Identification and Analysis of Axolotl Homologs for Proteins Implicated in Human Neurodegenerative Proteinopathies.

Neurodegenerative proteinopathies such as Alzheimer's Disease are characterized by abnormal protein aggregation and neurodegeneration. Neuroresilience or regenerative strategies to prevent neurodegeneration, preserve function, or restore lost neurons may have the potential to combat human proteinopathies; however, the adult human brain possesses a limited capacity to replace lost neurons. In contrast, axolotls (Ambystoma mexicanum) show robust brain regeneration. To determine whether axolotls may help identify potential neuroresilience or regenerative strategies in humans, we first interrogated whether axolotls express putative proteins homologous to human proteins associated with neurodegenerative diseases. We compared the homology between human and axolotl proteins implicated in human proteinopathies and found that axolotls encode proteins highly similar to human microtubule-binding protein tau (tau), amyloid precursor protein (APP), and ß-secretase 1 (BACE1), which are critically involved in human proteinopathies like Alzheimer's Disease. We then tested monoclonal Tau and BACE1 antibodies previously used in human and rodent neurodegenerative disease studies using immunohistochemistry and western blotting to validate the homology for these proteins. These studies suggest that axolotls may prove useful in studying the role of these proteins in disease within the context of neuroresilience and repair.

Transcriptome Profiling after Early Spinal Cord Injury in the Axolotl and Its Comparison with Rodent Animal Models through RNA-Seq Data Analysis.

BACKGROUND: Traumatic spinal cord injury (SCI) is a disabling condition that affects millions of people around the world. Currently, no clinical treatment can restore spinal cord function. Comparison of molecular responses in regenerating to non-regenerating vertebrates can shed light on neural restoration. The axolotl (Ambystoma mexicanum) is an amphibian that regenerates regions of the brain or spinal cord after damage. METHODS: In this study, we compared the transcriptomes after SCI at acute (1-2 days after SCI) and sub-acute (6-7 days post-SCI) periods through the analysis of RNA-seq public datasets from axolotl and non-regenerating rodents. RESULTS: Genes related to wound healing and immune responses were upregulated in axolotls, rats, and mice after SCI; however, the immune-related processes were more prevalent in rodents. In the acute phase of SCI in the axolotl, the molecular pathways and genes associated with early development were upregulated, while processes related to neuronal function were downregulated. Importantly, the downregulation of processes related to sensorial and motor functions was observed only in rodents. This analysis also revealed that genes related to pluripotency, cytoskeleton rearrangement, and transposable elements (e.g., Sox2, Krt5, and LOC100130764) were among the most upregulated in the axolotl. Finally, gene regulatory networks in axolotls revealed the early activation of genes related to neurogenesis, including Atf3/4 and Foxa2. CONCLUSIONS: Immune-related processes are upregulated shortly after SCI in axolotls and rodents; however, a strong immune response is more noticeable in rodents. Genes related to early development and neurogenesis are upregulated beginning in the acute stage of SCI in axolotls, while the loss of motor and sensory functions is detected only in rodents during the sub-acute period of SCI. The approach employed in this study might be useful for designing and establishing regenerative therapies after SCI in mammals, including humans.

The Dynamic Landscapes of Circular RNAs in Axolotl, a Regenerative Medicine Model, with Implications for Early Phase of Limb Regeneration.

Circular RNAs (circRNAs) are of relevance to regenerative medicine and play crucial roles in post-transcriptional and translational regulation of biological processes. circRNAs are a class of RNA molecules that are formed through a unique splicing process, resulting in a covalently closed-loop structure. Recent advancements in RNA sequencing technologies and specialized computational tools have facilitated the identification and functional characterization of circRNAs. These molecules are known to exhibit stability, developmental regulation, and specific expression patterns in different tissues and cell types across various organisms. However, our understanding of circRNA expression and putative function in model organisms for regeneration is limited. In this context, this study reports, for the first time, on the repertoire of circRNAs in axolotl, a widely used model organism for regeneration. We generated RNA-seq data from intact limb, wound, and blastema tissues of axolotl during limb regeneration. The analysis revealed the presence of 35,956 putative axolotl circRNAs, among which 5331 unique circRNAs exhibited orthology with human circRNAs. In silico data analysis underlined the potential roles of axolotl circRNAs in cell cycle, cell death, and cell senescence-related pathways during limb regeneration, suggesting the participation of circRNAs in regulation of diverse functions pertinent to regenerative medicine. These new observations help advance our understanding of the dynamic landscape of axolotl circRNAs, and by extension, inform future regenerative medicine research and innovation that harness this model organism.

Establishing an Efficient Electroporation-Based Method to Manipulate Target Gene Expression in the Axolotl Brain.

The tetrapod salamander species axolotl (Ambystoma mexicanum) is capable of regenerating injured brain. For better understanding the mechanisms of brain regeneration, it is very necessary to establish a rapid and efficient gain-of-function and loss-of-function approaches to study gene function in the axolotl brain. Here, we establish and optimize an electroporation-based method to overexpress or knockout/knockdown target gene in ependymal glial cells (EGCs) in the axolotl telencephalon. By orientating the electrodes, we were able to achieve specific expression of EGFP in EGCs located in dorsal, ventral, medial, or lateral ventricular zones. We then studied the role of Cdc42 in brain regeneration by introducing Cdc42 into EGCs through electroporation, followed by brain injury. Our findings showed that overexpression of Cdc42 in EGCs did not significantly affect EGC proliferation and production of newly born neurons, but it disrupted their apical polarity, as indicated by the loss of the ZO-1 tight junction marker. This disruption led to a ventricular accumulation of newly born neurons, which are failed to migrate into the neuronal layer where they could mature, thus resulted in a delayed brain regeneration phenotype. Furthermore, when electroporating CAS9-gRNA protein complexes against TnC (Tenascin-C) into EGCs of the brain, we achieved an efficient knockdown of TnC. In the electroporation-targeted area, TnC expression is dramatically reduced at both mRNA and protein levels. Overall, this study established a rapid and efficient electroporation-based gene manipulation approach allowing for investigation of gene function in the process of axolotl brain regeneration.

A scATAC-seq atlas of chromatin accessibility in axolotl brain regions.

Axolotl (Ambystoma mexicanum) is an excellent model for investigating regeneration, the interaction between regenerative and developmental processes, comparative genomics, and evolution. The brain, which serves as the material basis of consciousness, learning, memory, and behavior, is the most complex and advanced organ in axolotl. The modulation of transcription factors is a crucial aspect in determining the function of diverse regions within the brain. There is, however, no comprehensive understanding of the gene regulatory network of axolotl brain regions. Here, we utilized single-cell ATAC sequencing to generate the chromatin accessibility landscapes of 81,199 cells from the olfactory bulb, telencephalon, diencephalon and mesencephalon, hypothalamus and pituitary, and the rhombencephalon. Based on these data, we identified key transcription factors specific to distinct cell types and compared cell type functions across brain regions. Our results provide a foundation for comprehensive analysis of gene regulatory programs, which are valuable for future studies of axolotl brain development, regeneration, and evolution, as well as on the mechanisms underlying cell-type diversity in vertebrate brains.

NANOG is required to establish the competence for germ-layer differentiation in the basal tetrapod axolotl.

Pluripotency defines the unlimited potential of individual cells of vertebrate embryos, from which all adult somatic cells and germ cells are derived. Understanding how the programming of pluripotency evolved has been obscured in part by a lack of data from lower vertebrates; in model systems such as frogs and zebrafish, the function of the pluripotency genes NANOG and POU5F1 have diverged. Here, we investigated how the axolotl ortholog of NANOG programs pluripotency during development. Axolotl NANOG is absolutely required for gastrulation and germ-layer commitment. We show that in axolotl primitive ectoderm (animal caps; ACs) NANOG and NODAL activity, as well as the epigenetic modifying enzyme DPY30, are required for the mass deposition of H3K4me3 in pluripotent chromatin. We also demonstrate that all 3 protein activities are required for ACs to establish the competency to differentiate toward mesoderm. Our results suggest the ancient function of NANOG may be establishing the competence for lineage differentiation in early cells. These observations provide insights into embryonic development in the tetrapod ancestor from which terrestrial vertebrates evolved.

Downregulation of Yap1 during limb regeneration results in defective bone formation in axolotl.

The Hippo pathway plays an imperative role in cellular processes such as differentiation, regeneration, cell migration, organ growth, apoptosis, and cell cycle. Transcription coregulator component of Hippo pathway, YAP1, promotes transcription of genes involved in cell proliferation, migration, differentiation, and suppressing apoptosis. However, its role in epimorphic regeneration has not been fully explored. The axolotl is a well-established model organism for developmental biology and regeneration studies. By exploiting its remarkable regenerative capacity, we investigated the role of Yap1 in the early blastema stage of limb regeneration. Depleting Yap1 using gene-specific morpholinos attenuated the competence of axolotl limb regeneration evident in bone formation defects. To explore the affected downstream pathways from Yap1 down-regulation, the gene expression profile was examined by employing LC-MS/MS technology. Based on the generated data, we provided a new layer of evidence on the putative roles of increased protease inhibition and immune system activities and altered ECM composition in diminished bone formation capacity during axolotl limb regeneration upon Yap1 deficiency. We believe that new insights into the roles of the Hippo pathway in complex structure regeneration were granted in this study.

Leukocyte Tyrosine Kinase (Ltk) Is the Mendelian Determinant of the Axolotl Melanoid Color Variant.

The great diversity of color patterns observed among amphibians is largely explained by the differentiation of relatively few pigment cell types during development. Mexican axolotls present a variety of color phenotypes that span the continuum from leucistic to highly melanistic. The melanoid axolotl is a Mendelian variant characterized by large numbers of melanophores, proportionally fewer xanthophores, and no iridophores. Early studies of melanoid were influential in developing the single-origin hypothesis of pigment cell development, wherein it has been proposed that all three pigment cell types derive from a common progenitor cell, with pigment metabolites playing potential roles in directing the development of organelles that define different pigment cell types. Specifically, these studies identified xanthine dehydrogenase (XDH) activity as a mechanism for the permissive differentiation of melanophores at the expense of xanthophores and iridophores. We used bulked segregant RNA-Seq to screen the axolotl genome for melanoid candidate genes and identify the associated locus. Dissimilar frequencies of single-nucleotide polymorphisms were identified between pooled RNA samples of wild-type and melanoid siblings for a region on chromosome 14q. This region contains gephyrin (Gphn), an enzyme that catalyzes the synthesis of the molybdenum cofactor that is required for XDH activity, and leukocyte tyrosine kinase (Ltk), a cell surface signaling receptor that is required for iridophore differentiation in zebrafish. Wild-type Ltk crispants present similar pigment phenotypes to melanoid, strongly implicating Ltk as the melanoid locus. In concert with recent findings in zebrafish, our results support the idea of direct fate specification of pigment cells and, more generally, the single-origin hypothesis of pigment cell development.

Characterization of immunoglobulin loci in the gigantic genome of Ambystoma mexicanum.

Background: The axolotl, Ambystoma mexicanum is a unique biological model for complete tissue regeneration. Is a neotenic endangered species and is highly susceptible to environmental stress, including infectious disease. In contrast to other amphibians, the axolotl is particularly vulnerable to certain viral infections. Like other salamanders, the axolotl genome is one of the largest (32 Gb) and the impact of genome size on Ig loci architecture is unknown. To better understand the immune response in axolotl, we aimed to characterize the immunoglobulin loci of A. mexicanum and compare it with other model vertebrates. Methods: The most recently published genome sequence of A. mexicanum (V6) was used for alignment-based annotation and manual curation using previously described axolotl Ig sequences or reference sequences from other vertebrates. Gene models were further curated using A. mexicanum spleen RNA-seq data. Human, Xenopus tropicalis, Danio rerio (zebrafish), and eight tetrapod reference genomes were used for comparison. Results: Canonical A. mexicanum heavy chain (IGH), lambda (IGL), sigma (IGS), and the putative surrogate light chain (SLC) loci were identified. No kappa locus was found. More than half of the IGHV genes and the IGHF gene are pseudogenes and there is no clan I IGHV genes. Although the IGH locus size is proportional to genome size, we found local size restriction in the IGHM gene and the V gene intergenic distances. In addition, there were V genes with abnormally large V-intron sizes, which correlated with loss of gene functionality. Conclusion: The A. mexicanum immunoglobulin loci share the same general genome architecture as most studied tetrapods. Consistent with its large genome, Ig loci are larger; however, local size restrictions indicate evolutionary constraints likely to be imposed by high transcriptional demand of certain Ig genes, as well as the V(D)J recombination over very long genomic distance ranges. The A. mexicanum has undergone an extensive process of Ig gene loss which partially explains a reduced potential repertoire diversity that may contribute to its impaired antibody response.

Establishing a New Research Axolotl Colony.

The field of regenerative biology has taken a keen interest in the Mexican axolotl (Ambystoma mexicanum) over the past few decades, as this salamander successfully regenerates amputated limbs and injured body parts. Recent progress in research tool development has also made possible axolotl genetic manipulation and single-cell analysis, which will help understand the molecular mechanisms of complex tissue regeneration. To support the growing popularity of this model, we describe how to set up a new axolotl housing facility at a research laboratory. We also review husbandry practices for raising axolotls and using them in biological research, with a focus on diet, water quality, breeding, and anesthesia.

Hybridization Chain Reaction Fluorescence In Situ Hybridization (HCR-FISH) in Ambystoma mexicanum Tissue.

In situ hybridization is a standard procedure for visualizing mRNA transcripts in tissues. The recent adoption of fluorescent probes and new signal amplification methods have facilitated multiplexed RNA imaging in tissue sections and whole tissues. Here we present protocols for multiplexed hybridization chain reaction fluorescence in situ hybridization (HCR-FISH) staining, imaging, cell segmentation, and mRNA quantification in regenerating axolotl tissue sections. We also present a protocol for whole-mount staining and imaging of developing axolotl limbs.

Fluorescence In Situ Hybridization of DNA Probes on Mitotic Chromosomes of the Mexican Axolotl.

Fluorescence in situ hybridization (FISH) is used extensively for visual localization of specific DNA fragments (and RNA fragments) in broad applications on chromosomes or nuclei at any stage of the cell cycle: metaphase, anaphase, or interphase. The cytogenetic slides that serve as a target for the labeled DNA probe might be prepared using any approach suitable for obtaining cells with appropriate morphology for imaging and analysis. In this chapter, we focus on the application of molecular cytogenetic methods such as DNA labeling, slide preparation, and in situ hybridization related to cells from Mexican axolotl.

The Use of Small Molecules to Dissect Developmental and Regenerative Processes in Axolotls.

The axolotl provides an interesting model organism to study different biological processes that are of interest to basic biological sciences and biomedical research. Although axolotls have been in labs for close to 160 years, genetic manipulations still represent a major challenge for most labs. The use of small molecules to target specific signaling pathways allows studies to proceed in animals that are difficult to manipulate genetically. This chapter provides a description of how we administer these chemicals to axolotls.

COMET Assay for Detection of DNA Damage During Axolotl Tail Regeneration.

The purpose of this chapter is to evaluate DNA damage during axolotl tail regeneration using an alkaline comet assay. Our method details the isolation of cells from regenerating and non-regenerating tissues and the isolation of peripheral blood for single-cell gel electrophoresis. Also, we detail each of the steps for the development of the comet assay technique which includes mounting the isolated cells on an agarose matrix, alkaline electrophoresis, and DNA damage detection.

Bead Implantation and Delivery of Exogenous Growth Factors.

Genetic methods in axolotls (Ambystoma mexicanum) remain in their infancy which has hampered the study of limb regeneration. There is much room for advancement, especially with respect to spatiotemporal regulation of gene expression. Secreted growth factors play a major role in each stage of regeneration. The use of slow-release beads is one of the most effective methods to control the spatiotemporal expression of secretory gene products. The topical administration of secreted factors by slow-release beads may also prove effective for future applications in non-regenerative animals and for medical applications in humans, in which genetic methods are not available. In this chapter, we describe a methodology for using and implanting slow-release beads to deliver exogenous growth factors to salamanders.

Axolotl Transgenesis via Injection of I-SceI Meganuclease or Tol2 Transposon System.

The axolotl (Ambystoma mexicanum ) has been widely used as an animal model for studying development and regeneration. In recent decades, the use of genetic engineering to alter gene expression has advanced our knowledge on the fundamental molecular and cellular mechanisms, pointing us to potential therapeutic targets. We present a detailed, step-by-step protocol for axolotl transgenesis using either I-SceI meganuclease or the mini Tol2 transposon system, by injection of purified DNA into one-cell stage eggs. We add useful tips on the site of injection and the viability of the eggs.

Applying a Knock-In Strategy to Create Reporter-Tagged Knockout Alleles in Axolotls.

Tetrapod species axolotls exhibit the powerful capacity to fully regenerate their tail and limbs upon injury, hence serving as an excellent model organism in regenerative biology research. Developing proper molecular and genetic tools in axolotls is an absolute necessity for deep dissection of tissue regeneration mechanisms. Previously, CRISPR-/Cas9-based knockout and targeted gene knock-in approaches have been established in axolotls, allowing genetically deciphering gene function, labeling, and tracing particular types of cells. Here, we further extend the CRISPR/Cas9 technology application and describe a method to create reporter-tagged knockout allele in axolotls. This method combines gene knockout and knock-in and achieves loss of function of a given gene and simultaneous labeling of cells expressing this particular gene, that allows identification, tracking of the "knocking out" cells. Our method offers a useful gene function analysis tool to the field of axolotl developmental and regenerative research.

A Practical Guide for CRISPR-Cas9-Induced Mutations in Axolotls.

Clustered regularly interspaced short palindromic repeats (CRISPR) is a powerful tool that enables editing of the axolotl genome. In this chapter, we will cover how to retrieve gene sequences, confirm annotation, design CRISPR targets, analyze indels, and screen for Crispant axolotls. This is a comprehensive guide on how to use CRISPR on your favorite gene and gain insights into its function.

Baculovirus Production and Infection in Axolotls.

Salamanders have served as an excellent model for developmental and tissue regeneration studies. While transgenic approaches are available for various salamander species, their long generation time and expensive maintenance have driven the development of alternative gene delivery methods for functional studies. We have previously developed pseudotyped baculovirus (BV) as a tool for gene delivery in the axolotl (Oliveira et al. Dev Biol 433(2):262-275, 2018). Since its initial conception, we have refined our protocol of BV production and usage in salamander models. In this chapter, we describe a detailed and versatile protocol for BV-mediated transduction in urodeles.

Artificial Insemination in Axolotl.

In axolotls (Ambystoma mexicanum), fertilization takes place internally. After courtship, the male axolotl deposits spermatophores, which the female takes up into her cloaca in order to fertilize eggs internally. The success of axolotl breedings is subject to several poorly understood factors including age, pairing, and genotype. In some cases, individuals are unable to breed naturally despite having significant scientific value. Assisted reproductive technologies represent one approach to maintaining stocks of such individuals, as well as supplementing natural breedings of laboratory stocks.Here, we describe a protocol for artificial insemination--an assisted reproductive technology in which sperm is extracted from a male and transferred into the female cloaca, thus mimicking natural fertilization in axolotls. We believe that this simple method can be applied to other salamander species that have internal fertilization and also help restore endangered wild populations.