Newt Hoxa13 has an essential and predominant role in digit formation during development and regeneration.

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.

Amphibian newts as experimental models for studying weight gain after castration.

Vertebrate animals often exhibit sexual dimorphism in body shape. In mammals, decreases in sex hormones caused by testicular castration can affect body shape and occasionally lead to pathologies such as obesity. Post-castration obesity can also be problematic for the health of companion animals, including non-mammals. In order to understand the mechanism of post-castration obesity in vertebrates other than mammals, experimental models are required. We examined whether the Iberian ribbed newt, which has recently become a popular experimental model for amphibian research, could serve as a model for analyzing changes in body shape after castration. In newts, new testes can be regenerated after removal of differentiated testes. We analyzed changes in body shape by removing the testes under conditions in which they could regenerate or conditions in which they could not regenerate. Removal of the testes reduced blood testosterone levels. The body weight and abdominal girth of the newts were increased compared with normal male newts. Transcriptome analysis of the liver showed that a set of genes related to lipid metabolism was continuously up-regulated in castrated newts. Our study suggests that changes in body shape after castration are common in vertebrates. Iberian ribbed newts are thus a suitable model for comparative studies of the long-term physiologic- and endocrine-level effects of castration.

Phenotype-genotype relationships in Xenopus sox9 crispants provide insights into campomelic dysplasia and vertebrate jaw evolution.

Since CRISPR-based genome editing technology works effectively in the diploid frog Xenopus tropicalis, a growing number of studies have successfully modeled human genetic diseases in this species. However, most of their targets were limited to non-syndromic diseases that exhibit abnormalities in a small fraction of tissues or organs in the body. This is likely because of the complexity of interpreting the phenotypic variations resulting from somatic mosaic mutations generated in the founder animals (crispants). In this study, we attempted to model the syndromic disease campomelic dysplasia (CD) by generating sox9 crispants in X. tropicalis. The resulting crispants failed to form neural crest cells at neurula stages and exhibited various combinations of jaw, gill, ear, heart, and gut defects at tadpole stages, recapitulating part of the syndromic phenotype of CD patients. Genotyping of the crispants with a variety of allelic series of mutations suggested that the heart and gut defects depend primarily on frame-shift mutations expected to be null, whereas the jaw, gill, and ear defects could be induced not only by such mutations but also by in-frame deletion mutations expected to delete part of the jawed vertebrate-specific domain from the encoded Sox9 protein. These results demonstrate that Xenopus crispants are useful for investigating the phenotype-genotype relationships behind syndromic diseases and examining the tissue-specific role of each functional domain within a single protein, providing novel insights into vertebrate jaw evolution.

Appendage-restricted gene induction using a heated agarose gel for studying regeneration in metamorphosed Xenopus laevis and Pleurodeles waltl.

Amphibians and fish often regenerate lost parts of their appendages (tail, limb, and fin) after amputation. Limb regeneration in adult amphibians provides an excellent model for appendage (limb) regeneration through 3D morphogenesis along the proximodistal, dorsoventral, and anteroposterior axes in mammals, because the limb is a homologous organ among amphibians and mammals. However, manipulating gene expression in specific appendages of adult amphibians remains difficult; this in turn hinders elucidation of the molecular mechanisms underlying appendage regeneration. To address this problem, we devised a system for appendage-specific gene induction using a simplified protocol named the "agarose-embedded heat shock (AeHS) method" involving the combination of a heat-shock-inducible system and insertion of an appendage in a temperature-controlled agarose gel. Gene expression was then induced specifically and ubiquitously in the regenerating limbs of metamorphosed amphibians, including a frog (Xenopus laevis) and newt (Pleurodeles waltl). We also induced gene expression in the regenerating tail of a metamorphosed P. waltl newt using the same method. This method can be applied to adult amphibians with large body sizes. Furthermore, this method enables simultaneous induction of gene expression in multiple individuals; further, the data are obtained in a reproducible manner, enabling the analysis of gene functions in limb and tail regeneration. Therefore, this method will facilitate elucidation of the molecular mechanisms underlying appendage regeneration in amphibians, which can support the development of regenerative therapies for organs, such as the limbs and spinal cord.

Step-by-step protocol for alternative injury models in newt cardiac regeneration.

Although the heart is one of the most important organs for animal survival, its regenerative capacity varies among animal species. Notably, adult mammals cannot regenerate their hearts after damage such as acute myocardial infarction. In contrast, some vertebrate animals can regenerate the heart throughout their lives. Cross-species comparative studies are important to understand the full picture of cardiac regeneration in vertebrates. Among the animal species able to regenerate the heart, some urodele amphibians, such as newts, possess a remarkable capacity for this process. Standardized methods of inducing cardiac regeneration in the newt are needed as a platform for studies comparing newts and other animal models. The procedures presented here describe amputation and cryo-injury techniques for the induction of cardiac regeneration in Pleurodeles waltl, an emerging model newt species. Both procedures consist of simplified steps that require no special equipment. We additionally show some examples of the regenerative process obtained using these procedures. This protocol has been developed for P. waltl. However, these methods are also expected to be applicable to other newt and salamander species, facilitating comparative research with other model animals.

Cryo-injury procedure-induced cardiac regeneration shows unique gene expression profiles in the newt Pleurodeles waltl.

BACKGROUND: Cardiac regeneration in the adult mouse is not substantial. Some vertebrates, such as newts and zebrafish, regenerate the heart throughout their lives. To understand how regenerative abilities differ among animal species, comparative research has been conducted in animals like mouse, zebrafish, and newt. For those purposes, cryo-injury is suitable as an experimental model for the pathological condition of human myocardial infarction. In fact, cryo-injury procedures are common in mouse and zebrafish. RESULTS: In the present study, we induced cryo-damage on the ventricle in Iberian ribbed newts using a liquid nitrogen-chilled probe. We observed that the injured area recovered within 8 weeks, with remodeling of scar tissue and proliferation of cardiomyocytes. We investigated the subsequent recovery of cryo-injured and amputated tissues by comparative analysis of the gene expression profiles following these two procedures. CONCLUSIONS: Notably, we established a cryo-injury procedure for the newt and confirmed that regeneration of the cryo-damaged myocardial tissue is achieved by changes in gene expression that are milder than those observed in the amputation model. Our results suggest that the cryo-injury method is suitable for comparing the process of cardiac regeneration in the newt with that in other animal models.

Insights regarding skin regeneration in non-amniote vertebrates: Skin regeneration without scar formation and potential step-up to a higher level of regeneration.

Skin wounds are among the most common injuries in animals and humans. Vertebrate skin is composed of an epidermis and dermis. After a deep skin injury in mammals, the wound heals, but the dermis cannot regenerate. Instead, collagenous scar tissue forms to fill the gap in the dermis, but the scar does not function like the dermis and often causes disfiguration. In contrast, in non-amniote vertebrates, including fish and amphibians, the dermis and skin derivatives are regenerated after a deep skin injury, without a recognizable scar remaining. Furthermore, skin regeneration can be compared with a higher level of organ regeneration represented by limb regeneration in these non-amniotes, as fish, anuran amphibians (frogs and toads), and urodele amphibians (newts and salamanders) have a high capacity for organ regeneration. Comparative studies of skin regeneration together with limb or other organ regeneration could reveal how skin regeneration is stepped up to a higher level of regeneration. The long history of regenerative biology research has revealed that fish, anurans, and urodeles have their own strengths as models for regeneration studies, and excellent model organisms of these non-amniote vertebrates that are suitable for molecular genetic studies are now available. Here, we summarize the advantages of fish, anurans, and urodeles for skin regeneration studies with special reference to three model organisms: zebrafish (Danio rerio), African clawed frog (Xenopus laevis), and Iberian ribbed newt (Pleurodele waltl). All three of these animals quickly cover skin wounds with the epidermis (wound epidermis formation) and regenerate the dermis and skin derivatives as adults. The availability of whole genome sequences, transgenesis, and genome editing with these models enables cell lineage tracing and the use of human disease models in skin regeneration phenomena, for example. Zebrafish present particular advantages in genetics research (e.g., human disease model and Cre-loxP system). Amphibians (X. laevis and P. waltl) have a skin structure (keratinized epidermis) common with humans, and skin regeneration in these animals can be stepped up to limb regeneration, a higher level of regeneration.

Photoperiod-independent testicular development in the model newt Pleurodeles waltl.

Urodele amphibian newts have unique biological properties in male gametogenesis, in addition to their extreme regenerative capacity. Male newts are able to regenerate new testes even after reaching sexual maturity and can possess multiple testes. Notably, these animals maintain primordial germ cell-like cells in a tissue adjacent to the testis. Spermatogenesis proceeds while synchronizing in a region-specific manner in the testis. However, the newt species that have been used most commonly require 2-3 years to achieve sexual maturity, and spermatogenesis in these species shows seasonality. These traits have restricted the use of newts for studies on testicular development and spermatogenesis, and testis development in newts remains poorly characterized. Recently, the Iberian ribbed newt Pleurodeles waltl has been established as an emerging model organism. P. waltl reaches sexual maturity more quick after birth than do other newts and is capable of breeding year-round. Thus, P. waltl is expected to serve as an appealing experimental model for studying the mechanisms of male gametogenesis in the urodeles. In the present study, we use P. waltl to describe the entire developmental process of the newt testis from primordial gonad to maturity. Notably, the mature testes show synchronized progression of spermatogenesis along the anteroposterior axis. Additionally, we demonstrate that the process of spermatogenesis in P. waltl proceeds irrespective of day length. Our results show that P. waltl newts are a suitable model for investigating the process of testicular development. We also expect that these results will be useful for the maintenance of P. waltl bioresources.

Cas9 ribonucleoprotein complex allows direct and rapid analysis of coding and noncoding regions of target genes in Pleurodeles waltl development and regeneration.

Newts have remarkable ability to regenerate their organs and have been used in research for centuries. However, the laborious work of breeding has hampered reverse genetics strategies in newt. Here, we present simple and efficient gene knockout using Cas9 ribonucleoprotein complex (RNP) in Pleurodeles waltl, a species suitable for regenerative biology studies using reverse genetics. Most of the founders exhibited severe phenotypes against each target gene (tyrosinase, pax6, tbx5); notably, all tyrosinase Cas9 RNP-injected embryos showed complete albinism. Moreover, amplicon sequencing analysis of Cas9 RNP-injected embryos revealed virtually complete biallelic disruption at target loci in founders, allowing direct phenotype analysis in the F0 generation. In addition, we demonstrated the generation of tyrosinase null F1 offspring within a year. Finally, we expanded this approach to the analysis of noncoding regulatory elements by targeting limb-specific enhancer of sonic hedgehog, known as the zone of polarizing activity regulatory sequence (ZRS; also called MFCS1). Disruption of ZRS led to digit deformation in limb regeneration. From these results, we are confident that this highly efficient gene knockout method will accelerate gene functional analysis in the post-genome era of salamanders.

Newts can normalize duplicated proximal-distal disorder during limb regeneration.

BACKGROUND: Urodele animals can regenerate their limbs from the blastemas. The previous results of grafting proximal blastemas to distal limb levels (P to D transplantation) led to serial duplication of limb segments. However, it is unknown whether grafting to any distal levels in P to D transplantation causes serial duplication. In other words, it is unknown whether or not newt limbs can normalize such a kind of duplicated type of positional disorder in the proximal-distal axis. Therefore, we grafted the most proximal blastemas to various distal levels of the proximal-distal axis using newts (Pleurodeles waltl). The transgenic newts expressing green fluorescent protein or mCherry were used to clearly distinguish between donor and host tissues. RESULTS: Normal segmental formation without duplication occurred in P to D transplantation within the stylopod. In addition, donor blastemas lost the fates of the stylopods, and the missing portion in the stylopod by amputation was restored by the insertion of host cells. In contrast, the blastemas from the stylopod formed whole limbs after transplantation to the tail. CONCLUSIONS: These results showed that urodele limbs can normalize the duplicated type of positional disorder within the stylopod by erasing a part of the fate in the blastemas. Developmental Dynamics 247:1276-1285, 2018. © 2018 Wiley Periodicals, Inc.

Effects of cadmium exposure on Iberian ribbed newt (Pleurodeles waltl) testes.

To characterize the histomorphologic effects of cadmium on adult newt testes, male Iberian ribbed newts (6 months post-hatching) were intraperitoneally exposed to a single dose of 50 mg/kg of cadmium, with histologic analysis of the testes at 24, 48, 72, and 96 h. Beginning 24 h after cadmium exposure, apoptosis of spermatogonia and spermatocytes was observed, and congestion was observed in the interstitial vessels of the testes. Throughout the experimental period, the rates of pyknotic cells and TUNEL and cleaved caspase-3 positivity were significantly higher in the spermatogonia and spermatocytes of cadmium-treated newts compared with control newts. There were no significant differences between cadmium-treated and control newts in phospho-histone H3 positivity in the spermatogonia and spermatocytes. These results suggest that spermatogonia and spermatocytes in adult Iberian ribbed newts are highly sensitive to cadmium. This is the first report of the histomorphologic characteristics of cadmium-induced testicular dysfunction in newts.