ATP-mediated increase in H+ efflux from retinal Müller cells of the axolotl.

Previous work has shown that activation of tiger salamander retinal radial glial cells by extracellular ATP induces a pronounced extracellular acidification, which has been proposed to be a potent modulator of neurotransmitter release. This study demonstrates that low micromolar concentrations of extracellular ATP similarly induce significant H+ effluxes from Müller cells isolated from the axolotl retina. Müller cells were enzymatically isolated from axolotl retina and H+ fluxes were measured from individual cells using self-referencing H+-selective microelectrodes. The increased H+ efflux from axolotl Müller cells induced by extracellular ATP required activation of metabotropic purinergic receptors and was dependent upon calcium released from internal stores. We further found that the ATP-evoked increase in H+ efflux from Müller cells of both tiger salamander and axolotl were sensitive to pharmacological agents known to interrupt calmodulin and protein kinase C (PKC) activity: chlorpromazine (CLP), trifluoperazine (TFP), and W-7 (all calmodulin inhibitors) and chelerythrine, a PKC inhibitor, all attenuated ATP-elicited increases in H+ efflux. ATP-initiated H+ fluxes of axolotl Müller cells were also significantly reduced by amiloride, suggesting a significant contribution by sodium-hydrogen exchangers (NHEs). In addition, α-cyano-4-hydroxycinnamate (4-cin), a monocarboxylate transport (MCT) inhibitor, also reduced the ATP-induced increase in H+ efflux in both axolotl and tiger salamander Müller cells, and when combined with amiloride, abolished ATP-evoked increase in H+ efflux. These data suggest that axolotl Müller cells are likely to be an excellent model system to understand the cell-signaling pathways regulating H+ release from glia and the role this may play in modulating neuronal signaling.NEW & NOTEWORTHY Glial cells are a key structural part of the tripartite synapse and have been suggested to regulate synaptic transmission, but the regulatory mechanisms remain unclear. We show that extracellular ATP, a potent glial cell activator, induces H+ efflux from axolotl retinal Müller (glial) cells through a calcium-dependent pathway that is likely to involve calmodulin, PKC, Na+/H+ exchange, and monocarboxylate transport, and suggest that such H+ release may play a key role in modulating neuronal transmission.

The Accessory Limb Model Regenerative Assay and Its Derivatives.

When the Accessory Limb Model (ALM) regenerative assay was first published by Endo, Bryant, and Gardiner in 2004, it provided a robust system for testing the cellular and molecular contributions during each of the basic steps of regeneration: the formation of the wound epithelium, neural induction of the apical epithelial cap, and the formation of a positional disparity between blastema cells. The basic ALM procedure was developed in the axolotl and involves deviating a limb nerve into a lateral wound and grafting skin from the opposing side of the limb axis into the site of injury. In this chapter, we will review the studies that lead to the conception of the ALM, as well as the studies that have followed the development of this assay. We will additionally describe in detail the standard ALM surgery and how to perform this surgery on different limb positions.

Axolotl: A resourceful vertebrate model for regeneration and beyond.

The regenerative capacity varies significantly among the animal kingdom. Successful regeneration program in some animals results in the functional restoration of tissues and lost structures. Among the highly regenerative animals, axolotl provides multiple experimental advantages with its many extraordinary characteristics. It has been positioned as a regeneration model organism due to its exceptional renewal capacity, including the internal organs, central nervous system, and appendages, in a scar-free manner. In addition to this unique regeneration ability, the observed low cancer incidence, its resistance to carcinogens, and the reversing effect of its cell extract on neoplasms strongly suggest its usability in cancer research. Axolotl's longevity and efficient utilization of several anti-aging mechanisms underline its potential to be employed in aging studies.

Hydrogen peroxide is necessary during tail regeneration in juvenile axolotl.

BACKGROUND: Hydrogen peroxide (H2 O2 ) is a key reactive oxygen species (ROS) generated during appendage regeneration among vertebrates. However, its role during tail regeneration in axolotl as redox signaling molecule is unclear. RESULTS: Treatment with exogenous H2 O2 rescues inhibitory effects of apocynin-induced growth suppression in tail blastema cells leading to cell proliferation. H2 O2 also promotes recruitment of immune cells, regulate the activation of AKT kinase and Agr2 expression during blastema formation. Additionally, ROS/H2 O2 regulates the expression and transcriptional activity of Yap1 and its target genes Ctgf and Areg. CONCLUSIONS: These results show that H2 O2 is necessary and sufficient to promote tail regeneration in axolotls. Additionally, Akt signaling and Agr2 were identified as ROS targets, suggesting that ROS/H2 O2 is likely to regulate epimorphic regeneration through these signaling pathways. In addition, ROS/H2 O2 -dependent-Yap1 activity is required during tail regeneration.

Descripción morfométrica de la anatomía externa e interna del Ambystoma mexicanum / Morphometric description of the external and internal anatomy of Ambystoma mexicanum

RESUMEN: El Ambystoma mexicanum o comúnmente llamado axolote, es un anfibio miembro del género Ambystoma que comprende 32 especies presentes desde el sur de Canadá hasta la región central de México. Actualmente esta especie se encuentra en peligro de extinción debido a cambios fisicoquímicos en su medio ambiente y la depredación por organismos introducidos en su hábitat. Dentro de las múltiples estrategias para su conservación se encuentran el desarrollo de programas de investigación, educación y desarrollo del manejo clínico. El objetivo del presente trabajo estuvo enfocado en obtener y analizar datos morfométricos acompañados por una descripción de la anatomía externa e interna de la especie Ambystoma mexicanum con el propósito de contribuir a su conservación. Por lo anterior, cinco ejemplares de la especie Ambystoma mexicanum de 2 años, criados en cautiverio, fueron estudiados para obtener valores morfométricos externos e internos utilizando un calibrador vernier, balanzas analíticas y rayos X. Los resultados de la observación externa mostraron el dimorfismo sexual característico de estas especies presentes en la zona peri cloacal, además, el análisis radiográfico permitió observar y señalar algunas estructuras óseas del esqueleto axial y apendicular. Los ejemplares presentaron un peso promedio de 31,6 g y una longitud de 15,7 cm. Los miembros anteriores mostraron una longitud de 2,92 cm y 2,8 cm en los miembros posteriores. El análisis de los órganos internos mostró que el corazón tiene un peso de 0,036 g y una longitud de 0,75 cm, los pulmones un peso de 0,019 g y una longitud de 2,6 cm, el estómago arrojó una longitud de 4,86 cm y el intestino 10,88 cm. En conclusión, los valores presentados en el presente trabajo sirven de referencia en futuros trabajos de investigación clínica veterinaria y manejo zootécnico del axolote para su conservación y preservación.

SUMMARY: The Ambystoma mexicanum commonly known as axolotl, is an amphibian and member of the genus Ambystoma which includes 32 species that can be found from southern Canada to central Mexico. Currently this species is in danger of extinction due to physicochemical changes in its environment and predation by organisms introduced into its habitat. Among the multiple strategies to aid in its conservation are the development of research programs, education and development of clinical management. The objective of this work is focused on obtaining and analyzing morphometric data accompanied by a description of the external and internal anatomy of the species Ambystoma mexicanum with the purpose of contributing to the conservation of this species. Therefore, five 2-year-old specimens of the Ambystoma mexicanum species, raised in captivity, were studied to obtain external and internal morphometric values using a vernier caliper, analytical balances and X-rays. The results of external observation showed the characteristic sexual dimorphism of these species present in the pericloacal zone. Furthermore, the radiographic analysis allowed to observe and point out some bony structures of the axial and appendicular skeleton. The specimens presented an average weight of 31.60 g and a length of 15.70 cm. The forelimbs showed a length of 2.92 cm and 2.8 cm in the hindlimbs. Analysis of the internal organs showed that the heart had a weight of 0.036 g and a length of 0.75 cm, the lungs a weight of 0.019 g and a length of 2.6 cm, the stomach had a length of 4.86 cm and the intestine 10.88 cm. In conclusion, the values presented in this work serve as a reference for future veterinary clinical research and zootechnical management of the axolotl for its conservation and preservation.

Excisional biopsy and radiotherapy for management of an olfactory neuroblastoma in an axolotl (Ambystoma mexicanum).

CASE DESCRIPTION: A 4-year-old sexually intact male leucistic axolotl (Ambystoma mexicanum) was presented with a 2-week history of dysrexia and difficulty swallowing. CLINICAL FINDINGS: Physical examination revealed a 1-cm-diameter intraoral mass on the rostral aspect of the palate and swelling of the left nasal fossa. Local invasion into the left nasal fossa was suspected during oral examination. The lesion was marginally excised, and an incompletely excised olfactory neuroblastoma was diagnosed histologically. Five weeks later, physical examination revealed persistent erythema, delayed healing of the rostral portion of the palate, and a mild facial deformity associated with a white mass in the nasal cavity. TREATMENT AND OUTCOME: 6 weeks after excision, adjuvant electron (6-MeV) beam radiotherapy was initiated for treatment of the incompletely excised olfactory neuroblastoma and likely presence of a recurrent mass. The protocol consisted of 4 weekly fractions of 8 Gy each (total, 32 Gy) with the axolotl under anesthesia. No acute adverse radiation effects were noted following radiotherapy. The oral erythema resolved after the third session. No recurrence was observed 2 months after treatment, and the owners reported no abnormal signs at home. The axolotl died 3.5 months after radiotherapy was completed (8 months after marginal excision of the tumor) secondary to an environmental management failure. Postmortem histologic evaluation showed no evidence of neoplasia. CLINICAL RELEVANCE: In axolotls, olfactory neuroblastoma should be considered in the differential diagnosis of intraoral palatal masses. This report describes the first application of radiotherapy for treatment of an olfactory neuroblastoma in an axolotl.

Axolotls' and Mices' Oral-Maxillofacial Trephining Wounds Heal Differently.

The Ambystoma maxicanum (axolotl) regenerates strikingly from wounds and amputations. Comparing its healing ability to non-regenerative species such as the mouse should help narrow in on mechanisms to improve human wound healing. Here, the tongue and intermandibular soft tissues of both mice (C57BL/6NCrl) and axolotls were wounded with a 2-2.5 mm punch biopsy. The study aimed to compare the differences between these 2 species following surgical resection with regard to the macroscopic and histological characteristics. These include wound closure times, epithelial wound sealing and thickness as well as acute immune marker myeloperoxidase (MPO) response over 30 days. Post surgery, mice visually showed greater haemorrhage; their wounds immediately collapsed while it took 14 days for the axolotls mandibular void to close. The epithelium sealed the axolotls' wound margins within 24 h with a maximal mean thickness of 0.42 ± 0.13-fold normalized to unwounded skin. In mice, the epithelium separately sealed the ventral and dorsal sides, respectively at 7 and 7-30 days with mean maximal epithelial thicknesses reaching 13 ± 5.6 and 3.0 ± 0.63-fold. Mean MPO-positive cell values peaked in axolotls at 14 ± 1.5-fold between hours 6-12; while in mice, it peaked at 8.7 ± 0.9-fold between hours 24-96. We conclude that axolotls form smaller blood clots, have a faster and thinner epithelial cell migrating front, and a shorter MPO-positive cell response in comparison to mice. These observations may help refine future oral and facial wound-healing research and treatment.

Himalaquinones A-G, Angucyclinone-Derived Metabolites Produced by the Himalayan Isolate Streptomyces sp. PU-MM59.

Himalaquinones A-G, seven new anthraquinone-derived metabolites, were obtained from the Himalayan-based Streptomyces sp. PU-MM59. The chemical structures of the new compounds were identified based on cumulative analyses of HRESIMS and NMR spectra. Himalaquinones A-F were determined to be unique anthraquinones that contained unusual C-4a 3-methylbut-3-enoic acid aromatic substitutions, while himalaquinone G was identified as a new 5,6-dihydrodiol-bearing angucyclinone. Comparative bioactivity assessment (antimicrobial, cancer cell line cytotoxicity, impact on 4E-BP1 phosphorylation, and effect on axolotl embryo tail regeneration) revealed cytotoxic landomycin and saquayamycin analogues to inhibit 4E-BP1p and inhibit regeneration. In contrast, himalaquinone G, while also cytotoxic and a regeneration inhibitor, did not affect 4E-BP1p status at the doses tested. As such, this work implicates a unique mechanism for himalaquinone G and possibly other 5,6-dihydrodiol-bearing angucyclinones.

Fibroblast dedifferentiation as a determinant of successful regeneration.

Limb regeneration, while observed lifelong in salamanders, is restricted in post-metamorphic Xenopus laevis frogs. Whether this loss is due to systemic factors or an intrinsic incapability of cells to form competent stem cells has been unclear. Here, we use genetic fate mapping to establish that connective tissue (CT) cells form the post-metamorphic frog blastema, as in the case of axolotls. Using heterochronic transplantation into the limb bud and single-cell transcriptomic profiling, we show that axolotl CT cells dedifferentiate and integrate to form lineages, including cartilage. In contrast, frog blastema CT cells do not fully re-express the limb bud progenitor program, even when transplanted into the limb bud. Correspondingly, transplanted cells contribute to extraskeletal CT, but not to the developing cartilage. Furthermore, using single-cell RNA-seq analysis we find that embryonic and adult frog cartilage differentiation programs are molecularly distinct. This work defines intrinsic restrictions in CT dedifferentiation as a limitation in adult regeneration.

Cardiac regeneration in the axolotl is unaffected by alterations in leukocyte numbers induced by lipopolysaccharide and prednisolone.

OBJECTIVE: Cardiac regeneration in the axolotl has been found to rely on the innate immune system, and especially macrophages have been demonstrated to play a vital role in regulating the regenerative process. In this study we wanted to induce a pro- and anti-inflammatory milieu in the axolotl during heart regeneration to test the resilience of the regenerative response. RESULTS: This was induced via repeated intrapericardial injections of lipopolysaccharide or prednisolone during a 40-day regeneration period in order to challenge the presumably fine-tuned inflammatory response that normally facilitates regeneration. We observed a local and systemic leucocyte response to pro- and anti-inflammatory stimulation, but we found cardiac regeneration to be structurally and functionally unaffected.

The diversity in sensitivity of TRPA1 and TRPV1 of various animals to polyphenols.

The perception of tastes is sensed by the receptors that stimulate sensory cells. We previously reported that TRPA1 and TRPV1 channels expressed in the oral cavity of mammals, are activated by the auto-oxidized product of epigallocatechin gallate (oxiEGCG), a major astringent catechin in green tea. Here, we investigated and compared the sensitivity of TRPA1 and TRPV1 from various animals to astringent polyphenols. We selected three polyphenols, oxiEGCG, tannic acid and myricetin. HEK293T cells expressing TRPA1 or TRPV1 from mammal, bird, reptile, amphibian, and fish, were analyzed for their activation by the Ca2+-imaging. We found the apparent diversity in the polyphenol-sensitivity among various animals. Mammalian TRPs showed relatively higher sensitivity to polyphenols, and especially, human TRPA1 and TRPV1 could be activated by all of three polyphenols at 20 µM. Reptile TRP channels, however, were insensitive to any polyphenols examined. Moreover, the polyphenol-sensitivity of zebrafish TRPA1 and TRPV1 was quite different from that of medaka TRP channels. Since many polyphenols are present in plants and the sensing of polyphenols using TRP channels in the oral cavity might cause astringent taste, the observed diversity of the polyphenol-sensitivity of TRP channels might be involved in the divergence in the food habit of various animals.

Optogenetic manipulation of cellular communication using engineered myosin motors.

Cells achieve highly efficient and accurate communication through cellular projections such as neurites and filopodia, yet there is a lack of genetically encoded tools that can selectively manipulate their composition and dynamics. Here, we present a versatile optogenetic toolbox of artificial multi-headed myosin motors that can move bidirectionally within long cellular extensions and allow for the selective transport of GFP-tagged cargo with light. Utilizing these engineered motors, we could transport bulky transmembrane receptors and organelles as well as actin remodellers to control the dynamics of both filopodia and neurites. Using an optimized in vivo imaging scheme, we further demonstrate that, upon limb amputation in axolotls, a complex array of filopodial extensions is formed. We selectively modulated these filopodial extensions and showed that they re-establish a Sonic Hedgehog signalling gradient during regeneration. Considering the ubiquitous existence of actin-based extensions, this toolbox shows the potential to manipulate cellular communication with unprecedented accuracy.

Lung injury in axolotl salamanders induces an organ-wide proliferation response.

BACKGROUND: Ambystoma mexicanum, the axolotl salamander, is a classic model organism used to study vertebrate regeneration. It is assumed that axolotls regenerate most tissues, but the exploration of lung regeneration has not been performed until now. RESULTS: Unlike the blastema-based response used during appendage regeneration, lung amputation led to organ-wide proliferation. Pneumocytes and mesenchymal cells responded to injury by increased proliferation throughout the injured lung, which led to a recovery in lung mass and morphology by 56 days post-amputation. Receptors associated with the Neuregulin signaling pathway were upregulated at one and 3 weeks post lung amputation. We show expression of the ligand, neuregulin, in the I/X cranial nerve that innervates the lung and cells within the lung. Supplemental administration of Neuregulin peptide induced widespread proliferation in the lung similar to an injury response, suggesting that neuregulin signaling may play a significant role during lung regeneration. CONCLUSION: Our study characterizes axolotl lung regeneration. We show that the lung responds to injury by an organ-wide proliferative response of multiple cell types, including pneumocytes, to recover lung mass.

Celiotomia e gastrotomia para remoção cirúrgica de corpos estranhos em axolote (Ambystoma mexicanum): relato de caso / Celiotomy and gastrotomy for surgical removal of foreign bodies in axolotl (Ambystoma mexicanum): case report

A ingestão de corpos estranhos em aquários artificiais é uma ocorrência frequente observada na clínica cirúrgica de anfíbios criados como pets, o que inclui os axolotes (Ambystoma mexicanum). O presente relato descreve um caso de ingestão de sete corpos estranhos em um axolote macho, de cinco meses de idade, com histórico de abaulamento irregular e irredutível da superfície corpórea ventral, de consistência firme. Na anamnese foi informado que o animal habitava um aquário com substrato de cascalhos. Ao exame físico, o axolote apresentou bom estado geral e parâmetros vitais dentro da normalidade para a espécie. Os materiais deglutidos foram identificados em região gástrica por meio de exame radiográfico corpóreo total, e suas características condiziam com o substrato utilizado no aquário do paciente. Como protocolo anestésico, foi priorizada a imersão em Isofluorano e gás oxigênio, com o objetivo de atingir a via branquial e, ocasionalmente, transdérmica. A remoção cirúrgica foi feita através de celiotomia e gastrotomia em ambiente aquático com temperatura, pH e coleção bacteriana controlados, conforme literatura disponível e tendo em consideração a natureza, diâmetro e localização dos corpos estranhos. Após cinco e quinze dias do procedimento, acompanhou-se a cicatrização da ferida cirúrgica, sendo possível constatar bom restabelecimento da continuidade dos tecidos e bom estado geral do paciente.

The ingestion of foreign bodies in artificial aquariums is a frequent occurrence observed in the surgical clinic of amphibians raised as pets, which includes axolotls (Ambystoma mexicanum). The present report describes a case of ingestion of seven foreign bodies in a male axolotl, five months old, with a history of irregular and irreducible bulging of the ventral body surface, with firm consistency. In the anamnesis, it was reported that the animal inhabited an aquariums with gravel substrate. On physical examination, the axolotl showed good general condition and vital parameters within the normal range for the species. The swallowed materials were identified in the gastric region by means of total body radiographic examination, and their characteristics were consistent with the substrate used in the patient's aquarium. As anesthetic protocol, immersion in Isofluorane and oxygen gas was prioritized, in order to reach the branchial and, occasionally, transdermal route. Surgical removal was performed through celiotomy and gastrotomy in an aquatic environment with controlled temperature, pH and bacterial collection, according to available literature and taking into account the nature, diameter and location of foreign bodies. After five and fifteen days of the procedure, the healing of the surgical wound was monitored, showing a good restoration of tissue continuity and a good general condition of the patient.

Celiotomia e gastrotomia para remoção cirúrgica de corpos estranhos em axolote (Ambystoma mexicanum): relato de caso / Celiotomy and gastrotomy for surgical removal of foreign bodies in axolotl (Ambystoma mexicanum): case report

A ingestão de corpos estranhos em aquários artificiais é uma ocorrência frequente observada na clínica cirúrgica de anfíbios criados como pets, o que inclui os axolotes (Ambystoma mexicanum). O presente relato descreve um caso de ingestão de sete corpos estranhos em um axolote macho, de cinco meses de idade, com histórico de abaulamento irregular e irredutível da superfície corpórea ventral, de consistência firme. Na anamnese foi informado que o animal habitava um aquário com substrato de cascalhos. Ao exame físico, o axolote apresentou bom estado geral e parâmetros vitais dentro da normalidade para a espécie. Os materiais deglutidos foram identificados em região gástrica por meio de exame radiográfico corpóreo total, e suas características condiziam com o substrato utilizado no aquário do paciente. Como protocolo anestésico, foi priorizada a imersão em Isofluorano e gás oxigênio, com o objetivo de atingir a via branquial e, ocasionalmente, transdérmica. A remoção cirúrgica foi feita através de celiotomia e gastrotomia em ambiente aquático com temperatura, pH e coleção bacteriana controlados, conforme literatura disponível e tendo em consideração a natureza, diâmetro e localização dos corpos estranhos. Após cinco e quinze dias do procedimento, acompanhou-se a cicatrização da ferida cirúrgica, sendo possível constatar bom restabelecimento da continuidade dos tecidos e bom estado geral do paciente.

The ingestion of foreign bodies in artificial aquariums is a frequent occurrence observed in the surgical clinic of amphibians raised as pets, which includes axolotls (Ambystoma mexicanum). The present report describes a case of ingestion of seven foreign bodies in a male axolotl, five months old, with a history of irregular and irreducible bulging of the ventral body surface, with firm consistency. In the anamnesis, it was reported that the animal inhabited an aquariums with gravel substrate. On physical examination, the axolotl showed good general condition and vital parameters within the normal range for the species. The swallowed materials were identified in the gastric region by means of total body radiographic examination, and their characteristics were consistent with the substrate used in the patient’s aquarium. As anesthetic protocol, immersion in Isofluorane and oxygen gas was prioritized, in order to reach the branchial and, occasionally, transdermal route. Surgical removal was performed through celiotomy and gastrotomy in an aquatic environment with controlled temperature, pH and bacterial collection, according to available literature and taking into account the nature, diameter and location of foreign bodies. After five and fifteen days of the procedure, the healing of the surgical wound was monitored, showing a good restoration of tissue continuity and a good general condition of the patient.

The first report on circulating microRNAs at Pre- and Post-metamorphic stages of axolotls.

MicroRNAs (miRNAs) are endogenously coded small RNAs, implicated in post-transcriptional gene regulation by targeting messenger RNAs (mRNAs). Circulating miRNAs are cell-free molecules, found in body fluids, such as blood and saliva, and emerged recently as potential diagnostic biomarkers. Functions of circulating miRNAs and their roles in target tissues have been extensively investigated in mammals, and the reports on circulating miRNAs in non-mammalian clades are largely missing. Salamanders display remarkable regenerative potential, and the Mexican axolotl (Ambystoma mexicanum), a critically endangered aquatic salamander, has emerged as a powerful model organism in regeneration and developmental studies. This study aimed to explore the circulating miRNA signature in axolotl blood plasma. Small RNA sequencing on plasma samples revealed 16 differentially expressed (DE) circulating miRNAs between neotenic and metamorphic stages out of identified 164 conserved miRNAs. Bioinformatics predictions provided functional annotation of detected miRNAs for both stages and enrichment of DE miRNAs in cancer-related and developmental pathways was notable. Comparison with previous reports on axolotl miRNAs unraveled common and unique members of the axolotl circulating miRNome. Overall, this work provides novel insights into non-mammalian aspects of circulating miRNA biology and expands the multi-omics toolkit for this versatile model organism.

A double fluorescence chimeric limb regeneration model reveals muscle fiber reconnection during axolotl limb regeneration / Modelo de regeneración quimérica de doble fluorescencia del miembro revela la reconexión de la fibra muscular durante la regeneración del miembro axolotl

SUMMARY: Axolotl limb regeneration is a fascinating characteristic that has attracted attention for several decades. Our previous studies on axolotl limb regeneration indicated that the satellite cells in the remnant muscles move distally into the blastema to regenerate new muscles that are separated by a gap from remnant muscles. Thereafter, the regenerative muscle fibers start to reconnect with remnant ones. In this study, the reconnection at the individual muscle fiber level was elucidated to test the hypothesis that this reconnection happens synchronously among involved muscles. Three pairs of EGFP+ mid-bud stage blastemas were transplanted onto freshly amputated stumps of RFP+ axolotls at the same thigh position to generate double fluorescence chimeric regenerative hindlimbs. These regenerative limbs were harvested very late far beyond they had reached the late differentiation stage. Fluorescence imaging of these limbs in cross sections revealed that in the proximal remnant part of the muscle fiber, reconnection occurred at a different pace among the muscles. In the major thigh muscle gracilis, the reconnection started from the periphery before it was completed. Furthermore, RFP+ muscle fibers contributed to muscle regeneration in the distal regenerative parts. Intriguingly, this red cell contribution was limited to ventral superficial muscles of the calf. This kind of double fluorescence chimeric limb regeneration model may help increase the understanding of the patterning of axolotl limb regeneration in late stages.

RESUMEN: La regeneración del miembro de Axolotl es una característica fascinante que ha llamado la atención durante varias décadas. Nuestros estudios previos sobre la regeneración del miembro del Axolotl indicaron que las células satélite en los músculos remanentes se mueven distalmente hacia el blastema para regenerar nuevos músculos que están separados por una brecha de músculos remanentes. A partir de entonces, las fibras musculares regenerativas comienzan a reconectarse con las restantes. En este estudio, se aclaró la reconexión a nivel de fibra muscular individual para probar la hipótesis de que esta reconexión ocurre sincrónicamente entre los músculos involucrados. Se trasplantaron tres pares de blastemas EGFP+ en la etapa de yema media en tocones recién amputados de axolotls RFP+ en la misma posición del muslo para generar miembros posteriores regenerativos quiméricos de fluorescencia doble. Estos miembros regenerativos se cosecharon muy tarde mucho más allá de haber alcanzado la etapa de diferenciación tardía. Las imágenes de fluorescencia de estos miembros en secciones transversales revelaron que en la parte remanente proximal de la fibra muscular, la reconexión se produjo a un ritmo diferente entre los músculos. En el músculo grácil, la reconexión comenzó desde la periferia antes de completarse. Además, las fibras musculares RFP+ contribuyeron a la regeneración muscular en las partes regenerativas distales. Curiosamente, esta contribución de glóbulos rojos se limitó a los músculos superficiales ventrales de la pantorrilla. Este tipo de modelo de regeneración quimérica de doble fluorescencia del miembro puede ayudar a aumentar la comprensión del patrón de la regeneración del miembro del Axolotl en etapas tardías.

Axolotl Ambystoma mexicanum extract induces cell cycle arrest and differentiation in human acute myeloid leukemia HL-60 cells.

Acute myeloid leukemia is the most common form of acute leukemia in adults, constituting about 80% of cases. Although remarkable progress has been made in the therapeutic scenario for patients with acute myeloid leukemia, research and development of new and effective anticancer agents to improve patient outcome and minimize toxicity is needed. In this study, the antitumor activity of axolotl (AXO) Ambystoma mexicanum crude extract was assessed in vitro on the human acute myeloid leukemia HL-60 cell line. The anticancer activity was evaluated in terms of ability to influence proliferative activity, cell viability, cell cycle arrest, and differentiation. Moreover, gene expression analysis was performed to evaluate the genes involved in the regulation of these processes. The AXO crude extract exhibited antiproliferative but not cytotoxic activities on HL-60 cells, with cell cycle arrest in the G0/G1 phase. Furthermore, the AXO-treated HL-60 cells showed an increase in both the percentage of nitroblue tetrazolium positive cells and the expression of CD11b, whereas the proportion of CD14-positive cells did not change, suggesting that extract is able to induce differentiation toward the granulocytic lineage. Finally, the treatment with AXO extract caused upregulation of CEBPA, CEBPB, CEBPE, SPI1, CDKN1A, and CDKN2C, and downregulation of c-MYC. Our data clearly show the potential anticancer activity of Ambystoma mexicanum on HL-60 cells and suggest that it could help develop promising therapeutic agents for the treatment of acute myeloid leukemia.

Inhibition of Wound Epidermis Formation via Full Skin Flap Surgery During Axolotl Limb Regeneration.

Classical experiments in salamander regenerative biology over the last century have long established that the wound epidermis is a crucial signaling structure that forms rapidly post-amputation and is required for limb regeneration. However, methods to study its precise function at the molecular level over the last decades have been limited due to a paucity of precise functional techniques and genomic information available in salamander model systems. Excitingly, the recent plethora of sequencing technologies coupled with the release of various salamander genomes and the advent of functional genetic testing methods, including CRISPR, makes it possible to re-visit these foundational experiments at unprecedented molecular resolution. Here, I describe how to perform the classically developed full skin flap (FSF) surgery in adult axolotls in order to inhibit wound epidermis formation immediately following amputation. The wound epidermis normally forms via distal migration of epithelial cells in the skin proximal to the amputation plane to seal off the wound from the outside environment. The surgery entails immediately suturing full thickness skin (which includes both epidermal and dermal layers) over the amputation plane to hinder epithelial cell migration and contact with the underlying damaged mesenchymal tissues. Successful surgeries result in the inhibition of blastema formation and limb regeneration. By combining this surgery method with contemporary downstream molecular and functional analyses, researchers can begin to uncover the molecular underpinnings of wound epidermis function and biology during limb regeneration.