Material properties in regenerating axolotl limbs using inverse finite element analysis.

BACKGROUND: The extracellular mechanical environment plays an important role in the skeletal development process. Characterization of the material properties of regenerating tissues that recapitulate development, provides insights into the mechanical environment experienced by the cells and the maturation of the matrix. In this study, we estimated the viscoelastic material properties of regenerating forelimbs in the axolotl (Ambystoma mexicanum) at three different regeneration stages: 27 days post-amputation (mid-late bud) and 41 days post-amputation (palette stage), and fully-grown time points. A stress-relaxation indentation test followed by two-term Prony series viscoelastic inverse finite element analysis was used to obtain material parameters. Glycosaminoglycan (GAG) content was estimated using a 1,9- dimethyl methylene blue assay. RESULTS: The instantaneous and equilibrium shear moduli significantly increased with regeneration while the short-term stress relaxation time significantly decreased with limb regeneration. The long-term stress relaxation time in the fully-grown time point was significantly lower than 27 and 41 DPA groups. The GAG content was not significantly different between 27 and 41 DPA but the GAG content of cartilage in the fully-grown group was significantly greater than in 27 and 41 DPA. CONCLUSIONS: The mechanical environment of the proliferating cells changes drastically during limb regeneration. Understanding how the tissue's mechanical properties change during limb regeneration is critical for linking molecular-level matrix production of the cells to tissue-level behavior and mechanical signals.

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.

Click Chemistry-Enabled Conjugation Strategy for Producing Dibenzodiazepinone-Type Fluorescent Probes To Target M2 Acetylcholine Receptors.

The development of fluorescently labeled receptor-targeting compounds represents a powerful pharmacological tool to study and characterize ligand-receptor interactions. Despite significant advances in developing sub-type-specific antagonists for muscarinic acetylcholine receptors (mAChRs), reports on antagonists feasible for click chemistry are less common. Here, we designed and synthesized an antagonist suitable for probe attachment through click chemistry, namely, dibenzodiazepinone (DIBA)-alkyne, based on a previously reported DIBA scaffold with a high binding affinity to type-2 mAChR (M2R). To demonstrate the versatility of DIBA-alkyne as a building block for bioconjugates, we assembled DIBA-alkyne with Cyanine5 fluorophores (Cy5) and polyethylene glycol (PEG) biomolecules to obtain fluorescent DIBA antagonist (DIBA-Cy5) and fluorescent DIBA PEG derivatives. Flow cytometric analysis showed that DIBA-Cy5 possessed a high binding affinity to M2R (Kd = 1.80 nM), a two-order magnitude higher binding affinity than M1R. Fluorescent DIBA PEG derivatives maintained a potent binding to the M2R (Kd ≤ 4 nM), confirmed by confocal microscopic imaging. Additionally, DIBA-Cy5 can serve as a fluorescent ligand in the receptor-ligand competitive binding assay for other mAChR ligands, an attractive alternative to the traditional radioligand-based assay. The competitive binding mode between DIBA-Cy5 and orthosteric antagonist atropine/allosteric modulator LY2119620 indicated a dualsteric binding mode of the DIBA-type antagonist to M2R. Lastly, we demonstrated the direct staining of DIBA-Cy5 to M2R receptors in the sinoatrial node of a mouse heart. The adaptability of the clickable DIBA antagonist to a wide range of fluorophores and biomolecules can facilitate its use in various biomedical applications such as binding assays that screen compounds for M2R as the receptor target.

Usability of deep learning pipelines for 3D nuclei identification with Stardist and Cellpose.

Segmentation of 3D images to identify cells and their molecular outputs can be difficult and tedious. Machine learning algorithms provide a promising alternative to manual analysis as emerging 3D image processing technology can save considerable time. For those unfamiliar with machine learning or 3D image analysis, the rapid advancement of the field can make navigating the newest software options confusing. In this paper, two open-source machine learning algorithms, Cellpose and Stardist, are compared in their application on a 3D light sheet dataset counting fluorescently stained proliferative cell nuclei. The effects of image tiling and background subtraction are shown through image analysis pipelines for both algorithms. Based on our analysis, the relative ease of use of Cellpose and the absence of need to train a model leaves it a strong option for 3D cell segmentation despite relatively longer processing times. When Cellpose's pretrained model yields results that are not of sufficient quality, or the analysis of a large dataset is required, Stardist may be more appropriate. Despite the time it takes to train the model, Stardist can create a model specialized to the users' dataset that can be iteratively improved until predictions are satisfactory with far lower processing time relative to other methods.

Local mechanical stimuli correlate with tissue growth in axolotl salamander joint morphogenesis.

Movement-induced forces are critical to correct joint formation, but it is unclear how cells sense and respond to these mechanical cues. To study the role of mechanical stimuli in the shaping of the joint, we combined experiments on regenerating axolotl (Ambystoma mexicanum) forelimbs with a poroelastic model of bone rudiment growth. Animals either regrew forelimbs normally (control) or were injected with a transient receptor potential vanilloid 4 (TRPV4) agonist during joint morphogenesis. We quantified growth and shape in regrown humeri from whole-mount light sheet fluorescence images of the regenerated limbs. Results revealed significant differences in morphology and cell proliferation between groups, indicating that TRPV4 desensitization has an effect on joint shape. To link TRPV4 desensitization with impaired mechanosensitivity, we developed a finite element model of a regenerating humerus. Local tissue growth was the sum of a biological contribution proportional to chondrocyte density, which was constant, and a mechanical contribution proportional to fluid pressure. Computational predictions of growth agreed with experimental outcomes of joint shape, suggesting that interstitial pressure driven from cyclic mechanical stimuli promotes local tissue growth. Predictive computational models informed by experimental findings allow us to explore potential physical mechanisms involved in tissue growth to advance our understanding of the mechanobiology of joint morphogenesis.

Wnt Signaling Coordinates the Expression of Limb Patterning Genes During Axolotl Forelimb Development and Regeneration.

After amputation, axolotl salamanders can regenerate their limbs, but the degree to which limb regeneration recapitulates limb development remains unclear. One limitation in answering this question is our lack of knowledge about salamander limb development. Here, we address this question by studying expression patterns of genes important for limb patterning during axolotl salamander limb development and regeneration. We focus on the Wnt signaling pathway because it regulates multiple functions during tetrapod limb development, including limb bud initiation, outgrowth, patterning, and skeletal differentiation. We use fluorescence in situ hybridization to show the expression of Wnt ligands, Wnt receptors, and limb patterning genes in developing and regenerating limbs. Inhibition of Wnt ligand secretion permanently blocks limb bud outgrowth when treated early in limb development. Inhibiting Wnt signaling during limb outgrowth decreases the expression of critical signaling genes, including Fgf10, Fgf8, and Shh, leading to the reduced outgrowth of the limb. Patterns of gene expression are similar between developing and regenerating limbs. Inhibition of Wnt signaling during regeneration impacted patterning gene expression similarly. Overall, our findings suggest that limb development and regeneration utilize Wnt signaling similarly. It also provides new insights into the interaction of Wnt signaling with other signaling pathways during salamander limb development and regeneration.

A constitutively expressed fluorescent ubiquitination-based cell-cycle indicator (FUCCI) in axolotls for studying tissue regeneration.

Regulation of cell cycle progression is essential for cell proliferation during regeneration following injury. After appendage amputation, the axolotl (Ambystoma mexicanum) regenerates missing structures through an accumulation of proliferating cells known as the blastema. To study cell division during blastema growth, we generated a transgenic line of axolotls that ubiquitously expresses a bicistronic version of the fluorescent ubiquitination-based cell-cycle indicator (FUCCI). We demonstrate near-ubiquitous FUCCI expression in developing and adult tissues, and validate these expression patterns with DNA synthesis and mitosis phase markers. We demonstrate the utility of FUCCI for live and whole-mount imaging, showing the predominantly local contribution of cells during limb and tail regeneration. We also show that spinal cord amputation results in increased proliferation at least 5 mm from the site of injury. Finally, we use multimodal staining to provide cell type information for cycling cells by combining fluorescence in situ hybridization, EdU click-chemistry and immunohistochemistry on a single FUCCI tissue section. This new line of animals will be useful for studying cell cycle dynamics using in situ endpoint assays and in vivo imaging in developing and regenerating animals.

A matter of nerves.

Regrowing new body parts requires neural input to restore appropriately sized limbs in salamanders.

Imaging in vivo acetylcholine release in the peripheral nervous system with a fluorescent nanosensor.

The ability to monitor the release of neurotransmitters during synaptic transmission would significantly impact the diagnosis and treatment of neurological diseases. Here, we present a DNA-based enzymatic nanosensor for quantitative detection of acetylcholine (ACh) in the peripheral nervous system of living mice. ACh nanosensors consist of DNA as a scaffold, acetylcholinesterase as a recognition component, pH-sensitive fluorophores as signal generators, and α-bungarotoxin as a targeting moiety. We demonstrate the utility of the nanosensors in the submandibular ganglia of living mice to sensitively detect ACh ranging from 0.228 to 358 µM. In addition, the sensor response upon electrical stimulation of the efferent nerve is dose dependent, reversible, and we observe a reduction of ∼76% in sensor signal upon pharmacological inhibition of ACh release. Equipped with an advanced imaging processing tool, we further spatially resolve ACh signal propagation on the tissue level. Our platform enables sensitive measurement and mapping of ACh transmission in the peripheral nervous system.

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.

HDAC Inhibitor Titration of Transcription and Axolotl Tail Regeneration.

New patterns of gene expression are enacted and regulated during tissue regeneration. Histone deacetylases (HDACs) regulate gene expression by removing acetylated lysine residues from histones and proteins that function directly or indirectly in transcriptional regulation. Previously we showed that romidepsin, an FDA-approved HDAC inhibitor, potently blocks axolotl embryo tail regeneration by altering initial transcriptional responses to injury. Here, we report on the concentration-dependent effect of romidepsin on transcription and regeneration outcome, introducing an experimental and conceptual framework for investigating small molecule mechanisms of action. A range of romidepsin concentrations (0-10 µM) were administered from 0 to 6 or 0 to 12 h post amputation (HPA) and distal tail tip tissue was collected for gene expression analysis. Above a threshold concentration, romidepsin potently inhibited regeneration. Sigmoidal and biphasic transcription response curve modeling identified genes with inflection points aligning to the threshold concentration defining regenerative failure verses success. Regeneration inhibitory concentrations of romidepsin increased and decreased the expression of key genes. Genes that associate with oxidative stress, negative regulation of cell signaling, negative regulation of cell cycle progression, and cellular differentiation were increased, while genes that are typically up-regulated during appendage regeneration were decreased, including genes expressed by fibroblast-like progenitor cells. Using single-nuclei RNA-Seq at 6 HPA, we found that key genes were altered by romidepin in the same direction across multiple cell types. Our results implicate HDAC activity as a transcriptional mechanism that operates across cell types to regulate the alternative expression of genes that associate with regenerative success versus failure outcomes.

Combinatorial Transcriptional Profiling of Mouse and Human Enteric Neurons Identifies Shared and Disparate Subtypes In Situ.

BACKGROUND & AIMS: The enteric nervous system (ENS) coordinates essential intestinal functions through the concerted action of diverse enteric neurons (ENs). However, integrated molecular knowledge of EN subtypes is lacking. To compare human and mouse ENs, we transcriptionally profiled healthy ENS from adult humans and mice. We aimed to identify transcripts marking discrete neuron subtypes and visualize conserved EN subtypes for humans and mice in multiple bowel regions. METHODS: Human myenteric ganglia and adjacent smooth muscle were isolated by laser-capture microdissection for RNA sequencing. Ganglia-specific transcriptional profiles were identified by computationally subtracting muscle gene signatures. Nuclei from mouse myenteric neurons were isolated and subjected to single-nucleus RNA sequencing, totaling more than 4 billion reads and 25,208 neurons. Neuronal subtypes were defined using mouse single-nucleus RNA sequencing data. Comparative informatics between human and mouse data sets identified shared EN subtype markers, which were visualized in situ using hybridization chain reaction. RESULTS: Several EN subtypes in the duodenum, ileum, and colon are conserved between humans and mice based on orthologous gene expression. However, some EN subtype-specific genes from mice are expressed in completely distinct morphologically defined subtypes in humans. In mice, we identified several neuronal subtypes that stably express gene modules across all intestinal segments, with graded, regional expression of 1 or more marker genes. CONCLUSIONS: Our combined transcriptional profiling of human myenteric ganglia and mouse EN provides a rich foundation for developing novel intestinal therapeutics. There is congruency among some EN subtypes, but we note multiple species differences that should be carefully considered when relating findings from mouse ENS research to human gastrointestinal studies.

Spiny mice (Acomys) exhibit attenuated hallmarks of aging and rapid cell turnover after UV exposure in the skin epidermis.

The study of long-lived and regenerative animal models has revealed diverse protective responses to stressors such as aging and tissue injury. Spiny mice (Acomys) are a unique mammalian model of skin wound regeneration, but their response to other types of physiological skin damage has not been investigated. In this study, we examine how spiny mouse skin responds to acute UVB damage or chronological aging compared to non-regenerative C57Bl/6 mice (M. musculus). We find that, compared to M. musculus, the skin epidermis in A. cahirinus experiences a similar UVB-induced increase in basal cell proliferation but exhibits increased epidermal turnover. Notably, A. cahirinus uniquely form a suprabasal layer co-expressing Keratin 14 and Keratin 10 after UVB exposure concomitant with reduced epidermal inflammatory signaling and reduced markers of DNA damage. In the context of aging, old M. musculus animals exhibit typical hallmarks including epidermal thinning, increased inflammatory signaling and senescence. However, these age-related changes are absent in old A. cahirinus skin. Overall, we find that A. cahirinus have evolved novel responses to skin damage that reveals new aspects of its regenerative phenotype.

3D visualization of macromolecule synthesis.

Measuring nascent macromolecular synthesis in vivo is key to understanding how cells and tissues progress through development and respond to external cues. Here we perform in vivo injection of alkyne- or azide-modified analogs of thymidine, uridine, methionine, and glucosamine to label nascent synthesis of DNA, RNA, protein, and glycosylation. Three-dimensional volumetric imaging of nascent macromolecule synthesis was performed in axolotl salamander tissue using whole-mount click chemistry-based fluorescent staining followed by light sheet fluorescent microscopy. We also developed an image processing pipeline for segmentation and classification of morphological regions of interest and individual cells, and we apply this pipeline to the regenerating humerus. We demonstrate our approach is sensitive to biological perturbations by measuring changes in DNA synthesis after limb denervation. This method provides a powerful means to quantitatively interrogate macromolecule synthesis in heterogenous tissues at the organ, cellular, and molecular levels of organization.

Cells often respond to changes in their environment by producing new molecules and building new cell components, such as proteins, which perform most tasks in the cell, or DNA and RNA, which carry genetic information. Complex tissues ­ such as limbs, which are made up of muscles, tendons, bones and cartilage ­ are difficult to see through, so studying when and where cells in these tissues produce different types of molecules is challenging. New approaches combining advanced three-dimensional microscopy and fluorescent labelling of molecules could provide a way to study these processes within whole animal tissues. One application for this is studying how salamanders regrow lost limbs. When salamanders such as axolotls regrow a limb, some cells in the limb stump form a group called the blastema. The blastema contains cells that are specialized to different purposes. Each cell in the blastema produces many new proteins as well as new DNA and RNA molecules. Fluorescently labeling particular molecules and taking images of the regenerating limb at different times can help to reveal how these new molecules control and coordinate limb regrowth. Duerr et al. developed a three-dimensional microscopy technique to study the production of new molecules in regenerating axolotl limbs. The method labeled molecules of different types with fluorescent markers. As a result, new proteins, RNA and DNA glowed under different colored lights. Duerr et al. used their method to show that nerve damage, which hinders limb regrowth in salamanders, reduces DNA production in the blastema. There are many possible applications of this microscopy method. Since the technique allows the spatial arrangement of the cells and molecules studied to be preserved, it makes it possible to investigate which molecules each cell is making and how they interact across a tissue. Not only does the technique have the potential to reveal much more about limb regrowth at all stages, but the fluorescent markers used can also be easily adapted to many other applications.

Glycosaminoglycans compositional analysis of Urodele axolotl (Ambystoma mexicanum) and Porcine Retina.

Retinal degenerative diseases, such as age-related macular degeneration (AMD) and retinitis pigmentosa (RP), are major causes of blindness worldwide. Humans cannot regenerate retina, however, axolotl (Ambystoma mexicanum), a laboratory-bred salamander, can regenerate retinal tissue throughout adulthood. Classic signaling pathways, including fibroblast growth factor (FGF), are involved in axolotl regeneration. Glycosaminoglycan (GAG) interaction with FGF is required for signal transduction in this pathway. GAGs are anionic polysaccharides in extracellular matrix (ECM) that have been implicated in limb and lens regeneration of amphibians, however, GAGs have not been investigated in the context of retinal regeneration. GAG composition is characterized native and decellularized axolotl and porcine retina using liquid chromatography mass spectrometry. Pig was used as a mammalian vertebrate model without the ability to regenerate retina. Chondroitin sulfate (CS) was the main retinal GAG, followed by heparan sulfate (HS), hyaluronic acid, and keratan sulfate in both native and decellularized axolotl and porcine retina. Axolotl retina exhibited a distinctive GAG composition pattern in comparison with porcine retina, including a higher content of hyaluronic acid. In CS, higher levels of 4- and 6- O-sulfation were observed in axolotl retina. The HS composition was greater in decellularized tissues in both axolotl and porcine retina by 7.1% and 15.4%, respectively, and different sulfation patterns were detected in axolotl. Our findings suggest a distinctive GAG composition profile of the axolotl retina set foundation for role of GAGs in homeostatic and regenerative conditions of the axolotl retina and may further our understanding of retinal regenerative models.

Investigating Nrg1 Signaling in the Regenerating Axolotl Spinal Cord Using Multiplexed FISH.

Amputation of a salamander tail leads to functional spinal cord regeneration through activation of endogenous stem cells. Identifying the signaling pathways that control cell proliferation in these neural stem cells will help elucidate the mechanisms underlying the salamander's regenerative ability. Here, we show that neuregulin 1 (Nrg1)/ErbB2 signaling is an important pathway in the regulation of neural stem cell proliferation in the spinal cord of the axolotl salamander (Ambystoma mexicanum). Simultaneous localization of nrg1 mRNA and Nrg1 protein was performed by utilizing a hybridization chain reaction fluorescence in situ hybridization (FISH) methodology in tissue sections. Multiplexed FISH also permitted the phenotyping of multiple cell types on a single fixed section allowing the characterization of mRNA expression, protein expression, and tissue architecture. Pharmacological inhibition of ErbB2 showed that intact Nrg1/ErbB2 signaling is critical for adult homeostatic regeneration as well as for injury-induced spinal cord regeneration. Overall, our results highlight the importance of the NRG1/ErbB2 signaling pathway in neural stem cell proliferation in the axolotl.

Spinal Cord Regeneration in Amphibians: A Historical Perspective.

In some vertebrates, a grave injury to the central nervous system (CNS) results in functional restoration, rather than in permanent incapacitation. Understanding how these animals mount a regenerative response by activating resident CNS stem cell populations is of critical importance in regenerative biology. Amphibians are of a particular interest in the field because the regenerative ability is present throughout life in urodele species, but in anuran species it is lost during development. Studying amphibians, who transition from a regenerative to a nonregenerative state, could give insight into the loss of ability to recover from CNS damage in mammals. Here, we highlight the current knowledge of spinal cord regeneration across vertebrates and identify commonalities and differences in spinal cord regeneration between amphibians.

Tracking neural crest cell cycle progression in vivo.

Analysis of cell cycle entry/exit and progression can provide fundamental insights into stem cell propagation, maintenance, and differentiation. The neural crest is a unique stem cell population in vertebrate embryos that undergoes long-distance collective migration and differentiation into a wide variety of derivatives. Using traditional techniques such as immunohistochemistry to track cell cycle changes in such a dynamic population is challenging, as static time points provide an incomplete spatiotemporal picture. In contrast, the fluorescent, ubiquitination-based cell cycle indicator (Fucci) system provides in vivo readouts of cell cycle progression and has been previously adapted for use in zebrafish. The most commonly used Fucci systems are ubiquitously expressed, making tracking of a specific cell population challenging. Therefore, we generated a transgenic zebrafish line, Tg(-4.9sox10:mAG-gmnn(1/100)-2A-mCherry-cdt1(1/190)), in which the Fucci system is specifically expressed in delaminating and migrating neural crest cells. Here, we demonstrate validation of this new tool and its use in live high-resolution tracking of cell cycle progression in the neural crest and derivative populations.