A chromatin code for limb segment identity in axolotl limb regeneration.

The salamander limb correctly regenerates missing limb segments because connective tissue cells have segment-specific identities, termed "positional information". How positional information is molecularly encoded at the chromatin level has been unknown. Here, we performed genome-wide chromatin profiling in mature and regenerating axolotl limb connective tissue cells. We find segment-specific levels of histone H3K27me3 as the major positional mark, especially at limb homeoprotein gene loci but not their upstream regulators, constituting an intrinsic segment information code. During regeneration, regeneration-specific regulatory elements became active prior to the re-appearance of developmental regulatory elements. In the hand, the permissive chromatin state of the homeoprotein gene HoxA13 engages with the regeneration program bypassing the upper limb program. Comparison of regeneration regulatory elements with those found in other regenerative animals identified a core shared set of transcription factors, supporting an ancient, conserved regeneration program.

Navigation and Use of Custom Tracks within the Axolotl Genome Browser.

The availability of the chromosome-scale axolotl genome sequences has made it possible to explore genome evolution, perform cross-species comparisons, and use additional sequencing data to analyze both genome-wide features and individual genes. Here, we will focus on the UCSC genome browser and demonstrate in a step-by-step manner how to use it to integrate different data to approach a broad question of the Fgf8 locus evolution and analyze the neighborhood of a gene that was reported missing in axolotl - Pax3.

Gene and transgenics nomenclature for the laboratory axolotl-Ambystoma mexicanum.

The laboratory axolotl (Ambystoma mexicanum) is widely used in biological research. Recent advancements in genetic and molecular toolkits are greatly accelerating the work using axolotl, especially in the area of tissue regeneration. At this juncture, there is a critical need to establish gene and transgenic nomenclature to ensure uniformity in axolotl research. Here, we propose guidelines for genetic nomenclature when working with the axolotl.

Distinct toll-like receptor signaling in the salamander response to tissue damage.

BACKGROUND: Efficient wound healing or pathogen clearance both rely on balanced inflammatory responses. Inflammation is essential for effective innate immune-cell recruitment; however, excessive inflammation will result in local tissue destruction, pathogen egress, and ineffective pathogen clearance. Sterile and nonsterile inflammation operate with competing functional priorities but share common receptors and overlapping signal transduction pathways. In regenerative organisms such as the salamander, whole limbs can be replaced after amputation while exposed to a nonsterile environment. In mammals, exposure to sterile-injury Damage Associated Molecular Patterns (DAMPS) alters innate immune-cell responsiveness to secondary Pathogen Associated Molecular Pattern (PAMP) exposure. RESULTS: Using new phospho-flow cytometry techniques to measure signaling in individual cell subsets we compared mouse to salamander inflammation. These studies demonstrated evolutionarily conserved responses to PAMP ligands through toll-like receptors (TLRs) but identified key differences in response to DAMP ligands. Co-exposure of macrophages to DAMPs/PAMPs suppressed MAPK signaling in mammals, but not salamanders, which activate sustained MAPK stimulation in the presence of endogenous DAMPS. CONCLUSIONS: These results reveal an alternative signal transduction network compatible with regeneration that may ultimately lead to the promotion of enhanced tissue repair in mammals.

The giant axolotl genome uncovers the evolution, scaling, and transcriptional control of complex gene loci.

Vertebrates harbor recognizably orthologous gene complements but vary 100-fold in genome size. How chromosomal organization scales with genome expansion is unclear, and how acute changes in gene regulation, as during axolotl limb regeneration, occur in the context of a vast genome has remained a riddle. Here, we describe the chromosome-scale assembly of the giant, 32 Gb axolotl genome. Hi-C contact data revealed the scaling properties of interphase and mitotic chromosome organization. Analysis of the assembly yielded understanding of the evolution of large, syntenic multigene clusters, including the Major Histocompatibility Complex (MHC) and the functional regulatory landscape of the Fibroblast Growth Factor 8 (Axfgf8) region. The axolotl serves as a primary model for studying successful regeneration.

Giant lungfish genome elucidates the conquest of land by vertebrates.

Lungfishes belong to lobe-fined fish (Sarcopterygii) that, in the Devonian period, 'conquered' the land and ultimately gave rise to all land vertebrates, including humans1-3. Here we determine the chromosome-quality genome of the Australian lungfish (Neoceratodus forsteri), which is known to have the largest genome of any animal. The vast size of this genome, which is about 14× larger than that of humans, is attributable mostly to huge intergenic regions and introns with high repeat content (around 90%), the components of which resemble those of tetrapods (comprising mainly long interspersed nuclear elements) more than they do those of ray-finned fish. The lungfish genome continues to expand independently (its transposable elements are still active), through mechanisms different to those of the enormous genomes of salamanders. The 17 fully assembled lungfish macrochromosomes maintain synteny to other vertebrate chromosomes, and all microchromosomes maintain conserved ancient homology with the ancestral vertebrate karyotype. Our phylogenomic analyses confirm previous reports that lungfish occupy a key evolutionary position as the closest living relatives to tetrapods4,5, underscoring the importance of lungfish for understanding innovations associated with terrestrialization. Lungfish preadaptations to living on land include the gain of limb-like expression in developmental genes such as hoxc13 and sall1 in their lobed fins. Increased rates of evolution and the duplication of genes associated with obligate air-breathing, such as lung surfactants and the expansion of odorant receptor gene families (which encode proteins involved in detecting airborne odours), contribute to the tetrapod-like biology of lungfishes. These findings advance our understanding of this major transition during vertebrate evolution.

Introducing www.axolotl-omics.org - an integrated -omics data portal for the axolotl research community.

Genomic resources are indispensable for biological investigations in model organisms. In recent years, a number of genomic resources including a full genome assembly, extensive transcriptomic data, as well as genome editing has been developed for the axolotl, a classical model organism for developmental, neurobiological and regeneration studies, making the axolotl a highly versatile system. Here we describe the Axolotl-omics website that allows rapid ortholog searches, and access to genome and transcriptomic resources.

Towards computerized diagnosis of neurological stance disorders: data mining and machine learning of posturography and sway.

We perform classification, ranking and mapping of body sway parameters from static posturography data of patients using recent machine-learning and data-mining techniques. Body sway is measured in 293 individuals with the clinical diagnoses of acute unilateral vestibulopathy (AVS, n = 49), distal sensory polyneuropathy (PNP, n = 12), anterior lobe cerebellar atrophy (CA, n = 48), downbeat nystagmus syndrome (DN, n = 16), primary orthostatic tremor (OT, n = 25), Parkinson's disease (PD, n = 27), phobic postural vertigo (PPV n = 59) and healthy controls (HC, n = 57). We classify disorders and rank sway features using supervised machine learning. We compute a continuous, human-interpretable 2D map of stance disorders using t-stochastic neighborhood embedding (t-SNE). Classification of eight diagnoses yielded 82.7% accuracy [95% CI (80.9%, 84.5%)]. Five (CA, PPV, AVS, HC, OT) were classified with a mean sensitivity and specificity of 88.4% and 97.1%, while three (PD, PNP, and DN) achieved a mean sensitivity of 53.7%. The most discriminative stance condition was ranked as "standing on foam-rubber, eyes closed". Mapping of sway path features into 2D space revealed clear clusters among CA, PPV, AVS, HC and OT subjects. We confirm previous claims that machine learning can aid in classification of clinical sway patterns measured with static posturography. Given a standardized, long-term acquisition of quantitative patient databases, modern machine learning and data analysis techniques help in visualizing, understanding and utilizing high-dimensional sensor data from clinical routine.

Publisher Correction: Application and optimization of CRISPR-Cas9-mediated genome engineering in axolotl (Ambystoma mexicanum).

In the version of this protocol originally published, the recipe for CAS9 buffer was incorrectly identified as a recipe for sodium acetate solution, and vice versa. These errors have been corrected in the PDF and HTML versions of the paper.

Application and optimization of CRISPR-Cas9-mediated genome engineering in axolotl (Ambystoma mexicanum).

Genomic manipulation is essential to the use of model organisms to understand development, regeneration and adult physiology. The axolotl (Ambystoma mexicanum), a type of salamander, exhibits an unparalleled regenerative capability in a spectrum of complex tissues and organs, and therefore serves as a powerful animal model for dissecting mechanisms of regeneration. We describe here an optimized stepwise protocol to create genetically modified axolotls using the CRISPR-Cas9 system. The protocol, which takes 7-8 weeks to complete, describes generation of targeted gene knockouts and knock-ins and includes site-specific integration of large targeting constructs. The direct use of purified CAS9-NLS (CAS9 containing a C-terminal nuclear localization signal) protein allows the prompt formation of guide RNA (gRNA)-CAS9-NLS ribonucleoprotein (RNP) complexes, which accelerates the creation of double-strand breaks (DSBs) at targeted genomic loci in single-cell-stage axolotl eggs. With this protocol, a substantial number of F0 individuals harboring a homozygous-type frameshift mutation can be obtained, allowing phenotype analysis in this generation. In the presence of targeting constructs, insertions of exogenous genes into targeted axolotl genomic loci can be achieved at efficiencies of up to 15% in a non-homologous end joining (NHEJ) manner. Our protocol bypasses the long generation time of axolotls and allows direct functional analysis in F0 genetically manipulated axolotls. This protocol can be potentially applied to other animal models, especially to organisms with a well-characterized transcriptome but lacking a well-characterized genome.

Single-cell analysis uncovers convergence of cell identities during axolotl limb regeneration.

Amputation of the axolotl forelimb results in the formation of a blastema, a transient tissue where progenitor cells accumulate prior to limb regeneration. However, the molecular understanding of blastema formation had previously been hampered by the inability to identify and isolate blastema precursor cells in the adult tissue. We have used a combination of Cre-loxP reporter lineage tracking and single-cell messenger RNA sequencing (scRNA-seq) to molecularly track mature connective tissue (CT) cell heterogeneity and its transition to a limb blastema state. We have uncovered a multiphasic molecular program where CT cell types found in the uninjured adult limb revert to a relatively homogenous progenitor state that recapitulates an embryonic limb bud-like phenotype including multipotency within the CT lineage. Together, our data illuminate molecular and cellular reprogramming during complex organ regeneration in a vertebrate.

Author Correction: The axolotl genome and the evolution of key tissue formation regulators.

In the originally published version of this Article, the sequenced axolotl strain (the homozygous white mutant) was denoted as 'D/D' rather than 'd/d' in Fig. 1a and the accompanying legend, the main text and the Methods section. The original Article has been corrected online.

The axolotl genome and the evolution of key tissue formation regulators.

Salamanders serve as important tetrapod models for developmental, regeneration and evolutionary studies. An extensive molecular toolkit makes the Mexican axolotl (Ambystoma mexicanum) a key representative salamander for molecular investigations. Here we report the sequencing and assembly of the 32-gigabase-pair axolotl genome using an approach that combined long-read sequencing, optical mapping and development of a new genome assembler (MARVEL). We observed a size expansion of introns and intergenic regions, largely attributable to multiplication of long terminal repeat retroelements. We provide evidence that intron size in developmental genes is under constraint and that species-restricted genes may contribute to limb regeneration. The axolotl genome assembly does not contain the essential developmental gene Pax3. However, mutation of the axolotl Pax3 paralogue Pax7 resulted in an axolotl phenotype that was similar to those seen in Pax3-/- and Pax7-/- mutant mice. The axolotl genome provides a rich biological resource for developmental and evolutionary studies.

Planar cell polarity-mediated induction of neural stem cell expansion during axolotl spinal cord regeneration.

Axolotls are uniquely able to mobilize neural stem cells to regenerate all missing regions of the spinal cord. How a neural stem cell under homeostasis converts after injury to a highly regenerative cell remains unknown. Here, we show that during regeneration, axolotl neural stem cells repress neurogenic genes and reactivate a transcriptional program similar to embryonic neuroepithelial cells. This dedifferentiation includes the acquisition of rapid cell cycles, the switch from neurogenic to proliferative divisions, and the re-expression of planar cell polarity (PCP) pathway components. We show that PCP induction is essential to reorient mitotic spindles along the anterior-posterior axis of elongation, and orthogonal to the cell apical-basal axis. Disruption of this property results in premature neurogenesis and halts regeneration. Our findings reveal a key role for PCP in coordinating the morphogenesis of spinal cord outgrowth with the switch from a homeostatic to a regenerative stem cell that restores missing tissue.