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1.
Proc Natl Acad Sci U S A ; 121(11): e2314911121, 2024 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-38442169

RESUMO

In amniote limbs, Fibroblast Growth Factor 10 (FGF10) is essential for limb development, but whether this function is broadly conserved in tetrapods and/or involved in adult limb regeneration remains unknown. To tackle this question, we established Fgf10 mutant lines in the newt Pleurodeles waltl which has amazing regenerative ability. While Fgf10 mutant forelimbs develop normally, the hindlimbs fail to develop and downregulate FGF target genes. Despite these developmental defects, Fgf10 mutants were able to regenerate normal hindlimbs rather than recapitulating the embryonic phenotype. Together, our results demonstrate an important role for FGF10 in hindlimb formation, but little or no function in regeneration, suggesting that different mechanisms operate during limb regeneration versus development.


Assuntos
Fator 10 de Crescimento de Fibroblastos , Animais , Fator 10 de Crescimento de Fibroblastos/genética , Fator 10 de Crescimento de Fibroblastos/metabolismo , Membro Posterior/crescimento & desenvolvimento , Regeneração , Pleurodeles/genética , Pleurodeles/crescimento & desenvolvimento , Pleurodeles/metabolismo
2.
PLoS One ; 19(2): e0299661, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38416753

RESUMO

Epigenetics is an emerging field of research because of its involvement in susceptibility to diseases and aging. Hypoxia and hyperoxia are known to be involved widely in various pathophysiologies. Here, we compared the differential epigene expression pattern between Pleurodeles waltl and Mus musculus (commonly known as Iberian ribbed newt and mouse, respectively) exposed to hypoxia and hyperoxia. Adult healthy newts and mice were exposed to normobaric hypoxia (8% O2) and hyperoxia (80% O2) for 2 hours. We collected the lungs and analyzed the expression of hypoxia-inducible factor 1 alpha (Hif1α) and several key epigenes from DNA methyltransferase (DNMT) family, histone deacetylase (HDAC) family, and methyl-CpG binding domain (MBD) family. The exposure to hypoxia significantly increased the mRNA levels of DNA methyltransferase 3 alpha (Dnmt3α), methyl-CpG binding domain protein 2 (Mbd2), Mbd3, and histone deacetylase 2 (Hdac2) in lungs of newts, but decreased the mRNA levels of DNA methyltransferase 1 (Dnmt1) and Dnmt3α in lungs of mice. The exposure to hyperoxia did not significantly change the expression of any gene in either newts or mice. The differential epigene expression pattern in response to hypoxia between newts and mice may provide novel insights into the prevention and treatment of disorders developed due to hypoxia exposure.


Assuntos
Hiperóxia , Pleurodeles , Animais , Camundongos , Pleurodeles/genética , Hiperóxia/genética , Hipóxia/genética , Salamandridae/genética , Pulmão , RNA Mensageiro/genética , DNA , Metiltransferases
3.
Int J Mol Sci ; 24(23)2023 Nov 29.
Artigo em Inglês | MEDLINE | ID: mdl-38069262

RESUMO

The reprogramming of retinal pigment epithelium (RPE) cells into retinal cells (transdifferentiation) lies in the bases of retinal regeneration in several Urodela. The identification of the key genes involved in this process helps with looking for approaches to the prevention and treatment of RPE-related degenerative diseases of the human retina. The purpose of our study was to examine the transcriptome changes at initial stages of RPE cell reprogramming in adult newt Pleurodeles waltl. RPE was isolated from the eye samples of day 0, 4, and 7 after experimental surgical detachment of the neural retina and was used for a de novo transcriptome assembly through the RNA-Seq method. A total of 1019 transcripts corresponding to the differently expressed genes have been revealed in silico: the 83 increased the expression at an early stage, and 168 increased the expression at a late stage of RPE reprogramming. We have identified up-regulation of classical early response genes, chaperones and co-chaperones, genes involved in the regulation of protein biosynthesis, suppressors of oncogenes, and EMT-related genes. We revealed the growth in the proportion of down-regulated ribosomal and translation-associated genes. Our findings contribute to revealing the molecular mechanism of RPE reprogramming in Urodela.


Assuntos
Pleurodeles , Descolamento Retiniano , Animais , Humanos , Descolamento Retiniano/genética , Descolamento Retiniano/metabolismo , Retina/metabolismo , Epitélio , Urodelos , Transcriptoma , Epitélio Pigmentado da Retina/metabolismo
4.
Dev Growth Differ ; 65(5): 266-271, 2023 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-37155321

RESUMO

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.


Assuntos
Pleurodeles , Salamandridae , Animais , Vertebrados , Mamíferos
5.
Dev Growth Differ ; 65(5): 255-265, 2023 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-37209318

RESUMO

Pleurodeles waltl is coming to light as a model animal, especially in regeneration studies, but deep studies on the molecular mechanisms have been limited due to the absence of primary tissue cells for wide usage. Therefore, we aimed to grow primary cells from limb tissue of P. waltl for in vitro experiments. Limb tissues were cut into small pieces and seeded as "explants" on culture dishes coated with fibronectin and gelatin. Compared to the control without coating, both fibronectin and gelatin supported quicker outgrowth of cells from explants and faster cell adhesion, and fibronectin showed significantly better performance than gelatin. Interestingly, the doubling time of cells on fibronectin- and gelatin-coated surfaces was almost the same (42.39 ± 2.79 h vs. 42.91 ± 3.69 h) and was not significantly different from that on non-coated plates (49.64 ± 3.63 h). The cryopreserved cells were successfully recovered and showed a multiplication capacity that was similar to that of fresh cells. Senescent cells were barely detected even after long-term sub-culture (>15 passages). Moreover, enhanced fluorescence of MitoSOX™ Red in cells under H2 O2 exposure confirmed the respondence to chemical stimuli. Collectively, our results show that we are able to grow enough good-quality cells from P. waltl limb tissue for in vitro experiments, and fibronectin coating provides the best biocompatible environment for cell outgrowth and attachment.


Assuntos
Fibronectinas , Pleurodeles , Animais , Fibronectinas/farmacologia , Fibronectinas/metabolismo , Pleurodeles/metabolismo , Gelatina/farmacologia , Gelatina/metabolismo
6.
Differentiation ; 132: 15-23, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37055300

RESUMO

BACKGROUND: Aging and regeneration are heavily linked processes. While it is generally accepted that regenerative capacity declines with age, some vertebrates, such as newts, can bypass the deleterious effects of aging and successfully regenerate a lens throughout their lifetime. RESULTS: Here, we used Spectral-Domain Optical Coherence Tomography (SD-OCT) to monitor the lens regeneration process of larvae, juvenile, and adult newts. While all three life stages were able to regenerate a lens through transdifferentiation of the dorsal iris pigment epithelial cells (iPECs), an age-related change in the kinetics of the regeneration process was observed. Consistent with these findings, iPECs from older animals exhibited a delay in cell cycle re-entry. Furthermore, it was observed that clearance of the extracellular matrix (ECM) was delayed in older organisms. CONCLUSIONS: Collectively, our results suggest that although lens regeneration capacity does not decline throughout the lifespan of newts, the intrinsic and extrinsic cellular changes associated with aging alter the kinetics of this process. By understanding how these changes affect lens regeneration in newts, we can gain important insights for restoring the age-related regeneration decline observed in most vertebrates.


Assuntos
Cristalino , Pleurodeles , Animais , Salamandridae , Matriz Extracelular , Divisão Celular
7.
Methods Mol Biol ; 2637: 341-357, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36773159

RESUMO

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.


Assuntos
Sistemas CRISPR-Cas , Pleurodeles , Animais , Humanos , Xenopus laevis/genética , Xenopus/genética , Sistemas CRISPR-Cas/genética , Pleurodeles/genética , Edição de Genes/métodos
8.
Dev Growth Differ ; 65(2): 86-93, 2023 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-36680534

RESUMO

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.


Assuntos
Pleurodeles , Medula Espinal , Animais , Xenopus laevis/genética , Pleurodeles/genética , Sefarose , Expressão Gênica , Mamíferos
9.
Methods Mol Biol ; 2562: 259-270, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36272082

RESUMO

Salamanders have been used as research models for centuries. While they exhibit a wide range of biological features not seen in mammals, none has captivated scientists like their ability to regenerate. Interestingly, axolotl macrophages have emerged as an essential cell population for tissue regeneration. Whether the same is true in other salamanders such as newt species Notophthalmus viridescens, Cynops pyrrhogaster, or Pleurodeles waltl remains to be seen. Unfortunately, regardless of the species, molecular tools to study macrophage function in salamanders are lacking. We propose that the readily available, terminally differentiated peritoneal macrophages from newts or axolotls could be used to validate molecular reagents in the study of macrophage function during tissue regeneration in salamanders.


Assuntos
Macrófagos Peritoneais , Urodelos , Animais , Pleurodeles , Mamíferos , Salamandridae
10.
Dev Growth Differ ; 64(9): 494-500, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36308507

RESUMO

Urodele amphibians have exceptional regeneration ability in various organs. Among these, the Iberian ribbed newt (Pleurodeles waltl) has emerged as a useful model organism for investigating the mechanisms underlying regeneration. Neural stem cells (NSCs) are an important source of regeneration in the central nervous system (CNS) and their culture method in vitro has been well established. NSCs form spherical cell aggregates called neurospheres and their formation has been demonstrated in various vertebrates, including some urodele species, but not in P. waltl. In this study, we reported neurosphere formation in brain- and spinal cord-derived cells of post-metamorphic P. waltl. These neurospheres showed proliferative activity and similar expression of marker proteins. However, the surface morphology was found to vary according to their origin, implying that the characteristics of the neurospheres generated from the brain and spinal cord could be similar but not identical. Subsequent in vitro differentiation analysis demonstrated that spinal cord-derived neurospheres gave rise to neurons and glial cells. We also found that cells in neurospheres from P. waltl differentiated to oligodendrocytes, whereas those from axolotls were reported not to differentiate to this cell type under standard culture conditions. Based on our findings, implantation of genetically modified neurospheres together with associated technical advantages in P. waltl could reveal pivotal gene(s) and/or signaling pathway(s) essential for the complete spinal cord regeneration ability in the future.


Assuntos
Células-Tronco Neurais , Pleurodeles , Animais , Pleurodeles/anatomia & histologia , Pleurodeles/metabolismo , Salamandridae , Medula Espinal , Neurônios
11.
Science ; 377(6610): eabp9186, 2022 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-36048957

RESUMO

The evolution of advanced cognition in vertebrates is associated with two independent innovations in the forebrain: the six-layered neocortex in mammals and the dorsal ventricular ridge (DVR) in sauropsids (reptiles and birds). How these innovations arose in vertebrate ancestors remains unclear. To reconstruct forebrain evolution in tetrapods, we built a cell-type atlas of the telencephalon of the salamander Pleurodeles waltl. Our molecular, developmental, and connectivity data indicate that parts of the sauropsid DVR trace back to tetrapod ancestors. By contrast, the salamander dorsal pallium is devoid of cellular and molecular characteristics of the mammalian neocortex yet shares similarities with the entorhinal cortex and subiculum. Our findings chart the series of innovations that resulted in the emergence of the mammalian six-layered neocortex and the sauropsid DVR.


Assuntos
Evolução Biológica , Neurônios , Pleurodeles , Telencéfalo , Animais , Atlas como Assunto , Neocórtex/citologia , Neocórtex/fisiologia , Neurônios/metabolismo , Pleurodeles/fisiologia , Telencéfalo/citologia , Telencéfalo/fisiologia , Transcriptoma
12.
Dev Dyn ; 251(5): 864-876, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-34964213

RESUMO

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.


Assuntos
Pleurodeles , Peixe-Zebra , Animais , Camundongos , Pleurodeles/genética , Regeneração/genética , Salamandridae/genética , Transcriptoma , Peixe-Zebra/genética
15.
Dev Growth Differ ; 63(6): 277-284, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34133763

RESUMO

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.


Assuntos
Fotoperíodo , Pleurodeles , Animais , Células Germinativas , Masculino , Salamandridae , Testículo
16.
Sci Rep ; 10(1): 7798, 2020 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-32385319

RESUMO

Marine tetrapods quickly diversified and were established as marine top predators after the end-Permian Mass extinction (EPME). Ichthyosaurs were the forerunner of this rapid radiation but the main drivers of the diversification are poorly understood. Cartorhynchus lenticarpus is a basal ichthyosauriform with the least degree of aquatic adaptation, holding a key to identifying such a driver. The unique specimen appeared edentulous based on what was exposed but a CT scanning revealed that the species indeed had rounded teeth that are nearly perpendicular to the jaw rami, and thus completely concealed in lateral view. There are three dental rows per jaw ramus, and the root lacks infoldings of the dentine typical of ichthyopterygians. The well-developed and worn molariform dentition with three tooth rows supports the previous inference that the specimen is not of a juvenile. The premaxilla and the corresponding part of the dentary are edentulous. Molariform dentition evolved three to five times independently within Ichthyosauriformes in the Early and Middle Triassic. Convergent exploitation of hard-shelled invertebrates by different subclades of ichthyosauriforms likely fueled the rapid taxonomic diversification of the group after EPME.


Assuntos
Evolução Biológica , Dentição , Extinção Biológica , Fósseis , Paleontologia , Pleurodeles , Animais , Pleurodeles/anatomia & histologia , Dente/anatomia & histologia , Dente/diagnóstico por imagem
17.
Biochem Biophys Res Commun ; 521(4): 914-920, 2020 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-31711646

RESUMO

The thermosensation mechanism plays critical roles in various animals living in different thermal environment. We focused on an axolotl, which is a tailed amphibian originally from Lake Xochimilco area in the Vally of Mexico, and examined its behavior response to heat stimulation. Mild heat at 33 °C induced noxious locomotive activity to axolotls, but the noxious response of another tailed amphibian, Iberian ribbed newt, was not observed at 33 °C. To explore the mechanism for the temperature sensitivity of axolotls, we isolated a cDNA of TRPV1. Using the degenerate primer PCR method, we identified the DNA fragment encoding axolotl TRPV1 (axTRPV1), and then cloned a full-length cDNA. We studied the chemical and thermal sensitivities of axTRPV1 by two-electrode voltage clamp method using Xenopus oocyte expression system. Capsaicin, acid, and 2-aminoethoxydiphenylborane apparently activated axTRPV1 channels in a dose-dependent manner. The analysis of thermal sensitivity showed that axTRPV1 was significantly activated by heat but not by cold. The average temperature threshold for heat-activation was 30.95 ±â€¯0.12 °C. This thermal activation threshold of axTRPV1 is unique and significantly low, when compared with the known thresholds of TRPV1s from various animals. Further, this threshold of axTRPV1 is well consistent with the observation of heat-induced behavior of axolotls at 33 °C, demonstrating that axolotl shows noxious response to mild heat mediated through axTRPV1.


Assuntos
Ambystoma mexicanum/fisiologia , Canais de Cátion TRPV/genética , Canais de Cátion TRPV/metabolismo , Animais , Regulação da Temperatura Corporal/fisiologia , Compostos de Boro/farmacologia , Capsaicina/farmacologia , Clonagem Molecular , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Temperatura Alta , Locomoção , Oócitos/efeitos dos fármacos , Oócitos/fisiologia , Técnicas de Patch-Clamp , Filogenia , Pleurodeles/fisiologia , Reação em Cadeia da Polimerase , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Xenopus
18.
Int J Dev Biol ; 63(6-7): 281-286, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31250911

RESUMO

Urodele amphibian newts have an outstanding history as experimental animals in various research fields such as developmental biology and regeneration biology. We have reported a model experimental system using the Spanish newt, Pleurodeles waltl, and it enables reverse/molecular genetics through gene manipulation. Microinjection is one of the core techniques in gene manipulation in newts. In the present study, we examined the conditions of the microinjection method, such as egg preparation, de-jelly solution, and formulation of injection medium. We have successfully optimized the injection protocol for P. waltl newts, and our improved protocol is more efficient and lower in cost than previous methods. This protocol can be used for the microinjection of plasmid DNA with I-SceI or mRNA, as well as genome editing using the CRISPR-Cas9 system. This protocol will facilitate research through gene manipulation in newts.


Assuntos
Animais Geneticamente Modificados/genética , Técnicas de Transferência de Genes , Microinjeções/métodos , Óvulo/metabolismo , Pleurodeles/genética , RNA Mensageiro/administração & dosagem , Espermatozoides/metabolismo , Animais , Animais Geneticamente Modificados/fisiologia , Masculino , Óvulo/crescimento & desenvolvimento , Plasmídeos/administração & dosagem , Plasmídeos/genética , Pleurodeles/fisiologia , RNA Mensageiro/genética , Regeneração , Espermatozoides/crescimento & desenvolvimento
19.
DNA Res ; 26(3): 217-229, 2019 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-31006799

RESUMO

Urodele newts have unique biological properties, notably including prominent regeneration ability. The Iberian ribbed newt, Pleurodeles waltl, is a promising model amphibian distinguished by ease of breeding and efficient transgenic and genome editing methods. However, limited genetic information is available for P. waltl. We conducted an intensive transcriptome analysis of P. waltl using RNA-sequencing to build and annotate gene models. We generated 1.2 billion Illumina reads from a wide variety of samples across 12 different tissues/organs, unfertilized egg, and embryos at eight different developmental stages. These reads were assembled into 1,395,387 contigs, from which 202,788 non-redundant ORF models were constructed. The set is expected to cover a large fraction of P. waltl protein-coding genes, as confirmed by BUSCO analysis, where 98% of universal single-copy orthologs were identified. Ortholog analyses revealed the gene repertoire evolution of urodele amphibians. Using the gene set as a reference, gene network analysis identified regeneration-, developmental-stage-, and tissue-specific co-expressed gene modules. Our transcriptome resource is expected to enhance future research employing this emerging model animal for regeneration research as well as for investigations in other areas including developmental biology, stem cell biology, and cancer research. These data are available via our portal website, iNewt (http://www.nibb.ac.jp/imori/main/).


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Pleurodeles/genética , Regeneração/genética , Transcriptoma , Animais , Feminino , Perfilação da Expressão Gênica , Masculino , Especificidade de Órgãos , Filogenia , Análise de Sequência de RNA
20.
Dev Biol ; 443(2): 127-136, 2018 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-30213538

RESUMO

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.


Assuntos
Proteína 9 Associada à CRISPR/genética , Pleurodeles/genética , Regeneração/genética , Animais , Animais Geneticamente Modificados , Cruzamento/métodos , Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas , Biologia do Desenvolvimento/métodos , Técnicas de Inativação de Genes , Fenótipo , Pleurodeles/metabolismo , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Análise de Sequência de DNA/métodos
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