Visit to SABITA
Max H. YunSalamander models in regeneration and beyond: new resources and paradigms
Exhibiting the widest repertoire of regenerative abilities among vertebrates, which extend to complex organs and entire limbs, salamanders have long served as research models for understanding the basis of vertebrate regeneration. Recent technical advances have enabled salamander systems such as the axolotl (Ambystoma mexicanum) and the Spanish ribbed newt (Pleurodeles waltl), resulting in a significant expansion of research areas incorporating these models and a wealth of biological insights, from regeneration through development, genome organization and evolution. Here, I will discuss recent advances in salamander resources, including the giant P. waltl genome, and their contributions towards understanding principles of regeneration and beyond. Further, I will introduce a new paradigm of de novo organ regeneration, the axolotl thymus, with significant implications for our understanding of cellular transitions. Lastly, I will discuss the potential of salamander models to illuminate links between regeneration and ageing.
Session 1 – Moderated by Sven Vilain
Jessica Whited (Online)
17.15 - 18.00Amputation-induced systemic stem cell activation in axolotl
Regenerative abilities vary dramatically across animal lineages. Yet, how systems-level injury responses in individual species shape ultimate regenerative outcomes is not well understood. We recently discovered a body-wide stem cell activation response that occurs following limb amputation in axolotls. Here, we present evidence that this systemic activation primes appendages distant to the original injury for their own future regeneration events. Using single-cell RNAseq, we identified the repertoire of systemically-activated cells to be the same cells likely to fuel organismal growth and cell replenishment during homeostasis. We investigated the mechanism of systemic activation in axolotls and found the process requires innervation by the peripheral nervous system at distant responding sites. We used transcriptomics to identify several candidate molecular pathways for controlling systemic activation. We found inhibiting adrenergic signaling is sufficient to block systemic activation, and we found that activating adrenergic signaling can promote early phases of regeneration. Inspired by previous reports of mTOR activity being required for systemic cell cycle re-entry following injury in both highly-regenerative species and species with modest regenerative abilities, we also tested mTOR signaling in axolotl. Our studies also implicate mTOR signaling as required for axolotl systemic activation and priming following amputation. Together, these results demonstrate a direct link between systemic stem cell responses and localized regeneration of an appendage, and they also highlight roles for peripheral nerves, adrenergic signaling, and the mTOR pathway in systemic injury responses in axolotls. They also suggest a possible model whereby common initial injury responses, acting on stem cells also used for growth and homeostasis across species, are followed by differing instructions that result in regeneration versus repair.
Jesus Chimal-Monroy (Online)
18.00-18.20Endogenous RA induces proximo-distal limb duplications during limb regeneration
During limb regeneration, cells of the distal end of the stump are released from their tissue organization and acquire a mesenchymal phenotype. These cells will proliferate to give rise to a blastema from which all limb tissues will differentiate. It is known that RA binds and activates heterodimers formed by RARs and RXRs. It is catabolized in the cytoplasm by CYP26 enzymes yielding polar metabolites that are inactive or less active than RA, consequently controlling intracellular levels of active RA. Thus, the inhibition of Cyp26 results in an increase in endogenous retinoic acid. In this study, we used a specific inhibitor to determine the role of Cyp26 during limb regeneration.
Our results showed that by inhibiting the enzyme Cyp26, PD duplication is observed as occurs after the exogenous RA treatment. Transplanting a blastema from amputated limbs treated with Inh-Cyp26 to a non-treated amputated limb cannot produce a PD duplication in the recipient limb, regenerating only the missing part of the amputated limb. Surprisingly, the Inh-Cyp26-treated donor limb, from which the blastema was removed, regenerated with a new PD duplication from the cut site. Results suggest that instructive information to induce proximal-distal duplications is not retained in blastema but in the stump. Furthermore, we observed that RAR gamma rescues normal regeneration.
Olena Zhulyn (Online)
18.20- 18.35Evolutionary divergence of mTOR signaling drives translational remodeling underlying rapid wound closure and regeneration
One of the greatest outstanding questions in biology is why some species can scarlessly heal and regenerate tissues while others cannot. Here, we demonstrate that rapid activation of protein synthesis is a unique, and previously uncharacterized, feature of the injury response critical for limb regeneration in the highly regenerative axolotl (A. mexicanum). By applying polysome sequencing, we identify hundreds of transcripts, including antioxidants and ribosome components, which do not change in their overall mRNA abundance but are selectively activated at the level of translation from pre-existing mRNAs in response to injury. In contrast, we show that protein synthesis is not activated in response to digit amputation in the non-regenerative mouse. We further identify the mTORC1 pathway as a key upstream signal that mediates this regenerative translation response in the axolotl. Inhibition of this pathway is sufficient to suppress translation, wound closure and regeneration in the axolotl. Surprisingly, although mTOR is highly conserved across evolution, we discover novel expansions in key functional regions of mTOR kinase that are unique to highly regenerative urodele amphibians. By engineering an axolotl mTOR in human cells, we demonstrate that these expansions create a hypersensitive kinase that may allow axolotls to maintain this pathway in a highly labile state primed for rapid activation. We hypothesize that fundamental differences in the sensitivity and function of mTOR kinase may underlie metabolic differences and nutrient sensing between regenerative and non-regenerative species that are key to regeneration. Together, these findings highlight the unanticipated impact of the translatome on orchestrating the early steps of wound healing in highly regenerative species and provide a missing link in our understanding of vertebrate regenerative potential.
Session 2 – Moderated by Elif Eroğlu
Ji-Feng Fei (Online)
10.15 - 10.45Applying stereo-seq technology to dissect the mechanisms of brain regeneration
Promoting brain regeneration is challenging due to low inherent regenerative capacity of the mammalian brain and limited understanding of cellular and molecular mechanisms guiding this process. Here, by analyzing the highly regenerative axolotl brain with Stereo-seq, we reconstructed its telencephalon architecture with spatially resolved gene expression profiles at single-cell resolution, and fine cell dynamic maps throughout development and regeneration. We demarcated major mammalian types of excitatory, inhibitory neurons, and ependymoglial cells (EGCs) in axolotl brain. Intriguingly, we identified a regeneration-unique EGC cluster, named as reactive EGC (reaEGC) that is rapidly induced upon injury in the wound area. It appears to be the major source participating in regeneration to give rise to lost neurons, via intermediate progenitors, which largely resembles the development process. Furthermore, we uncover an injury-induced nerve response in regeneration. Upon injury, spatial-defined neurons at lesion respond to wounding and switch to an immature-neuron-like status determined by their transcriptome signature. Altogether, our work resembles the first spatial-temporal single cell brain atlas of anamniote tetrapod, decodes the complex cellular and molecular dynamics of axolotl telencephalon in development and regeneration. It lays the foundation for mechanistic studies of brain regeneration and evolution in future.
Takashi Takeuchi (Online)
10.45-11.05Multiple disruption of newt Hox13 paralogs revealed that Hoxa13 has an essential and predominant role in digit formation during limb development and regeneration
The 5’Hox genes play crucial roles in limb development and specify regions in the proximal-distal axis of limbs. However, there is no direct genetic evidence that Hox genes are essential for limb development in non-mammalian tetrapods or for limb regeneration. Here, we produced single to quadruple Hox13 paralog mutants using the CRISPR/Cas9 system along with germline mutants in newts (Pleurodeles waltl), which have strong regenerative capacities. We showed that Hox13 is essential for digit formation in development, as it is in mice. In addition, Hoxa13 has a predominant role in digit formation, unlike in mice. The predominance is probably due to the restricted expression pattern of Hoxd13 in limb buds and the strong dependence of Hoxd13 expression on Hoxa13. Finally, we indicated that Hox13 is also necessary for digit formation in limb regeneration. Our findings reveal that the general function of Hox13 is conserved between limb development and regeneration, and across taxa. The predominance of Hoxa13 function both in newt limbs and fish fins, but not in mouse limbs, suggests a potential contribution of Hoxa13 function in fin-to-limb transition.
Anastasia Polikarpova (Online)
11.05-11.20Cellular and molecular profile of axolotl large bone fractures
Understanding of the cellular and molecular mechanisms of fracture callus formation and bone healing is needed to treat bone fracture non-unions. Axolotls cannot heal bone critical-size defects (CSD, >30% of the bone length), but are able to regenerate the amputated limb by producing blastema, a transient progenitor cell mass. A substantial part of blastema is formed by the soft connective tissue (SCT) cells which build the distal part of the skeleton in the regenerate. Restricted ability of SCT cells to contribute to bone callus and lack of blastema factors may cause bone non-union in CSD. Aim: We aim to understand if SCT cells migrate to CSD and identify the factors, inducing SCT to contribute to bone. Material and methods: We use polyolefin tube-stabilized femur fracture model in combination with CT cell tracing, bulk and single-cell RNA sequencing, EdU labelling, immunofluorescence and histological stainings. Results: Whereas in small fracture (SF) fracture bridging by cartilaginous callus took place at 3-6 weeks, and bone formed at 12 weeks post-surgery, CSD gap persisted at 12 weeks post-surgery. We found Prrx1+ progenitor cells accumulating in the CSD gap, but no callus formed and proliferation was diminished in CSD in comparison to blastema. To trace SCT cells, skin, bones or muscles from axolotls with fluorescently labelled SCT cells were transplanted to wildtype animals. The labelled SCT cells were migrating to the fracture site. RNAseq analysis shows changes in proliferation- and blastema-specific gene expression. scRNAseq analysis showed similar connective tissue cell phenotype at the early stages of CSD and BL healing (3-5 dpi). However, at 8-11 dpi, CSD and BL CT cells differ in their gene expression, in particular, CSD cells are less de-differentiated and proliferative. This could be also influenced by differences in wound epidermis and macrophage profiles. Conclusions and outlook: SCT cells are able to migrate to the CSD, indicating potency to heal the fracture, however the signals inducing blastema-like condition or cartilage formation are absent in fracture, resulting in CSD non-union. We suggest to provide the proliferation- and de-differentiation-promoting factors to CSD to induce blastema-like phenotype to promote bone bridging.
Session 3 – Maria del Rosario Sanchez-Gonzalez
11.40 - 12.10Stable Limb Morphogenesis Regardless of the Blastema Size in Limb Regeneration
Axolotls can regenerate their limbs throughout their life. Smaller animals having perfectly patterned limbs are about 3 cm and larger ones are about 30 cm. Regardless of the animal size, they exhibit perfect limb regeneration ability after limb amputation. It has been considered that limb regeneration is mostly a recapitulation of limb developmental processes. However, limb development always takes place in the identical size of the limb field. On the other hand, limb regeneration occurs in the various sizes of a limb. Those arose the question of how the developmental gene system is controlled in various sizes of a limb blastema. We focused on Shh and Fgf8 expression patterns in various sizes of axolotl limb blastemas. We investigated those expression patterns by in situ hybridization and found the expression domains of Shh and Fgf8 are varied in accordance with the blastema size. We found that the effects of the variation of gene expression patterns are buffered by the SHH diffusion range taking a relatively constant distance regardless of the blastema sizes. We concluded that the constant SHH diffusion contributes to creating consistent limb morphogenesis in various sizes of an axolotl limb. We also found that the constant SHH diffusion range creates an active proliferative zone (aPZ) within the constant range. Actually, the aPZ is under the influence of two mitogens, SHH and FGF8. We also found that the first phalangeal cartilage (digit II) emerged from the aPZ. Then, the aPZ is gradually shifted toward the posterior end, which contributes to posterior digit formation. This digital morphogenetic system also contributes to consistent limb morphogenesis. In the meeting, I would like to explain this complex and beautiful system to make consistent limb morphogenesis in various sizes of the field.
Tatiana Sandoval-Guźman (Online)
12.10-12.40Biomechanics of skeletal formation in development and regeneration
Lizbeth A. Bolaños-Castro
12.40- 12.55Signaling Pathways Regulating Thymus Regeneration and Development in the Axolotl, Ambystoma Mexicanum
The thymus constitutes the main site for T cell development and maturation, and is thus central for the physiology of adaptive immunity. A hallmark of immune system aging is thymic involution, which has been observed across vertebrates and is regarded as a non-reversible process, highlighting the need for strategies promoting thymus regeneration. We have recently found that, exceptionally, the Mexican axolotl (A. mexicanum) is capable of de novo thymus regeneration. Here, we address the impact of diverse molecular pathways on thymus regeneration and development through pharmacological treatments, coupled with live imaging and whole-mount immunofluorescence. Through this approach, we find that the BMP and ERK pathways are necessary for thymus regeneration. Furthermore, by analyzing single cell RNAseq data from regenerating thymic nodules, we uncover a critical role for the Midkine pathway in thymus regeneration as well as development. This work broadens our understanding of thymus organogenesis and salamander immunity, laying the foundations for further comparative approaches across vertebrates.
Lunch by Bosphorus
Session 4 – Moderated by Ryan Kerney
Maria Antonietta Tosches (Online)
14.45-15.30Cell Type Profiling in Salamanders Identifies Innovations in Vertebrate Forebrain Evolution
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 novelties arose in vertebrate ancestors remains unclear. To reconstruct forebrain evolution in tetrapods, we took advantage of the phylogenetic position and of the simple brain architecture of salamanders. Using single-cell RNA sequencing, we built a cell-type atlas of the telencephalon of Pleurodeles waltl. Moreover, we used bioinformatic approaches to compare these cell-type data to similar datasets from other vertebrate species. This analysis reveals the series of innovations that resulted in the emergence of the sauropsid DVR, and the mammalian six-layered neocortex. General implications on the value of salamanders in evolutionary studies will be discussed.
15.30 - 15.50Do Non-Canonical Protein Secretion Mechanisms Contribute to Regeneration Signaling?
Cell-cell communication is essential for biological phenomena. Extracellular signaling molecules, for example, growth factors and cell surface proteins, are one of the substances of intercellular communication. During organ regeneration, signaling molecules are involved in critical cellular events such as cell migration, proliferation, differentiation, patterning and size control. Previously, we identified a molecule, Axolotl MARCKS Like Protein (AxMLP) as an extracellular factor that can initiate initial proliferative response in the axolotl (Ambystoma mexicanum) appendage regeneration (Sugiura, T., et al., Nature 2016). Importantly, we found that AxMLP was expressed in the wound epidermis (WE) that is an essential tissue to promote the process of appendage regeneration in salamanders (Tornier G. 1906. Arch. fur Entwmech). AxMLP was released into extracellular space in vitro. This was surprising, as MARCKS family proteins had been described to be intracellular proteins (El Amri, M., et al., J Biomed Sci. 2018). In the current study, we focus on the mechanisms of AxMLP secretion. Our preliminary results suggest that: 1. AxMLP is not secreted via the canonical protein secretion pathway
2. Extracellular vesicles containing AxMLP can induce proliferation of cultured myotubes
3. Membrane association of AxMLP is related to its secretion
4. Pharmacological screening identify promising molecules that regulate AxMLP secretion
5. MLP reporter transgenic axolotl allows us to investigate MLP secretion in vivo. Taken together with our previous study, these results imply that MLP released extracellularly from the WE via a non-canonical protein secretion pathway initiates proliferative response to the cells underneath the WE and contributes to forming the blastema.
Mehmet Anıl Oguz
15.50 - 16.10Salamander Species in Turkey: Their Populations and Biology
Aim: Turkey has high amphibian variation as a consequence of its geographic and climatic diversity. Every salamander species in Turkey belong to Salamandridae family. The aim is to present and introduce the populations and biology of salamander species in Turkey with the photos in their own habitats.
Material and Methods: As a result of vast number of fieldwork and excursions conducted throughout many years, I have photographed as well as researched salamander species in Turkey. Due to they are mostly nocturnal and they hide under the stones in rainy seasons in the day, I have found them at night or under the stones in the day.
Results: Turkey currently has sixteen salamander species. These species are: Salamandra infraimmaculata (Turkish Fire Salamander), Mertensiella caucasica (Caucasian Salamander), Lyciasalamandra antalyana (Antalya Salamander), L. atifi (Atıf’s Lycian Salamander), L. billae (Bille’s Lycian Salamander), L. flavimembris (Marmaris Salamander), L. luschani (Luschan’s Lycian Salamander), L. fazilae (Fazıla’s Lycian Salamander), Neurergus crocatus (Urmia Newt), Neurergus strauchii (Anatolian/Strauch’s Spotted Newt), Lissotriton vulgaris (The Smooth Newt), Ommatotriton vittatus (Southern Banded Newt), Ommatotriton ophryticus (North-Eastern Banded Newt), Ommatotriton nesterovi (North-Western Banded Newt), Triturus ivanbureschi (Balkan-Anatolian Crested Newt), Triturus anatolicus (Anatolian Crested Newt). The taxonomic status of salamander species in Turkey changes continuously due to their genetic variations. Hence, the taxonomic status should always be followed.
Conclusions: Turkey hosts many salamander species, resulted by its climate and geography. In addition, to the existing phenotypes, their phenotypes show lots of variety among the populations because of the mutations. Salamander species are mostly used as model organism in regeneration and neuroscience researches. Salamanders in Turkey have been understudied in the field of regeneration and neuroscience areas so far. Hence, they have a great potential to discover novel field of medicine, biology and pharmacology.
16.10- 16.25Metabolic Adaptations During Cardiac Regeneration in the Axolotl
Aim: Illuminate the interplay of metabolic processes in the heart and capacity for cardiac regeneration by assaying metabolism during different time points of cardiac regeneration. The axolotl represents a unique animal model to this end, as the vertebrate most closely related to humans known to be capable of true intrinsic cardiac regeneration. Metabolism has been proven to play a key role in regenerative processes. In adult and neonatal mice as well as zebrafish, studies have shown that specific metabolic adaptations are favorable to heart regeneration, although the exact mechanisms involved remain unclear.
Materials/methods: In this ongoing study, we use a cryoinjury to model myocardial infarction and investigate the metabolic profile accompanying different stages of regeneration – from initial injury response, transient scar formation and finally regeneration and maturation into new fully functioning myocardium. To this end, we are utilizing echocardiography, respirometry, metabolite analysis in blood and cardiac tissue, quantitative histology as well as autoradiography with radiolabeled FDG (glucose analog) and acetate (correlates with OXPHOS in heart) tracers to gather a detailed picture of metabolic processes in vivo that underlie a successful regenerative response.
Results: Preliminary results indicate a global increase in oxygen consumption during the instigation of the regenerative response, and a general up-regulation of cardiac metabolism in response to injury, the details of which will be highly informative in understanding how specific pathways may support or inhibit regenerative processes. Further data analysis is currently ongoing. A temperature based experiment in which animals were housed at different temperatures (10, 20 and 30 ̊C), shows key adaptations in metabolism including oxygen consumption and blood glucose, ketone and lactate; accompanied by inhibition of regenerative ability at colder temperatures and indications of increased regeneration rate at warmer temperatures.
Conclusion: cardiac regeneration in the axolotl is associated with distinct adaptations in systemic and local cardiac metabolic processes.
Session 5 – Moderated by Hasan Körkaya
James Godwin (Online)
16.45-17.30Immunological Control of Regeneration
17.30 - 17.50Heart Regeneration in Pleurodeles Waltl
The contribution of the epicardium, the outermost layer of the heart, to cardiac regeneration has remained controversial due to a lack of suitable analytical tools. By combining genetic marker-independent lineage-tracing strategies with transcriptional profiling and loss-of-function methods, we report here that the epicardium of the highly regenerative salamander species Pleurodeles waltl has an intrinsic capacity to differentiate into cardiomyocytes. Following cryoinjury, CLDN6+ epicardium-derived cells appear at the lesion site, organize into honeycomb-like structures connected via focal tight junctions and undergo transcriptional reprogramming that results in concomitant differentiation into de novo cardiomyocytes. Ablation of CLDN6+ differentiation intermediates as well as disruption of their tight junctions impairs cardiac regeneration. Salamanders constitute the evolutionarily closest species to mammals with an extensive ability to regenerate heart muscle and our results highlight the epicardium and tight junctions as key targets in efforts to promote cardiac regeneration.
17.50 - 18.10Macrophages Orchestrate Successful Lens Regeneration in Newts
Macrophage involvement during tissue regeneration has been a subject of great discussion over recent years. Lens regeneration in newts involves the transdifferentiation of iris pigmented epithelial cells (iPECs) into lens cells. The elegant simplicity of this process serves as a great platform to uncover mechanism by which macrophages promote scar-free healing in regeneration competent animals. Here we used clodronate liposomes to deplete macrophages in the eye from two newt species Notophthalmus viridescens and Pleurodeles waltl. We found that macrophages depletion inhibited lens regeneration in both species. Furthermore, early clodronate administration resulted in a significant decrease in iPECs proliferation, induced an unresolved cellular accumulation in the aqueous chamber, prolonged inflammation, generated a fibrotic like response and caused abnormalities in ECM remodeling. Even though macrophages were allowed to return after clodronate administration was stopped, ramifications caused by early macrophage depletion persisted for 100 days. Interestingly, secondary injury in the eye, alleviated the effects of macrophage depletion and restarted the regeneration process. These results add to the growing evidence on the importance of macrophages during tissue regeneration. Delineating the mechanisms by which newt macrophages promote cell cycle re-entry, resolve inflammation, and promote ECM remodeling will present a vital step towards our goal of inducing scar-free healing in other vertebrates.
Michael J Raymond (Online)
18.10- 18.25Investigation of the Mechanism Promoting Positional Plasticity in Axolotl Limb Regeneration
Following limb amputation in the Mexican Axolotl, a regenerative organ called the blastema forms at the amputation site and recreates the missing limb pattern of the regenerate. For this new pattern to form, the pattern information within cells contributing to the blastema becomes flexible, a property that we call positional plasticity. While the induction and maintenance of positional plasticity is dependent on the limb nerves, the underlying mechanisms are not well understood. To elucidate how uninjured limb cells transition to the positionally plastic state, we developed a new assay called the Plasticity-ALM (P-ALM) to characterize the underlying regulation of positional plasticity. We demonstrated that the re-expression of limb patterning genes coincides with the induction of positional plasticity and that newly generated positional information stabilizes after 3-7 days. Using the P-ALM system, we identified the nerve-dependent signaling molecules that promote positional plasticity by grafting growth factor-soaked beads into a wound site. Cooperative BMP and FGF signaling was shown to be sufficient to induce positional plasticity in mature limb cells. We also investigated the mechanisms behind the induction of positional plasticity at the genomic level. Using quantitative microscopy and custom bioimage analysis algorithms, we investigated changes to nucleus architecture and H3K27me3 modifications in limb wounds cells as they transition into positionally plastic cells. We showed that while a loss of heterochromatin correlates with positional plasticity, it is insufficient for its induction or maintenance. We detected a global loss of H3K27me3 abundance in positionally plastic cells and a reaccumulation of this mark as cells stabilize their positional information. To investigate this modification at the individual gene-level, we performed ChIP sequencing on cells from uninjured tissue, lateral wound sites, and blastemas. Surprisingly, GSEA demonstrated a significant increase in the abundance of H3K27me3 in gene sets that are associated with limb patterning in the blastema relative to wounds. In future studies, we will investigate the role of FGF and BMP signaling in mediating these modifications in wounds.
Session 6 – Moderated by Süleyman Yıldırım
09.00- 09.45Egg capsule microbial symbionts attenuate salamander tail tip regeneration
The amphibian egg capsule is unique in harbouring symbiotic prokaryotic and eukaryotic microbiota. We have been exploring the diversity of this microbiota in Ambystoma maculatum through amplicon sequencing and culturing, and testing the potential benefits of these microbes through selective co-culturing with sterilized embryonic hosts. By modifying a tail tip regeneration assay developed by the Voss lab at the University of Kentucky, we have found that none of the cultured bacteria affect rates of tail tip regeneration. However, the mutualistic algae Oophila amblystomatis does increase the rate of tail tip regeneration in controlled trials. We are exploring the mechanistic basis of this increase by modifying pO2 levels and a chemical fraction screen of Oophila-derived metabolites.
Maria del Rosario Sanchez-Gonzalez
09.45-10.05Axolotl, a new animal model for a “leaky” blood brain barrier
The blood brain barrier (BBB) represents a physical interface that tightly regulates the trafficking of molecules between the blood and the neural tissue, thereby maintaining the physiological homeostasis of the brain. Little is known about how regenerative-competent vertebrates such as amphibians establish the BBB, in particular in view of the lack of protoplasmic astrocytes in these animals, a key cell type regulating BBB permeability in mammals. Here, we analyzed the BBB permeability in the brain of Xenopus laevis and Ambystoma mexicanum (Axolotl). Surprisingly, we observed remarkable differences between the two species. While the BBB was similarly tight for 1kDa molecules in Xenopus as in zebrafish and mammals, Axolotl showed a complete leakiness for the 1kDa tracer and an increased endothelial transcytosis, despite the absence of any obvious neurological deficits. This suggests that Axolotl might have implemented specific mechanisms to protect the brain from detrimental consequences of “leaky blood vessels”.
10.05-10.20Mining axolotl microbiome: Novel insights into axolotl limb regeneration by using machine learning
Aim: The axolotl (Ambystoma mexicanum) is a common vertebrate model organism in regeneration research. Previously, Demircan et al. (2019) published research on axolotl limb regeneration by monitoring the temporal microbiome dynamics of this process. Interestingly, the three main phases of axolotl limb regeneration were reflected in the uncovered longitudinal microbiome profile. The present study followed up on this observation by mining the resulting ASV abundance dataset. The main purpose was to examine in more detail variation in microbiome structure and detect elusive microbial aspects.
Material and methods: The aforementioned microbiome dataset was retrieved from a dedicated repository and bioinformatically analyzed. Hereby, data mining was performed using the following consecutive steps: explanatory data analysis (EDA), unsupervised ML (including hierarchical clustering and dimension reduction) and supervised ML (using a Random Forest classifier).
Results: The EDA and Unsupervised ML steps revealed grouping of samples according to the respective regeneration phases. Interestingly, the combined analysis of alpha and beta diversity delineated a hitherto hidden overarching microbiome pattern, characterized by concurrent changes in the microbial community structure in the course of axolotl limb regeneration, namely simultaneous microbiome reshaping and alpha diversity reduction. The separation of samples based on regeneration phase was readily evident and statistically significant (p<0.05). Concordantly, the Random Forest-based classification model successfully predicted the phase labels in the subsequent supervised ML step. Importantly, the classification highlighted a distinct list of bacterial taxa as important features (predictors), which can be considered as new biomarker candidates for distinguishing between the phases.
Conclusions: In this work, we evaluated patterns in the microbime variation in the context of axolotl limb regeneration using a machine learning approach. Furthemore, we identified novel potential discerning microbial biomarkers of regeneration phase. Overall, the insights gained from the microbime data minig complement available experimental results and contributes to the progress in this research field.
Session 7 – Moderated by Maximina Yun
10.45- 11.00Non-canonical primary body axis segmentation through a multi-potent progenitor during axolotl tail regeneration
The ability of the axolotl to regenerate its tail represents a striking ability of vertebrate primary body axis regeneration, but how this process is regulated remains largely unknown. Current models of regeneration as seen in for example axolotl limb regeneration suggest a resident connective tissue population undergoes dedifferentiation after which developmental programs are re-deployed. In the context of tail regeneration this imply redeployment of somitogenesis, however previous work has suggested myomere are the earliest segmenting structures and are added in a non-linear fashion. Here we describe a resident progenitor in the axolotl tail which contributes to all regenerating paraxial mesodermal lineages. Moreover tail patterning as exemplified by vertebral segmentation is dependent on a somite independent non-canonical program. We find resident progenitors are in an inter-myomeric region and express a unique set of marker genes including Lfng, Scx, and Meox. Both during development and regeneration these cells contribute to myogenic, sclerogenic, and dermogenic lineages. While lineage contributions during regeneration closely recapitulate development, tissue patterning during regeneration follows a non-canonical somite independent program. In the absence of somites, there is no rostro-caudal polarity and resegmentation as a consequence is lost Moreover vertebrae are accurately re-established both after tail amputation as well as after vertebral extirpation. Finally, somitic mutant animals recover from vertebral malformations during regeneration, showing that even on a genetic level there are significant differences in tissue patterning programs during development and regeneration. Taken together these results show that even within a single organism multiple tissue specific modes of regeneration can exists: while limb regeneration is dependent on the dedifferentiation of connective tissue cells followed by the redeployment of developmental programs, tail regeneration is dependent on a resident progenitor cell population followed by the deployment of a patterning program which is distinct from canonical developmental programs.
11.00-11.20Cardiac Regeneration, Newt
Since cardiac regeneration in vertebrates was first reported in the newt in 1974, the research field has expanded to teleosts (zebrafish) and mammals (mouse, and potentially human). In order to elucidate the principle of cardiac regeneration, comparative research among these vertebrates will be important. In the present study, we developed a cryo-injury procedure for the newt. We showed the gene expression profiles in cardiac regeneration induced by cryo-damage differed from the profiles induced by conventional ventricular amputation. 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.
Marko Pende (Online)
11.20-11.40Tissue-clearing and light-sheet imaging for in toto histological interrogation of organs and organisms
Tissue clearing in combination with light-sheet microscopy enabled scientists to observe regeneration, development, or the connectome in entire animals without the necessity of histological sectioning. However, the vast variety of different tissue clearing methods often ponders the question which protocol to use for an organ, animal, or animal developmental stage of interest. After mastering tissue clearing, the decision for the right imaging system introduces another level of difficulty. In this technical presentation we try to show the best tissue clearing approaches for different axolotl development stages raging from early embryo to 5cm animals. Further, we show different ways to image such tissue-cleared samples.
James Monaghan (Online)
11.40-12.25Regeneration of the axolotl lung
Mammals have a limited capacity to repair their lung after injury. In contrast, we show here that axolotls are capable of lung regeneration after partial pneumonectomy. We show that injury induces an organ-wide proliferative response that supports regrowth of the missing lung tissue rather than generating a blastema. To identify epithelial cell populations involved in axolotl lung regeneration, we performed single-cell RNA-seq on uninjured lungs. We identified seven potential epithelial cell types in the axolotl lung, corresponding to most of the major cell types in the mammalian lung. We then used 12-plexed fluorescence in situ hybridization comprised of candidate cell type markers. This spatial analysis showed that the axolotl lung epithelium has a complex tissue architecture consisting of multiple specialized cell types.
Session 8 – Moderated by Prayag Murawala
Stephen Randal Voss (Online)
14.00- 14.45Leukocyte Tyrosine Kinase is the Mendelian Determinant of the Axolotl Melanoid Color Variant
The great diversity of color patterns observed among animals is largely explained by the differentiation of relatively few pigment cells during development. Mexican axolotls present a variety of color phenotypes that span the continuum of leucitic to highly melanistic. The melanoid axolotl is a Mendelian variant characterized by large numbers of melanophores, relatively fewer xanthophores, and no iridophores. Early studies of melanoid were influential in developing the single origin hypothesis of pigment cells from a common stem cell and the potential role of pigment metabolites in directing the development of pigment organelles that define different pigment cell types. Specifically, these studies identified 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 clone the causative locus. Significantly different frequencies of single nucleotide polymorphisms were identified between pooled RNA samples of wildtype and melanoid sibs for a chromosome 14q genomic region. This region harbored Gephyrin (Gphn), an enzyme that catalyzes the synthesis of the molybdenum cofactor that is required for XDH activity, and Leucocyte Tyrosine Kinase (Ltk), a phosphatase that is required for iridophore differentiation in zebrafish. Wildtype Ltk crispants exactly phenocopied the melanoid phenotype, thus confirming Ltk as the melanoid locus. We discuss these results within the contexts of the single origin of pigment cells hypothesis and current developmental genetic models of pigment cell differentiation.
14.45-15.05Single-cell analyses of cell type diversity, neurogenesis and regeneration in the axolotl telencephalon
Salamanders, such as the axolotl (Ambystoma mexicanum), play a role in the study of tetrapod-conserved traits. Cell type diversity in salamander brains and their relation to other vertebrate brains has until now been studied mainly histologically. Axolotl brains grow during postembryonic life and also regenerate after injury through the neurogenic activity of ependymoglia cells. It is still unclear how similar postembryonic and regenerative neurogenesis programs of ependymoglia cells are and whether neuronal connections are accurately recovered after regeneration is completed.
Here, we delineate the cell populations in the axolotl telencephalon during homeostasis and regeneration using single-nuclei genomic methods and spatial profiling and define their similarities to amniote telencephalic cell types. Among these, we identify a population of glutamatergic excitatory neurons with transcriptional similarity to amniote olfactory cortical neurons. These neurons receive input projections from the olfactory bulb, indicating a conserved role in olfactory processing.
After targeted brain injury, removing olfactory cortex-like glutamatergic neurons, we uncover that they are efficiently regenerated through a transcriptional program highly similar to homeostatic neurogenesis. Finally, we find that regenerated olfactory cortex-like neurons receive input projections from the olfactory bulb, suggesting re-establishment of the circuit.
15.05-15.25The Effects of Yap1 Downregulation During Early Phase of Limb Regeneration
The Hippo pathway is crucial to regulating vital cellular processes such as differentiation, regeneration, cell migration, organ growth, apoptosis, and cell cycle. Activation of the transcription coregulator component of this pathway, YAP1, promotes gene expression to drive proliferation, migration, differentiation, and suppress apoptosis. Although YAP1 functions in mammalian organ regeneration are well-established, its role in axolotl limb regeneration is understudied. The axolotl is an astonishing model organism to explore the molecular basis of organ and extremity regeneration due to its exceptional functional restoration capacity. This study examined Yap1 function in the early phase of limb regeneration by combining molecular, histological, and proteomics methods. Knock-down of Yap1 resulted in impaired regeneration fidelity, evident by bone formation defects. The altered expression level of proteases, ECM components, immune system elements, and osteogenesis-chondrogenesis regulators may explain the diminished bone formation capacity during axolotl limb regeneration upon Yap1 deficiency. Further functional studies aiming to modulate the expression level or activity of the candidate proteins described in this study would provide new insights into axolotl limb regeneration regulation.
15.25-15.45Sleeping beauty: The effect of long-term anesthesia on the regenerative ability of the axolotl
a) Aim Whole animal experiments in the regenerative field often require repeated use of anesthesia during injury procedures and at follow up examinations. Usually, anesthesia lasts only from minutes to a few hours before animals are reawakened. This limits the level of acceptable invasiveness of procedures, and it makes it difficult to untangle behavioral changes caused by injury to physiological processes involved in the regenerative response. We aimed to uncouple behavior from the regenerative response by developing a model of continuous anesthesia in the axolotl for 60 days spanning the majority of a full regenerative cycle following limb amputation or cryoinjury to the heart.
b) M&M Extensive piloting was performed to optimize the type of anesthetic, dose, route of delivery and osmolality of the housing medium. Subsequently, a study was conducted in which 18 age-matched axolotls (BM=20.4±3.4 g) were separated into five groups: three groups (each n=4) of continuously anesthetized animals receiving either no injury, limb amputation or cryoinjury to the heart, respectively, and two groups (each n=3) of animals receiving the same injuries, but maintained awake after the initial injury. Benzocaine (ethyl 4‐aminobenzoate, anesthetic and analgesic) was used during surgery and propofol (2,6-diisopropylphenol, anesthetic) was used for long-term anesthesia. During the regenerative progression both metabolic rate (respirometry), limb regeneration rate (photo), heart function and regeneration rate (echocardiography), and kidney function (creatinine) were repeatedly measured. Sixty days post injury, heart injury animal were sacrificed for quantitative histology, whereas animals with regenerated limbs were reawakened and subjected to behavioral analysis.
c) Results A total of 75% of continuously anesthetized animals survived until experimental end. Limb and heart regeneration progression was not significantly different between anesthetized and awake control groups. Reawakened animals did not show behavioral changes. d) Conclusions Axolotls can tolerate prolonged exposure to propofol anesthesia (60 days) and regenerate both limb and heart at the same rate in a comatose state as control animals. Dosage must be carefully monitored to avoid propofol toxicity.
Session 9 – Moderated by Mehmet Anıl Oğuz
Laura Muzinic (Online)
16.15- 17.00Animal Husbandry
The Ambystoma Genetic Stock Center (AGSC) is a research resource center that sustains and provides axolotl stocks to researchers globally. The AGSC maintains a variety of axolotl stocks of all life stages. The care and husbandry of axolotls require specific knowledge of water quality, temperature, and multiple housing parameters. I will illustrate how the axolotl life history operates in the laboratory with husbandry variables.
Session 10 – Moderated by Gürkan Öztürk
17.10- 18.40Community Issues and PI Meeting