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Three POU family class V gene homologues are expressed in the development of Xenopus. In contrast to the expression of Pou5f3.1 and Pou5f3.2 in organogenesis, Pou5f3.3 is expressed during oogenesis in ovary. We investigated the expression and function of Pou5f3.3 in organogenesis of Xenopus laevis. RT-PCR and immunohistochemical analysis indicated that Pou5f3.3 was expressed in a small number of adult liver cells and blood cells. Immunocytochemical investigation proved that Bmi1, a marker for hematopoietic progenitor cells, was co-expressed in Pou5f3.3-expressing small spherical cells in the peripheral blood. In anemic induction by intraperitoneal injection of phenyl hydrazine, the number of Pou5f3.3-expressing cells significantly increased within 3 days after phenyl hydrazine injection. In CRISPR/Cas mutagenesis of Pou5f3.3, Bmi1-positive hematopoietic progenitor cell count decreased in the hematopoietic dorsal-lateral plate (DLP) region, resulting in a considerable reduction in peripheral blood cells. CRISPR/Cas-induced hematopoietic deficiency was completely rescued by Pou5f3.3 supplementation, but not by Pou5f3.1 or Pou5f3.2. Transplantation experiments using the H2B-GFP transgenic line demonstrated that DLP-derived Pou5f3.3-positive and Bmi1-positive cells were translocated into the liver and bone through the bloodstream. These results suggest that Pou5f3.3 plays an essential role in the establishment and maintenance of hematopoietic progenitor cells during Xenopus development., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Vertebrates have unique head structures that are mainly composed of the central nervous system, the neural crest, and placode cells. These head structures are brought about initially by the neural induction between the organizer and the prospective neuroectoderm at early gastrula stage. Purinergic receptors are activated by nucleotides released from cells and influence intracellular signaling pathways, such as phospholipase C and adenylate cyclase signaling pathways. As P2Y receptor is vertebrate-specific and involved in head formation, we expect that its emergence may be related to the acquisition of vertebrate head during evolution. Here, we focused on the role of p2ry4 in early development in Xenopus laevis and found that p2ry4 was required for the establishment of the head organizer during neural induction and contributed to head formation. We showed that p2ry4 was expressed in the head organizer region and the prospective neuroectoderm at early gastrula stage, and was enriched in the head components. Disruption of p2ry4 function resulted in the small head phenotype and the reduced expression of marker genes specific for neuroectoderm and neural border at an early neurula stage. Furthermore, we examined the effect of p2ry4 disruption on the establishment of the head organizer and found that a reduction in the expression of head organizer genes, such as dkk1 and cerberus, and p2ry4 could also induce the ectopic expression of these marker genes. These results suggested that p2ry4 plays a key role in head organizer formation. Our study demonstrated a novel role of p2ry4 in early head development., (© 2018 The Authors. Development, Growth & Differentiation published by John Wiley & Sons Australia, Ltd on behalf of Japanese Society of Developmental Biologists.)

Recent advances in genome editing using programmable nucleases have revolutionized gene targeting in various organisms. Successful gene knock-out has been shown in Xenopus, a widely used model organism, although a system enabling less mosaic knock-out in founder embryos (F0) needs to be explored in order to judge phenotypes in the F0 generation. Here, we injected modified highly active transcription activator-like effector nuclease (TALEN) mRNA to oocytes at the germinal vesicle (GV) stage, followed by in vitro maturation and intracytoplasmic sperm injection, to achieve a full knock-out in F0 embryos. Unlike conventional injection methods to fertilized embryos, the injection of TALEN mRNA into GV oocytes allows expression of nucleases before fertilization, enabling them to work from an earlier stage. Using this procedure, most of developed embryos showed full knock-out phenotypes of the pigmentation gene tyrosinase and/or embryonic lethal gene pax6 in the founder generation. In addition, our method permitted a large 1 kb deletion. Thus, we describe nearly complete gene knock-out phenotypes in Xenopus laevis F0 embryos. The presented method will help to accelerate the production of knock-out frogs since we can bypass an extra generation of about 1 year in Xenopus laevis. Meantime, our method provides a unique opportunity to rapidly test the developmental effects of disrupting those genes that do not permit growth to an adult able to reproduce. In addition, the protocol shown here is considerably less invasive than the previously used host transfer since our protocol does not require surgery. The experimental scheme presented is potentially applicable to other organisms such as mammals and fish to resolve common issues of mosaicism in founders.

Transcription activator-like effector nucleases (TALENs) have previously been used for targeted genome editing in various organisms including Xenopus laevis. However, because of genomic polyploidization, X. laevis usually possess homeologous genes (homeologs) with quite similar sequences that make the analysis of gene function difficult. In the present study, we show methodological examples of targeted gene modification of X. laevis homeologs. The X. laevis cytoglobin gene (cygb) consists of two homeologs (xlcygba and xlcygbb), and molecular phylogenetic analysis suggested that they have potentially different functions. Thus, there is a need to establish a method of homeolog-specific gene disruption to clarify gene functions in detail. Here, we show successful examples of homeolog-specific and simultaneous gene disruption for xlcygba and xlcygbb. We found that selective digestion can be performed with at least three mismatches in TALEN target sites in both homeologs. This report paves the way for the functional analyses of X. laevis homeologs, even those containing nearly identical sequences.

Xenopus laevis tadpoles can completely regenerate their appendages, such as tail and limbs, and therefore provide a unique model to decipher the molecular mechanisms of organ regeneration in vertebrates. Epigenetic modifications are likely to be involved in this remarkable regeneration capacity, but they remain largely unknown. To examine the involvement of histone modification during organ regeneration, we generated transgenic X. laevis ubiquitously expressing a fluorescent modification-specific intracellular antibody (Mintbody) that is able to track histone H3 lysine 9 acetylation (H3K9ac) in vivo through nuclear enhanced green fluorescent protein (EGFP) fluorescence. In embryos ubiquitously expressing H3K9ac-Mintbody, robust fluorescence was observed in the nuclei of somites. Interestingly, H3K9ac-Mintbody signals predominantly accumulated in nuclei of regenerating notochord at 24 h postamputation following activation of reactive oxygen species ( ROS). Moreover, apocynin (APO), an inhibitor of ROS production, attenuated H3K9ac-Mintbody signals in regenerating notochord. Our results suggest that ROS production is involved in acetylation of H3K9 in regenerating notochord at the onset of tail regeneration. We also show this transgenic Xenopus to be a useful tool to investigate epigenetic modification, not only in organogenesis but also in organ regeneration. [ABSTRACT FROM AUTHOR]

Transcription activator-like effector nucleases (TALENs) have been extensively used in genome editing in various organisms. In some cases, however, it is difficult to efficiently disrupt both paralogous genes using a single pair of TALENs in Xenopus laevis because of its polyploidy. Here, we report targeted mutagenesis of multiple and paralogous genes using two pairs of TALENs in X. laevis. First, we show simultaneous targeted mutagenesis of three genes, tyrosinase paralogues ( tyra and tyrb) and enhanced green fluorescent protein (egfp) by injection of two TALENs pairs in transgenic embryos carrying egfp. Consistent with the high frequency of both severe phenotypic traits, albinism and loss of GFP fluorescence, frameshift mutation rates of tyr paralogues and egfp reached 40-80%. Next, we show early introduction of TALEN-mediated mutagenesis of these target loci during embryogenesis. Finally, we also demonstrate that two different pairs of TALENs can simultaneously introduce mutations to both paralogues encoding histone chaperone with high efficiency. Our results suggest that targeted mutagenesis of multiple genes using TALENs can be applied to analyze the functions of paralogous genes with redundancy in X. laevis. [ABSTRACT FROM AUTHOR]