6 nfatc3 nipbl pck1 phf10 prdx2 rxrb sall4 smad10 smad4 sqstm1 trappc13 ube2d3 urod ventx1
GO keywords: AP-2 adaptor complex [+]
???displayArticle.antibodies??? B3gat1 Ab3 Lama1 Ab1
Lines/Strains:

Xtr.legmZimml Xtr.tupmZimml Xtr.dstmZimml Xtr.bunmZimml Xtr.bxemZimml Xtr.mammZimml Xtr.smad4mZimml Xtr.mlomZimml Xla.Tg(Xtr.ins:dnthra-eGFP)Brown Xtr.Ivory Coast TGA (homozygous blue) Xla.Tg(Xtr.col2a1:RTTA;TRE:Cre;cryga:GFP3)Buchz

???displayArticle.disOnts??? kidney lipoma
???displayArticle.omims??? BLOOD GROUP, KIDD SYSTEM; JK

???attribute.lit??? ???displayArticles.show???

	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. doi:10.1371/journal.pgen.0020091.g001
	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. doi:10.1371/journal.pgen.0020091.g002
	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. doi:10.1371/journal.pgen.0020091.g003
	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. doi:10.1371/journal.pgen.0020091.g004
	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. doi:10.1371/journal.pgen.0020091.g005
	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).
	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. doi:10.1371/journal.pgen.0020091.g007
	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). doi:10.1371/journal.pgen.0020091.g008
	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.
	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).
	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.
	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)

References [+] :

Amaya, Frog genetics: Xenopus tropicalis jumps into the future. 1998, Pubmed, Xenbase