Transcription activator‐like effector nucleases efficiently disrupt the target gene in Iberian ribbed newts (<i><scp>P</scp>leurodeles waltl</i>), an experimental model animal for regeneration

Hayashi, Toshinori; Sakamoto, Kousuke; Sakuma, Tetsushi; Yokotani, Naoki; Inoue, Takeshi; Kawaguchi, Eri; Agata, Kiyokazu; Yamamoto, Takashi; Takeuchi, Takashi

doi:10.1111/dgd.12103

Cited by 38 publications

(27 citation statements)

References 25 publications

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“…Forward genetic screens to identify mutations affecting these processes cannot be carried out within a reasonable time frame, and reverse genetics using homologous recombination is particularly challenging in anamniotic vertebrates. Recent innovations in targeted mutagenesis using DNA-modifying enzymes have enabled the generation of frogs, fish and newts with mutations in targeted genes (Hwang et al, 2013a;Ishibashi et al, 2012;Lei et al, 2012;Hayashi et al, 2014), and here we provide the first demonstration of the successful application of such a technique in the axolotl. As a wealth of information about genes implicated in urodele limb regeneration has been compiled over decades of research using the axolotl, DNA-modifying enzymes and RGNs now make possible a reverse genetic approach for the characterization of these genes in vivo.…”

Section: Discussionmentioning

confidence: 92%

Highly efficient targeted mutagenesis in axolotl using Cas9 RNA-guided nuclease

et al. 2014

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Among tetrapods, only urodele salamanders, such as the axolotl Ambystoma mexicanum, can completely regenerate limbs as adults. The mystery of why salamanders, but not other animals, possess this ability has for generations captivated scientists seeking to induce this phenomenon in other vertebrates. Although many recent advances in molecular biology have allowed limb regeneration and tissue repair in the axolotl to be investigated in increasing detail, the molecular toolkit for the study of this process has been limited. Here, we report that the CRISPR-Cas9 RNA-guided nuclease system can efficiently create mutations at targeted sites within the axolotl genome. We identify individual animals treated with RNA-guided nucleases that have mutation frequencies close to 100% at targeted sites. We employ this technique to completely functionally ablate EGFP expression in transgenic animals and recapitulate developmental phenotypes produced by loss of the conserved gene brachyury. Thus, this advance allows a reverse genetic approach in the axolotl and will undoubtedly provide invaluable insight into the mechanisms of salamanders' unique regenerative ability.

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Section: Discussionmentioning

confidence: 92%

Highly efficient targeted mutagenesis in axolotl using Cas9 RNA-guided nuclease

et al. 2014

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“…Similar to white, we reasoned that albino might map to a locus associated with albinism in other amphibians 37,38 , including axolotl 39 , and so focused our efforts on tyr and oca2 . tyr encodes an enzyme that functions in melanin synthesis while oca2 encodes an integral membrane protein that functions to maintain melanosomal pH 40,41 .…”

Section: Resultsmentioning

confidence: 99%

Identification of Mutant Genes and Introgressed Tiger Salamander DNA in the Laboratory Axolotl, Ambystoma mexicanum

Woodcock

Vaughn-Wolfe

Elias

et al. 2017

Sci Rep

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The molecular genetic toolkit of the Mexican axolotl, a classic model organism, has matured to the point where it is now possible to identify genes for mutant phenotypes. We used a positional cloning–candidate gene approach to identify molecular bases for two historic axolotl pigment phenotypes: white and albino. White (d/d) mutants have defects in pigment cell morphogenesis and differentiation, whereas albino (a/a) mutants lack melanin. We identified in white mutants a transcriptional defect in endothelin 3 (edn3), encoding a peptide factor that promotes pigment cell migration and differentiation in other vertebrates. Transgenic restoration of Edn3 expression rescued the homozygous white mutant phenotype. We mapped the albino locus to tyrosinase (tyr) and identified polymorphisms shared between the albino allele (tyr a) and tyr alleles in a Minnesota population of tiger salamanders from which the albino trait was introgressed. tyr a has a 142 bp deletion and similar engineered alleles recapitulated the albino phenotype. Finally, we show that historical introgression of tyr a significantly altered genomic composition of the laboratory axolotl, yielding a distinct, hybrid strain of ambystomatid salamander. Our results demonstrate the feasibility of identifying genes for traits in the laboratory Mexican axolotl.

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“…Construction of omics data has already been reported in the newt Nothophtalmus viridescens, and the presence of many newtspecific transcripts as well as genes conserved in vertebrates has been revealed (Looso et al, 2013). Furthermore, advances in gene technology allowed DNA targeting in newts using TAL effector-like nuclease (Hayashi et al, 2014;Flowers et al, 2014). These technologies will contribute to the further elucidation of the molecular mechanisms underlying fertilization events in nonmodel amphibian species.…”

Section: Cysteine Rich Secretory Proteins (Crisps)mentioning

confidence: 99%

In silico identification of the genes for sperm-egg interaction in the internal fertilization of the newt Cynops pyrrhogaster

Watanabe¹,

Takayama‐Watanabe²

2014

Int. J. Dev. Biol.

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A specific sperm-egg interaction in the oviductal matrix is crucial for internal fertilization of the red-bellied newt, Cynops pyrrhogaster. An understanding of the molecular basis of this interaction is expected to elucidate the evolutionary history of internal fertilization in amphibians. Recently, deep sequencing technology has provided global gene information even in nonmodel animals, allowing us to understand specific features of the molecular mechanisms underlying fertilization in C. pyrrhogaster. In the present study, we screened de novo assembled RNAseq from ovary, testis, and oviduct samples in C. pyrrhogaster and identified the base sequences encoding zona pellucida (ZP) proteins, voltage-dependent Ca 2+ channels, and cysteine-rich secretory proteins (CRISPs), which respectively are sperm receptors for egg envelopes, major mediators of sperm intracellular signaling, and expected extracellular modulators for sperm function in the female reproductive tract. In the ovary, ZP homologues of all six subgroups were found, including a ZP1 homologue that was newly found in amphibians, a ZP4 homologue, and six ZPC homologues. The unique combination of ZP proteins suggests a new mechanism for sperm binding to egg envelopes in the internal fertilization of C. pyrrhogaster. In the testis, CaV1.1, 1.2, and 3.2, which are L-and T-type voltage-dependent Ca 2+ channels, were found as potential mediators for the internal fertilization-specific sperm-egg interaction. We also found CRISP 2 in the oviduct, which is speculated to participate in the sperm-egg interaction. These results indicate that the de novo assembled RNAseq is a powerful tool allowing analysis of the specific sperm-egg interactions in C. pyrrhogaster.

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Transcription activator‐like effector nucleases efficiently disrupt the target gene in Iberian ribbed newts (Pleurodeles waltl), an experimental model animal for regeneration

Cited by 38 publications

References 25 publications

Highly efficient targeted mutagenesis in axolotl using Cas9 RNA-guided nuclease

Highly efficient targeted mutagenesis in axolotl using Cas9 RNA-guided nuclease

Identification of Mutant Genes and Introgressed Tiger Salamander DNA in the Laboratory Axolotl, Ambystoma mexicanum

In silico identification of the genes for sperm-egg interaction in the internal fertilization of the newt Cynops pyrrhogaster

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