Sperm MMP-2 is indispensable for fast electrical block to polyspermy at fertilization in Xenopus tropicalis.

Sperm matrix metalloproteinase-2 (MMP-2) is necessary for frog fertilization. Monospermy is ensured by a fast, electrical block to polyspermy mediated by a positive fertilization potential. To determine the role of the MMP-2 hemopexin domain (HPX) in a fast block to polyspermy during fertilization of the frog, Xenopus tropicalis, we prepared mutant frogs deficient in mmp2 gene using the transcription activator-like effector nuclease method. mmp2 ΔHPX (-/-) sperm without MMP-2 protein were able to fertilize wild-type (WT; +/+) eggs. However, polyspermy occurred in some eggs. The mutant sperm generated a normal fertilization potential amounting to 10 mV, and were able to fertilize eggs at 10 mV, at which WT sperm never fertilized. Sensitivity during voltage-dependent fertilization decreased in mutant sperm. This study demonstrates for the first time that the genetic alteration of the MMP-2 molecule in sperm causes polyspermy during fertilization of a monospermic species. Our findings provide reliable evidence that sperm MMP-2 is indispensable for the fast, electrical block to polyspermy during Xenopus fertilization.

Tail Resorption During Metamorphosis in Xenopus Tadpoles.

Tail resorption in anuran tadpoles is one of the most physically and physiologically notable phenomena in developmental biology. A tail that is over twice as long as the tadpole trunk is absorbed within several days, while concurrently the tadpole's locomotive function is continuously managed during the transition of the driving force from the tail to hindlimbs. Elaborate regulation is necessary to accomplish this locomotive switch. Tadpole's hindlimbs must develop from the limb-bud size to the mature size and the nervous system must be arranged to control movement before the tail is degenerated. The order of the development and growth of hindlimbs and the regression of the tail are regulated by the increasing levels of thyroid hormones (THs), the intracellular metabolism of THs, the expression levels of TH receptors, the expression of several effector genes, and other factors that can modulate TH signaling. The tail degeneration that is induced by the TH surge occurs through two mechanisms, direct TH-responsive cell death (suicide) and cell death caused by the degradation of the extracellular matrix and a loss of cellular anchorage (murder). These pathways lead to the collapse of the notochord, the contraction of surviving slow muscles, and, ultimately, the loss of the tail. In this review, I focus on the differential TH sensitivity of the tail and hindlimbs and the mechanism of tail resorption during Xenopus metamorphosis.

Developmental gene expression patterns in the brain and liver of Xenopus tropicalis during metamorphosis climax.

Thyroid hormones (THs) induce metamorphosis in amphibians, causing dynamic changes, whereas mammalian newborns undergo environmental transition from placenta to open air at birth. The similarity between amphibian metamorphosis and the mammalian perinatal periods has been repeatedly discussed. However, a corresponding developmental gene expression analysis has not yet been reported. In this study, we examined the developmental gene expression profiles in the brain and liver of Xenopus tropicalis during metamorphosis climax and compared them to the respective gene expression profiles of newborn rodents. Many upregulated genes identified in the tadpole brain during metamorphosis are also upregulated in the rodent brain during the first three postnatal weeks when the TH surge occurs. The upregulation of some genes in the brain was inhibited in thyroid hormone receptor α (TRα) knockout tadpoles but not in TRß-knockout tadpoles, implying that brain metamorphosis is mainly mediated by TRα. The expression of some genes was also increased in the liver during metamorphosis climax. Our data suggest that the rodent brain undergoes TH-dependent remodeling during the first three postnatal weeks as observed in X. tropicalis during the larva-to-adult metamorphosis.

Homeotic transformation of tails into limbs in anurans.

Anuran tadpoles can regenerate their tails after amputation. However, they occasionally form ectopic limbs instead of the lost tail part after vitamin A treatment. This is regarded as an example of a homeotic transformation. In this phenomenon, the developmental fate of the tail blastema is apparently altered from that of a tail to that of limbs, indicating a realignment of positional information in the blastema. Morphological observations and analyses of the development of skeletal elements during the process suggest that positional information in the blastema is rewritten from tail to trunk specification under the influence of vitamin A, resulting in limb formation. Despite the extensive information gained from morphological observations, a comprehensive understanding of this phenomenon also requires molecular data. We review previous studies related to anuran homeotic transformation. The findings of these studies provide a basis for evaluating major hypotheses and identifying molecular data that should be prioritized in future studies. Finally, we argue that positional information for the tail blastema changes to that for a part of the trunk, leading to homeotic transformations. To suggest this hypothesis, we present published data that favor the rewriting of positional information.

Thyroid Hormone Receptor α- and ß-Knockout Xenopus tropicalis Tadpoles Reveal Subtype-Specific Roles During Development.

Thyroid hormone (TH) binds TH receptor α (TRα) and ß (TRß) to induce amphibian metamorphosis. Whereas TH signaling has been well studied, functional differences between TRα and TRß during this process have not been characterized. To understand how each TR contributes to metamorphosis, we generated TRα- and TRß-knockout tadpoles of Xenopus tropicalis and examined developmental abnormalities, histology of the tail and intestine, and messenger RNA expression of genes encoding extracellular matrix-degrading enzymes. In TRß-knockout tadpoles, tail regression was delayed significantly and a healthy notochord was observed even 5 days after the initiation of tail shortening (stage 62), whereas in the tails of wild-type and TRα-knockout tadpoles, the notochord disappeared after ∼1 day. The messenger RNA expression levels of genes encoding extracellular matrix-degrading enzymes (MMP2, MMP9TH, MMP13, MMP14, and FAPα) were obviously reduced in the tail tip of TRß-knockout tadpoles, with the shortening tail. The reduction in olfactory nerve length and head narrowing by gill absorption were also affected. Hind limb growth and intestinal shortening were not compromised in TRß-knockout tadpoles, whereas tail regression and olfactory nerve shortening appeared to proceed normally in TRα-knockout tadpoles, except for the precocious development of hind limbs. Our results demonstrated the distinct roles of TRα and TRß in hind limb growth and tail regression, respectively.

Vitamin A induced homeotic hindlimb formation on dorsal and ventral sides of regenerating tissue of amputated tails of Japanese brown frog tadpoles.

When anuran tadpoles are treated with vitamin A after tail amputation, hindlimb-like structures can be generated instead of the lost tail part at the amputation site. This homeotic transformation was initially expected to be a key to understanding the body plan of vertebrates. Unfortunately, homeotic limb formation has been reproduced in only some Indian frog species and a European species, but not in experimental anurans such as Xenopus laevis or Rana catesbeiana. Consequently, this fascinating phenomenon has not been well analyzed, especially at the molecular level. In addition, the initial processes of ectopic limb development are also unclear because morphological changes in the early phases have not been analyzed in detail. In this study, we report the induction of homeotic transformation using Japanese brown frogs and present a detailed morphological analysis. Unexpectedly, the ectopic limbs developed not only at the ventral sites, but also at the dorsal sites of the tail regenerates of vitamin A-treated tadpoles. The relationship between position and axial orientation of ectopic limbs suggested the double duplication of positional value order along the rostral-caudal axis and the dorsal-ventral axis of the tail regenerates.

An Inhibitor of Thyroid Hormone Synthesis Protects Tail Skin Grafts Transplanted to Syngenic Adult Frogs.

Tail regression in amphibian tadpoles during metamorphosis is one of the most dynamic morphological changes in animal development and is induced by thyroid hormone (TH). It has been proposed that tail resorption is driven by immunological rejection in Xenopus laevis, based on experimental evidence showing that larval skin grafts become atrophic on syngenic recipient adult frogs. This led to the hypothesis that tail regression is induced by an immunological rejection against larval skin-specific antigens called Ouro proteins. However, our group has demonstrated that ouro-knockout tadpoles undergo normal metamorphosis, including tail resorption in Xenopus tropicalis, which indicates that the expression of ouro genes is not necessary for tail regression. In the present study, we showed that an inhibitor of TH synthesis promotes the survival of larval tail skin grafts on syngenic adult Xenopus tropicalis frogs. The levels of endogenous THs in adult frogs were also comparable to those in metamorphosing tadpoles of Xenopus laevis with a regressing tail, and TH induced the regression of tadpole tail tips of Xenopus tropicalis in organ culture. Taken together, these results strongly suggest that endogenous THs in the recipient adult frog induce the degeneration of syngenic tail skin grafts.

Mechanisms of tail resorption during anuran metamorphosis.

Amphibian metamorphosis has historically attracted a good deal of scientific attention owing to its dramatic nature and easy observability. However, the genetic mechanisms of amphibian metamorphosis have not been thoroughly examined using modern techniques such as gene cloning, DNA sequencing, polymerase chain reaction or genomic editing. Here, we review the current state of knowledge regarding molecular mechanisms underlying tadpole tail resorption.

no privacy, a Xenopus tropicalis mutant, is a model of human Hermansky-Pudlak Syndrome and allows visualization of internal organogenesis during tadpole development.

We describe a novel recessive and nonlethal pigmentation mutant in Xenopus tropicalis. The mutant phenotype can be initially observed in tadpoles after stage 39/40, when mutant embryos display markedly reduced pigmentation in the retina and the trunk. By tadpole stage 50 almost all pigmented melanophores have disappeared. Most interestingly, those embryos fail entirely to make pigmented iridophores. The combined reduction/absence of both pigmented iridophores and melanophores renders these embryos virtually transparent, permitting one to easily observe both the developing internal organs and nervous system; accordingly, we named this mutant no privacy (nop). We identified the causative genetic lesion as occurring in the Xenopus homolog of the human Hermansky-Pudlak Syndrome 6 (HPS6) gene, combining several approaches that utilized conventional gene mapping and classical and modern genetic tools available in Xenopus (gynogenesis, BAC transgenesis and TALEN-mediated mutagenesis). The nop allele contains a 10-base deletion that results in truncation of the Hps6 protein. In humans, HPS6 is one of the genes responsible for the congenital disease HPS, pathological symptoms of which include oculocutaneous albinism caused by defects in lysosome-related organelles required for pigment formation. Markers for melanin-producing neural crest cells show that the cells that would give rise to melanocytes are present in nop, though unpigmented. Abnormalities develop at tadpole stages in the pigmented retina when overall pigmentation becomes reduced and large multi-melanosomes are first formed. Ear development is also affected in nop embryos when both zygotic and maternal hsp6 is mutated: otoliths are often reduced or abnormal in morphology, as seen in some mouse HPS mutations, but to our knowledge not described in the BLOC-2 subset of HPS mutations nor described in non-mammalian systems previously. The transparency of the nop line suggests that these animals will aid studies of early organogenesis during tadpole stages. In addition, because of advantages of the Xenopus system for assessing gene expression, cell biological mechanisms, and the ontogeny of melanosome and otolith formation, this should be a highly useful model for studying the molecular mechanisms underlying the acquisition of the HPS phenotype and the underlying biology of lysosome-related organelle function.

Generation of Albino Cynops pyrrhogaster by Genomic Editing of the tyrosinase Gene.

Albino animals are useful for in situ hybridization experiments that demonstrate gene expression in embryos and organs, for the immunological rejection of skin grafts transplanted to host animals, and to identify tissues with regenerative ability during limbs and retina regeneration processes. Cynops pyrrhogaster has extensive regenerating capacities. To facilitate regenerative research, in the present study, we produced albino C. pyrrhogaster using genomic editing. The DNA fragment containing part of the tyrosinase gene from C. pyrrhogaster was amplified using degenerate primers corresponding to evolutionarily conserved nucleotide sequences among several species, and the nucleotide sequence was determined. We designed a transcription activator-like effector nuclease (TALEN) that targets a candidate of the C. pyrrhogaster tyrosinase gene. Fertilized eggs were injected with TALEN mRNA, and albinos of C. pyrrhogaster were obtained. The results of the present study demonstrated that TALEN can be used effectively for genomic editing in C. pyrrhogaster and that the candidates of the tyrosinase gene that were cloned by us are essential for melanin synthesis. The albino newts created in the present study can be used as versatile experimental material.

Ouro proteins are not essential to tail regression during Xenopus tropicalis metamorphosis.

Tail regression is one of the most prominent transformations observed during anuran metamorphosis. A tadpole tail that is twice as long as the tadpole trunk nearly disappears within 3 days in Xenopus tropicalis. Several years ago, it was proposed that this phenomenon is driven by an immunological rejection of larval-skin-specific antigens, Ouro proteins. We generated ouro-knockout tadpoles using the TALEN method to reexamine this immunological rejection model. Both the ouro1- and ouro2-knockout tadpoles expressed a very low level of mRNA transcribed from a targeted ouro gene, an undetectable level of Ouro protein encoded by a target gene and a scarcely detectable level of the other Ouro protein from the untargeted ouro gene in tail skin. Furthermore, congenital athymic frogs were produced by Foxn1 gene modification. Flow cytometry analysis showed that mutant frogs lacked splenic CD8(+) T cells, which play a major role in cytotoxic reaction. Furthermore, T-cell-dependent skin allograft rejection was dramatically impaired in mutant frogs. None of the knockout tadpoles showed any significant delay in the process of tail shortening during the climax of metamorphosis, which shows that Ouro proteins are not essential to tail regression at least in Xenopus tropicalis and argues against the immunological rejection model.

Characterization of myosin II regulatory light chain isoforms in HeLa cells.

Myosin II regulatory light chain (MRLC) is canonically known as a subunit of conventional myosin (myosin II), which tunes cytoplasmic contractility in cells. Recent studies have also revealed the noncanonical functions of MRLC, such as engagement with other proteins including unconventional myosins. Three MRLC isoforms (MRLC1, MRLC2, and MRLC3) are known in humans. The characteristics of MRLC2 are well known, but those of MRLC1 and MRLC3 are unclear; therefore, the properties of the three MRLC isoforms were investigated. The MRLCs were all phosphorylated at Thr18/Ser19, which is required for myosin II stimulation. MRLC mRNAs were expressed at the same level throughout the cell cycle in HeLa cells. The MRLCs colocalized with each other and their turnover rate was similar to that of myosin II heavy chain. Depletion of all the MRLCs perturbed cell spreading. The overproduction of MRLC2 or MRLC3, but not MRLC1, could effectively compensate for this defect, suggesting that MRLC2 and MRLC3 play dominant roles in cell spreading. Finally, computer simulations of the three-dimensional protein structures indicated that the location of the N-terminus of MRLC1 differs from that of MRLC2 or MRLC3, depending on its sequence. Thus, these MRLC isoforms have overlapping but distinct functions have been proposed.

Xenopus pax6 mutants affect eye development and other organ systems, and have phenotypic similarities to human aniridia patients.

Mutations in the Pax6 gene cause ocular defects in both vertebrate and invertebrate animal species, and the disease aniridia in humans. Despite extensive experimentation on this gene in multiple species, including humans, we still do not understand the earliest effects on development mediated by this gene. This prompted us to develop pax6 mutant lines in Xenopus tropicalis taking advantage of the utility of the Xenopus system for examining early development and in addition to establish a model for studying the human disease aniridia in an accessible lower vertebrate. We have generated mutants in pax6 by using Transcription Activator-Like Effector Nuclease (TALEN) constructs for gene editing in X. tropicalis. Embryos with putative null mutations show severe eye abnormalities and changes in brain development, as assessed by changes in morphology and gene expression. One gene that we found is downregulated very early in development in these pax6 mutants is myc, a gene involved in pluripotency and progenitor cell maintenance and likely a mediator of some key pax6 functions in the embryo. Changes in gene expression in the developing brain and pancreas reflect other important functions of pax6 during development. In mutations with partial loss of pax6 function eye development is initially relatively normal but froglets show an underdeveloped iris, similar to the classic phenotype (aniridia) seen in human patients with PAX6 mutations. Other eye abnormalities observed in these froglets, including cataracts and corneal defects, are also common in human aniridia. The frog model thus allows us to examine the earliest deficits in eye formation as a result of pax6 lesions, and provides a useful model for understanding the developmental basis for the aniridia phenotype seen in humans.

Development of a new approach for targeted gene editing in primordial germ cells using TALENs in Xenopus.

A gene of interest can be efficiently modified using transcription activator-like effector nucleases (TALENs) (Christian et al., 2010;Li et al., 2011). However, if a target gene is essential for development, growth and fertility, use of TALENs with high mutagenic activity in F0 frogs could result in developmental disorders or sterility, which would reduce the number of F1 progeny and make F1 phenotypical analysis difficult. We used the 3' untranslated region of DEADSouth gene (DS-3') of Xenopus tropicalis to solve this problem, because the addition of the DS-3' to mRNA is known to induce primordial germ cell (PGC)-specific expression and reduce the stability in somatic cells of mRNA in Xenopus laevis. At first, we inserted the X. tropicalis DS-3' downstream of the EGFP termination codon and confirmed that the EGFP expression was specifically detected in PGCs for three weeks. Therefore, we inserted the DS-3' downstream of the termination codon of the TALEN coding sequence. The tyrosinase gene was selected as the target gene for TALEN because the bi-allelic mutation of this gene is easily discernible by the albino phenotype. When fertilized eggs were microinjected with TALEN mRNAs fused to the DS-3', their sperm and oocytes had a high rate (84-100%) of target-gene modification in contrast to the lower rate (0-45%) of nucleotide alteration observed in somatic cells.

Highly efficient gene knockout by injection of TALEN mRNAs into oocytes and host transfer in Xenopus laevis.

Zinc-finger nucleases, transcription activator-like effector nucleases (TALENs) and the CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins) system are potentially powerful tools for producing tailor-made knockout animals. However, their mutagenic activity is not high enough to induce mutations at all loci of a target gene throughout an entire tadpole. In this study, we present a highly efficient method for introducing gene modifications at almost all target sequences in randomly selected embryos. The gene modification activity of TALEN is enhanced by adopting the host-transfer technique. In our method, the efficiency is further improved by injecting TALEN mRNAs fused to the 3'UTR of the Xenopus DEADSouth gene into oocytes, which are then transferred into a host female frog, where they are ovulated and fertilized. The addition of the 3'UTR of the DEADSouth gene promotes mRNA translation in the oocytes and increases the expression of TALEN proteins to near-maximal levels three hours post fertilization (hpf). In contrast, TALEN mRNAs without this 3'UTR are translated infrequently in oocytes. Our data suggest that genomic DNA is more sensitive to TALEN proteins from fertilization to the midblastula (MBT) stage. Our method works by increasing the levels of TALEN proteins during the pre-MBT stages.

Comparison of TALEN scaffolds in Xenopus tropicalis.

Transcription activator-like effector nucleases (TALENs) are facile and potent tools used to modify a gene of interest for targeted gene knockout. TALENs consist of an N-terminal domain, a DNA-binding domain, and a C-terminal domain, which are derived from a transcription activator-like effector, and the non-specific nuclease domain of FokI. Using Xenopus tropicalis (X. tropicalis), we compared the toxicities and somatic mutation activities of four TALEN architectures in a side-by-side manner: a basic TALEN, a scaffold with the same truncated N- and C-terminal domains as GoldyTALEN, a scaffold with the truncated N- and C-terminal domains and an obligate heterodimeric nuclease domain, and a scaffold with the truncated N- and C-terminal domains and an obligate heterodimeric Sharkey nuclease domain. The strongest phenotype and targeted somatic gene mutation were induced by the injection of TALEN mRNAs containing the truncated N- and C-terminal domains and an obligate heterodimeric nuclease domain. The obligate heterodimeric TALENs exhibited reduced toxicity compared to the homodimeric TALENs, and the homodimeric GoldyTALEN-type scaffold showed both a high activity of somatic gene modification and high toxicity. The Sharkey mutation in the heterodimeric nuclease domain reduced the TALEN-mediated somatic mutagenesis.

Targeted gene disruption in the Xenopus tropicalis genome using designed TALE nucleases.

Transcription activator-like effector nucleases (TALENs) are attractive and powerful molecular tools for targeted gene disruption because of their simple design and quick assembly. To evaluate the utility of TALENs in genome editing in Xenopus tropicalis, we prepared nine pairs of TALENs for the tyrosinase, noggin and MMP-9TH genes. All of the TALENs had some activity in a single-strand annealing assay using a cultured frog cell line, suggesting double-stranded DNA cleavage activity by the TALENs at their target site. The injection of mRNAs encoding TALENs into fertilized X. tropicalis embryos resulted in Cel-1 cleavage of the PCR fragment containing the target site amplified from embryo genomic DNA, indicating that a mutation in the target gene had occurred during embryogenesis. These mutations were confirmed by the sequencing of clones derived from the PCR fragments of genomic DNA. Patches of vitiligo were observed in tadpoles raised from fertilized eggs that had been injected with mRNAs of TALENs for the tyrosinase gene. TALENs containing the repeat variable di-residue (RVD) NN appeared to show more activity than TALENs containing RVD NK, although both RVD NN and NK preferentially associate with a G nucleotide.

Expression of the amelogenin gene in the skin of Xenopus tropicalis.

Anuran skin contains a calcified dermal layer, referred to as the Eberth-Kastschenko (EK) layer, which is found between the stratum spongiosum and the stratum compactum. Although it is established that some anuran species possess the EK layer, little is known about this layer from the standpoint of evolutionary and developmental biology. We conducted a morphological analysis by staining the dorsal skin from many species with alizarin red S to investigate the calcified layer. This layer was observed in all of the anurans tested, as well as in fishes and one species of caecilian with dermal scales, but not in urodeles, amniotes, or a scaleless caecilian. All of the investigated species with dermal scales exhibited a calcified layer in their dermis, while the anurans showed the EK layer, but no scales. We also analyzed the expression of genes related to scale formation (sparc, mmp9, and mmp2) in the dorsal skin of X. tropicalis. These genes were highly expressed at the metamorphic climax stage, which preceded the deposition of calcium. Furthermore, we examined the gene expression profile of amelogenin, the major protein found in the enamel matrix of the developing tooth. In X. tropicalis, amelogenin was upregulated in the skin at the climax stage and was expressed in the adult dermis at a high level. These data provide the first experimental evidence of the expression of amelogenin in the skin. These findings will lead to a better understanding of the developmental formation of the EK layer and the function of amelogenin.

Generation of albino Xenopus tropicalis using zinc-finger nucleases.

To generate albino lines of Xenopus tropicalis, we injected fertilized eggs with mRNAs encoding zinc-finger nucleases (ZFNs) targeting the tyrosinase coding region. Surprisingly, vitiligo was observed on the skin of F0 frogs that had been injected with ZFN mRNAs, indicating that both tyrosinase genes in the genome were disrupted in all melanocytes within the vitiligo patches. Mutation analysis using genomic DNA from the skin revealed that two mosaic F0 frogs underwent spatially complex tyrosinase gene mutations. The data implies that the ZFN-induced tyrosinase gene ablations occurred randomly over space and time throughout the entire body, possibly until the young tadpole stage, and that melanocyte precursors lacking functional tyrosinase proliferated and formed vitiligo patches. Several albino X. tropicalis, which are compound heterozygotes for biallelic tyrosinase mutations, were obtained by mating the mosaic F0 frogs. To our knowledge, this is the first report of the albino vertebrates generated by the targeted gene knockout.

Regulation of thyroid hormone sensitivity by differential expression of the thyroid hormone receptor during Xenopus metamorphosis.

During amphibian metamorphosis, a series of dynamic changes occur in a predetermined order. Hind limb morphogenesis begins in response to low levels of thyroid hormone (TH) in early prometamorphosis, but tail muscle cell death is delayed until climax, when TH levels are high. It takes about 20 days for tadpoles to grow from early prometamorphosis to climax. To study the molecular basis of the timing of tissue-specific transformations, we introduced thyroid hormone receptor (TR) expression constructs into tail muscle cells of Xenopus tadpoles. The TR-transfected tail muscle cells died upon exposure to a low level of thyroxine (T4). This cell death was suggested to be mediated by type 2 iodothyronine deiodinase (D2) that converts T4 to T3-the more active form of TH. D2 mRNA was induced in the TR-overexpressing cells by low levels of TH. D2 promoter contains a TH-response element (TRE) with a lower affinity for TR. These results show that the TR transfection confers the ability to respond to physiological concentrations of TH at early prometamorphosis to tail muscle cells through D2 activity and promotes TH signaling. We propose the positive feedback loop model to amplify the cell's ability to respond to low levels of T4.