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The 5'Hox genes play crucial roles in limb development and specify regions in the proximal-distal axis of limbs. However, there is no direct genetic evidence that Hox genes are essential for limb development in non-mammalian tetrapods or for limb regeneration. Here, we produced single to quadruple Hox13 paralog mutants using the CRISPR/Cas9 system in newts (Pleurodeles waltl), which have strong regenerative capacities, and also produced germline mutants. We show that Hox13 genes are essential for digit formation in development, as in mice. In addition, Hoxa13 has a predominant role in digit formation, unlike in mice. The predominance is probably due to the restricted expression pattern of Hoxd13 in limb buds and the strong dependence of Hoxd13 expression on Hoxa13. Finally, we demonstrate that Hox13 genes are also necessary for digit formation in limb regeneration. Our findings reveal that the general function of Hox13 genes is conserved between limb development and regeneration, and across taxa. The predominance of Hoxa13 function both in newt limbs and fish fins, but not in mouse limbs, suggests a potential contribution of Hoxa13 function in fin-to-limb transition. [ABSTRACT FROM AUTHOR]

Recent advances in genome editing using programmable nucleases, such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system, have facilitated reverse genetics in Xenopus tropicalis. To establish a practical workflow for analyzing genes of interest using CRISPR-Cas9, we examined various experimental procedures and conditions. We first compared the efficiency of gene disruption between Cas9 protein and mRNA injection by analyzing genotype and phenotype frequency, and toxicity. Injection of X. tropicalis embryos with Cas9 mRNA resulted in high gene-disrupting efficiency comparable with that produced by Cas9 protein injection. To exactly evaluate the somatic mutation rates of on-target sites, amplicon sequencing and restriction fragment length polymorphism analysis using a restriction enzyme or recombinant Cas9 were performed. Mutation rates of two target genes (slc45a2 and ltk) required for pigmentation were estimated to be over 90% by both methods in animals exhibiting severe phenotypes, suggesting that targeted somatic mutations were biallelically introduced in almost all somatic cells of founder animals. Using a heteroduplex mobility assay, we also showed that off-target mutations were induced at a low rate. Based on our results, we propose a CRISPR-Cas9-mediated gene disruption workflow for a rapid and efficient analysis of gene function using X. tropicalis founders. [ABSTRACT FROM AUTHOR]

A large number of chemicals are routinely detected in aquatic environments, and these chemicals may adversely affect aquatic organisms. Accurate risk assessment requires understanding drugmetabolizing systems in aquatic organisms because metabolism of these chemicals is a critical determinant of chemical bioaccumulation and related toxicity. In this study, we evaluated mRNA expression levels of nuclear receptors and drug-metabolizing enzymes as well as cytochrome P450 (CYP) activities in pro-metamorphic tadpoles, froglets, and adult frogs to determine how drug-metabolizing systems are altered at different life stages. We found that drug-metabolizing systems in tadpoles were entirely immature, and therefore, tadpoles appeared to be more susceptible to chemicals compared with metamorphosed frogs. On the other hand, cyp1a mRNA expression and CYP1A-like activity were higher in tadpoles. We found that thyroid hormone (TH), which increases during metamorphosis, induced CYP1A-like activity. Because endogenous TH concentration is significantly increased during metamorphosis, endogenous TH would induce CYP1A-like activity in tadpoles. [ABSTRACT FROM AUTHOR]

Amphibians have made many fundamental contributions to our knowledge, from basic biology to biomedical research on human diseases. Current genome editing tools based on the CRISPR-Cas system enable us to perform gene functional analysis in vivo, even in non-model organisms. We introduce here a highly efficient and easy protocol for gene knockout, which can be used in three different amphibians seamlessly: Xenopus laevis, Xenopus tropicalis, and Pleurodeles waltl. As it utilizes Cas9 ribonucleoprotein complex (RNP) for injection, this cloning-free method enables researchers to obtain founder embryos with a nearly complete knockout phenotype within a week. To evaluate somatic mutation rate and its correlation to the phenotype of a Cas9 RNP-injected embryo (crispant), we also present accurate and cost-effective genotyping methods using pooled amplicon-sequencing and a user-friendly web-based tool., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Amphibians provide an ideal model to study the actions of thyroid hormone (TH) in animal development because TH signaling via two TH receptors, TRα and TRβ, is indispensable for amphibian metamorphosis. However, specific roles for the TRβ isoform in metamorphosis are poorly understood. To address this issue, we generated trβ- disrupted Xenopus tropicalis tadpoles using the CRISPR-Cas system . We first established a highly efficient and rapid workflow for gene disruption in the founder generation (F0) by injecting sgRNA and Cas9 ribonucleoprotein. Most embryos showed severe mutant phenotypes carrying high somatic mutation rates. Utilizing this founder analysis system, we examined the role of trβ in metamorphosis. trβ -disrupted pre-metamorphic tadpoles exhibited mixed responsiveness to exogenous TH. Specifically, gill resorption and activation of several TH-response genes, including trβ itself and two protease genes, were impaired. However, hind limb outgrowth and induction of the TH-response genes, klf9 and fra-2 , were not affected by loss of trβ Surprisingly, trβ- disrupted tadpoles were able to undergo spontaneous metamorphosis normally, except for a slight delay in tail resorption. These results indicate TRβ is not required but contributes to the timing of resorptive events of metamorphosis., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

The nuclear positioning and chromatin dynamics of eukaryotic genes are closely related to the regulation of gene expression, but they have not been well examined during early development, which is accompanied by rapid cell cycle progression and dynamic changes in nuclear organization, such as nuclear size and chromatin constitution. In this study, we focused on the early development of the sea urchin Hemicentrotus pulcherrimus and performed three-dimensional fluorescence in situ hybridization of gene loci encoding early histones (one of the types of histone in sea urchin). There are two non-allelic early histone gene loci per sea urchin genome. We found that during the morula stage, when the early histone gene expression levels are at their maximum, interchromosomal interactions were often formed between the early histone gene loci on separate chromosomes and that the gene loci were directed to locate to more interior positions. Furthermore, these interactions were associated with the active transcription of the early histone genes. Thus, such dynamic interchromosomal interactions may contribute to the efficient synthesis of early histone mRNA during the morula stage of sea urchin development., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2017. Published by The Company of Biologists Ltd.)