Rapid gynogenetic mapping of <i>Xenopus tropicalis</i> mutations to chromosomes

Khokha, Mustafa K.; Krylov, Vladimír; Reilly, Michael J.; Gall, Joseph G.; Bhattacharya, Dipankan; Cheung, Chung Yan J.; Kaufman, S.; Lam, Dang Khoa; Mácha, J; Ngo, Catherine; Prakash, Neha; Schmidt, Philip; Tlapáková, Tereza; Trivedi, Toral; Tůmová, Lucie; Abu-Daya, Anita; Geach, Timothy J.; Vendrell, Elisenda; Ironfield, Holly; Sinzelle, Ludivine; Sater, Amy K.; Wells, Dan E.; Harland, Richard M.; Zimmerman, Lyle B.

doi:10.1002/dvdy.21965

Cited by 42 publications

(79 citation statements)

References 67 publications

(53 reference statements)

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“…These were siblings of the germ-line mutated males from clutches that showed a high frequency of somatic mutation. Gynogenesis diploidizes activated eggs by preventing the extrusion of the second polar body and serves to homozygose recessive mutations (25). Although off-target mutations would result in high frequencies of mutant embryos in gynogenotes produced from these females, we did not detect such mutations.…”

Section: Resultscontrasting

confidence: 62%

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Efficient targeted gene disruption in the soma and germ line of the frog Xenopus tropicalis using engineered zinc-finger nucleases

Young

Cherone

Doyon

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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127

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The frog Xenopus , an important research organism in cell and developmental biology, currently lacks tools for targeted mutagenesis. Here, we address this problem by genome editing with zinc-finger nucleases (ZFNs). ZFNs directed against an eGFP transgene in Xenopus tropicalis induced mutations consistent with nonhomologous end joining at the target site, resulting in mosaic loss of the fluorescence phenotype at high frequencies. ZFNs directed against the noggin gene produced tadpoles and adult animals carrying up to 47% disrupted alleles, and founder animals yielded progeny carrying insertions and deletions in the noggin gene with no indication of off-target effects. Furthermore, functional tests demonstrated an allelic series of activity between three germ-line mutant alleles. Because ZFNs can be designed against any locus, our data provide a generally applicable protocol for gene disruption in Xenopus .

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Section: Resultscontrasting

confidence: 62%

“…Female X. laevis were injected with 500 U HCG, and eggs were fertilized with macerated testes and injected at the four-cell stage with 10 nL maximum volume. Diploid gynogenotes were generated according to previously described methods (25).…”

Section: Methodsmentioning

confidence: 99%

Efficient targeted gene disruption in the soma and germ line of the frog Xenopus tropicalis using engineered zinc-finger nucleases

Young

Cherone

Doyon

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

127

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“…After feeding stage 45/46, ecl embryos declined rapidly and never reached metamorphosis. To determine the genetic lesion in ecl mutants, we genotyped genomic DNA from mutants and WT siblings using published or selfdesigned simple sequence-length polymorphism (SSLP) markers (11,20). Linkage analysis using pools of genomic DNA from gynogenetic mutants and WT siblings assigned the ecl mutation to linkage group 6 (LG6) (old LG2), corresponding to scaffold 6 in X. tropicalis genome assembly 7.1 (www.xenbase.org) (Fig.…”

mentioning

confidence: 99%

“…Earlier studies of lineage analysis and spatiotemporal expression of transcription factors during inner ear development led to construction of an inner ear fate map in Xenopus and this fate map allows us to interpret gene expression patterns within the context of the anatomy (8,9). In recent years, genetic and genomic approaches have been developed in Xenopus tropicalis (10)(11)(12)(13). To advance our understanding of inner ear development, we have screened N-ethyl-N-nitrosourea (ENU) mutagenized X. tropicalis colonies and recovered an inner ear mutant named eclipse (ecl).…”

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confidence: 99%

Sp8 regulates inner ear development

Chung

Medina-Ruiz

Harland

2014

Proc. Natl. Acad. Sci. U.S.A.

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A forward genetic screen of N-ethyl-N-nitrosourea mutagenized Xenopus tropicalis has identified an inner ear mutant named eclipse (ecl). Mutants developed enlarged otic vesicles and various defects of otoconia development; they also showed abnormal circular and inverted swimming patterns. Positional cloning identified specificity protein 8 (sp8), which was previously found to regulate limb and brain development. Two different loss-of-function approaches using transcription activator-like effector nucleases and morpholino oligonucleotides confirmed that the ecl mutant phenotype is caused by down-regulation of sp8. Depletion of sp8 resulted in otic dysmorphogenesis, such as uncompartmentalized and enlarged otic vesicles, epithelial dilation with abnormal sensory end organs. When overexpressed, sp8 was sufficient to induce ectopic otic vesicles possessing sensory hair cells, neurofilament innervation in a thickened sensory epithelium, and otoconia, all of which are found in the endogenous otic vesicle. We propose that sp8 is an important factor for initiation and elaboration of inner ear development.T he vertebrate inner ear is a sensory organ responsible for balance and sound detection. Dysfunction of the inner ear is among the most common congenital disorders, affecting at least 1 in 500 births (1) and ∼40% of sensorineural deafness is associated with inner ear malformations (2). However, the study of hearing and balance impairment in humans is limited by the inability to follow inner ear development. Vertebrates share similarities in the sequence of developmental events that form the inner ear: the formation of an otic placode from an ectodermal thickening, morphogenesis to form the otocyst, and regional patterning of the otic vesicle (OV), resulting in the 3D membranous labyrinth (3, 4). Multipotent sensory progenitor cells are induced in the ectoderm surrounding the anterior neural plate, a domain termed the preplacodal region (PPR), and six1 has been characterized as marking this panplacodal domain. Signals from hindbrain and the regional expression of different transcription factors differentiate the PPR into the otic placode. The OV is partitioned by asymmetrical expression of various developmental regulators to pattern subdomains of the developing inner ear.The abilities of Xenopus to elucidate the cellular and molecular aspects of developmental processes position it as a valuable model organism (5-7). Earlier studies of lineage analysis and spatiotemporal expression of transcription factors during inner ear development led to construction of an inner ear fate map in Xenopus and this fate map allows us to interpret gene expression patterns within the context of the anatomy (8, 9). In recent years, genetic and genomic approaches have been developed in Xenopus tropicalis (10-13). To advance our understanding of inner ear development, we have screened N-ethyl-N-nitrosourea (ENU) mutagenized X. tropicalis colonies and recovered an inner ear mutant named eclipse (ecl). The ecl mutant perturbs specificity protei...

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“…Labeled chromosomes were identified using their p/q arm ratios and relative sizes. The total length of the largest chromosome, chromosome 1, was used as an internal standard (34). Two slides per probe and over Ϸ30 metaphase spreads per slide were analyzed.…”

Section: ϫ⌬⌬Ctmentioning

confidence: 99%

Characterization of a Xenopus tropicalis Endogenous Retrovirus with Developmental and Stress-Dependent Expression

Sinzelle

Carradec

Paillard

et al. 2011

J Virol

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We report on the identification and characterization of XTERV1, a full-length endogenous retrovirus (ERV) within the genome of the western clawed frog (Xenopus tropicalis). XTERV1 contains all the basic genetic elements common to ERVs, including the classical 5-long terminal repeat (LTR)-gag-pol-env-3-LTR architecture, as well as conserved functional motifs inherent to each retroviral protein. Using phylogenetic analysis, we show that XTERV1 is related to the Epsilonretrovirus genus. The X. tropicalis genome harbors a single full-length copy with intact gag and pol open reading frames that localizes to the centromeric region of chromosome 5. About 10 full-length defective copies of XTERV1 are found interspersed in the genome, and 2 of them could be assigned to chromosomes 1 and 3. We find that XTERV1 genes are zygotically transcribed in a regulated spatiotemporal manner during frog development, including metamorphosis. Moreover, XTERV1 transcription is upregulated under certain cellular stress conditions, including cytotoxic and metabolic stresses. Interestingly, XTERV1 Env is found to be homologous to FR47, a protein upregulated following cold exposure in the freeze-tolerant wood frog (Rana sylvatica). In addition, we find that R. sylvatica FR47 mRNA originated from a retroviral element. We discuss the potential role(s) of ERVs in physiological processes in vertebrates.

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Rapid gynogenetic mapping of Xenopus tropicalis mutations to chromosomes

Cited by 42 publications

References 67 publications

Efficient targeted gene disruption in the soma and germ line of the frog Xenopus tropicalis using engineered zinc-finger nucleases

Efficient targeted gene disruption in the soma and germ line of the frog Xenopus tropicalis using engineered zinc-finger nucleases

Sp8 regulates inner ear development

Characterization of a Xenopus tropicalis Endogenous Retrovirus with Developmental and Stress-Dependent Expression

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