Preclinical Molecular Signatures of Spinal Cord Functional Restoration: Optimizing the Metamorphic Axolotl (Ambystoma mexicanum) Model in Regenerative Medicine.

Regenerative medicine offers hope for patients with diseases of the central and peripheral nervous system. Urodele amphibians such as axolotl display an exceptional regenerative capacity and are considered as essential preclinical model organisms in neurology and regenerative medicine research. Earlier studies have suggested that the limb regeneration ability of this salamander notably decreases with induction of metamorphosis by thyroid hormones. Metamorphic axolotl requires further validation as a negative control in preclinical regenerative medicine research, not to mention the study of molecular substrates of its regenerative abilities. In this study, we report new observations on the effect of experimentally induced metamorphosis on spinal cord regeneration in axolotl. Surprisingly, we found that metamorphic animals were successful to functionally restore the spinal cord after an experimentally induced injury. To discern the molecular signatures of spinal cord regeneration, we performed transcriptomics analyses at 1- and 7-days postinjury (dpi) for both spinal cord injury (SCI)-induced (experimental) and laminectomy (sham) groups. We observed 119 and 989 differentially expressed genes at 1- and 7-dpi, respectively, while the corresponding mouse orthologous genes were enriched in junction-, immune system-, and extracellular matrix-related pathways. Taken together, our findings challenge the prior notions of limited regenerative ability of metamorphic axolotl which exhibited successful spinal cord regeneration in our experience. Moreover, we report on molecular signatures that can potentially explain the mechanistic substrates of the regenerative capacity of the metamorphic axolotl. To the best of our knowledge, this is the first report on molecular responses to SCI and functional restoration in metamorphic axolotls. These new findings advance our understanding of spinal cord regeneration, and may thus help optimize the future use of axolotl as a preclinical model in regenerative medicine and integrative biology fields.

Longitudinal 16S rRNA data derived from limb regenerative tissue samples of axolotl Ambystoma mexicanum.

The Mexican axolotl (Ambystoma mexicanum) is a critically endangered species and a fruitful amphibian model for regenerative biology. Despite growing body of research on the cellular and molecular biology of axolotl limb regeneration, microbiological aspects of this process remain poorly understood. Here, we describe bacterial 16S rRNA amplicon dataset derived from axolotl limb tissue samples in the course of limb regeneration. The raw data was obtained by sequencing V3-V4 region of 16S rRNA gene and comprised 14,569,756 paired-end raw reads generated from 21 samples. Initial data analysis using DADA2 pipeline resulted in amplicon sequence variant (ASV) table containing a total of ca. 5.9 million chimera-removed, high-quality reads and a median of 296,971 reads per sample. The data constitute a useful resource for the research on the microbiological aspects of axolotl limb regeneration and will also broadly facilitate comparative studies in the developmental and conservation biology of this critically endangered species.

Experimentally induced metamorphosis in highly regenerative axolotl (ambystoma mexicanum) under constant diet restructures microbiota.

Axolotl (Ambystoma mexicanum) is a critically endangered salamander species and a model organism for regenerative and developmental biology. Despite life-long neoteny in nature and in captive-bred colonies, metamorphosis of these animals can be experimentally induced by administering Thyroid hormones (THs). However, microbiological consequences of this experimental procedure, such as host microbiota response, remain largely unknown. Here, we systematically compared host bacterial microbiota associated with skin, stomach, gut tissues and fecal samples, between neotenic and metamorphic axolotls based on 16S rRNA gene sequences. Our results show that distinct bacterial communities inhabit individual organs of axolotl and undergo substantial restructuring through metamorphosis. Skin microbiota among others, shifted sharply, as highlighted by a major transition from Firmicutes-enriched to Proteobacteria-enriched relative abundance and precipitously decreased diversity. Fecal microbiota of neotenic and metamorphic axolotl shared relatively higher similarity, suggesting that diet continues to shape microbiota despite fundamental transformations in the host digestive organs. We also reproduced the previous finding on reduction in regenerative capacity in limbs of axolotl following metamorphosis, highlighting the need to investigate whether shifts in microbiota is causally linked to regenerative capacity of axolotl. The initial results on axolotl microbiota provide novel insights into microbiological aspects of axolotl metamorphosis and will establish a baseline for future in-depth studies.

A comparative study of fixatives for axolotl blastema tissue / Estudio comparativo de fijadores para el tejido blastema del axolotl

Regeneration is defined as tissue renewal and functional restoration process of the damaged parts of the body after an injury. Ambystoma mexicanum, commonly named the Axolotl, is one of the unique vertebrates, which has a remarkable ability to regenerate their extremities following the amputation. Although the process of regeneration includes several periods, it can be divided into two main phases; blastema formation and dedifferentiation. In the couple of hours following the amputation, wound closure occurs by migration of epithelial cells around the amputation site followed by macrophage infiltration and dedifferentiation of cells to turn into stem cells. Accumulated stem cells form a very authentic tissue type called blastema, which is crucial for successful regeneration. In order to evaluate this exceptional tissue and acquire high quality images, it is crucial to employ specific procedures to prepare the tissue for imaging. Here, in this study, we aimed to investigate success of various fixative solutions (Carnoy's, Bouin's, % 10 NBF, Clarke's, Alcoholic Formaline and AFA) to monitor the fixed blastema. Our data reveals that integrity of the blastema tissue differs among used fixatives and a significant difference is observed between the samples in terms of staining quality.

La regeneración se define como la renovación del tejido y el proceso de restauración funcional de las partes dañadas del cuerpo después de una lesión. Ambystoma mexicanum, comúnmente llamado Axolotl, es uno de los únicos vertebrados que tiene una notable capacidad para regenerar sus miembros después de una amputación. Aunque el proceso de regeneración incluye varios períodos, se puede dividir en dos fases principales: formación del blastema y desdiferenciación. En el par de horas después de la amputación, el cierre de la herida ocurre por la migración de células epiteliales alrededor del sitio de la amputación seguido por una infiltración de macrófagos y la desdiferenciación de las células para convertirse en células madre. Las células madre acumuladas forman un tipo de tejido muy diferenciado denominado blastema, que es crucial para una exitosa regeneración. Para evaluar este tejido y adquirir imágenes de alta calidad, es crucial emplear procedimientos específicos para la obtención de imágenes. En este estudio, se intentó investigar el éxito de varias soluciones fijadoras (Carnoy, Bouin, % 10 NBF, Clarke, Formalina Alcohólica y AFA) para monitorear la fijación del blastema. Nuestros datos revelan que la integridad del tejido del blastema difiere entre los fijadores utilizados y una diferencia significativa observada entre las muestras se da en términos de la calidad de tinción.

Detailed tail proteomic analysis of axolotl (Ambystoma mexicanum) using an mRNA-seq reference database.

Salamander axolotl has been emerging as an important model for stem cell research due to its powerful regenerative capacity. Several advantages, such as the high capability of advanced tissue, organ, and appendages regeneration, promote axolotl as an ideal model system to extend our current understanding on the mechanisms of regeneration. Acknowledging the common molecular pathways between amphibians and mammals, there is a great potential to translate the messages from axolotl research to mammalian studies. However, the utilization of axolotl is hindered due to the lack of reference databases of genomic, transcriptomic, and proteomic data. Here, we introduce the proteome analysis of the axolotl tail section searched against an mRNA-seq database. We translated axolotl mRNA sequences to protein sequences and annotated these to process the LC-MS/MS data and identified 1001 nonredundant proteins. Functional classification of identified proteins was performed by gene ontology searches. The presence of some of the identified proteins was validated by in situ antibody labeling. Furthermore, we have analyzed the proteome expressional changes postamputation at three time points to evaluate the underlying mechanisms of the regeneration process. Taken together, this work expands the proteomics data of axolotl to contribute to its establishment as a fully utilized model.